ZB 37 / f/^ Digitized by Microsoft® CORNELL UNIVERSITY LIBRARY S^ 924 104 224 831 Digitized by Microsoft® This book was digitized by Microsoft Corporation in cooperation witli Cornell University Libraries, 2007. You may use and print this copy in limited quantity for your personal purposes, but may not distribute or provide access to it (or modified or partial versions of it) for revenue-generating or other commercial purposes. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® DIAGNOSTIC METHODS WEBSTER Digitized by Microsoft® Digitized by Microsoft® DIAGNOSTIC METHODS CHEMICAL, BACTERIOLOGICAL AND MICROSCOPICAL A Text-book for Students and Practitioners BY RALPH W. WEBSTER, M. D., Ph. D. ASSISTANT PROFESSOR OF PHARMACOLOGICAL THERAPEUTICS AND INSTRUCTOR IN MEDICINE IN RUSH MEDICAL COLLEGE, UNIVERSITY OF CHICAGO; DIRECTOR OF CHICAGO LABORATORY, CLINICAL AND ANALYTICAL FIFTH EDITION, REVISED AND ENLARGED WITH 37 COLORED PLATES AND 171 OTHER ILLUSTRATIONS "'■o I \ PHILADELPHIA BLAKISTON'S SON & CO, 1012 WALNUT STREET Digitized by fVHcrosoft® No.5:^i°i FiEST Edition, Copyright, 1909, by P. Blakiston's Son & Co. Second Edition, Copyright, 191 2, by P. Blakiston's Son & Co. Thibd Edition, Copyright, 1913, by P. Blakiston's Son & Co. Fourth Edition, Copyright, 1914, by P. Blakiston's Son & Co. Fifth Edition, Copyright, 1916, by P. Blakiston's Son & Co. W 3S THE MAPLE PRESS YORK PA ^ Digitized by Microsoft® V/ TO THE MEMORY OF MY PATHER DR. JOHN RANDOLPH WEBSTER THIS VOLUME IS LOVINGLY DEDICATED Digitized by Microsoft® Digitized by Microsoft® PREFACE TO THE FIFTH EDITION During the last year, investigation along all scientific lines has been greatly curtailed for self-evident reasons. However, sufficient new material, both as regards methods and general points of attack, is at hand to warrant quite extensive changes in the subject matter of this work. Among the additions will be found Petroff's method of obtaining pure cul- tures of tubercle bacilli; a discussion of the Endameba gingivalis; description and methods of study of Bacillus rhinitis; Wolff-Junghans' test for gastric carcinoma; Wagner's "dry test" for occult blood; Phenoltetrachlorphthalein test of hepatic activity; Urease method for urea; Folin's new method for crea- tinin in urine; Benedict and Murlin's method for amino-acids in urine; Folin and Denis' micro-chemical methods for non-protein nitrogen, urea and uric acid in the blood; Schick diphtheria toxin reaction; Bronfenbrenner's modifi- cation of Abderhalden's test; and tests of blood before transfusion. The subject matter throughout the text has been brought up to date by the inclu- sion of a large number of references to the original literature. The author wishes to express his extreme gratification at the reception of his former editions and, also, his deepest thanks for any and all criticisms which he has obtained from his reviewers and friends. He desires, especially, to acknowledge his indebtedness to Dr. F. B. Moorehead for his kindly criti- cism of the section on Endameba gingivalis. Ralph W. Webster. 25 E. Washington St., Chicago. Digitized by Microsoft® Digitized by Microsoft® PREFACE TO THE FOURTH EDITION Although less than a year has elapsed since the appearance of the third edi- tion of this work, the author has felt justified in adding considerable new material in this revision. This has come about largely through the sugges- tions of teachers and general practitioners regarding the inclusion of certain subjects not hitherto discussed. Thus, the new matter embraces, among other subjects, a discussion of the Negri bodies recently successfully cultivated by Noguchi; Weisz's test for urochromogen; Dorrance's method for coagulation of the blood; Kowarsky's method for glucose in the blood; Levaditi and Manouelian's as well as Nogu- chi's methods for staining spirochsete in tissue; Lange's colloidal-gold test for congenital syphilis; a discussion of the organism of anterior poliomyelitis recently cultivated by Flexner and Noguchi. The Wassermann test has been brought up to date by inclusion of considerable new matter; while the complement-fixation test in gonorrhea has been elaborated. The Her- man-Perutz reaction for syphilis is fully described. The application of Abderhalden's dialyzation method to the diagnosis of cancer and dementia praecox has been freely discussed. Further, an entirely new chapter on Clinical Bacteriology has been added, in which are . discussed the differen- tiation of the more important pathogenic organisms and, also, the methods of preparation of vaccines. The subject matter throughout the text has been brought up to date by the inclusion of a large number of references to the original literature. The author wishes to express his great satisfaction at the reception of the third edition of his work and, also, his full appreciation of the kind sugges- tions offered him regarding the general matter of the book. He wishes, especially, to thank his colleagues Drs. T. L. Dagg and C. C. Croy for their many valuable suggestions and criticism. Ralph W. Webster. 2$ E. Washington St., Chicago. Digitized by Wlicrosoft® Digitized by Microsoft® PREFACE TO THE FIRST EDITION In the present work the writer has attempted to bring together, for the use of the student and practitioner, the generally accepted facts regarding the various phases of clinical medicine, which may be rather more closely studied by the application of laboratory methods than without their use. It is to be especially emphasized that laboratory work must go hand in hand with the more direct clinical examination of the patient, as the former can be inter- preted only in the light of the latter. While it is true that, in some cases, the laboratory findings may be of even more value than those of physical ex- amination, yet it is to be understood that the function of the Clinical Labora- tory is, more largely, perhaps, an accessory one, the application of methods of physical diagnosis usually pointing out the way toward a successful solution of the clinical problem by special laboratory methods, which yield numerous confirmatory or differential points not at all clearly defined by the methods of direct clinical examination. The aim of the author has been to present the direct bearing of the various methods outlined upon the clinical history of the case and to point out the special interpretation of the findings in any given examination. Particular attention has been directed to the selection of methods, both for the simpler clinical and more complex scientific requirements. As no method, no matter how exact it may be or how sound its basic principle, will yield reliable re- sults in the hands of the inexperienced, the writer has paid much attention to the details of such methods and has endeavored to direct the thought of the worker to the possible obstacles to be overcome before he is able properly to perform these examinations and interpret his results. The writer makes no claim for originality except, possibly, in the matter of arrangement of subject-matter and selection of methods and ideas, which have been established by others after years of earnest research. His endeavor has been to be as catholic as possible in his reading and as selective as his judgment permitted, to the end that the student or practitioner be saved the burden of sifting the wheat from the chaff. • Numerous text-books, monographs, and special articles have been freely used in the preparation of the text, the writer attempting in each case to give due credit for such reference. It Is possible that some direct use of material has been made which has not received deserved recognition. If so, the writer here acknowledges his indebtedness to them as well as to those whom he has directly quoted. It has seemed desirable to omit extensive reference to the literature, as a bibliography to be of working value must be much more extensive and com- xi Digitized by Microsoft® Xll PREFACE plete than is possible within the scope of this book. The writer has, however, inserted at the end of each chapter a list of the more important larger works which he has found useful in correlating the general subjects included in the several sections. In conclusion, the writer wishes to express his deep obligation to his col- leagues for their many valuable suggestions as to the subject-matter of the text: to Prof. W. S. Haines for assistance in revising the section on Urine; to Dr. J. M. Washburn for many additions and revisions of the section on Blood; to Dr. O. J. West for many practical points throughout the whole work; to Dr. W. A. Pusey for photographs of Blastomycetes under various conditions; to Dr. Brown Pusey for slides showing various organisms in the conjunctival exudates; and to Dr. N". Gildersleeve, of the University of Pennsylvania, for illustrations of megalosporon and microsporon. Further, the writer wishes to express his thanks to Miss Hill for the excellent work done by her in pre- paring the original drawings which appear throughout the work. Ralph W. Websteb. Digitized by Microsoft® TABLE OF CONTENTS CHAPTER I The Sputum Page I. General Considerations i II. Physical and Chemical Characteristics 2 Amount ,, 2 Consistency 2 Reaction. 3 Color 3 Odor 4 Character 4 Chemistry 5 III. Macroscopic Examination 6 Cheesy masses 6 Dittrich's plugs 6 Curschmann's spirals 7 Fibrinous casts 8 Concretions 8 Bronchioliths 8 Pneumoliths 8 ' Echinococcus membranes . . ... 9 Foreign bodies . 9 IV. Microscopic Examination 9 Pus-cells 10 Red blood-cells .... . 11 Epithelial cells . 11 Elastic tissue . . . . 12 Crystals 13 Bacteria. 14 Saprophytes 14 Pathogenic types 17 Tubercle bacillus 17 Lepra bacillus 24 Smegma bacillus 24 Timothy bacillus . 24 Pneumococcus .25 Friedlander's bacillus .... 25 Influenza bacillus . 26 BacOlus pertussis .... ... . . 26 Bacillus typhosus . . ... .... 26 Staphylococcus and streptococcus pyogenes . . . .26 Bacillus pestis . . .... 27 Bacillus anthracis ... 27 Bacillus mallei . . . .... .... 27 Actinomyces hominis .... 27 xiii Digitized by Microsoft® XIV TABLE OF CONTENTS Page Animal parasites . ^° Amebae ... 28 Flagellates . . ... 28 Cestodes. . . . ... • 29 Tfematodes . . ... 29 V. The Sputa in Disease . . . 30 Pulmonary tuberculosis ... • 3° Croupous pneumonia 3i Broncho-pneumonia . ... 31 Acute bronchitis . ... 31 Chronic bronchitis ■ ■ • 32 Simple type 32 Putrid type ■ 32 Fibrinous type 32 Bronchial asthma . . 33 Influenza . . 33 Gangrene of the lung 33 Abscess of the lung . 33 Perforating empyema 34 Pneumonoconioses . . 34 CHAPTER II Oral, Nasal, Aural and Conjunctival Secretions I. Oral Secretion . . 35 General considerations. . . 35 Microscopic examination . . . ... -36 Pathologic changes ... ... 37 Pharyngomycosis leptothrica . . 38 Diphtheria . . . . . . 38 Vincent's angina . . . 40 Streptococcic sore throat. . 40 Gonorrheal stomatitis . Thrush . Oral endamebiasis II. Nasal Secretion . . ... General considerations. Rhinitis 41 41 42 44 44 Pathologic changes . . . ... 44 45 Hay fever . ...... . . 45 Meningitis . . . ... .45 III. Aural Secretion . .• . ■ . 46 General considerations. . 46 Pathologic changes . . _ .5 IV. Conjunctival Secretions .^ General considerations. . . . > .y Pathologic changes . .... ... 47 Diphtheritic conjunctivitis . . .... 4,7 Infectious conjunctivitis . . .» Gonorrheal conjunctivitis . . .g Trachoma .... . .g Vernal cogp^g^|j^^.^^^^^^@ [[ [ [ ^^ TABLE OF CONTENTS XV CHAPTER III Gastric Contents Page I. General Considerations 50 II. Methods of Obtaining Gastric Contents 52 Stomach-tube 52 Test meals . 54 Ewald meal . . 54 Boas meal . 55 Riegel meal 55 Fischer meal . . 55 Salzer meal . 56 Sahli meal . . . . 56 III. Macroscopic Examination . 57 Amount . 57 Color . . 58 Odor . . . 59 Consistency . . . 59 Contents from fasting stomach ... 60 Vomitus 60 Contents after test meals . . 61 IV. Microscopic Examination 62 General 62 Pood remnants 62 Boas-Gppler bacillus . . 62 Sarcinas ventriculi 63 Protozoa. .... 63 Tissue fragments 63 Crystals 63 V. Chemical Examination. . 63 General . 64 Total acidity . 64 Free hydrochloric acid . . 65 Qualitative tests 66 Topfer's test . 66 Gunzburg's test . . 66 Boas' test . ... 67 Tropeolin test . . 67 Quantitative tests. 67 Mintz's method 68 Topfer's method . . 68 Amount of free hydrochloric acid 69 Euchlorhydria 70 Hypochlorhydria 70 Ana-chlorhydria . 7° Hyperchlorhydria 71 Combined hydrochloric acid 71 Method of Martius and Luttke " 71 Method of Topfer 72 Hydrochloric acid deficit .' 73 Organic acids. . 73 Total organic acids 73 Lactic acid-. ........ . ... x„-„«/k\ 74 ''hiOitiz&dby Microsoft® Imann s test < Uflfelmanh s tfest ' . . . 75 XVI TABLE OF CONTENTS Page 75 75 76 77 77 77 Kelling's test. ... Strauss' method ... . Butyric acid . Acetic acid . . . Gastric ferments . . . . Pepsin Qualitative methods 7* Quantitative examination 79 Hammerschlag's method 79 Mett's method . 79 Method of Thomas and Weber 80 Chymosin . . 81 Leo's method. . . 81 Riegel's method ... 81 Lipase ... 8^ Products of protein digestion 82 Products of carbohydrate digestion 82 Blood . . . . . . . Gases . . ' , Function of the stomach and contents . VI. Motility of the Stomach Leube's method . . Boas' method Method of Ewald and Sievers . Winternitz' test . .... VII. Absorptive Power of the Stomach . . . Potassium iodide test . VIII. Indirect Examination of the Stomach Contents , Giinzburg's method . . . . . Sahli's desmoid reaction . . . IX. Gastric Juice in Disease . . Hyperchlorhydria . .... Hypersecretion . . ... Achylia gastrica Acute gastritis . . . . Chronic gastritis Nervous dyspepsia . Ulcer of the stomach . . Carcinoma of the stomach Salomon's test ... . . . . . Neubauer and Fischer's test . . . . Wolff and Junghans' test CHAPTER IV 83 83 84 84 85 85 85 86 86 86 87 87 87 83 88 89 89 90 90 90 91 91 93 93 95 The Feces I. General Considerations. . . Normal feces. . . Diet of Schmidt and Strasburger DietofPolin. . Obtaining intestinal juice Functions of i Estimation orintestinal" XX TABLE OF CONTENTS Determination of inorganic sulphates Purdy's centrifugal method Carbonates Sodium and potassium . Calcium and magnesium Iron Organic constituents . Nitrogenous bodies Total nitrogen Kjeldahl's method Urea ... Determination of urea. Knop-Hufner method Doremus ureometer . Polin's method . Morner-Sjoqvist method Urease method .... Ammonia . Quantitative determination Method of Schlosing Polin's method . Formalin method . Uric acid. . Quantitative determination .. Folin's method . Salkowski-Ludwig method . . Method of Rudisch and Kleeberg Ruhemann's method Purin bases . Creatinin . Qualitative tests Weyl's test. Jaffa's test . Quantitative determination . Polin's methods. Undetermined nitrogen Amino acids Hippuric acid Oxyproteic and alloxyproteic acids AUantoin . Fatty acids . Oxalic acid . Quantitative determination . Baldwin's method. . Ferments ... Pepsin . Diastase . Lipase . Mucin-Hke bodies Mucin. . . . . Nucleo-albumin . . . Pigments and, chrqmogens ^^ Ri3M§ffnQy IViia-osoft® Uroerythrin Page . 210 . 210 . 211 211 211 213 . 213 213 213 , 217 , 219 221 222 222 223 225 225 227 228 229 230 231 232 235 235 236 237 239 240 241 243 243 243 243 243 246 246 248 248 248 249 250 251 251 251 251 251 252 252 252 252 254 254 254 TABLE OF CONTENTS XXI Page Urobilin 255 Indican ... 256 Tests for indican 257 Jaffa's test 257 Obermayer's test 257 Rosenbach's test . . 258 Quantitative determination . ... .... 259 Wang's method . ... . . . . 259 Polin's method 260 Uroroseinogen 260 Abnormal Composition . . .... 260 Proteins . . 260 Serum-albumin. . 261 Albuminuria 261 Functional. 261 Febrile . . ... 264 Traumatic . . .... 265 Hematogenous . . . . . . 265 Toxic . 265 Neurotic. . . . 265 With definite renal lesions . ... .265 Qualitative tests . . . .... 266 Heat and acid test '. . 267 Heller's nitric acid test . . . 268 Perrocyanide test . . . . . .271 Sulpho-salicylic acid test . . . . . 271 Spiegler's test . . 271 Quantitative methods . ......... . . 272 Scherer's method . . . 272 Esbach's method ... . . 272 Method of Tsuchiya . . . . . 273 Purdy's centrifugal method . . . . 274 Removal of albumin . . . . .274 Serum-globulin . . . 274 Qualitative tests . -275 Quantitative method . ... 276 Proteoses . . .... 276 Primary proteoses ... 276 Bence- Jones' protein . . 276 Secondary proteoses . 278 Tests . . . . . . 279 Bang's method . . . . 279 Clinical significance . . 279 Peptone . ... . . 281 Hemoglobin . . ... .281 Heller's test . . . .282 Donogany's test . . . 282 Fibrin. . ... 283 Carbohydrates 283 Glucose . . .284 Glycosuria . . . 284 Qualitative tests . . . . . 288 '^^Efism'^^ -Microsoft® ^88 XXll TABLE OF CONTENTS Benedict's test . . . Fehling's test. . Haines' test Alm^n-Nylander's test Fermentation test . . Phenyl-hydrazin test Quantitative tests . . Fehling's method Bang's method . Purdy's method Haines' method. Polariscopic method Fermentation method Roberts' method Levulose. . . . Levulosuria Seliwanoff's test Phenyl-methyl-hydrazin test Pentose . ... Pentosuria . . . QuaUtative tests Tollen's reaction Orcin test . Quantitative test . . . Diphenyl-hydrazin method Cammidge's reaction Lactose . . ... - Lactosuria Rubner's test. . Maltose ... Maltosuria . . Glycuronic acid. Neuberg's quantitative method . Acetone bodies Acetone Qualitative tests Legal's test Lieben's test Gunning's test . . Frommer's test . Quantitative methods Huppert-Messinger method Polin's method Diacetic acid . Qualitative tests Gerhardt's test . Arnold's test . Lipliawsky's test . . /S-oxybutjrric acid . Quantitative determination Black's method. . Shaffer's method . AbnorrriSygs^S^^y Microsoft® Blood pigments . TABLE OF CONTENTS XXIU Page Hemoglobin . . . 328 Hematoporphyrin . . . . 328 Biliary pigments ...... . . . 329 Qualitative tests ... . 329 Smith's test . 33° Gmelin's test . . 330 Rosenbach's test . . 330 Nakayama's test . . . 330 Hammarsten's test . , • • 33i Bile acids . 331 Hay's test . . 331 Oliver's test . 332 Melanin . . . . 332 Phenol derivatives . . 332 Alkapton .... . .... 333 Ehrlich's diazo reaction . . . 334 Russo's reaction .... ... 335 Dimethyl-amino-benzaldehyd reaction 336 IV. Microscopic Examination . . 336 Unorganized sediments , . .... 338 Those appearing in acid urine 338 Uric acid . .... 338 Sodium acid urate . . . . . 339 Potassium acid urate . . .... 340 Xanthin. . . .... 341 Calcium oxalate . . 341 Cystin. . . . . 342 Cystinuria . . 342 Leucin. . . 343 Tyrosin . . . . 344 Calcium sulphate . . . 345 Bilirubin 345 Hippuric acid . 345 Neutral calcium phosphate . . 346 Pat . . . . . 346 Chyluria. ... . ... 346 Those appearing in alkaline urine . . ... 347 Ammonium urate . ... 347 Calcium tri-phosphate . . . 347 Magnesium phosphate . . 348 Magnesium-ammonium phosphate 348 Calcium carbonate 349 Organized sediments . 349 Mucoid material . 349 Epithelial cells 349 Pus-cells. . . 351 Pyuria. . 351 VitaU's test . . 353 Donne's test 354 Enumeration of pus-cells . . 354 Red blood-cells . 354 Hematuria. . 354 Casts . . . Digitized by Microsoft® 356 True casts . . . 356 XXIV TABLE OF CONTENTS Hyaline casts. . Granular casts . Waxy casts . . Fibrinous casts . Epithelial casts . Fatty casts . Blood-casts . Pus-casts . Cylindroids . . Pseudo-casts . Cylindruria Spermatozoa . Tissue fragments Bacteria . . BaciUuria . , Parasites ... V. Calculi Heller's table for analysis Uric acid calculi Calcium oxalate calculi Phosphatic calculi Calcium carbonate calculi . . Cystin calculi . Xanthin calculi . . Urostealith calculi . VI. Functional Diagnosis . Cryoscopy . Electric conductivity Chlorid excretion . Methylene blue test . . . Phloridzin test . . Phenolsulphonephthalein test . Page • 356 ■ 357 358 • 359 ■ 359 • 359 ■ 359 359 360 361 361 363 363 363 366 366 367 368 368 368 368 370 370 370 370 370 371 371 372 372 373 373 CHAPTER VII Secretion of the Genital Organs I. Male Secretions . . . . General considerations. . . . . Microscopic examination. ... Pathologic variations . . , . . Medico-legal aspects. .... Florence's test Barberio's test . . ... II. Female Secretions . Vaginal secretions. . . Microscopic examination. Pathology . Blenorrhea Purulent secretions . . . Fetid secretions Uterine secretions. ,, „,.. . „^. Menstrfiwecf by M/croso/?® The lochia ... ... . 376 . 376 378 378 ■ 379 379 380 350 . 380 . 381 . 381 . 382 . 382 . 382 . 382 ■ 383 TABLE OF CONTENTS XXV Page Amniotic fluid ... .... 383 Abortion 384 Vesicular mole 384 Carcinoma 384 CHAPTER VIII The Blood I. General Considerations . . . . 385 II. Physiology and Chemistry . 386 Blood formation and blood-forming organs . .... 386 Total volume of blood ... ... . . . . 387 Volume relations of cells to plasma . . 390 Methods of obtaining blood ... . .... 392 Physical properties . . .... 393 Color ... 394 Odor . . •394 Reaction. . . .... 395 Specific gravity . . 399 Viscosity. . . . . 401 Coagulation . . .401 Osmotic pressure and cryoscopy . . . . 405 Electric conductivity . . . ... 406 Chemical properties . . 407 Total solids 409 Blood pigments . 409 Hemoglobin . 409 Pseudo-hemoglobin 411 Oxy-hemoglobin . ..411 Met-hemoglobin 411 Carbon monoxid-hemoglobin .... . 412 Carbon-dioxid-hemoglobin . . .... 412 Sulph-hemoglobin . . ... 412 Decomposition products . . . . ... 413 Hematin. . . 413 Hematoporphyrin . . 414 Hematoidin . . . .... 414 Hemosiderin 415 Malarial pigment 415 Estimation of hemoglobin 415 Direct methods . 416 Indirect methods . . 416 Hemometer of Fleischl-Miescher 417 Hemoglobinometer of Dare . . . . .420 Hemometer of Sahli. . . ... 421 Hemoglobinometer of Oliver. . 422 Hemoglobinometer of Tallqvist 424 Variations in amount of hemoglobin . ... . .425 Ohgo-chromemia . . ... . . 426 Color index .... . 426 Proteins of the blood .... 427 Other riitro^^^xQ^y^jf^soft® 430 Total nitrogen .... 430 XXVI TABLE OF CONTENTS Total non-protein nitrogen . Urea Uric acid . . . Ammonia Creatinin Amino-acids Carbohydrates Pats and fatty acids . . Acetone Biliary constituents . Inorganic constituents . Blood gases . . Ferments of the blood . . Enumeration of the cells. . ... Hemocytometer of Thoma-Zeiss . Hemocytometer of Durham Hemocytometer of Oliver . III. Morphology of the Blood Examination of fresh blood Preparation of smears . Fixation of smears . Staining methods . Erythrocytes ... Appearance and structure . . . Size and shape . Nucleation Number ... ... Normal variations . . Pathological variations Oligo-cythemia . Poly-cythemia Staining properties . . . Degenerations Isotonicity and resistance . . Variations in childhood and old age . Functions Leucocytes , .... Appearance . . Types in normal blood . Lymphocytes. . . Large mononuclears Polymorphonuclear neutrophiles Polymorphonuclear eosinophiles Polymorphonuclear basophiles . Types in pathological blood Myelocytes Irritation forms . Degeneration forms Differential counting Number . . Leucocytosis Physiological . . Eas^'a^fciiy. Microsoft® Mixed leucocytosis . . Page 430 435 439 443 444 445 445 449 451 451 452 454 454 • 455 • 456 • 467 . 468 • 469 470 471 473 476 486 486 488 490 492 492 495 495 495 496 498 499 500 501 502 502 502 502 503 504 506 506 507 507 509 509 510 511 512 513 515 518 TABLE OF CONTENTS XXVU Page Lymphocytosis 518 Eosinophilia 519 Mast-cell type 52 1 Leucopenia . . . . 521 Variations in infancy and childhood . . 521 Functions ... . 522 Blood-plates ... ... 523 Appearance ... 523 Size . . 524 Number . . . . 524 Staining properties 524 Function ... .... 525 Hemoconien . . 525 Morphology of the blood-forming organs 525 IV. Pathology of the Blood . 528 Special . ... 528 Anemia . 528 Primary . 528 Simple primary anemia . 528 Chlorosis . . 529 Progressive pernicious anemia 531 Splenic anemia . 532 Anemia infantum pseudo-leukemica 533 Leukanemia ... . 534 Aplastic anemia 534 Secondary 534 Acute hemorrhage . . . 535 Chronic hemorrhage 536 Inanition .... 536 Intestinal parasites 537 Fever 538 Blood poisons . . . . 538 Leukemia 539 Spleno-myelogenous type 539 Lymphatic type 543 Acute type . 543 Pseudo-leukemia ... .... 544 Hodgkin's disease 544 Tuberculosis of the lymph glands . . . 545 Lympho-sarcoma . 545 Gummatous lymphoma . 545 General 546 Blood changes following surgical intervention 546 Constitutional diseases ... . 547 Diabetes mellitus ... . . . 547 Gout . 548 Addison's disease . . . ... .... 548 Rickets . . 549 Myxedema 549 Acute infections . . . 549 Pneumonia. ... 550 Typhoid fever . . . 552 Scarlet fever 553 MeaslQigiti^e.d .by Microsoft® 554 XXVIU TABLE OF CONTENTS Variola . . Diphtheria . . Pertussis . . . Rheumatism . Chronic infections' Tuberculosis . Syphilis . Leprosy . Carcinoma ... Effects of splenectomy. V. Parasitology of the Blood . . Malaria Examination of fresh blood . Tertian parasite . Quartan parasite . . Estivo-autumnal parasite Stained specimens Sporogony General hematological changes Relapsing fever . Sleeping sickness Kala-azar . . Pilariasis Syphilis . Cultivation of treponema pallidum Yellow fever . . Rocky mountain spotted fever Distomiasis VI. Bacteriology of the Blood Technic Organisms found in the blood VII. Serum Pathology Ehrlich's side-chain theory Phagocytosis Opsonins . Diagnostic use of vaccines Tuberculin reactions Luetin reactions Schick reaction . VIII. Sero-Diagnosis . . Agglutination reactions Gruber-Widal test . Method of Bass and Watkins. Diseases other than typhoid Precipitin reaction ... Complement-fixation test Wassermann reaction Noguchi method . . Diseases other than syphilis Abderhalden's sero-diagnosis . Of pregnancy. . . Of other conditions . Herman-Perutz reaction . Tests heiB^iiai&SakiyirMicrosoft® 555 55& 55& 557 557 557 55& 559 559 56a 561 561 563 56J 565 566 568 57» 572 574 575 576 577 578 581 58J 584 585 585 585 587 59a 594 598 598 60a 601 603. 605 606 607 607 611 61J 614. 617 620- 629 636 638 63» 649 651 653 TABLE OF CONTENTS XXIX Page IX. Medico-Legal Aspects 654 Red cells 654 Guaiac test 655 Schaer's test 655 Phenolphthalin test 656 Teichmann's test 657 Spectroscopic examination 658 Precipitin test ' 658 X. Value and Limitations of Blood Examinations 659 CHAPTER IX Transudates and Exudates I. General Considerations ... 662 II. Physical and Chemical Properties 663 Serous exudates 664 Chylous exudates 665 Chyloid exudates 665^ Hemorrhagic exudates . . 665 Purulent exudates. . 666 Putrid exudates 666 III. Bacteriology . ... 667 Tubercle bacilli 667 Inoscopy. . 667 Gonococci 667 Smegma bacilli . : . . . 669 Ducrey's bacilli . 669 Spirochete pallidas . ... .... 669 IV. Cytology . ... 672 Technic . . . .... .... 672 Cytology of normal fluids .... . 673 Cytology of pathological fluids 673 Pleural exudates 674 Primary tubercidar pleurisy 674 Secondary tubercular pleurisy . . 674 Pneumococcus pleurisy . . 674 Streptococcus pleurisy . . 675 Typhoid pleurisy 675 Malignant pleurisy . . 675 Nephritic and cardiac pleurisy .... 675 Peritoneal exudates ... . . .... 675 V. Cyst Fluids 676 Ovarian cysts 676 Serous cysts 676 Myxoid or colloid cysts ... . 676 Papillary cysts 677 Dermoid cysts 677 Parovarian cysts ... 677 Hydrocele . . 677 Spermatocele . ... .... 678 Hydronephrosis 678 Hydatid cysts ^. ... . ,., . ,,.. . . ,,^ . 678 Pancreatic cysl^'S'^'zeCpy/W/croso/?® 678 XXX TABLE OF CONTENTS Page VI, Cerebrospinal Fluid ^78 Lumbar puncture . v ... "79 Microscopic examination. ... ' °°2 Epidemic cerebrospinal meningitis . , 682 Tubercular meningitis . . . 684 Acute anterior poliomyelitis 684 Cerebrospinal syphilis 685 ' Noguchi's butyric acid test. 685 Nonne's test 685 Ross- Jones test . . . 686 Lange's colloidal-gold test 686 CHAPTER X Secretion of the Mammary Glands I. General Considerations . . . . . 688 II. Physical and Chemical Properties . 689 Appearance and color . . . . 690 Specific gravity . 690 Reaction. ... . 691 Coagulation . . 691 Total solids . . . . 691 Ash. . . . ..... 691 Protein . . . . . . '. 691 Total protein . . 692 Method of Sebelien . 692 Method of Boggs . 692 Casein. . . 693 Albumin and globulin ... 693 Pat . 693 Babcock's method . . ... 693 Extraction method 694 Lactose . . . . ... 695 Preservatives in cow's milk ... 695 Sodium carbonate. . . . 695 Salicylic acid ... 696 Formaldehyde ^ 696 Boric acid and borax 696 III. Bacteriological Examination of Milk . 696 CHAPTER XI Clinical Bacteriology I. General Considerations 699 II. Sterilization . . ... 700 III. Preparation of Culture Media . . 701 IV. Incxjbation . ... 704 V. Preparation of Cultures . . 705 VI. Staining *■••«. 707 VII. Identification fi^'fi(^^4M#'crosp/?® ! ^ ". 709 TABLE OP CONTENTS XXXI Page VIII. Vaccines 723 Preparation of vaccines 724 Stock vaccines . .' 724 Mixed vaccines . . 725 Autogenous vaccines . . -725 Anti-typhoid vaccination 726 Index. . . .... . 728 Digitized by Microsoft® Digitized by Microsoft® LIST OF ILLUSTRATIONS PLATES I. Tubercle Bacilli in Sputum . II. Streptococcus Pyogenes . III. Leptothrix and Spirocheta Buccalis (Unstained) IV. Diphtheria Bacilli Showing Polar Staining . V. Endamebae Gingivalis (Gros.) . . VI. Koch- Weeks Bacillus. Morax-Axenfeld Diplobacillus . VII. Trachoma Bodies of Prowazek-Greeff VIII. Vegetable Cells found in Feces . IX. Osazons . .... X. Ammonium Urate Crystals XI. Waxy Casts ... XII. Mucous Threads in Urine (Unstained) XIII. Cystitis Due to Colon' Bacillus . ... XIV. Staphylococcus Cystitis XV. Absorption Spectra ... XVI. Absorption Spectra ... XVII. Fresh Normal Blood XVIII. Types of Red Cells . . XIX. Ring Bodies in Red Cells XX. The Leucocytes ... XXI. lodophilia XXII. Polynuclear Leucocytosis XXIII. Chlorotic Anemia . . . XXIV. Blood in Pernicious Anemia . XXV. Blood in Leukanemia ... XXVI. Blood in Spleno-myelogenous Leukemia XXVII. Lymphatic Leukemia XXVIII. The Tertian Parasite (Unstained) . XXIX. The Quartan Parasite (Unstained) . . . XXX. The Estivo-autumnal Parasite (Unstained) XXXI. Tertian Parasite (Stained) ... XXXII. Estivo-autumnal Parasite (Stained) . . XXXIII. Gonococci in Urethral Discharge .... XXXIV. Spirochete 'P^^^^'^^^ftj^Bp Microsoft® ■ To Face Page 20 26 35 39 42 47 47 48 iSS 294 347 359 361 366 367 410 411 470 488 498 502 509 512 529 531 534 540 543 563 565 567 568 570 668 670 XXXIV LIST or ILLUSTRATIONS To Face Page XXXV. Exudate from Tubercular Pleurisy 674 XXXVI. Exudate in Pneumonic Pleurisy 675' XXXVII. Exudate in Malignant Pleurisy . . 676 FIGURES 1. Curschmann's Spirals . . . 2. Objects Found in the Sputum . 3. Aspergillus Fumigatus 4. Micrococcus Catarrhalis. . . . 5. Budding Forms of Blastomycetes 6. Diplococcus Pneumoniae. . 7. Friedlander's Bacillus . 8. Bacillus Influenzae . g. Actinomyces 10. Paragonimus Westermanii .... 11. Ovum of Paragonimus Westermanii 12. Vincent's Spirillum and Bacillus . 13. Oidium Albicans . . 14. Stomach Tube 15. Turck's Aspiration Apparatus . . 16. Boas-Oppler Bacillus . . 17. Strauss' Separatory Funnel . 18. Sahh's Desmoid Bag . . . 19. Normal Feces . . ... 20. Boas' Stool-Sieve 21. Schmidt's Fermentation Apparatus 22. Cholera Spirilla 23. Bacillus Typhosus 24. Amceba Coli. 25. Coccidium Hominis . 26. Trichomonas Intestinalis 27. Cercomonas Hominis 28. Megastoma Entericum 29. Balantidium Coli. . 30. Taenia Solium 31. Taenia Saginata 32. Taenia Cucumerina 33. Taenia Nana. 34. Taenia Diminuta 35. Ovum of Taenia Diminuta 36. Taenia EchinococcusD/g/feecf-Jby Microsoft® Page 7 10 IS IS 16 25 2S 26 28 29 30 40 41 52 53 63 76 88 100 104 124 129 130 135 136 137 13& 13& 139 140 141 141 14? 142- 14? 143- LIST OF ILLUSTRATIONS XXXV Page 37. Hydatid Cyst 143 38. Bothriocephalus Latus ' . . . 144 39. Dibothriocephalus Cordatus 144 40. Ascaris Lumbricoides 145 41. Asc'aris Mystax 146 42. Oxyuris Vermicularis 147 43. Strongyloides Intestinalis 148 44. Trichiuris Trichura. 149 45. Trichinella Spiralis 150 46. Tail of Uncinaria Duodenalis . .• 151 47. Anterior End of Uncinaria Duodenalis 151 48. Tail pf Uncinaria Americana 153 49. Anterior End of Uncinaria Americana 153 50. Parasitic Bodies, Ova, and Larvae 154 51. Fasciola Hepatica . . . 157 52. Fasciolopsis Buski 158 53. Opisthorchis Felineus : 159 54. Opisthorchis Sinensis 160 55. Eustrongylus Gigas " . . . . 161 56. Acarus Scabiei 162 57. Demodex FoUiculorum 163 58. Leptus Autumnalis 163 59. Pediculus Capitis 164 60. Pediculus Vestimenti 164 61. Pediculusi Pubis 164 62. Pulex Irritans 165 63. Pulex Penetrans 166 64. Achorion Schonleinii 167 65. Normal Hair. . 168 66. Trichophyton Endo-ectothrix 168 67. Microsporon Audouini ■ 169 '68. Mycelial Threads of Blastomycetes 170 69. Budding Forms of Sporothrix Schenckii 171 70. Urinometer and CyUnder .... 188 71. Volumetric Flasks . ... 203 72. Kjeldahl's Nitrogen Apparatus 218 73. Doremus' Ureometer 222 74. Doremus-Hinds Ureometer 223 75. Folin's Urea Apparatus 224 76. Schlosing's Ammonia Apparatus . . 229 77. Folin's Ammonia Apparatus 230 78. Folin's Absorption Bulb 231 79. Ruhemann's Uricometer 239 80. Sargent's Colorimeter 244 81. Conical Test-glass Digiti-zed by Microsoft® 269 82. Horismascope 269 XXXVl LIST OF ILLUSTRATIONS 83. Esbach's Albuminometer 84. Laurent Polariscope . .... ... 85. Diagrammatic Representation of the Course of Light through the Laurept Polariscope 86. Einhorn's Saccharometer . . 87. Lohnstein's Fermentation Tube for Undiluted Urine . . 88. Lohnstein's Fermentation Tube for Diluted Urine . ... 89. Purdy Electric Centrifuge . 90. Sediment Tube 91. Percentage Centrifuge Tube 92. Various Forms of Uric Acid 93. Acid Sodium Urate .... 94. Xanthin . . 95. Calcium Oxalate 96. Cystin . . 97. Pure Leucin 98. Impure Leucin. . . . 99. Tyrosin . . . 100. Calcium Sulphate loi. Bilirubin ... ... 102. Cholesterin . . . . ... 103. Magnesium-Ammonium Phosphate 104. Calcium Carbonate . . . . 105. Urinary Epithelium . 106. Pus Corpuscles. 107. Hyaline Casts . ... 108. Granular Casts 109. Epithelial Casts no. Fatty Casts 111. Blood, Pus, Hyaline, and Epithelial Casts . 112. Cylindroids ... 113. Scolex and Booklets of Taenia Echinococcus in Urine 114. Ova and Miracidium of Schistosomum Hematobium 115. Normal Semen. . . 116. Chorionic Villi . 117. Daland's Hematocrit 118. Hematocrit Tube . . 119. Blood Needle . 120. Dare's Hemoalkalimeter ... 121. Pycnometer ... 122. Boggs' Coagulometer ... . . 123. Beckmann Apparatus . 124. Direct- vision Spectroscope. . 125. Hemometer of Fleischl-Miescher . 126. Hemoglobinometer (Qiw^§d by Microsoft® 127. Method of Filling the Dare Blood Pipet . . . Page 273 300 301 202 304 304 337 337 339 340 340 341 342 343 344 344 345 34S 347 348 349 350 352 356 358 359 360 361 362 366 367 377 384 390 391 393 396 400 404 405 410 417 420 421 LIST OF ILLUSTRATIONS XXXVll Page 128. Hemometer of Sahli . . . 422 129. Hemoglobinometer of Oliver . . . 423 130. Tallqvist's Hemoglobinometer . 424 131. Thoma-Zeiss Counting Chamber ... 456 132. Diluting Pipets . ... . . 457 133. Ruled Surface of Thoma-Zeiss Counting Chamber . . 458 134. Tiirk's Ruling of Counting Chamber . 458 135. Plan" of Counting the Cells . 464 136. Cross-section of Durham's Blood Pipet . 467 137. Oliver's Hemocytometer . . .... . 468 138. Preparation of Blood-smears with Glass Slides .... . 471 139. Preparation of Blood-smears with Cigarette Paper ... 472 140. Ehrlich Forceps . .... 472 141. Pinch Forceps . . .... . 473 142. Oven for Fixing Blood-films ... . 474 143. Normal Blood Showing Rouleaux Formation and Fibrin Network . 486 144. Cycles of the Malarial Parasite . . . . 571 145. Spirillum of Obermeier . ... 574 146. Trypanosoma Gambiense . . ... 575 147. Filaria Bancrofti . . ..... 577 148. Spirochetae Pallidse and Refringens . 580 149. Ultra-condenser of Reichert . 581 150. Schistosomum Hematobium 584 151. Illustrating the Mechanism of Toxin-cell Union . . 594 152. Illustrating the Elaboration and Action of Antitoxin 595 153. Illustrating the Mechanism of Hemolysis . . 596 154. Illustrating the Mechanism of Antihemolysis . . . 597 iSS- Bacillus Typhosus at Beginning of Widal Test . ... 607 156. A Pseudo- Widal Reaction . . . 607 157. A Positive Widal Reaction . . 608 158. Hemin Crystals from Human Blood . 657 159. Lumbar Puncture ... 680 160. Diplococcus In tracellularis Meningitidis . . 683 161. Normal Milk and Colostrum. . . 688 162. Babcock Milk and Cream Bottles . 694 163. Bottle for Human Milk .... 694 164. Soxhlet Apparatus . 695 165. Wolfhiigel's Colony Counter 697 166. Lautenschlager Hot Air Sterilizer. . 699 167. Autoclave . 7°° 168. Arnold Sterilizer . . . 701 169. Incubator ... . . . 705 170. Platinum Needles . . . . . . ... 706 171. Apparatus for Anaerobic Cultivation ... 707 Digitized by Microsoft® Digitized by Microsoft® DIAGNOSTIC METHODS CHAPTER I THE SPUTTTM I. General Considerations At the present time the examination of the sputum is more or less Um- ited to the search for various specific organisms, especially the baciUi of tuber- culosis and of pneumonia. This is very much to be regretted, as frequently the appearance, amount, consistency, color, and other characteristics are of great aid and have led out older brothers to correct diagnoses before the days of microscopic examination. "^ The sputum, strictly speaking, should be considered as the material which comes from the respiratory passage anywhere along its course. It may be, therefore, of laryngeal, bronchial, or alveolar origin. More commonly, how- ever, we find the sputum considered by the general practitioner and by the laity as anything which is expectorated, so that specimens which consist of nothing more than salivary secretion are frequently sent to laboratories for examination. In some cases, especially of inflammation of the naso-pharynx or perforation from neighboring organs, this buccal secretion may be mixed with material from the nose, mouth, ear, or esophagus. For these reasons, if for no others, it is absolutely that frequent examinations of sputum, in the strict sense, be made before a negative diagnosis of a suspected condi- tion may be given. As a rule, any sputum at all should be considered pathological, as normal persons raise little or nothing from the lungs at any time. By this is not meant that serious disease may obtain when a small amount of sputum exists, as patients suffering with catarrhal conditions of the naso-pharynx frequently have an accumulation of material, which has settled in the bronchial tubes over night and is raised in the morning. Those of us who are unfortunate enough to live in atmospheres which are loaded with soot and dirt frequently raise a certain amount of sputum which arises from an increased activity of the mucous membrane of the respiratory passages to compensate for the dryness and irritation which these foreign substances have caused. This morning sputum is small in amount and is in the form of large, tough, elastic masses which very much resemble boiled sago. These masses are at times extremely dark in color, due to the dirt which has been taken in with the inhaled air. In such sputum we find much mucus, degenerated epithelium, pus cells and various micro-organisms. These organisms are 1 See Laird, Jour. Am. Med. Assn., 1915, LXIV, 427. Digitized by Microsoft® 2 DIAGNOSTIC METHODS rarely of pathologic significance, although many pathogenic types may be present. The sputum should be collected in receptacles which may be completely and easily disinfected. The best receivers for the sputum are the ordinary paper spit-cups which can be burned as occasion may demand. The practice of expectorating upon cloths is only to be advised when these cloths are imme- diately burned. In the spit-cup should always be contained a certain amount of disinfectant, such as dilute carbolic acid or corrosive sublimate solution, so that no chance of transference of infection may arise. Neglect of such precautions has frequently led to serious consequences in the case of the healthy members of the family. When sputum is to be collected for exami- nation, the material raised by coughing should be received in a wide-mouthed bottle which contains no disinfectants, as these agents coagulate the protein material of the infecting organism and in some cases change its staining characteristics. Immediately after examination of such material it should, of course, be treated with the disinfecting solution. II. Physical and Chemical Characteristics Amount. Some general idea of the amount of sputum expectorated is always advis- able. It is rarely necessary, however, to make any collection of the material during the 24 hours ' period, as one can usually gain the information by ques- tioning the patient or nurse as to the amount passed from time to time. The amount of sputum expectorated in 24 hours depends very much upon the nature of the pathologic condition. In cases of so-called dry bronchitis, dif- fuse bronchitis, early tuberculosis, and occasionally of lobar pneumonia, the sputum is so viscid that there is practically none obtained. In cases of chronic bronchitis, tuberculosis with cavity formation, and bronchiectasis we find large amounts ; while in cases of lung abscess or of perforating pleurisy, blood or pus may flow from the mouth in very large quantities. The absolute amount of sputum may, therefore, vary from a cubic centimeter to a liter or more. An extensive expectoration will naturally have more or less serious effect upon the patient's general nutrition, so that we are not surprised to find cases in which as high as 5 per cent, of the total nitrogen eliminated passes from the system in the sputum (Lenz). Consistency. As a rule, the consistency of the sputum varies from that of a liquid to a highly tenacious material, inversely as the amount of sputum. This latter statement holds in most cases with the expection of pneumonia, in which we have an extremely tenacious sputum and, also, a very abundant one. Just what substance induces this extreme tenacity is uncertain,. but it may be gener- ally said that mucin is the causative faptor, although the tenacious pneumonic sputum shows very httle mucin. According to Kossel, the tenacity may be due to the presence of nuclein derivatives. In the early stages of acute bron- chitis, or bronchial asthnOp^^Si toM^^-?Sf^h the sputum is usually very THE SPUTUM 3 tenacious and ropy; while in edema of the lungs, pulmonary abscess, putrid bronchitis, and pulmonary gangrene it is very watery and contains large numbers of pus-cells. Reaction. The fresh specimen of sputum is usually alkaUne in reaction. However, in cases in which the sputum has remained in the' lungs for some time, as in cavity formation, the reaction is acid. Color. The color of the sputum may range from that of a colorless material to one showing any of the tints of the rainbow. These colors are due to admixtures of various abnormal products with the sputum. A bright red sputum is sig- nificant of the presence of blood, according to Traube, unchanged red blood- cells necessarily being present. The amount of blood may vary from a light streaking of the sputum to one showing a deep red, rusty, or prune-juice color. Such bloody sputa are found after trauma, pneumonia, gangrene, hemorrhagic infarction of the lungs, chronic passive congestion, as well as adventitious mix- ture of sputum with nasal or pharyngeal material containing blood. The blood may be due to rupture of a vessel and may then constitute the condition known as pulmonary hemorrhage. This condition of hemoptysis' is fre- quently confounded with that of hematemesis and is differentiated by the fact that the blood in hemoptysis is frothy, bright red in color, alkaline in reaction, and usually associated with mucopurulent material; while the blood in hematemesis is frequently dark and grumous, usually clotted and acid in reaction. In sputa which are tinted by changed hemoglobin, the color is most varied. Many oxidation products of hemoglobin are found in the sputum, depending upon the time which the sputum has lain in the lungs. Thus, for instance, in pneumonia we find a rusty, prune-juice colored sputum, whose color seems to be due to an unknown derivative of hemoglobin. The shade of color in pneumonia may, however, range from red through brown to green. In cases of mitral disease associated with passive congestion of the lungs we find, frequently, a light brown color, due to the presence of hematoidin gran- ules in the epithelial cells. In many cases of abscess of the liver which perforates into the lung or in catarrhal jaundice, bile pigments may be found in the sputum which may give rise to various colored sputa from red through blue to green. Although, as shown in a later table, the bile pigments and hemoglobin are very closely related chemically, yet in the chnical significance of the appearance of bile pig- ment in such cases as the above is very important. In many cases of true jaundice the sputum, in case any exists, may show a distinct grass-green color due to the presence of oxidized bile pigments. This same green color frequently appears in the sputum, however, in cases of croupous pneumonia during lysis. In this latter case the color is due, probably, to the same pig- ment which is oxidized before expectoration. In these cases of pneumonia with green sputum and no jaundice, a fresh involvement of the lung is usually 1 See van der Hoeven-L6^|jayj^^j^y^jJ^j^g^^yngol., 1913, XXXV,'83i. 4 DIAGNOSTIC METHODS associated with a rusty sputum. Besides jaundice and pneumonia we may- have, as causes of green sputum, certain chloromata of the lungs as well as the development of chromogenic bacteria within the lungs. This action of chromogenic bacteria is not always observed when the sputum is expectorated, but may appear only after the sputum has stood. The presence of the bacillus pyocyaneus may give a sputum which is brilhant blue or greenish in tint. Sputa very frequently show changes in color as well as consistency due to various substances inhaled. Thus we find a distinctly black sputum, in cases of anthracosis, in coal-miners and in many city residents. So frequent in this occurrence that the lung tissue may even be invaded by the coal pig- ment. ^ Workers in bronze and brass as well as other metals frequently show a sputum tinged red with ferric oxide, arising from a condition of the lung known as siderosis.^ Stone-cutters frequently show much stone-dust in the sputum which is characteristic of the condition of chalicosis, stone-cutters' phthisis, or grinders ' rot. Workers in flour mills and in bakeries frequently expectorate doughy masses, while those in cottom mills show the presence of cotton fibers in their sputum. Finally it may be said that the color of the sputum may be changed by the presence of certain foods, such as milk, eggs, and chocolate, while tobacco users frequently have a sputum tinged dark brown. Odor. Ordinarily the sputum has no odor unless it has stagnated either in the receiving cup or in the system. Such old sputum sometimes has a very dis- tinct putrefactive odor. The odor of sputum in tuberculosis and bronchiec- tasis is peculiarly heavy and sweet, while that in putrid bronchitis is often extremely offensive. In cases of perforating empyema a pecuUar cheese-Hke odor is observed. While these odors are not characteristic in themselves, they are usually more or less significant of the condition with which they are commonly associated. Character of the Sputum. The character of the sputum has reference more to the apparent com- position of the sputum that to its consistency. Air is usually present in the sputum in various amounts, so that one may judge, from the size of the air- bubbles, of the size of the bronchi from which the sputum came. The sputuin from cavities and large bronchi contains no air and, therefore, sinks in water, This is the so-called sputum fundum petens. Sputum known as mucoid sputum is glairy, transparent, ' and tenacious, becoming cloudy on the addition of acetic acid due to the precipitation of mucin. This type of sputum is found particularly in acute bronchitis and in asthma. A mucopurulent sputum is one containing both pus and mucoid material. Small amounts of pus give a whitish color, either to the whole or to portions of the sputum, the pus being observed in masses or in streaks through the mucoid material. Larger quantities of pus give a yellowish or occasionally a 'See Filadoro/Med. nuov^'?9i3, iV.i'^a; aS'o°?Payaorn, Jour. Med. Research loi^, XXIX,2S9; Klotz, Am. Jour. Pub. Health, 1914, IV, 887. . y i> 2 See Gigon, Beitr. z. path. anat. u. z. allg. Path., 1912, LV, 46. THE SPUTUM 5 yellowish green tinge to the sputum. In this latter type the pus and mucus seem to be mixed homogeneously. In the sputum from cavities we find the mucopurulent material arranging itself flatly like a coin, constituting the so- called "nummular" sputum. Purulent sputum is found in cases of ruptured empyema, abscess of the lung, and in some cases of bronchiectasis. This purulent sputum differs from the mucopurulent type in the fact that the pus is much more abundant and is almost in the pure state, being mixed with a small amount of tenacious mucus. In some cases, especially in edema of the lungs, a sputum is obtained, known as serous sputum, which is colorless and quite frothy. This sputum resembles very closely the ordinary salivary secretions and should not be confused with it. In many cases, especially in putrid bronchitis, gangrene of the lung, and bronchiectasis, the sputum on being voided into a cylinder will separate into three distinct layers, occasionally into four. The upper layer is of frothy mucus; a second layer, which is not always present, consists of certain albu- minous material which hangs in long shreds down into the third layer, which consists of the sero-pus and is usually opaque and watery. The bottom layer contains the morphological elenients, pus, tissue shreds, and bacteria. Besides the varieties of sputum named above, the admixture of blood may give rise to sputum which is known as sanguinous sputum or as sanguino- mucopurulent or purulent sputum. Chemical Properties. The chief chemical examination is appUed to the detection and estimation of the amount of albumin and of mucin in the sputum. The method is as follows:^ Treat the sputum with an equal quantity of 3 per cent, acetic acid, in order to precipitate the mucin. Shake thoroughly for a few minutes and allow to stand for 15 minutes. Filter through filter-paper until a perfectly clear filtrate is obtained, adding a known amount of water to facilitate filtration if necessary. Apply to this filtrate any of the tests for albumin given in the section on urine. If the quantitative determination is desired, use Tsuchiya 's reagent as the precipitant, multiplying the result by the di- lution of the sputum to obtain the true percentage of albumin. A large amount of albumin indicates, of course, such conditions as pneu- monia, pulmonary edema or perforating empyema. While the early workers regarded anything more than a slight opalescence as pathological, a trace of albumin indicating a pulmonary tuberculosis in their opinion, later investiga- tors showed that albumin is present in the sputum whenever there is active exudation into the alveoli as well as into and under the pleura. Many more recent studies have proven that albumin may not be present in the sputum of true pulmonary tuberculosis, but is frequently seen in benign affections and when present is due, in the majority of cases, to occult blood. For these reasons it is more than probable that such chemical tests are to be given merely a presumptive interpretation.^ 'Lesieur and Prires, Paris m&l., 1911, IV, 29. 2See Wanner, Deutsch. AKh-i-Jdin. Jyjkdj.rjpQ 3471 Roger and Levy- Valensi, Presse m6d., 1910, XVIII, aS^i^imf.HjiY, XlxPi^f igfeeraerd. Jour, de m6d., de chir. et 6 DIAGNOSTIC METHODS Besides these albuminous principles, the sputum, especially in cases of gangrene and putrid bronchitis, contains a ferment very much resembling in its action the trypsin of the pancreatic juice (Stolnikow) . ^ This ferment seems to indicate a highly destructive process in the lung tissue. Other chemical, substances, such as glycogen and fatty acids, " are frequently found in the sputum but may be passed with mere mention. The so-called myelin granules or globules, which appear in the alveolar cells of the sputum, consist largely of fatty principles, such as protagon, lecithin and cholestrin. III. Macroscopic Examination While much that is included in the previous section would come properly under the head of the macroscopic examination of the sputum, the writer has reference more, in this connection, to the appearance of macroscopic elements as distinguished from those which are purely microscopic. (i) Cheesy Masses. Frequently one finds in the sputum small cheese-like particles which vary in size from that of a pin-point to that of a pea, the large majority being about the limit of ordinary vision. These cheesy masses are fragments of necrotic tissue and appear in the larger form in cases of abscess or gangrene of the lung, while in tuberculosis they are always small unless the cavity, from which the material is derived, is markedly necrotic. The color of these masses varies from a yellow to a black. Those fragments which come from an abscess are of yellow color due to the presence of much pus, the darker ones contain decom- position products of hemoglobin, while many of them are tinged a deep black with coal pigment. If the sputum be squeezed between two glass plates these cheesy particles or fragments can sometimes be more distinctly seen. They are present in largest numbers in the so-called "nummular" sputum from a tuberculous cavity. (2) Dittrich's Plugs. These masses are similar to the small caseous particles above mentioned and are frequently expectorated by perfectly normal individuals. The true de Pharmacol., 1910, XV, 505; Raymond, Presse mM., 1911, XIX, 675; Goodman, Arch., Int. Med., 191 1, VIII, i63;f ishberg and Felberbaum, Med. Record, i9ii,LXXX, 870; Fish- berg, Arch. Diagnosis, 19,12, V, 220; Works, Jour. Am. Med. Assn., 1912, LIX, 1537; Scott, Ibid., 1913, LX, 440; Acs-Nagy, Wien. Wchnschr., 1912, XXV, 1904; Kauflfmann, Beitr. z. Klin. d. Tuberk., 1913, XXVI, 269; Schmitz, Med. Klin., IQ13, IX, 1163; Berkovits and Rudas, Berl. klin. Wchnschr., 1913, L, 1752; Ridge and .Treadgold, Lancet, 1913, II, 382; Schneider, Zentralbl, f. inn. Med., 1913, XXXIV, 1025; Gelderblom, Deutsch. med. Wchnschr., 1913, XXXIX, 1987; Maliwa, Deutsch. Arch. f. klin. Med., 1913, CXII, 231; Hohn and Himmelberger, jour. Am. Med. Assn., 1914, LXII, 20; Lewis, New York Med. Jour., 1914, XCIX, 1031; Melikjanz, Med. Obozr., -19x4, LXXX, 819; Beitr. z. Klin. d. Tuberk., i9i4,XXX, 81; Wien. klin. Wchnschr., 1914, XXVII, 653; Cocke, Am. Jour. Med. Sc, 1914, CXLVIII, 724; Hafemann, Deutsch. med. Wchnschr., 1914, XL, 1715; Benzler, Beitr. z. Klin. d. Tuberk., 1914, XXXII, 363; Glover, Brit. Jour. Tuberc, i9i4,VIII, 217; Holm and Chambers, Jour. Michigan Med. Soc, 1914, XIII, 226; Callender, Med. Record, 1914, LXXXVI, 783; Fantoni, Gazz. d. osp., 1914, XXXV, 417; Lowenbein, Ztschr. f. Tuberk., 191S, XXIII, 122; Szaboky, Ibid., 352; Snijders, Nederl. Tijdschr. v. Geneesk., 1915, I, 349; Lockwood, Jour. Am. Med. Assn., 191S, LXIV, 574. 'See Eiselt, Ztschr. f. klin. Med. 1912, LXXV, 71; also, Maliwa, Deutsch. Arch. f. THE SPUTUM 7 plugs are distinct cases of the bronchi or bronchioles and vary in size from pin-point to that of a bean, the majority being about the size of a mustard seed. The smaller ones are opaque and yellowish-white in color, while the larger ones have a distinct gray tinge. They are usually expectorated free from pus or mucus, so that they frequently give rise to anxiety, especially in those of a hypochondriac tendency. These plugs have a distinctly disagree- able odor, which is more evident if they are crushed on the glass plates. Microscopic examination of these mases shows large clumps of bacteria, fatty acid crystals, free fat globules, and cellular detritus. Occasionally a few leucocytes are found, but these are rare, while pigment granules, either of hematogenous or extraneous origin, are sometimes observed. While such plugs are especially numerous in cases of putrid bronchitis and bronchiectasis, they are frequently found in the crypts of the normal tonsil as well as in cases of follicular tonsillitis or of ozena. mjQ I. II. Fig. I. — Curschmann's spirals (Tyson after Curschmann). II and III, enlarged; a, a, central thread. III. I, Natural size; (3) Curschmann's Spirals. These structures are found in the sputum in practically every case of true bronchial asthma and have been reported in acute bronchitis, croupous pneumonia, chronic pulmonary tuberculosis, and occasionally in chronic bronchitis. They are not present in every paroxysm of asthma, but are more frequently found just at the end of the paroxysm and are absent when the spu- tum becomes mucopurulent. They seemto be derived through true exuda- tibn frojn the bronchioles, as Curschmann says, a bronchiolitis exudativa. These structures are recognizable, to a certain extent, by the naked eye, but for their absolute identification a microscopic examination is essential. They are composed of a spirally-twisted network of very deUcate fibrils, in the meshes of which are numerous epithehal cells and eosinophile leucocytes. Along with these cellular bodies one frequently find large numbers of diamond- shaped crystals, known as the Charcot-Leyden crystals, which will be dis- cussed later. This spirally- twisted mass (the mantle) seems to be wound Digitized by Microsoft® 8 -DIAGNOSTIC METHODS around a central light thread. While this is the structure of the complete spiral, we frequently find variations pointing apparently to two distinct forms. The first is the spirally-twisted strand of mucus with the enclosures above mentioned. The second is the tight spiral mass of mucus wound around a central fibre. This central fibre is very refractive and is homogeneous in structure, varying in size from J^ to i8 microns in diameter. The length of^ these spirals is from i to 2 cm. and their breadth about i mm. (4) Fibrinous Casts. By the term fibrinous cast we refer more directly to true bronchial casts, which are composed of fibrin. These are observed in pneumonia, in which case they are derived from the smaller bronchioles and are brownish or reddish in color and contain many red and white blood-cells. These smaller casts vary from J^ to 3 cm. in length. In the chronic fibrinous bronchitis we find the so- called arborescent casts which are usually whitish in color and contain many epithelial cells. These casts vary in size from i to 15 cm. in length by several mm. in thickness. We may have an acute form of fibrinous bronchitis accom- panying various febrile conditions, so that similar casts may appear in almost any of the infectious diseases. These larger casts are fairly firm, usually have a lumen, and branch dichotomously five to ten times. Microscopic ex- amination shows them to consist of large numbers of longitudinal fibers con- taining blood and epithehal cells in their meshes. They may be stained with the Weigert fibrin stain in a very beautiful way. Such staining methods show that not all the material in such casts is fibrin, so that it may be neces- sary to rename these structures as simple bronchial casts rather than as fibrinous forms. Besides these fibrinous casts, one occasionally may find distinct bronchial casts composed of the mycehum of various fungi. Thus mycelial threads of the aspergillus have been reported by Osier, Devillers and Renon. (5) Concretions. This name is applied to anything, expectorated with the sputum, which has the appearance or consistency of a stone. These concretions are formed in dilated portions of the bronchi or in cavities by the calcification of the stag- nated contents. Although concretions consisting of cartilaginous or osseous material are frequently found postmortem, yet there seems to be httle men- tion in the hterature of any such formations being expectorated.^ (ff) Bronchioliths. These concretions are formed by the deposition of calcium salts in the stagnated contents of the bronchus or of a cavity. They may be derived from the smaller or larger bronchi, but rarely form arborescent shapes, being usu- ally irregular and varying in size from apin-head to that of a walnut. They are usually single, but may be multiple. They vary in consistency from that of chalk to a stone, and may be expectorated in small numbers over long periods of time. '■ See Cade and Gu6rm, Lyi THE SPUTUM 9 (6) Pneumoliths. These lung stones are in the majority of cases of tuberculous origin. They usually arise by the calcification of caseous areas which later ulcerate into a bronchus and are expectorated unless too large. They may, also, arise by the calcification of a pulmonary cavity or of a bronchial lymph-gland. These lung stones consist of the carbonates, phosphates, and sulphates of cal- cium and magnesium, in some one or more salts predominating, while in others still different combinations may exist. These pneumoliths either have a chalky or a clacareous consistency and vary in size from that of a pin-head to that of a tennis-ball. These lung stones are usually expectorated en masse or in the form of smaller portions of a larger stone. In some cases these smaller stones may reach the number of 500 (Portal). (6) Echinococcus Membranes. Rarely one may find in the sputum fragments of the walls of echinococcus cysts or their contents. These may come from a perforating cyst of the liver, kidney, or lung. The presence of the laminated membrane and of the parasitic scoUces and hooks makes it possible to arrive at an absolute diagnosis of the origin of such material. The membrane is thick, tough, and of a porcelain- Uke color and may show a laminated or fibrillated structure. The parasite is discussed in a later section. (7) Foreign Bodies. Examination of the sputum may reveal the presence of material which has lodged in the air-passages and been retained for long periods of time. Such bodies are coins, fish-bones, and cherry-stones. Heyfelder reports a case of the expectoration of a wooden cigar-holder 11 3'^ years after its disappearance. * IV. Microscopic Examination The microscopic examination of the sputum is almost the only one to which it is subjected at the present day. This is to be regretted, as much may be learned from a careful macroscopic examination. However, a microscopic examination reveals evidence which points to an absolute diagnosis more fre- quently than does the macroscopic examination. Before making the micro- scopic examination, it is wise to place the sputum in a flat-bottomed dish (Petri dish) which has half of its base blackened, so that the more suspicious particles may be selected for microscopic investigation. Some experience in this work is necessary, as one is frequently called upon to recognize material which is purely extraneous and has absolutely nothing to do with the sputum, such material practically always comes from the buccal cavity and consists of fragments of various food-stuffs, such as bread, fruit pulp, meat fibers, vege- table tissue, and portions of tobacco leaf. Naturally, such material should not mislead one, but it very frequently does. The fragments of meat tissue contain elastic tissue fibers and may lead one to state that such material is present in the sputum, thus giving expression to the possibiUty of a diagnosis of incipient tuberculosis. A portion of the sputum selected for examination is taken up with a plati- Digitized by Microsoft® U±J\\jSSKJO±X\^ iKUC^ j.xx\jx^o num loop and spread in a thin layer upon a glass slide. It is then dried by passing the slide several times through the flame care being taken not to burn, the specimen. The smear is allowed to cool and is then stained either with LofHer's methylene blue for general purposes or with special stains for the various specific organisms, (a) Pus-cells. (Leucocytes). There is practically no specimen of sputum which does not contain leuco- cytes in larger or in smaller numbers. The true pus-cell is the polymorpho- nuclear neutrophile and appears in the sputum frequently filled with fat globules or pigment granules. In cases of asthma, the eosinophile cells are very abundant, while basophile cells may occasionally obtain. Although Fig. 2. — Objects found in the sputum (Landois). i, Detritus and dust-particles; 2, pig- mented alveolar epithelium; 3, fatty degenerated and partially pigmented alveolar epithe- lium; 4, alveolar epithelium showing myelin-degeneration; 5, free myelin forms; 6, 7, des- quamated ciliated epithelium, partly changed and deprived of its cilia; 8, squamous epithe- lium from the mouth; 9, leucocytes; 10, elastic fibers; 11, fibrinous cast of a small bronchus; 12, leptothrix buccalis, together with cocci, bacilli, and spirochete; li, fatty acid crystals and free fatty granules; b, hematoidin; c, Charcot's crystals; d, cholesterin. these cells are so frequent in asthma, a diagnosis should not rest upon such evidence alone, as there seems to be a form of bronchitis, possibly of the tuber- cular variety, which has been named "eosinophilic bronchitis" from the large number of such cells observed. In pulmonary tuberculosis, which is not associated with a mixed infection, it is very common to find the small mono- nuclear leucocyte (lymphocyte) in place Of the polynuclear type. This finding is of such frequent occurrence that the writer is often led to search many slides for tubercle bacilli in case the organisms have not been found in the earlier specimens examined. Basophilic cells are not infrequently observed in the sputum. Pottenger^ reports the presence of macro-phagocytes in a case of chronic hemoptysis. The cytology of the sputum shows little of diagnostic value. Digitized by Microsoft® 'Jour. Am. Med. Assn., i9i2,LIX, 1794. THE SPUTUM II The thin smears of sputum may be treated like blood-smears and stained with the same stains when one desires to study the cellular types present. (6) Red Blood-cells. These cells are frequently found in the sputum and may have much signifi- cance. They may occasionally arise from contamination with nasal or buccal discharges, but the true bronchial or pulmonary sputum usually shows them only in cases of hemorrhage or exudation. The rusty sputum of pneumonia contains large numbers of such cells and the hemorrhage from a tubercular cavity may be very extensive. These cells are occasionally well-preserved, but at times they are difficultly recognizable. They may be very much distorted in shape, so that their color and staining properties must be relied upon for differentiation. (c) Epithelial Cells. Various types of epithelial cells are found in the sputum. Pavement epithelium may come from the mouth, pharynx, and upper larynx. Cylin- drical epithelium may be derived from the nose or the bronchi. These cylin- drical cells may be ciliated, but rarely does one find these ciliated forms except incases of asthma and bronchitis. Alveolar epithelial ceUs are present in nor- mal sputa as they are constantly desquamated from all the epithelial sur- faces. These cells assume a large variety of forms and frequently show various types of degeneration. They are very numerous in bronchitis and in general inflammatory conditions of the lungs, but may occur in almost any condition, associated with irritation along the respiratory tract. These cells contain large numbers of granules which are probably referable to coal pig- ment. Such cells give the sputum a grayish or green color. An abundance of such cells in the sputum was designated in earlier times as "phthisis melan- otica." Occasionally these alveolar cells are filled with fat globules. In other cases one finds the so-called myelin globules, which are irregular in shape, often showing concentric lines, with very little refractility, and of a dull greenish or blue appearance. The cells may be invisible and only the myelin appear as a large irregular mass. Much doubt exists as to the origin of these globules, but it is more probable that they represent simply the fatty prod- ucts of degenerated protoplasm. These cells are especially frequent in the normal morning sputum, in acute or chronic influenza, and in the so-called desquamative catarrhal pneumonia, when they appear as small lumps re- sembling boiled sago. Occasionally free myelin globules are found in the sputum. These globules stain poorly with the aniline dyes, are stained yellow with iodin, but do not stain 'black with osmic acid or red with Sudan III. Frequently these alveolar cells contain pigment granules, derivatives of hemoglobin. This material is in the form of amorphous granules of a brown color and seems to be identical with hemosidern, but may become iron-free, when it more closely resembles hematoidin. Cells containing such granules are especially numerous in chronic heart disease and are styled, therefore, "heart- disease cells." They occur, however, in any condition in which red blood-cells Digitized by Microsoft® 12 DIAGNOSTIC METHODS escape into the alveoli and are found, therefore, in pneumonia, infarction of the lung, and hemorrhagic pulmonary tuberculosis. (d) Elastic Tissue. The presence of elastic tissue in the sputum is indicative of destruction of the lung tissue and in many cases is found in the sputum before one can detect tubercle bacilli. It has, therefore, some importance in the early diagnosis of tuberculosis. When this elastic tissue is grouped in masses it is usually vis- ible to the naked eye. However, one more frequently relies upon microscopic examination for its detection. The method of Clark, as applied to the de- tection of elastic tissue, is usually the best one to follow. The sputum is placed upon a glass plate about 14 inches square and pressed out by a smaller one, about 6 inches square, into a thin layer. The plates are then placed upon a dark background and examined with a hand lens. Instead of these glass plates one may use Petri dishes. The elastic tissue fibers appear either in the form of distinct strands some- times grouped in an elongated network, in that of the alveolar type, in which the fibers preserve the outline of the alveoli and are long and branching, or in that from the arteries in which we may have a distinct sheet-like arrangement. These fibers are characterized by their undulating outline, their curling ends, their sharp edges and uniform diameter, their frequent branching, and their intense refractility. These characteristics are brought out both with the low and with the high power of the microscope. The fibrous tissue fibers are dif- ferentiated from elastic fibers by the fact that the former are present inbundles of fine wavy lines without the coarse black refractive appearance of elastic tissue. Chains of bacteria, especially the leptothrix forms, frequently inter- lace in such a way as to simulate the alveolar structure of elastic tissue. These chains differ, however, from elastic tissue in their refractility, in the absence of the •wavy outline, and in their denser arrangement. The elastic tissue fibers derived from food substances have the same characteristic as the lung elastic fiber, with the exception that they are not arranged in the alveolar form and may at times be coarser and more irregular in outline than the pulmonary elastic tissue. Vegetable cells and fibers, as well as fatty acid crystals, which may occur in the sputum, should not mislead one into assuming the presence of elastic tissue. Should one wish to stain elastic tissue, he may use the orcein stain of Unna-Tanzer. This stain consists of one gram of orcein dissolved in a mixture of 80 c.c. of 95 per cent, alcohol and 35 c.c. of distilled water, 40 drops of strong hydrochloric acid being added after solution is complete. In using this stain the elastic fibers are treated with a few c.c. of the dye and then warmed for five minutes, after which the preparation is decolorized with acid alcohol. The elastic tissue fibers will be stained a brownish violet by this process. If the elastic tissue is very small in amount we usually resort to chemical means for the isolation of this material. Ten c.c. of sputum are mixed with an equal volume of 10 per cent, sodium hydrate solution and the mixture is boiled until it becomes homogeneous. Four volumes of water are then added, the entire mass well mixed, and either allowed to stand or to settle by cen- trifugation. In this wa)£lfeli^®efi5l&MiMS'■ See von Teckman, Zur Lehrevon derPneumonomykosis asergillina, Inaug. Dissert, p. '''Isee'weidman, Proc. ^.^^(^^1^%°?^^^! 6. THE SPUTUM 17 any of the pus-forming organisms. More frequently, however, when these organisms are present in excessive numbers a contamination or a direct patho- genic influence should be suspected. A form known as the micrococcus te- tragenus occurs both as a pathogenic and a harmless organism. It consists, as its name implies, of four cocci arranged in a square within a mucous capsule. It stains with the ordinary dyes and is Gram-positive. This organism is found, in its pathogenic state, in bronchitis, tubercular cavities, and hemor- rhagic infarctions.^ The harmless form differs from the pathogenic type in the fact that it cannot be cultivated. The sarcinas are rarely found in the sputum. These organisms are somewhat smaller than those occurring in the stomach and are probably purely saprophytic in the sputum. They are found, however, in cases of putrid bronchitis, especially when this occurs in emphysematous lungs, in gangrene, tuberculosis, and pneumonia. The micrococcus catarrhalis is found frequently in the sputum as a sapro- phyte, but may become pathogenic especially in some epidemics of la grippe.^ it is larger than the ordinary staphylococcus, while its arrangement in lateral pairs and its Gram-negative properties obscure its differentiation from the gonococcus and meningococcus unless cultural methods be employed. (See last chapter.) In cases of general systemic blastomycosis, Eisendrath and Ormsby have found the blastomycetes in the sputum. They recommend the examination of the unstained specimens after the addition of 10 per cent, sodium hydrate solution. In such preparations examined with a high-power dry lens, the typical refractile blastomycetes are observed. (See Parasitic Diseases.) (2) Pathogenic Types. (a) Tubercle Bacillus. This organism is the most important pathogenic type found in the sputum. Its detection is usually easy and should be attempted in all suspicious cases, as an early diagnosis may frequently save the life of the patient. In the days before the organism was recognizable, physicians based their diagnosis of con- sumpiion upon the macroscopic appearance of the sputum. While such examinations frequently lead to a presumptive diagnosis of tuberculosis, nothing can settle the question except the microscopic examination of the sputum. This statement needs some modification in several ways. In the first place, specimens of tubercular sputum may not show the presence of the bacilH so that several examinations of sputum, collected at different periods, must be made. In the second place, the sputum may be examined in the very early stage of the disease and no tubercle baciUi be found, but in such cases the presence of elastic tissue fiber would be very significant of tubercular changes. In these days we have, fortunately, recourse to other diagnostic measures in case tubercle bacilli cannot be found in the sputum. I have reference here to the use of tuberculin, either introduced in the form of an 1 Steele (Jour. Am. Med. Assn. ,1914, LXII, 930) reports a case of general septicemia due to this organism. ^Hastings and Niles, Jour. Exper. Med., 1911, XIII, 638. Digitized by Microsoft® DIAGNOSTIC li. HjXXXKJJ^iJ injection as done by Koch, dropped into the eye as advocated by Calmette, as advised by Pirquet applied after the manner of vaccination, or used as an inunction as suggested by Moro. This test is usually certain and can be relied upon to settle the diagnosis. In examining the sputum microscopically the fine cheesy particles pre- viously mentioned are selected and smears of such material made upon glass slides. It is always advisable to make at least five such smears to insure definite results. It has frequently been found that many suspicious-looking sputa show no tubercle bacilli when subjected to the ordinary methods of examination. In order, therefore, to increase the possibility of positive re- sults, the sputum should be rendered homogeneous and fluid. The best method of accomplishing this is, in the writer's opinion, Loeffler 's^ modification of the antiformin process of VUenhuth. The technic is as follows: s, lo, or more c.c. of the sputum are placed in a flask and mixed with an equal quan- tity of a 50 per cent, solution of antiformin (a 10 per cent, solution of sodium hypochlorite containing 5 to 10 per cent, of sodium! hydrate) and boiled for a period not exceeding 15 minutes. Solution occurs associated with consider- able foaming and browning of the mixture. For every 10 c.c. of this solution are now added 1.5 c.c. of a mixture of i part of chloroform and 9 parts of alcohol. After thoroughly shaking to produce a fine emulsion, portions of the fluid are placed in sedimenting tubes, the tubes are corked, and centri- f uged for fifteen minutes. The heavier elements collect in a film just above the chloroform, which film holds the tubercle bacilU owing to the marked affinity of chloroform for the fatty and waxy material in these organisms.^ The supernatant liquid is poured off and the film above mentioned is removed and placed upon a glass slide, the excess of fluid being taken up with filter-paper. As a fixative a drop of egg-albumin, preserved with 3^ per cent, carbolic acid, is added and a thin spread made by means of a second slide. This smear is allowed to dry and is then stained by one of following methods. This enrich- ment process of Loeffler furnishes preparations which often show a remarkable increase in numbers of tubercle bacilli as compared with those found by the usual smear methods. A further advantage of this method is that practically all organisms, with the exception of those of the acid-fast type to which the tubercle bacillus belongs, are destroyed. If the mixture be allowed to stand for a few hours instead of being boiled, one may obtain material for pure-cul- ture or for inoculation purposes, as the secondary invaders are eliminated. For these latter purposes it is necessary to wash the sediment frequently with sterile water by means of centrifugation and decantation in order to remove the excess of alkali present. This may, of course, diminish the number of tubercle bacilli and, at the same time, cause contamination of the specimen. In consequence, inoculation and cultural experiments with such material frequently give negative results in known tubercular cases. A somewhat different method of eliminating the secondary invaders and of 'Deutsch. med. Wchnschr., 1910, XXXVI, 1987. 2 See Panzer, Ztschr. f. physiol. Chem., 1912, LXXVIII, 414; Wells, Interstate Med. Jour., 1914, XXI, 221. ' Digitized by Microsoft® THE SPUTUM 19 obtaining the tubercle bacillus in pure culture from the sputum (as well as other material) has been introduced by Fetrof- and promises to become a Very valuable aid in the study of the presence and characteristics of the tubercle bacillus. By the employment of a special culture medium to which gentian violet is added, PetrofiE takes advantage of the fact that this dye will inhibit the growth of many organisms but will not retard the development of the tubercle bacillus at the dilution at which it is used. Further, he adopts, as his homogenizing agent, an alkaline solution to which the tubercle bacillus is resistant while the viability of most other organisms is quite strongly affected. . Preparation of the Cxilture Medium Five hundred grams of beef or, preferably, veal are infused in 500 c.c. of a 15 per cent, solution of glycerin in water. Allow the infusion to stand for 24 hours and squeeze the meat in a sterile press, collecting the juice in a sterile beaker. Sterilize the shells of one dozen fresh eggs by pouring hot water upon them. Carefully break the eggs into a sterile beaker and thoroughly mix the white and yolk with a sterile rod. Filter through sterile gauze into a sterile graduated cylinder. To two parts of the egg add one part of the meat infusion and add to this mixture sufficient i per cent, alcoholic solution of gentian violet to make a dilution of i to 10,000. Tube about 5 c.c. of this mixture in each sterile test-tube and inspissate the slanted tubes for three successive days: on the first day at 8s°C. until all the medium is soldified; on the second and third days for not more than i hour at 7S°C. (If this medium is to be used for the isolation of the bovine type of tubercle bacillus, the glycerin may be omitted in making the meat infusion.) Method Equal parts of fresh sputum (about 5 c.c.) and 3 per cent, sodium hydrate solution are well shaken and placed in the incubator for 20 to 30 minutes until the sputum is fairly well digested. Neutralize this mixture to sterile litmus paper with normal hydrochloric or sulphuric acid. Centrifugalize at high speed for 10 minutes. Decant the supernatant fluid and spread the sediment over several slants of the culture medium. Incubate for 7 to 12 days. The cultural characteristics may be more or less variable, some of the colonies being of small pin-point size, while others are large and flat. Some of the types may decolorize the medium, while others appear violet. Mor- phologically, the organisms may vary from cocci to long rods. .By this method of Petroff tubercle bacilli may be detected in a compara- tively short time in cases which have show repeated negative results by the usual microscopic methods, as well as in cases which have not yielded positive results with animal inoculation following the antiformin method. It is simple, 'Jour. Exper. Med., igis, XXI 38; Bull. Johns Hopkins Hosp., 1915, XXVI, 275; also Mitchell and Simmons, Jour. Am. Med. Assn., iqis, LXV, 245. Digitized by Microsoft® 20 DIAGNOSTIC METHODS practical and reliable. It is especially recommended as it tends to increase the possibility of diagnosis in obscure cases and may, even, supplant animal inoculation tests. Staining Characteristics. The methods of staining the tubercle bacillus depend upon the property, possessed by this organism, of taking up the aniline dyes with great difficulty, but, when once stained, of becoming just as resistant against decolorization. Breskman (Jour. Am. Med. Assn., 1910, LIV, 1591), Dixon (Ibid., 1913, LX, ' 993) and, more recently. Wherry (Jour. Infect. Dis., 1913, XIII, 144) have shown that the tubercle bacillus varies its acid-fast properties in its develop- ment on culture media, becoming more acid-fast or non-acid-fast according as the cultural conditions are or are not favorable for the synthesis of fat. Tamura (Ztschr. f. physiol. Chem., 1913, LXXXVII, 85) demonstrates that this acid-fast property is due to the presence in the bacterial cell of an alco- hol, mykol (C29H66O), which is partially bound to some higher fatty acid in the form of an ester. Ziehl-Neelsen Method. The smears are made upon glass slides and are fixed by passing several times through a flame. The smear is covered with carbol-fuchsin solution' (a mixture of 90 parts of 5 per cent, carbohc acid water and 10 parts of a con- centrated alcohohc solution of f uchsin) , and is then heated over a flame for one to three minutes in such a way that the staining solution steams, but does not boil. If the staining solution is heated too strongly the smear decol- orizes less readily so that it is very good practice never to boil the staining solution. Where several sUdes are to be examined, the writer has found the copper plate of Ehrlich very useful.^ The sUdes are laid upon the plate, are covered with the stain, and allowed to heat for 10 minutes. The usual tech- nic is, however, to heat a single slide at one time, more of the stain being added as the first evaporates. Some workers find that the immersion of the smear in cold carbol-fuchsin for 24 hours gives somewhat clearer pictures, but the time is too long for the ordinary laboratory diagnosis. Having thus stained the smear with the carbol-fuchsin solution, it is then decolorized. The tubercle bacillus is not only acid-fast, but also alcohol-fast, so that we use decolorizing agents containing both acid and alcohol. There are many acid-fast organisms known, such as the. bacillus lepras, the smegma baciUus, the timothy bacillus, the butter bacillus and many saprophytic bacilh found in water, soil, and manure.' Few or none of these organisms are absolutely both alcohol and acid-fast, so that the use of the combined decolorizer wiU usually differentiate the tubercle bacillus. Many decolorizing agents have advised, but the writer finds the use of a 10 per cent, solution of sulphuric acid in 95 per cent, alcohol very reliable. This decolorizing agent does not burn the specimen nor does it prevent the morphological characteristics from appearing in a clear-cut way. Some workers advise the use of a 2 per cent. ^See Veihoeff, Jour. Am. Med. Assn., 1912, LVIII, 1355. 2 See Schmidt, Jour. Am. Med. Assn.. 191S, LXIV, 823. ^Lycopodium spores, coicl£>i^^em^y:MilBIWiBOfflS>otheT substances not inffequently found in the sputum are acid-fast and may prove very misleading. PLATE I. ■< ! s 1/ ■■'^<. S, ^~ . ^ jv?\ / ' f'^'^'S >^ ,!^.: >fatHarine Mil! Tubercle Bacilli in Sputum. Ziehl-Neelsen Method. Digitized by Microsoft® Digitized by Microsoft® THE SPUTUM 21 hydrochloric acid in 80 per cent, alcohol, while others use 25 per cent, nitric acid, followed by alcohol. The technic of decolcrization is as follows: Wash the smear, which has been stained with carbol-fuchsin, in water and flood the specimen with the decolorizing solution until only the faintest pink color is seen in the smear. It frequently happens that the thicker portions of the smear resist this decolorizing so that it may be necessary either to make a new smear or to examine merely the portions which have been decolorized. After decolorization is complete the specimen is washed in water and counter- stained with Loffler's methylene blue (saturated alcoholic solution of methy- lene blue 30 c.c, 100 c.c. of a I to 10,000 aqueous potassium hydrate solution) for a few seconds, after which the specimen is washed with water, dried be- tween filter-paper, and examined with the oil immersion lens. In such preparations the tubercle bacilli are seen as bright red rods, some- what bent, sometimes much curved and occasionally showing distinct branch- ing forms. ^ In a few preparations one may find the curves of the bacillus so marked that a very close resemblance to the spirillar forms obtains. These organisms occasionally show a distinct beading, giving the appearance of bright red cocci. ^ The size of these organisms varies from iH to 3 J^ microns and about %o microns in width. They may be single or arranged in clumps, sometimes in the form of distinct crosses, sometimes parallel, and very fre- quently forming acute angles by the joining of two bacilli. Gabbet's Method. This method is much more simple than the preceding, but is not as reliable. By this method the decolorization and counter-staining are carried out in one operation. The smears are prepared as previously described and stained with the carbol-fuchsin solution. The excess of the staining solution is drained off without washing and is replaced by Gabbet's methylene blue solution (methylene blue 2 grams, sulphuric acid 25 c.c, water 75 c.c). This solution is allowed to act for one to three minutes and is then washed off with water and the specimen dried and examined. The tubercle bacilli will appear as bright red rods as previously described, while the other organisms as well as the various cellular types will be stained blue. This method is not as reliable as is the former, owing to the fact that the alcohol-fast bacilli resist decolorization and may confuse one in making the diagnosis. Moreover, the use of the strong acid may cause decolorization of some of the tubercle baciUi and will, therefore, give rise to wrong ideas. These methods are the ones usually followed in routine laboratory work for the detection of the tubercle bacillus. Pappenheim's Method. The technic of this method is as follows: The preliminary staining is carried out with carbol-fuchsin solution as previously outlined. The speci- men is then drained and covered with the decolorizing solution, which is made by dissolving i gram of rosolic acid in 100 c.c. of absolute alcohol, saturating the mixture with methylene blue and adding 20 parts of glycerin. This solu- 1 See Dixon, Jour. Am. Med. Assn.. 1913, LX, 993 and 1294. 2 See Babes, Berl. klin. wEk0kefk^Jm?l!^SOn® 2 2 DIAGNOSTIC METHODS tion is drained off slowly and the process repeated several times. The slide in then washed in water, dried between blotting-paper and examined with the immersion lens. The tubercle bacilH are stained red and the other organ- isms blue. Much's Method. As is well known, certain specimens of undoubted tubercular sputum as well as tissues frequently do not show tubercle bacilli when any of the above methods of staining are used, owing to the facts that neither is every acid- alcohol-fast bacillus the tubercle bacillus nor is every tubercle bacillus abso- lutely acid-fast. It is to Spengler ^ and, more especially, to Much ^ that we are indebted for a method which will demonstrate these non-acid-fast types of tubercle bacilli, which are distinctly granular, frequently appearing as mere granules rather than as true bacilli. These granules, under certain unknown conditions, change into true bacillary types and vice versa.^ Much's method is a modified Gram method, the tubercle bacilli being Gram-positive.* Pre- pare smears as described above. Cover this smear with a carbol-methyl vio- let solution (lo c.c. of a saturated alcoholic solution of Gruebler's methyl vio- let B. N. mixed with 90 c.c. of 2 per cent, aqueous carbolic acid solution) and heat to boiling several times. Wash off stain with water and cover smear withLugol's solution (iodine i gram, potassium iodide 2 grams, water 300 c.c.) for 5 minutes. Wash with water and treat with 5 per cent, nitric acid for one minute and follow this with 3 per cent, hydrochloric acid for 10 seconds. Without washing place the slide in a mixture of equal parts of acetone and ab- , solute alcohol until the smear is colorless. Wash with distilled water and counter-stain with i per cent, aqueous solution of safranin for a few seconds. Wash in water, dry thoroughly, and examine with the immersion lens. The tubercle bacilli and the granular forms appear bluish while the other organ- isms are red.^ Value of Examinations. Brown" in an able manner has summed up the value of the sputum exami- nation for tubercle baciUi. He gives as his reasons for beheving that one should be guarded in forming an opinion of the prognosis of certain cases the following points: (i) Many of the tubercle bacilli may not be stained at all. (2) Old foci may give off very few and young foci no bacilli at all. (3) By the occlusion of a bronchus the contents of a focus may be shut off entirely for a time and thus the expelled sputum may contain a large number of tubercle bacilli. (4) The organisms may be present one day and not again for months. (S) The organisms may be abundant in one part of a specimen and none be found in others. (6) Some patients \fith fatal tuberculosis (caseous pneumo- nia or acute mihary tuberculosis) may have no bacilli in the sputum, while in other cases the organisms are present even before physical signs obtain. (7) iDeutsch. med. Wchnschr., 1905, XXXI, 1228 and 1353; Ibid., 1007, XXXIII, 557. ^Beitr.z.Klin.d.Tuberk., 1907, VIII, 8s and 357. 'See Koiber, Deutsch. med. Wchnschr., 191 2, XXXVIII, 1494; Weiner, Munch, med. Wchnschr., 1914, LI, 1838; Bruckner, Ztschr. f . klin. Med., 1914, LXXX, 360; Mircoh, Gazz. d. osp., 1914, XXXV, 617; Tallo, Gazz. internaz. di med., 115, XVIII, 241. 'See Benians, Jour. Path, and Bacteriol., 1912, XVII, 199. ^See Mas y Magro, Rev. ^f|fflfc*t/:fey- Wfe^OSSft^V, 37. « Montreal Med. Jour., 1901,'XXX, 769; Jour. Am. Med. Assn., 1903, XL, 514. THE SPUTUM 23 In severe cases with bronchitis the secretion of the bronchus will dilute the sputum and give the appearance of a reduction in the number of organisms. While the number of bacilli in the sputum may thus vary, it is usually in direct ratio to the severity of the disease, although for the reasons above men- tioned too much rehance should not be placed upon the number of organisms found. Brown recommends the use of a somewhat modified Gaffky's table in judging of the prognosis in any particular case. The cases are classified as follows, being designated by the Roman numerals : I. Only one to four bacilli in whole preparation. II. Only one on an average in many fields. III. Only one on an average in each field. IV. Two to three on an average in each field. V. Four to six on an average in each field. VI. Seven to twelve on an average in each field. VII. Thirteen to twenty-five on an average in each field. VIII. About fifty on an average in each field. IX. About 100 on an average in each field. There has been some attempt to base a prognosis upon the form and grouping of the tubercle bacilH, the short rods indicating a rapid growth while the longer form shows a slower development. "^ The continued expectoration of large numbers of baciUi would indicate a cavity, while the sudden increase in numbers associated with an increase in the cellular elements would point to lung disintegration. A steady decrease over a long period of time would indi- cate improvement. It should be stated as a working rule that the finding of a single or a very few organisms in the sputum should be looked upon with sus- picion, but that an absolute diagnosis should be made only after repeated ex- amination has shown the presence of the tubercle bacilli.^ The worker will find in the study of preparations stained as above that artefacts are very com- mon so that a hurried examination is never warranted. The sputum in tubercular cases rarely shows tubercle bacilli in pure cul- ture. One usually finds large numbers of streptococci, staphylococci, micro- cocci catarrhalis, and frequently influenza bacilli and pneumococci. Pus cells may be few or many. Blood-cells may or may not be present, while elastic tissue fiber is very frequent, appearing in many cases before tubercle bacilli can be demonstrated. It should be remembered that mixed infections may be so marked that few, if any, tubercle bacilli appear in the sputum. The writer has frequently found sputa showing fairly large numbers of tubercle bacilli becoming practically negative for these organisms if the sputum be al- lowed to stand exposed to the air for 48 hours. The explanation is that the other organisms so far outgrow the tubercle bacillus that they prevent any fur- ther development of this latter organism and bring about such degeneration 'See Wilson, Lancet, 1914, II, 1198; Cohn, Beitr. i.. Klin. d. Tuberk., 1914, XXXI, i; Kirchenstein, Ibid., n. ^ The type of tubercle bacillus found in the sputum is practically always the hlman strain Only seven cases are reported in the literature of infection with the bovine type (see Gri- fith, Brit.Med. Jour., 1914, DflgjfEel!^<]&5F'"lWJ©P9Si©lfif@iIed.Wchnschr., 1914, XL, 1299). 24 DIAGNOSTIC METHODS that the staining quaUties of the tubercle bacillus are markedly affected. This fact has been taken advantage of in the chnical use of injections of pus organ- isms into tubercular joints. Q>) Lepra Bacillus. The bacillus lepvss, first described by Hansen, is a small slender bacillus from 4 to 6 microns in length and surrounded by a shmy envelope. These bacilh behave toward staining reagents very niuch Hke the tubercle bacillus, but are less resistant toward acid and alcohol than is the tubercle bacillus, so that a differentiation is possible provided decolorization is rather severe. The stained bacilli often show clear spots or appear as if made up of stained granules. These organisms may be found in many cases of leprosy in the sputum or nasal secretion, so that in doubtful cases a differentiation is necessary. While these organisms stain much more easily than do the tubercle bacilli and are more easily decolorized, it may be necessary to resort to inoculation ex- periments to make the absolute differentiation.^ (c) Smegma Bacillus. This bacillus may be found normally in the saUva, coating of the tongue, the tartar of the teeth, and in the crypts of the tonsils. Pathologically, it may be found in cases of simple bronchitis, in the sputum in cases of gangrene of the lungs, and in the suppurative discharges from the ears. While these organisms are much more commonly confused with the tubercle bacillus when the urine is examined than when the feputum is investigated, yet they must be borne in mind in every sputum examination. It may be necessary to resort to inoculation experiments to decide the question, but ordinarily the use of the differentiating stain of Pappenheim (previously described) as well as the fol- lowing method of Bunge and Trantenroth may be used. After fixation of the smear, the fat is removed by soaking the specimen in absolute alcohol. The preparation is now covered with a 5 per cent, solution of chromic acid for 15 minutes, after which it is washed with water. The smear is stained with car- bol-fuchsin, decolorized with 16 per cent, sulphuric acid for three minutes, and is then counter-stained for five minutes in a concentrated alcoholic solution of methylene blue. This method shows the tubercle bacillus as distinct red, while the smegma bacillus is blue. While both of these methods of differ- entiation are usually applicable, yet one occasionally finds the smegma bacil- lus resisting the action of 16 per cent, sulphuric acid for 30 minutes and occa- sionally of strong alcohol for 12 hours. (d) The Timothy Bacillus. This organism is present in the mouth reaching it through the medium of butter and milk, which may contain it in large numbers. These baciUi re- sist the decolorizing action of both alcohol and acid to almost the same extent as do the tubercle bacilli, but they usually appear as somewhat longer and iSee Duval, Jour. Exper. Med., igii, XIII, 365; also Duval and Wellman, Jour. Am. Med. Assn., 1912, LVIII, 1427; Clegg cultivates this bacillus in symbiosis with amcebas and cholera vibrios (Philip. Jour. Sc, Sec. B., 1909, IV, 403); Smith, Lynch and Rivas have demonstrated the transmissibility of the lepra bacillus by the bed-bug (Am. Jour. Med. Sc, 1913, CXLVI, 671). See, also, Wplbach and, Honeij, Jour. Med. Research, 1913, XXIX, 367; Honeij and Parker, Ibidi?/0f«@C6qr,MJ^/'OSO/r® THE SPUTUM 25 thicker rods. Strangely enough, this organism produces a lesion in guinea- pigs which resembles very closely that of true tuberculosis, so that the inocu- lation test will not always be conclusive unless other animals are inoculated with material from the first one, in which case no lesions develop in the later Fig. 6. — Diplococcus pneumonise. (Williams.) animals. Fortunately, the cultural peculiarities of this organism are markedly different from those of the tubercle bacillus, as the former develops readily on the ordinary culture media. (e) The Pneumococcus (Diplococcus pneumoniae). This organism, discovered by Frankel and elaborated by Weichselbaum, is generally recognized as the etiologic factor in cases of acute croupous pneu- monia, although other organisms not infrequently give rise to this condition. It is found in large numbers in the sputum and other exudates, appearing as a small slighty elongated conical or lance-shaped coccus, which shows a marked tendency to occur in pairs (diplococci) with the broader ends in apposition. Occasionally it is arranged in short chains resembling streptococci.' In exudates and in the blood this organism is usually enveloped by a well-defined hyaline zone or capsule. In clinical work it is rarely necessary to stain this capsule to identify the organism but if this be desirable it is best done by the method of Rosenow.^ It stains well with the ordinary dyes and is Gram-positive. Friedlander's Bacillus (bacillus mucosus capsulatus) is occasionally found in some cases of lobar and lobular pneumonia and occasionally may be con- 'See Dochez and Avery (Jour. Exper. Med., 1915, XXI, X14; Ibid., XXII, 105) and Lyall (Ibid., 1915, XXI, '146) for studies of the types of pneumococci occurring in nor- mal and pathological throats. 2 Jour. Infect. Dis., 191 1, J^fized by Microsoft® Fig. 7. — Fried- lander's bagillus (above) : pneumococ- cus (below) (Greene). 26 DIAGNOSTIC METHODS sidered the etiologic factor in such conditions, although it is usually a secondary im'ader. These bacilli grow readilj' on artificial media, are encapsulated and stain easily with the ordinary dyes but are Gram-negative. (/) The Influenza Bacillus. This bacillus, known as Pfeiffer's bacillus, is found in the bronchial sputum, especially in the pulmonary type of this disease. ^ The most char- acteristic sputum is greenish-yellow in color with lumps of pus in num- mular form. The organisms are found in such sputum as small, short bacilli measuring '^lo to ■^iq micron in breadth by ^lo micron in length. They usually occur singly, but may form chains. In the stained specimens these organisms show distinct polar stainings, appearing frequently as diplo- cocci. They are stained with dilute carbol-fuchsin solution, faintly with the ordinary methylene blue solution, or are identified by their Gram-negative characteristics. The best counter- stain used in the Gram method is either Bismarck brown or safraiiin, the organisms appearing both intra- and extra-cellular. ig) The Bacillus Pertussis. This organism, discovered by Bordet and Gengou, and elaborated by Klimenko, has been frequentlv found in the sputum in cases of whooping-cough. It resembles very Fig. S.-BaciUus influenza in sputum. '^^''^''^y ^^^ influenza bacfllus, appear- (Ahboii.) ing as short, plump, ovoid baciUi, with rounded ends and lying singly or in small groups between the pus and epithelial cells. It stains feebly with the usual d}-es often showing bipolar staining and is Gram-negative. This organism is rarely intra-cellular and may thus be distinguished from the influenza bacillus. (//) Typhoid Bacillus. This organism has been found in the sputum in typhoid fever cases show- ing a coexistent bronchitis or pneumonia. The sputum is usuallv hemor- rhagic in character and shows the bacilli as short, thick rods, staining with the ordinary dyes and negative to Gram's stain. (i) Staphylococcus and Streptococcus Pyogenes. These organisms are found in practically every sputum examined and can be identified only by the use of cultural methods. They stain well with any iSc-e Davis (Jour. .\m. Me4^;):Ssn jIqi^, I^V. iSi^U a discussion of the role of tliis organism, as well as other men«M'A?¥M: WrrfB^WlMflSiifMc group as secondary invaders in respiratory infections. .\lso, WoUstein, Jour. Exper. JVIed., 1915, XXII, 44s ) PLATE II. )CarWarine Hill Streptococcus Pyogenes. (Methylene Blite Stain.) Digitized by Microsoft® Digitized by Microsoft® THE SPUTUM 27 of the aniline dyes and are Gram-positive. Their presence in the sputum has little pathologic significance.^ (j) The Bacillus Pestis. This bacillus of bubonic plague was discovered by Kitasato and Yersin in 1894. It is a short, thick bacillus, measuring from 0.8 to 2 microns in length and from 0.4 to 0.8 micron in thickness. A capsule may be usually made out and the stained organism frequently resembles a diplococcus, owing to the intense polar staining with intermediate faint staining. These organisms are Gram-negative. The bacillus pestis is found in the sputum of persons suffering from the pneumonic type of this disease and should be recognized owing to the markedly infective character of the material.^ It may be necessary abso- lutely to identify the organism by inoculation and cultural experiments. (k) The Bacillus Anthracis. The sputum of cases of pulmonary anthrax may contain large numbers of these bacilli. These organisms are from 5 to 10 microns in length and from I to 1 3*^ in breadth. They are frequently grouped in long segmented threads, the segments varying in length, but usually being two or three times as long as broad. Occasionally these bacilli may be single, but are usually multiple. They form oval spores in the middle of the short segments. The organism stains with the ordinary dyes and is also Gram-positive. For absolute identification cultural and inoculation experiments, the latter into white mice, may be necessary, but the morphological characteristics will usually identify it. (/) The Bacillus Mallei. This organism of glanders is found in the sputum in the pulmonary form of this disease. Morphologically, there is nothing characteristic in the ap- pearance of this organism beyond the fact of the presence of faintly staining areas in the protoplasm of the rather long bacilli. These organisms stain by Gram's method as well as with the ordinary aniline dyes. For a final diagnosis inoculation into a guinea-pig should be made. (w) Actinomyces Hominis (Ray Fungus). This fungus, which gives rise to the condition known as lumpy jaw in cattle, occasionally infects man, causing pulmonary conditions designated streptothricosis. The mucopurulent sputum in such conditions contains elastic tissue and small sulphur-yellow granules which are visible to the naked eye and are the characteristic findings of such cases. Macroscopically these granules are yellowish, grayish, or brownish in color, and are sometimes abundant and sometimes scarce.* They are very friable, and when gently ' The streptococcus viridans is not infrequently found in association with infections of the upper respiratory tract. See Cecil, Arch. Int. Med., 1915, XV, 150. Leuetscher (Ibid., 1915, XVI, 657) discusses the importance of cultural methods in non-tubercular infections. 2 See Strong and Teague, Philip. Jour. Trop. Med., 1912, VII , 187. ' Davis (Jour. Infect. Dis., 1914, XIV, 144) calls attention to actinomyces-like granules found in the crypts of tonsils and composed of bacilli, streptococci and spirilla. See, also, RuUmann, Munch, med. Wchnschr., 1914, LXI, 1890: Claypole, Arch. Int. Med., 1914, XIV, 104; Davis, Ibid., I, C^lbmdjb^MiGm^^lll, ss: 28 DIAGNOSTIC METHODS crushed beneath the cover-glass and examined microscopically appear to have broken up into hyaline rounded masses at the margins of which, on close inspection, fine radial striations or filaments or hyaline club-shaped bodies, all closely set together, may be seen. The club-shaped bodies are variable in size and are composed of a hyaline refringent substance. In the granules obtained from the lesions in man the club-shaped formations are much less frequently observed than those obtained from the lesions in cattle (Mallory and Wright). If cover-glass preparations be made and stained with Gram's method, one will usually find isolated and matted Fig. 9. — Actinomyces. (Williams.) filaments, many of which may be seen to branch, in addition to longer and shorter fragments of filaments and fine detritus of the same. If clubs are present in the granules they may be found scattered throughout the prepara- tions. In this connection see Bridge. "^ (g) Animal Parasites. (o) Amebse. Artault has described a unicellular ameboid body which resembles very closely a leucocyte when stained, but, while motile, differs in refractility and staining quality. This he calls the ameba pulmonalis. In cases of perforat- ing liver abscess the true amebae coli may be found in the sputum, and, ac- cording to Flexner, in cases of abscess of the jaw communicating with the mouth. ^ These amebae will be discussed fully in the chapter on Feces, to which the reader is referred. It should be noted here that these organisms may not be numerous so that many slides may have to be examined. (6) Flagellates. Flagellated organisms, such as the trichomonas pulmonalis and the cercomonads, are found in the sputum associated with Dittrich's plugs in cases of gangrene, putrid bronchitis, and tubercular cavity formation. The trichomonas is probably identical with the trichomonas vaginalis or intestinalis. 1 Jour. Am. Med. Assn., 1911, LVII, 1501; also, Schlegel, Kolle and Wassermann's Handb. d. path. Mikroorg., 1912, V, 301. 2 These are probably identical with the endamebae gingivalis found in the mouth in cases of pyorrhea. (See next chapE^)'f;zecf by Microsoft® THE SPtTTUM 29 (c) Cestodes. Not infrequently the lung is the seat of infection with the taenia echino- coccus.^ In such cases various foreign bodies, such as fragments of mem- branes, scolices, booklets, and cysts, may be found in the sputum. Such formations may, also, be found in cases of liver abscess perforating into the lung. Any one of the above formations is characteristic of this condition. The parasite will be discussed fully in the section on Feces, to which the reader is referred. The sputum in such cases is usually purulent or mucopurulent and may be tinged with blood. This sputum may be expectorated over a long period Fig. 10. — Paragonimus westermanii (ventral view); 10 X 1. A, oral sucker; B, ceca; D, acetabulum; E, genital pore; F, uterus; G, ovary; H, testicles; /, vitelline glands; K, excretory canal; L, excretory pore. {Tyson after Braun.) of time and may even contain tubercle baciUi from a coexistent tuberculosis. A distinguishing point between the sputum of pulmonary echinococcus dis- ease and that of a perforating liver abscess is that the sputum in the latter is usually bile stained.'' {d) Trematodes. The most common of this class of animal parasites is the ordinary "lung fluke," which has been called also distoma pulmonale, distoma Westermanii disfoma Ringeri, and Paragonimus Westermanii. The eggs of this parasite are much more frequently found in the sputum than are the parasites them- 'See Filia, Pensiero med., 1914, IV, 741; also, Ortali, Gazz. d. osp., igis, XXXVI, 225. ^ See Maliwa, Munch, med. Wchnschr., 1914, LXI, 2367. Digitized by Microsoft® 30 DIAGNOSTIC METHODS selves, so that the diagnosis will rest with the finding of these ova. These eggs measure from 80 to 100 microns in length and 40 to 60 microns in width. They are brownish in color, oval in shape, have a smooth thin shell and a lid near one end which is quite characteristic. The parasite is from 8 to 10 mm. long, 4 to 6 mm. wide, and is very markedly rounded anteriorly, being nearly as thick as broad. The sputum in such cases is usually small in amount, is very tenacious, and is reddish or rusty due to admixture of blood with the mucus. Frequently no blood is found, in which case the sputum will still be of a yellowish or brown color due to the eggs themselves. The sputum, also, contains many spirals, which resemble very closely the Curschmann spiral and, also, the Charcot-Leyden crystals. The eggs of another species, of distoma, the dis- toma hematobium, has been found in the sputum by Manson. Gage^ has reported the finding of the larvae of strongyloides intestinalis in the sputum. Fig. II. — Ovum of paragonimus wester- manii, from sputum: 1000 X I. {Tyson after Braun.) V. The Sputa in Disease (i) Pulmonary Tuberculosis. It has been truly said by Brown that pulmonary tuberculosis has no char- acteristic form of sputum. The amount voided may vary from the very slight type of fibroid tuberculosis to the very abundant sputum of cavity formation. It is to be said that the amount of pus will usually depend upon the extent of the secondary infection, although caseous degeneration may lead to the expectoration of large amounts of material resembling pus. In the early cases of pulmonary tuberculosis we may find a small amount of sputum which is expectorated only in the morning. This may be very tenacious and resemble very much the sago-like sputum previously mentioned. Sooner or later depending upon the extension of the disease, there will appear small caseous particles which are very suggestive. As ulcerative processes proceed, the sputum becomes more profuse, yellowish or greenish in color, and muco-purulent in character. In any stage of this ulcerative tuberculous con- dition Are may find blood in amounts ranging from a few blood-cells to a sputum loaded with blood from a hemorrhagic focus. Likewise we will find elastic tissue in more or less amount and tubercle bacilli varying from a few to many in each field. The color of the tubercular sputum may range through all the shades of the spectrum, the greenish shade being associated with a most marked decomposition. As stated previously, the most sus- picious looking sputa frequently contain no tubercle bacilli, so that fre- quent search must be made for these organisms which are the only diagnostic point of the sputum in this condition. 'Arch. Int. Med., 191 1, vQ(gilsed by Microsoft® THE SPUTUM 31 (2) Croupous Pneumonia. The early sputum of acute lobar pneumonia is usually yellowish-red in color and very tenacious in consistency. In some cases the sputum is mucoid and abundant for a few days, but soon takes on the characteristic reddish color from the presence of unchanged red blood-cells. Its consistency is so great that the receptacle may be inverted without allowing any material to run out. The characteristic rusty sputum, which is found when the exudation into the alveoli is taking place, is homogeneous, glairy, very tenacious, and deep red in color. This rusty sputum, while characteristic of pneumonia, is sometimes replaced by one ranging in color from a yellow to a green. These colors are due to different oxidation products of hemoglobin, and are, perhaps, more fre- quently observed in the stage of resolution when the sputum becomes less tenacious and more abundant. The greenish sputa in pneumonic conditions have some importance. This coloration may be due to a coincident jaundice or may arise from delayed resolution, especially when the exudate has been particularly hemorrhagic. It is, moreover, sometimes an indication that a true tubercular condition has intervened and, hence, that the prognosis must be guarded. The so-called prune-juice sputum> usually indicates a severe type of the •disease, while at times it may signify merely a beginning resolution. ^ Fibrinous coagula are found, according to Osier, in every case in which search is made. These may vary from very small bronchial casts to very large branching t)^es. Curschmann's spirals as well as the Charcot-Leyden crystals are frequently observed. The characteristic organism of this con- dition, the diplococcus lanceolatus of Frankel, is usually found, but has only incidental importance, as it is so frequently present in the sputum of normal individuals.^ (3) Bronchopneumonia. The sputum of this disease is rarely characteristic. It partakes of both the type of a bronchitic and a pneumonic sputum. It may, therefore, contain much mucus and pus, may be viscid, may be streaked with blood, but is rarely so distinctly rusty as in the croupous type of this disease. As the disease is so limited in extent it is more or less rare to find an abundant sputum or to observe fibrinous coagula. Microscopical examination shows various organisms, but nothing diagnostic. (4) Acute Bronchitis. The sputum in this condition is very scanty in the early stages, is usually very tenacious and is expelled with difficulty. This early bronchitic sputum is known as "sputum crudum" and consists of practically pure mucin, con- taining within its meshes a few leucocytes, red cells, bronchial epithelial cells, and a few myelin drops. 'Pacini (Interstate Med. Jour., 1912, XIX, 536) has advanced the following reaction as characteristic of early pneumonic sputum: Mix i volume of sputum with 10 volumes of distilled water, agitate for 5 minutes and filter. To a test-tube containing 10 c.c. of distilled water add 5 drops of a i per cent, aqueous methyl-violet solution. Add to this latter 10 drops of the sputum filtrate. A positive reaction is shown by the appearance of a distinct red color. ^See Rosenberger and ^ogJ^^^^J^^ Y^^Med^^^r., 1913, XCVII, 532. 32 DIAGNOSTIC METHODS After a few days the sputum is increased in amount, becomes less viscid, and assumes the type of a distinct muco-purulent sputum. This sputum, called the sputum coctum, contains numerous pus-cells, is yellow or yellowish- green in color, shows the presence of large numbers of red cells, as a rule, and an increase of the polynuclear leucocytes over the mononuclear form. These mononuclear forms are more characteristic of the sputum of true tubercular conditions. Fat may be found, either in isolated drops or in large masses. As improvement in the condition occurs, the sputum becomes more abundant, and more distinctly purulent, and then gradually diminishes until it ceases. The sputum of acute bronchitis may give much information as to the course of the disease, as the transition from the viscid mucoid sputum through the abundant purulent stage to the final cessation is quite characteristic, (s) Chronic Bronchitis. (a) Simple Chronic Bronchitis. In most of these cases the sputum is either very little in amount or is much more abundant than in the acute forms. Such cases of simple chronic bron- chitis are usually those following the acute type of the disease in which we find the expectoration, for long periods of time, of a tenacious, viscid, and scanty sputum. Later it may become more abundant and muco-purulent, and may have a dark color and a distinctly foul odor. In the type of chronic bronchitis associated with cardiac disease we find large amounts of blood which may be fresh or changed, giving the typical prune-juice appearance. In such sputum we frequently find large numbers of the so-called "heart-disease cell" which have been previously described. (J) Putrid Bronchitis. This condition is brought about by dilatation of the bronchial tubes fol- lowing a chronic bronchitis. The sputum lies stagnant in these dilated bronchi so that it decomposes to a great extent. The sputum in such cases is very abundant, is of an ash-gray or brown color, is markedly purulent, and has a very disagreeable odor. On standing it separates into the three layers which have been previously discussed. In such conditions no elastic tissue fiber is found, so that we have here a differentiation from gangrenous or tuberculous pulmonary conditions. The sputum in this condition is very similar to that found in bronchiectasis, which is usually associated with decomposition of the sputum. Whether a diagnosis is possible between a straight putrid bronchitis and bronchiectasis is doubtful, if one relies merely upon the sputum. The sputum in bronchiectasis occurs usually in the morning and is then very pro- fuse. It shows, however, the characteristics of the sputum of putrid bron- chitis, but is more commonly associated with the presence of pus, while 50 per cent, of cases show more or less profuse hemorrhage. (c) Fibrinous Bronchitis. The chief characteristic of this condition is the expectoration of more or less perfect bronchial casts. These maybesingle or may be distinctly branch- ing, showing the arrangement of the entire bronchial tree. This condition occurs quite frequently Mml^Sl'%m%%fWorAe diseases, but in the dis- THE SPUTUM 33 cussion at this point we have reference to the idiopathic type of the disease. The sputum in this latter class of diseases is mucoid and very abundant in the earlier stages. After a few days there is expectorated, following a severe coughing spell, a bronchial cast. This expectoration is usually tinged with blood. Such casts may be expectorated over long periods of time and their form may vary as previously described. (6) Bronchial Asthma. The sputum in bronchial asthma is, perhaps, more characteristic than that of any other pulmonary condition. During the paroxysm of asthma there may be no sputum, or it may be scanty, consisting of the glairy mucoid plugs known as the pearls of Laennec. The sputum contains many eosino- phile cells and many alveolar epithelial cells with myelin degeneration. The mucoid sputum usually contains large numbers of the spirals of Curschmann along with the Charcot-Leyden crystals. In some cases of asthma one finds small cylindrical casts of bronchi. Some of these branch while the majority are straight and may taper at one end into the central fiber of a true spiral. (7) Influenza. The sputum of the pulmonary type of this condition shows in the early stages as a very scanty tenacious expectoration. Later it increases in amount, becomes muco-purulent and often blood-streaked, and is greenish-yellow in color. This sputum contains large numbers of Pfeiffer's bacilli, which have been previously discussed. (8) Gangrene of the Lung. The sputum in this condition is very profuse, is greenish-brown in color, is very offensive in odor, and is extremely fluid in character. It contains shreds of elastic tissue which serve to distinguish it from the sputum of putrid bron- chitis or bronchiectasis. This sputum separates, as do other forms of sputum which have undergone stagnation and decomposition, into three distinct layers. Microscopic examination shows fragments of necrotic tissue varying from very minute particles to those several cm. in length. Very few epithelial cells or leucocytes are found, but red blood-cells are more or less frequent. The bacterial content is usually very high, but nothing characteristic is found among these organisms. (9) Abscess of the Lung. The most characteristic feature of true abscess of the lung or of liver abscess which has perforated into the lung is the sudden appearance of a large amount of pure pus containing fragments of lung tissue. This material usually has the normal odor of pus, but may become offensive, although never as markedly so as in gangrene or putrid bronchitis. The sputum of *the perforating liver abscess is usually distinguished from that of the true lung abscess by the so-called "anchovy-sauce" appearance. The color may vary, due to the presence of various types of bile pigment. Microscopically, bilirubin crystals may b^gj^g^.^ ^^y Microsoft® 34 DIAGNOSTIC METHODS (lo) Perforating Empyema. The sputum of such conditions is composed almost entirely of pus and is thin and liquid. It contains many hematoidin crystals, but very little elastic tissue fiber or other tissue fragments. The odor is usually described as that of old cheese in the beginning, but soon becomes offensive owing to decomposition. (ii) Pneiimonoconioses. The sputum in these various conditions will depend upon the pigment with which the lung has been infiltrated. The expectoration is usually muco- purulent, very profuse, and is laden with coal-dust (anthracosis), iron-dust (siderosis), with stone-dust, chalk-dust or plaster of Paris (chalicosis), and with starch granules (amylosis). BIBLIOGRAPHY Andre. La grippe ou influenza. Paris, 1908. AuFRECHT. Die Lungenentzundungen. Wien, 1901. Cornet. Die Tuberkulose. Wien, 1901. De Jong. .Etude histo-chimique et cytologique des crachats. Paris, 1907. Hoffmann. Die Kranklieiten des Bronchien. Wien, 1900. Sticker. Lungenblutungen. Wien, 1901. Digitized by Microsoft® Digitized by Microsoft® PLATE III. Leptothrix and Spirocheta BirccALis. (Unstained Specimen.) Digitized by Microsoft® CHAPTER II ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS I. Oral Secretions (i) General Considerations. The oral secretion is a mixture derived from the various buccal glands, the submaxillary, sublingual,' parotid, and mucous glands. To this secretion has been given the name saliva. It is a colorless, odorless, and tasteless fluid, which appears somewhat stringy and frothy, separating on standing into two layers, the upper one of which is clear and the lower one cloudy. The function of this secretion is to moisten the mouth and throat and, also, to aid in swallow- ing the food as well as partially to digest the starchy food through the action of a specific ferment (ptyalin) which it contains. The normal daily amount of saliva secreted is usually about 1,500 c.c, this quantity varying under the influence of many factors, both physiologic and pathologic. The specific gravity ranges between 1,002 and 1,009 giving a total solid content of 3 to 12 grams. Its reaction is alkaline, corresponding to 0.006 to 0.48 per cent, of sodium hydrate. While the reaction of the saliva is normally always alkaline, we occasionally find an acid reaction, especially in children and in the early morning hours, due to the production of lactic acid by the bacteria which are always present in the mouth. Likewise we find an acid reaction especially in conditions associated with acidosis, such states very frequently leading to dental caries and to many other irritative conditions of the mouth. The recent work of Talbot along this line is especially interesting. The chemical composition of the saliva does not have ary great clinical significance'^ with the exception of the presence of the sulphocyanates, the nitrites, and the characteristic ferment ptyalin. These substances seem to have some importance both from a diagnostic and symptomatic standpoint, so that a few remarks may be timely. The presence of potassium sulphocyanate (KCNS) is more or less characteristic of normal saliva and may be detected as follows: Collect a few c.c. of saliva before meals and allow this to filter. Add a few drops of hydrochloric acid and then a drop or two of ferric chlorid solution, when a distinct red color will be observed, whose depth will depend upon the amount of sulphocyanate present. It has been stated that heat should be apphed in this test, but the writer has never found it necessary as the characteristic reaction almost invariably appears in the cold. This color disappears on the addition of mercuric chlorid solution, which fact may serve to differentiate it from the similar one given by the sahva of opium habitues and due to meconic acid. Very little pathologic significance has been at- 1 See Herzfeia and Stocker (Zentralbl. f. inn. Med., 1913, XXXIV, 753) for a discussion of uric acid in saliva. Digitized by Microsoft® 36 DIAGNOSTIC METHODS tached to variations in the amount of the sulphocyanate in the saUva, but it is interesting to note that in many cases of diabetes as well as in cases of severe stomatitis this substance is frequently absent. The nitrites may be detected by the more delicate tests used in water analysis, as their amount is usually not sufficient for the ordinary quaUta- tiVe tests. A very good test is the use of the Griess-Ilosvay reagent (J^ gram of sulphaniUc acid is dissolved in 150 c.c. of dilute acetic acid and treated with }io gram of naphthylamin dissolved in 20 c.c. of boiling water. On standing, a blue sediment forms which is separated and dissolved in 150 c.c. of dilute acetic acid). On treating 10 c.c. of saliva with a few drops of this reagent and heating, a red color will develop in the presence of nitrites. The' most important constituent of saliva is, the ptyalin which has a defi- nite hydrolytic action upon starch, converting this polysaccharid into maltose through the intermediate stages of erythrodextrin and achroodextrin. This action may be readily seen by treating a little starch paste with a few c.c. of filtered sahva and placing the vessel in the incubator for 10 to 15 minutes. At the end of this time iodin solution is added when a distinct red color or an entire absence of color will be noticed. It is to be remembered here that starch, treated with iodin, is colored blue, so that a change of the starch is evident by the color reaction. Careful work by Litmanowicz^ has shown that the diastatic power of saliva is unaffected by physiologic or pathologic variations in general body functions.^ (2) Microscopic Examination. On allowing saliva to stand it separates into two distinct layers, the upper one clear and containing the liquid portion, while the lower is cloudy and con- tains the morphological elements. In the microscopic examination of this lower layer we observe many epitheUal cells in the form of large, irregluar, squamous cells which are derived from the mucous membrane of the mouth and tongue. The number of these cells present depends, of course, upon the erosion to which the mouth has been subjected by various irritants either of the food or of disease. The characteristic cells of the saliva are the sahvary corpuscles, which resemble the leucocytes, but are larger and more granular. Occasionally red blood-cells are seen, but these have no direct significance other than to denote ulcerative or markedly irritative conditions somewhere in the nasopharynx. Beside these constituents of the saliva, we find, micro- scopically, many micro-organisms of the mold, yeast, and bacterial types. The bacteria are always present in the mouth as they are taken in with the air, food, and drink. Few of these have any direct significance, although the spirochaeta buccahs and microdentium should be borne in mind, especially when an examination is being made for the spirochaeta pallida.' The former is differentiated from the latter by the fact that its ends lie upon a line drawn longitudinally through the center of its spirals, while such a line drawn through the paUida Hes above and below its ends. Moreover, it should be re- 1 Zentralbl. f. d. ges. Physiol, u. Path, des Stoffw., 1909, IV, 81. * See Hirata, Biochem. Ztschr., 1912, XL VII, 167 ; van Haeff, Nederl. Tydschr. v. Geneesk., 191S, LIX, 307; Lubimoff, Russk. Vrach., 1915, XIV, 269. ' See Thibaudeau, Jourr^yJ^^^jftj^^tfty^^^, 446. ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 37 membered that the smegma bacillus is an occasional habitant of the mouth and throat and may occur in specimens of sputum, giving rise to the assump- tion of the presence of tubercle bacilli unless proper means of identification are used. Simon has pointed out an interesting fact that the majority of the micro-organisms which are constantly present in the mouth cannot be culti- vated on artificial media, while the temporary invaders easily develop. Many pathogenic bacteria have been found in the mouth of the healthy sub- ject.^ This is interesting clinically as showing the constant danger to which we are all subject, in case our resistance becomes lowered. The writer recalls that the most virulent culture of pneumococci obtained from 200 throats, both diseased and normal, was from his own at the time when he was in perfect condition and showed no symptoms thereafter. Beside the pneumonia organism, streptococci and diphtheria bacilli are frequently found in the mouths of perfectly healthy individuals. Molds and yeast fungi are rarely found in the saliva during health, but they are frequently present in patho- logical conditions. (3) Pathologic Changes. The normal daily secretion of the saUva is, as stated above, about 1,500 c.c. The composition of the secretions of the various glands, which' contribute to the mixed secretion, differs rather widely, the one from the other. We may, therefore, have changes, not only in amount of saUva, but, also, in the quality, depending on the diseased condition of one or more of these glands. The quantity of saliva is diminished in inflammation of the saUvary glands, such as in parotitis, in all febrile diseases, in diabetes, and in nephritis. The secretion is also diminished by the therapeutic use of preparations of bella- donna and of opium. It is increased by certain poisons, such as pilocarpin and mercury, by excessive irritation with acids and alkalies, and, also, by irri- tations arising from carious teeth. Occasional cases have been reported of a greatly increased amount of saliva through some obscure nervous reflex, while such a condition is not unusual in pregnancy. An increased flow of sahva is known as saUvation of ptyalism. In determining whether or not salivation really exists, observation will frequently show increased amounts of sahva at all times. In some cases, however, it is necessary to measure the amount and, also, to make later chemical examinations of the saliva. The best way of ob- taining sahva free from contamination is to wash the mouth thoroughly with a solution of sodium bicarbonate, brush the teeth thoroughly with the same solution, and then rinse out the mouth with cold water. On now touch- ing the inner surface of the teeth or the edge of the tongue with a glass rod that has been dipped into dilute acid, saliva will be seen pouring into the mouth from many points. This saliva is then coUected in clean receptacles and the quantity measured. Variations in the reaction of the saliva are not uncommon in pathologic conditions. In various intestinal diseases with which we may have an associ- ated stomatitis, an acid reaction is frequently noted. Also in fevers, diabetes, starvation, and other conditions giving rise to acidosis (overloading of the sys- >See Kuster, KoUe and Wg^^#fe©a|ij&j^JW/feWfe»iB. VI, 435- 38 DIAGNOSTIC METHODS tern with acid products) the reaction of the saliva is always acid. Strauss and Cohn beheve that the saliva is practically always alkahne, even under pathological conditions. Coating of the Tongue. A coating of the tongue is practically always abnormal, as the normal appearance is a bright reddish color with no visible deposits.^ A change in the normal appearance of the tongue has so long been indicative, in the minds of the profession, of disturbed conditions not only in the mouth, but in the stomach and the bowels, that one should always take into consideration any such change. In severe infectious fevers a brownish coating with a furred ap- pearance is practically always seen. This consists of remnants of food and of incrusted blood, along with large numbers of micro-organisms and dark des- quamated epithehal cells. The white coating contains no blood and is more indicative of simple gastro-intestinal disturbance than is the brown coating. The so-called " tartar" which forms upon the teeth seems to consist of depos- ited calcium carbonate and contains many actively motile spirochete as well as large segmented leptothrices, along with leucocytes and epithelial cells. Pharyngomycosis Leptothrica. In many pathological conditions of the throat, such as tonsillitis, diphthe- ria, and thrush, we frequently find the tonsillar and other buccal structures covered with a coating which is, in many cases, a distinct membrane contain- ing the pathogenic organisms in large numbers. Many perfectly normal sub- jects complain of the formation, in the tonsillar crypts, of plugs of material which are easily removed by pressure. These are frequently found in patients subject to tonsillitis, but, also in those showing no pathological conditions of the tonsils and are closely related to Dittrich's plugs, which have been discussed. In the pyoid masses of phar)'ngomycosis leptothrica, one finds large num- bers of lymphocytes, epithelial cells and long segmented fungi, the lepto- thrices buccalis,! which are colored bluish-red by a solution of iodopotassic iodid. In such conditions the polynuclear neutrophiles are present in only small numbers. In some cases patches of these fungi extend over quite an area of the tonsils so that the appearance may be one of the formation of a diphtheritic membrane, although microscopic examination will at once clear up the diagnosis.^ Diphtheria. One of the most important examinations of the oral cavities consists in the detection of the diphtheria bacillus (Klebs-Loffier bacillus), as an early diagnosis of this disease frequently enables the physician to institute anti- toxin treatment. Such an examination should never be omitted in any case of suspected sore throat, especially where any membraneous patches are pre- sent.' By means of a stout platinum loop or a swab of cotton a piece of mem- brane or a portion of the exudate is scraped from the throat. This material ^See Schilling, Deutsch. Arch. f. klin. Med., 1914, CXIII, 622. 'Conlon (Jour. Am. Med. Assn., igis.LX, 900) has reported the aspergiUus niger as a causative factor in infection of the pharynx. » See Neisser and Gins, Kog^^^^^^^yipyy^;^^^ d. path. Mikroorg., 1912, V, 931- PLATE rV. t Ya+harine >^i/(. Diphtheria Bacilli Showing Polar Staining. (Neisser Method, Counter Stained WITH Sapranin.) Digitized by Microsoft® Digitized by Microsoft® ORAL, NASAL, AURAL, AKD CONJUNCTIVAL SECRETIONS 39 is then spread over the surface of Loffler's blood serum and is allowed to incu- bate at 37°C. for six to eight hours. This period of incubation is of some im- portance as it has been definitely shown that at the end of six to eight hours the diphtheria organism is the only one which will attract much attention, while if left for a longer time, other organisms, especially the streptococcus and staphylococcus, will so far outgrow the diphtheria bacillus that this latter may be unrecognizable unless the incubation be carried 36 hours, when the diphtheria bacillus then assumes the ascendency. From this culture, cover- glass preparations are then made and stained for one to five minutes in Loffler's alkaline methylene blue solution. They are then rinsed in water, dried, and examined with the immersion lens. Neisser's Stain. This stain is supposed to differentiate the diphtheria bacillus from all others. The smear is stained for five minutes with a methylene blue solution (methylene blue, Grtibler, i gram, 20 c.c. of 96 per cent, alcohol, glacial acetic acid 50 c.c, and water 950 c.c). The stain should be filtered before use. The specimen is heated gently during the staining process and the dye re- newed as the stain evaporates. Wash in water^ and stain for two minutes with an aqueous solution of Bismarck brown or, better, a dilute solution of safranin. The polar bodies will be stained a deep blue, while the body of the bacillus will take a light brown or red color. ^ Microscopically, the stained organism appears as a slightly curved rod, but especially characteristic are the bizarre forms, such as rods with alternate staining and nonstaining portions, rods with distinct deeply staining polar bodies, club-shaped or " narrow- waisted " rods, many of which lie together in distinctly parallel lines. Diphtheria baciUi may be found in the throat for weeks after all symptoms have disappeared^ so that it is wise to enforce isolation of the patient until a negative examination for these organisms is obtained." In causing the dis- appearance of these bacilli from the throats of " carriers" the method of spray- ing the nose and throat every three hours with a 24-hour bouillon culture of staphylococcus aureus, as advanced by Schiotz (Ugesk. f. Laeger, 1909, LXXVI, 1373), has proven very satisfactory. However, Bissell (Jour. Am. Med. Assn., 1913, LXI, 1393) believes the method is questionable as it leads to false security owing to the fact that the few diphtheria baciUi actually left in the deeper structures are masked by the numerous organisms of the sprayed culture.* Occasionally in examination of smears from the throat, true diphtheria ^Gins (Deutsch. med. Wchnschr., 1913, XXXIX, 502) advises a short (2 to 3 seconds) trSatment at this point with Lugol's solution containing i per cent, of lactic acid. See, also, Tamura, Ztschr. f . physiol. Chem., 1914, LXXIX, 289; Neilsen, Hospitalstid., 1915, LVIII, 225. ^See Raskin, Deutsch. med. Wchnschr., 1911, XXXVII, 2384; also, Ponder, Lancet, 1912, CLXXXIII, 22; Kinyoun, Am. Jour. Pub. Health, 1915, V, 246; Mood, Southern Med. Jour., 1915, VIII, 482. « See Markl and PoUak, Wien. klin. Wchnschr., 1913, XXVI, 1617. *See Womer, Jour. Am. Med. Assn., i9i3,LXI, 2293. Hektoen and Rappaport (Jour. Am. Med. Assn., 1915, LXIV, 1985) advise the use of kaolin, either swallowed or given as an insufHation for this purpose. Digitized by Microsoft® 4° DIAGNOSTIC METHODS bacilli may be confounded with pseudo-diphtheria bacilli, and in examination of other specimens, such as those taken from the eye, the bacillus xerosis may be confusing. These different organisms are best differentiated by the study of their action in fermenting or not fermenting certain sugars. According to Knapp, the pseudo-bacilli will ferment none of the sugars, the diphtheria bacilli will ferment dextrose, mannite, maltose, and dextrin, but not saccharose, while the xerosis bacillus ferments dextrose, mannite, maltose and saccharose (cane sugar), while it does not ferment dextrin. The details of the methods for these tests must be found in bacteriological works. (See Chapter XI.) Vincent's Angina (Ulceromembranous Angina and Stomatitis). In this coridition, smears taken from the throat, as well as the free sahva will be found to contain many organisms of two special types, the first, spirilla, and the second, long fusiform bacilli. Usually both of these types are found together, but occasionally the spirilla are absent. The spirilla usually meas- J^ '^ ^ \)i ' hL ^^^ %>- Fig. 12. — Vincent's spirillum and bacillus. {Coplin.) sure from 36 to 40 microns in length and )-^ micron in breadth, while the ba- cilli are 6 to 1 2 microns in length and are somewhat thicker in the center than at the end. These organisms may be readily stained withLoffler's methylene blue, gentian violet, or dilute carbol-fuchsin, but they decolorize with Gram's method.^ They are regarded by some as representing definite stages in the development of a trypanosome. Streptococcic Sore Throat. During recent years several milk-borne epidemics of sore throat with se- vere constitutional symptoms have occurred, especially in Boston, Chicago and Baltimore, of which the causative organism is a peculiar streptococcus (the streptococcus epidemicus). In smears from the throat and tonsillar exu- dates this highly virulent organism occurs in short chains, the spherical cocci appearing in twos in the chain. They are strongly Gram-positive and are sur- ' The organism has been recently cultivated, but inoculation experiments have been unsuccessful. See Gins, KoUe and Wassermann's Handb. d. path. Mikroorg., 1912, V, 1003. Digitized by Microsoft® ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 41 rounded by a definite capsule. They produce a relati^•ely narrow zone of hemolysis on blood agar but little or no greenish color.' Gonorrheal Stomatitis. In this condition the usual changes of infection are observed along with the appearance of the gonococci in the smears. Boston reports several cases of supposed gonorrheal stomatitis in which cultural methods showed the absence of this organism, although the smears showed the presence of intra- cellular Gram-negative diplococci. Such reports are not surprising in view of the fact that so many saprophytic diplococci (such as the diplococcus flavus and crassus) are found which may or may not stain by Gram's method. The almost constant presence (in the throat) of the micrococcus catarrhalis must be borne in mind. Thrush. This is a condition most commonly seen in children, but ma^' occur in adults, especially in those with tubercular tendencies. The saliva in this con- dition is'usually acid and somewhat increased in amount. Microscopic ex- FiK -Oidium albicans. [Kolle and W'u sserinaiiti.) amination of the membrane shows many epithehal cells, leucocytes, and much granular detritus with a network of branching band-like formations, showing distinct segments.- The contents of the segments are clear and usually con- tain two highly refracti\'e granules, one at each pole. This organism is known as the Oidium albicans. It stains well with the ordinary aqueous methylene blue solution. • See Davis and Rosenow, Jour .\m. lied. .\5sn., 1912, LVIII, 773; Hamburger, Ibid., p. 1109; Davis, Ibid., pp. 1283 and 1852; Capps and iSIiller, Ibid., p. 1848; Heinemann, Ibid., 1912, LIX, 71O; Leutscher, Ibid., p. 809; l-rost, Pub. Health Reports, 1912, XXVII, 18S9; Stokes and Hachtel, Ibid., p. 1923; Winslow, Jour. Infect. Dis., 1912, X, 73; Hamburger, Bull. Johns Hopkins Hosp., 1913, XXIV, i; Capps, Jour. Am. Med. Assn., i9i3,LXI, 723; Mann, Jour. Infect. Dis., 1913, XII, 4S1; North, White and Avery, Ibid., 1914, XIV, 124; Capps and Davis, Jour. Infect. Dis., 1914, XV, 130; Arch. Int. Med., 1914, XIV, 650; Rosenow and Moon, Jour. Infect. Dis., 1915, XVII, 69; Kirkland (Brit. Med. Jour., 1914, I, 419) reports a case from Cheltenham which belongs in this group; Bray, Jour. Am. Med. Assn., i9i5,LXIV, 1127. ^Ashford (Jour. Am. Med. Assn., 1915, LXIV, 810 and 1893) reports the linding, in most cases of this type, of a monilia (yeast) not hitherto described. Digitized by Microsoft® 42 DIAGNOSTIC METHODS Oral Endamebiasis. The condition, known as pyorrhea alveolaris or Riggs' disease, has for years been regarded as a form of bacterial infection in which local irritative processes and systemic disturbances are looked upon as predisposing factors. Many types of bacteria, such as streptococci, pneumococci and staphylococci, have been found and isolated from the pus pockets about the roots of the teeth, but these cannot be regarded as the only etiologic factors in this condi- tion, at least in the majority of cases. ^ While it has been known for years that parasites, especially those of the ameba group, were very frequently found in the mouth and in the soft tartar about the teeth, these were regarded as innocuous invaders, except in a few isolated cases. During the last year a great amount of work, following the first communication of Barrett, has centered about these amebse and their association with the lesions of pyorrhea, until at present their pathogenic im- portance is being freely discussed. There is little question, according to Smith and Barrett, that we must now "regard these parasites as either di- rectly causative of a large class of gingival and alveolar pyorrheas or as impor- tant members of a symbiotic chain with one or other of the numerous asso- ciated vegetable micro-organisms, in the production of these lesions." It is to be said that the proof of the pathogenic importance of the endamebas in Riggs' disease rests upon their almost constant presence in the suppurating pockets of pyorrhea (while they are rarely found in healthy mouths), and the prompt removal of both suppuration and of the endamebas when emetin (first advo- cated by Roger) is administered locally or generally. The organisms have not yet been cultivated, so that the question of conformity with the classic postu- lates of Koch has not been investigated. Further, many of the cases supposedly cured by emetin show recurrences after a few weeks' freedom from treatment. While there is no question of the presence of these amebae in cases of pyorrhea, it has not been firmly established, as yet, that they are truly pathogenic rather than merely " members of the symbiotic chain." It is evident, therefore, that more detailed study is necessary before the question of pathogenicity is settled. Several forms of endamebae have been described as habitants of the buccal and dental tissues, some of which were supposed to be pathogenic. These various amebae are more or less similar in their general characteristics, so that it is more than probable that the one, now first recognized by Barrett and a little later by Bass and Johns as almost constantly associated with pyorrhea, is the same as was long ago described by Gros, Steinberg, Grassi, Flexner and Prowazek. It is no more than just, therefore, that this ameba, whose patho- genicity is being investigated, should be styled Endameba^ gingivalis (Gros) and not Entameba buccalis (Prowazek), as some call it. These endamebse, as studied by Smith and Barrett, show the following characteristics: "Naked parasitic amebae of usual diameter in resting exam- ^See Nodine, Dental Cosmos, 1914, LVI, 969; Gilmer and Moody, Jour. Am. Med. Assn., 1914, LXIII, 2023; Billings, Ibid., 2024; Mayo, Ibid., 2025; Rosenow, Ibid., 2026; Craig, Ibid., 2027; Hartzell and Henrici, Ibid., 1915, LXIV, 1055. ^ The name endamebg. was first suggested by Leidy in 1879 and should take precedence of the term entameba advocated by Casigrandi and Barbagallo in 1897. Digitized by Microsoft® PLATE V. «^' a. ■^m ^i.'. ^, ». m > ' - « (•J v^. c / . , '/!(.;,■»; «t#ir.^#:*;'^;,-- ,_■ 0.0/Ml^m/. Camera Lucid a Drawings of Endameba Gingivalis (Gros), Stained with Iron Hematoxylin. {A. J. Smith and, M. T. Barrett, Jul. of Parasitology.) Digitized by Microsoft® Digitized by Microsoft® ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 43 pies of 0.030 to 0.035 ^i^- fwith exceptional instances reaching 0.040 or slightly above) ; with refractile and faintly greenish-tinted hyahne ectosarc well defined from the granular endosarc, b.ut sometimes so thin as to be easily overlooked; endosarc granular, colorless and in all but the more minute exam- ples containing few of many digestion vacuoles in which globular detritus of leucocytic nuclei and red blood cells are commonly found along with bacteria; with a small (0.002 to 0.005 mm.) rounded nucleus, invisible or at best un- certainly distinguishable in the unstained specimen, usually central or sub- central in position, but at times eccentric (when the ingesta push it to one side). The nucleus is very poor in chromatic substance, vesicular, with a small "binnenkorper," sometimes showing a minute centriole, a clear space^jbe- tween it and the nuclear border containing no chromatin or at most a very few incomplete threads; and the border is represented by a thin but somewhat irregular Hne of chromatin, about which the writers are unable to recognize a further membrane. The degree of motility manifested by the parasite is fairly comparable to that of the dysenteric endameba. The pseudopods as a rule are few (one, two or three), usually broadly lobose and commonly attain- ing a maximum length of the diameter of the endosarc. The pseudopods are composed practically entirely of the ectosarc, the granular endosarc terminat- ing at the base or extending but a Uttle into the larger ones." The material for examination is best obtained by scraping the bottom of the lesion in the peridental membrane between the tooth and alveolar process, as the endamebae do not live on the normal gum margin but at the edge of the tissue which they are invading. As the secondary infection with pyogenic organisms causes a profuse flow of pus which distends the pocket, very few endamebae can reproduce and live in this necrotic material. For this reason it is absolutely essential for success in this examination that most of this purulent material be removed from the old lesions and the specimen for examination he obtained from the bottom of the lesions. By means of a tooth- pick or, preferably, a Younger scaler No. 8 or 9, remove some of the material from the bottom of the lesions. If the specimen is to be examined in the fresh condition, mix the material with a drop or two of slightly warm normal saline solution on a warm slide and place over this a cover-glass. Examine at once with the high-power dry lens. Careful study will reveal the motile organisms showing the characteristics mentioned above. If the organisms are not actively motile, it is not the simplest matter to recognize them in the fresh state unless one is fairly familiar with them. For general work the stained specimen is, probably, more satisfactory for examination, as differentiation is more certain. Obtain the material as above mentioned and scrape it onto a clean slide with a toothpick, spreading it out into a thin layer with the flat side of the toothpick. Care should be taken not to rub the material back and forth as this may break up the amebae. Allow the shde to dry in the air and fix by passing once or twice through the flame. Cover the smear with carbol-f uchsin for 10 to 1 5 seconds, wash off the excess of stain with water and stain withLoffler's methylene blue for 30 seconds. Wash in water, dry an(^^^Qej^^i^p)^|j^ersion lens. Properly pre- 44 DIAGNOSTIC METHODS pared specimens should have a deep purple color. The red blood cells stain a deep red. Pus cells show a bright purple nucleus with a light pink proto- plasm. Tissue cells are larger than the pus cells and appear reddish with a small purple nucleus. Bacteria may be stained either red or blue, depend- ing on their type. The endamebae vary in size from cells somewhat smaller than a pus cell to those several times as large. The endoplasm stains a deep blue, while the irregular border of ectoplasm stains purple. A small round or oval centric nucleus is observed staining a deep port-wine color, while many inclusion bodies (contained in vacuoles) may be seen staining a deep purple. Usually the entire endameba appears to be surrounded by a clear zone, due to retraction during drying.^ II. Nasal Secretion This secretion does not present many points for study and seems to be of pathologic significance only in infectious conditions. Normally, the nasal secretion is comparatively scanty, clear, tenacious, odorless, salty in taste, and alkaUne in reaction. It is largely composed of mucus, showing squamous and ciliated epithelium in abundance,^ occasionally leucocytes, large numbers of bacteria and Charcot-Leyden and triple phosphate crystals. The bacterial content of the nasal secretion is made up of both pathogenic and non-patho- genic organisms, the tubercle bacilli having been frequently obtained from a normal mucous membrane.^ Pathologic Changes. In most acute infections, as well as in the so-called acute colds, the nasal secretion is at first diminished in amount, but soon becomes very profuse. This secretion shows the same appearance as does the normal fluid, but as ulceration ensues may be heavily loaded with pus-cells and bacteria. The chronic suppurative process in the nose may affect any or all of the accessory sinuses, so that we may have very severe conditions arising from simple ulceration. Frequently the ulcerative and membranous conditions spoken of above may extend from the mouth to the nose, so that distinct diphtheritic 'Gros, Bull. Soc. imp. nat. de Moscow, 1849, XXII, 549; Steinberg, Souremenaya meditsina, Dissert. Kiev, 1862; Grassi, Gazz. med. Ital.-Lomb., 1879, XXXIX, 446; Flex- ner, BuU. Johns Hopkins Hosp., 1892, III, 104; Kartulis, Ztschr. f. Hyg., 1893, XIII, g; Prowazek, Arbeiten a. d. Kais. Gesundheitsamte, 1904, XXI, 42; Leyden and Lowenthal, Charite Ann., 1905, XXIX, 3; Verdun and Bruyant, L'Echo m6d., du Nord, 1907, XI, 375; Bruyant and PeUssier, Ibid., 1909, XIII, 301; Brumpt, Pr^is de Parasitol., Paris, 1910; Barett, Dental Cosmos, 1914, LVI, 948 and 1345; Chiavaro, Editorial in Dental Cosmos, 1914, LVI, 1089; Austral. Jour. Dent., 1914, XVIII, 343; Dental Review, 1914, XXVIII, 1122; Bass and Johns, New Orleans Med. and Surg, jour., 1914, LXVII, 456 and 671; Smith, Middleton and Barrett, Jour. Am. Med. Assn., 1914, LXIII, 1746; Evans and Mid- dleton, Ibid., 19x5, LXIV, 422; Bass and Johns, Ibid., 553; Howe, Dental Cosmos, 1915, LVII, 307; Brit. Dental Jour., 1915, XXXVI, 464; Talbot, Dental Cosmos, 1915, LVII, 485; Jour. Am. Med. Assn., 1915, LXIV, 928; Rogers, Indian med. Gaz., 1915,1, 121; Stewart, Med. Record, 1915, LXXXVII, 798; Dental Cosmos, 1915, LVII, 60s; Stur- ridge, Ibid., 780; Johns, Interstate Med. Jour., 1915, XXII, 529; Smith, Jour. Am. Med. Assn., 1915, LXIV, 1567; Smith and Barrett; Jour. Parasitol., 1915, 1, 159; Brunelles and Ginsberg, New York Med. Jour., 1915, CII, 554; Williams, von Shelly and Rosenberg, Proc. New York Path. Soc, 1915, XV, 334; Bass and Johns, Alveodental Pyorrhea, Saun- ders, Phila., 1915; Hecker, Am. Jour. Med. Sc, 1915, CXLIX, 889. ^Proskauer (Berl. klin. Wchnschr., 1915, LI, 172 and 255) has shown that some of the cells may be melanotic. '.See Kuster, KoUe and Wassermann's Handb. d. Path. Mikroorg., 1913, VI, 450. Digitized by Microsoft® ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 45 membranes are frequently found in the nasal cavities. Molds may develop to such an extent that true pathological lesions obtain.' Rhinitis. At present the infectious nature of the catarrhal conditions known as "colds" is generally accepted, many organisms, such as micrococcus catar- rhalis, pneumococcus, streptococcus, influenza bacillus and others, having been associated in difEerent cases with the etiology. It would seem that these organisms are, however, to be regarded as simply secondary invaders, in many cases at least, and become of importance only when the discharge is purulent. TunniclifE'' has found, in the mucoid stages of acute and chronic rhinitis, a deUcate, curved. Gram-negative bacillus, which appears to have a direct pathogenic association with these conditions. While this Bacillus rhinitis of Tunnicliff is found in only 6 per cent, of normal noses, it was present in 98 per cent, of cases of acute rhinitis and in 90 per cent, of cases of chronic non- purulent rhinitis. It is, apparently, not found at all in purulent rhinitis, the other organisms mentioned above being the causative factors in this latter condition. There is Uttle doubt that this organism has an etiologic relation- ship to acute and chronic rhinitis on account of its almost constant presence in the nose in such cases, its general absence from the normal nose, its ability to produce rhinitis experimentally with recovery in pure culture, and on ac- count of the production, in cases of acute and chronic rhinitis and in persons injectedwiththebacillus, of specific opsonins and complement-binding bodies. This organism may be found by spreading a fairly large amount of the nasal discharge (mucus) on a glass slide, drying and staining with carbol- f uchsin. Often a large number of microscopic fields may have to be examined before the organisms are found. They vary in length from 5 to 8 microns and from 3^^ to }4 micron in width. The ends are pointed or slightly rounded; generally the bodies are slightly curved, but may be straight, wavy or bent over at one end. A ring and an enlargement in the form of a ball are occasion- ally seen at one extremity. The organism is a strict anaerobe growing at 37°C. slowly, the colonies on slightly alkaline goat-blood agar appearing usually in about seven days as small, dull, round growths. The organism does not grow on plain agar. Specimens from cultures stain a little more deeply than those taken directly from the mucus, but they retain their Gram-negative characteristics. Hay Fever. In this condition the nasal secretion is found to be increased to a large ex- tent at certain times of the day and much diminished at others, depending upon the paroxysms of the disease. Nothing of pathological importance has been found, however, in the examination of the nasal secretion in this condition, no specific organism having been identified. Meningitis. In some cases of meningitis the cerebrospinal fluid passes into the nasal cavity as a result of caries of the bones of the skull. This fluid may be dis- ' See Tilley, Jour. Laryngol., Rhinol. and Otol., 1915, XXX, 145. "Jour. Infect. Dis., 19^3, ^miZ^'tf^y^M^oM^' 493- See HoweU, Ibid., 456; also, Kruse, Munich med. WchflSchr.,,igni., LXI, 1547. 46 DIAGNOSTIC METHODS tinguished by the fact that it contains practically no albumin, but does show the presence of a reducing substance which may or may not be sugar. Tliis fluid may also contain the diplococcus intracellularis meningitidis of Weichsel- baum. While this organism is not found in all cases of epidemic meningitis, yet it is found in many, so that the nasal secretion may be of some diagnostic importance from this standpoint. The above diplococcus is similar, both in morphological and staining characteristics, to the gonococcus, and presents many difficulties in differentiation (see cerebrospinal fluid). In the course of glanders, leprosy, plague, pneumonia, typhoid' fever, influenza, and many other infectious diseases, the characteristic organisms of these conditions may be found in the nasal secretion. Recently Goldberger and Anderson^ have demonstrated the presence of the unknown virus of measles in the mixed buccal and nasal secretions.' Occasionally concretions are found in the nose, but these rarely reach a large size and do not have very great pathologic significance. They are largely composed of vegetable fibers taken in by inhalation and cemented by mucus which is hardened by the deposition of lime salts. In the condition known as ozena, the nasal secretion and fetid crusts, so characteristic of this disease, are found to contain numerous organisms of various types, among which one observes a peculiar cocco-bacillus which is difficult of cultivation and isolation. This organism was first noted by Perez, but it was not until the later studies of Hofer and his associates that it was practically established as the etiologic factor in this disease. To this micro-organism has been given the name coccobacillus fcetidus ozena (Perez).' III. The Aural Secretion Normally no secretions appear in the external ear, with the exception of that of cerumen, while the secretion of the middle ear and of the internal ear is normally inaccessible to examination. We find, therefore, that the chief importance which is attached to the clinical examination of the aural secre- tions, is entirely a pathological one. In catarrhal and inflammatory condi- tions of the external auditory canal, one finds naturally very large numbers of organisms, with which the disease may or may not be associated. In the chronic inflammatory processes of the middle ear, the more important organ- isms found are the pneumococcus, streptococcus pyogenes, staphylococcus pyogenes, bacillus pyocyaneus, the bacillus of Friedlander, the bacillus coli communis, the diplococcus intracellularis, the typhoid bacillus, and especially the diphtheria bacillus. As disease of the middle ear is so commonly asso- ciated with disease of the naso-pharynx, it is possible to find in the discharge from the ear any organism which is causing trouble either in the nose or in the throat. Hamilton has shown the almost constant presence of the pseudo- •Jour. Am. Med. Assn., igii, LVII, 476 and 971; Am. Jour. Dis. Child., 1912, IV, 20. 2 See also Lucas and Prizer, Jour. Med. Research, 1912, XXVI, 181. 3 See Hofer, Wien. klin. Wchnschr., 1913, XXVI, ion; also, Abel and Hallwachs, KoUe and Wassermann's Handbuch, 1913, VI, 534; McGowan, Jour. Laryngol., Rhinol. and Otol., 191S, XXIX, 57; Guggenheim, Interstate Med. Jour., 1915, XXII, 129; Horn, Jour. Am. Med. Assn., LXV, 788. Digitized by Microsoft® Digitized by Microsoft® PLATE VI. ^-^ ' , '' • r ,y /'' / X J^/ ■.■*s. _s\, '^r;. Kafbapinj Mill MORAX-AXENFELD DiPLOBACILLUS. (GRAM'S StaIN.) Courtesy of Dr. Brown Pusey. J '■^Ji'-—^.. J.':^ fh^^ Cthar.pe H in Koch-Weeks Bacillus. (Gram's Stain.) Courtesy of Dr. Brown Pusey. Digitized by Microsoft® ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 47 diphtheria bacillus in the discharge of the running ears following scarlet fever. It is not an uncommon thing to find certain inflammatory processes of para- sitic origin in the external auditory canal. This condition is known as otomycosis and is frequently caused by the aspergillus niger. Besides this organism, many other fungi belonging to this group have been found in the external ear. Among these we find the aspergillus flavus and fumigatus, the aspergillus nidumus, the mucor septatus, the eurotium malignum, and the penicillium minimum. These parasites are very readily detected by remov- ing a small portion of the mycotic mass and spreading it thinly on a slide. Add a small drop of water, apply a cover-glass, and examine under a high- power dry lens. For the details of structure of such organisms, bacteriological works should be consulted. Besides these fungi we occasionally find in the external auditory canal larvae of various insects. These larvae may develop later into the full-grown insect and may be removed from the ear by the movements of the animal. In other cases such larvae have incited inflammatory processes which are occasionally troublesome, as in the case reported by Richardson. IV. Thk Conjunctival Secrktions Under normal conditions the secretion of the conjunctiva and of the lacri- mal gland concerns us only very little. It is in the course of an inflammatory process in which these secretions may be greatly increased and greatly changed by the inflammation that any clinical importance attaches to them. In in- flammatory conditions of the conjunctiva we find certain organisms which require identification in order that proper treatment may be instituted and the proper prognosis given.^ It is to be recalled that the pseudo-diphtheria bacUlus is practically always found in smears made from the conjunctival secretion, yet it is rarely, if ever, pathogenic in this situation. Pathologic Changes. Diphtheritic Conjunctivitis. In cases of conjunctivitis which are traceable to infection with the diph- theria organism, we frequently find the formation of an extensive membrane which consists of epithelial cells, leucocytes, and large numbers of streptococci along with the diphtheria bacilli. Clinically, a membrane formation on the conjunctiva may arise from infection of this tissue with organisms other than the diphtheria bacillus; hence, it is wise in all suspicious cases to submit a portion of the membrane to both direct microscopic and cultural examinations. The appearance of the organism has been previously described. Infectious Conjunctivitis. In acute infectious conjunctivitis, various organisms have been found, the most common ones being the Koch- Weeks bacillus, the pneumococcus, and the gonococcus. Occasionally one finds the staphylococcus, strepto- ^Wilder and McCuUough (Jour. Am. Med. Assn., 1914, LXII, 1156) report a case of sporotrichosis of the eye in which the organisms were observed, in direct smears, as Gram- positive oval bodies and in <=^]Q^£§SW'kd?b°ihm- 48 DIAGNOSTIC METHODS COCCUS, colon bacillus, influenza bacillus, Morax-Axenfeld bacillus, the diphtheria baciUus, and other organisms. ^ In some regions the Koch-Weeks bacillus is frequently found as the etiologic factor in acute infectious conjunctivitis, while in others it is rarely, if ever, observed. This is an organism of the influenza group, a small, thin, Gram-negative bacillus, which is, so far as known, pathogenic only for the human conjunctiva (see cut). It grows best on media containing a slight amount of human blood, especially in symbiosis with the xerosis bacillus. This latter organism is differentiated from the Klebs-Loffler bacillus only by the application of the fermentation tests spoken of under Examination for Diphtheria Bacilli in the Throat. The most common bacterial cause of chronic conjunctivitis is the bacillus of Morax-Axenfeld, usually seen as a diplobacillus, several groups of which may at times be arranged in chains (see Plate V). It is a Gram-negative or- ganism and grows well onLoffler's blood-agar, which it digests, forming on the surface at the beginning of its growth, very characteristic small pits. Gonorrheal Conjunctivitis. This form of conjunctivitis is much more common than is generally sup- posed, so that the identification of the organism is of great importance. In the development of this type of conjunctivitis, large or small amounts, of pus are invariably present between the folds of the conjunctiva. A portion of this pus may be collected by means of a cotton swab or a platinum loop and smeared thinly over a slide. This smear is then fixed in the flame and is stained for the gonococcus by both the methylene blue and Gram stains. The technic of this latter staining process, as well as the characteristic appearance of the organism, will be discussed in a later section. Trachoma. Lately, a great deal of attention has been given to some bodies, which are found in trachoma and which are, possibly, the long-sought cause of this infec- tious disease of the conjunctiva. These organisms are known as the Prowa- zek-Greeff bodies and are shown in the accompanying cut. They are best stained by the Giemsa stain (see Blood), smears being made in the usual manner. The present status of these bodies is that they are almost always found in the acute stages of this disease and are, occasionally, observed in other conditions which do not present the clinical features of trachoma. Noguchi and Cohen (Jour. Exper. Med., 1913, XVIII, 572) report the cultivation of an organism, resembling the trachoma body, from cases of trachoma with and without cell inclusions. Whether or not they are the true etiologic factor must still be considered an undecided question. ^ iSee Axenfeld, Kolle and Wassermann's Handbuch, igij, VI, Gregersen (Arch. f. Verdauungskr., 1913, XIX, 43) advocates the use of Schmidt's test of administering 100 grams of very slightly cooked meat land the later study of the stools for connective-tissue fiber. 2 See Friedrich, Berl. klin. Wchnschr., 1913, L, 32; also, Rehfuss, Am. Jour. Med. Sc, 1914, cxLVii, 848. Digitized by Microsoft® GASTRIC CONTENTS 89 chlorhydria, we very frequently note a diminished motility due to spasm of the pylorus. This condition brings about a stagnation of the stomach con- tents and a consequent increase in the fermentative processes. The acidity in such cases may run as high as 200° or over and the digestive powers of the gastric juice, as regards protein substances, may be much increased, the car- bohydrate digestion being correspondingly diminished. These facts point to the reason for the administration of an increased protein diet in such cases, the protein combining with the hydrochloric acid and thus taking a portion of the excess from the field of action. While this condition is not a distinct entity, yet we find many cases which come under the heading of idiopathic hyperchlorhydria and which are not associated with other pathologic conditions. Some of these cases are purely functional and clear up promptly under proper treatment, while others are of nervous origin and are remedied only when the etiologic factor is eliminated. In this latter type of hyperchlorhydria the degree of acidity varies with the nervous symptoms, giving rise to the term " heterochylia." (2) Hypersecretion (Gastrosuccorrhea). By hypersecretion is meant an excessive secretion of gastric juice which is out of proportion to the physiologic stimulus. This hypersecretion occurs even when no stimulus is present, is always pathological, and, according to Riegel, always produces pathological results. A hyper- or continuous secre- tion may be determined by finding a fairly large amount of gastric juice in the fasting stomach under conditions which rule out stenosis and stagnation. The stomach is washed out before the patient retires, the contents being with- drawn the following morning. Tf a quantity of highly acid fluid is obtained, a hypersecretion is proven. The quantity taken from the fasting stomach should never be more than 100 c.c. This secretion, to be called a hypersecre- tion, must contain no food remnants, no sarcinae nor yeast cells, but should be distinctly acid. This condition is probably a functional neurosis, being constant or inter- mittent and a part of a general neurosis, a secretory neurosis, or the result of organic nervous disease, such as the gastric crises of tabes dorsalis. Reich- mann has reported cases of the periodic or intermittent type, during the inter- vals between the attack the digestion of the patient being normal. Such cases are known as Reichmann's disease. The chronic cases are of long duration and have a gradual onset. The patient complains of much discomfort, feeling of weight or depression in the stomach, pain during digestion, vomiting, especially at night, and a gastric contents with a large amount of free hydrochloric acid. Dilatation of the stomach sooner or later comes on as the result of spasm of the pylorus ind uced by the hyperacidity. In these dilated stomachs we find, of course, products of fermentation and many yeast cells and sarcinse. (3) Achylia Gastrica. This condition may arise either from a functional disturbance of the mucosa or a true atrophy of the mucosa. This latter state, known as a trophic Digitized by Microsoft® 90 DIAGNOSTIC METHODS gastritis, may be the end stage of a chronic gastritis or the result of carcinoma. When this condition is not due to direct gastric disturbance, it is more fre- quently seen in connection with pernicious anemia, in which the general nutri- tion is very much below par. The local condition may not be suspected, as the hypermotility, so common in achylia gastrica, may prevent attention being drawn to the stomach. For a diagnosis of achylia, the test meal of Ewald gives very good results. Examination of the gastric contents shows that the food is little changed, the total acidity very low (i to 6°), no free hydrochloric acid, gastric ferments much diminished or entirely absent, and lactice acid only in small amounts. The motility of the stomach is usually little impaired, so that the retention of food is unusual. "^ (4) Acute Gastritis. The stomach contents of acute gastritis shows a diminished total acidity, little or no free hydrochloric acid, organic acids relatively increased, much mucus and undigested food. This condition is usually brought on by direct irritation and is generally easily remedied by total abstinence. The material for chemical examination is usually obtained in these cases from the vomitus, as the passing of the stomach-tube is very rarely tolerated. (s) Chronic Gastritis. All grades of this condition may exist up to complete atrophy of the mucosa. Examination of the stomach contents shows practically no diges- tion of the food material, much mucus intimately mixed with the food parti- cles, the secretion usually diminished, free hydrochloric acid diminished or absent, ferments much reduced, protein digestion small, starch digestion Uttle affected, microscopic examination showing the presence of many epithelial cells and leucocytes. There are some cases of chronic gastritis in which a hyperacidity of the juice is evident, but these are rare, the usual finding being one of diminished acidity. The motility of the stomach in these cases is sometimes normal, sometimes increased, or may be diminished. One of the most characteristic findings in this condition is the presence of a large amount of mucus containing either leucocytes or their nuclei and epithelial cells from the walls of the stomach. If there is little acid present in the gastric contents the mucus may swell up and appear greater in volume. As a general rule, it may be said that the mucus and the hydrochloric acid vary inversely as their amounts. (6) Nervous Dyspepsia. This condition of nervous dyspepsia is part of a general neurosis and may show no characteristic findings in the stomach contents. The degree of acidity may range from a normal to either a hyper- or a h5T>oacidity, while the amount of ferments present will not usually vary. The findings in nervous dyspepsia are not at all constant, varying at different examinations. We have, therefore, more or less distinct methods of differentiation between this con- » Disqu6 (Arch. f. Verdauungskr., 1914, XX, 366); Orlofif (Russk Vrach., 1914, XIII, 1006) believes there eventually occurs a vicarious increased secretion of trypsin in this condition. See, also, Rehfusg^ijf^^^^^j^^^ggjjSg) CL, 72. GASTRIC . CONTENTS 9 1 dition and chronic gastritis. In the first place, the acidity of chronic gastritis remains constant for several examinations while that of nervous dyspepsia is variable. The ferments are diminished in cases of gastritis, while they are normal in nervous dyspepsia. Much mucus is found in gastritis while little or none is present in dyspepsia. The cases of nervous dyspepsia are partially associated with distinct errors in eating. The influence of nervous conditions over gastric function has been very well expressed by Emerson when he says, "a neurasthenic will often worry his subliminal gastric sensations into the sphere of consciousness." (7) Ulcer of the Stomach. The diagnosis of ulcer of the stomach depends, to a arge extent, upon the clinical symptoms of the disease rather than upon the examination of the stomach contents.^ While the symptoms of this disease, increasing dyspepsia, pain, vomiting, blood in the vomitus, and hyperacidity of the vomitus, are well- known, the stomach-tube should rarely ever be used in obtaining the con- tents of the stomach, owing to the danger of perforation in such cases. ^ The vomitus of such cases is usually ejected from one to three hours after a meal and contains well-digested food. Blood may or may not be present and may be either fresh red blood or dark in color from the formation of hematin. The total acidity of the gastric contents is usually increased, hydrochloric acid constituting a large part of this total acidity. . A single examination of the gastric contents will rarely determine anything about an ulcer, so that re- peated examinations of the vomitus must be made to obtain a general idea of the acidity. In these cases blood is usually present in tJie feces and may be detected as outlined later. ' When an ulcer is complicated by a beginning car- cinoma, we may find all tjrpes of variation in the acidity of the stomach contents. (8) Carcinoma of the Stomach. In no other condition of the stomach is an absolutely certain early diagno- sis to be so much desired as in carcinoma of the stomach. The chemical features of the gastric juice in this condition may be very suggestive or may be negative. The clinical history of the case along with the age of the patient are probably of more importance in making a diagnosis of carcinoma than are variations in the chemical composition of the juice. The local symptoms of carcinoma of the stomach are sometimes as variable as are the changes in the gastric juice, so that every possible point in diagnosis should be taken advantage of, if for no other purpose than to exclude this condition.* Perhaps the most important sign of carcinoma is the absence of free hydro- chloric acid. Although this condition is present in about 85 per cent, of cases, > See Verbrycke, Am. Jour. Med. Sc, 1913, CXLVI, 742. ' Rosenow (Jour. Am. Med. Assn., 1913, LXI, 1947) has shown the close relationship of streptococcic invasion to gastric ulcer. See, also, Rosenow and Sanford, Jour. Infect. Dis., 191S, XVII, 219. ' See Lichty, Am. Jour. Med. Sc, 1914, CXLVIII, 680; Smith, Jour. Med. Research, 1914, XXX, 147; Smithies, Arch. Diagnosis, 1915, VIII, 147; Interstate Med. Jour., 1915, XXII, 207; Boas, Arch. f. Verdauungskr., 1915, XXI, 94; Sippy, Jour. Am. Med. Assn., 1915, LXIV, 1625; Mayo, Ibid., 1915, LXV, 1069; Cheney, Ibid., 1227. * See Schutz, Wien. klin. Wchnschr., 1913, XXVI, 1053. Digitized by Microsoft® 92 DIAGNOSTIC METHODS it cannot always be traceable to carcinoma, as it may occur in atrophic gastri- tis and advanced chronic gastritis. This lack of free hydrochloric acid is due to the union of this acid with some body which in itself does not show an alkahne reaction. Von de Velden suggests that the secretion from the cancer is the active agent in neutralizing the hydrochloric acid. Moreover, the pro- ducts of protein digestion might have some power in neutralizing the acid Emerson having shown that hexone bases are present as a result of the action upon the protein of a ferment^ derived from the tumor itself. Certainly, in' cases of carcinoma the total nitrogen of the stomach contents is much increased, so that such bodies are probably a very great factor in the diminution of the hydrochloric acid. This reduction in hydrochloric acid is also influenced by changes in the mucosa to such an extent that the active secretion is diminished. The failure of free hydrochloric acid is usually a very early symptom, but it must be remembered that hydrochloric acid may be present in normal amounts or even in increased amounts when the carcinoma is small and occupies the pyloric region or when this growth develops on the base of an old ulcer. ^ The acidity may vary markedly from day to day, sometimes showing no free hydro- chloric acid and sometimes a considerable amount. This variation is of con- siderable practical importance. In cases of carcinoma of the esophagus the disappearance of hydrochloric acid from the gastric contents seems necessarily to be the result of the secretion of the tumor neutralizing the gastric juice. Along with this diminution in free hydrochloric acid the total acidity is also diminished.^ The presence of an increased amount of lactic acid is a very valuable sign in cancer. '' About 90 per cent, of patients show the presence of lactic acid when there is no free hydrochloric acid, but when there is a large amount of combined HCl pointing to a free secretion of this acid. Lactic acid may not be present in cases of carcinoma, especially those in which the growth is upon the base of an old ulcer, or it may be present in conditions other than carci- noma, such as chronic gastritis, associated with atrophy of the mucosa and dilatation of the stomach, especially when a benign stenosis of the pylorus exists. It must be stated, however, that we usually find an increased secre- tion of hydrochloric acid in cases of simple benign pyloric stenosis, so that lactic acid cannot be formed in the presence of this increased hydrochloric acid. According to Riegel, the chief cause of the lactic acid formation is the combination of motor insufficiency with a hypoacidity associated with 'a diminished secretion, both of acids and ferments. This diminution in the amount of ferments is in no way specific for cancer, as it is in reality due to the chronic gastritis which is set up by the tumor. As lactic acid cannot be formed in the presence of an appreciable amount of free HCL, we do not al- 1 Kriloff (Med. Obozr., 1913, LXXIX, i) believes trypsin is always present in cancer, especially of the pylorus. See, also, Halpern, Mitt. a. d. Grenzgeb. der Med. u. Chir., 1914, XXVIII, 709. 2 See Levy, Beitr. z. klin. Chir., 1914, XCIII, 696; Ochsner, Jour. Am. Med. Assn., 1915, LXV, 1073. ' See Friedenwald, Am. Jour. Med. Sc, 1914, CXLVIII, 660; Smithies, Jour. Am. Med. Assn., 1914, LXIII, 1839; Ibid., 1915, LXIV, 643. ^ See Schryver and Sing^/Qgetf.flyiVW/fef^^lfljKg, VI, 71 and 309. GASTRIC CONTENTS 93 ways find after a test meal a large excess of this acid, especially in those cases in which considerable combined HCl is present. It seems advisable, there- fore, in testing for the presence of lactic acid, to examine the contents of the fasting stomach in the morning after it has been well washed out the pre- ceding evening. Whether we are to assume that lactic acid is formed by the action of the organisms in the stomach upon food material, whether it be a normal product of digestion, or whether it is a product of the activity of a ferment derived from the tumor, must be left for future investigation. The vomited material or the material obtained by washing out the stom- ach shows very little digestion of the protein elements, while the carbo- hydrates are well hydrolyzed. The amount of material in the stomach will be large or small depending upon the degree of stenosis, so that it is nothing unusual to obtain several pints of material containing undigested protein residue. Microscopic examination of this material may show cellular masses washed off from the tumor or may show these fragments embedded in masses of blood. ^ Blood is a usual finding in cases of carcinoma and may be detected by the methods discussed under Feces. Sarcinae and yeasts are rare, the former occurring more frequently in cases of marked dilatation. The Boas- Oppler bacilli are more or less constant findings in carcinoma, occurring in about 9c per cent, of the cases and only very rarely in any other condition. Salomon's Test. This test is based upon the fact that albumin is secreted from the car- cinoma itself and passes into the gastric contents. The patient is placed upon an absolutely protein-free diet for 24 hours and the stomach carefully washed out at the end of this time with 400 c.c. of physiological salt solution. A few hours thereafter the contents of the stomach are removed and the remain- ing material washed out with 400 c.c. of physiological salt solution. The total nitrogen and the albumin are then estimated, the former by the Kjeldahl method and the latter by the Esbach method (see Urine). The nitrogen ranges from 10 to 70 mg. per 100 c.c. in cases of carcinoma, while in other conditions it varies from o to 16 mg. per 100 c.c. The Esbach reaction gives a distinctly appreciable precipitate for albumin, anything over 0.5 parts per 1 000 being considered indicative of carcinoma. While this test is no t infallible, yet the writer has found it present in so many cases of carcinoma that he is in- clined to make a very strong presumptive diagnosis on the basis of this test.'' Neubauer and Fischer's Test. These investigators,' working in Miiller's clinic, have taken advantage of the well-known fact that normal peptic digestion does not proceed to the formation of amino-acids but stops at the peptone or peptid stage. Further, it has been shown by Emerson, Fischer and others that digestion of protein goes somewhat further in carcinomatous stomachs than in normal ones, owing to the fact that carcinomatous tissue, as well as various body tissues, contains proteolytic ferments capable of producing more complete hydrolysis than does ' See Simon and Caussade, Presse mSd., 1914, XXII, 265; Loeper and Binet, Arch, des Mai. de I'App. digestif, ig 14, VIII, 181; Caussade, Rev. de Med., 1914, XXXIV, 428. ^ See Hohlbaum, Arch. f. klin. Chir., 1914, CIV, 1069. ' Deutsch. Arch, f . klin. M^igM^^J^idmsoft® 94 DIAGNOSTIC METHODS pepsin (see Lewin^). Neubauer and Fischer, following the investigations of Erdmann and Winternitz, Glaessner, and Volhard, use this hydrolysis of peptids by such proteolytic ferments for diagnostic purposes. Their method is as follows : The contents of the stomach are withdrawn one-half to three- fourth hour after an Ewald meal; lo c.c. of the filtered material are mixed with a little gylcyl-trytpophan^ and a little toluol is added to prevent bacterial ac- tion. The mixture is placed in the incubator at 37°C. for 24 hours. At the end of this period, withdraw 2 or 3 c.c. of the mixture from beneath the toluol layer, place this in a test-tube, add a few drops of 3 per cent, acetic acid and then allow the fumes of bromine to pass into the tube from an open bottle of bromine. This part of the technic must be carried out very carefully as an excess of bromine will obscure and even destroy the reaction. After shaking the tube a positive reaction is shown by the appearance of a rose-red or intense red color. If no red color appears, carefully add more bromine vapor and shake. This addition may be continued until an excess of bromine is evi- denced by a light yellow color of the mixture. These workers have shown that errors may arise in this test due to the presence of (i) tryptophan in the gastric contents, (2) peptid-splitting bacteria, (3) trypsin or pancreatic juice, and (4) blood. To do away with these sources of error they advise the rejection o; gastric contents showing any of these substances. Filtering the contents removes the peptid-splitting bacteria to some extent while the addition of toluol prevents their development. Tryptophan, as such, may be tested for as usual, while the presence of pan- creatic juice may be assumed if bile be present. Blood is shown by tests outlined under Feces. Warfield' has recently shown another fallacy of this test, which may render the test of little clinical value. He has found that the saliva of many patients is capable of decomposing glycyl-tryptophan into its constituent elements. He concludes, therefore, that swallowed alkaline saliva, when mixed with neutral or faintly acid (not over 0.05 per cent. HCl) gastric contents, may introduce sufficient error to account for the discordant results obtained. Koelker* has found a di- and tripepid-splitting ferment in the saliva. Lyle and Kober^ believe that a negative result with this test is of far greater value than is a positive one. Weinstein^ believes that the presence of tryptophan in the gastric con- tents, as withdrawn, is more positive as a test for carcinoma than is the glycyl- tryptophan test, providing intestinal fluid has not regurgitated into the stomach. He has confirmed the findings of Warfield and asserts that this latter fallacy can not be applied to his "tryptophan test" as the salivary proteolytic action is not such as to produce tryptophan from protein in non- cancerous gastric contents. He, therefore, suggests the following modifica- • Ergebn. d. inn. Med., igo8, II, 212. " Glycyl-tryptophan, as prepared for the test, is a clear solution. It is furnished by KaUe & Co., Biebrich a. Rhein, under the name of " Fermentdiagnostikum." It comes in smaU bottles which are used directly for the test by simply adding the 10 c.c. of filtered gastric contents and incubating for 24 hours. = Bull. Johns Hopkins Hosp., 191 1, XXII, 150. * Ztschr. f. physiol. Chem., 1911, LXXVI, 27. '• New York Med. Jour,, loio, XCI, 115 " iMew lorK iviea. jour,, loio, A.U1, 1151. ' Jour. Am. Med. Assn., t^fiZ9qiby5(iimO,S&ff^hVlI, 1420. GASTRIC CONTENTS 95 tion. A portion of the gastric contents is withdrawn 4 to 5 hours after a full dinner and the filtered contents tested directly for tryptophan after acidifying the filtrate with acetic acid. If no reaction appears, incubate for 24 hours and repeat the test. Owing to the large number of possible fallacies and the discordant results obtained with this test, it seems probable that it, like so many others, must be regarded as of little diagnostic value. ^ Freidman and Hamburger (Arch. Int. Med., 1913, XII, 346) believe that the proteolytic cleavage of stomach contents is due, in most instances, to regurgitated trypsin, although leucocytes and bacteria play some r61e. They advocate a combined method of proteolysis and peptolysis, which they believe preferable to other tests advanced. A high peptolysis with low proteolysis speaks for carcinoma; while a high peptolysis and high proteolysis speaks against carcinoma. This test promises to be a reliable one but confirmatory work must establish its status. Wolff and Junghans' Test. These workers^ have originated a test for the diSerentiation of benign and malignant achylias, which test seems to have considerable diagnostic value if properly controlled and interpreted. The test is based on the following ob- servations. Normal aspirated test meals show a relatively large amount of soluble albumin, which arises from the digestion of the protein of the test meal by the gastric ferments in the presence of free hydrochloric acid. This albu- min increases in amount in proportion to the time which it remains in contact with the normal gastric juice. In conditions associated with achylia or with- marked diminution of hydrochloric acid in the stomach, it would be reasonable to suppose that little or no protein digestion would occur. This was found to be the case in benign achylias but not true in the malignant types. In these latter cases the soluble albumin (probably a proteose) was found to be much increased. This albumin may arise (i) from protein retention from a pre- vious meal, due to stasis, lack of motor tone, or true obstruction, as shown by Clarke and Rehfuss; (2) from mixing of the test meal with so-caUed "cancer juice" exuded from the malignant growth; (3) from the presence of blood or pus arising from ulcerative lesions ; (4) from the presence of swallowed saliva or, especially, sputum; and (5) from the action of a specific peptid-splitting ferment arising from the malignant growth and capable of splitting the pro- tein into lower groupings of aminoacids. This latter source is the most important, although the others must be excluded before proper interpretation is possible. In other words, one must be sure (i) that the stomach is free ' In this connection see Taylor and Hall, Jour. Path, and Bacteriol, 1912, XVII, 121; Sanford and Rosenbloom, Arch. Int., Med., 1912, IX, 445; Smithies, Ibid., 1912, X, 357 and 521; Jacque and Woodyatt, Ibid., 560; Hamburger, Jour. Am. Med. Assn., 191 2, LIX, 847; and Smithies, Ibid., 1913, LXI, 1793. . * Wolff and Junghans, Berl. klin. Wchnschr., 1911, XL VIII, 978; Wolff, Magen and Darmkrankheiten, Berlin, 1912, 217. See, also, Thiele, Berl. klin. Wchnschr., 1912, XLIX, 544; Einstein, Med. Klin., 1912, VIII, 484; Roubitschek and Weiser, Arch. f. Verdauungskr., 1913, XIX (Erganz. Bd.), 146; Rolph, Med. Record, 1913, LXXXIII, 848; Smithies, Am. Jour. Med. Sc, 1914, CXLVII, 713; Trallero, Deutsch. med. Wchnschr., 1914, XL, 1428; Kahn and Jacobowitz, Biochem. Bull., 1915, IV, 214; Lenskaia, Russk. Vrach., 1915, XIV, 267; Katznelson, Ibid., 292; ClarkeandRehfuss, Tour. Am. Med. Assn., r9i5, LXIV, 1737. 96 DIAGNOSTIC METHODS from remnants of previous protein meals, as in some cases this gastric re- siduum may be sufficient to give erroneous results;^ (2) that blood and pus are absent, at least in more than traces; and (3) that sputum especially is not swallowed during the period of retention of the test meal, as some types of sputum are relatively rich in soluble albumins. While, theoretically, typical cases of advanced gastric carcinoma, at least, should show little or no free hydrochloric acid, it should be remembered that such is actually not the case, so that this test applied to cases in which free hydrochloric acid is present to an appreciable extent may, of course, give high albumin values due to ordinary gastric digestion. However, the studies of Clarke and Rehfuss, by means of their fractional method of gastric exami- nation, show that the curve of protein digestion under normal conditions follows the acid curve rather sharply, while in malignant states the protein curve diverges from the acid curve and, as digestion progresses, this dispro- portion increases until there is a marked separation of the curves. From these facts, these workers argue that the fractional study of both the curves of gastric acidity and protein digestion should be simultaneously studied before the proper interpretation can be reached. Technic. In order to avoid the possibility of retention of protein residues in the stomach, the patient is allowed a light meal with very little protein on the evening before the examination is to be made. A light cathartic is adminis- tered the same evening. The next morning administer an Ewald test break- fast. Withdraw this meal within 45 to 60 minutes. Filter the contents through filter-paper until clear. While filtration is proceeding, arrange a series of six clean test-tubes in a rack, numbering the tubes serially. By means of a 1 c.c. pipet, which is grad- uated in divisions of i/ioo c.c, place in the respective tubes i c.c, 0.5 c.c, 0.25 c.c, 0.1 c.c, 0.05. c.c, and 0.025 c.c. Now make up the volume in each tube to 10 c.c. by the addition of distilled water from a finely graduated pipet. The dilutions in these tubes will be, then, i to 10, i to 20, i to 40, i to 100, i to 200, and I to 400. Intermediate and extra dilutions may be made if it is desired to work the reaction down to a much finer point. Mix the contents of the tubes thoroughly by shaking and inverting the tubes. Overlay the contents of each tube -with i c.c. of the reagent to precipitate the albumin and note the appearance of a white ring at the point of contact of the fluid and reagent in the various tubes. A black background will bring out these rings more distinctly. Wolff's reagent is as follows: Phosphotungstic acid 0.3 gram. Concentrated Hydrochloric Acid i .0 c.c. Alcohol (96 per cent.) 20. o c.c. 1 See Rehfuss, Bergeim anAHww^Jow./^m^Med^^sn., 1914, LXIII, 11. GASTRIC CONTENTS 97 Interpretation. Normally a distinct ring of albumin will be seen at dilutions of i to lo, i to 20 and, occasionally, at i to 40. This reaction, which is called negative at these dilutions, may even reach, in very rare cases, as high as i to 200 due to various factors spoken of above, especially to the presence of blood and retention of protein residues. The importance of excluding extrinsic sources of albumin is thus evident. Generally speaking, however, the diagnosis of cancer becomes more probable the higher the dilution at which the albumin precipitation occurs, although cancer cannot be excluded in some cases when the precipitation does not appear in the last tubes. A precipitation unit of 100 or over speaks rather markedly for a diagnosis of cancer, presumption be- coming almost a relative certainty when the unit is 200 to 400. This im- plies, of course, that all the points mentioned above have been taken into consideration. Smithies regards a reaction at a dilution of 100 as doubtful. He states that this test " was a more constant finding in gastric extracts than were absent free hydrochloric acid, the presence of lactic acid and the gly- cyltryptophan test. It was rather more constant than tests for occult blood and the demonstration of motor inefficiency. It was not so consistent in its manifestation as the demonstration of organisms of the Boas-Oppler group or the increase in the formol index." It will be seen, therefore, that this test, of positive precipitation of albumin in dilutions of the gastric extract from i to 200 up, is of considerable value in the differentiation of the benign and malignant achylias or achlorhydrias, especially if the former are not associated with motor insufficiency. In cases of ulcer, especially if pyloric stenosis obtains, positive Wolff- Junghans' tests are not infrequent, indicating that symptomatology, gastric acidity and other findings must be taken into consideration. Like many other tests for cancer, this must not be relied upon to settle the diagnosis, to the exclusion of other laboratory and clinical data. With proper technic and care in excluding extrinsic factors, this test has certainly more than a presumptive value, giving definitely positive findings in fully 80 per cent, of disputed cases. BIBLIOGRAPHY I 2 3 4 5 6, 7 8 9 ID, II 12 Boas. Die Erkrankungen des Magens. Berlin, 1906. Billings. Diseases of the Digestive System. New York, 1906. Debove, Achard, et Castaigne. Manuel des Maladies du Tube Digestif. Paris, 1907. EwALD. Die Erkrankungen des Magens. Berlin, 1900. Harley and Goodbody. The Chemical Investigation of Gastric and Intestinal Diseases. London, 1906. Hemmeter. Diseases of the Stomach. Philadelphia, 1906. LoEPER. Manuel des Maladies du Tube Digestif. Paris, 1907. Lyon. Diagnostic et Traitement des Maladies de L'Estomac. Paris, 1909. Reed. Diseases of the Stomach and Intestines. New York, 1907. RiEGEL. Die Erkrankungen des Magens. Wien, 1903. RoESER. La Chimie Alimentaire. Paris, 1906. Sigaud. Traite clinique de la digestion. Paris, 1907. ^ Digitized by Microsoft® 98 DIAGNOSTIC METHODS 13. Smithies and Ochsner. Gastric Cancer. Philadelphia, 1915. ' 14. Stockton. Diseases of the Stomach and Their Relation to Other Diseases. New York, 19 14. 15. ZuNz. Methodik der Mageninhaltuntersuchung, Handh. d. Biochem. Arbeits- Methoden, 1914, VIII, 44. 16. ZwEiG. Diagnose und Therapie der Magen- und Darmkrankheiten. Wien, 1913- Digitized by Microsoft® CHAPTER IV THE FECES I. General Considerations The feces are composed of substances of different origin, which may be divided as follows: (i) Food remnants, either undigestible constituents or digestible but unabsorbed elements; (2) secretions of the alimentary tract; (3) decomposition products and bacteria; (4) formed and unformed elements derived from the intestinal wall, and (5) foreign bodies, such as hair, wood fiber, parasites, parasitic ova, and enteroliths. It is not possible to draw a sharp line between a truly physiologic and pathologic composition of the feces. In each individual case this division will depend upon a number of factors, among which may be mentioned composition of the food, method of taking the food, individual functional capacity of the intestines, frequency of the bowel movements, and general systemic conditions. The condition of the food remnants will give much information regarding the functional capacity of the bowel. Under normal conditions, about one-third of the dry substance of the feces arises from the bacteria, this amount increasing under certain pathologic conditions which will be later discussed. As products of bacterial activity we observe the formation of hydrobilirubin from bilirubin as well as the reduction of certain medicaments and the rare formation of certain diamins, which will be treated later. This bacterial activity will vary much under pathologic conditions and may have much to do with the symptoma- tology of the case investigated. ^ The ordinary decomposition products are derived from the carbohydrates and proteins of the food; from the former are produced, by fermentation, volatile fatty acids, lactic acid, succinic acid, alcohol, carbon-dioxid, hydrogen, and methane, while from the latter are formed by putrefaction indol, skatol, phenol, ammonia, and hydrogen sul- phid. The fats are decomposed only to a very slight extent. The normal products of digestion in the intestinal tract as well as the factors bringing about these changes will be discussed later. ^ Normal Feces. For proper comparisons between the feces of various individuals the food must be the same. Starvation feces, meat feces, and milk feces are typical types, but are not normal in the strict sense. For comparison, however, an arbitrary norm must be established in order to judge of slight variations which have to do with special differences in utilization of food and which are not observable by the eye except under certain conditions (as, for instance, fatty ' See von Noorden, Jour. Am. Med. Assn., 1913, LX, loi. 2 See Sanford, Wisconsin Med. Jour., 1914, XII, 281; also, McClanahan and Moore, Am. Jour. Med. Sc, 1915, CXLIX, 815. Digitized by Microsoft® lOO DIAGNOSTIC METHODS stools).. Through the work of Praussnitz and of Schmidt and Strasburger a new field for study of normal and abnormal feces has been opened up. A normal feces should be one which consists almost entirely of remnants df the digestive juices and intestinal secretion arising from a purely digestible, properly prepared, and assimilable food. It contains approximately 8.6 per' cent, of nitrogen, i6 per cent, of ether extract, and 15 per cent, of ash calcu- lated on the dry basis. Any appreciable variation from this composition would indicate a diminution of the functional activity of the bowel. In the investigation of pathological cases this composition is scarcely to be assumed, as.the diet is more or less restricted, the appetite is capricious, the intestinal activity is variable, and the feces are, therefore, different from those of the healthy. It must not be understood from this that a feces showing the Fig. 19. — Normal Feces. [Landois.) a, Muscle fibers; b, tendon; c, epithelial cells; d, leucocytes, e-i, various forms of plant- Cells, among which are large numbers of bacteria; between h and b are yeast-cells; k, ammonium-magnesium phosphate. above composition is necessarily normal in other respects. One may not rely entirely on the chemical composition, but must largely consider the macroscopic and microscopic findings. In order that we may have a sound basis upon which to judge of intestinal activity it seems wise to have some sort of test diet which may be given to suspected cases. Diet of Schmidt and Strasburger. This diet' is so selected that it can be used by the healthy as weU as by those with intestinal trouble; its amount is sufficient to satisfy the maximal calorie requirement of the individual while at rest; it contains the three chief groups of food material in definite relation to each other; is as free as possible from remnant-leaving food, and can be easily obtained and prepared. The starchy food present is of the amount and kind which have been shown most favorable for the prevention of excessive fermentation in the bowel. The daily diet is as follows: One and five- tenths liters of milk, 100 grams of zwieback, two eggs, 50 grams of butter, 125 grams of beef (raw weight), 190 grams cooked potato, and a gruel of 80 grams of oatmeal. This is distributed 1 Die Faeces des Menschen, Berlin, 1915. , Digitized by Microsoft® THE FECES lOI through the day as may best suit the patient. This diet contains 102 grams of protein, in grams of fat, and 191 grams of carbohydrate, yielding 2,234 raw calories. In cases which show a diarrhea, due to the milk, one may sub- stitute instead of J^ Uter of milk the same amount of cocoa made from 20 grams of cocoa powder, 10 grams of sugar, 100 grams of milk, and 400 grams of water. Small variations in the amount of milk, sugar, butter, and even of eggs may be permitted, but the outhne, as regards meat, zwieback, potato, and gruel should be rigidly adhered to. This diet should be administered for three days or longer if necessary to obtain a stool which comes from it. In order to judge of the first appearance of the stool from this diet, the patient should be given a capsule containing 0.3 gram of powdered carmin, both preceding and following the diet.^ Instead of carmin, one may use cork, charcoal, or silicic acid. Folin's Diet. This diet^ is especially serviceable in case one wishes to follow the metabo- lism in any special case. Its easy application and its fairly constant values for nitrogen, phosphorus, chlorin, and sulphur make it invaluable. The standard diet, which is given to the patient daily for several days, is as follows: Whole milk, 500 c.c. Cream (18 to 22 per cent, fat), 300 c.c. Eggs (whole), 450 gm. Horlick's m,alted milk, 200 gm. Sugar, 20 gm. Sodium chlorid, 6 gm. Water, 2,100 c.c. This diet contains approximately 119 gm. of protein, 148 gm. of fat, and 225 gm. of carbohydrate, yielding 2787 raw calories. The intake of one day is nitrogen 18.9 gm., 5.9 gm. of P2O5, 3.8 gm. of SO3, and 6.2 gm. of CI. It is not to be compared with that of Schmidt and Strasburger for estimating the intestinal activity, but is more reliable if metaboUsm relations are to be studied. The normal motility of the feces is from 6 to 20 hours, whUe on a milk diet it may vary from 36 to 48 hours. This factor may be of importance in judg- ing of intestinal obstruction. The isolation of a stool under a special diet is of great importance, as the success of an investigation will depend upon this. Obtaining Intestinal Juice. Boas has shown that it is often possible, by the use of the stomach tube, to obtain sufficient intestinal secretion to permit of examination. His technic is, however, rather difficult, is trying to the patient and is often unsatisfactory. Boldyreff, Volhard, Faubel and, more recently, Koziczkowsky' have shown ^ See Basch, Jour. Am. Med. Assn., 1913, LXI, 1295; Strauss, Arch. f. Verdauungskr., 1914, XX, 299. 2 Jour. Physiol., 1905, XIII, 45. 'Ztschr. f. klin. Med., 1909, LXVIII, 261. Digitized by Microsoft® I02 DIAGNOSTIC METHODS that the intestinal juice may easily be obtained by the following method.' The patient is allowed to take 150 c.c. of pure olive oil when the stomach is empty, preferably in the morning. At the end of three-fourth hour, during which time the patient must remain quiet in a sitting posture, the gastric contents are removed by means of a stomach tube. The material, obtained by means of this oil test-breakfast,^ readily separates into two layers, an upper of oil and a lower of the mixed gastric and intestinal secretions. This may then be tested for the various ferments in the usual ways. Trypsin is best detected by the method of Gross. ^ Either the fluid ob- tained as above or aportion of the feces, rubbed up with three times its amount of a I to 1,000 NajCOs solution andfiltered, maybe used for the test. 100 c.c. of a 0.5 per thousand solution of casein in i to 1,000 Na2C03 solution are treated with 10 c.c. of fluid mentioned above and the mixture placed in the in- cubator for eight to twelve hours. At the end of this period the addition of a few drops of a dilute (i per cent.) acetic acid should produce no precipitate in cases digestion is complete, that is if trypsin is present. Functions of the Intestinal Juices. The pancreatic juice as excreted into the intestine is an alkaUne fluid con- taining three ferments, trypsin which hydrolyzes protein, amylopsin which acts upon the carbohydrates, and lipase (steapsin) which aids in the digestion of fat. The trypsin appears to be excreted in the form of a zymogen which is activated by a second ferment, enterokinase, derived from the intestinal mucosa, while the lipase and amylopsin are active when secreted. It has been shown by Pawlow that the passage of free hydrochloric acid into the duode- num is a direct stimulant to the excretion of pancreatic juice. Bayhss and Starling believe, however, that the stimulus to the pancreatic secretion is not the free acid but is a ferment, secretin, which is formed by the action of the hydrochloric acid upon the intestinal mucosa. While the trypsin acts upon protein bodies, splitting them through various stages into the ultimate prod- ucts, amino-acids and hexone bases, there is a second ferment, erepsin, dis- covered in the intestinal mucosa by Cohnheim, which acts upon the interme- diate splitting products of protein, such as the albumoses and pepton, carrying this conversion to the same lengths as does trypsin.' It is interesting to note that we find in the intestinal juice of the infant a ferment, lactase, which hydrolyzes lactose into the simpler saccharids. The presence of the bile, which reaches the duodenum through the common duct, is necessary for the proper digestion and absorption of the fatty substances of the food. Variations in this constituent may reflexly cause disorders in the secretion of the stomach, one finding very frequently hyperacidity associated with obstructive jaundice. Whether the bile may be assumed to have a disinfecting power must be left for the future. Were this influence actually present, it is hard to understand, why, in cases of long- ' See Matko, Med. Klin., 1913, IX, 1546 and 1587. ^ Deutsch. med. Wchnschr., 1909, XXXV, 706. 'See Schlecht and Wittmund, Deutsch. arch. f.klin.Med., 1912, CVI, 517; Friedmann, Med. Record, i9i2,LXXXI, 355; Abderhalden, Ztschr. f. physiol. Chem., 1912, LXXVIII, 344- Digitized by Microsoft® THE FECES I03 standing exclusion of bile, we do not find catarrhal conditions as we do in cases of marked putrefactive processes in the bowel. Estimation of Intestinal Digestion. A careful chemical examination of the feces, coupled with a macroscopic and microscopic investigation, will give much information regarding the degree of intestinal digestion and absorption. However, such methods are time-consuming and not easily performed by the general practitioner. For this reason Sahli has introduced a method, similar to his stomach method, of investigating such activity. He employs glutoid capsules, which are made from gelatin hardened with formaldehyd. These capsules either do not dissolve in the gastric juice at all or only after considerable time, although they are quickly soluble in the intestinal juice. In these capsules is placed material which will not diffuse through the capsule wall and whose absorption may be studied from an examination of the saliva or urine. Sahli uses either iodoform or salol. In the former case o. 15 gram of iodoform is placed in a glutoid capsule, and given with an Ewald test meal. Under the best condi- tions (normal gastric motility, normal intestinal digestion, and normal intes- tinal absorption) the iodin reaction may be expected to appear, according to Sahli, in the saUva within four to six hours; that is, within one and one-fourth hours after the capsule has been dissolved by the pancreatic juice. Instead of iodoform salol may be used, being given in the amount of one-half gram along with an Ewald test meal. The reaction for salicyluric acid may be obtained in the urine within one and one-half hours after the capsule has been taken. Sahli gives the following results obtained by the use of this capsule: When the stomach contains neither free hydrochloric acid nor pepsin, the reaction is not delayed so long as gastric motility is good. In cases of diarrhea due only to an increased peristalsis without any marked disturbance of intestinal diges- tion, the reaction is either normal or even somewhat hastened. In other types of diarrhea characterized by an involvement of the intestinal chemistry or intestinal absorption, the reaction is either absent or distinctly delayed or the capsules may be found undigested in the feces. This method also aids in differentiating an icterus due to occlusion of the ductus choledochus at its point of entrance into the intestine, in which case the digestion of the capsule may be interfered with, from one where the obstruction to the bile flow is higher up near the liver. This test may be of some presumptive evidence in the diagnosis of pancreatic carcinoma, although all cases of pancreatic car- cinoma do not necessarily occlude the duct, in which case a positive result would obtain. Galli reports a case of carcinoma of the pancreas in which the glutoid capsule was dissolved, although no pancreatic juice was present in the bowel. II. Macroscopic Examination (i) Method. The macroscopic examination of the feces embraces not only the study of the physical characteristics, but also a recognition of various normal and Digitized by IVIicrosoTt® I04 DIAGNOSTIC METHODS abnormal substances. It is convenient, when examining the feces, to employ some form of a washing apparatus to separate the coarser from the finer particles. Boas has introduced a special sieve for such work which, however, does not have much advantage over the ordinary flour-sifter which Einhorn advises. Strauss' method of washing the feces by a current coming from below seems to be much the best of any of these types of apparatus. In the absence of any other equipment, an ordinary household sieve of various-sized mesh will answer the purposes. Small amounts of mucus or connective tissue frag- ments may be recognized by rubbing up a portion of the stool in a mortar with a little water, when these substances will float upon the surface. In some cases it may even be advisable to place the feces in tall glass jars in which the stool is mixed with water and allowed to arrange itself in layers. If the stool is of ordinary consistency, it should be spread out in a more or less thin \^''^^^^^^^^rfS^Hf layer so that the larger particles may be ^^■■[^^^^^^ easily recognized. If it be, however, a watery stool the contents are thoroughly mixed and examined as such. Fig. 20. — Boas' stool-sieve. (Hemmeter.) (2) Amount. The amount of feces excreted will de- pend upon (i) the quantity and quality of the food; (2) the remnants of intestinal juices and debris; (3) the condition of the digestive organs, and (4) the bacteria. These factors are all important, each being dependent to a certain extent upon the other. The average moist weight may be considered as varying from 100 to 250 grams with a dry weight of 20 to 40 grams. This amount may, however, reach as high as 20 kilograms as in a case reported by Lynch. Number of Stools. The number of stools which may be passed in 24 hours is subject to very wide variation, even under physiological conditions, but is usually constant in the same individual. At least one stool a day should be considered normal, although many persons are accustomed to have only one movement of the bowels in 48 hours and others inlonger periods. We must, therefore, consider each individual case before judging as to abnormality in the number of stools, Lynch^ has reported a case of a patient having only one stool each 100 days, while Geib and Jones^ discuss a case in which there was no stool during aij entire year the patient at the end of that time voiding 32 liters of feces. . THE FECES I05 A diarrhea is said to exist when the stools are frequent and fluid. The frequency may vary from 2 to 50 in 24 hours, although a single liquid stool in 24 hours may constitute a diarrhea in a patient unaccustomed to having a movement every day. A normal stool is never fluid, so that the character of the stool is of more importance than the number, as individual peculiarities must be considered. A diarrhea may be due to increased peristalsis, to in- creased intestinal secretion, a diminished gastric secretion, or a decreased absorption from the bowel. The most extreme grades of diarrhea are ob- served in Asiatic cholera, dysentery and the summer diarrhea of infants, al- though a marked diarrhea does occur in enteritis, peritonitis, intestinal tuberculosis, and uremia. Some infectious diseases are more frequently associated with diarrhea than are others, but little of diagnostic importance is obtained from this symptom. By constipation isi meant the infrequent and irregular movement of the bowels, associated with symptoms which are relieved by administration of laxatives. The habits of the individual must be taken into consideration before judging as to a real or apparent constipation. This condition is physio- logically a result of a sedentary life or of a diet lacking in elements which will stimulate intestinal peristalsis. ^ Pathologically, we find constipation in cases of dilated stomach, occulsion of the bowel, atony of the bowel wall, in con- ditions causing increased cerebral pressure, and in obstruction by pressure from without the bowel. It must be stated here that we frequently have an apparent diarrhea merely as a symptom of constipation high up in the bowel. Such being the case, it is better practice to administer a laxative in such conditions than it is to give an astringent. (3) Consistency and Fonn. The consistency and form of the normal stool vary considerably depending upon the nature of the food ingested. The stool is much softer with a purely vegetable diet, of which about 85 per cent, is water, than with an animal diet, of which only 65 per cent, is water. One differentiates, as regards consistency, a well-formed, a mushy, and a fluid stool, between which types there are many gradations, some stools being partly formed and partly fluid. Many factors may influence the amount of water present in the stool, such as (i) a lessened absorption of water from the intestinal canal; (2) intake of a large amount of water, and (3) an increased secretion from the intestinal glands. The consistency of the normal stool varies from day to day and can be constant only when the patient is placed upon a standard diet. It may be abnormally too fluid or too solid, in the latter case being frequently voided in the form of very hard masses, known as " scybala." As a general rule, it may be stated that the greater the absorption of water from the intestine the more firm will the feces be and, in consequence, the more frequently will these scybala form. Stools are frequently observed of which the consistency is normal but of which the size of the cylinder is quite small. Such small "lead- pencU" sized stools have been supposed to be indicative of stricture of the lower bowel, but this condition is not necessarily present. ' See Cannon, Jour. Am. J5f^^A|s^bJ^J|^yLD|o^/^^ Io6 DIAGNOSTIC METHODS Besides the water-content of the feces, the amount of fat, mucus, and vegetable residue has much to do with the consistency of the stool. One may differentiate the fat from water in the stool by placing a small portion of the feces upon slide and pressing a cover-slip down upon it. If the cover-glass remains when the pressure is removed increased fat may be assumed, while if the softness of the feces be due to increased water the cover-glass will spring away from the feces. In some cases, especially those associated with achyha gastrica, the stools are very frothy, indicating a marked bacterial decomposition. Such stools should not be confused with those of the ordinary diarrhea or with the char- acteristic "rice-water" stools of Asiatic cholera, in which particles of mucus are readily detected. Such stools are never found associated with large fat contents. (4) Odor. The peculiar odor of the normal stool is referable to the presence of indol and skatol, which arise from the putrefaction of protein material in the large intestine. Along with these we may find other substances, such as hydrogen sulphid, methane, and phosphine. The odor of the- stool is much more marked following a meat diet than that after a vegetable diet; it is very slight on a milk diet and is practically lost in the fasting condition. If the processes in the intestine are of such a nature that fermentation of the carbohydrates exceeds the putrefaction of the proteins, the stool shows a distinct sour odor traceable to the presence of butyric or acetic acid. The odor in cases of acute and chronic diarrhea is frequently very slight, while that of the loose, watery discharges of cholera is peculiar and sperm-like, referable to the presence of cadaverin. In the diarrhea of children a distinct putrid odor may be present, although this is not necessarily the case. The so-called acholic stools have in themselves, according to Schmidt, very little odor, showing this property only when complicated by decomposition processes arising from the lack of bile. In cases of severe dysentery and carcinoma of the large intestine an intensely disagreeable odor is observed which differentiates these conditions from those associated with other types of decomposition. (5) Color. The color of the stool varies from a light brown to even a black, depending upon the kind of food, the residue of the intestinal secretions, the presence of pathologic products from the intestinal wall, and the administration of thera- peutic agents. The dark color of the normal stool is due to the presence of hydrobilirubin, which is formed from the secreted bilirubin by the reducing processes in the intestines. Bilirubin is found normally in the stool of a nursing child, being converted either into biliverdin or hydrobilirubin under abnormal' conditions. This change from a light yellow infantile stool to a greenish one has much importance to the pediatrician. A well-formed stool is always darker in color than is the thin stool, which is equivalent to saying that the stool is darker the longer it remains in the intestine. The color of a stool under a meat diet is dark brown, with a vegetable diet a lighter brown, and following a milk dietQ/S!ffeie52fetlj!(AM/c/PBft/if®lor which is traceable to the THE FECES 107 diet is shown best in those cases in which bile is excluded from the bowel. The dark color of the meat feces is probably traceable to the conversion of the blood- coloring matter into hematin and not to the formation of sulphid of iron which is so commonly stated. Food products may color the feces a characteristic shade. Thus, coffee may give a dark brown color, cocoa a brownish-red color, red wine a smoky black-brown color which has a shade of green. Chloropyll- containing plants, such as spinach and lettuce, give rise to greenish shades. Occasionally the reduction process in the intestines may go as far as to convert the bilirubin into leucohydrobilirubin instead of into hydrobilirubin. This hydrobilirubin is identical with the urobilin found in the urine. Such stool's may be practically colorless when voided, but will be converted into the dark brown normal-colored stool on contact with the air. Besides these pig- ments, biliprasin has been isolated from the feces by Fleischer, while Miiller obtained cholecyanin. The variations in color of the stool following administration of therapeutic agents is frequently characteristic. Thus, after the use of calomel one ob- serves a distinct green coloration due to the conversion of the bilirubin into bUiverdin. Bismuth preparations color the stool a distinct black, due to the formation of the oxid or sulphid of bismuth. Rhubard, senna, santonin, and gamboge cause a distinctly yellow coloration which will change to a reddish tone in the presence of alkali. Iron compounds produce shades rang- ing from dark brown to black while methylene blue gives rise to the produc- tion of a blue-green color and sandal-wood a reddish-violet color. Pathologic Variations in Color. Cases showing the presence of large amounts of mucus or of pus in the feces are characterized by a gray-white or yellowish-gray colbration of the feces. Such cases are seen in membranous colitis and rupture of an abscess, especially of the appendiceal variety, into the intestinal tract. In cases of syphilitic or carcinomatous ulceration of the colon or rectum this character- istic color tone may be more or less influenced by the presence of blood. Stools showing the presence of ajarge amount of fat are clay colored. That this coloration is due to the excess of fat, rather than to the absence of bUe, may be shown by extracting the feces with alcohol and ether, in which extraction the bile will be taken up along with the fat and will color the sol- vents. These acholic stools as they have been called occur both in cases associated with biliary obstruction and in those showing no obstruction. It would be better practice, therefore, to style these stools colorless, instead of acholic stools. Striimpell was able to obtain stools of a light brown color by feeding patients a diet containing small amount of fat, thus proving that the increased fat was more important than the dimunition of bile, although this latter does account for some cases. This clay-colored stool may also be found in diarrhea, while in Asiatic cholera and dysentery the stools may be absolutely colorless. They have been also found in cases of leukemia, carci- noma of the stomach or intestine, tuberculous enteritis, and chronic tuber- culous peritonitis. The cause of this lack of color may be the same unknown cause that produces the forniation of leucohydrobilirubin spoken of above. Io8 DIAGNOSTIC METHODS In some cases we find a distinct golden-yellow or even a green color of the feces. This is due to the presence of unaltered bile, on the one hand, and bih- verdin, on the other. Biliverdin is usually found in abnormal decomposition processes in the intestine of the infant, while unaltered bile may appear in cases of increased intestinal peristalsis, in which the contents of the bowel are rushed onward before absorption can take place. This is probably the partial explanation of the green stool following the administration of calomel. Normally, bilirubin is not found in the intestine below the ascending colon. Such being the case, it may be possible to judge of the point of irritation in a diarrheal attack by the fact that the higher up in the bowel the pdnt of dis- turbance the more of this pigment will be found in the feces. Bilirubin in- dicates, therefore, an enteritis especially of the small, but also of the large intestine. This bile pigment may be found most frequently on cellulose material, mucus, muscle fibers, and fat. It is readily detected by rubbing up 2 or 3 c.c. of the feces with a concentrated aqueous solution of pure mercuric chlorid. This mixture is allowed to stand 24 hours and is examined micro- scopically thereafter. The fragments to which the pigment is attached will stain red if due to hydrobilirubin, while those stained with bilirubin will show a green color. Naturally, in this examination chlorophyll-containing fragments must be excluded. The most favorable material for such exami- nation is the mucus. A green color of the stools may also be observed due to infection with the bacillus pyocyaneus. Blood may give rise either to a distinct red color, a brownish-red shade, or even a black tint. If the blood be adherent to the scybalous masses or to the well-formed feces, it is usually derived from the rectum or anus and indi- cates hemorrhoids; if it be evenly distributed with the food material and is changed from a bright red to a brownish color, it indicates a hemorrhage in the stomach or high up in the small intestine, especially if the stool be solid; while an evenly mixed bloody feces of fluid character will usually point to the colon as the seat of the trouble. As a general rule, it may be said that the darker the color the more remote from the rectum will be the hemorrhage. Tarry black blood is seldom of low origin, usually indicating trouble in the stomach or duodenum, while fluid scarlet blood usually arises from the colon or rectum, although in some cases of typhoid fever the blood may be a bright red, although the hemorrhage may be fairly well up in the bowel. In cases of intussusception the blood may appear mixed with serum, but with no fecal matter. In deciding as to the importance of blood in the feces one must naturally exclude that arising from food or from hemorrhages above the gastro-intes- tinal tract. Thus blood coming from the mouth, nose, throat, or lungs may pass into the stomach and out with the feces, making a mistake in diagnosis very possible. Further, blood arising from vaginal discharges, which may be mixed with the feces at the time of defecation, should be excluded. The detection of blood in the feces is more or less simple and direct in the fresh state, but when intimately mixed with the feces its recognition is a matter of some diflaculty^jy^^^g^gv ^^^^^^^J^i^en or occult bleeding blood is never detected microscopically or macroscopically. It is practically THE TECES I09 useless to search for blood-cells in the feces, as rarely are perfect cells found, unless the blood is present in very large amount, many specimens showing no cells. Guaiac Test (Van Deen's Test). A small portion of the stool is rubbed up with water and one-third of its volume of glacial acetic acid added. This mixture is well shaken in a test- tube and a few cubic centimeters of ether added. After thoroughly shaking this 'mixture, it is allowed to settle, when the ether, in the presence of blood, will have assumed a brownish color. In case the ethereal extract is not clear, a few drops of alcohol may be added. On adding to this ethereal extract a mixture consisting of equal parts of fresh tincture of guaiac and ozonized turpentine, a blue ring will form at the point of contact or a blue coloration will be seen throughout the mixture if the tube be shaken. This test is much more reliable in its negative phase than in its positive phase. The writer has frequently found positive tests for blood arising from the employment of tubes previously used with copper solutions or with nitric acid, so that he would advise the worker invariably to use either new or thoroughly clean test-tubes when testing for the presence of blood. More- over, this test is given by other substances, which are enumerated in the sec- tion on Blood and may react positively in the absence of blood. It the patient eats watermelon, prunes, potatoes or rice, has been taking iron, or the feces contain much pus, a distinct reaction may be present. "■ In this test as well as in all of the other tests outlined, the presence of hematin arising from the meat of the diet must be excluded. This can be done only by placing the .patient upon an absolutely meat-free diet for several days preceding the examination. The Schaer-Klunge Test. This test is very similar to the preceding, but is much more deUcate, being positive after the ingestion of only three grams of blood. It is even more im- portant when using this test to exclude all hemoglobin and chlorophyll- containing foods for some days preceding the examination. The stool is rubbed up with water and treated as in the preceding test with acetic acid and ether. To this ethereal extract is then added a mixture of i c.c. of ozonized turpentine and 3^ c.c. of fresh 3 per cent, alcoholic aloin solution. This may be prepared by dissolving what aloin will lie on the point of a spatula in J^3 of a test-tube of 95 per cent, alcohol. At the line of contact a distinct red ring will be observed in the presence of blood in from three to five minutes. Care- ful work "with this test has shown that fat interferes to some extent with its delicacy. It is, therefore, customary to treat the feces with an equal volume of ether and to shake thoroughly to remove all the fat present. The ethereal solution is then poured off and the remaining fecal material mixed with one- third its volume of glacial acetic acid and 10 c.c. of ether, being then thoroughly shaken and set aside. A portion of this brownish ethereal extract, which contains the hematin 1 Boiling the watery suspension of the feces and allowing it to cool before adding the acid and ether will prevent reactions arising from ferments and oxygen transmitters other than blood. Digitized by Microsoft® no DIAGNOSTIC METHODS formed by the action of the glacial acetic acid upon the hemoglobin of the blood, is poured into a thoroughly clean test-tube and treated as above de- scribed with the turpentine and aloin solution. If the tube be shaken after the red contact ring has formed the whole mixture will assume a cherry-red color. If the tube is allowed to stand for a few minutes the aloin solution may sink to the bottom, forming a distinct red layer beneath that of the ether and turpen- tine. A reaction to be positive should appear within lo minutes as the aloin itself will gradually turn red under the conditions of the experiment if left for a much longer period. Charcoal instead of carmin should be used to mark the feces. The writer has found this test very reliable and very easy of application. Weber's Test. This test has the advantage of excluding practically every other factor which might influence the tests previously given.^ A portion of the feces is extracted with ether to remove the fat and is then separated from the ethereal solution. This fat-free feces is then rubbed up with water and treated with glacial acetic acid and ether. The ether, as in the preceding test, takes Up the hematin which is now detected by the spectroscope, showing the char- acteristic spectrum, namely, an intense narrow band in the red between C and D and a somewhat more definitely marked group of three broader bands, (i) in the yellow, (2) at the boundary between yellow and green, and (3) at the boundary between the green and blue, this last band being difficultly recognizable. In order to avoid confusion with the spectrum of methemoglobin or of chlorophyll, one may convert the hematin into reduced hematin (hemochro- mogen) by the addition of alcohohc potassium hydrate, water, and ammonium sulphid solution. The spectrum of this latter substance is characterized by the two bands in the green. This test is probably the most sensitive and should be more frequently employed. It is very simple of application, is very reliable and has practi- cally no fallacy, especially when the hematin is converted into the hemo- chromogen. This test is, however, not so delicate as the aloin test, so that small amounts of blood, as found, in occasional cases of occult bleeding, may escape detection. Weber originally used the guaiac test with a modification of extracting the fat from the feces before testing with guaiac and turpentine. Schumm states that if the feces be thoroughly extracted with alcohol and ether most of the fat and urobilin will be removed and that under these circumstances Weber's test, either with the guaiac tincture and turpentine or with the spec- troscope, becomes much more valuable. Adler's Test. O. and R. Adler have introduced the use of benzidin as a test for the presence of blood. The stool is extracted with a mixture of alcohol and ether for the reasons above mentioned. It is then treated with glacial acetic acid and with ether as described in the other tests. This acid ethereal extract, 1 Queisser (Therap. Mon£©to/fBe6l( l^?^MRsr0Sdff®nd blood by this test in cases fed upon the Lenhartz gastric ulcer"aiet, such cases being free from gastro-intestinal ulceration. THE FECES III which contains the hematin, is then treated with 2 c.c. of a saturated alcoholic benzidin solution and 2 c.c. of hydrogen peroxid ("3 per cent.). In the pres- ence of blood a greenish-blue color appears. Wagner' advocates a so-called "dry test" of the stool which is dehcate, reliable and extremely simple. He uses as reagent the Schlesinger-Holst mixture of a knife-point of benzidin, 2 c.c. of glacial acetic acid and 20 drops of a 3 per cent, solution of hydrogen peroxid. If a few drops of this mixture be poured over a little of the soUd feces spread on a clean glass sUde, a greenish- blue color will appear almost immediately in the presence of blood. This benzidin test is very sensitive and can be recommended for general use. For a discussion of the PhenolphthaUn (Meyers' or Boas') test, see Blood. (6) Mucus. From the diagnostic standpoint the recognition of much mucus in the feces is of the greatest importance. Any amount of visible mucus should be considered pathological, although it is to be remembered that mucus may be increased physiologically as the result of hypersecretion, in which case it wiU appear as a slimy coating of the scybalous masses or as small adherent par- ticles. B'oas regards the mucus found after strong cathartics as normal, but this is to be questioned as the irritation may be suflScient to set up a mild hyper- secretion. The mucus expelled with the meconium and, according to Lynch, even that passed by infants up to the second week of life should be considered normal. The fecal mucus is a true mucin, being precipitated by acetic acid, but dissolving in 10 per cent. HCl. The larger portions of mucus maybe easily recognized with the naked eye, but the smaller bits are more clearly brought out by rubbing up the feces with water and holding the material, in a thin layer, toward the light. If the feces be well formed, the mucus may be separated from the exterior of the cylinder, as it is never found in the interior of a firm feces. In mushy stools the mucus is intimately mixed with the fecal material, usually in the form of smaller particles, the exterior type of mucus being of much larger flakes. Nothnagel has reported a jelly-like consistency of a mushy stool in a case of jejunal diar- rhea, in which the mucus was not derived from the intestinal wall, but came probably from the bile. It is important that one be able to recognize mucus, as mistakes have been made in the presence of swollen vegetable tissue, fruit pulp, echinococcus membranes, and even of parasites. The ordinary form in which mucus appears in the feces is in clumps, flocculi, or shreds with irregular margins. These pieces may vary in size from those just visible to those several inches in length. In some cases strips, tubes, ribbons, or macaroni-like pieces are observed, which are especially frequent in enteritis membranacea or mucous coHtis. The amount of mucus passed may vary from a few flakes to an enormous mass, Bories having seen 120 grams in one movement. Occasionally one may see, especially in the stools of infants, masses resembling cooked sago granules or "frog spawn" which Kitagawa as identified as mucinous material. ' Zentralbl. f. Chir., 1914, XLI, 1182; Arch. f. Verdauungskr., 1914, XX, 552. See, also, Lyle, Curtman and Marshall, Jour. Biol. Chem., 1914, XIX, 445; Zengerle, Med. Klin., I9I4,X, 179s; Indemans, NederL-.Tjidschr. )f«;fiSl?fi9?fefKNi9iS. LIX, 2077; Roberts, Jour. Am. Med. Assn., 1915, LXV, V4S?'«w'^fl«/E5^A'««ff?iQi4, XL VI, 313, advocates the use of hematin as a reagent for occult blood. 112 DIAGNOSTIC METHODS The consistency of fecal mucus varies from that of a jelly-hke mass to one having the density of thin leather. The larger the piece the firmer it appears, although exceptions do occur. Pure mucus is usually glassy or jelly-like, certain inclusions changing its consistency. Cellular inclusions in the mucus change it to a paper-like mass, while some specimens appear tenacious due to absorption of protein material or to a diminution of its water-content. These inclusions, as well as those of fat or bacteria, will diminish the usual transpar- ency of the mucus. On remaining long in the bowel, mucus may take on the normal brown color of the feces due to hydrobilirubin, a dark orange shade from bilirubin, a greenish hue from biliverdin, or a red to reddish-brown tinge from blood pig- ments. The usual state of the mucus, however, is colorless. It may be stated generally that the most of the macroscopically recogniz- able mucus of the feces arises from the large intestine. Owing to its easy digestibility the mucus from the mouth, stomach, and upper intestines passes out only under conditions of great motiUty. The secretion of mucus in the small bowel is much less than in the large intestine. Of all recognizable forms of fecal mucus only the smallest particles arise from the small bowel; and then only when they are found in a fluid feces. The higher the secreting point the smaller will the particles be, as a rule. These upper intestinal flecks contain much detritus of digestion and half-digested cells or free nuclei and crystals in cellular form, frequently a few bilirubin granules being observed. The mucus from the large intestine, especially from the sigmoid, is large in amount, is jelly-like in appearance and has ordinarily no inclusions. In cases of mucous colitis the mucus may be passed in the forms of the large strips or bowel-casts, appearing as pure transparent material grayish-white or bloody in color and having no inclusions. No fecal matter may be present. Such a condition is a pure secretion neurosis and becomes a true inflammation only when cell-inclusions are observed. It is to be said that mucus, even with a small inclusion of pus or blood, does not necessarily point to ulceration. On the other hand, one must not judge from the absence of mucus that no catarrh exists, as one frequently finds variations in such excretions. Microscopic mucus is much rarer than the macroscopic type, although both may be present at the same time. Microscopically, mucus appears as a structureless mass, characterized by irregular Unes running through it and by a difiicultly recognizable margin, showing a more or less transparent ground substance in which may or may not be found epithelial cells, pus-ceUs, blood- cells, bacteria, protozoa, food remnants, and crystals. If the mucus comes from the higher sections of the bowel the food remnants will predominate, while in that from the lower bowel the cellular elements are in excess. Mucus is stained with difficulty. For a successful stain the reaction must be neutral and very little admixture with foreign material is permissible. Thionin colors mucin a specific violet, while the other tissue elements are stained blue. Methylene blue and methyl violet stain it but slightly, while other aniline dyes color only the enclosed cells. lodin may give a diffuse ^^^^°^' Digitized by Microsoft® THE FECES 113 (7) Pus. Macroscopically visible masses of pus of a gray- white color are occasionally found in the stools, but these may hardly be distinguished from mucus par- ticles without the microscope. If in large amounts, attention should be directed to the perforation of an abscess into the bowel. Such pus is more or less intimately mixed with the fecal material. The passage of pus from the small intestine and even from the cecum is associated with such a marked decomposition that it can be recognized only with the greatest difficulty. The nuclei may still persist, but these resemble very closely those of the food cells. A few isolated leucocytes are usually present in the feces, as a result of diapedesis through the mucous membrane. A pure pus excretion is never seen in cases of uncomplicated catarrh, but in ulcerative processes of the large intestine and in many affections of the small bowel (dysentery, ulcerative colitis, syphiUs, carcinoma, tuberculosis, and typhoid) pus-cells may be found in small masses. Casein flocculi should be differentiated from pus masses by microscopic examination, which wUl show fat droplets mixed with the albumin. (S) Food Remnants. Thie appearance of macroscopic amounts of food remnants in the feces is known as lientery and is dependent upon the nature and amount of food, its method of preparation, the degree of its mastication, and upon the condition of the digestive organs. Much less undigested material remains on a meat diet than after a vege- table ration. In the former case we may find small bits of bone, cartilage, tendon, hair, feathers, skin, fish scales, and connective tissue; whUe in the latter we may observe cellulose-containing cells, such as those of cereal, cotyledonous and leguminous vegetables, skins of fruits, nuts, and vegetables, grape seeds, cherry stones, etc. Cooking has a great influence upon the digestibility of any food. Boiling seems to be the best method, although much of the nutritive material is taken up by the cooking water. The method of roasting is such as to produce a more nutritive but a less digestible substance, unless it is carried only to the point of slight coagulation of the protein. The outside layers of roasted meats seem to be practically indigestible, according to the work of van Ledden Hulsebosch. Smoking seems to be the least desirable form of preparing meats. Vegetables become more digestible through the cooking process, owing to the bursting of the indigestible cellulose membrane. Those vege- tables, such as lettuce, cucumbers, onions, turnips, cabbage, and radishes, which are eaten raw, appear in the feces absolutely unchanged. While great individual differences exist in the power of utilizing digestible or undigestible food, yet we must assume that any appreciable residue, espe- cially following the Schmidt-Strasburger diet, is pathological. The digestive insufficiency may begin in the mouth as a result of too little mastication. The importance of thorough mastication has been especially emphasized by Fletcher. Lack of digestive power of the stomach will have but little influ- g Digitized by Microsoft® 114 DIAGNOSTIC METHODS ence on intestinal digestion, providing the pancreatic secretion is sufl&ciently active, with the exception that raw or smoked connective tissue will be undigested and will appear in the feces. Lientery is much less frequent in motor disturbances of the stomach than in those of the intestines. Perhaps the gre^itest amount of undigested residue is seen in cases of perforation of an ulcer or carcinoma of the stomach into the intestine, a direct communication being established between the stomach and colon. Naturally, in cases of increased intestinal motility, one will find more food residue in the feces than under normal conditions. This motor insuf- ficiency is especially answerable for the appearance of undigested starch remnants, as, even under the most severe conditions, starch digestion is not interfered with to any extent in the bowel, providing mastication and prepa- ration of food have been sufficient and the succus entericus has not been so diminished by catarrhal processes as to permit of fermentation. The direct irritation from the hard particles of cellulose may cause an increased peris- talsis and, hence, directly lead to hentery. Marked disturbance of protein and fat digestion, evidenced by an intense lientery, will occur if the pancreatic secretion be insufficient. This is even more marked if gastric disturbance coexists. A lack of bile will cause an exclusive disturbance of fat digestion, which is seldom manifested by the appearance of macroscopic particles of fat, but is characterized by the typical clay color of the stool. The final factor which influences lientery is the insufficiency of the ab- sorptive power of the intestine. This increases the food residue by retarding digestion, according to the law that an accumulation of the products of fer- ment activity will prevent further action of the ferment, as well as by holding within the bowel the products already digested. Only in exceptional cases can one judge of the specific factor at the bottom of a lientery, a boundary line between normal and abnormal being drawn with difficulty. It is, however, important that one be able to recognize food remnants both macroscopically and microscopically. The writer recalls a case in which the residue of orange and banana pulp was mistaken for a new parasite. This recognition of the nature of a fecal residue is especially im- portant in the examination of the infantile stool. Normally, the nursing child shows no food residue in the feces, but frequently white flecks or clumps are observed, which may consist of casein but perhaps more frequently of fat or of soaps. The proper recognition of such particles will require both chemical and microscopic examination. The following discussion of the appearances of the various food residues . will include both the macroscopical and microscopical examinations, as these are inseparable in practical work. (A) Protein Residues. (a) Muscle Fibers. The appearance of muscle fibers in increased amount in the stool is known as azotorrhea} The muscle fibers appear as isolated pieces of different size 'Also called creatorrlioea._,See.WerUieinjisr. ZtsdM^s^ Win. Med., 1912, LXXVI, S7; Tileston, Arch. Int. Med., igi^'Se/^^^ SXo'^mSS^ZWhr. i. klin. Med., igis.LXXVH, 40. THE FECES I15 and shape. The smallest pieces have a circular or oval contour, the medium- sized particles are jagged, while the larger masses have parallel sides and angular surfaces. These fibers are colored yellow or yellowish-brown by the hydrobilirubin, but may be tinged with bilirubin or by foreign pigments. The color will depend both upon the amount of pigment and upon the time the fibers have lain in the bowel. The smaller pieces may be entirely homogeneous, although these as well as the larger bits show both transverse and longitudinal striations. If. the fibers are well digested, the longitudinal striations may be the only ones show- ing, and even these may disappear. No nucleus is visible unless the pancre- atic secretion is entirely lacking.^ No specific micro-chemical reactions are known for these fibers. They may be tested with any of the color reactions for protein material. Connective tissue and elastic tissue fibers are occasionally associated with muscle fiber and may be recognized by their appearance. (&) Casein. This is found especially in the infant stool and is always pathological. These masses, known as curds, are more or less round clumps which vary in size from that of a pin head to that of a hazel-nut. They are either pale or golden-yellow, the larger masses being a pure white inside. These curds always have a distinct yellow tone exteriorly, but the interior is pure white. If these particles are pressed between a slide and cover-glass, they spread out like white cheese and show absolutely no structure microscopically. These masses show the protein reactions. Leiner's Test. A small amount of fecal matter containing these curds is spread on a slide and dried in the air. It is then fixed by heat and stained with a mixture of equal parts of a 0.75 per cent, solution of acid fuchsin and methyl green in 50 per cent, alcohol (dilute mixture i o times with water) . At the end of 1 5 minutes the slides are placed in distilled water and left for one hour. Casein and para- casein will take a pale blue or violet color, while similar substances will show a greenish tone. Microscopically, one may differentiate from these true casein particles (curds), certain products which are more or less normal in the stools of the child. These masses are smaller than the curds, being not usually over pin- head size, are more yellow in color, and appear under the microscope as clumps of fatty acid crystals or of fat droplets and bacteria bound together by mucus. ^ (B) Fat Residues. Fatty substances are present in all stools, if there be any in the diet, either in the form of neutral fat, free fatty acids, or soap, especially of calcium and magnesium. If present in macroscopically recognizable amounts, the con- dition is known as steatorrhea. Neutral fat may be present in the form of white colorless clumps of differ- ^ See Atchley (Arch. Int. Med., 1915, XV, 634), whose experiments tend to disprove the value of Schmidt's nuclear test. 2 See Courtney, Am. Jour./^/fg^^^/J^/cfggg^® Il6 DIAGNOSTIC METHODS ent size and irregular in shape, some being globular while others are distinctly- angular. The more usual form is the refractive, opaque, more or less yellow globule. Occasionally the fat may appear as a melted oil which hardens over the surface of a formed stool or gives the appearance of vagelin to a semi- solid stool. Neutral fat may be recognized by its ready solubility in ether and its black color on treatment with osmic acid and deep red color when acted upon by Sudan III ,or Scharlach R. Fatty acids appear either as scales or as crystals of varying form. The scales may not be distinguishable from those of pure fat except by their easy solubility in cold alcohol. The crystals are thin, delicate, curved needles, which run to a distinct point and are grouped in thick masses. ^ Other types of fatty acid needles may occur, such as the small lancet-shaped plate or those closely resembling the soap crystal. These crystals are colorless and are soluble in cold alcohol, showing no stain with the above reagents for free fat. The fatty acid scales are, however, stained by these dyes. The soaps Ukewise appear in the feces as scales or as crystals. The ^scales are less refractive, more firm, usually more angular, and may be color- less or yellow-brown from hydrobilirubin or yellow from bilirubin. The crystals appear most frequently as uncolored needles, which are shorter, plumper, not so pointed as the fatty acid crystals, and are arranged in clusters. Schmidt has described a peculiar form of crystal, which he styles the "crack- nel" form, a round type with a sunken center- and a raised border. These crystals are insoluble in ether, do not melt on warming, and are uncolored with stains. Treated with acids they form fatty acid crystals. Any condition whch interferes with the proper absorption of fat by the mucosa or lymphatics will lead to steatorrhea. This condition may be ob- served, physiologically, after the ingestion of large amounts of fat, as in the oil treatment of gall-stones, but we are little concerned with such findings. It is seen, pathologically, in cases of atrophy of the mucosa, in amyloid disease of the intestines, tuberculous ulceration, tubercular peritonitis, tabes mesen- terica, and even in catarrhal enteritis. The peculiar, glistening, gray-white, pasty, acholic stool of steatorrhea is seen more frequently, however, in cases of biliary obstruction and of pancre- atic disease. The fat in these cases of biliary stasis is in the usual form, that is three-fourths of the ether extract of the bile-free feces is present as fatty acids and soaps and one-fourth as neutral fat. In cases of acholia 50 to 80 per cent, of the fat will be unabsorbed instead of the normal 5 to 10 per cent.^ In the fatty stools of pancreatic disease, the fat is largely present as fatty acid, no soap being found. The association of steatorrhea with the absence of decomposition products, few bacteria, and the presence of maltose in the feces is much more indicative of pancreatic disease than is steatorrhea alone (Le Nobel). (C) Carbohydrate Residues. Starch may be present, in the normal feces, enclosed within plant cells which have resisted digestion, but well-preserved starch granules are never 1 See von Hoesslin and Kg^/fj|K^ g^j|^^^^gj^(^ klin. Med., 1912, CV, 576. THE FECES 117 found normally. A few partially digested or complete colorless granules mc^y indicate no abnormality, but many will point to a hyperacidity or to a disturb- ance in the small intestine, especially to an insufficiency of the succus en- tericus leading to the so-called "fermentative dyspepsia" (Schmidt). Starch may be detected by the addition of Lugol's iodin solution to a small portion of the feces spread upon a slide. A blue coloration will in- dicate starch, while a red tone will show the presence of erythrodextrin. The varieties of cellulose-containing substances found in the feces are as numerous as are the types of such food material. These have been discussed previously and cannot be here elaborated. Certain microchemical tests may prove valuable in the identification of such cellulose material. Cellulose treated with sulphuric acid and then with iodin solution gives a blue color due to the conversion of cellulose into amyloid. If a solution of zinc chlorid be allowed to act upon a suspected mass of cells, which have been previously treated with Gram's iodin solution, a piurple coloration will be produced. If a neutral or weakly alkaline solution of Congo- red be added to cellulose, it will be stained a distinct red. Cellulose is soluble only in an ammoniacal solution of cupric oxid, known as Schweitzer's reagent. (9) Biliary Constituents. As a rule, the unchanged biliary acids, glycocholic, taurocholic, and cholalic acids, are absorbed from the bowel so that they do not appear in appreciable amounts in the feces. Schmidt believes that cholalic acid is present in slight amounts in all feces and that all the bile acids may be in- creased in pathologic conditions. Bile pigments, on the other hand, are always present, chiefly in the form of hydrobilirubin, although bilirubin may occur. In pathologic conditions we may find, beside these two pigments, biliverdin, bilifuscin, bilicyanin, and biUhumin. Stercobilin (urobilin) in the feces is usually an index of blood dis- integration.^ The tests for these pigments as well as for the free bile acids -and their salts will be considered in detail in the section on Urine. ^ In case of cholelithiasis gall-stones of varying size may be found in the feces, although not in every case. It is, therefore, of the greatest importance that the feces be carefully examined in suspected cases for the presence of these concretions. This is especially the case whenever a severe, colicky, abdomi- nal pain of doubtful origin exists. The feces should be well mixed with water and passed through a fine sieve which will retain any of the suspected particles. A single examination of the feces is not sufficient in such cases unless the stones be found. In cases of negative findings, the stool should be searched for at least two weeks following a suspected attack. According to Naunyn, only the very firm stones will leave the bowel, the softer ones break- ing up into small bits in the bowel. These gall-stones vary from the size of a pin-head to that of a pigeon's egg and are found as small crumbling masses or as hard stones which either have a ' See Robertson, Arch. Int. Med., 1915, XV, 1072; Ibid., 1915, XVI, 429. * See, however, Jovine, Gazz. d. osp., 1913, XXXIV, 1071. For the chemical composi- tion of human bile consult Czyhlarz, Fuchs and von Fiirth, Biochem. Ztschr., 1913, XLIX, 120; also, Rosenbloom, Jour. Biol. Chem., IQ13, XIV, 241. Digitized by microsoft® Il8 DIAGNOSTIC METHODS jagged surface or a smooth surface with characteristic facets, indicating the presence of many such stones. These stones consist for the most part of cholesterin mixed with the biUary pigment, or of compounds of calcium with the biliary pigments. These calcareous stones may be combinations of the various bile pigments and are always the hard facetted type, while the choles- terin stones are softer and may be colorless or tinged with bile pigment. The nucleus of these stones is usually a mass of organic detritus, in some cases being made up of clumps of bacteria, such as the typhoid bacillus and bacillus coli communis. A gall-stone may be usually recognized from its fractured surface, but frequently it becomes necessary to submit it to chemical examina- tion for purposes of identification. In this examination for gall-stones the worker must not be deceived by the presence of extraneous substances such as seeds, cherry-stones, fats and soaps of high melting point and of masses of impacted feces. In the treat- ment of gall-stones by the use of olive oil many of these soft fatty translucent masses appear in the feces which are in no way associated with gall-stones. (lo) Intestinal Sand and Concretions. By intestinal sand we have reference to the small granules or masses of inorganic salts which appear in the feces. These masses are very small, may be spherical or irregular, are usually hard, usually have a reddish-brown or green color and consist of inorganic compounds mixed with organic detritus, especially with fat and bacteria. While most of this material is made up of ingested substances, we may have in cases of neurasthenia and especially in those associated with mucous colitis the excretion of large numbers of these particles as the result of a true secretory neurosis. Myer and Cook^ have recently shown that the banana may be a source of this intestinal sand.^ The massing together of this intestinal sand may form intestinal concre- tions or enteroliths. As a rule, however, these intestinal concretions have as a nucleus some foreign body around which calcium and magnesium salts of phosphoric and carbonic acid or ammonium magnesium phosphate have been deposited. These enteroliths are usually hard, heavy, and round, being colored, as a rule, brownish. ^ A second form of intestinal calculus, known as coproliths, are irregular in shape, usually softer than the enteroliths, and consist of inorganic material, mixed with inspissated feces. Neither of these types of intestinal concretion have any great pathologic significance, although the possibihty must be granted that some of them might either form or lodge 'in the appendix and thus be accountable for an acute appendicitis. (ii) Tissue Fragments. The examination of feces for tissue fragments is more or less unsatisfactory, owing to the fact that th^se fragments are diflELcultly recognizable after being partially digested by the juices of the intestinal canal. It not infrequently happens, however, especially in carcinoma of the lower bowel, that such f rag- 1 Am. Jour. Med. Sc, igog, CXXXVII, 383. « Talbot (Jour. Am. Med. Assn., 1913, LXI, 238) shows that the banana is not always the source. 3 See Coerr, Jour. Am. ^^tn^Med'^fWiFdi^® THE FECES IIQ ments may be obtained and a diagnosis made possible by microscopic exami- nation. A diagnosis of malignancy would better not be made unless these tissue fragments show typical cellular arrangement, or at least, typical ar- rangement of the nuclei. The writer has seen a diagnosis of carcinoma made on the basis of a strip of mucus with cell enclosures and would, therefore, warn the worker to be on his guard against the possibility of such an absurd mistake. III. Microscopic Examination This examination includes the search for many substances which have been described un'der the macroscopic examination, so that these need not be here considered. There are, however, some elements, both morphological and crystalline, which have not been treated. For any success whatever in the microscopic examination of the feces, great care must be taken in the selection of rhe material for examination. This is especially true when searching for parasites or their ova, as well as for the differentiation between various food particles and secretions from the intestinal wall. If the stool be soft and mushy it should be thoroughly mixed by stirring and suspicious particles looked for macroscopically. Fresh speci- mens as well as stained preparations are then made and examined both with ■the low and high power. In examining feces for bacteria it is always well to mix the feces with water and centrifuge for a few minutes. The bacteria will remain suspended in this process and the coarser fecal material wiU be depos- ited. The fluid is then poured off and mixed with equal parts of alcohol, the mixture being again centrifuged when the organisms wiU be found in the ■sediment. This procedure is the one adopted by Strasburger in estimating the proportion of bacteria in the dry stool. Various microchemical reactions are carried out with isolated fecal material and frequently are of great importance. In this procedure a few ■drops of reagent are allowed to flow under a cover-glass which covers the specimen. The staining material may be drawn under the cover-glass by •placing a piece of ordinary filter-paper on the opposite side of the cover-shp. If the examination for amebfe is to be made it is essential that the stool be kept warm, as the characteristic differentiating point of an ameba is its -active motiUty. This can be done by placing the feces in a bottle which is surrounded by warm water'until the examination is made. It is wise, also, to make the examination upon a warm stage, although this is not absolutely -essential unless extended search has to be made for the parasite. There are no specific stains for the feces, the ordinary Loffler's methylene blue serving very well for a general staining of bacteria, while characteristic stains must be used when searching for special organisms. Morphological Elements. Epithehal cells are present in every specimen of feces. These may be squamous in form and are usually found in the mucoid particles of the stool. "They are especially present in rectal carcinoma and in ulcerative conditions -of the lower bowel. The cyUndrical type of epithelium is much the commonest iorm and is found, also, in association with the mucus. Many well preserved Digitized by Microsoft® I20 DIAGNOSTIC METHODS cells may be present if their source is in the lower bowel, but, as a rule, these cells show all types of degeneration. They occur in catarrhal inflammations of the intestinal mucosa and are rarely associated with pus-cells unless ulcer- ation has taken place. If the irritation is in the small bowel these cells are always more or less digested and contain bilirubin particles in cellular arrange- ment, while if from the lower bowel the cell is usually intact. The presence of red blood-cells is determined more by the chemical ex- amination than by the microscopical. It may occasionally happen that these cells may be seen, but a good result will depend upon a fortunate selection of the material for examination . A few scattered leucocytes are practically always found in the feces,i and assume pathologic importance only when present in large amounts as pus-cells, whose occurrence has been previously discussed. Crystals. Besides the crystals of free fatty acids and. soaps previously mentioned we find large numbers of other crystals in the feces. Among these we observe neutral phosphates of calcium and of magnesium which appear as wedge- shaped crystals occasionally forming rosettes in the former case while in the latter the crystals are more in the form of rhombic plates. The ammonium magnesium phosphate crystals are practically always present in the feces, appearing either as the typical coflSn-lid crystal or as irregular fern-like masses. These triple phosphate crystals were at one time supposed to be characteristic of typhoid stools, but like so many other things their importance has been exaggerated. Various other crystals of calcium compounds are observed, such as calcium carbonate, calcium sulphate, and calcium oxalate, along with calcium salts of unknown fatty acids, which have been described by Nothnage . as irregular, oval, or circular masses, either fissured or showing concentric striations, and being always bile-stained . The lactate of calcium is seen in the form of radiating needles arranged in sheet-like masses in the stools of children on a milk diet. Cholesterin crystals are found as thin, transparent, rhombic plates with notched corners. These crystals do not always appear in the typical shape, so that they should be tested by the addition of concentrated sulphuric acid when the cholesterin crystals will change from a yellow to a blood-red, violet, green, and finally blue color. The Charcot-Leyden crystal appears in typical form in the feces as a colorless, diamond, double pyramid-shaped crystal. The presence of these crystals is practically characteristic of helminthiasis, al- though they may indicate the presence of any parasite from the least harmful to the most pernicious. Hematoidin crystals occur as reddish-yellow rhombic plates or as groups of needles or amorphous masses in stools showing the presence of blood chemically. These have no special signifiance and are not found as frequently as are the other blood pigments. After the use of bismuth preparations we find the oxid of bismuth appear- ing in the form of black irregular rhombic crystals with notched edges. Char- 'Albu and Werzberg (Ztschr. f. klin. Med., 1912, LXXIV, 394) call attention to the frequent presence, in the fjces of amebic dysentery, of eosinophile leucocytes and myelocytes. Digitized by Microsoft® THE FECES 121 coal appears in the form of irregular black masses which are larger and not so rhombic as are the bismuth crystals. The examination of the feces for the various bacteria as well as the para- sites and parasitic ova will be discussed in a special section. Various sub- stances appearing in the feces have been mistaken for parasites or their ova and these also will be discussed in the later sections. IV. Chemical Examination The chemical examination of the feces would naturally embrace both the qualitative and quantitative estimation of the products of digestion and de- composition as well as the estimation of the undigested portion of the food along with the products derived from the gastrointestinal canal itself. Clin- _ ically, such work is rarely carried out and has questionable value from a diagnostic standpoint. However, in following the metabolism in any special case it is essential that the absorbed and unabsorbed portion of the food be known. This applies more particularly to the proximate food principles, protein, fat, and carbohydrate, although in some cases the estimation of the inorganic intake and excretion is of first importance.^ The writer cannot attempt in the scope of this work to go into great details regarding the chem- ical examination of the feces, but must limit himself to a few selected topics. Reaction. The normal reaction of the feces does not vary much from a neutral one, although it has a tendency to be slightly alkaline, owing to the presence of the alkaUne secretions of the intestinal tract. The alkalinity of these secretions is diminished both by the combination of the alkali salt with the digestion products and by the absorption of some of this alkaline material into the blood. This reaction may at times be acid owing to the formation of lactic and butyric acids in the fermentative processes, while an increased alkalinity may be observed in cases of markedly increased ammoniacal decomposition. A pure meat diet gives, as a rule, an alkaline feces, while a pure carbohydrate or fat diet will have a tendency to form an acid feces. The reaction of the feces under pathologic conditions is of little impor- tance, although in typhoid fever the reaction is somewhat more strongly alka- line than in almost any other condition. So much depends upon the diet as well as upon the condition of the digestive organs that no conclusions at present may be drawn from the reaction of the feces. Total Solids. The normal amount of feces passed in 24 hours ranges from 100 to 250 grams, of which about 75 per cent, is water and 25 total solids. Although the determination of the total solids of the feces has little of importance in itself, it is essential that one should know the dry weight of the feces in order that he may properly calculate the amount of the various substances in the dry feces. In determining the total solids in the feces, the specimen, preferably the 24-hour specimen or each movement separately, is placed in an evaporating dish, covered with a small amount of alcohol, and heated over a water-bath • See Hoh, Courtney andJlales, Am, Tour.. Dis. Child-, iQiSi IX, 213; von der Heide, Biochem. ztschr, 1914, l,x^Pm^^ed by Microsoft® ■122 DIAGNOSTIC METHODS with frequent stirring. Small amounts of alcohol should be added as evaporation proceeds in order to hasten the drying process. It is, moreover, necessary to add a small amount (lo to 15 c.c.) of dilute sulphuric acid and to mix it thoroughly with the feces. This combines with the free ammonia, forming a non-volatile salt, and thus prevents loss of nitrogen. If the stool be rich in fats, it is wise to add a weighed amount of dry washed sand to make the mass more porous and thus permit of quicker drying. When the water has been driven off on the water-bath, the specimen may be placed in the dry- ing oven at 105° and left for several hours, after which it is placed in the desic- cator and dried to constant weight. Knowing the weight of original substance taken and the weight of the dry substance, it is very easy to determine the percentage of total sohds. An increased total solids of the feces is observed in most cases of constipa- tion, while in diarrhea the solid residue is much diminished. Increased sepa- ration of fluid into the bowel and diminished absorption from the bowel are the factors regulating the fluid content. Nothing can be determined, how- ever, by the examination of the amount of total sohds, as regards the patho- logic conditions accountable for an increase or a decrease. Total Nitrogen. The determination of the total nitrogen must always be made in estimating the metabolism in any given case. In so doing it is likewise essential that one should know the absolute nitrogen intake, as without such a factor nothing can be learned from the excretion either in the urine or in the feces. The daily excretion of nitrogen in a fasting condition varies from i to 4 grams, on a mixed diet this may run from 3 to 7 grams, while on a vegetable diet the nitro- gen of the feces may be as high as 10 grams, a pure meat diet yielding between 2 and 6 grams. In pathologic conditions this amount is practically always increased, due both to lack of digestive power and to decrease in the absorp- tive function of the intestines. Outside of metaboUc experiments the absolute amount of nitrogen of the feces has no great diagnostic importance beyond showing some perversion of intestinal activity. ^ In insufficiency of the pancre- atic secretion, we are apt to find the greatest loss of nitrogen by way of the feces. The method of determination of total nitrogen will be given in detail in the section on Urine to which the reader is referred. The constituents which go to make up this total nitrogen are the various undigested protein bodies, such as albumin, globuhn, nucleoprotein, nucleo- albumin, and gelatin, along with partially or completely digested products of protein origin, bacteria, and secretions from the intestines. These latter bodies embrace albumoses, peptone, the various amino-acids, and the hexone bases along with a certain amount of ammonium salts. Tde chemicrl prop- erties of these substances, as well as the methods of their chamical detection and estimation, must be looked for in works on physiological chemistry. The bacteria form a large percentage of the total nitrogen. ^ It is rare, even in 1 See McCrudden and Fales, Jour. Exper. Med., 1912, XVII, 20 and 24; also Koplik and Crohn, Am. Jour. Dis. Children, 1913, V, 36; Gamble, Ibid., 1915, IX, 519; Van Slyke, Courtney and Fales, Ibid., 533. 2 Mattill and Hawk (JourdB^DfjJjaodw. TOH&^S/?®3) shows that the bacterial nitro- gen constitutes 53.9 per cent. oFtne" total-lecal nitrogen. THE TECES I 23 metabolic work, that the nitrogen partition of the feces is determined. Much more importance attaches, with our present knowledge, to such division of the nitrogenous material of the urine. Likewise the products of abnormal decomposition of protein material, taking place in the bowel with the forma- tion of such products as indol, skatol, and phenol, are rarely searched for in the feces. ^ Our clinical knowledge of the importance of these substances is con- fined largely to their detection and estimation in the urine. In the study of the metaboUsm of various cases of cystinuria, which is associated with abnor- mal protein disintegration, certain diamines, such as cadaverin and putrescin, have been isolated from the feces by Udransky and Baumann.^ Fat. The chemical estimation of the amount of fat is of importance only in metabolic work, as in the more direct clinical examinations this substance is detected by macroscopic and microscopic methods. It is, however, essential that the loss of fat by way of the feces should be known before a proper meta- bolic balance can be struck. The dried feces is treated with a small amount of i per cent, acid-alcohol and evaporated to dryness in order to convert any soaps which maybe present into the fatty acids. The dry residue thus treated is then placed in a Soxhlet apparatus and extracted with ether for at least 72 hours. The ether, which has taken up the free fat and the fatty acids, is then evaporated and the resi- due of fats weighed. From this weight the percentage of fat may be deter- mined, and, knowing the original dry weight of the total 24-hour feces, the total fat lost by way of the feces may be readily calculated.' The amount of fat in the feces wiU depend much upon the amount and quahty of the diet. In fasting conditions the amount is usually about i gram. On diets poor in fat the amount in the feces may exceed that of the diet, thus indicating a loss of fat from the body. It seems to be a general rule that the higher the melting point of the fat of the diet the greater will be the loss in the feces. Thus on a diet containing only butter as a fat about 4 per cent, of the intake will be lost, while with pork fat the loss may be as high as 13 per cent. It is impossible to say, a priori, just what the fat content of a normal feces would be. With the ordinary diet of our country it would range from 2 to 7 per cent, of the intake, which should be somewhere about 100 grams of fat per day, representing a loss of from 2 to 7 grams daily. Carbohydrates. As previously stated, carbohydrates may appear in the feces either in the form of starch or of cellulose. Only in exceptional conditions do we find any of the monosaccharids present. Disaccharids, such as lactose and maltose, may be occasionally found, but only when there is a combination of insuffici- ' See Moewes, Ztschr. f. exp. Path, und Ther., 1912, XI, 555. See, also, Rodella, Arch. f. Verdauungskr., 1914, XX, 657; Whipple (Jour. Am. Med. Assn., igis, LXV, 476) believes that the intoxication of intestinal obstruction is due to the absorption of a primary proteose. 2 See Hopkins (Arch. Int. Med., 1913, XI, 300) for a discussion of the presence of hemo- lysins in fecal extracts. « See Cowie and Hubbard, Am. Jour. Dis. Child., 1913, VI, 192; also, Bloor, Jour. Biol. Chem., 1913, XV, 105; Ibid.vi.913, XVL 517;. Saxon, Ibid., 1914, XVII, 99; Gephart and Csonka, Ibid., 1914, XIX, siimized by Microsoft® 124 DIAGNOSTIC MKTHODS ency, both of a salivary and pancreatic ferment. The presence of any ap- preciable amount of starch granules must be considered pathological, while the amount of cellulose will depend upon the amount in the food as well as upon the preparation of the food and its mastication. Nothing is to be learned, from the clinical standpoint, by the determina- tion of the absolute amount of carbohydrate in the feces. In the study of the utilization of food substances by the system and in general metabolic work it is, however, necessary to know just how much of the carbohydrate intake is absorbed. This may be determined indirectly by subtracting from the weight of the dry feces the sum of the protein, fat, and ash. This result will represent a much higher figure for carbohydrates than the one obtained by direct determination. In the direct determination much difference exists between the soluble forms, and the insoluble cellulose. In the latter case we have to do with a useless form of carbohydrate and should, therefore, direct our attention rather toward the estimation of the amount of undigested or partially digested starch. We should determine the amount of cellulose and subtract this factor from the total carbohydrate obtained in the previous subtraction. This corrected figure will represent more nearly the true amount of carbohydrate than will the former, al- though for scientific purposes a direct determination is essential. The starch may be estimated by taking a weighed amount of the dry feces and treating with 50 c.c. of 10 per cent. HCl. This is then boiled for one-half hour in orderto convert the starch and digested portions of starch into the monosaccharids. This acid solution is then filtered and washed with sufficient water to make the total approximately the same as that of the original solution. The filtrate is then neutralized with sodium hydrate and made up to exactly 100 c.c. in a volumetric flask. The sugar in this solution is then determined by the methods outUned under Urine. This method, although not absolutely accurate, will yield results sufficiently correct for ordinary purposes. More accurate results will be given by the Volhhard-Pfliiger mehod to be discussed later. The amount of glucose, as deter- mined by either of these methods, will give, if multiplied by 0.94, the amount of starch in the feces taken. The percent- age may then be readily calculated and the total quantity of the 24-hour excretion of starch determined. The fermentation method of Schmidt was advanced to permit of rough estimation of the presence of pathological amounts of starch, which, under ordinary circumstances, should have been digested. The principle of the method is the estimation of the amount of gas produced by the action, of the intestinal bacteria upon the sugar which is formed from the starch by the intestinal amylolytic ferments. P^^irt^^th^^deJ^^^lj^ the patient is placed upon Fig 21. Schmidt's fermen- tation apparatus. THE FECES I 25 the test diet outlined on page 100. The test is made as follows: Approxi- mately 5 grams of the moist stool are placed in the vessel a which is then filled with water and the contents thoroughly mixed (see cut) . The stopper is then placed in the fermentation flask in such a way that no air-bubbles are left. The tube h is filled with tap-water and closed with a small stopper without the inclusion of any air-bubbles. The tube c is now placed in position as shown in cut and the whole apparatus put in an incubator for 24 hours. The tube c has a small opening in the top so that water may be readily forced from the tube' b into c by pressure of the gas produced. According to Schmidt o.i gram of starch will cause the tube h to show about one-half its volume of gas. Normally, a positive result is said to occur when the tube is one-fourth to one- third filled with gas. The material in the fermentation flask a should be tested with litmus-paper after the test is complete to obtain the reaction of the mixture. If the gas formation is, as it should be, due to carbohydrate fermen- tation there will be a slight increase in the acidity of the mixture, while if it be due to protein putrefaction the reaction will show a slightly increased alkalinity. The various decomposition products of carbohydrate digestion and fer- mentation are rarely of importance in fecal examinations. These products consist of volatile fatty acids, lactic acid, saccharic acid, alcohol and aldehyde, and the conjugated glycuronic acids. Tests for these various substances will be found in various parts of this work or in works on physiological chemistry to which the reader is referred. ' From the clinical standpoint the gases produced by the processes of fer- mentation and putrefaction in the bowel have little value. The same is to be said of the quantitative estimation of the inorganic constituents of the feces. These latter may be determined by methods outlined in quantitative analysis. The various ferments found in the intestinal canal may be detected in the feces, but such examinations are at present of little clinical value. Methods of isolating these ferments and of determining their activity belong more to physiological chemistry than to clinical diagnosis and will, therefore, be neglected.^ Phenoltetrachlorphthalein Test. There is ho question but that the liver has a more varied and manifold functional activity than any other organ of the body. Slight disturbances in hepatic activity are a matter of relatively frequent occurrence, but these are no attended with any marked perversion of function as the larger portion of the parenchyma of the liver may not be involved. In disease, however, of the liver the character of the functional changes will vary with the nature of the underlying pathological condition. There is not so much difficulty in deter- mining, from clinical and laboratory data, that hepatic disease exists as there is in estimating the intensity of the disease and the degree of disturbance of the functional activity. For this reason various attempts have been made to ' See Fischer, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 179. - See Crohn, Am. Jour. Med. Sci., 1913, CXLV, 393; also, Chace and Myers, Arch. Int. Med., 1913, XII, 628; Brown, Bull. Johns Hopkins Hosp., 1914, XXV, 200; Einhorn, Am. Jour. Med. Sc, 1914, ^XLVgl |^o^^g3h^IMd.^8^Arch. Int. Med., 1915, XV, 581. 126 DIAGNOSTIC METHODS devise tests which would indicate such functional variations sharply and per- mit of a judgment concerning their intensity. While many of these tests have been proven of questionable value, some of them, such as the levulose and galactose tolerance test as well as the urobihnogen estimation, are of some importance in diagnosis and prognosis and will be discussed in the section on Urine. These tests have, for the most part, lacked a quantitative element, indicating more the presence than the extent of the disease. Rowntree, Hurwitz and Bloomfield have introduced a test for the functional activity of the Uver, which is probably the best at our disposal. They take advantage of the fact, discovered by Abel and Rowntree, that phenolte- trachlorphthalein, a drug first prepared by Orndoff and Black, escapes from the body only in the bile and may be recovered from the feces to a consider- able extent. Under normal conditions the drug is not excreted to any ap- preciable extent in the urine. In other words, the liver exerts a specific action in excreting this dye, just as does the kidney in eliminating phenolsulphone- phthalein. With this fact as a basis, these workers were able to show that a decreased capacity for its excretion was a resultant of lowered functional activity due to disease, and that minimal amounts of the dye escaped with the urine in pathological conditions of the liver. By direct estimation of the amount of this dye excreted by the bile and carried into the intestines one may readily estimate the presence and, to some extent, the degree of the functional disturbance of the liver. The solution used for the injection is an aqueous solution of the disodium salt of phenol tetrachlorphthalein. This is prepared as follows: 2.5 grams of the phenoltetrachlprphthalein are placed in a 200 c.c. flask with 5 c.c. of an 8 per cent. (2N) sodium hydrate solution and 45 c.c. of freshly distiUed water. Boil for 20 minutes under a reflux condenser. Filter the solution into a 100 c.c. flask, when it is ready for use. It is an intense purplish-red solution, which does not keep for many days owing to the precipitation of the dye by the CO2 of the air. Hence relatively fresh solutions must be used. Method of Administration. Eight c.c of the above solution, representing 400 ijig. of phenoltetrachbr- phthalein, are administered intravenously to the patient, using all the anti- septic and aseptic precautions which this method involves. The funnel and tubes are filled with sterile physiologic salt solution and, after the flow is es- tablished, the solution of the dye is added. Fifty to 100 c.c. of salt solution are used, the dye being washed in with the salt solution until the fluid enter- ing the vein is colorless. Collection of Material. Before beginning the administration of the dye, active purgation of the patient should be induced and should be continued throughout the time of observation. If little feces can be obtained, enemata should be employed. Collect all the fecal material which passes for 48 hours and the urine for 24 hours. Place the total48-hour specimen of feces in a large bottle and dilute it with I to 1 .5 liters of water, denea^ng an tbaamoua^of feces. Place this mixture THE FECES 127 in a shaking machine for 5 to 20 minutes. Without allowing time for sedi- mentation, one- tenth of the total mixture is placed in a liter flask and to this is added 5 c.c. of 40 per cent, sodium hydrate, which causes the mixture to take on a dirty red color. Now dilute the mixture to i liter with water, stopper the flask, and shake thoroughly. One hundred c.c. of this preparation are placed in a 200 c.c. flask and 5 c.c. of a saturated solution of basic lead acetate are added. Now add 5 c.c. of 40 per cent, sodium hydrate solution to elicit the maximum intensity of red color. If the amount of NaOH added does not seem sufi&cient, add a little more, being careful not to redissolve the precipi- tate present.^ Make up the contents of the flask to 200 c.c, shake and filter a small portion for comparison with a standard solution of phenoltetrachlor- phthalein. This comparison is made in a colorimeter, either of the Duboscq type or of the type of Rowntree and Geraghty's modification of the colori- meter of ^Autenrieth-Konigsberger. For comparison one may use 0.4 c.c. of the original solution, making this up to a liter and adding enough NaOH to insU'Te the maximum color. There are certain elements connected with this test that are not very de- sirable. It involves intravenous injection, which is followed in many cases by thrombosis at the site of injection. This is not sufficient to occasion serious discomfort, but the fact should be remembered and its possibilities taken into consideration. Further, it necessitates a careful collection of feces for 48 hours. The quality of the red color obtained in the fecal extract in some cases is such that accurate quantitative determinations are difficult. The results with this test show that a phenoltetrachlorphthalein output in the feces below 30 per cent, or its appearance in the urine is quite infrequent in health. The output of a less amount is relatively frequent in hepatic disease, although a normal output of 30 per cent, does not exclude liver lesions. The output of the dye in the feces is independent of the quantity of bile excreted, although it is evident that none of the dye will reach the intestines in cases of obstruction of the duct. Functional changes, as evidenced by a lessened out- put of the dye in the feces, have been most marked in cirrhosis, carcinoma and in cachexias associated with severe anemias. Chronic passive congestion does not give rise to a much changed excretion, although marked hepatic con- gestion associated with insufficiency of the myocardium is usually accompanied by a lowered output. Although this test is not be regarded as diagnostic in all cases of perversion of hepatic activity, yet it offers much evidence which can be obtained by very few other tests so far advanced.^ ' If difficulty is experienced on account of the quality of the color at this point, add to the 100 c.c. of mixture 5 to 10 c.c. or more of a calcium clilorid mixture (CaClj, 90 grams; cone. NH4OH, 10 c.c; water, 5° c.c.) until the best color appears. Dilute to 200 c.c, allow the mixture to stand from i to 24 hours and decant the supernatant fluid for comparison. " Orndoff and Black, Jour. Am. Chem. Soc, 1901; XLI, 349; Abel and Rowntree, Jour. Pharmacol, and Exper. Therap., 1909, I, 233: Whipple, Mason and Peightal, Bull. Johns Hopkins Hosp., 1913, XXIV, 207; Rowntree, Hurwitz and Bloomfield, Ibid., 327; Whippl e, Peightal and Clark, Ibid., 343; Whipple and Christman, Jour. Exper. Med., 1914, XX, 297; Chesney, Marshall and Rowntree, Jour. Am. Med. Assn., 1914, LXIII, 1533; Sisson, Arch. Int. Med., 1914, XIV, 804; Whipple and Speed, Jour. Exper. Med., 1915, XXI, 203; McLester and Frazier, Jour. Am. Med. Assn., 1915, LXV, 383; Kahn and Johnston, New York Med. Jour., 1915, CII, 848. Digitized by Microsoft® 128 DIAGNOSTIC METHODS V. Bacteriology of the Fkces The bacteria of the intestinal canal are of many types, many of which are purely saprophytic, while others may or may not be pathogenic.^ The number of these bacteria is usually enormous. One may determine the amount of these bacteria by the method of Strasburger, who uses the following technic: Two c.c. of feces are rubbed up in a porcelain mortar with 30 c.c. of y'2 per cent, hydrochloric acid. This mixture is then placed in centrifugal tubes and whirled for one minute. The bacteria will remain in suspension in the liquid which is poured off from the sediment. The sediment is again rubbed up with a little hydrochloric acid and again centrifuged, the liquid being added to the first portion. This procedure may be repeated until the fluid no longer becomes turbid on centrifuging. This acid solution, holding in suspension the bacteria, is then mixed with an equal portion of ordinary alcohol and placed in a beaker which is allowed to remain on a constant water-bath at 40° for 24 hours. At the end of this period a portion of the fluid may be evaporated and more alcohol added. This mixture is then placed in the centrifuge tubes and whirled for several minutes. The bacteria are now deposited and the supernatant fluid is poured off and mixed so that it may be again centrifuged. The bacterial sediment is washed with alcohol and ether in the centrifuge tube and is then placed in a weighed dish. This is dried at 100° and again weighed. In order to determine the amount of bacteria as compared with the total feces, the dry weight of 2 grams of the fresh feces is determined as previously outlined. Knowing these factors, the percentage of bacteria in the dry feces may be easily calculated. Stras- burger found that this was approxiihately one-third the weight of the dry stool and represented about 8 grams per day.^ As this dry feces is used for the determination of total nitrogen, we must bear in mind that the bacteria of the feces will represent about one-half of the total nitrogen of the feces. This fact is especially important in metabolic work. It is to be said that the bacterial flora of the intestine is so varied that very careful and long-continued work is necessary to isolate a special organism. From the standpoint of preventive medicine it should always be remembered that the feces contain large numbers of typhoid bacilli and cholera spirilla, so that measures should be taken to treat properly such ejecta as soon as voided. While the majority of the bacteria of the intestines are saprophytic, being in- troduced with the food or drinking-water, yet we find the Bacillus coU communis as a normal habitant of the intestine. This organsism is usually harmless, but may, under certain conditions, become distinctly pathogenic, many obscure cases of gall-bladder infection, for instance, being traceable to this organism. The presence of non-pathogenic organisms seems to be an essential for proper performance of intestinal function. This idea, orignally advanced by Pasteur, has been denied by Schottelius, Nuttall, and Thierfelder, although their experiments extended only over periods of 17 days. These intestinal 1 See MacNeal and Chace, Arch. Int. Med., 1913, XII, 178; also, MacNeal, Am. Jour. Med. Sc, 1913, CXLV, 801; Rahe, Jour. Infect. Dis.j^rnis, XVI, 210. 2 See Mattill and Hawk, PmtS^^jmm.gr^$QTmy , 433. THE FECES 129 bacteria not only aid digestion, but also prevent a certain amount of abnormal decomposition, owing to the fact that they inhibit the development of foreign types to large extent, especially under normal conditions. These normal bacteria may under certain conditions, however, become pathogenic, but only when the intestinal wall loses its continuity. In case the normal bacteria of the intestine become too numerous, the products formed by their activity upon protein material may be absorbed and bring about certain toxic efiects.^ It is interesting in this connection to find that the bacteria are diminished in amount in chronic constipation, according to Strasburger. The toxic symptoms must, therefore, be referable to the absorption of other than bac- terial products of decomposition, or the products have increased toxicity. For the best conditions to exist the symbiotic relations of the intestinal bac- teria should be such that there are neither too few nor too many bacteria. 22. — Cholera spirilla. [Pilfield.) The normal adult stool shows a preponderance of Gram-negative organ- isms (colon bacilli). Pathologically an increase in this type may be noted (cholera spirilla) or Gram-positive organisms may prevail. This Gram- staining variation in the bacteria of the feces is. also, largely influenced by changes in diet, bacteria of the Bacillus acidophilus and bifudus types be- coming quite prominent under a substitution of a low protein for a high protein diet." (a). The Cholera Spirillum. This organism, known as the comma bacillus, is about 2 microns long and J-2 rnicron thick. It is very actively motile and has a single delicate flagellum at one end. It stains easily with the ordinary bacterial stains and is decolorized by Gram's method. The cultural peculiarities of this spirillum will be discussed in the last section of this book. This organism is usually recognizable in the stools of Asiatic chioera, which are the characteristic " rice- water" stools. The blood serum of patients affected with cholera will show ' See Zuntz, Die Naturnissenschaften, 1913, I, 7. - See Hertcr and Kendall, Jour. Biol. Chem., 1910; VII. 203; Rettger and Horton, Cen- tralbl. f. Bakteriol., Orig., 1914, LXXIII, 362; Osborne and Mendel, Jour. Biol. Chem., 1914, XVIII, 177; Logan, Jour. Path, and BacterioL. 1014. XVIII, 527; Rettger, Jour. Exper. Med., 1915, XXI, 365; Torrey, Jour. Infect. Dis., roij, XVI, 72. ' Digitized by Microsoft® I30 DIAGNOSTIC METHODS very characteristic agglutination of cultures of these organisms. The blood is usually used in a dilution of i to 15, the reaction being observed in from 5 to 20 minutes. Closely related to this comma bacillus of Koch is the bacillus of Finkler- Prior. This latter organism may be distinguished from the spiriUum of Asiatic cholera by its morphology, the organism being larger and thicker than the comma bacillus, and by the appearance of the stab cultures on gelatin. The cholera spirillum forms a typical funnel-shaped depression, while the bacillus of Finkler-Prior shows a stocking-Uke depression. The Finkler- Prior bacillus is found in cases of cholera nostras. It may be necessary for the absolute identilication of these organisms to apply bacteriolytic tests with serum of animals immunized against a specific type of organism. ' (See Chapter XI.) Fig. 23. — Bacillus typhosus, stained to show flagella. (Ocrld a/ll-r FrankH and Pjciffcr.) (b) Typhoid Bacillus. This organism, discovered by Eberth, is so similar in morphology to numerous other organisms, especially to the Bacillus coli communis, that simple staining methods do not suffice for its detection. The characteristic stool of typhoid fever is a copious watery stool, having a strong odor and an alkaline reaction. This stool is known as the " pea-soup" stool, and may be tinged with blood and contain many pus-cells. The typhoid bacilli are medium-sized organisms with rounded ends, generall)' short, but sometimes long or thread-like and frequently showing faintly stained, sharply defined areas in their protoplasm. They are actively motile and have both polar and lateral flagella. This organism stains with the ordinary dyes and is decolorized by Gram's method. Method of Drigalski and Concradi.^ Three pounds of minced beef are mixed with 2 liters of water and allowed ' See Public Health Reports, 1912, XXVII, 371. = Ztschr. f. Hyg. u. Infektii^n^|rank]i^_i9£^,,^XXXJJ^ 2S3 of benzin in differentiating mm For a discussion of the use enians, Ztschr. f. Chemotherap., 1913, II, 28; Bierast, Centralbl. f. Bakteriol, I. Abt., Orig., 1914, LXXIV, 348; Schmitz, Miinch. med, Wchnschr., i9i4,LXI, 2115; Jaffe, Wien. klin. Wchnschr., 19 15, XXVIII, 418. THE FECES I31 to stand overnight. The beef is then pressed and the juice boiled for one hour and filtered. To the filtrate are added 20 grams of Witte's peptone, 20 grams of nutrose, and 10 grams of sodium chlorid. Boil this mixture one houriand filter. To the filtrate 60 grams of agar are added and the mixture boiled for three hours, one of which should be in the autoclave. Slightly alkalinize the mixture to litmus-paper, filter, and boil lor one-half. hour. To this hot agar solution, which should be now about 60°, add the following litmus- lactose solution. Two hundred and sixty c.c. of litmus solution are boiled for 10 minutes, after which 30 grams of chemically pure lactose are added and the mixture boiled for 1 5 minutes longer. This litmus-lactose solut on is added while boiling to the hot agar solution, the mixture being well shaken and again faintly alkalinized to litmus. Four c.c. of a hot sterile 10 per cent, solution of sodium carbonate and 20 c.c. of a freshly prepared o.i per cent, solution of crystal- violet B-. (Hochst) in warm sterile distilled water are then mixed in. This medium may be poured directly into plates or kept in flasks. It soon hardens to a firm mass and does not become dry readily. The principle upon which the use of this medium depends is that in th'e presence of both lactose and protein the colon bacillus will first attack the milk-sugar, while the typhoid bacillus will act upon the protein. In the presence of litmus the colonies of colon bacilli become distinctly red, while those of the typhoid bacillus are blue. The crystal-violet inhibits the growth of many of the other organisms, especially of the acid-producing type. If the stool be fluid, as is usually the case, one seriesof two plates is inocu- lated with the undiluted stool, another with a stool diluted with 10 volumes of sterile normal salt solution, while other dilutions, such as i to 100 and i to 1000 may also be made. If the stool is solid it is rubbed up into a homogene- ous mass with sterile salt solution and the various dilutions made as above. In making the inoculations from the stools, the material is rubbed over the surface of the medium, the plates being left open to allow the surface to dry. The dry plates are then placed in the incubator at 37" and examined at the end of 24 hours. Any contamination of the media will be killed by the crys- tal-violet. It frequently happens that certain strains of the paratyphoid bacillus develop blue colonies in this medium. It is, therefore, necessary for absolute differentiation that the agglutination test described under blood be carried out. In this way the typhoid bacillus may be absolutely identified, especially in the presence of its well-known morphological characteristics. Method of Kendall and Day. This method' permits of rapid diagnosis and is especially of importance in the examination of stools of suspected "carriers." The culture medium is a modification of that of Endo. Plain nutrient sugar-free agar is prepared as follows: 1000 c.c. of cold tap water, 15 grams of powdered agar,io grams of Witte's peptone, and 3 grams of Liebig's extract of beef are mixed and cooked in the double boiler for one hour. Just alkalinize to litmus by careful addition of sodium hydrate solution, cook for 15 minutes and filter through 1 Tour. Med. Research, iaijt,.XXV, SS- , See, also^Schmidt, Deutsch. med. Wchnschr., 1 9 1 5 , XLI, 33 ; mrschhmMimi^ilpibm(^^0ft® 132 DIAGNOSTIC METHODS absorbent cotton. This filtrate is then stored in flasks containing known amounts (100 c.c.) and is sterilized in the autoclave. When it is desired to use this media, add i gram of chemically pure lactose to each 100 c.c. and heat in the sterilizer until the media is melted and the lactose thoroughly distri- buted. Now prepare a mixture of i c.c. of a filtered 10 per cent, alcoholic solution of fuchsinandio c.c. of a fresh 10 per cent, aqueous solution of sodium sulphite. Add i.c.c of this sterilized and decolorized fuchsin mixture to each 100 c.c. of the liquid lactose media and mix thoroughly. Plates are poured and allowed to harden (with the covers removed) in the incubator for 30 minutes, when they are ready for inoculation. A small portion of feces is emulsified in 10 c.c. of sugar-free^ bouillon and incubated for i hour at 37°C. This supension is rubbed gently but firmly over the surface of the agar plates by means of sterile hooked glass rods and incubated for 18 hours at 37°C. The translucent, colorless, "dew- drop" colonies are removed to small tubes, which contain i c.c. of sugar-free bouillon and have been keptat 37°C. prior to use.^ Incubate for 2 hours and make agglutination tests, using a known serum of high agglutinating power (see blood). (c) The Bacillus of Dysentery. Bacillary dysentery is distinctly different from the amebic type of dysen- tery. The bacillus dysenteriae, or Shiga's bacillus, is now generally recog- nized as the specific organism of this type of dysentery. Flexner found a similar bacillus in the dysentery of the Philippines, while Kruse has found .practically the same type in Germany. In the United States Flexner and Harris find an organism which answers the description of the ordinary Shiga bacillus. The variation between the different organisms described is very slight, so that one may generally consider them as varieties of the same species, although certain cultural and agglutinating differences are observed. The Shiga bacillus is a short rod with rounded ends, much resembling the typhoid bacillus but much plumper. It does not seem to have very active motility as far as progression is concerned, although it does show a high degree of molecular motility. It stains with the usual dyes and is decolorized by Gram's method. The only sure method of identification of the various types of the dysentery bacillus seems to be the agglutination test. The fecal material is best obtained by curettage of the rectum, as these bacilli seem to be present in the mucus and are thus more easily concentrated. The cultural peculiarities of this organism will be found in a later section." (d) The Tubercle Bacillus. The examination of the stools for tubercle bacilli is not always satis- factory. The enormous number of bacteria of the feces may prevent a recog- nition of the tubercle bacillus even though it is present. In the feces we find' 1 This sugar-free broth is specially prepared to remove the muscle-sugar. Meat extract is inoculated with colon bacilli and incubated for 16 hours. Biol and strain through cloth. The filtrate is then made up as ordina.ry bouillon. 2 Or the colonies may be transferred to Russell's medium (litmus-lactose-glucose agar) as recommended by Lumsden (^^/g^^lfo^^fi^t^OiS^©^^^"' '^^^- THE FECES 133 certain organisms which are acid-fast, such as the timothy bacillus, which is very closely related in morphology and staining characteristics to the tubercle baciUus. We should be on our guard, therefore, lest we make a wrong diagnosis. If these organisms are present on repeated examination and there are clinical symptoms pointing to such a trouble of the bowel, one may give a presumptive diagnosis of tuberculosis, remembering that the tubercle bacilli may have come from swallowed tubercular sputum. ^ In selecting the material for examination, pick out the particles of mucus, especially those which are blood-stained or purulent. One may be much more certain of definite results if the antiformin or PetrofE method be used upon the feces, which has been rubbed up with distilled water (see p. i8). The staining methods are as usual. The Bacillus aerogenes capsulatus (Bacterium Welchii; gas bacillus) is fast coming into prominence as an etiologic factor in many cases of intestinal disturbance associated with diarrhea, although it is possibly a normal habi- tant of the intestinal tract of children.^ VI. Parasitology of the Feces In the examination of the feces for parasites one should obtain the feces as fresh as possible. This is especially the case where the examination for protozoa such as the ameba is to be made. The feces should be kept in a warm vessel prior to the examination, as the motility of these unicellular organisms is shown only with great difficulty after they have become cold. The other types of protozoa are less sensitive to changes in temperature and show their active motility, providing the feces be examined soon after voiding. It sfeems to be generally accepted that these organisms are the more easily found the more fluid, more mucoid, and more alkaline the feces. The par- ticles for examination should be preferably the masses of mucus which can be found by careful search in the liquid stool. The organisms are best examined in their fresh condition, as the staining agents usually require fixation of the specimen and consequent death of the parasite.^ In the examination of the feces for parasitic ova the following method of Yaoita (Deutsch. med. Wchnschr., 1912, XXXVIII, 1540) is, in my opinion, the best. From several different parts of the fecal mass take portions the size of a pea. Place in a test-tube and add 5 c.c. of 25 per cent, antiformin solution. Mix thoroughly and warm but do not boil. Allow to cool, add 5 c.c. of ether and shake well. Filter the mixture through one layer of gauze and cetrifuge the filtrate for one minute. Four layers are formed, in the lower of which are the ova along with the resistant cellulose, connective tissue 1 See Laird, Kite and Stewart, Jour. Med. Research, 1913, XXIX, 31;' Bergstrand, Hygiea, 1915, LXXVII, 97; Petroff, Jour. Exper. Med., 1915, XXI, 38. ^ See Kendall and Smith, Boston Med. and Surg. Jour., 1911, CLXIV, 306; Kendall. Ibid., 1912, CLXV, 75; Orton, Jour. Med. Research, 1913, XXIX, 287; Knox and Ford, Bull, Johns Hopkins Hosp., 1915, XXVI, 27; Morse, Am. Jour. Med. Sc, 1915, CXLIX, 17. ' Ross (Further Researches into Induced Cell-reproduction and Cancer, Philadelphia, 191 1 and 1912) has advocated the method of embedding the suspected feces in agar-jelly which may be impregnated with the stain. See, also. Smithies, Arch. Int. Med., 191 2, I^, 736. Digitized by Microsoft® 134 DiAGNOSTIC METHODS and muscle fibers and salts. Pour off the supernatant fluid and treat the sedi- ment with dilute HCl and a little ether. Shake well and centrifuge again. This latter process is repeated two or three times. The ova are thus freed from most of the other fecal material and may be detected readily by micro- scopic examination of the sediment.' (i) Protozoa. The protozoa are unicellular animal organisms. These, although living occasionally symbiotically, are more usually found as isolated single organ- isms. A few of these organisms are sufficiently large to be detected by the naked eye, but the majority are minute and require the finer microscopic detection. They consist essentially of a mass of protoplasm (cytoplasm or sarcode), with differentation for functional purposes (organelles) of a variable character, constancy, and prominence. In the ameba, for instance, the sarcode may be separable into an internal distinctly granular portion known as the endosarc, and a peripheral clearer portion known as the ectosarc; a cell-membrane in some instances is a well-marked feature, while in others it is absent; and in some of the free-Uving protozoa special external coverings of chitinous, siliceous, or chalky composition enclose the protozoon. Of the various parts the nucleus is, after the cytoplasm, the most constant, varying much in appearance, shape, size and number in the individual form (single nucleus of variable size and shape; double or dimorphic nucleus, a macronu- cleus of vegetal character, a micronucleus with- creative function; polymor- phous nucleus, multiple nuclear granules more or less widely distributed in the cytoplasm). Not uncommon examples of specialization are met in the contractile vacuoles, in pigment spots, in mouth-like ingestion foci and their pits on the surface of many forms with relatively firm cell membrane, in the anus-like excretory points of the same form, or the peripheral motor organelles, in the sucking tubes of the suctoria, and in the hook-like fixation apparatus of the gregarines. Motile protozoa move in a variety of ways. The naked rhizopods move by a peculiar rolling due to currents in the internal substance of the cell or by the protrusion of the cell-substance as extensions or pseudopoda, these move- ments being always accompanied by change in the cellular shape of the animal. Ciliates and flagellates move through the activity of the special cuticular appendages known as cilife and flagella. (a) Rhizopoda (Sarcodina). Amoebina. (a) Amoeba coli (endamoeba histolytica). The ameba was first discovered in the large intestine by Lambl, although we are indebted to Losch for the first accurate description of this organism. He did not believe it to be the cause of dysentery, but regarded it as a sec- ondary invader. Since that time much work has been done, a controversy hav- ing arisen regarding its specificity. In a great many cases of dysentery this organism is not found, but in its stead bacteria, especially the Shiga bacillus, 1 See Fauntleroy and ^^^f^aj^e^ ^y^Sfi^M^- "^^^' ^^' ^^- THE FECES 135 are present in large numbers. This bacillary dysentery is something entirely different from the amebic type of this disease. It is ty ue that in certain cases of dysentery as well as in normal individuals, amebae are found which are differentiated with great difficulty from the true amaeba coli. There seem to be two distinct types : one pathogenic, to which Councilman and Lafleur give the name amoeba dysenteriae, which Losch styles amoeba coli, and which Schaudinn designates endamoeba histolytica; a second type, which is non- pathogenic, has been styled by the first writers amoeba coli Losch and by Schaudinn endamoeba coli. Viereck^ has described a third form, which is pathogenic and is known as the endamoeba tetragena. This form is now be- lieved to be identical with the endamoeba histolytica. These forms have not been cultivated. The cultivated forms are non-pathogenic to man.^ Fig. 24. — Amceba coli. [Hemmeter.) One may sum up the points relative to the pathogenicity of the endomoeba histolytica as follows: (i) It appears in a form of dysentery which is anat- omically characterized by peculiar ulcerations which are markedly different from the diphtheritic inflammatory processes of the bacillary dysentery. (2) The more recent the case the more numerous are these parasites; (3) they are deposited in the dysenteric ulcers and tend to pass into the deeper tissue appearing as true tissue parasites; (4) they frequently deposit themselves in the liver causing abscesses which contain these organisms in large numbers and practically no other infectious material; (5) by injection of amebae- ' Beiheft z. Arch. f. Schiffs. -u. Tropenhyg., 1907, I, i. =" See Craig, Arch. Int. Med., 1911, VII, 362; Am. Jour. Med. Sc, 1912, CXLV, 83; Jour. Med. Research, 1912, XXVI, i; War Dept., Office of Surgeon General, Bull. 2, 1913, 95; Jour. Am. Med. Assn., I9I3,LX, 1353; South. Med. Jour., 1913, VI, 370; Jour. Infect. Dis., 1913, XIII, 30; Whitmore, Arch. Int. Med., 1912, IX, 515; Darling, Arch. Int. Med., 1913, XI, I and 49s; Jour. Am. Med. Assn., 1913, LX, 1220; James, New York Med. Jour., 1913, XCVIII, 702; Couret and Walker, Jour. Exper. Med., 1913, XVIII, 252; Williams and Calkins, Jour. Med. Research, 1913, XXIX, 43; Giffin, Jour. Am. Med. Assn., 1913, LXI, 67s; Walker and Sellards, Editorial, Jour. Am. Med. Assn., 1914, 300; James, Ann. Trop. Med. and Parasitol., 1914, VIII, 133; Craig, Arch. Int. Med., 1914, XIII, 737 and 917; Sellards and Baetjer, Am. Jour. Trop. Dis. and Prev. Med., 1914, II, 231; Bull. Johns Hop- kins Hosp., 191S, XXVI, 4S£fe,Yfz^^6y^crosc^(§'^' ^^'^ ^^^^' ^^^'' ^^^^' "' '^^°' 136 DIAGNOSTIC METHODS containing feces into the large intestine of animals typical amebic dysentery- may be caused. It is to be borne in mind that symbiosis with bacteria is apparently necessary for the development of amoebae. The endamoeba histolytica is an actively motile, roundish, pear-shaped, oval, or irregular unicellular organism having an endosarc which is typically granular and may contain lebcocytes, red blood-cells, bacteria, particles of food, or pigments which the parasite has ingested, and shows the clear hyaUn ectosarc, which is, perhaps, best seen in the pseudopoda. The pseudopoda are the typical motile portions of the parasites, these projections being thrown out from any point of the periphery, the protoplasm seeming to flow into them and drawing the animal after it. The parasite may not move, but 1 4 Fig. 25. — Coccidium hominis, from intestine of rabbit: 1, A degenerate epithelial cell containing two coccidia; 2, free coccidium from intestinal contents; 3, coccidium with four spores and residual substance; 4, an isolated spore; 5, spore showing the two falciform bodies — X 1140. {Tyson after RailUet.) will change its external appearance by throwing out these pseudopoda in various directions. The nucleus is a homogeneous, little ref ractile, chromatin- poor, spherical mass, about 6 microns in diameter (the diameter of the organ- ism itself ranging between 10 and 50 microns, the average being 35). This nucleus is not always clearly visible, appearing much more frequently in the animal killed by corrosive sublimate. In the granular endosarc one fre- quently sees several vacuoles which may or may not pulsate, the general opinion being that pulsation is absent, although change in shape is frequent.^ The stools of this amebic dysentery are thin and watery, show an alkaline ^ If one wishes to stain such preparations, the method of Darling (Jour. Am. Med. Assn., 1912, LIX, 292, and Science, 1913, XXXVII, 58) is reliable. Stain with Wright's stain in the usual way and follow this with Giemsa's stain until the film has a purple cast. Then plunge this into 60 per cent, alcohol to which lo to 20 drops of aqua ammoniae have been added. Differentiate in this way until the film has a violet color. Dry and examine. See, also, Craig, War Dept., Office of Surgeon General, Bull. ±, 1913, 11. Digitized by Microsoft® THE FECES 137 reaction, and have a peculiar lime-like odor. Much mucus, blood, and occa- sionally many pus-ceUs are found for which reason the mucus should be selected for examination in case these organisms are suspected. "^ Frequently one may obtain better specimens for examination by examining mucus which is obtained with a rectal tube. (jS) Endamoeba coli (amoeba cell Losch). This parasite varies in size between 10 and 15 microns. The hyahne pro- toplasm of the pseudopoda is not distinctly differentiated from the ectoplasm. It is opaque, gray in color, and its nucleus is sharply defined, being character- ized by its richness in chromatin. The movements of this organism are not as rapid as those of the endamoeba histolytica, which does not show as active phagocytic power. According to Craig, about 65 per cent, of normal persons show these non-pathogenic endamoeba coU in the feces. (6) Sporozoa. Coccidium hominis (coccidium perforans; cystospeimium hominis). This organism appears in the feces as an oval or spherical parasite about 22 microns long and showing a thin periphery. A large number of nuclei are usually observed. The infection with these organisms seems to arise from rabbits in whose intestines these parasites develop in large numbers, (c) Flagellates. (a) Trichomonas intestinalis. This organism was first studied by Marchand and Zunker and later elaborated by Grassi, Roos, and Janowski. It is proably identical with the one known as trichomonas vaginalis which may live in the vagina, the urethra, large and small intestine, the stomach, and may be found in the sputum. Various forms have been described as being found in the intestine, but they are in all probability the same organism; among these we find protoryxo- myces coprinarius, monocercomonas hominis, cimanomonas hominis, trichomonas hominis, cercomonas coli hominis, and cercomonas seu Bodo urinarius. This is a colorless protozoon of a pyriform or spindle shape, rounded in front and bearing three flagella which are apt to be merged at the base and easily lost, the posterior end pointed but not bearing a flagellum. It is from 20 to 25 microns in length and 8 to 12 broad. Along the body, starting from the pj^ 26.— base of the flagella, runs an undulating membrane in a some- Trichomonas what spiral manner to the posterior end. It has a finely (Tyson.) granular cytoplasm and at its anterior end a vesicular nucleus, behind which one or more non-pulsating vacuoles may be seen. At times this organism may be observed to assume an ameboid form, the movements of the flagella having then ceased and projections-resembling pseudopoda being observed". ' Musgrave and Clegg advise the administration of a saline cathartic and the later ex- amination of the fluid portion of the stool. 2 See Castellani, Jour. TrQjgiMzAcfi&^Hfer090#®VII, 65; Lynch, New York Med. Jour., iQis, CI, 886. 138 DIAGNOSTIC METHODS (/3) Cercomonas hominis. This organism was first studied by Davine and has been found by many other workers. It is known under the names of cercomonas intestinalis , monocercomonas hominis, cinanomonas hominis. The adult organism is a small, colorless, pyriform parasite, with round anterior end provided with one long flagellum and a pointed posterior end. It is 8 to lo microns long and has no undulatory membrane, as has the trichomonas intestinalis. ^^.,5^ V./«6^ (7) Megastoma entericum. This organism was first found by Lambl in the feces of children. It is known under Fig. 27. — Cercomonas hominis; several names among which are Lamblia in- fiysmT ^"'^ ^' '™^^^" varieties, testinalis, hexamitus duodenalis, dimorphous muris, and megastoma intestinale. This para- site is a colorless pear-shaped protozoon with a rounded anterior end and a pointed posterior end bearing a pair of flagella. The anterior end has one side concave with a raised border or lip, one pair of flagella, arising at the anterior border of this disk-like concavity, and two pairs together from its posterior margin. The cytoplasm is finely granular and the dumb-bell shaped nucleus is anteriorly about the middle level of the concavity. Vacuoles are absent and solid inclusions are never observed. The length of these organ- isms is from 15 to 16.5 microns, while the width is from 10 to 12.5 microns. The number of these organisms found in the feces may be very large. The surest points for their diagnosis seem to be the concavity and the dumb-bell shaped nucleus. The source of infection for man is the drinking of infected water. {d) Infusoria. Balantidium coli (paramoecium coli) . This parasite is colorless, ovoid in shape, 70 to 100 microns long and 50 to 70 microns broad, having a finely granular cytoplasm containing frag- ments taken from the intestinal ma- terial, and a clear ectoplasm showing numerous longitudinal striations. It is covered completely with actively ^^^tes'td three epithelial cells with -Megastoma entericum, showing and lateral views in larger ,- o , three epithelial cells with at- motlle Cilia which are more dense t ached examples to the right. {Tyson) about the funnel-shaped mouth which extends about one-fourth the length of the body. The nucleus is kid- ney shaped and is usually accompanied by one or more accessory nuclei, while two or more contractile vacuoles are seen which pulsate to a slight extent. This parasite has been found in connection with various types of diarrheal affection and also in persons entirely free from intestinal symptoms. It may be present in the stools in very large numbers, being found especially in the colon, but in severe cases also in the jejunum. The hog is the most common Digitized by Microsoft® THE FECES 139 host of this parasite, which at times shows marked pathogenicity (see Bowman'). (2) Entozoa (Enthelmintha). {A) Platodes (FlatWorms). (a) Cestodes (Tape-worms). The cestodes are naked, flat worms of elongated ribbon shape, endopara- sitic, at least in their adult stage, and in many instances in all stages, without a digestive canal, and always more or less distinctly divided into segments. The entire parasite, or strobile, may be looked upon as a colony of individuals united in ribbon fashion from their mode of origin, for convenience in their development and functional performance; the various segments being derived by a process of constriction from the originally acquired parasite, which is spo- ken of as the head, nurse, or scolex of the strobile. A characteristic of the ces- todes is the differentiation of two developmental stages: the first, cysiicercus stage, in which the connective tissue or parenchymatous organs are invaded, and, secondly, the development of the sexually ' t mature animal in the intestine. The scolex ob- tains entrance as a larva into the intestinal tract of the host, becomes attached by a special fixation ap- paratus to the mucous membrane, and there de- velops into the adult parasite, forming the anterior extremity of the strobile in the developed worm. The head is usually a very small and inconspicuous object of globular, pyriform or club shape with a short posterior extension, spoken of as the neck. In the middle of the frontal face of the head there is pj^ j. Balantidiumcoli: often a small prominence, known as the rostellum, a, Nucleus'; -b, vacuoles; 1 . 1 • i_ r J ■ t, cytostome, with pit and about which may be arranged m one or more rows, peristome; d, ingested as one of the means of fixation of the parasite, small material. {Tyson after booklets as a crown. As more constant rneans of fixation the head is provided with two or four suckers, rounded or linear depressions with more or less definite lips. Back of the head by a process of constriction from the neck, the segments, also known as links or proglottids, arise, the newest form always being placed between the neck and the next older link. Thus the older segments are always separated more and more from the head by each newly formed proglottid, each as it grows older and recedes further from the neck developing in size; the length of the strobile being thus dependent upon the two factors, growth of the individual length and the new formation of segments. These new segments as they are first formed are usually very short and proportionately broader, but as they in- crease in size with age they generally enlarge especially in their long diameter, and come to be more or less square. The number of these links may vary from three or four to several thousands, the length of some worms being ido or more feet. The structure of each link and hence of the whole strobile includes ' Jour. Am. Med. Assn., 1911, LVII, 1814. See, also, Hinkelmann (New York Med. Jour., 1915, CI, 200), who finds these parasites present in water supplies. Digitized by Microsoft® I40 DIAGNOSTIC METHODS an interior or matrix of an indeterminate connective reticular material, from which the various organs appear to develop and in which they are imbedded; over which are to be recognized exteriorly a delicate cuticle and beneath the latter, two layers of so-called muscle, the outer layer longitudinal and the inner transverse and circular. ^ Aside from the common parts the various links may be looked upon as in- dividuals. There is no digestive canal, all nutrition being obtained by the parasite from absorption of dissolved material from the fluids in the infested intestine. The only really highly organized parts are the generative organs, each link containing both male and female organs. The terminal links are the ones containing the most ova, while the links nearest the head are usually but partially developed. In their development the ova fiU the canal of the oviduct more and more often causing the appearance of side pockets of more or less branching character. The terminal ripe links either actually contain- ing the ova or, after discharge of more or less of the original number, are apt to •become separated from the strobile and be carried with the fecal matter from the intestine. Either with or without intermediate development the embryo is in some way, by water or in solids, carried into the alimentary canal of a second host. Arrived in this situation, both by its own activity and by passive convection by blood and lymph streams, the embryo penetrates the intestinal waU and becomes deposited in one ror other situation as a larva, bladder-worm, or / cysticercus. This larval or cysticercus form is surrounded by a dehcate connective tissue outer wall, derived from the host by a process of reac- tive inflammation, within which lies the true bladder-worm. This is essentially the head of ^ a^^ead and neck, and ^^^. ^!^^"^^ parasite. This cysticercus, after re- ovum X300, of t£enia solium, maining a variable period in the tissues, is de- ^^r''iTysoZj?ri:L.)'' -^-^^ -^^h the aesh of its host by a third (definitive) host. (a) Taeniidse. (i) Taenia solium. This parasite, which is derived from infection through the cysticercus cellulosas of pork, has been also named icmia cucurbitina, taenia dentata, cystotoenia solium, and pork tape-worm. The average length of the strobile is 2 to 3 meters, occasionally reaching twice this measurement. The head is somewhat spherical or slightly tetragonal from the four rather prominent cup-like suckers with thick lips. . The head varies from }i to i mm. in di- ameter, while the suckers range from 34 to ^^ mm. in diameter. It is pro- vided with a short, thick, rostellum bearing a double crown of booklets, usu- ally 28 in number. The neck is thin, about 3 cm. in length and is unsegmented. The proglottids number between 800 and 900, the fully grown and ripe seg- ments measuring from 9 to 12 mm. long and 5 to 6 mm. broad. The uterus 1 See Douthitt, III. Biologj^^^^ggpjf^lj^.^pj^^^o. 3. THE FECES 141 consists of a large, median, longitudinal trunk with from 7 to 10 coarsely den- dritic branches on each side. The ova are round or oval, the shell very thin but surrounded by an embryonic layer which is thick and shows distinct radiating lines. These eggs are usually of a brownish color a:nd may show on their interior the hooklets of the embryo. This parasite in its adult stage is practically hmited to the small intestine of man, while the larval form has been found in swine, monkeys, dogs, etc. It has been shown that careful cooking and prolonged and thorough salting and drying of the meat will destroy the vitality of the cysticercus cellulosae. It is, therefore, plain that any meat should be more or less thoroughly cooked before being eaten. (2) Taenia saginata. This parasite is the heef tape-worm and is also known as the tcmia medio- canellata, tania inermis, and tania dentata. The adult worm varies from 3 to 8 meters in length, has a head from i to 2 mm. in diameter, tetragonal in shape without hooklets or rostellum, with four cup-shaped suckfers each 0.8 mm. in diameter and placed at the corner of the frontal face. The ripe seg- ments are from 18 to 20 mm. long and S to 7 mm. broad. The uterus shows a distinct median longitudinal trunk with 20 to 35 lateral single or dichoto- mously branching and slender diverticula. The eggs are spherical with a thin shell surrounded by a thick radially striated embryonic shell. These eggs are from 30 to 40 microns long by 20 to 30 microns wide. (3) Taenia cucunjerina. Infection with this parasite is relatively rare in the United States. It is found almost exclusively in children, the infection occurring through dogs and cats, the larval form of the parasites being found in the body lice and fleas. ^ This organism has other synonyms, among them being tcenia canina, tania moniliformis, tania elliptica, dipylidium caninum, and dipylidium cucumerinum. The parasite is from 15 to 35 cm. in length. The head is small, rhomboidal, with a clavate rostellum surrounded by three or four crowns of hooklets (48 to 60 in number), suckers rather large with radially marked borders, and neck very short. The segments are from 80 to 150 in number, the older ones being 8 to 11 mm. in length and i to 3 mm. in breadth Head and Fig. 32.- neckoftffini'a saginata: A, and often showing a reddish-brown color. Ta:nia retracted; B extended, -phe links frequently swell out in the cumerina. (Tyson after Gould.) 4.1. 7 ^v u -l ■ (Tyson after middle so that the parasite has an ap- Leuckart.) pearance not unlike a chair of beads. A single uterus is common to the two oviducts, consisting of a network of tubes in which the ova lie in groups, fiUing small saccules, each containing 10 or 15 1 See Lins, Wien. klin. ^'^'^■^ l^^/wB^&«^- 142 DIAGNOSTIC METHODS ova and surrounded by a reddish material which gives the color to the worm. The ova are spherical, 43 to 50 microns in diameter, and have a double wall. Within the wall one observes an embryo armed with booklets. (4). Taenia nana. This worm is known as the " dwarf tape-worm" and is perhaps best known in Italy and Southern Europe, although it has been found in many cases in the Fig. 33. — Taenia nana:Xio. {Tyson after Gould.) eastern and southern portions of the United States. It has been called the tcenia agyptica, hymenolepis nana, hymenolepis murina, a,nd diplacanthus nana. The infection with the ova is probably through the use of unfiltered water tainted with human or murine dejecta. It is most frequently seen in children, and inhabits the ileum, usually from the middle toward the ileocecal valve. The parasite is from 10 to 15 mm. in length and from 0.5 to 0.7 mm. broad, is provided with a subglobular head measuring 0.2 to 0.3 mm. in transverse diameter, shows four large rounded suckers and a large rostellum retractile into an infundibulum. The rostellum is sur- rounded by a single row of characteristic booklets, 24 to 30 in number and 14 to 18 microns in length. The neck is rather long and slender, being followed by about 150 small proglottids, which are broader than long (0.4 to 0.9 mm. broad by 0.14 to 0.3 long). The ova are characteristic. They are round or oval in shape, 32 to 36 by 42 to 56 microns in size, and have two distinct membranes. At each pole of the inner membrane is seen a small protuberance from which springs Fig. 34.— Head a number of clear refractile threads, and neck of taenia , . , i- , -i , 1 . . diminuta. ^ {Tyson which are distributed m a waving „f t^nia diminuta. after Braun.) fashion through the substance inter- {Tyson after Braun.f mediate to the outer and inner walls. (5). Taenia diminuta. Synonyms. — Hymenolepis diminuta; hymenolepis flavopunctata; tcmia leptocephala; tcenia flavopunctata; tcenia minima; and tcenia varerina. The parasite is 10 to 60 mm. long, head small, globular, with four globose suckers situated close to apex, rostellum small, pyriform, and devoid of booklets. The segments number 800 to 1300 and are broader than long. Gravid uterus nearly fills the segments, showing as transverse line when not ripe. Digitized by Microsoft® THE PECES 143 The ova are round or slightly oval, yellowish in color, double-walled, inner wall showing slight protuberances at poles, a layer" of albuminous material being seen between the walls. (6). Taenia echinococcus. This parasite in its adult stage is met with in the upper part of the small intestine of dogs, wolves, and jackals. The larval form, known as the hydatid cyst, is found in man, although more frequently in the ox, hog, horse, dog, cat, rabbit, etc. This disease is rare in America, and is acquired through association with the dog more frequently than in other ways. The most common seat of hydatid disease is in the liver. * Fig. 36. — Taenia echinococcus: a, Adult; b, head from echinococ- cus cyst. On left a detached hook- let, as seen in fluid from cyst. ( Ty- son after Coplin and Bevan.) Fig. 37. — Hydatid cyst, showing daughter cysts. In the lower part of field is a whitish mass containing parts of the walls of ruptured daughter cysts. The thick wall of the mother cyst is well shown. From liver of man, X %. {Coplin.) The parasite is 2 to 5 mm. long, has a small subglobular head measuring 0.3 mm. in transverse diameter and bearing a rostellum with a double row of very characteristic booklets (28 to 50 in number) and four prominent cup- shaped suckers. The neck is short and rather thick; proglottids three or four in number, the last of which is usually longer than the rest of the worm put together. Uterus consists of a thick longitudinal median trunk wiUi a few short lateral branches. Ova spheroidal with thin radially striated shells and containing a granular hexacanthus embryo. Length of ova 30 to 60 microns, transverse diameter 25 to 30 microns. 1 See Phillips (Jour. Am. Med. Assn., 1913, LXI, 1981), who reports such a cyst in the pancreas. Digitized by Microsoft® 144 DIAGNOSTIC METHODS (b). Bothriocephaloidea. (i). Bothriocephalus latus. Synonyms. — Dibothriocephalus latus; tania lata; dibothrium latum; bothriocephalus latissimus; fish tape-worm. This parasite is most commonly met in the human intestine, but may be found in dogs and cats. It is most common in central Europe and in the maritime countries of Europe, British Islands, and Japan. The examples found in America occur in foreigners, as a rule. The ova, which are usually' in large numbers in the feces, re- quire for their future development immersion in water. The liberated embryo is then taken up by fresh- water fish and conveyed to man. Strobile 2 to 10 meters (20 in a few cases) in length, marked in ripe segments by brownish central rosette (uterus with ova). Head elongated, almond shaped, 2 to 5 , mm. long and 0.7 mm. trans- versely, with two lateral grooves or bothridia as suckers. Neck variable according to degree of con- traction. The segments number 3,000 to 4,000 and begin about 50 cm. from the head.' The anterior links are poorly- defined, in their growth increasing slowly in length but markedly in breadth. The TT „ o T) . u ■ i^ips liiiks measure 2 to 4 mm. in ^ ^., , Fig. 38.— Bothrio- ^ , , . . ,, Fig. 39.— Diboth- cephalus latus., (Tyson length and 10 to 12 m Width, rioceplialus cordatus: after Leuckart.) ^^f^ opaque brownish rosettes in ^^"1'- (Tyson after .1 • 1 11 1. TT - , . Leuckart.y the middle line. Uterus formed of a number of plicated tubes in the form of a rosette. The ova are brownish in color, ellipsoidal in shape, 68 to 71 microns m length and 44 to 45 in transverse diameter, have a thin shell, and a lid which may be opened or closed. The contents of the ova are coarsely granular or mulberry-like. Infection with this worm is not always single, as high as 100 worms hav- ing been reported in the same individual. Many cases of infection with this parasite are associated with a high-grade anemia, which is distuiguishable from pernicious anemia only by the effects of removal of the parasite. (2) Dibothriocephalus cordatus. This is a tape-worm of the sanie genus as the above and is parasitic in seals, being transmitted from them to man. It varies in length from 80 to IIS cm. Proglottids about 600 7 to. 8 mm. broad. The head is heart- Digitized by Microsoft® THE FECES 145 shaped, 2 mm. long and broad. The ova are similar to those of the latus, but are a little larger in size, (3) Bothriocephalus sp. Ijima et Kuri- moto. Synonyms. — Diplogonoporus grandis; Krab- ^ fj / bea grandis. Strobile measures up to 10 meters in length. Proglottids short and broad, head, neck, and number of segments unknown. Uterus rosette- shaped with several loops on each side. Ova brownish, operculated, oval, 63 microns in length and 48 to 50 in width. Intermediate host un- known, probably fish. (i8) Trematodes (Fluke-worms). The various forms of distoma, which belong to this class, are more properly hepatic parasites, although they and their ova may at times appear in the intestines and feces. A detailed discus- sion of these parasites will be taken up in the chapter on Parasites. (B) Nematodes (Round Worms). The nematode worms are unsegmented, elongate, circular or nearly so in their transverse section, cylindrical or more or less delicately fusi- form and tapering toward each end. They are with but few exceptions parasitic and include many important examples, which are parasitic in man. Intermediate hosts, so essential for the in- termediate development of the flukes and tape- worms, are practically absent in the nematodes. (a) Ascaridse. (i) Ascaris lumbricoides. This is the common round worm or maw- worm seen so frequently in children. The num- ber in a single host is usually small, but may be very large. Its habitat is the small intestine, Fig. 40-— -Ascaris lumbri- , , . ,, . „ coides: to left, male m lateral but the eggs may occur in the vomitus as well as aspect; to right, female, ventral in the feces aspect, natural size. (Tyson The male worm is whitish to reddish-yellow in color; 15 to 17 cm. long, 3 to 3.5 mm. thick; elongate, fusiform; cuticle finely ringed; oral orifice terminal with three lips (one dorsal and the other two meeting in the median ventral Une), each with fine denticulations on margins; '" Digitized by Microsoft® 146 DIAGNOSTIC METHODS at base of superior lip two papillae, one only at base of other two lips; pos- terior end terminating conically, curved ventrally, with two slightly curved, short, equal spicules projecting from the subventral cloaca; 70 to 75 papillae on the ventral face of posterior end, of which seven pairs are postanal. The female parasite is 20 to 25 cm. long, 5 to 5.5 mm. thick; anterior end and general appearance as in male; posterior end tapering, ending in a conical, pointed, straight tail, vulva at level of first third "of body length (in a slightly depressed annular band) ; anus subterminal. The ova are elliptical in shape, 50 to 75 microns long and 40 to 58 mi- crons broad; shell thick and transparent; stained yellowish with fecal ma- terial when in feces, but colorless in uterus; protoplasm unsegmented and coarsely granular; covered with a mammilaled albuminous envelope which may be lost.'- Fig. 41. — Ascaris mystax. A, Male; B, female; C, anterior extremity, enlarged and shown from dorsum to eijchibit the lateral wing-like cuticular expansions; D, same showing in profile. (Tyson after Railliet.) (2) Ascaris mystax. Synonyms. — Ascaris cams; ascaris lumhricus canis; ascaris teres; ascaris caniculcB; ascaris cati; ascaris tricuspidata; ascaris felis; ascaris werneri; ascaris marginata; ascaris alata; and fusaria mystax. The male parasite is whitish or slightly brownish; 40 to 60 mm. long, i mm. thick; anterior end usually curved, with lateral cuticular expansions making the end look somewhat arrow-like; mouth terminal with three nearly equal lips with denticulate margins; at base of superior lip two papilte, on inferior hp one ordinary and two minute papillae; posterior end curled and with lateral cuticular alar expansions, and on each side of cloacal aperture 26 papillae, of which five are postanal. ^ See Foster, Jour. Parasitol., 1914, I, 31. Digitized by Microsoft® THE FECES 147 o. The female worm is 120 to 180 mm. long; anterior end and general appear- ance as in male; posterior end straight, terminating obtusely; vulva at an- terior fourth of body length; anus subterminal. The ova are almost spherical; 68 to 72 microns in diameter; shell thin with thin albuminous envel- ope showing an alveolated surface. "■ (3) Oxyxiris vermicularis. Synonyms. — Ascaris vermicularis; fusaria ver- micularis; ascaris gracorum; pin-worm; thread- worm; seat-worm. The male is whitish in color; 3 to 5 mm. long, 0.3 to 0.4 mm. thick; cuticle transversely striated and at head end showing a vesicular swelling along the dorsal and ventral median lines; lateral Unes distinct; mouth terminal, .with three retractile lips; esophagus with distinct bulb; posterior end conical, curved ventrally', with six pairs of papillae and slight cuticular expansion on each side; one spicule hooked at free end. The female is 10 mm. long, 0.6 mm. thick; anterior end and general appearance as in male; posterior end straight, extended to a long mucro- nate tail; vulva at anterior third of body length. The ova are oval, flattened on one side with a characteristic asymmetry, 50 microns long and 16 to 20 broad; shell thin; colorless. This parasite inhabits the rectum and colon, but it may travel even into the stomach. The ova are rarely found in the feces, except in the mucus or about the anus.^ {b) Angiostomidae. Strongyloides intestinalis. Fig. 42.— Oxyuris vermi- Synonyms. — Anguillula intestinalis et stercoralis; rj^ht^ male^ (con^derably leptodera intestinalis et stercoralis; pseudorhabditis enlarged); A, anus; O, stercoralis; rhabdonema strongyloides; rhabdonema ^x^^^ Braun ) ' ■ intestinalis. This organism is found in two different forms: the first dioic and free, the second parasitic, as parthogenetic females. The parasitic form lives in the upper intestinal tract of man; is 2.5 mm. long, cylindrical, with pointed tail end, cuticle smooth; mouth simple with four lips; long, slender, cylin- drical esophagus reaching one-fourth of the length of the worm; anus close 1 See Beisele, Miirich. med. Wchnschr., 191 1, LVIII, 2391; Flury, Arch. f. exper. Path, u. Pharmakol., 1913, LXVII, 294. ^ See Crowell and Hammack, Philippine Jour. Sc, 1913, VIII (B), 157; WiUets, Ibid., 1914, IX, 233; Fracker, Jour. Parasitol., 1914, I, 22; Aschoff, Berl. klin. Wchnschr., 1914, LI, 1504. Digitized by Microsoft® 148 DIAGNOSTIC METHODS to tail; vulva at posterior third, containing yellowish-green oval ova, 50 to 58 microns long and 30 to 34 microns broad. The larvae develop in the intestine and are passed in the fecal material. These larvae are at fijrst from 200 to 240 microns in length, but increase to two or three times this length. The larvffi differ essentially from the parent in having a rhabditiform esophagus. In the discharged feces at about 3o°C. these develop with one moulting of the cuticle to a free-living generation with separate sexes. Fig. 43. — Strongyloides intestinalis; on the left, a gravid female from human intestine (natural size 2.5 mm.). In the middle, a rhabditiform larva from fresh fecal matter, X120; to the right, a filariform larva from culture, X120. {Tyson after Braun.) In this free sexual generation the worms are smooth, cylindrical, and tapering, with pointed tail-ends; the mouth is the same as in the parasitic form; esophagus rhabditiform with its anterior portion long and with the pos- terior pyriform and containing a Y-shaped chitinous armatxire; anus at base of tail; male with tail curved and two spicules, body length 0.7 mm.; female I mm. long, with straight pointed tail, vulva a little back of the middle; ova few, yellowish, ellipsoid, thin-shelled, 70 by 45 microns in size, sometimes hatching in the uterus. _The larvae of this generation look much as their ine larvae 01 tnis g;,ene Digitized by Microsoft® THE FECES 1 49 free parents, are at first 0.22 mm. in length, but grow to 0.55 mm., then moult and assume a filariform or strongyloid character like that of the parasitic grandparent. These gain access to the intestine of a new host in an unknown manner or shortly die. These worms may be found throughout the upper gastrointestinal tract, especially in the duodenum and upper part of the jejunum. The time elaps- ing between infections with the filariform larvae of the sexual generation and the appearance of the rhabditiform embryos of the parasitic type in the stools is between two and three weeks. The parthogenetic female types are usually called the strongyloides intestinalis, while the free sexual form is styled strongyloides stercoralis. This form is found widely distributed in Indo-China, the East Indies, Africa, Europe, and North and South America. The mode of transmission to the second host is probably through the means of unfiltered water or of unclean, uncooked vegetables. (c) Trichotrachelidse. (i) Trichiuris trichiura. Synonyms. — Ascaris trichiura; trichocephalus trichiurus; trichocephalus hominis; trichocephalus dispar; trichocephalus mastigodes; whip-worm. The male worm is 35 to 45 mm. long; whitish; anterior three-fifths slender and thread-like; posterior two-fifths thicker, cylindrical, terminally rounded and curled; anus terminal; single spicule in a tubular sheath containing small spinules. The female parasite is 35 to 50 mm. long; shape as in male^ for front and body; posterior extremity straight, bluntly pointed terminally; vulva at beginning of thick pos- terior portion of body. The ova are very characteristic, brown, oval, thick- ^V^- 44-— 7Tri- walled, with a colorless shining button-like protuberance at natural size: a' each pole. The eggs are 50 to 54 microns long and 23 *^^'^; ^1 female, microns wide, with an unsegmented yolk. Occasionally these eggs show variations in shades of brown, some being very much lighter than others. (2) Trichinella spiralis (trichina spiralis). The male worm is 1.4 to 1.6 mm. long and 0.04 mm. thick; cylindrical in shape; anterior end tapering, posterior end gradually and slightly thicken- ing and terminating in a bifid extremity with two lateral somewhat conical tail appendages; cloacal aperture between these, which form a sort of bursa; back of cloacal aperture two pairs of papillae. The female worm is 3 to 4 mm. long; anterior end as in the male; posterior end nearly of same thickness to tail, which is rounded; anus terminal; vulva at anterior fifth of body; viviparous. The larvae when born are 90 to 100 microns in length, obtuse anteriorly, posteriorly prolonged to a pointed tail; when encysted as "muscle trichinje" the larvae measure about i mm. ,lpng, and o.qa mm. in thickness, tapering Digitized byivlicrosofm ^ ^ ISO DIAGNOSTIC METHODS anteriorly, more thick and obtuse posteriorly with complete organization as in the adult and showing the characters of the different sexes. This parasite or its larvae are rarely if ever found in the stools. In its adult sexual stage it infests the intestinal tract of man and a number of mam- FiG. 45. — Trichinella spiralis: a, Gravid female, "intestinal trichiura"; E, embryos; G, vulva; Ov, ovary; b, adult male, "intestinal trichiura"; T, testicles; C, young larva; d, larva in musculature; e, encapsulated larva in muscle. {Tyson after Braun.) malians, gives origin to a large number of larval worms, after which the adults die. , The larvae pass through the muscular wall of the intestine and are car- ried by the blood-current into various muscles of the host. Here they pass an Digitized by Microsoft® THE FECES 151 indefinite encysted stage, a capsule forming around them and becoming calcified.' Herrick and Janeway,^ Packard,' and Cross* found the embryo in the blood, the latter in blood from an ear puncture. The blood was laked with 3 per cent, acetic acid, was centrif uged, and the sediment examined as outlined by Staubli.^ The chief means of infection of man with the trichinella spiralis is through the eating of insufficiently cooked pork, especially ham. {d). Strongylidse. (i). Uncinaria duodenalis. Synonyms. — Ancylostoma duodenale; strongylus quadridentatus; dochimus ancylostomum; sclerostoma duodenale; strongylus duodenalis; dochmius duo- denalis, and European hook-worm. The male parasite is whitish or blotched posteriorly with brownish when the intestine contains blood; 8 to 10 mm. long; cuticle finely striated trans- FiG. 46. — Tail, with expanded bursa, of male uncinaria duodenalis. (Tyson) Fig. 47. — Anterior end, showing mouth parts, of uncinaria duodenalis , dorsal view. (Tyson.) versely; tapering to a blunt point anteriorly and with head curved upon the dorsum so as to give a sHghtly hooked anterior end; on each side of the median line on the ventral side of oral border two hook-Uke chitinous teeth and on dorsal border on each side of the median line one less curved chitinous tooth; with a dorsal conical tooth extending along back of oral cavity from the base of the cavity; in the oral cavity about the esophageal opening a delicate arma- ture consisting of two dorsal and two ventral lancet-like pieces; posteriorly the body ends in an abruptly pointed tail in a copulatory bursal expansion of the cuticle, this having one dorsal and two lateral lobes; in the folds of the bursa one dorsal subdivided muscular ray, each division ending tridigitally, and on each side symmetrically placed an undivided dorsolateral, a divided lateral, undivided lateroventral, subdivided ventral and undivided small subventral muscular rays; cloacal aperture superterminal; two equal spicules. • See Flury, Arch. f. exper. Path. u. Pharmakol., 1913, LXIII, 164; Gruber, Miinch. med. Wchnschr., 1914, LXI, 645; Van Cott and Lintz, Jour. Am. Med. Assn., 1914, LXII, 680. 2 Arch. Int. Med., rgog, III, 263. 'Jour. Am. Med^Assn., 1910, LIV, 1297. * Arch. Int. Med., 1910, VI, 301. 6 KoUe and Wassermann'j)|^|y^^^^jy^g;yly^^roorg., 1913, VIII, 73. t2 DIAGNOSTIC METHODS The female worm has the general appearance of the male and is shaped anteriorly like it; 12 to 18 mm. long; posteriorly tapering to a finely pointed tail; anus subterminal; vulva about the posterior third of the body length; two uterine and ovarian tubes. The ova are colorless, elliptical, thin-shelled with unsegmented or early segmenting material, 50 to 60 microns long and 30 microns broad. This worm has a wide distribution both in tropical and subtropical coun- tries. Its habitat is in the duodenum, jejunum, and upper part of the human ileum. Infection with this organism is known as uncinariasis or ancylostomia- sis} A particularly severe type of anemia is set up by this parasite both through the influence of loss of blood and the elaboration of toxic hemolytic material by the parasite. The eggs of this organism are frequently found in the stools and should be carefully searched for in every case of severe anemia. The mode of infection by these organisms may be direct ingestion of dirty water or unclean vegetables, but it is probable that the most frequent method of infection is through the skin, these larvae attaching themselves to the feet of people walking in infested sand or water. The larvae penetrate the skin and make their way through the blood- and lymph-currents to the lungs, whence they penetrate to the air-passages and are supposed to be carried upward to- ward the mouth by the bronchial mucus and are then swallowed. (2). Uncinaria americana^ (American Hook-worm). The male parasite differs from the former organisms discussed in being of smaller size (6 to 9 mm. long and more slender than the duodenalis), in the smaller size and more conical shape of the head, in having no booklets on the oral rim, but instead on each side a large ventral and smaller dorsal chitinous lip, extending from the rim toward the median line; in a greater prominence and projection into the oral cavity of the dorsal conical tooth; in the smaller size of the copulatory bursa, its dorsal lobe being subdivided and the ventral margin being extended so as to form an indefinite ventral lobe; and showing the dorsal muscular ray of the bursa divided, each division ending in a bipar- tite tip. The female worm differs from the duodenalis in being shorter and more slender (8 to 15 mm. long), with similar differences of the anterior end as above outUned for the male; vulva just in front of the middle of body length instead of at the posterior curve as in the uncinaria duodenalis. The ova of this parasite are somewhat larger (68 to 70 microns long and 38 to 40 microns broad) than those of the uncinaria duodenalis, but are other- wise similar. Stiles has found that these parasites^ are the common cause of the frequent " anemia of the South." Smith believes that uncinariasis exists in every case in which "ground-itch" has occurred within eight years and that the disease is rarely ever present in those who have not had this condition during that ^ See Bruns, Kolle and Wassermann's Handbuch, 1913, VIII, 41. ^ Also called Necator americanus. See Stitt, Jour. Am. Med. Assn., 1Q12, LIX, 1706; also Stiles, Pub. Health Reports, 1913, XXVIII, 7; also, Ashford, War Dept., Office of Surgeon General, Bull. 2, 1913, 59 and 72; FerreU, Jour. Am. Med. Assn., i9i4,LXII, 1937. 2 See Glover, Jour. Am. M^^^^y )W^VS^¥iol^> ^°^ "• discussion of uncinariasis in oriental immigrants. ^ ' THE FECES 153 period. This would point to the general transmission of infection through the skin. The uncinaria duodenalis has long been known as the cause of the so-called Egyptian chlorosis, tunnel-workers' anemia, brick-layers' anemia, and other conditions necessitating work in low-lying watery places. Pseudoparasites. It not infrequently happens that extraneous substances are found in the feces which very closely simulate a parasite or its ova in appearance. These substances are, for the most part, food residues and should be carefully differ- entiated by applying tests for cellulose, which will show in all vegetable cells. Stiebel has described, under the name of diacanthos polycephalus, a frag- ment of the woody portion of a bunch of raisins. Sultzer describes a mul- berry seed as a vesicular worm under the name of ditrackyceros rudis. Basti- FiG. 48. — Tail, with expanded bursati^ of male uncinaria ameri- cana. {Tyson.) Fig. 49. — Anterior end of uncinaria americana, showing mouth parts (dorsal view). (Tyson.) ani considers the larynx of a bird which he found in the fecal material as being a biped worm, under the name of sagiiula hominis. ScopoU regards a frag- ment of the trachea of a bird as an entozoon form under the name of physis intestinalis. A pupil of Moquin-Tandon describes a strip of lettuce under the name of striatula, regarding it as a worm intermediate between the ascaris and the oxyuris (Guiart and Grimbert).^ Perhaps the most frequent pseudoparasite of the feces is the pulp of an orange, it shows in the feces in the form of large oblong masses terminated by two slender extremities, one of which ends in a sort of parenchyma. These vesicular masses are the large cells which secrete the orange juice and which are generally found intact in the fecal material. These have been frequently mistaken for hydatids or for parasitic ova. Certain spores, such as those of the truffle or of lycopodium, have not infrequently been mistaken for para- sitic eggs. Moreover, one may find in the fecal material the pollen of the coniferous plants which very closely simulates parasitic ova. The spines which form the down on certain fruits, such as raspberries, strawberries, peaches, and quinces, so closely resemble parasites that careful study is in some cases essential. 1 Francaviglia (PoUclinico, 191S) XXII, 1052), reports the presence of blood clots simu- lating parasites. ^.^.^^^^ ^^ Microsoft® 1 54 DIAGNOSTIC METHODS lO.M aO. 30. 40. 60. 60. 70. 80. so. 100. 110. t20. IdO. 140. ISO t60. I70. ISO. 180 ZO. Fig. . SO. — Parasitic bodies, ova, and larvse met in human feces; color approximate only. (Tyson.) 1. Larval strongyloides intestinalis. 2. Ovum of fasciola hepatica. 3. Ovum of taenia nana. 4. Ovum of uncinaria duodenalis. 5. Ovum of uncinaria americana. 6. Ovum of taenia saginata. 7. Ovum'of taenia solium. 8. Ovum of opisthorchis sinensis. 9. Ovum of opisthorchis felineus. 10. Ovum of cotylogonimus heterophyes. 11. Ovum of taenia cucumerina. 12. Ovum of ascaris lumbricoides. 13. Ovum of dicrocoelium lanceatum. 14. Ovum of bothriocephalus latus. 15. Ovum of trichiuris trichiura. 16. Ovum of oxyuris vermicularis. Digitized by Microsoft® Digitized by Microsoft® PLATE VIII. Katharine HiU Vegetable Cells pound in Feces. (After Schmidt and Strasburgee.) Digitized by Microsoft® THE FECES 155 It is, therefore, wise in all cases before pronouncing a finding as one of a parasite to be perfectly sure of your ground. BIBLIOGRAPHY 1. Braun. Die tierischen Parasiten des Menschen. Wiirzburg, 1908. 2. Briancon. De L'Ankylostomiase. Lyon, 1904. 3. Cammidge. Feces of Children and Adults. New York, 1914. ■ 4. Craig. The Parasitic Amebas of Man. Philadelphia, 1911. 5. Hecht. Die Faeces des Sauglings und des Kindes. Wien, 1913. 6. Hemmeter. Diseases of the Intestines. Philadelphia, 1902. 7. Lynch. Coprologia. Buenos Ayres, i8g6. 8. MusGRAVE AND Clegg. Amebas: Their Cultivation and Etiologic Significance. Manila, 1904. 9. NoTHNAGEL. Beitrage zur Physiologie und Pathologie des Darmes. Wien, 1895. Krankheiten des Darmes. Wien, 1900. 10. Von Oefele. Technik der chemische Unterschung des menschlichen Kothes. Leipzig, 1908. 11. Schmidt und Strasburger. Die Faeces des Menschen. Berlin, 1915. 12. Van Ledden Hulsebosch. Makro- und mikroskopische Diagnostik der mensch- chen Exkremente. Berlin, 1899. Digitized by Microsoft® CHAPTER V PARASITES I. Geneeal Considerations In the previous portions of the work, the writer has introduced those parasites which are more particularly related to the patts under discussion. There are, however, a large number of organisms which do not fall naturally within the scope of any of the chapters outlined for this book. These will, therefore, be discussed in general without much regard to distinct classifica- tions, as many of the subdivisions have been treated previously. This discus- sion is taken, for the most part, from Tyson. ^ II. TSEMATODES (FlXJKE-WORMS) Flukes are naked and unsegmented flat worms, usually of the shape of a leaf or of the tongue (occasionally pyramidal or elongated and more or less cylindrical), provided with incomplete digestive canal (without anus), pos- sessing one or more suckers and occasionally booklets; with but few exceptions hermaphroditic and, as a rule, presenting a complicated series of metamor- phoses in their development. In structure it is customary to speak of the surface upon which the genital pore opens as the ventral; this surface commonly shows also the orifices of the mouth and one or more suckers. On the dorsal surface in many occurs the opening of a small canal, spoken of as Laurer's canal, of uiiknown func- tion. The surface of the body is covered by a fairly thick and firm cuticle, often provided over variable areas with small spines or tubercles. Beneath the cuticle over its internal surface is spread the superficial muscular layer (not showing the structure of muscle of higher animals, however), with longi- tudinal, circular, and diagonal fibers, while within this along the borders is met the parenchymatous muscle. The general internal tissue of the body, spoken of as the parenchyma, is a fine reticular connective tissue which closely surrounds the various organs. The suckers of trematodes vary in number and arrangement on the ante- rior and posterior extremities, the ventral surface and its borders, and in a few cases also on the dorsal surface. Usually the oral opening is surrounded by such a sucker and in addition, in the forms likely to be met with in man, on the ventral surface some distance posterior to the oral sucker is a second, known as the ventral sucker or acetabulum, in the median line. Not infrequently in the lining of these suckers, on their lips, or on the cuticle close to the hps, chitinous booklets are to be found. 1 See Ward, Illinois Med. Jour., 1912, XXII, 417; Cort, 111. Biological Monographs, 191S, I, No. 4. Digitized by Microsoft® 156 PARASITES 157 The alimentary system consists of a mouth, opening in the oral sucker and situated terminally or on the ventral surface of the anterior end of the worm. This cavity continues into a dilated tube with thick walls, the pharynx, this extending posteriorly by a short, straight, and usually narrow esophagus, which divides in the anterior portion of the body into the two intestinal tubes or ceca. At the posterior extremity of the body is a small orifice, the excre- tory pore, which serves as outlet for a series of more or less complex canals for the con- vection of the fluid waste, the arrangement representing a low nephridial apparatus, while the mouth serves as an anus. The reproductive system is highly developed, showing numerous minor variations in the different genera and species. Adult flukes are parasitic upon a wide range of animal life, including the higher animals, fish, amphibia, reptiles, and birds, living as ectoparasites and endoparasites. The varieties afiecting man are compara- tively few. The most common parts of the human body to be infested are the intes- tines, gall-ducts, respiratory tubes, and blood-vessels. (a) Fasciolidae. (i) Fasciola hepatica. Synonyms. — Distomum hepaticum; dis- tomum cainee; fasciola humana, cladoccelium hepaticum; common liver-fluke. A comparatively large fluke, measuring 20 to 50 mm. long and 8 to 13 mm. wide, of leaf shape, with anterior extremity pro- Fig. 51.— Showing the sexual glands longed into a small cone; greatest width of °LkSr?inte?tffiecifx)'<>, ^'temnl body about the anterior third of length; glands; Dr, ovary; 0», uterine canal; light brown color; cuticle provided with verl? vitemfe'duafl^t^i'mt^i p^^^^ alternating transverse rows of spines, ex- 5, ventral sucker. (Tyson after Braun.) tending on ventral surface to the posterior level of testes, but not as far posteriorly on the dorsal surface; the oral sucker at the anterior end of cephalic cone, inclining to ventral surface, I mm. in diameter; ventral sucker near anterior end behind cephalic cone, 1.6 mm. in diameter; well developed larynx and short esophagus; intestinal branches extending nearly to the posterior extremity of the worm, approach- ing the median line posteriorly with few median and numerous lateral branches; excretory pore at posterior extremity, with well-developed system of excretory tubes; genital pore in the median line anterior to the ventral sucker; two large highly branched testes, mostly posterior to the transverse Digitized by Microsoft® IS8 DIAGNOSTIC METHODS vitelline duct; ovary single, branched, lying in front of testes and to one side of the median line; the uterus coiled into a rosette, showing as a brown spot just back of the ventral sucker on the ventral surface; vitelline glands numer- ous, ranging along each lateral border from the level of the ventral sucker to the pos- terior extremity of the worm; vitelline ducts running transversely at about the end of the anterior third of the body. The ova are yellowish-brown, oval, operculated, and measure 130 to 145 microns in length and 70 to 90 microns in width. This fluke is a common one in most mammalians and has a wide geographical distribution over the world, being found not infrequently in America. Its usual habitat is the gall-ducts, but it has been seen in the gall-bladder, intestines, in the portal and other venous channels and in subcutaneous cysts. The ova appear in the feces and are the chief means of diagnosis of this condi- tion. The embryo which develops from the ovum completes its developmental cycle in the body of a snail, the Limnaea trunca- tula. From the body of the snail the cer- carise escape in the water and become at- tached to grass or other aquatic material, which is taken in by the animal. Infec- tion in man is quite rare, about 32 cases having been reported in the literature. Fig. 52.— Fasciolopsisbuski;o,oral f^) Fasciolopsis buski. sucker; b, acetabulum; c, cirrus pouch; t-.. 7 i . t- x d, vitelline glands; e, "shell gland"; SYNONYMS. — Dtstomum buskt; Mstomum f and g, posterior and anterior testicles; crassutn. h, ovary; i, cecum; %, uterus. {Tyson ~, ', , ,■ , . . . .11 after Braun.) The length of this organism is variable, ranging between 24 and 70 mm.; breadth 5.5 to 14 mm.; lance-shaped; narrowing more rapidly anteriorly than poste- riorly, maximal width about the middle of the length; no cephalic cones; brownish in color; cuticle without spines; oral sucker small and placed on ventral surface of anterior extremity; ventral sucker two or three times as large as the oral, placed near anterior end and showing a saccular distention extending posteriorly; very short esophagus, back of the fairly developed pharynx; the two ceca without branches; genital pore at the anterior quarter of the acetabulum; cirrus pouch large; testicles branched; in posterior part of body one back of the other; uterus in anterior half of body, tortuously coiled; ovary at middle of length of body, to the right of median line; Laurer's canal present; vitelline follicles numerous along lateral margin from the level of the Digitized by Microsoft® PARASITES 159 ventral sucker to the posterior extremity; the transverse vitelline ducts at the equator of body. The ova are brownish, ovoid, operculated, and measure 125 microns in length and 75 in width. Very little is known of the intermediate stage and of the host of Busk's intestinal fluke. This has been found in the small intestine of man and probably arises from eating infected food. (3) Opisthorchis felineus. Synonyms. — Distomum conus; distomum lanceo- latum (Siebold) ; distomum sibiricum; distomum tenui- colle; the European cat-fluke. Variable in size according to the state of contrac- tion, 8 to II mm. long and 1.5 to 2 mm. broad; yellow- ish-red and nearly transparent; flat and lanceolate; anterior end constricted and attenuated into a cone; posterior end more obtuse; cuticle without spines; oral sucker toward the ventral surface at anterior extremity ; ventral sucker at base of cone about one-fourth of body length; pharynx and esophagus of equal length; ceca comparatively straight and unbranched, reaching nearly to the posterior extremity and often seen filled with blood; excretory pore terminal; its tubular vesicle winding in the median line between the testes and branching in front of the anterior testes; testes in the posterior part of body, the anterior four-lobed, the other five-lobed; cirrus and pouch absent; genital pore n ■ 1, in the median line in front of the ventral sucker; chis felineus; from liver slightly lobate ovary in the median line anterior to the °{ cat; 10 x 1. (Tyson , . . . after Braun.) testes; receptaculum semims promment; uterus an- terior to the ovary and testes coiled in the middle third of the body; vitelline follicles occupy about the middle third of the body, be- ginning anteriorly at the level of the ventral sucker. The ova are oval in shape, operculated, and measure 30 microns in length and 11 in width. This worm has been found in the gall-ducts of man and other animals, especially in Russia, Siberia, Hungary, and Japan. (4) Opisthorchis sinensis. Synonyms. — Distomum sinense; distomum spathulatum; distomum hepatis endemdcum seu perniciosum; distomum hepatis innocuum; distomum japonicum; the Japanese or Chinese liver-fluke. This parasite measures 10 to 20 mm. in length and 2 to 5 mm. in breadth; long and lanceolate; reddish and nearly transparent when fresh; cuticle with- out spines; oral larger than ventral sucker, on the ventral face of anterior extremity; ventral sucker about one-fourth of body length posterior to former; pharynx and esophagus small and a bifurcation of the latter close to oral sucker; ceca unbranched, reaching close to the posterior extremity: excretory system as in the previous species; in the posterior fourth of body the two Digitized by Microsoft® i6o DIAGNOSTIC METHODS third of the body. testes, one in front of the other, with from four to six dendritic branches; no cirrus or pouch; genital pore in the median line just in front of the aceta- bulum; ovary tri-lobed and placed just anterior to a large gourd-shaped receptaculum seminis both in the median line and anterior to testes; uterus well-developed and coiled in the middle area of the body between the ovary and ventral sucker; vitelline follicles in the marginal fields along the middle The ova are brown, oval, operculated, and measure 27 to 30 microns in length and 15 to 17 in width. This fluke is comparatively common in Japan and eastern Asia and has been reported in America. It infests the gall-ducts and has been found both in the pancreatic duct and intestine. Little is known of the intermediate hosts. Many other types of these distomata are met with in the liver of various animals and are occa- sionally found in man. Among these we find the distomum lanceolatum, distomum heterophyes, distomum conjunctum, and amphistomum hominis (gastrodiscus hominis). III. Nematodes (Round Worms). Eustrongylus gigas. Synonyms. — Ascaris canis et martis; ascaris visceralis et renalis; strongylus gigas; sirongylus renalis; eustrongylus visceralis. The male worm is red in color; 14 to 40 cm. in length and 4 to 6 mm. in thickness; slightly tapering anteriorly; mouth terminal; with a hex- agonal orifice surrouiided by six lips bearing papilla; cuticle thin and transparent, finely Fig. 54.-Opisthorchis sin- striated transversely; about 150 papUls along ensis; ventral surface, stretched; the longitudinal lines laterally; caudal extremity V.T^^^r^^^'J *' "^^; ^' g«°i- with an oval plate-like expansion serving as a tal pore; a, acetabulum; . -^ ^ ° e, uterus; /, vitelline glands; bursa, its margin bearing small papillae and fnis7riatVe"stanT?T '^^- '^^^^'^^ ^!^^^"'^'^ '^"'^^"^^^ ^^'^ ^^^^"^^"^^ ^^^^^^ icles; k, excretory canal; m, sexual spicule. B™r ^°''' ^^^'"^ "■^'"' ^^^ ^^^^^^ parasite shows the general ap- pearance and head end as in the male; 20 to 100 cm. in length and 5 to 12 mm. in thickness; caudal extremity obtuse, straight, with anus subterminal; vulva 50 to 70 mm. posterior to mouth; single ovarian and uterine tube plicated from near the anterior end along the in- testine nearly to the anus, then returning to the vulva near the anterior end. The ova are brown, ellipsoid, with thick shell marked by external cribri- form depressions, 64 to 68 microns in length and 40 to 44 microns in breadth. Digitized by Microsoft® PARASITES i6i This worm, which is more common in the dog, has been reported a number of times in man. It is the largest of the nematode worms and has its habitat in the pelvis of the kidney. Little is known of its life history. IV. Parasites of the Skin Arthropoda. These are bilaterally symmetrical segmented animals whose segments do not correspond, but vary in structure, and which primitively bear upon each segment a pair of jointed appendages. The segments are often more or less fused, thus forming special body-regions which may themselves be more or less fused together as well. The covering of these animals is a com- FiG. ss- — Eustrongylus gigas: female, natural size, in kidney of dog. {Tyson after Railliet.) paratively thick and strong cuticle which remains pliable between the seg- ments of the body and of the jointed appendages, but which commonly be- comes hard and shell-like from chitinous or calcareous material directly over the different body-segments and internodes of the jointed appendages. This arrangement requires that in the growth of the individual the firm ex- ternal covering should from time to time be shed, such changes taking place periodically and being known as moults. While each segment in the primi- tive animal is provided with a pair of jointed appendages, these in the indi- vidual species are often lost from this or that part of the body, or may remain rudimentary and inconspicuous or may take on special features of structure from the assumption of special function which causes their wide departure from the original and common type used for locomotion. The arthropods 1 1 Digitized by Microsoft® l62 DIAGNOSTIC METHODS commonly reproduce by ovulation, the development of the embryo to the adult often showing more or less complicated metamorphoses. The true parasite forms of the arthropoda thus far met in man are limited to the Arachnoids and Insects. (A) Arachnoidea. (a) Sarcoptes or acarus scabiei (the Itch Parasite). This parasite is oval in shape, is provided with horns and bristles, is barely visible to the naked eye, the male being from 0.2 to 0.3 mm. in length by 0.145 to 0.19 mm. in breadth; the female is somewhat larger, showing a length of 0.33 to 0.45 mm. and a breadth of 0.25 to 0.35 mm. The female lies at the end of a burrow in the epidermis, in situations A . B Fig. 56. — Acarus scabiei: ^4, female, dorsal view; B, portion of human epidermis, show- ing burrows with contained . ova and young acarians. {Gould.) where the skin is most delicate, as between the fingers, at the elbows, under the knees, and in the groin. In this burrow, which varies from a few mUh- meters to a centimeter in length, the female deposits her eggs, after which she dies. The eggs hatch in from 4 to 8 days, and in about 14 days the larvae are sufficiently matured to make their own burrows. The disease is communicated either by the clothing or by personal con- tact. To demonstrate the parasite, the burrow is opened with a needle and the female pressed out on a slide, which is then covered and examined. (J) Demodex foUiculorum. This parasite is very small, varying in length from 0.3 to 0.4 mm. It is somewhat cylindrical, tapering to an obtuse point at the posterior end. This parasite has its habitat in the sebaceous follicles, especially of the face and nose. (c) Leptus autumnalis (Harvest-bug). . This parasite is also known as trombidium irritans or red jigger. This is a minute red parasite, from 0.3 to 0.5 mm. long, which has three pairs of legs, with rows of bristles upon its back and belly. It prevails in summer on Digitized by Microsoft® PARASITES 163 grass and plants and attaches itself to the skin of man by its hooklets. The condition of the skin due to the infection with this parasite is known as trombidiosis.i (B) Insecta. (o) Hemiptera. (i) Pediculus capitis (Head-louse). The male is from i to 1.5 mm. long, the female 1.8 to 2 mm. long. The color of the parasite varies some- what with the race of its host. In the Caucasian it is gray with a dark border, in the Negro and Chinamen it is much darker in color. The. eggs are 0.6 mm. in length and are attached to the hairs, forming the so-called "nits." These nits are whitish oval masses which are easily visible. This parasite, while usually found upon the hair of the head, may be found in other portions of the body.^ The symptoms may be severe or very slight. Fig. 57. Demodex folli- culorum: from dog, enlarged. {Tyson after Braun.) vestimenti Fig. 58. — ^Leptus autum- nalis; enlarged. {Tyson after Braun.) (2) Pediculus (Body-louse). This parasite is consider- ably larger than the former, being from 2 to 5 mm. long and whitish-gray in color, the back part of the body being wider than the thorax. The antennae are longer than are those of the head- louse. The eggs are from 0.7 to 0.9 mm. in length, about 70 being laid by each female. This parasite is found upon the clothing in which it deposits its eggs, especially about the neck, back and abdomen. (3) Pediculus pubis (phthirius inguinalis or Crab-louse). This parasite is smaller than the head-louse, grayish-yellow or grayish- white in color, the male being from 0.8 to i mm. in length, the female about 1. 1 2 mm. in length. The eggs are pear-shaped, from 0.8 to 0.9 mm. in length and from 0.4 to 0.5 mm. in breadth. This parasite infests the parts of the body covered by the shorter hairs, such as the pubis, axilla, eye-brows, and chest. ' See Olson, Jour. Am. Med. Assn., 1915, LXIV, 2060. 2 Goldberger and Anderson (Pub. Health Rep., 1912, XXVII, 297) show that typhus fever may be transmitted by this parasite as, also, by the body louse. See, also, Nicolle, Comte and Conseil, C. R. Acad. Sc.,,1909, CXLIX, 486; Ricketts and Wilder, Jour. Am. Med. Assn., 1910, LIV, 1304; Goldberger and Anderson, Pub. Health Rep., 1910, XXV, 177; Ibid., 1912, XXVII, 83s; and Jour. Am. Med. Assn., 1912, LIX, 514- Digitized by Microsoft® 164 DIAGNOSTIC METHODS (4) Cimex lectularius (acanthia lectularia or Bed-bug). While, strictly speaking, the bed-bug is not a parasite of man, yet as its habitat is the bed, bedding, and walls of the sleeping apartment of man, it may be considered as indirectly parasitic. It usually emerges at night from Fig. sg. — -Pediculus capitis, Xis- (Tyson after Braun.) Fig. 60. — Pediculus vesti- menti, Xio. (Tyson after Braun.) its lodging for the purpose of securing its nourishment in the blood of its victims. This parasite is reddish-brown in color, oval in shape, from 4 to 5 mm. in length and 3 mm. in breadth. These insects, if crushed between slides Fig. 61. — Pediculus pubis. (Tyson after Braun.) or as more usual between the hand and a part of the victim's body, have a characteristic odor very much resembling kerosene. The blood is drawn from the victim by means of a long proboscis. The eggs are approximately 1.12 mm. in length and require about 11 months for their development to the sexually ripe insect. These eggs are retained in the crevices of the bed, floors, Digitized by Microsoft® PARASITES i6s furniture, wall-paper, and other parts of the dwelling so that the complete removal of these eggs and parasites is a matter of some difficulty.^ That these insects have more or less importance from the standpoint of transmission of disease from one person to another must be remembered. Individuals vary in their susceptibility to the bite of the bed-bug, some being indifferent to it while others are markedlv affected bv it. '' -rTT — - --, ^^ ."' w^^ % \ ^ 1 ^^ f % Fig. (\2. — Pulex irritans, X14. (Tyson ajlcr Braun.) (h) Diptera. (i) Pulex irritans (Common Flea). The male is from 2 to 2.5 mm. in length, the female as much as 4 mm. It is a red or brownish-red insect, having a laterally compressed body, an oral haustellum, serrated soft mandibles, a tongue sheathed in an inferior labium, and a pair of labial four-jointed palpi. Each of the triple segments of the thorax bears a pair of five-jointed double-clawed legs. The female deposits her eggs, not on the human being, fortunately, but in the fissures, crevices, or holes of garments or furniture which may be accessible. (2) Pulex penetrans (Sand-flea or Jigger). This parasite is a minute, brownish-red, egg-shaped insect which pene- trates the skin of man. The female is the infecting insect and produces pain- ful irritation and even suppuration. Vegetable Parasites. ( I ) Achorion Schonleinii. This organism is the cause of the disease known as favus or tinea favosa. This fungus invades the root sheaths, the bulbs, and the shafts of the hair filaments of the scalp, but it also occurs upon the "non-hairy" portions of the skin and upon the nails. ^ The spores gain access to the deeper layers of the skin and develop around the hair-shaft, forming a characteristic yellowish cup-shaped crust which has a peculiar mouse-like odor. ' See Rucker, Pub. Health Reports, 1912, XXVII, 1854; Thomson, Ann. Trop. iled. and Parasitol., 1914, VIII, 19. - See Foster, Jour. Am. Med. .\ssn., 1914, LXIII, 640. Digitized by Microsoft® 1 66 DIAGNOSTIC METHODS In searching for this parasite, a favus crust is softened by the addition of a few drops of water or dilute sodium hydrate solution and placed upon a slide and examined with the high-power dry lens. The hairs may also be examined in the same manner or may be stained by methods outlined in the discussion on Tinea tricophytina. The mycelial threads appear as narrow, flattened, ramifying, short or elongated, linear cells or tubes, which may be simple and empty, or be divided more or less regularly by transverse partition walls transforming the longer and simple into shorter and compound cells. The latter often contain in their cavities sporules clinging to either side, in which case the mycelial threads are termed sporophores. The conidia are encapsulated or are strung together %. 'fSVv, Fig. 63. — Pulex penetrans: young female, enlarged. {Tyson ajicr Braun.) like the beads of a necklace, and appear as round, oval, angular, or very irregularly contoured bodies. These mycelial threads branch at right angles, the spores measure from 3 to 10 microns in diameter (Hyde). (2) Trichophyton megalosporon endothrix. This organism is the cause of tinea circiiiata (herpes tonsurans, ring- worm of the body), and of tinea sycosis (hyphogenous sycosis, tinea barbffi, ringworm of the beard, barber's itch). The trichophyton is composed of spores which vary greatly in size, but which, as a rule, are somewhat larger than those of the type next to be dis- cussed. '^ They are frequently cuboidal, oval, or irregularly rounded, but their chief characteristic lies in their arrangement in lines or chains, extending up and down the hair shaft. The mycelium is found without, but never within the hairs (Hyde). These fungi may be stained by the method of Morris and Calhoun. The hair is first washed in ether to remove all fatty debris; it is then put for one ' Priestley (Ann. Trop. INjQ^^/lS^Ak'itM'pfPi^?^!, 113) reports the presence of the microsporon scorleum in a case of ringworm. PARASITES 167 or two minutes in Gram's iodin solution and is stained after drying for from one to five minutes in gentian-violet. It is again dried and treated for a min- ute or two with the iodin solution and for an equal length of time in aniline oil containing pure iodin, after which it is cleared with aniline oil, washed in xylol, and mounted in Canada balsam. (3) Microsporon audotiini (Trichophyton Microsporon). This parasite appears under the microscope chiefly in the form of a large number of round spores, irregularly grouped or massed about the follicular portions of the hair. Mycelial threads, large and branching, are often seen Fig. 64. — Achorion schonleinii, X 500 diameters. {Van Harlingen.) within the hair. The sheath of spores surrounding the hair is often con- tinued upward for Ke to J^ inch above its exit from the follicle and may be recognized as a whitish or grayish coating of the hair. These mycelial threads are all within the hair proper, thus differing from those of the tricho- phyton which are never within the hair; after repeatedly dividing and sub- dividing they terminate on the outer surface of the shaft in fine filaments, at the extremities of which are the spores. This parasite is the cause of the dis- ease tinea tonsurans, or ringworm of the scalp. (4) Microsporon furfur. This parasite is readily recognized by the microscopic examination of the scales scraped from the skin. Innumerable clustered spores, highly refractive and resembling in their circular and oval contours droplets of oil, are quite characteristic. The mycelial threads are not usually branched, but lie in a close network, among which sporophores are distinguishable, with conidia Digitized by Microsoft® 1 68 DIAGNOSTIC METHODS Im km 111 vm Fig. 65.— Normal hair, X 900. Wtt Fig. 66.— Hair showing trichophyton endo-ectothryx, X 900. Digitized by Microsoft® PARASITES 169 and terminal elements emerging at one extremity of the spore case. Both elements of this organism are more readily stained by the aniline dves than are those of the trichophyton or favus. This organism is the cause of tire con- dition known as tinea versicolor. (5) Microsporon minutissimum. This organism is the etiologic factor of erythrasma. It is characterized by the extreme delicacy and fineness of its threads and very minute spores. The threads are either simple, cylindrical bodies of variable size or they may exhibit partition septa, may divide dichotomously, and may terminate in hooked or knobbed expansions. The largest transverse diameter is 0.6 micron; in length the mycelium presents the greatest variations. VAH Fig. 67. — Hair showing microsporon audouini, X 000. (6) Blastomycetes. These organisms may be found in the cutaneous eruptions of the skin in blastomycosis and may be described as follows, according to Montgomery and Ormsby. In unstained preparation the organisms appear as round or oval bodies with a double-contoured highly refractive capsule. Within the cap- sule, in many instances, granules or spore-like bodies can be distinguished. The addition of a i to 10 per cent, solution of potassium hydrate to the specimen under examination facilitates the recognition of these bodies. In stained sections the double contoured, homogeneous capsule is usually sepa- rated from a finely or coarsely granular protoplasm by a clear space of vary- ing width. Vacuoles of different sizes are found in some organisms. In both pus and tissue, organisms in pairs or in various stages of budding are commonly seen.^ The parasite, as a rule, varies in size from 7 to 20 microns, though slightly smaller and much larger forms occur in some cases. ^ See Whitman, Jour. Infect. Dis., 1913, XIII, S5. See a long series of articles by various authors in .-Vrch. Int. Med., 1914, XIII, 509 ff. Digitized by Microsoft® 170 DIAGNOSTIC METHODS The organisms are readily obtained in pure culture from unbroken ab- scesses, from miliary abscesses in the borders of the cutaneous lesions, and from the miliary nodules and abscesses in the deep-seated organs. The peculiarities of the cultures of blastomycetes must be looked for in other works. Microscopically, the organism obtained in culture appears at first as a fine, branching mycelium with a few small spore-like bodies. Later a Fig. 68. — Mycelial threads of blastomyces from old agar culture. (From photograph by W. A. Pusey.J large, segmented, often pod-like mycelium appears, together with large, round, or oval bodies with bud-like projections. The condition known as coccidioidal granuloma, which is especially ob- served in California, has often been confused in the literature with blasto- mycosis. It is, however, now well established that the etiologic factor of this disease is the coccidinidcs. impiitist,,^n org ,.„ . ,. ^. , . DigifizeabyWlicrosi. dinermg distnrctly in many respecfs from tr ism somewhat similar to but Blastomyces. This organism PARASITES 171 was first reported by Posadas and Wernicke, but was later classified by Rixford and Gilchrist cultivated by Ophuls."- (7) Sporothrix Schenckii. This organism is the cause of sporotrichosis and was first recognized by Schenck, Hektoen and Perkins.- Since its discovery a large number of cases have be.en identified in all parts of the world, especially in the middle- western portions of the United States. The lesions in this condition re- semble, at times, those of tuberculosis, while, occasionally, they appear more like those of syphilis. The chief characteristics are the sharply de- fined, painless, cutaneous or subcutaneous abscesses which follow the lym- phatics and do not yield to the ordinary surgical procedures. In living tissues the sporothrix organisms appear as elongated or oval bodies fairly uniform in Fig. 69. — Budding forms of Sporotlirix Schenckii in pus, X 1200. Gram's Stain. (Cour- tesy of Dr. D. J. Davis.) size and' often showing distinct budding processes. The arrangement is usu- ally single, but frequently two or more are found end to end or arranged radially about a central spore. Branched mycelial filaments are not found in the living tissues but they develop abundantly on artificial media, form- ing, at their sides and extremities, numerous spores, oval in shape and differ- ing decidedly in appearance from the typical tissue forms. These latter forms may be found, however, in small numbers in artificial cultures (Davis). Smears from the lesions are made in the usual way. To obtain cultures proceed as follows: Some of the light yellow pus is taken, by aseptic methods, from the suspected lesion, is transferred either to glucose or maltose agar, or blood agar, and is kept at room temperature. After a few days small points appear from which circular growths with marked striations develop in ID to 12 days. Later these may show a deep black pigmentation. Smears made from these cultures show a typical sporothrix, the mycelium being coarse (2 n broad) and forming a dense, branching, septate network. Spores 1 See MacNeal and Taj'lor, Jour. Med. Research, ior4, XXX, 261; Cooke, .\rch. Int. Med., 191S, XV, 479; Brown and Cummings, Ibid., 60S. ^ See Gougerot, in Kolle and Wassermann's Handb. d. path. Mikroorg., 1012, V, 211. Digitized by Microsoft® 172 DIAGNOSTIC METHODS (4 to s M long and 2 to 3 /t broad) develop by budding either from lateral or terminal filaments or from the sides of the thread. The organism stains readily with the ordinary dyes and is, also, Gram-positive.^ (8) Negri Bodies. Considerable discussion has centered about these bodies as the etiologic factor or rabies (hydrophobia), but now, thanks to the successful experiments of Noguchi^ in cultivating them, the subject must be regarded as practically settled in the affirmative. These bodies are found in almost all of the nerve cells of the central nervous system but are more numerous and more easily found in the cells of Ammon's horn, cortical tissue in region of the fissure of Rolando, and in the Purkinje cells of the cerebellum. They are also present in the salivary glands of the infected animal but are not readily detected therein. Their demonstration in the cells of the brain is regarded as equiva- lent to a positive diagnosis of rabies. As they have never been found in any condition other than hydrophobia, they must be regarded as specific. These bodies were originally described by Negri,' although Galtier* and Pasteur^ had previously demonstrated the infectious character of rabies. Since that time many workers have confirmed their findings and have added much to our knowledge of these bodies.^ They are found lying in the cytoplasm of the nerve cells or in their branches, occasionally outside, and appear as round, oval, triangular, pear- shaped, spindle- or sausage-shaped bodies. They vary in size from 0.5 >i to 20 11 in diameter, the size increasing with the stage of the disease. They show a hyaline cytoplasm with an entire margin and with one or more inner bodies (nuclei) having a more or less complicated and regular structure. There do not appear to be any vacuoles in the smear preparations but in the tissue specimens vacuoles are quite distinct (probably artefacts). Most of the bodies show chromatoid granules of varying size and number and ar- ranged about the nucleus like a ring. In stained specimens these -granules are seen as basophilic granules, rods or circles. They are, therefore, properly classified as protozoan parasites. To demonstrate these Negri bodies in the brain cells of a suspected animal, the following method of Williams andLowden^ is recommended for diagnostic purposes. A fragment of the gray substance from the cerebral cortex in the region of the fissure of Rolando, another from Ammon's horn and a third from 1 See Hamburger, Jour. Am. Med. Assn., 1912, LIX, 1590; Sutton, Ibid., 1913, LX, 115; Chipman, Jour. Cutan. Dis., 191 2, XXX, 339; Adams, Journal Lancet, I9r2, XXXII, 39s; Jour. Am. Med. Assn., 1913, LX, 1784; Taylor, Ibid., 1913, LX, 1142; Davis, Jour. Infect. Dis., 1913, XII, 4S3; Hamburger, Illinois Med. Jour., 1914, XXV, 99; Dominguez, Med. Record, 1914, LXXXV, 608; Davis, Jour. Infect. Dis., 1914, XV, 483; Sutton, Jour. Am. Med. Assn., 1914, LXIII, 1153 ; Meyer and Aird, Infect. Infect. Dis., 1915, XVI, 399; Davis, Ibid., 1915, XVII, 174; Champana, Riforma Med., igts, XXX, 925. 2 Jour. Exper. Med., 1913, XVIII, 314. See, also, Levaditi, C. R. Soc. de Biol., 1913, LXXV, 505; Kraus and Barbara, Deutsch. med. Wchnschr., 1914, XL, 1507; Noguchi, Berl. klin. Wchnschr., 1914, XLI, 1931; Proescher, New York Med. Jour., 1914, C, 953. ^Ztschr. f. Hyg., 1903, XLIII, 507; XLIV, 520; 1909, LXIII, 421. ■> C. R. Acad. d. sc, 1879, LXXXIX, 444. ' Ibid., 1881, XCII, 159. « See Babes, Traite de la rage, Paris, 191 2; Proescher, Berl. klin. Wchnschr., I9I3,L, 633; Poor and Steinhardt, Jour. Infect. Dis., 1913, XII, 202; Luzzani, Ann. de I'lnst. Pasteur, 13, XXVII, 907 and 1039;," - - ' Jour. Infect. Dis., 1906, I9I3,^XXVII, 907_and i°39;/^^^ jJyi^J^y^^^y^Assn., 1914, LXIII, 1658. PARASITES 1 73 the cerebellum, is taken from a section made at right angles to the surface and placed on slides about i inch from their ends. A cover-glass is now pressed upon it until it is spread out in a moderately thin layer; the cover-glass is moved slowly and evenly over the slide, leaving the first portion of the slide clean. Use slight pressure in making the smear, pressing rather more on the edge of the cover-glass away from the end of the slide toward which the cover- glass is moving, thus driving more of the nerve tissue along the smear and producing more well-spread nerve cells. Dry in air and stain as follows. . Fix the air-dried smears in methyl alcohol for five minutes. Pour on the slide the staining solution and allow to stand for one-half to three hours. The staining solution is made by adding lo drops of Giemsa's stain to lo c.c. of dis- tilled water made alkaline by the previous addition of i drop of i per cent, potassium carbonate solution. The longer period of staining is preferable, as overstaining does not occur even after 24 hours. Pour off the stain, wash in running tap-water for one to three minutes and dry with filter paper. If the smear be thick, dip it in 50 per cent, methyl alcohol before it is washed in water. The cytoplasm of the Negri bodies stains blue, while the central bodies and chromatid granules stain a blue-red or azur. The larger bodies have usually a somewhat darker blue tint than the smaller ones. The cytoplasm of the nerve cells stains blue, while the nuclei are red, the nucleoli are a dull blue and the red cells a pink-yellow. If it is desired to make a quick examination proceed as follows, although this method is not to be advised. Fix in methyl alcohol for five minutes and add equal parts of Giemsa's stain and distilled water for ten minutes. Wash, dry and examine. Mallory^ recommends the following technique. The smears made as above described are fixed in Zenker' solution for one-half hour; after being rinsed in tap-water they are placed successively in 95 per cent, alcohol, con- taining suflBicient iodine to make a port-wine color, one-fourth hour, 95 per cent, alcohol one-half hour, absolute alcohol one-half hour, 10 per cent, aqueous solution of eosin for 20 minutes, rinse in tap-water, place in alkaline methylene-blue solution for 15 minutes, differentiate in 95 per cent, alcohol from one to five minutes, dry with filter paper and examine. The cytoplasm of the Negri bodies is a magenta, the central bodies and chromatoid granules are a very dark blue, the nerve cell cytoplasm a light blue, the nucleus a darker blue and the red cells a brilliant pirik. Van Gieson^ recommends the following: To 10 c.c. of distilled water add 3 drops of a saturated alcoholic solution of rose-aniline violet and 6 drops of LoefHer's methylene-blue solution. Fix the smears in methyl alcohol, pour the above stain on them and warm until steam rises. Pour off the stain, rinse in water, and allow to dry. The stain must be prepared fresh each time as it is not permanent. The cytoplasm of the bodies is a deep and distinctive red, their inner structures are a dark blue, the nerve cell light blue and the blood cells a pale salmon red.' ' Pathological Technique, Philadelphia, 1911. 2 Centralbl. f. Bakteriol., I. Abt., 1907, XLIII, 205. ' See Kotsevalofif, Russky Vrach., 1914, XIII, 616. Digitized by Microsoft® 174 DIAGNOSTIC METHODS Noguchi^ has succeeded in cultivating these bodies from infected animals and has, in turn, reproduced rabies by inoculating cultures containing the granular, pleomorphic, or nucleated bodies. He has, then, recovered the granular and nucleated bodies from the brain of the inoculated animals. The specificity of these bodies would seem, therefore, to have been definitely established. See, however, Moon^ and Williams.' BIBLIOGRAPHY Carpenter. The Microscope and its Revelations. Philadelphia, 1901. Daniels. Laboratory Studies in Tropical Medicine. Philadelphia, 1903. Emery. Bacteriology and Hematology. Philadelphia, 1905. Hyde and Montgomery. Diseases of the Skin. Philadelphia, 1902. Jackson. Tropical Medicine. Philaddphia, 1907. Oertel. Medical Microscopy. Philadelphia, 1902. PusEY. Diseases of the Skin. New York, 1907. ' Jour. Exper. Med., 1913, XVIII, 314. 'Jour. Infect. Dis., 1913, XIII, 232. ' Jour. Am. Med. Assn., 1913, LXI, 1509. Digitized by Microsoft® CHAPTER VI THE URINE I. General Considerations The examination of the urine is one of the most important features of clinical diagnosis. So constant are the physical and chemical properties of the normal urine that any marked abnormality is easily detected. The relation between the kidneys and the blood is so close that the kidneys soon excrete any abnormal substances which have found their way into the blood-current. For this reason we find in the urine the abnormal products of perverted metabo- lism of the system or of special organs. It is true that the urinary findings may be, in any special case, secondary to those of the blood or of clinical ex- amination, but in such conditions we may detect substances in the urine, which put us on our guard against making a specific diagnosis or point out the way to a correct difierentiation. More or less marked changes in the character of the urine will occur whenever a pathologic condition exists any- where in the system. These changes may not always be sufficient to be of direct diagnostic value, but in many cases may settle a differential diagnosis.* In the urine we find excreted the products arising from the metabolism of the various proximate principles, both of the tissues and the food. Know- ing the intake of such material, we are able from our examination of the urine to judge of the manner in which the system is handling the material brought to it. In recent years the study of the metabolism in various conditions has been so extended that determinations which a few years ago were unusual are now matters of almost daily routine. An examination of the urine will fre- quently reveal the presence of irregular digestive and absorptive powers of the intestines through the appearance of certain abnormal products of protein decomposition. Moreover, the study of the nitrogen partition of the urine is taking on increasing importance from day to day, so that the estimation of the factors determining this division should be possible by any one attempting to follow the metabolic activity of the system in any specified condition. When the oxidative powers of the system are lessened, we find the urine showing abnormal products as an indication of such deficiency. These products are more or less characteristic and may be determined with a great degree of exactitude. Thanks to the work upon metabolism in diabetes, for instance, we now know that the glycosuria is clinically not of as great im- portance as is the presence of many other abnormal products associated with the sugar; in other words, a glycosuria must not be considered as identical with diabetes. ' See Freeman, Jour. Am. Med. Assn., 1914, LXIII,^i8o2; Diner, New York Med. Jour., 191S, CI, 1007. Digitized byWcrosoft® 176 DIAGNOSTIC METHODS The system has a definite disintoxicating power toward certain rloxious substances, whether introduced from without or formed within. While the blood is of special importance in such processes as far as bacterial products are concerned, an examination of the urine will frequently reveal much informa- tion regarding the metabolic toxins or the medicinal poisons. The estima- tion of the conjugated glycuronic and sulphuric acids of the urine will throw much light on the degree of this activity. In this connection it may be mentioned that indican, a product of bacterial decomposition of protein in the intestinal canal, may be taken as a direct indicator of the degree of such decomposition, but that we must not assume that all of the conjugated acids have such an origin. Besides these general indirect points of interest, an examination of the urine will often reveal a direct anatomical lesion of the kidneys. Time was when we regarded the mere presence of albumin in the urine as indicative of a kidney lesion, but we know that a thorough clinical examination is necessary before a diagnosis is possible. Albumin may or may not mean kidney trouble and may even be purely physiologic. Too much^stress can hardly be laid upon the necessity of closely associating the urinary findings in any condition with the clinical symptoms of the case. The writer will have much to say later regarding the various abnormalities of the urine, but he wishes to im- press at this point the fact that no finding, no matter how abnormal it may seem, should be considered absolutely pathognomonic, without taking into consideration the clinical manifestations of the case. An albumimuria, gly- cosuria, cylindruria, or pyuria may mean one thing at one time and another at a second period, so that the worker is cautioned against jumping at con- clusions. The urine will yield much information, providing the worker knows how to interpret his findings. A laboratory worker must be cautious in his attitude and report his findings without any attempt at interpretation, unless he is aware of the clinical history of the case in point. The writer must refer to works on physiology for the various theories which have arisen from time to time regarding the mechanism of secretion of the urine. Suffice it to say at this point that the urine is excreted through the activities of the kidneys, the water and salts being secreted by the glomeruli, while the majority of the excretory products are eliminated by the vital or selective activity of the epithelium of the renal tubules. It is evident, there- fore, that the physical and chemical characteristics of the urine will depend both upon the blood-pressure'- within the capillaries and the rate of flow through these vessels as well as upon the .condition of the secreting epithelium. Collection and Preservation of the Urine. In all urinary examinations in which quantitative relations are to be studied it is necessary that a portion of the total 24-hour specimen be ex- amined. This should be thoroughly mixed and carefully measured. The composition of the different voidings is so variable that no definite idea regarding the elimination can be gained from a single specimen. 1 See Lawrence (Am. Jour. Med. Sc, 1912, CXLIV, 330) who shows that the amount of blood m a unit of time is of much more importance than the pressure. See, also, Leschke Ztschr f. klin. Med., i9i4,LXg^^ J^jjj j^^^;^^j^:^ur. Biol. Chem., 1915, XXII, 321. THE URINE 177 If a mere qualitative examination is to be made, a single specimen may be studied. In chronic nephritis, for instance, the morning urine may show points of interest as compared with that voided in the evening after a day's activity. In diabetes it may be desired to study the effect of a carbohydrate meal upon the sugar excretion. This may best be done by an examination of the urine passed three or four hours after such a meal. The microscopic examination is best made as soon as possible after voiding, but it is to be remembered that much variation may be noted in the sediment of the different voidings. In making the 24-hour collection, the patient is instructed to empty his bladder at a specified time, preferably at 7 a. m. This portion is thrown away and all urine passed from that time until the bladder is emptied at 7 a. m. the next day is saved. If one desires to separate the day and night urine, the voidings from 7 A. m. to 7 p. m. may be kept in one container and those from 7 p. M. to 7 A. M. in a second vessel properly labeled. A thoroughly clean bottle of one-half to one gallon in capacity should be used as the container.^ This should be well corked after each addition of urine and kept in a cool place. As urine undergoes decomposition more or less readily, depending upon bacterial activity, some preservative should be added to prevent such processes. The writer is accustomed to advise the use of a slight excess of chloroform. This may be removed by heating the urine and will not then interfere with the later reactions. If not removed, its presence will lead to a pseudocarbohydrate reaction. Three or four drops of formalin may be added for each pint of urine. This is an efficient agent, but it will lead to reactions simulating those for sugar and even albumin, and, moreover, will introduce a crystalline compound of formalin and urea into the sediment as well as. markedly interfering with the bile, urobilin^ and indican tests. Thymol may be added, but this may give a reaction similar to those for bile pigments, indican and albumin. Camphor,' chloral, and boracic acid have been used, but do not possess any virtues over the other preserva- tives mentioned. In any case, the worker should be on his guard in reporting abnormal findings without convincing himself that the reaction is not due to an added preservative. II. Physical Properties (i) Quantity. The amount of urine passed within 24 hours depends upbn several factors and varies both for individuals and for different races of people. It is self- evident that under normal conditions, the amount of urine will vary with the quaUty and quantity of the substances to be excreted, the condition of the renal parenchyma, the pressure and rate of flow of the blood-current, the vasomotor disturbances, the stage of digestion, the loss of fluid in the per- spiration as influenced by the surrounding temperature, amount of exercise, and extent of fluid intake; upon the weight of the subject, the sex, and age. ' See Folin and Denis, Arch. Int. Med., 1915, XVI, 195. ^ See Hausmann, Deutsch. med. Wchnschr., 1913, XXXIX, 1685; Schmiz, Ibid., 1914, XL, 128. ' See Rosenbloom, New ■iQ^/t®«^J!j)>fi/l(?*:?«??GD*©C, 735. 178 DIAGNOSTIC METHODS As a rule, the quantity of urine excreted varies between 1,200 and 1,500 c.c. (40 to 50 ounces), reaching a maximum two or three hours after a large fluid intake. Women excrete somewhat less than men, while children void relatively more than do adults, although the actual amount is less. In the adult we find the amount of urine is almost directly proportional to his weight, a normal large individual excreting nearer 1,800 than 1,500 c.c, the amount being about i c.c. per kilo and hour, while with a child the excretion is about 4 c.c. per kilo and hour. The physiologic limits of the urinary excretion are about 750 and 3,000 c.c. In cases showing as high an output as 3,000 c.c. one is justified, per- haps, in assuming the presence of some pathologic condition. The kidneys are not easily deranged by the excess work put upon them in excreting large quantities of urine, so that we may find a secretion of many liters per day continuing for an extended period without endangering the normality of the excreting organ. Normally, the amount of urine excreted during the day by far exceeds that voided during the night, while the afternoon urine is usually more than that of the morning. We find, however, that in edematous conditions either of hepatic, cardiac, or renal origin, the night urine usually exceeds that of the day. This condition is known as nycturia. As it is, perhaps, more fre- quently associated with cardiac insufficiency, it may have a diagnostic importance in these cases. Polyviria. By this is meant an excretion of an increased amount of urine. Just what amount of urine is to be considered as indicative of polyuria will deperjd much upon the habits of the patient as regards daily intake of fluid. As a rule, anything above 2,500 c.c. is at least suggestive of this condition. Just what factors are to be held accountable for the polyuria, is not always easy to decide in every case. An increased intake of fluid together with an •increased general blood-pressure will cause both an increased local renal pressure and an increased blood-flow through the kidney. While such a polyuria rarely exists in cases of ordinary chronic or active renal hyperemia, we find under the influence of drugs that a very decided increase in the urinary output may occur. A polyuria is .observed in the- convalescent stages of acute nephritis, in both chronic parenchymatous and interstitial nephritis, and in amyloid de- generation of the kidney. This excretion, especially in the chronic inter- stitial type, may be one in which the total sohds are normal or reduced, and is then known as hydruria. Diabetes meUitus is more frequently, perhaps, than other conditions associated with a polyuria. The quantity ehminated is dependent both upon the increased intake as a result of the polydipsia as well as upon the dehydrat- ing powers of the sugar. A certain relationship exists between the amount of fluid and the sugar, the polyuria being usually diminished by measures which decrease the amount of sugar excreted. This polyuria is not neces- Digitized by Microsoft® THE URINE 179 sarily continuous and may alternate with periods showing a normal or subnormal amount of urine. In cases of diabetes insipidus we find the daily excretion of as much- as 50 liters or more of urine. According to Meyer, this polyuria is due to the attempt on the part of the kidneys to secrete sufficient water to hold the solids in solution.' In cases associated with abnormal accumulations of fluid, such as pleuritis, ascites, and general edema, a polyuria will exist at the time of absorption of the exudates, owing to the presence of such large amounts of fluid in the blood-vessels.^ < The so-called "epicritic polyuria" is frequently observed during convales- cence from acute febrile attacks. This is probably indicative of the eUmina- tion of toxic products which have accumulated in the system during the progress of the disease. It is supposed to be of favorable import when occur- ring in a febrile condition, but it is to be recalled that this polyuria may be followed by a later oliguria which is of grave significance. As a rule, however, it may be said that as the case improves the urine is increased in amount. Polyuria may be observed in many nervous conditions, both functional and organic' The cause is probably some disturbance of the vasomotor apparatus as a result, perhaps, of irritation of the floor of the fourth ventricle, cerebellum, or cord. Hysteria, neurasthenia, elipepsy, and chorea are fre- quently associated, with a polyuria. A paroxysmal polyuria in the course of a suspected nervous disease is more indicative of a functional derangement, while a continuous polyuria is more frequently associated with true organic disease. Oliguria. This is a condition characterized by the excretion of a diminished amount of urine, 800 c.c. being given as the lower normal point of the urinary output. Here, again, the absolute figure must depend upon the patient and upon his customary excretion. A single examination is not sufficient to decide whether or not an oUguria exists. This condition is found, perhaps, most frequently in cases of broken compensation of the heart, where the blood-pressure is markedly diminished. It is present whether the cardiac incompetency be primary or secondary to hepatic, renal, or pulmonary lesions. Ohguria is noted in practically all acute febrile disorders, especially in typhoid fever. This is due, probably, to a combination of cardiac weakness with the increased loss of water by the skin and lungs. Moreover, we may have, in such states, a retention of fluid along with a direct contraction of the renal vessels. 1 See Lewis and Matthews (Trans. Chic. Path. Soc, 1913, IX, 16) who show the close relation of hypersecretion of the pars intermedia of the hypophysis to diabetes insipidus. See Socin, Ztschr. f., klin. Med., 1913, LXXVIII, 294; also, Forschbach, Ibid., 1913, LXXVII, 153; Goldzieher, Verhandl. d. deutsch. path. Gesellsch., 1913, XVI, 272; Ber- blinger. Ibid., 281; Simmonds, Munch, med. Wchnschr., 1914, LXI, 180; Hohlweg, Ibid., 927; Fitz, Arch. Int. Med., 1914, XIV, 706; Mathews, Ibid., 1914, XV, 451; Krikortz, Hygiea, 1915, LXXVII, 49. 2 See Baehr, Deutsch. Arch. f. klin. Med., 1913, CIX, 417; also, Leuret, Jour, de med. de Bordeaux, 1913, LXXXIV, 669. 'See Pirondini, Jour. d'Urol., 1914; V, 461; Mathieu, Bull. Acad, de Med., 1915, Lxxiii, 208. Digitized by Microsoft® l8o DIAGNOSTIC METHODS Acute nephritis as well as chronic parenchymatous nephritis are asso- ciated with a more or less extensive oliguria. This condition is probably referable to diminished functional activity of the glandular elements as well as to increased resistance within the tubules. A bilateral diffuse lesion is always necessary to cause much oliguria, as the sound kidney, if the trouble be unilateral, will take on vicarious activity. The more acute the condition, the greater the degree of oliguria. Oliguria may also occur following the administration of an anesthetic, in connection with eclampsia, hysteria, or epilepsy, after the loss of large quantities of fluid by hemorrhage, diarrhea, or vomiting, in cases of portal obstruction as seen in acute yellow atrophy or hepatic cirrhosis, or in cases in which pressure is exerted upon the vascular system, especially the vena cava, by tumors.^ Anuria. This oliguria may, in almost any case, proceed to complete anuria, which may or may not be of vital significance. Cases of anuria do occur without any preceding oliguria, as shown in acute nephritis and in some cases of hysteria. Anuria, per se, cannot be held responsible, however, for the uremic symptoms so frequently associated with it, as it may persist for many days, 19 in a case of Adams, without any uremic signs. Anuria may be due to obstructive, reflex, renal, and prerenal causes. We may have an occlusion of the urinary passages^ on one side and a reflex closure on the other. Tumors, prostatic hypertrophy, and toxic and nervous bladder disturbances may lead to a great degree of oliguria amounting, almost, to anuria. The so-called prerenal causes of anuria include scarlet fever, which may lead to a severe nephritis, phosphorus poisoning, action of ether and chloro- form, collapse, ureteral and urethral calculus, and cholera. (2) Appearance. Freshly voided urine should be clear and transparent. Only the faintest trace of any turbidity should be normally present, except soon after a meal rich in vegetable food, when a distinct turbidity may be noticed due to the precipitation of the phosphates in the alkaline urine. When allowed to stand for a short time, a light cloud is noted which gradually settles to the bottom of the container in the form of the so-called "nubecula." Tljis contains a few small granular cells and a few epithelial cells and is composed largely of mucus. On standing for a somewhat longer period, as for instance over night, at the ordinary temperature, distinct crystals of uric acid may separate and appear in the sediment. If the temperature of the urine is allowed to fall to a considerable extent during this period, a somewhat more marked turbidity will be produced owing to the precipitation of the acid urates. This sediment is particularly noticeable if a highly acid urine becomes very cold. If kept for a longer period at room temperature, or a shorter period during ' See Cohn, Berl. klin. Wchnschr., 1915, LII, 208. 2 See MacNider (Jour. Med. Research, 1912, XXVI, 79) for a discussion of the relation of sweUing of the tubular epithelium to the urinary output. Digitized by Microsoft® THE URINE lol the warmer months, a diffuse cloudiness will appear, due to the precipitation of the phosphates, owing to the lessened acidity or abnormal alkalinity of the urine. This alkalinity is due to the decomposition of the urea into am- monium carbonate. The crystals in this alkaline urine will be triple phosphates, calcium phosphates, ammonium urate, and calcium carbonate. Even before the urine becomes alkaline, a diffuse cloudiness may be present, due to the development of numerous saprophytic bacteria. This bacterial cloud is removed only with the greatest difficulty, as filtration of the urine has practically no effect upon it. Frequently, the addition of lead acetate to the urine will produce a voluminous precipitate, which may carry down the bacteria and permit of their filtration. This procedure is, however, not to be recommended, as other substances, if present in small amounts, may be carried down and thus escape detection. If the urine is cloudy when freshly voided, the turbidity may be the result of the precipitation of phosphates through the alkalinity or it may indicate the presence of an organized sediment, such as casts, epithelial cells, blood, and pus. The normal urine shows but very little viscosity, differing little from ordinary water in this respect. In certain conditions we find a marked degree of viscidity, which becomes especially apparent on attempting to filter the urine. In cases of chronic cystitis the excretion of a large amount of mucus may make the urine ropy and gelatinous. This increased viscosity may also be seen in cases of pyuria associated with decomposition.^ (3) Color. The color of the urine varies normally between various shades of yellow, the depth of color depending upon the concentration or specific gravity of the specimen. While the color is usually much paler in the urines of low specific gravity and very dark in those of high density, we find in diabetes melhtus a very pale urine with a high specific gravity. In cases of anemia the urine is always paler than normal, but in pernicious anemia the urine is highly colored owing to the marked destruction of the erythrocytes. As a rule, it may be said that an acid urine is more highly colored than an alkaline one, although many exceptions to this rule occur. There seems to be some difference between the urines passed at different periods of the day; thus the urine of the day is usually a distinct amber, while that of the night may take on a greenish tinge. To what this color in the latter case is due is at present unsettled. Several color scales have been introduced, such as those of Neubauer and Vogel and of Radde, but these are not sufficiently extensive to take in pathologic variations where they would be most impor- tant. As a rule, it is sufficient to divide the colors of the urine into those of the spectrum, making allowance for light, medium, and dark shades of each color. The pigments causing the normal and abnormal colorations of the urine will be discussed in detail in a later section. At this point the writer would say that normally these pigments are urochrome, uroerythrin, and urobihn, 1 See Steensma (Nederl. Tijdschr. v. Geneesk., 1914, LVIII, 24), who reports fluores- cence in certain types of urines. Digitized by Microsoft® l82 DIAGNOSTIC METHODS while the various conjugated glycuronic and sulphuric acids, blood pigments, biliary pigments, melanin, etc., are found in pathologic conditions. Pathologic Colorations. Deviating from the rule that the higher the specific gravity the more in- tense the color, diabetes mellitus shows an extremely light color with a high specific gravity. Owing to the. lack of pigment, in chlorosis we find a very pale urine. Chronic interstitial nephritis and amyloid degeneration of the kidneys are associated with an extremely pale urine. In febrile conditions the coloration may range from an orange-red to a distinctly red tint, owing to the increase in the amount of urobilin. This deep color is especially noticeable in cases of severe pneumonia. "^ This red- dish urine may also be due to an increase in the amount of uroerythrin, which is responsible for the deep color of the urate sediment so frequent in the con- centrated urines of febrile conditions, circulatory disturbance of the liver, and in cases 'associated with profuse perspiration. In cases of jaundice the urine may vary from a dark yellow or green to a brown or black, depending upon the concentration of the urine, the amount of bile pigments, and upon certain chemical activity which occurs in such urine. Not only will the color of the urine be deeper, but the foam which appears on shaking the specimen will take on a distinct yellowish-brown tint. If this biliary urine be allowed to stand in the cold for some time, crystals of bilirubin may separate out and be seen in the sediment. Urine which contains blood may have a violet shimmer, may appear smoky, blood red, brownish-black, or even deep black in color. These varia- tions depend both upon the amount and kind of pigment present. Hemo- globin gives a more reddish tint to the urine, while methemoglobin produces a brownish shade. Such urine is always cloudy, owing to the admixture of corpuscles and other organic material. The blood found in the urine may arise from any point in the genitourinary tract or may be of systemic origin. In the latter case conditions which give rise to hemolysis will cause the appear- ance of hemoglobin in the urine. The condition of chyluria is characterized by the presence of large numbers of highly refractile globules of fat along with many morphological constitu- ents. This gives rise to the appearance of a milky urine and is especially characteristic of infection with the filaria. It is not infrequent to find, in cer- tain cases of hysteria, a specimen of milky urine, owing to the fact that the pa- tient has added milk to the urine before sending it to be examined. The pres- ence of a large quantity of pus will also give the urine a milky appearance. The urine of patients suffering with melanotic tumors may be perfectly clear when freshly voided, but becomes black or dark brown on exposure to the air. This reaction is due to the transformation of the pigment melanogen to melanin, and may be hastened by the addition of oxidizing agents to the urine. This darkening of the urine extends characteristically from above downward. The condition known as alkaptonuria, which is characterized by the ex- ' See Hildebrandt, Ztschr.^,.^g^^ mr6M¥' '^^- THE "URINE 183 cretion of homogentisic and uroleucic acids, gives rise to the passage of a urine which is brownish-black in color and may be syrupy in consistency. This color is not always evident in the fresh specimen, but appears soon after being voided. In cases of peritonitis, suppuration anywhere in the system, gangrene, and marked intestinal putrefaction, the urine is frequently dark colored owing to the passage of certain aromatic products of decomposition, either indican or various derivatives of phenol. The coloration in these cases may vary from a dark brown or greenish-black to a distinct blue. These urines differ from those containing melanin in the fact that ferric chlorid does not blacken the urine as it does in the presence of melanin. This urine may contain a distinct amount of indigo, although the substance present is usually a different oxidation product. If indigo be present, a bluish-black scum will frequently rise to the surface of the specimen. Medicinal Coloration. After the use of carbolic acid either internally or externally, guaiacol, creosote, resorcin, naphthalin, salol, and various tar preparations the urine may vary from a dark brown to a black color. This coloration is due to the excretion of hydroquinon and of pyrocatechin, and may be evident only on allowing the urine to stand for some time. The urine containing pyrocate- chin may reduce alkaUne copper solutions, but will not affect such bismuth preparations. While a dark brown or black coloration of the urine may be found in cases of hemorrhage, melanosis, malaria, alkaptonuria, ochronosis, and chronic tuberculosis, one should be on his guard, as the medication of the case may be responsible for such coloration. Methylene blue will color the urine a greenish to deep blue shade, which may last for several days. Usually within an hour after this drug is taken the urine may show a faint tinge of green which may be more clearly brought out by acidifying with acetic acid and warming. The use of the hypnotics, trional, sulphonal, and tetronal, frequently gives rise to the voiding of a urine which has a deep red-wine color, due to the presence of hematoporphyrin. Pyramidon produces a urine of rose-red color, the pigment of which is soluble in ether, chloroform, and amyl alcohol. Anti- pyrin and purgatin both produce distinctly red urines. Chrysarobin, senna, rhubarb, cascara, and santonin produce a golden-yellow urine which becomes red in the presence of alkah. This coloration is due to the excretion of chrysophanic acid. According to Gorup-Besanez, the pigment of beets, huckleberries, blackberries, etc., may under' certain conditions be excreted in the urine and color it the corresponding shades. (4) Odor. The normal urine usually has a distinct aromatic odor which very much resembles that of beef broth. This odor is due to the presence of certain volatile acids and is more marked in urines of high concentration. ^ If the urine undergoes decomposition either within the bladder or on standing, a so-called "urinous odor" appears which is due to the decomposition of protein '■ Dehn and Hartman (Jour. Am. Chem. Soc, 1914, XXXVI, 2118 and 2136) believe the odor of urine to be due to a peculiar substance which they call "urinod." See, also, Hart- man, Arch. Int. Med., igi5,IX§tJsSd by Microsoft® 184 DIAGNOSTIC METHODS material. This odor is very markedly ammoniacal. Should such an odor appear in the freshly voided specimen, it is evidence of marked cystitis. Abnormal decomposition of the urine, as evidenced by changes in the odor, may be found in conditions associated with decomposition of pus and may be due to the presence of hydrogen sulphid along with the ammonia. This condition may be observed in cases of perforation of an abscess into the urin- ary tract, in which case the urine may have a distinctly fecal odor if the intes- tine be involved, while in carcinoma of the bladder this repulsive odor of the urine may also be noticed. A distinct fruity odor is often present in cases of diabetes mellitus, in many febrile conditions, and in some stomach and intestinal troubles, which may be directly traceable to the presence of acetone. Certain medicaments, such as oil of turpentine, give rise to a distinct odor of violets in the urine. Menthol causes an odor of peppermint, while cubebs, copaiba, sandal-wood oil, tolu, and saffron produce a peculiar spicy odor. Valerian and asafetida are excreted as such in the urine and produce their characteristic odor. Certain foods, such as meat, bouillon, and cofiee, produce a slight odor of the urine, while asparagus gives a peculiar char- acteristic odor due to the presence of methyl mercaptan. (5) Reaction. The normal urine has an acid reaction. According to the views formerly held, this acidity was directly due to the presence of acid salts, especially to sodium dihydrogen phosphate (NaH2P04), and not to the presence of any free acid. The recent work of Folin has shown that the phosphates in the clear urine are all of the monobasic (diacid) type. His figures indicate that the acidity of normal clear urines is ordinarily greater than the acidity of all the phos- phates present and that the excess must be due to free organic acids. For this reason the methods of Freund and of Lieblein for the determination of the acidity of the urine must be given up. To quote from Folin r^ "The current attractive, and in a measure plausible, belief that the acidity of urine is regu- lated by variations in the relative proportion of the two forms of 'acid phosphates ' is, therefore, erroneous. If urine does at no time contain com- paratively strong acids in the free form, the reason is in part the variability of the ammonia formation and in part the presence of salts of organic acids. In a mixture of salts containing an excess of acids it is the weakest which will re- main uncombined and the strongest organic acids will, therefore, exist as salts; but if the total amount of acidity becomes abnormally great, the quality (the strength) of the free acids may change." From the standpoint of physical chemistry the acidity of the urine, as of all other acid solutions, should represent the absolute number of dissociated hydrogen ions in a definite quantity of the urine. We are, therefore, face to face with the same problem confronting us in the examination of the alkalinity of the blood. In the case of the urine the question of indicator to be used in the titration test is a matter of great moment, as no two indicators will give 'Am. Jour. Physiol., 1905, XIII, 45. Digitizeaby Microsoft® THE URINE ' 185 the same degree of acidity. The one naturally to be selected would be that which will react to every possible substance of an acid nature. If we use the methods of physical chemistry we find, according to Hober/ that the urine is only about 30 times as acid as is distilled water and only about one ten-thousandth as acid as the titration figures would indicate. Such being the case, we must either entirely revise our figures for the acidity of the urine or employ methods which can be more easily carried out by the general worker than can those of physical chemistry.^ Folin has, therefore, introduced a method which uses direct titration of the urine and employs phenolphthalein as an indicator. This indicator reacts to all bodies of an acid nature, but cannot overcome certain diflSculties which are in the way of direct titration. These obstacles are (i) the occur- rence of calcium in the urine in the presence of the monobasic phosphates, and (2) the presence of ammonium salts. He has found that the addition of potassium oxalate to the urine will do away with these difficulties by holding in solution both the di- and tri-calcium phosphates and by preventing the dissociation of the ammonium compounds. Folin's Method. Total Acidity. Twenty-five c.c. of urine are treated with 15 to 20 grams of powdered potassium oxalate and one or two drops of a i per cent, alcoholic solution of phenol-phthalein. The mixture is shaken rapidly for one or two minutes and titrated at once with a tenth-normal sodium hydrate solution until a faint, distinct, permanent pink color is obtained. It is advisable to shake the flask during the titration so as to prolong the effects of the potassium oxalate. The acidity is expressed in terms of the amount of tenth-normal sodium hydrate solution necessary for neutralization of the 24-hour amount of urine. This is expressed as T, which is, on an average, 617. Free Mineral and Organic Acidity. Determine the amount of total phosphates present by titration with uranium nitrate solution as described later. Seven and one one-hundredth mg. of P2O6 have an acidimetric value equal to i c.c. of tenth-normal acid. The total acidimetric value of the phosphates of the 24-houj: urine- may be easily determined with the help of this factor, by converting the amount of phosphates into terms of N/io acid. From the total acidity (T) subtract the acidimetric value of the phosphates {P). The remainder is the acidity due to uncombined organic acids, and the difference, that obtained from calculating all the phosphoric acid as di- acid phosphate, is the free mineral acidity. For all ordinary studies of the acidity of the urine the direct titration of the total acidity and of the phos- phates gives the necessary information.' The excess of the total acidity '' Beitr. zur chem. Physiol, u. Path., 1903, III, 525. « See Henderson and Pahner, Jour. Biol. Chem., 1913, XIII, 393; Ibid., 1913, XIV, 81; Arch. Int. Med., 1913, XII, 153; also, Henderson, Science, 1913, XXXVII, 389; Henderson, Pahner and Newburgh, Jour. Exper. Pharmacol., 1914, V, 449; Henderson and Palmer, Jour. Biol. Chem., 1914, XVII, 305; Ibid., 1915, XXI, 37; Ibid., 57; Arch. Int. Med., 1915, XVI, 109; Host, Ztschr., f. klin. Med., 1915, LXXXI, 266. » See Walpole (BiocherjjyJ^g^igM^}^^^^^^)^^ for a discussion of various new indicators. ^ ' r86 DIAGNOSTIC METHODS above that calculated from the phosphates gives the total free acids present. If the acidity calculated from the total phosphates is greater than the titrated acidity, then there are practically no free organic acids present, and the ti- trated acidity represents the amount of phosphates present in the diacid form (Folin). While the acidity of the urine is best determined and expressed as outlined above, it seems wise to the writer to retain the same style of expression for the acidity as used in stomaich analysis. With this nomenclature one would state the acidity of the urine in degrees, that is, the amount of tenth-normal sodium hydrate necessary to neutralize loo c.c. Under normal conditions this will vary from 35 to 45°. It may be increased by a diet rich in meat, while it is decreased by a vegetable diet. There are many acids produced in the oxida- tion of protein, among which we find sulphuric, phosphoric, uric, and the oxyaromatic acids. Ordinarily, these play an indirect part in the acidity of the urine, although this phosphoric acid may exist in part as the dihydrogen phosphate and in consequence increase the acidity of the urine. The regula- tion of the metabolism is such that an increase of the acids produced in the system or taken into it from without is neutralized by an increased formation of ammonia, the salts appearing in the urine as the ammonium salts which do not, of course, increase the acidity of this fluid. This is the basis upon which one estimates the amount of ammonia in following a condition of acidosis.^ The reaction of the urine varies at different times of the day. The acidity appears to be highest in the morning before breakfast and is diminished after a meal, due to the secretion of hydrochloric acid into the stomach. The re- action of the urine may even be alkaline for a period of two or four hours after each meal, in which case the urine will be turbid from the precipitation of phosphates. This reaction of the urine following meals is known as the "alkaline tide" of the urine. Between meals the acidity of the urine will gradually increase until the next meal is taken. The reaction of the urine is modified to a great extent by the use of drugs.'* Thus, alkalies, such as carbonate and bicarbonate of sodium, will render the urine alkaline if taken between meals, while if taken just preceding a meal they will be neutralized by the gastric juice. All organic acids of the fatty series are oxidized in the system to carbonic acid and combine with bases forming basic salts which render the urine alkaline or less acid, providing these acids are not taken above the point of tolerance, as the writer has shown that large doses of such acids as citric acid will increase the acidity of the urine. The mineral and aromatic organic acids will, however, practically always increase the acidity. In many pathologic conditions we find the reaction of the urine variable.' 1 Fischer (Nephritis: An Experimental and Critical Study of Its Nature, Cause and the Principles of Its Relief, New York, 191 2) believes that the abnormal production or accumu- lation of acid in the cells of the kidney and the action of this acid on the colloidal structures of the kidney are accountable for all the changes which characterize nephritis. ^ See de Jager (Biochem. Ztschr., 1912, XXXVIII, 294), who shows that magnesium sul- phate increases the acidity, while sodium sulphate diminishes it. ' See Newburgh, Palmer and Henderson, Arch. Int. Med., 1913, XII, 146; also, Pertik (Virchow's Arch, f . path. Anat., 1913, CCXIII, 465) who finds a decreased acidity in tuber- culosis; Labb6 and Vitry, Pre£3^-g(^£/ij|^/\|^g^^)5f® THE URINE 187 Abnormal gastric activity may be either associated with an increase or a decrease in the acidity of the urine, depending upon a condition of h)rpo- or hyperacidity of the gastric juice. The rapid absorption of a transudate or exudate will lead to the excretion of an alkaline urine fron the presence of an increased amount of alkaline salts. An alkaline urine is not infrequently seen after intestinal hemorrhage, in certain cases of pneumonia, typhoid fever, chronic nephritis, and in cases in which exudates from the urinary tracts have become mixed with the urine. In certain cases of nervous diseases and in some cases of anemia we may also find an alkaline urine. The urine in all of the above cases will show, if tested by litmus-paper, an alkaline reaction in which the bluing of the red litmus-paper is permanent. This condition is known a.s fixed alkalinity and is quite distinct from the following type. In cases of decomposition of the urine within the urinary tract, through the influence of bacteria, the urea is decomposed into ammonium carbamate and carbonate. The alkaline reaction of the urine in such cases will be shown by a blue color of the red litmus-paper either held above it or placed in it, the blue color disappearing when the paper is dried. This condition is known as volatile alkalinity. If the urine shows this volatile alkalinity on being voided, the finding is significant of trouble somewhere along the urinary tract, especially within the bladder. Benedict has recently introduced the term "acid unit" into the study of the urinary output. One c.c. of urine with an acidity of i degree is equivalent to 100 acid units. In other words, one may determine the acid units in the 24-hour specimen by multiplying the number of cubic centimeters of the urine by the degrees of acidity. This is normally about 40,000. (6) Specific Gravity. The specific gravity of the normal urine ranges between 1,015 ^.nd 1,025, with an average of 1,020. This specific gravity will depend, of course, upon the amount of fluid intake, the quantity of the 24-hour specimen of urine, the degree of tissue activity, and the condition of the secreting organs. The intake of a large volume of water may reduce the specific gravity of the urine to a very low figure and, correspondingly, a small intake may lead to a urine of high gravity. We find, therefore, that perfectly normal urines may show specific gravities ranging from 1,010 to 1,030, with pathologic variations from 1,002 as high as 1,060 or more.^ In general routine work it is essential that the specific gravity of the 24-hour specimen be determined. Except in unusual cases; absolutely noth- ing of diagnostic value may be learned by the determination of the specific gravity of a single voiding of urine. The variations at different times of the day, under the influence of food, digestion, acidity of the skin and lungs, and exercise, may be so great that apparent pathologic figures may be ob- tained from a single specimen. Such variations are overcome for the most part in the 24-hour specimen unless pathologic conditions are present to keep up such variations. In some cases, especially in chronic diffuse nephritis, the morning specimen of urine almost invariably has a lower specific gravity 1 See Frey, Deutsch. ^rclQ^ffz^^ymibS^hmi'- i88 DIAGNOSTIC METHODS than that of the other periods of the day. For this reason one may determine the specific gravity of a single specimen of such urines. It is of especial importance that the total amount of urine in the 24-hour specimen be taken into consideration in judging of the value of a specific gravity. Thus in chronic interstitial nephritis we may find a large volume of urine with a low specific gravity, while in diabetes mellitus an even higher volume of urine may be present, showing a very high specific gravity. Technic. The most accurate method of determining the specific gravity is, of course, the use of the pycnometer. The prin- ciple of this method is the determination of the weight of a definite volume of urine as compared with that of the same volume of distilled water under the same conditions of temperature and atmospheric pressure. This method will be discussed in the section on Blood, to which the reader is referred. The cHnical method of estimating this factor is a dis- tinctly areometric one. The principle of this method is that a body immersed in a fluid will displace an amount of fluid equivalent to the loss in its own weight. By the use of instruments known as hydrometers or in the case of the urine as urinometers, this displacement is measured by immersing the hydrometer in the fluid and observing the point to which this instrument sinks. The stem of the hydrometer is graduated in divisions from 1,000 to 1,060 by differences of 1°, the 1,000 point being that to which the instrument sinks when immersed in distilled water at the temperature to which the instrument is calibrated. Any variation in the density of the solution in which this hy- / drometer is immersed will be evident by the depth to which it sinks, the more concentrated the solution the less will the instrument sink. The vessel in which the urine is poured should be cylindrical in shape, with parallel sides and wide base and sufficiently tall to permit of the complete sinking of the hydrometer. The forms of this cylinder with fluted sides are perhaps more desirable than the plain cylinder, as the bulb does not tend to stick to the sides of the vessel so readily. The vessel is filled about four-fifths full of urine, any foam being re- moved by the use of filter-paper. The hydrometer is placed in the urine with a twisting motion and allowed to come to rest. The depth to which the stem is immersed is then read off by observing the mark which coincides with the lower meniscus of the urine as seen from below. The worker should never attempt to read the specific gravity from above as a slight meniscus interferes with the accuracy of his reading. The worker must be absolutely Digitized by Microsoft® der. {Hawk.) THE URINE 189 sure that the urinometer neither rests upon the bottom of the cyUnder nor touches the sides, but should see that it floats perfectly free in the urine. The temperature at which the reading is taken is a matter of some moment as some of these instruments are graduated at i5°C. The ordinary model, as made by Squibb, is graduated at 2S°C., which is, perhaps, more nearly the working temperature of the room. A variation of 3° in tempera- ture between that of the room and that at which the instrument is calibrated, wiU give a difference of 1° of specific gravity; that is, a difference in the fourth place of the specific gravity. In ordinary clinical work corrections for varia- tions in temperature are usually unnecessary, as variations of two or three points in the fourth place of the specific gravity are of absolutely no impor- tance, as such changes might be attributed to chemical variations on stand- ing, even though the most accurate methods of estimating the specific gravity were used. If the quantity of urine by very small, it may be diluted with distilled water, so that the measuring cylinder may contain enough material to permit of a density estimation. The specific gravity of this diluted urine is then determined as above and the last two figures of the specific gravity are multipUed by the degree of dilution. Rough Estimate of Total Solids. As the degree of specific gravity is directly proportional to the amount of solids contained in the urine, one may roughly judge of the total solids by a simple calculation as follows: If the last two figures of the specific gravity be multiplied by 2.33 (Haeser's coefficient), the result will be the approximate number of grams of total solids in every 1,000 c.c. of urine. Knowing the quantity of urine passed in the 24 hours, a simple calculation will yield the total 24-hour excretion of solids. Long uses the coefficient 2.6. Instead of the above figure one may multiply the last two figures of the specific gravity by i.i (Haines' coefficient) and obtain the number of grains of solids in each fluidounce of the urine. This figure when multiplied by the total number of ounces of the 24-hour specimen will yield the excretion of solids in grains. This latter method has some advantage for the older practitioner who has not accustomed himself to the use of the metric system. It is to be said that neither one of the above methods can give anything but approximate results and in pathologic urines are absolutely unreHable. Highly albuminous urines invariably show a reduced specific gravity, while a high sugar content is associated with an increase in the density. In either one of these cases a calculation of the total solids by the above method will yield inexact figures. If the worker desires to know the exact amount of total solids in the 24-hour specimen, and this is sometimes advisable, recourse should be had to more exact methods of determination which will be discussed later. The specific gravity of a specimen of urine varies, of course, with the amount of total sohds.' Normally, these range from 60 to 70 grams with a '■ See Jacob, Deutsch. Arch. f. klin. Med., 1913, CX, 1. Digitized by Microsoft® igo DIAGNOSTIC METHODS 24-hour excretion of 1,500 c.c. of urine. The urea usually constitutes about one-half of the total solids. As the normal percentage of urea is approxi- mately two. with a specific gravity of 1,020, the writer has been struck with the usual close relationship of the percentage of the urea to the specific gravity. In watching this point in over 2,000 examinations of urine within the last year, the writer has observed that the percentage of urea will prac- tically parallel the last two figures of the specific gravity; in other words a specific gravity of 1,015, ^^^ instance, will normally be associated with a urea content of 1.5 per cent. This statement is true only in those cases which contain neither albumin nor sugar. As a rule, it may be said that the specific gravity of the urine is inversely proportional to the amount of fluid eliminated. It will, therefore, be evident that conditions leading to an oliguria will produce a high specific gravity, while those causing polyuria will give a low specific gravity. This state- ment must be modified when considering certain pathologic conditions, as we may find a diminished amount of urine with low specific gravity as in chronic nephritis in which the salts are diminished, although the organic albuminous bodies are much increased; while in diabetes mellitus we have an abundant urine of high specific gravity. The specific gravity of the urine is of more or less importance in judging of the activity of the kidneys. In acute nephritis we find a urine of high specific gravity, while in the chronic types of renal disease the specific gravity is low owing to the diminution of the salts. It is to be noted in this connec- tion that in the case of so-called "functional" albuminuria, the specific gravity of the urine is above the normal figures. A marked reduction in the specific gravity of any case of nephritis is of dangerous import. (7) Optical Activity. According to Haas,"^ the normal urine is slightly levorotatory, ranging from o.oi to 0.18. This optical activity is due to traces of the conjugated glycuronic acids which will be discussed later. An increase in this levorotatory power is observed due to the presence of increased amounts of glycuronic acid, |8-oxybutyric acid, albumin (in amounts over one-half part per thousand), and levulose. Dextrorotatory urines depend upon the presence of glucose, maltose, and lactose, while the presence of pentose usually gives rise to an optically inactive urine or one at least showing only a slight degree of dextrorotation. This optical activity under the influence of pathologic products will be discussed in the section on Carbohydrates, to which the reader is referred. III. Chemical Properties (A) Normal Composition. The tables given in most text-books showing the chemical composition of the urine cannot be regarded as absolutely indicative of the excretion as shown in the every-day specimens of urine. The composition of the urine ' Centralbl. f. d. Med. Wissensch., 1876. Digitized by Microsoft® THE URINE 191 is absolutely dependent upon the diet under normal conditions, so that a table to be exact must embrace the findings under a specified diet.^ Perhaps the most frequently quoted table is that of Parkes, which may be found in almost any text-book dealing with the urine. This table gives the various figures for the different substances excreted, but does not take into considera- tion the amount of the various types of foods used in the diet leading to this excretion. The writer, therefore, feels that it is wise to omit such a table at present, as we have no method of comparison of the excretion with the intake. The recent work of FoUn upon the urine of persons both under a mixed diet and a nitrogen-free diet gives an "approximately complete" determination of the urinary constituents under absolutely fixed dietary conditions. The figures for such diets will be given under the head of each individual substance discussed. The daily urine of a healthy adult will vary between 1,200 and 1,500 c.c. in amount and will contain from 60 to 70 grams of total solids, of which the inorganic constituents form from 25 to 30 grams and the organic between 35 and 40 grams.^ The inorganic constituents consist of the phosphates of sodium, potassium, calcium, and magnesium, the chlorids and sulphates of the alkali metals, various types of ammonium salts, traces of nitrates, cal- cium carbonate (especially under vegetable diet), and traces of iron com- piounds. While these inorganic substances have not hitherto been credited with much importance, to-day we are realizing more and more that much is to be learned by a careful study of the inorganic excretion.^ The organic substances are of especial importance both in metaboUc work and in the diagnosis of pathologic conditions. While we do not by any means know everything concerning the variations in excretion of these organic products, yet we do know much which is helpful in our direct diagnostic work as well as in our study of the progress of the disease. Among the organic substances which are more or less normal (although not always in large amounts) in the urine we find the lower and higher fatty acids, oxaUc acid, acetone, glycerophosphoric acid, a trace of glucose, lactose (especially in nursing mothers), carbamic acid, urea, oxaluric acid, allantoin (especially a few days after birth), creatinin, uric acid, purin bases, thiosulphuric acid, taurocarbamic acid, cystin, chondroitin-sulphuric acid, inosite, hippuric acid, benzoic acid, phenaceturic acid, p-oxyphenyl-acetic acid, hydro-p-cumaric acid, skatol-carbonic acid, conjugated sulphuric and glycuronic acids, oxy- proteic acid, pigments, organic iron compounds, traces of protein, and ferments. Under pathologic conditions we may find lactic acid, large amounts of acetone, aceto-acetic acid, ^-oxybutyric acid, fats, large amounts of glucose, levorotatory carbohydrates, r-arabinose, lecithin, cystin, putrescin, cadav- erin, ptomaines, oxymandelic acid, leucin, tyrosin, homogentisic acid, uro- ' See Long, Jour. Am. Med. Assn., 1912, LVIII, 757; ako, Long and Gephart, Jour. Am. Chem. Soc, 1912, XXXIV, 1229. Kinloch, Jour. Path, and Bacteriol., 1914, XIX, 77; Blatherwick, Arch. Int. Med., 1914, XIV, 409. ^ See Atkins and Wallace (Biochem. Jour., 1913, VII, 219) for a discussion of the critical solution point of urine. 'See Baumann and Howard (Arch. Int. Med., 191 2, IX, 665) for a discussion of the inorganic metabolism in scurvy. Digitized by Microsoft® 192 DIAGNOSTIC METHODS leucic acid, cholesterin, cholic acid, glycocholic acid, tauro-cholic acid, various derivatives of phenol, hematin, hematoporphyrin, methemoglobin, other blood pigments, bile pigments, melanin, and protein material. (a) Total Solids and Total Ash. The estimation of the total solids of the urine is a matter of considerable difficulty, owing to the fact that evaporation of the urine leaves a syrupy residue which is dried only with much trouble and constant loss of ammonia, formed through the action of diacid sodium phosphate upon urea in concen- trated solution. One may come very close to accurate results by placing a weighed amount of dry clean sand in a weighed platinum dish and adding lo to 15 c.c. of urine. This is then evaporated upon the water-bath and later in the drying oven at io5°C. The dish is then placed in the desiccator and allowed to remain until the weight becomes constant. Knowing the weight of the dish and sand, the amount of the urine added, and the weight of the dish after the urine is evaporated the total soHds may be easily calcu- lated. In case a large amount of residue is desired for quantitative work, recourse may be had to the method of Slagle,^ of adding 5 c.c. of concentrated H2SO4 to each liter of urine showing a specific gravity up to 1,020 and evaporating this to dryness. More H2SO4 must be used with the more concentrated urines. The estimation of the ash is as follows : Fifty c.c. of urine are evaporated to dryness over the water-bath in a weighed platinum or porcelain dish. The dish is then heated, while covered, over the free flame until gases cease to be evolved, especial care being taken not to permit sputtering of the con- tents. In some cases it is possible completely to incinerate the urine by long- continued heat. However, a more usual procedure is to treat the carbonized residue with distilled water, thoroughly stir the mixture and filter through a filter-paper whose ash is known. The contents of the dish should be washed onto the filter several times and the material upon the filter also washed with boiling water. The filter-paper and its contents are now placed in the dish and completely incinerated. After this procedure the filtrate and washings of the original carbonized material, which contain most of the inorganic con- stituents, are placed in the dish and evaporated at ioo°C. to dryness, and then incinerated over the free flame. The dish is now placed in a desiccator and dried to constant weight. Knowing the weight of the dish with its con- tents and the original weight of the dish a simple calculation wiU give the amount of ash in the 50 c.c. of urine taken. As originally stated the inorganic constituents will range between 25 and 30 grams under normal conditions. (b) Inorganic Constituents. (i) Chlorids. The chlorids are one of the most important groups of inorganic solids in the. urine. They are derived entirely from the food and, in consequence, the amount of excretion will depend upon the intake. The chlorids actually forming constituent parts of the food exist in combination with potassium and calcium, while those which are added as seasoning to the food are practically THE tJRINE 193 always in the form of sodium chlorid. As the amount contained in the food is trivial in comparison to that added, we are accustomed to regard practically all of the intake as sodium chlorid. This is a constituent of the serum of the blood and of other tissues, while the potassium salt is in more direct relation with the cellular elements. Under normal conditions from 10 to 15 grams of sodium chlorid are eliminated in 24 hours. The administration of a diet rich in salts will in- crease this amount, while a salt-poor diet will diminish the amount up to a certain point. If the diet be a starvation one, or an absolutely salt-free one, the chlorids will disappear almost entirely from the urine. The regulation of the metabolic activity of the system is such that a certain amount of salt must be retained in order to preserve the osmotic equilibrium. For this reason we find that withdrawal of salt from the diet does not lead to any appreciable diminution of the normal chlorid content either of the blood or tissues. An increase in the elimination of salt is practically always followed by a retention of salt unless a sufficient supply is furnished by the food. If food containing sodium chlorid be given after a period of salt-free diet, a portion of this salt will be retained. Conversely, we find, if the body has for pathologic reasons retained sodium chlorid, that an increased elimination will follow. This metabolic activity is intimately associated with the general pro- tein metabolism of the body. Any increase in the amount of circulating pro- tein as compared with the living protoplasm will be followed by an increased eUmination of the chlorids, which have been previously retained by the living or active protein material of the protoplasm. This fact is shown by the rela- tion between the elimination of the chlorids and the total nitrogen. With an ordinary diet this ratio is as one to one, but in disease it may be much disturbed owing to chlorid retention through renal insufficiency. The blood and tissues of patients with nephritis show a higher chlorid content than those of normal individuals. We should, therefore, expect, if the law of increased chlorid excretion being dependent upon increased circulating albumin were to hold, that the chlorids would be increased along with the albumin in nephritis. But we find that the kidney under those circumstances is unable to excrete the increased amounts of salts circulating in the blood. A further method of withdrawing sodium chlorid from the body consists in the administration of large quantities of alkaUne carbonates or of com- pounds of the alkahes with vegetable acids. As soon as these are given the body becomes poor, not only in acid substances and in HCl, but also at the same time in sodium and potassium. Practically speaking, the body becomes directly impoverished in NaCl. The body may also lose chlorin when vomit- ing is frequent, when absorption is diminished, when the stomach is jregularly washed out, and when diarrhea is marked. This loss may be especially noted in cases of hyperacidity of the gastric juice associated with vomiting. The chlorin in these conditions is withdrawn in the form of free acid, and in con- sequence the alkalinity of the tissues may be increased. Physiologic Variations. The amount of sodium chlorid excreted will depend directly upon the '3 Digitized by Microsoft® 194 DIAGNOSTIC METHODS amount ingested. We may find as high as 3-0 grams of salt in the 24-hour specimen or it may be as low as 2 grams, salt-free diet reducing the elimination to a mere trace. The elimination may be increased by active exercise, by increasing the water intake and hence the water output, and by the intake of a large amount of vegetable food. Much more chlorid is apparently excreted during the day than during the night. ^ Pathologic Variations. A marked diminution of the chlorids, which may in some cases be al- most complete, has been supposed to be pathognomonic of pneumonia. This, however, has been shown to be fallacious, as the same condition occurs in most acute febrile states, with a possible exception of intermittent fever. In a doubtful fever a large dinimution in the amount of urinary chlorids might be strongly presumptive of pneumonia, but would be conclusive only in the presence of distinct clinical signs of this disease. While the retention of these chlorids in the exudate of pneumonia may partially explain the diminution in the urine, it cannot explain the fact that the chlorids of the food are also retained. The explanation is more likely to be found, in the writer's opinin, in an existing renal insufficiency.^ This same retention of chlorids will be found also in any condition in which there is a transudate or exudate of any considerable bulk, so that this factor must play a certain r61e. As crisis approaches in pneumonia, the chlorids of the urine will in- crease in favorable cases, while in those of bad prognosis no such increase will, as a rule, be observed. Van der Bergh believes the explanation of the dimin- ished urinary chlorids in pneumonia to be an attempt on the part of the blood to maintain its osmotic pressure, the chlorids remaining &Ked in the tissues owing to the increase of the products of metabohsm in the plasma. The chlorids are diminished in all acute and chronic renal diseases associated with albuminuria. The work of Widal upon the influence of chlorids upon the progress of a renal disease has brought out the facts that not only do we have such a chlorid retention, but that the presence of chlorids in the food will increase both the albuminuria and the edema of these con- ditions. While these facts are incontrovertible, we must take into considera- tion, as Richter has shown, the amount of water intake as well.' This is such an important field to the clinician that the writer would refer to other works giving the details of the " dechloridization" treatment. A severe diarrhea will also diminish the amount of chlorids in the urine, as the chlorids of the food are carried off by the bowel too quickly to permit of absorption.. In cases of carcinoma of the stomach, in dilatation either from hypersecretion or stricture of the pylorus, and in some cases of ulcer of the stomach a diminution or even total absence of chlorids in the urine may be observed. In most chronic diseases, in anemic conditions, in rickets, and in marked 1 See Borelli and Girardi, Deutsch. Arch. f. klin. Med., 1914, CXVI, 216; Goldberg and Hertz, Ibid., 201. 2 See Snapper, Deutscli. Arch. f. klin. Med., 1913, CXI, 281; also, Hoff, Cor.— Bl. f. schweiz. Aerzte, 1913, XLIII, 1410. * See Fischer, Jour. Am. Med. Assn., 1915, LXIV, 325. Digitized by Microsoft® THE URINE 195 nervous diseases, such as melancholia or mania, the amount of chlorids may be greatly reduced. If the output of chlorids be very low in a chronic disease, the prognosis becomes grave unless the diet can explain the diminution.^ A diminution is observed in most febrile diseases, especially in the exanthe- mata, while in typhoid fever the reduction is not so marked. This slight diminution in typhoid fever may serve as a distinguishing point in the diagno- sis of meningitis from typhoid fever, in the former case the diminution being much more marked than in the latter. In acute yellow atrophy of the liver the chlorids are diminished, while in cirrhosis of the hver they are somewhat increased.^ The Chlorids are increased in all conditions which have previously shpwn a retention, according to the law which has been previously discussed. We find thus an increase in the period of convalescence from acute febrile diseases, especially pneumonia. Strangyle enough, the chlorids are found markedly increased in diabetes insipidus,^ which is associated also with the excretion of a large amount of urine. In epilepsy an increase may be observed follow- ing the attack. The chlorids are increased in the urine after the use of chloroform, whether administered internally or as an anesthetic* Some of the diuretics, especi- ally potassium acetate, produce an increase in the urinary chlorids. In metaboUc work it is frequently of advantage to study the effects of an ash-free diet upon the pathologic condition. Taylor^ has, therefore, intro- duced such a diet, consisting of the whites of 18 eggs, 120 grams of olive oil and 200 grams of crystallized sugar. The work of Goodall and JosUn' with this diet confirms the earlier views that it is practically impossible to diminish the chlorin of the body by more than 10 to 14 per cent, and that the loss of water is proportionate to this. Estimation of the Chlorids. For rough clinical purposes the amount of chlorids in the urine may be estimated as follows: A. few c.c. of clear, filtered urine, from which albumin if present is removed by heating with acetic acid, are placed in a test-tube and acidified with 10 drops of chemically pure nitric acid. This mixture is then treated with a few drops of 10 per cent, silver nitrate solution. If the chlorids are present in normal amount a distinct, curdy white precipitate will settle out. If the chlorids be increased a heavy precipitate will be observed, while if they diminised only a cloud without any flakes will be seen. Quantitative Determination. The best method for such determination is, in the writer's opinion, the ^See Barantschik, Deutsch. Arch. f. klin. Med., 1914, CXIV; Landsberg, Ztschr. f. Geburtsh. u. Gynak., 1914, LXXVI, 53; Levison, Jour. Am. Med. Assn., 1915, LXIV, 326. ^ Burnham (Jour. Am. Med. Assn., 1912, LVIII, 851) reports a case of alcoholic cirrhosis in which there was a constant decrease in the excretion of the urinary chlorids. 'See Forschbach and Weber, Ztschr. f. klin. Med., 1911, LXXIII, 221; also, Meyer, Ibid., 1912, LXXIV, 3S2. * See Graham, Jour. Exper. Med., 1915, XXII, 48. ' Univ. Cal. Pub., Pathology, 1904, 1, 71. ' Arch. Int. Med., i0o8, 1, 615. Digitized by Microsoft® 196 DIAGNOSTIC METHODS Arnold modification of Volhard's method.' The principle of the test is the precipitation of the chlorids in a definite amount of urine by a standard solution of silver nitrate in the presence of an excess of free nitric acid. If the precipitate of silver chlorid be filtered from the solution, the excess of silver nitrate may be determined in the filtrate by titration with a standard solution of potassium sulphocyanate, using a strong solution of iron-ammo- nium-alum as an indicator. The urine should be as fresh as possible and should contain no nitrites. Albumin, unless present in very large amount, need not be removed. It is wise, however, in case the urine shows a high albumin content, to acidify the urine with acetic acid, boil, and filter off the precipitated albumin. In doing this one should take a definite volume of urine, precipitate as above, and was the precipitate thoroughly with water in order to dissolve any chlorids which may have been retained by the al- bumin. The filtrate is made up to a definite volume, which represents the amount of urine originally taken. Thus, 20 c.c. of urine are treated as above and washed with sufficient water to make 50 c.c. In the test as outlined later, in which 10 c.c. of urine are used, 25 c.c. of this filtered albumin-free urine will represent'io c.c. of original urine. ^ Solutions Necessary. (i) A solution of silver nitrate of such a strength that i c.c. is equiva- lent to o.oi gram of NaCl or 0.00606 gram of CI. In making this solution 29.055 grams of pure anhydrous crystallized silver nitrate are dissolved in I liter of distilled water. The chemically pure AgNOs as found on the market is perfectly reliable and needs only an accurate chemical balance for weighing the exact amount. It is essential that this solution should contain exactly the amount specified, as the accuracy of the method depends upon the correct strength of the volumetric solutions. (2) A solution of potassium sulphocyanate of such a strength that 20 c.c. will correspond exactly to 10 c.c. of the silver solution or, in other words so that 2 c.c. of the cyanate solution be necessary to precipitate the silver, from each c.c. of the silver nitrate solution. Other workers use solutions of potassium sulphocyanate of somewhat different strength, but the simplicity of the calculations necessary to determine the chlorids of the urine is much increased by such a relation between the two volumetric solutions. As po- tassium sulphocyanate is very hygroscopic, it is impossible accurately to weight the exact amount necessary to make this solution. We, therefore, dissolve a slight excess (9 grams) of potassium sulphocyanate in approxi- mately I liter of water. In order to make this solution correspond exactly to the silver solution it is necessary to find out how much water must be added to make 20 c.c. of this neutralize 10 c.c. of the silver solution. The technic is as follows: ten c.c. of the known solution of silver nitrate are measured from a buret and diluted with 50 or 60 c.c. of distilled water. Five c.c. 1 See Bayne-Jones (Arch. Int. Med., 1913, XII, 90) for a simplification of this test; also, McLean and Selling, Jour. Am. Med. Assn., 1914, LXII, 1081; Blasucci, Ibid., 1399; Wunder, Munch. Med. Wchnschr., 1914, LXI, 2436; McLean and Van Slyke, Jour. Biol. Chem., 1915, XXI, 361. 2 If the urine be highly colored, it may be cleared with blood charcoal (see Larsson, Biochem. Ztschr., igi^, XLIX, A7g), , ^ 'DWded by Microsoft® THE URINE 197 of chemically pure nitric acid (specific gravity 1.2) and 5 c.c. of a strong solu- tion of iron-ammonium-alum are added and thoroughly mixed. This mix- ture is then titrated with the potassium sulphocyanate solution whose strength is to be determined. The principle of this titration is that the KC- NS first combines with the AgNOs, forming a white precipitate of silver sul- phocyanate. At the exact point at which this combination is complete the potassium sulphocyanate will combine with the iron of the indicator forming sulphocyanate of iron which is distinctly red in color. The titration is, there- fore, carried to the point at which a permanent faintly reddish-brown color ap- pears on shaking the mixture. The number of c.c. of the sulphocyanate solu- tion necessary to produce this end point is then read off from the buret and we are ready for our correction. As the sulphocyanate solution was inten- tionally made too strong, the titration should yield fewer than 20 c.c. of this solution. Supposing 18.5 c.c. of sulphocyanate solution were used we must obviously add to every 18.5 c.c. of the remaining sulphocyanate solution 1.5 N X d c.c. of water according to the equation C = in which C represents the number of c.c. of water which must be added to the remaining solution; N the total number of c.c. remaining after titration; n the number of c.c. consumed in one titration, and s the difference between the number of cubic centimeters theoretically required and that actually used in one titration. The calculation would, therefore, run as follows: C =- — -5 -^ = 79-58. We must, therefore, add to the remaining 1S.5 981.5 c.c. of potassium sulphocyanate solution 79.58 c.c. of water to make the sulphocyanate solution of such a strength that 20 c.c. will exactly precipitate the silver from 10 c.c. of the AgNOs solution. (3) A cold saturated solution of iron-ammonium-alum. This must be absolutely chlorin free. (4) Chemically pure nitric acid, chlorin-free and having practically no trace of nitrous acid. Specific gravity 1.2. Technic. Ten c.c. of urine or 25 c.c. of the diluted urine from which the albumin has been removed are accurately measured with a pipet and placed in a 100 c.c. volumetric flask. Five c.c. of nitric acid, 50 c.c. of water, and 20 c.c. of the standard silver solution are then added and the mixture thoroughly shaken. After this mixture has stood for about 10 minutes distilled water is added up to the graduating mark of the flask, after which the whole is thor- oughly mixed and the precipitated silver chlorid allowed to settle. This mixture is then filtered through a perfectly dry filter into a thoroughly dry 50 c.c. volumetric flask. This 50 c.c. of filtrate will represent, therefore, only 5 c.c. of urine, but the calculation made later will compensate for this. ■ This 50 c.c. is then poured into a beaker of about 250 c.c. capacity and the volumetric flask is thoroughly washed out with water, the washings being added to the solution in the beaker. Five c.c. of the alum solution are then added and the mixture titrated with the potassium sulphocyanate solution Digitized by Microsoft® 198 DIAGNOSTIC METHODS to the appearance of the first permanent reddish tinge of the solution. The number of c.c. of sulphocyanate solution, necessary to neutrahze the excess of silver remaining after the chlorid of silver has been filtered off, is then read off from the buret. Calculation. As 20 c.c. of the sulphocyanate solution are equivalent to 10 c.c. of the silver solution, it is evident that the number of c.c. of silver solution not used in the precipitation of the chlorids corresponds to the number of c.c. of sulpho- cyanate solution necessary to neutralize the 50 c.c. of the filtrate. We, there- fore, subtract the number of c.c. of sulphocyanate solution used from 20 (the number of c.c. of silver solution added) and obtain directly the number of c.c. of silver solution necessary to precipitate the chlorids in 10 c.c. of urine. As each c.c. of silver solution represents o.oi gram of NaCl or 0.00606 gram of CI, multiply these factors by the number of c.c. used, the percentage of chlorids in the urine being obtained by multiplying the amount of chlorids in 10 c.c. by 10, and the total amount by simply multiplying this figure by the number of hundreds of c.c. in the total 24-hour specimen of urine. If the urine is very highly colored it is advisable to add a few drops of a con- centrated solution of potassium permanganate before the titration. This will usually decolorize the urine so that the end point will be much more distinct. Purdy's Centrifugal Method. This method, while having nothing in common with the accuracy of the preceding one, is very convenient and has the advantage of yielding quick results which are clinically available. Ten c.c. of clear, filtered, albumin-free urine are placed in a centrifuge tube which is graduated to 15 c.c. One c.c. of strong nitric acid and 4 c.c. of a 5 per cent, solution of silver nitrate are then added. The tube is shaken by inversion and the mixture allowed to stand for a few minutes, after which it is placed in the centrifuge and whirled for three minutes at the rate of 1,200 revolutions per minute. The bulk percentage of silver chlorid is then read off, from which the percentage by weight both of sodium chlorid and of chlorin, equivalent to the precipitated silver chlorid, may be calculated. One per cent, by bulk represents 0.13 per cent, by weight of NaCl and 0.08 per cent, of CI. As previously stated, the amount of chlorin in the urine depends upon the amount ingested, ranging normally between 10 and 1 5 grams. By the use of Folin's standard diet, which contains 6.2 grams of CI, the excretion is found to be 6. 1 grams of CI in 24 hours. On the ash-free diet of Taylor the excretion at the end of 12 days of such a diet was 0.17 gram of CI in the 24 hours. (2) Phosphates. The phosphates occurring in the urine are the sodium, potassium, cal- cium, and magnesium salts of the tribasic orthophosphoric acid(H3P04). As previously stated, in the discussion on the reaction of the urine, normal, clear, acjd urine contains no diabasic monacid phosphates, but all of the phos- phates under these conditions are of the monobasic diacid type. If the urine becomes less acid or amphoteric in reaction we find, however, in addition Digitized by Microsoft® THE URINE 199 to the above, the disodium monohydrogen phosphate, the monocalcium phos- phate, and the monomagnesium phosphate; while if the urine be alkaline we may find the neutral phosphates in the ascendency. It must be remem- bered, therefore, that the nomal acidity of the urine is not strictly regulated by variations in the relative proportions of the monosodium dihydrogen phos- phate and of the disodium monohydrogen phosphate as usually stated. Be- sides these mineral phosphates, phosphoric acid is found in the urine in com- bination with glycerin as glycero-phosphoric acid, which is derived largely from the hydrolytic cleavage of lecithin compounds. According to Mandel and Oertel, Mathison,i and Yoshimoti,^ the output of organically bound phosphorus is not increased by a phosphorus-rich diet, while Kondo^ shows that, although absolutely increased in amount, its relation to total P2O6 is diminished. The larger portion of the urinary phosphoric acid is derived from the food, the smaller portion coming from the metabolism of the tissue protein, especially the nucleins. This endogenous phosphoric acid may be of special importance as variations will be found depending upon the degree of destruc- tion of the lecithin and nuclein compounds. It is to be remembered here that not all of the phosphoric acid ingested is excreted, as between a fourth and a third of the total quantity may remain in the feces in combination with calcium. In studying the effects of increased ingestion of phosphates, the feces must, therefore, be examined quite as closely as the urine. Physiologic Excretion. The amount of jjhosphoric acid excreted in the 24 hours is always ex- pressed in terms of PzOb. The normal P2O6 excretion of the adult varies from I to 5 grams with an average of about 3.5 grams. The figures of Folin, based upon a diet containing 5.9 grams of P2O6, show this excretion to average 3.87 grams in 24 hours, while patients on an ash-free diet eliminate approxi- mately 0.7s gram. In this excretion the phosphates of sodium and potas- sium usually exceed those of calcium and -magnesium, the former being ex- creted in the amounts of. 2 to 4 grams in the 24 hours, the latter from i to 1.5 grams. Little data exists regarding pathologic variations in the relation of these two types of phosphates so that no conclusion may at present be drawn. The excretion of PzOb will vary with the food, especially with the amount of calcium and magnesium of the food. These bases combine in the intestine with the phosphoric radical forming phosphates which are difficultly soluble.* This fact is taken advantage of by Croftan in the administration of calcium salts to precipitate the phosphates and thus diminish their activity in conditions attributable to uric acid. The phosphates are increased on an animal diet and diininished on a vegetable diet as Ziilzer has shown. During starvation an increase of the phosphates may be observed, as an indication of decomposition of the tissues. ' Biochem. Jour., 1909, IV, 274. " Ztschr. f. physiol. Chem., 1910, LXIV, 464. ' Biochem. Ztschr., 1910, XXVIII, 200. See, also, Plimmer, Biochem. Jour.,1913, VII,43. * See Wurtz, Biochem. Ztschr., 1912, XL VI, 103; also, Knox and Tracy, Am. jfour. Dis. Child., 1914, VII, 409. Digitized by Microsoft® 200 DIAGNOSTIC METHODS Administration of phosphates at this time will usually lead to a retention to counterbalance the previous loss. This same fact was observed in the discussion of the chlorids and may be stated as a general law, that an in- creased excretion is followed by a retention and a retention by an increased excretion. It must be stated, however, that an insufficient supply of phosphoric acid is not compensated for by such a great retention of phos- phates as of the chlorids. The organism eliminates even more phosphoric acid in starvation than in cases of deprivation of salt, as the decomposing protein sets free the salts bound up with it. Many attempts have been made to determine where and in what form phosphoric acid is retained in the body and where and from what sources the body draws upon it for excretion. It is a difficult matter to determine what amount of the phosphoric acid retained reaches the bones, what portion is devoted to the soft tissues, and how much of it remains organically combined in the body (Magnus-Levy). It has been found that the relation between the excretion of phosphoric acid and Ritrogen is normally about one to seven, the same relations which exist between the amount of nitrogen and phosphoric acid in the human muscular tissue. It is, therefore, plausible to assume that a retention of both nitrogen and phos- phoric acid will lead to a deposition of increased flesh. In starvation we find that this relation is markedly'disturbed, the phosphoric acid being both relatively and absolutely increased. Such being the case the loss of P2O5 must be largely sustained by the bones, which are relatively poor in nitrogen. For a full discussion of this subject the writer would refer to von Noorden's work on Metabolism and Practical Medicine, which gives great detail regarding all phases of metabolism. * The phosphates are increased during hard muscular exercise, while mental exercise seems to lead to a diminished excretion of the alkaline phosphates and an increased output of the earthy phosphates. The ingestion of large quantities of water is frequently associated with an increased elimination of the phosphates, although this is later followed by a slight retention. It not infrequently happens that a freshly voided urine shows a marked turbidity and even precipitation due to the deposit of earthy phosphates. This has been supposed to be due to an increased output of the phosphates, but it is now known to be nothing but the natural consequence of a change of urinary reaction from acid to alkaline. This condition which has been called " phosphaturia" would, therefore, much more appropriately be styled " alkalinuria." This subject will be discussed in a later section to which the reader is referred.^ Pathologic Variations. A diminished elimination may be observed in cases of acute febrile disease, especially at the height of pneumonia. The degree of diminution is usually proportionate to the severity of the disease and usually lessens as convalescence comes on. According to Gouraud, the earthy phosphates are considerably reduced in pneumonia, while in tuberculous conditions the phosphates are increased, an interesting point in differential diagnosis. This ' See Dunner, Deutsch. ^gf,^^-^<^-m^M^n- THE URINE 20 1 retention in pneumonia as well as in the other acute febrile diseases is pos- sibly due to the renal insufficiency which may be very great in such conditions. This diminished phosphatic excretion may not always obtain in the acute febrile conditions, in some cases a sudden increased output being observed. In typhoid fever Robin beUeves an increased elimination during the febrile rise to be an unfavorable sign, while an increase during defervescence indi- cates a favorable prognosis. The phosphates appear to be diminished in most chronic diseases. In all renal diseases, whether acute or chronic, a diminished excretion is present due to the renal insufficiency. This diminished phosphoric acid excretion is regarded by Purdy^ as a factor almost as constant as is the excretion of al- bumin. In gout the phosphoric acid excretion runs parallel to that of uric acid, decreasing immediately preceding the acute attack and rising as the attack subsides. In cases of pregnancy a diminished excretion is observed which is attributable to the withdrawal of phosphoric acid from the maternal organism for the purpose of the fetal bone formation. In certain bone dis- eases, such as osteomalacia, a diminished excretion is usually observed, although at times' an actual increase is seen. The earthy phosphates, espe- cially, are diminished in these latter conditions while the alkaline phosphates may be increased. In cases of myositis ossificans the excretion of inorganic phosphates does not seem to be much affected as one might expect from the new bone formation. In cases of hystero-epilepsy the phosphates are diminished, the diminu- tion usually being proportionate to the intensity of the attack, while in true epilepsy the phosphates appear to be more or less markedly increased. It is in just the nervous diseases that one would expect to find much variation in the phosphatic excretion, but very few data are found bearing on this sub- ject. Folin and Shaffer find that in the periods of nervous excitement the relative amount of phosphoric acid is diminished, but that the absolute amount is little changed. In Addison's disease, hepatic cirrhosis, acute yellow atrophy, and chronic lead-poisoning we may find an extensive decrease of phosphates in the urine. In certain cases which show most of the symptoms of diabetes mellitus without any sugar output, a phosphatic increase is observed in the urine. This condition has been called "phosphatic diabetes," and may be associated with the excretion of as high as 10 grams of PaOs within 24 hours. In true diabetes mellitus the phosphates may be increased at one time and diminished at another, as there seems to be an inverse ratio between the excretion of sugar and that of the phosphates. The phosphates seem to be increased in cases of pseudoleukemia, leu- kemia, hemorrhagic purpura, in cases of acute or chronic inflammatory proc- esses of the genito-urinary tract, and in cyclic vomiting of children. As previously stated, the output of urinary nitrogen bears a relation of about seven to one to that of the phosphate excretion. This relation has been termed the "relative value" of phosphoric acid and represents the amountof P2O6 cor- responding to 100 grams of N. Normally this ranges between 15 and 20, > Practical Urinalysis, PhlDtgit^&fJxb^eMicrOSOft® 202 DIAGNOSTIC METHODS Estimation of Phosphates. Ten c.c. of urine are rendered alkaline with ammonia. The earthy phos- phates are precipitated in the form of a flocculent precipitate and may be roughly estimated by the volume of the precipitate. If this alkalinized urine be filtered and the filtrate acidified with acetic acid, the addition of a few drops of ferric chlorid or of uranium nitrate solu- tion will precipitate the alkaline phosphates. These methods are purely qualitative and can have no clinical value beyond giving a general idea of the relative amounts of the earthy and alka- line phosphates. Instead of. ferric chlorid or uranium solution, magnesium mixture may be used for this purpose. Quantitative Determination. The best method for the estimation of the urinary phosphates is that of titration with uranium nitrate or acetate solution. The principle of this method is that phosphoric acid compounds in acetic acid solution give, on treatment with uranium nitrate, a yellowish-white flocculent insoluble pre- cipitate of uranium phosphate (UO2HPO4). As a means of recognizing the point at which an excess of uranium solution is present in the titrated fluid, one may use either a solution of ferrocyanid of potassium which gives a dis- tinct brownish color at the end point, or, preferably, a few drops of tincture of cochineal, which gives a grass-green color and has the advantage that it can be added directly to the titrated fluid, which is not the case with the ferro-cyanid of potassium. Necessary Solutions. (i) A solution of uranium nitrate or acetate of such a strength that 20 c.c. shall correspond to o.i gram of P2O6. It is a matter of absolute indifference whether the acetate or the nitrate be used, but the writer prefers the nitrate as this is more easily obtained in the pure state. In making up a solution of uranium nitrate of the above strength, one may not rely implicitly on the weighing, as the uranium nitrate may contain impurities or excess water and thus vitiate the results. It is, therefore, necessary to have a standard phosphate solution against which the uranium solution may be titrated. The usual solution recommended by various writers is one of disodium monohydrogen phosphate. This salt varies in its degree of hydration and its solutions do not keep well. Moreover, it is absolutely necessary when this salt be used that a definite amount of it be taken and converted into sodium pyrophosphate, after which a corresponding dilution of the solution must be made to make it of such a titer that every 50 c.c. shall be equivalent to o.i gram of PaOj. In view of these facts, the writer is accustomed to follow the suggestion of Giles^ and use chemically pure dihydrogen monopotassium phosphate. This salt crystalhzes well without any water of crystallization and does not alter on exposure to the air. This solution is to be made such a strength that 50 c.c. corresponds to O.I gram of P2O6, in other words a hter must contain 2 grams of PjOs. In ^ Sutton's Volumetric An^^s^Ph^^el^^j^o^^^ .94. THE TJRIKE 203 order to find out just how much of this salt must be dissolved in a Hter of water we must have recourse to a simple calculation. The formula of dihy- drogen monopotassium phosphate is KH2PO4, its molecular weight being 136. Two molecules of this salt are necessary to yield one molecule of P2O6 according to the equation 2KH2PO4 = P2O6 + K2O + 2H2O. If, therefore, one liter of the solution must contain 2 grams of P2O5, the amount of KH2PO4 which must be dissolved in a liter is easily calculated from the following proportion: 272 : 142 2. X = 3.83 We, therefore, dissolve 3.83 grams of dihydrogen monopotassium phosphate in I Eter of water and obtain directly a solution which contains 2 grams of P2O6 or one in which every 50 c.c. is equivalent to o.i gram of P2O6. It is Fig. 71. — Volumetric flasks. perhaps, needless to add that this solution should be made in an accurately standardized volumetric flask, and at the temperature at which the flask is calibrated. Having thus obtained our standard phosphate solution, we are now in a position to make up our standard uranium nitrate solution, the titer of which must be such that 20 c.c. corresponds to o.i gram of P2O6, or, in other words, one hter of which must be equivalent to 5 grams of P2O5. The formula of uranium nitrate is U02(N03)26H20, its molecular weight being 502.6. Uranium nitrate combines with dihydrogen potassium phosphate according to the following equation: 204 DIAGNOSTIC METHODS As seen above, two molecules of the dihydrogen phosphate are necessary to yield one molecula of P2O5. We will, therefore, have, when uranium nitrate acts upon the dihydrogen phosphate, only the equivalent of Yi molecule of P2O5; that is, 71 parts. As the uranium solution must contain 5 grams of P2O6 to the liter we may then calculate how much uranium nitrate is necessary to form the equivalent of such a solution by the following proportion: 502.6 :7i ::x :s. x = 35.39. Were we absolutely certain of the purity and state of hydration of our uranium nitrate, all that would be necessary would be to weigh out this exact amount. As this is not the case, we weigh out a slight excess (35.75 grams) and dissolve in one liter of distilled water. We are now ready to determine the strength of the uranium solution as follows: Fifty c.c. of the dihydrogen monopotassium phosphate solution are placed in a beaker and treated with a few drops of tincture of cochineal and 5 c.c. of acetic acid mixture (see below, solution 2). Some workers prefer the ad- dition of potassium ferrocyanid as an indicator, but this does not give as dis- tinct a contrast at the end point, and if tests are made by adding a drop of the mixture to the ferrocyanid solution on a white plate, loss of substance must occur. This mixture is then heated and titrated, as soon as the boiling point is reached, with the uranium solution until a trace of a distinct green color becomes permanent on stirring the mixture. Duplicate determinations are then made, the results of which should agree exactly with the original. The number of c.c. of uranium solution used is then read off and we are pre- pared for the calculation of the amount of water which must be added to standardize the solution. As 20 c.c. of this uranium solution should correspond exactly to 50 c.c. of the standard phosphate solution, we may, for the sake of example, use the same figures given for obtaining the dilution in the case of the sulphocyanate solution discussed under the heading of Chlorids. Thus if 18.5 c.c. of ura- nium solution were used we must add, according to the previous explanation, 79.58 c.c. of distilled water to the remaining 981.5 c.c. of uranium solution in order to make every 20 c.c. equivalent to o.i gram of P2O5. (2) An acetic acid mixture prepared by dissolving 100 grams of sodium acetate and 30 grams of glacial acetic acid in sufficient water to make 1000 c.c. This solution must be added in the determination of the urinary phos- phates in order to overcome the influence of the nitric acid liberated in the reaction and to convert any monacid phosphates into the diacid type. (3) An indicator, preferably tincture of cochineal prepared by digesting the ground cochineal bugs in 2 5 per cent, alcohol and filtering. This indicator has the advantage that it may be added directly to the solution to be titrated, while potassium ferrocyanid must be used by the plate method of adding a few drops of the solution to the indicator after each addition of uranium solution. Technic. Fifty c.c. of clear filtered, urine are placed in an Erlenmeyer flask and Digitized by Microsoff® THE TJRINE 205 treated with 5 c.c. of the acetic acid mixture, for the purpose of transforming any monacid phosphates into the diacid form and of neutralizing the nitric acid formed during the titration. A few drops (5 to 10) of tincture of cochineal are added, the mixture heated to the boiling-point, and then titrated as described above. It is wise invariably to run duplicate determina- tions. After each addition of the uranium nitrate the precipitate is allowed to settle.so that one may see more clearly the first trace of any green coloration or precipitate. The calculation is as follows: Supposing 10 c.c. of the uranium solution were used, the corresponding amount of P2O5 in the 50 c.c. of urine examined would then be found from the equation: 20 : 0.1 : : 10 : X. x = 0.05. The percentage of PaOs would, therefore, be o.i (2 X 0.05). If the total 24-hour urine were 1,500 c.c, the total P2O5 excretion would be obviously 1.5 grams. Total Phosphoric Acid. The above determination gives the total P2O6 excretion as far as the inorganic phosphates are concerned, but it does not take into consideration the organically bound phosphorus. This is usually very small in amount, but it is sometimes of value to know whether any variations exist. This may be determined by incinerating 50 c.c. of urine and determining the total phosphoric acid in the ash. This, however, is not as easily done as might be imagined, owing to the fact that the urinary residue is diflBicultly incinerated. The writer prefers, therefore, the method of Asboth-Folin which will be discussed in the section on Sulphates. The mass fused by this method is dissolved in hot water, the solution filtered and washed with hot water. This solution is then slightly acidified with HCl, treated with the acetic acid mixture, and titrated as in the previous determination. The difference between the figures obtained here and those obtained by direct titration of the urine will yield the amount of organically-bound phosphorus in terms of P2O6. The normal output of this organic phosphorus varies from o.oi to 0.1 gram in 24 hours, forming about 2 or 3 per cent, of the total P2O6. Phosphorus-rich diets increase the absolute amount but diminish the relative percentage. Purdy's Centrifugal Method. This method cannot be relied upon for accurate results in metabolic work, but may of be some service from the clinical standpoint. Ten c.c. of clear filtered urine are placed in a centrifuge tube graduated to 15 c.c. Two c.c. of 50 per cent, acetic acid, and 3 c.c. of 5 per cent, uranium nitrate solution are then added and thoroughly mixed with the urine by inversion of the tube. The tube is then placed in the centrifuge and operated at a speed of 1200 revolutions for three minutes. According to Purdy, i per cent, by bulk of uranium phosphate equals 0.04 gram of P2O5 in each 100 c.c. of urine. Each succeeding percentage by bulk increases by the figure o.oi. Thus a bulk percentage of five of uranium phosphate would equal 0.04 plus Digitized by Microsoft® 206 ' DIAGNOSTIC METHODS 0.04, or 0.08 gram of P2O5 in each 100 c.c. These figures are much at vari- ance with those of Ogden, who states that "he has found that each }{o of a c.c. of precipitate calculated as PsOb is equivalent to 0.0225 P^r cent, by- weight." Owing to these differences, the writer would suggest that the bulk percentage be stated as such rather than as parts by weight of P2O6. (3) Sulphur Compounds. The sulphur is present in the urine in three forms : ( i ) preformed or neutral sulphates; (2) ethereal or conjugated sulphates, sulphuric acid in combination with aromatic compounds, and (3) neutral, unoxidized, or organic sulphur. The total output of sulphur depends essentially upon the protein metabolism, both of that of the tissues and of the food.^ It is to be remembered that the sulphur elimination is much less accurate than that of the nitrogen as an indi- cation of the degree of protein metabolism, owing to the fact that different protein substances vary in their sulphur-content. The daily excretion of sulphur, in terms of SO3, varies from i to 3.5 grams, when the subject is upon a mixed diet. Ordinarily, the ethereal sulphates form about one-tenth of the total output. The neutral sulphur does not vary under normal conditions as far as its absolute amount is concerned, but we note, on changing the diet to one which is relatively free in protein material, that the relative amount of the neutral sulphur is markedly increased. Thus, Folin finds on a diet containing 18.9 grams of nitrogen and 3.8 grams of SO3 the daily excretion of total SO3 is 3.31 grams, of which the inorganic SO3 is 2.92 (87.8 per cent, of total), the ethereal SO3 is 0.22 (6.8 per cent.), and the neutral SO3 0.17 (5.1 per cent.). On a nitrogen-free diet, consisting of cream and arrowroot, a total SO3 excretion of 1.04 grams is noted, of which 0.63 gram (60.6 per cent.) is traceable to the inorganic SO3, 0.12 (11. 5 per cent.) to the ethereal SO3, and 0.29 (27.9 per cent.) to the neutral SO3. We are, therefore, con- fronted with the following f act, " the distribution of the sulphur in urine among the three chief normal representatives, inorganic sulphates, ethereal sulphates, and 'neutral sulphur,' depends on the absolute amount of sulphur present." On the ash-free diet of Taylor we find, according to Goodall and Joshn, a total SO3 secretion of 0.96. gram, of which the inorganic SO3 forms 0.71 gram (74 per cent.), the ethereal sulphates 0.05 gram (5.2 per cent.), and the neutral sulphur 0.2 gram (20.8 per cent.). It is doubtless true that practically all of the urinary sulphur is derived from protein metabolism, a definite relation being usually estabhshed be- tween the nitrogenous and sulphur output. Normally, N:S03::s:i, apparently regardless of whether the patient is on a nitrogen-rich or a nitro- gen-free diet; the absolute amounts of each, however, differ markedly, de- pending upon the diet. FoUn's figures show that as the total urinary sulphur is reduced, the percentage represented by the inorganic sulphates sinks from about 90 to 60 per cent. This fact has been expressed in the above quotation from Folhn. The reduction in the inorganic sulphates must be made up by a relative '■ See Cathcart and Green, Biochem. Jour., 1913, VII, i. Digitized by Microsoft® THE URINE 207 increase in the other forms of sulphur. The ethereal sulphates have for a long time been held to be an accurate index of thedegree of absorption of the products of intestinal protein decomposition. There can be no question but that increased intestinal decomposition is associated with increased output of the conjugated sulphuric acids, especially the indoxyl and skatoxyl sulphuric acids. These ethereal sulphates are diminished on a milk diet or- on the cream and arrowroot diet of FoUn or the vegetarian diet adopted by many of Chittenden's subjects. This is true especially as regards the urinary indican which is absolutely negative in such cases, while the total amount of ethereal sulphate is diminished only about 50 per cent. Such being the case we must have some other than intestinal origin for the large relative increase of the ethereal sulphates under a nitrogen-free diet. I quote from Folin:^ "(i) The urinary indican is not to any extent a product of the general protein metaboUsm, is therefore probably, as is generally supposed, a product of in- testinal putrefaction, and may consequently be assumed to indicate approxi- mately the degree of putrefaction in the intestinal tract. (2) The ethereal sulphates can only in part be due to intestinal putrefaction, and neither their absolute nor their relative amount can be accepted as an index of the extent to which the putrefaction is taking place in the intestines. (3) The ethereal sulphates, on the contrary, represent a form of sulphur metabolism which becomes more prominent when the food contains little or no protein." Here the sulphuric acid is conjugated with aromatic bodies formed from de- composition of tissue protein. Pathologic Variations. The sulphates, as a whole, must be increased in any condition associ- ated with increased protein catabolism. Thus we find in febrile conditions an increased output of sulphur corresponding to the intensity of the process, this increase being followed by a diminution as convalescence comes on. An increased elimination of sulphates has been observed in leukemia, diabetes mellitus and insipidus, progressive muscular atrophy, and following the use of such drugs as morphin, potassium bromid, sodium salicylate and acet- anihd. From the clinical standpoint the elimination of sulphates has little practical value, the variation in the amounts of ethereal sulphates and neu- tral sulphur being the chief factors of value. ^ While, as shown above, the ethereal sulphates are subject to great varia- tion, the indoxyl-potassium sulphate (indican) varying according to the de- gree of intestinal decomposition, we find some points of clinical interest; in their study. As the putrefactive processes normally occur below the ileo-decal valve, any condition increasing such decomposition with a consequent in- crease of urinary indican would indicate trouble in the lower bowel, more frequently of the chronic type. They are increased in cases showing abnor- mal intestinal absorption, as, for instance, in typhoid fever, intestinal tuber- culosis, peritonitis, and chronic intestinal catarrh. Obstructive jaundice is ^Loc. cit. ^ See Stadtmuller and Rosenbloom, Arch. Int. Med., 1913, XII, 276; Ross, Ibid., 1914, XIII, 889; Greenwald, Ibid., 1914, XIV, 374. Digitized by Microsoft® 208 DIAGNOSTIC METHODS usually associated with increase in the output of indican as the bile seems to have a great influence upon .putrefactive processes in the intestine. They ap- pear to be increased in cholera, while in ordinary diarrhea they are diminished in absolute amount, but may be relatively increased. In cases of gastric hypoacidity associated with bacterial decomposition in the stomach the urine may show a marked increase of indican. In acute nephritis a very intense indican reaction may be obtained, while in the chronic form the amount of indican is usually diminished. In cases of pus-formation almost anywhere within the system an increased elimination of ethereal sulphates may be ob- served due to the absorption of the products of decomposition of the pus. This point is of some importance in differentiating purulent from non-puru- lent affections of various organs. The writer has seen a persistent intense indicanuria, associated with middle-ear infection, which cleared up completely after thorough drainage. Neutral Sulphur. The neutral sulphur does not have a very definite relation to the amount of sulphur of the intake or to the amount formed in the decomposition of tissue protein. While it is said to vary in amounts representing from 12 to 1 5 per cent, of the total SO3 of the urine, this must be true only when the absolute amount of excreted SO3 is taken into consideration. By this is meant that although the absolute amount of neutral sulphur does not nor- mally vary to any great extent, its percentage relation to the total sulphur varies with the amount of sulphur. Thus Folin finds on an intake of 3.8 grams of SO3 an output of 0.17 gram of neutral SO3 while on a nitrogen-free diet the output is 0.2. The nature of the neutral sulphur of the urine is somewhat uncertain, as we seem to have only two well-established bodies, namely, the sulpho- cyanates and hydrogen sulphid. The sulphocyanates are derived largely from the absorption of material from the saliva and represent approximately one-third of the total neutral sulphur. The hydrogen sulphid may be re- garded as a decomposition product. Besides these we find cystein, which is an intermediary product of normal protein metabohsm, and tauro-carbamic acid, which is derived from the biliary material. In iases of jaundice we may find as high as 60 per cent, of the sulphur in the neutral form, which may be due to the absorption of this and other biUary material. Traces of chondroitin-sulphuric acid, oxyproteic acid, allox5:proteic acid, and uroferric acid contribute to the neutral sulphur-content of the urine. The greatest increase of neutral sulphur is probably associated with the presence of cystin which is not normally present in the lu-ine. This is undoubtedly derived from abnormal protein decomposition. The sulphur in the neutral form may reach as high as 30 or 40 per cent, of the total, due to the presence of cystin. The writer will refer- to a later section for a discus- sion of the subject of cystinuria. The variations in the neutral sulphur of the urine must be regarded as indicative of abnormal metabolic processes which are not associated with Digitized by Microsoft® THE URINE 209 variations in the other types of urinary sulphur. As FoUn has shown, "the neutral sulphur is not at all due to processes identical or similar to those which give rise to indican. The neutral sulphur represents products which in the main are independent of the total amount of sulphur ehminated or of pro- tein catabolized." Estimation of Total Sulphur (A). The method followed by the writer in determining the total urinary sulphur in terms of SO3 is the Fohn^ modification of the Asboth-Modrakow- sky method. Twenty-five c.c. of urine (50 c.c. if very dilute) are measured into a large nickel crucible of about 200 c.c. capacity and treated with 3 grams of sodium peroxid.^ The mixture is evaporated to a syrupy consistency and is then carefully heated to dryness. The latter part of this procedure requires about 15 minutes. The crucible is removed from the flame and allowed to cool. Moisten the residue with i or 2 c.c. of water, add about 7 grams of sodium peroxid and heat the mixture to complete fusion for about 10 minutes. Allow the fused mass to cool, add about 100 c.c. of water and heat to boihng for at least one-half hour to dissolve the alkali and decompose the peroxid. This mixture is transferred to an Erlenmeyer flask (about 400 c.c. capacity) by means of hot water and is diluted to about 250 c.c. Concentrated HCl is slowly added to the almost boiling solution until the oxid of nickel (formed in the process) just dissolves. This will require about 1 8 c.c. of acid for 8 grams of Na202. After boiling for a few minutes, the solution should be perfectly clear. If it is not clear too much water or too little peroxid were used in the last fusion. Allow the mixture to cool and filter from the insoluble residue, if there is any. To this clear acid solution add 5 c.c. of very dilute (i to 4) alcohol and boil for a few minutes to remove the last traces of chlorine, which are formed on acidifying the solution. Now add, drop by drop, 10 c.c. of a 10 per cent, barium chlorid solution and allow the precipitate of BaSOi to settle out for two days in the cold. At the end of this time the precipitate is filtered off, using either ashfree filters, or preferably, weighed porcelain Gooch crucibles with properly prepared asbestos mats. Wash the precipitate with about 250 c.c. of cold water, dry and ignite for 15 minutes. In this heat- ing of the Gooch crucible, the flame must touch neither its perforated bottom nor its sides. If the crucible be placed upright upon the cover of a platinum crucible, which rests upon a platinum triangle the flame may be applied directly to the bottom of the platinum. After the heating, the crucible is placed in the desiccator, is allowed to cool and is weighed. Knowing the weight of the crucible and mat befor6 and after this process, a simple calculation yields the amount of BaS04 obtained from the urine taken. If the filter-paper be used, the precipitate is washed as previously mentioned. Transfer the filter-paper and precipitate directly to a weighed platinum or porcelain crucible, add 3 or 4 c.c. of alcohol and ignite, to dry and partially burn the filter-paper. Heat the residue until complete incineration occurs ' Jour. Biol. Chem., 1906, 1, 131; Jour. Amer. Chem. Soc, 1908; XXXI, 284. See, also, Raiziss and Dubin, Jour. Biol. Chem., 1914, XVIII, 297. ^ Benedict, Denis, and Schmidt use, as the oxidizing agent, a solution of copper nitrate and potassium chlorate. 14 Digitized by Microsoft® 2IO DIAGNOSTIC METHODS and the ash becomes colorless. Cool the crucible in a desiccator and weigh. If the amount of BaS04 obtained by either of these methods be multiplied by 0.3429, the result is the amount of SO3 in the urine taken. A simple calculation yields the percentage and total amount excreted. Determination of Total Sulphates (B). Folin's Methods. Under this head we determine the neutral (preformed or inorganic) and ethe- real sulphates. Folin^ has advanced two methods, the first of which is preferable. 1. Precipitation in the Cold. — 25 c.c. of urine and 20 c.c. of dilute hydro- chloric acid (i to 4), or 50 c.c. of urine and 4 c.c. of concentrated HCl, are gently boiled in an Erlenmeyer flask (capacity about 200 c.c.) for 20 to 30 minutes (never less than 20). To prevent loss of material it is wise to keep the flask covered with a small watch-glass during the boiling. The flask is cooled for two or three minutes in running water and the contents are then diluted with cold water to about 150 c.c. To this solution are then added 10 c.c. of a 5 per cent, barium chlorid solution, care being taken not to shake or stir the contents during the addition. At the end of an hour or more, the mixture is shaken up and filtered. The rest of the determination is as out- lined above. 2. Precipitation in the Heat. — The boiling of the urine with hydrochloric acid is conducted exactly as in the preceding method. At the end of 20 to 30 minutes, the boiling urine is diluted to about 150 c.c. with hot water. The mixture is heated once more to the boiling point, is removed from the flame, and immediately precipitated by the addition of 5 c.c. of 10 per cent, barium chlorid solution, which must be added drop by drop. The mixture is allowed to stand for 2 hours in order to cool. The remainder of the process is as previously given. Determination of Inorganic (Neutral) Sulphates (C). About 100 c.c. of water (not less), 10 c.c. of dilute hydrochloric acid (i to 4), and 25 c.c. of urine (50 c.c. if dilute, when a correspondingly smaller amount of water is taken) are measured into an Erlenmeyer flask of about 200 c.c. capacity. 10 c.c. of a 5 per cent, solution of barium chlorid are added, using the drop method, the urine solution not being shaken or disturbed dur- ing this addition. At the end of a hour or more, the mixture is shaken and filtered. The remaining steps are given above. The figure obtained here represents the amount of SO3 referable to the preformed sulphates. The ethereal sulphates (D) need not be determined separately as the difference between the total sulphates (B) and the inorganic sulphates (C) will represent the amount of these ethereal sulphates in the specimen ex- amined. If the SO3 referable to the total sulphates (B) be subtracted from the SO3 of the total sulphur (A) the remainder represents the neutral sulphur in terms of SO3. Purdy's Centrifugal Method. Ten c.c. of clear urine are placed in a centrifuge tube and to it are added 5 c.c. of barium chlorid mixture, consisting of four parts of barium chlorid, ^Loc. cit. See, also, Rosenheim, Biochem. Jour. 1914, viii, 143, Digitized by Microsoft® THE URINE 211 one part of concentrated hydrochloric acid, and i6 parts of distilled water. The tube is inverted to insure mixing of the reagent and urine and is allowed to stand for a few minutes. It is then placed in a centrifuge and whirled three minutes at the rate of 1,200 revolutions per minute. Each per- centage of BaS04 by bulk represents approximately 0.25 per cent, of SO3 by weight. This result can, of course, not be as accurate as the preceding, but has some clinical advantage. (4) Carbonates. A freshly voided specimen of urine may contain small quantities of car- bonates and bicarbonates and some free carbonic acid. The amount of free carbonic acid varies with the degree of acidity of the urine and the amount of carbonate-forming material in the food. It has been found that vegetable foods are almost always productive of an alkaline urine, owing to the fact that the organic acids of the vegetables may be converted into carbonates and excreted as such. The carbonate which most frequently forms as a sedi- ment in the urine is calcium carbonate which will be treated in a later section. If the urine be acidified and a stream of air passed through this acidified urine into a vessel containing a solution of barium hydrate, the carbon-dioxid liberated by the acids will unite with the barium forming barium carbonate which may be filtered, dried, and weighed. (5) Sodium and Potassium. These metals exist in the urine in the form of the oxids NajO and K2O, the former being present in amounts of 4 to 7.5 grams, the latter varying from 2 to 4 grams. The normal relation between the excretion of these products is as 5 to 3. The sodium is largely derived from the addition of sodium chlorid to the diet, while the potassium is a constituent of most vegetable foods. We find, therefore, that both of these substances depend largely upon the diet in normal cases, while in pathologic conditions the potassium may be excreted in larger amounts.^ Thus we find in fever the potassium salts predominate over the sodium compounds up to the time of crisis, after which the sodium salts again assume their normal proportions. Increased exercise as well as increased decomposition of protein from pathologic causes will tend to increase the potassium of the urine. From the clinical standpoint the quantitative determination of the sodium and potassium of the urine has little significance. (6) Calcium and Magnesium. Both of these alkali-earth metals are excreted in the urine largely in the form of the phosphates. Calculated as the oxids, the calcium excretion (CaO) varies from o.i to 0.3 gram and the magnesium (MgO) from 0.15 to 0.4 gram in 24 hours, the relation between the calcium and magnesium excretion being about i to 1.5. The chief source of these compounds is the food, but it must be remem- bered that only a small portion of these substances is excreted in the urine. • See Blumenfeldt, Inaug. Dissert., Berlin, 1912; also, Ztschr. f. exp. Path. u. Therap., 1913, XII, 523. Digitized by Microsoft® 212 DIAGNOSTIC METHODS Calcium and, to a less extent, magnesium form compounds with phosphoric acid in the bowel and are excreted without being absorbed. Even if these compounds be injected subcutaneously the excretion is largely into the intes- tine. We should, therefore, study much more closely the calcium metabolism by examination of both the feces and urine. Little is known regarding the output of either of these metals, but what work has been done is more closely connected with the calcium excretion than that of magnesium. During starvation calcium oxid is increased both relatively and absolutely, its probable source being the bones. It is increased to some extent by exercise and is diminished after the administration of alka- lies. In chronic diseases we note an increase due, probably, to inanition. In tubercular conditions the calcium output is occasionally found to be dimin- ished, whil,e some cases of a marked increase have been noted. In diabetes meUitus, as in other conditions associated with an acidosis, the output of CaO may be greatly increased, thus running parallel to the ammonia output. In a case of myositis ossificans studied by the writer there seemed to be no great variation in the calcium output, while the magnesium content of the urine was markedly reduced. Austin has reported similar findings. Mc- Crudden and Fales report (Arch. Int. Med., 191 2, IX, 273) a loss of calcium and a retention of magnesium in non-puerperal osteomalacia. The same authors (Jour. Exper. Med., 191 2, XV, 450) find the calcium excretion in the urine, in cases of intestinal infantilism, to be almost negligible. Peabody (Jour. Exper. Med., 1913, XVII, 71) shows a retention of calcium and a normal excretion of magnesium in pneumonia.' Quantitative Determination. Calcium. McCrudden^ has recently introduced a method for this determination which, in my opinion, yields the best results. If the urine be alkaline it must be made neutral or sUghtly acid HCl and be filtered. If turbid or faintly acid to litmus, add 10 drops of concentrated HCl. Two hundred c.c. of the clear acid urine are treated with 10 c.c. of a 2.5 per cent, oxalic acid solution and 8 c.c. of 20 per cent, sodium acetate solution. Allow the mixture to stand over night at room temperature or shake vigorously for 10 minutes. Filter ofi the precipitate of calcium oxalate and wash it Cl-free with 0.5 per cent, ammonium oxalate solution. Save the filtrate for the determination of mag- nesium. The precipitate of calcium oxalate is then dried on the filter-paper at ioo°C. and is placed in a weighed platinum crucible and burned over the Bunsen flame until the filter-paper is completely ashed. The blast-lamp is then applied and the crucible and contents blasted for 15 to 20 minutes. The crucible is dried in the desiccator and weighed, the increase in weight repre- 1 See, also, Voorhoeve, Deutsch. Arch. f. klin. Med., 1913, CXI, 29. For various studies of the metabolism of calcium see Diller and Rosenbloom, Am. Jour. Med. Sc, 1914) CXLVIII, 6s; Bergeim, Stewart and Hawk, Jour. Exper. Med., 1914, XX, 218 and 225; Bookman, Am. Jour. Dis. Child., 1914, VII, 436; Rosenbloom and Cohn, ArcH. Int. Med., 1914, XIV, 263; Kleinschmidt, Berl. klin. Wchnschr., 1915, LH, 29. 2 Jour. Biol. Chem., 1910, VII, 83; Ibid., 1911, X, 187. See, also, Lyman, Ibid., 1915, XXI, SSI. Digitized by Microsoft® THE X7RINE 213 senting the calcium oxid (CaO) in the 200 c.c. of urine. A simple calculation wiU yield the amount of CaO in the 24-hour specimen. Magnesium. The filtrate and washings obtained in the above determination are treated with one-third the volume of 25 per cent, ammonium hydrate. A precipitate of magnesium ammonium phosphate (NH4MgP04) occurs, which is allowed to settle for several hours, is collected on an ash-free filter, is thoroughly washed with water containing one-third its volume of ammonia, and is dried in the oven at ioo°C. The filter-paper and precipitate are placed in a weighed platinum crucible and burned until the filter-paper is completely destroyed, after which the crucible and contents are heated over the blast- lamp for IS minutes. This heating converts the magnesium ammonium phosphate (NH4MgP04) into Magnesium pyrophosphate CMgzPzO?); one part of magnesium pyrophosphate represents 0.36243 part of MgO. All that is necessary, therefore, is to multiply the amount of magnesium pyrophos- phate by the above factor to obtain the amount of MgO in 200 c.c. of urine. (7) Iron. Iron is practically always present in the urine in organic combination. The amount actually present is very small, being given by Magnier as vary- ing between 3 and 11 mg. to the Uter, while Neumann and Mayer find the normal output to average 0.983 mg. The urinary iron has little clinical significance. It is increased in fever, in malaria, diabetes melUtus, and pernicious anemia, the amounts in some cases running as high as 20 mg. during the 24 hours. Any method for the estimation of the urinary iron must be very delicate and very accurate. The urine must be completely incinerated, the method of Neumann^ employing a mixture of concentrated sulphuric and nitric acid being by far the best.^ (c) Organic Constituents. (i) Nitrogenous Bodies. (o) Total Nitrogen. From the metabolic standpoint the estimation of the total nitrogen of the urine is one of the most important features of its chemical examination. The excretion of total nitrogen varies with the amount of nitrogen of the food and with the degree of tissue metabohsm. Normally, the system so adapts itself to the nitrogenous intake that the excretion of nitrogen, in the urine, feces, perspiration, etc., is equal to the intake. In other words, a normal person is in nitrogenous equilibrium. This subject of nitrogenous equilibrium is of such great importance and the factors which influence it so varied that the writer feels that it will be unwise to discuss it briefly for fear of not presenting it clearly to the student's mind. As a full discussion of this subject would be too extensive for the scope of this work the -writer must be content with reference to the admirable discussion by Magnus-Levy' in von Noorden's Hand-book of Pathology of Metabohsm. 1 Arch. f. Anat. u. Physiol., Phjrsiol. Abth., 1900, S. 159. 3 Cmcago'^^oS-^'^"'''' ^^9^zg(y bff^NbM^' *-^^ "™"y """^ ''' pneumonia. 214 DIAGNOSTIC METHODS The total nitrogen of the urine may be taken as a direct index of the pro- tein metabolism. Upon a starvation diet, or one from which the nitrogenous factors have been eliminated, we find a gradual reduction in the amount of urinary nitrogen. From about the fourth day of starvation the excretion becomes practically constant and continues until severe tissue-decomposition, as a sign of impending death, occurs. If, at this time, a diet rich in nitrogen be given, a certain amount of the intake will be retained, but not all. If this increased nitrogenous diet be continued a certain portion will be retained each day until the system again assumes a condition of nitrogenous equilib- rium, the output equaling the intake. It is to be remembered in this connec- tion that the carbohydrates and fats of the diet both have a certain direct influence in diminishing the protein metabolism. The normal amount of total nitrogen of the urine, with the subject upon a mixed diet, varies between lo and i6 grams per day. The work of Chit- tenden' has proven that this is much too high for individuals who desire to get the most out of their system with the least possible work. In other words "physiological economy" is much better subserved by a diet yielding from s to 6 grams of total nitrogen. The subjects of his experiments showed normal activities, both mental and physical, and at the end of the experiments felt much better than before them and had gained in weight. Folin^ in his work has shown that the normal excretion of six subjects, each observed for a period of five days, was i6 grams of total nitrogen on a diet of 119 grams of protein yielding 18.9 grams of nitrogen. On a nitrogen-free diet the excretion aver- aged 3.6 grams. He says: "It may, therefore, be positively stated, as a principle in the chemistry of metabolism, that the distribution of the nitrogen in urine among urea and the other nitrogenous constituents depends on the absolute amount of total nitrogen present." The subject of the distribution of the various nitrogenous products of the urine has been much changed by this work of Folin and of Chittenden. This distribution or, as it is better called, the "nitrogen partition of the urine," varies according to the diet. The following table shows the excretion of the various nitrogenous constituents and their percentage relations to the total nitrogen under a mixed diet and under one which is nitrogen free. Excretion in grams. Percentage of total N. Mixed N-Free Mixed N-Free Diet. Diet. Diet. Diet. Nitrogen, 16.00 3.60 100.00 100. Urea N, 13.90 2.20 86.87 61.7 Ammonia N, 0. 70 0.42 4-37 "-3 Creatinin N, 0.58 0.60 3.63 17.2 Uric Acid N, 0.12 0.09 0.7s 2.S Undetermined N, 0. 70 0.29 4-37 7-3 1 Physiological Economy in Nutrition, New York, 1905 ; Mendel, Jour. Am. Med. Assn., 1914, LXIII, 819; Benedict, Jour. Biol. Chem., 1915, XX, 263; Pepper and Austin, Ibid., 1915, XXII, 81; Osborne and Mendel, Ibid., 241. ' Loc. cit. Digitized by Microsoft® THE tTRINE 215 It will thus be seen that the total nitrogen excretion of the urine is made up of several factors. The principal points to be gained from the above table are that the urea, on a nitrogen-free diet, is markedly reduced. This reduc- tion must naturally be made up by increase in the other factors. We find the ammonia and especially creatinin markedly increased from the percentage standpoint, although both are absolutely diminished. We have, therefore, a distinctly endogenous nitrogen metabolism as well as an exogenous one. The urea content should, consequently, be considered as of direct importance in estimating the degree of protein tissue metabolism, although not as usually taught. This urea output cannot at the present time be considered as representing from 85 to 90 per cent, of the total endogenous nitrogen, but should be regarded more properly as representing between 60 and 65 per cent. It is true that the urea as found under mixed diets gives this higher figure, but at least 20 per cent, of this must be placed against useless activity on the part of the system. In other words, the intake of sufiicient nitrogen to yield a urea excretion amounting to 85 per cent, of the total N must be considered unnecessary. This is a somewhat enlarged expression of the fact that most people eat much more than is utilized by the system.'^ It is seen, therefore, that in the general run of urine examinations the urea output does not represent to us the extent of the tissue metabolism, as or- dinarily we have not sufficiently controlled the diet. The above table pre- sents a remarkable percentage increase in the amount of creatinin. The figures of Folin show that the absolute quantity of creatinin eliminated, whether upon a nitrogen-rich or on a nitrogen-free diet, is remarkably con- stant for the same individual. Although this is influenced to a slight extent by the diet, it is so slight that it can be disregarded (see Creatinin). Physiologic Variations. An increase in the total nitrogen is observed after a heavy protein meal. As previously stated, in starvation the nitrogen of the urine becomes constant after about the fourth day. This nitrogen excretion observed in starvation is, however, less than the minimum amount that rnust be given in the form of protein in order to maintain a nitrogenous equilibrium. As a rule, the amount of protein taken in is much in excess of the requirement of the sys- tem, so that the amount excreted probably represents protein which has never become a part of the system. For this reason the fallacy of considering urea as a direct representative of the protein metabolism of the tissues becomes evident. The intake of protein is, in reality, readjusted to suit the actual needs of the body, so that the urea can represent only a portion, about 60 per cent., of the total metabolic activity of the tissue protein. A physiological increase in the excretion of nitrogen is observed in the infant for four or five days after birth. The nitrogen excretion is increased when the intake of water has been greater than normal. This fact should be borne in mind in metabolic experiments in which the intake of water should be quite as much regulated and a well known as the intake of other substances. Physiologically, a diminished output of nitrogen is observed on a low I See Mendel and Lewis, ^w^Bfo^ ^he^.^^ig^^I, 19, 37 and SS- 2l6 DIAGNOSTIC METHODS nitrogen diet and also on a diet rich in carbohydrates and fat, as these latter substances provide the greater part of the necessary energy. The system must then utilize its own protein. Increased exercise is supposed to give a slight increase in the nitrogen output owing to increased muscular activity, but it is to be said that the loss of water through the increased perspiration may be such a factor in diminishing the urinary nitrogen that no increase may be observed. Certain medicaments as quinin and opium will usually diminish the output of nitrogen. Pathologic Variations. Increased Excretion. Perhaps the most marked increase in the urinary nitrogeri is observed in the acute febrile infections.^ This increase is not due to the temperature per se nor is there any parallelism between the urinary nitrogen and the degree of temperature. Whether or not this increase in nitrogen can be directly traced to the effects of the toxins produced by the organisms causing the dis- ease is still unsettled. That this cannot be the only element is proven by the fact that Krehl and Matthes have shown that more protein is destroyed in the so-called aseptic fever than is the case in the normal organism under simi- lar conditions, dietetic and physical. In some febrile conditions we find that the elimination of nitrogen may be reduced during the febrile period, while about the time of crisis a very marked output of urinary nitrogen may be ob- served. This is the well-known epicritical elimination of nitrogen. In the fever associated with acute nephritis the urinary nitrogen is not increased, but is rather diminished owing to the renal insufficiency as well as to the edema which occurs. A toxogenic decomposition of protein is found in cases of car- cinoma, pernicious anemia, chronic tuberculosis, leukemia, scurvy, and es- pecially in exophthalmic goiter. In cases of acute yellow atrophy and phos- phorous poisoning the total nitrogen may be increased, but the percentage of urea will be very much diminished. In cases of diabetes mellitus the nitrogen excretion is usually much in- creased, due more to the effect of the increased nitrogenous dist than to increased endogenous protein metabolism. Likewise, in diabetes insipidus a large increase in urinary nitrogen may be observed. An increase is oc- casionally observed in cases of nephritis owing to the large albumin content of the urine. During the progress of absorption of an exudate a very high excretion may be observed, the resolution of a pneumonic exudate, for in- stance, being easily followed by the variations in the urinary nitrogen.^ Diminished Excretion. A diminution in the nitrogen excretion is usually observed in convales- cence from acute and chronic conditions. This is probably due to the at- tempt on the part of the system to make up for the losses incurred during the active progress of the disease. It may be diminished in conditions in which the absorptive power of the intestine is much reduced. If the oxidative 1 See Sharpe and Simon, Jour. Exper. Med., 1914, XX, 282. 2 See Matthews and Miller, Jour. Biol: Chem., 1913, XV, 87; also, Gammeltoft, Habilita- tionschr., Kopenhagen, iQis-D/g/f/zecf by Microsoft® THE URINE 217 powers of the system are very much reduced as the result of chronic conditions, the urinary nitrogen will usually be much diminished. In cases of nephritis, both acute and chronic, a large diminution in the urinary nitrogen is observed. This is due primarily to the renal insuflBiciency and to the associated dropsy. In such conditions a marked increase may be observed in the fecal nitrogen, especially when marked diarrhea is a complicating factor. These periods of retention of nitrogen in nephritis may alternate with periods of increased elimination, so that examinations at different periods may show greatly con- flicting results. When the water output of the urine is largely diminished, as a result of transudation, of exudation, or of increased perspiration, the total nitrogen may be reduced.^ Estimation of Total Nitrogen (Kjeldahl). The principle of this method is as follows i'^ The nitrogenous constituents of the urine are oxidized by various oxidizing agents into ammonia. This ammonia is converted into ammonium sulphate by the sulphuric acid which is added at the same time as the oxidizing agents. After the preliminary de- composition and oxidation of the organic nitrogen into ammonium sulphate, free ammonia may be liberated by the action of strong sodium hydrate and distilled into a standard acid solution. Knowing the strength of the acid solution, one may then titrate the remaining acid with a standard alkali solu- tion and determine how much ammonia has combined with the acid. One c.c. of tenth-normal sulphuric acid, used by the ammonia liberated in the distillation, represents 0.001401 gram of nitrogen. Technic. Five c.c. of urine are accurately measured, either with a pipet or buret, into a Kjeldahl flask of Jena glass of 800 c.c. capacity. Ten c.c. of concentrated sulphuric acid and approximately i gram of copper sulphate are added, the flask placed in a hood and heated over a low flame until white fumes of sul- phuric acid are given off (Gunning's modification). Five grams of potassium sulphate are then added and the mixture heated with an increased flame to boiling for one-half to three-fourths of an hoiir. The solution should have lost every trace of a yellowish color and should have become by this time a clear bluish-green. The worker should be cautioned regarding the fumes given off in this process and conduct his work only in a hood with a good draft. It is frequently necessary to wash down the carbon from the sides of the vessel by shaking the fluid in such a way that the carbonized material is carried down to the bottom of the flask. One should be cautious lest he lose some of the liquid in this manipulation, which would not only throw out his determination but might result in a very severe burn should any of the material fall upon him. The mixture is allowed to cool completely before the further steps of the determination can be taken. Most workers advise at this juncture the trans- ference of the material from the first flask into a second distilling flask. The ' Minot, Bull. Johns Hopkins Hosp., 1914, XXV, 332; Hefter and Siebeck, Deutsch. Arch. f. klin. Med., 1914, CXIV, 497; Frothingham and Smillie, Arch. Int. Med., 1915, XV, 204; Murray, Brit. Med. Jour., 1915, 1, 131- " See Folin and Farmer (Jour. Biol. Chem., igi2, XI, 493) for a micro-chemical method based on this Kjeldahl process. ..TOsmetW is discu^d in the section on Blood. 2l8 DIAGNOSTIC METHODS writer has convinced himself that this procedure is not only unnecessary but is even unwise, as the transference may result in slight loss of material. He is therefore, accustomed to use the same flask both for the oxidation and distillation. After the mixture has cooled the neck of the flask is thoroughly washed with a stream of distilled water so that every trace of material may be carried from the neck into the body of the flask. Sufficient additional water is added to bring the total up to approximately 250 c.c. A Uttle talcum powder, a few pieces of pumice stone, or a few pieces of granulated zinc may then be added to prevent bumping of the contents when sodium sulphate separates out later in the process. Fifty c.c. of 40 per cent, sodium hydrate are then added for every 10 c.c. of sulphuric acid used in the original oxidation. Care should be Fig. 72. — Kjeldahl's nitrogen apparatus. taken in adding this strong alkali that none of it touches the upper portion of the neck of the flask. The alkalinized mixture is then shaken and connected with a Fresenius bulb which is attached to a Liebig condenser as shown in the accompanying cut. The outlet tube passes into an Erlenmeyer flask which should contain 50 c.c. of tenth-normal sulphuric acid. In cases with abnor- mally high nitrogen values it may be necessary to use a larger quantity than 50 c.c. of standard acid, but the writer has found only two instances in over 1,500 determinations in which an increased amount was necessary. The con- nection of the distilling flask to the bulb and condenser should be done rapidly to avoid any possible loss of ammonia. The distilling flask is heated slowly at first and the heat increased only after boiling has become regular. If heated too quickly, spurting of the liquid may occur and trapj^&fTa^^j/ljSfli^Fgpi^^to the bulb and thence over THE URINE 219 into the standard acid. The distillation should continue until about 150 c.c. have been distilled over, which will take from 20 to 30 minutes. The writer has observed that bumping of the mixture rarely occurs, under the conditions outlined above, before the ammonia is completely driven over. This can, however, not be taken as an absolute sign that every trace of ammo- nia has been distilled off. In order to see whether such is the case, one must test the outlet tube with a piece of moist red litmus-paper which will turn blue in the presence of traces of ammonia. If all ammonia has not been given off, the distillation must be continued until such is the case. If no more ammonia is being evolved, the distilling flask is disconnected from the bulb so that no suction may draw the standard acid into the condenser. The connecting bulb is removed and the tube of the condenser washed with a spray of distilled water so that any material adhering may be washed into the standard acid. The outlet tube is disconnected and washed both internally and externally into the standard acid solution.^ The standard acid solution is then titrated with tenth-normal sodium hy- drate solution, using cochineal, methyl-orange, alizarin-red, or methyl-red as an indicator. The writer prefers the use of the latter. As each c.c. of tenth- normal alkah is equivalent to each c.c. of tenth-normal acid, we subtract, from the original number of c.c. of acid (50), the number of c.c. of standard alkali used to neutralize the remaining standard acid. The difference gives us the number of c.c. of standard acid neutraUzed by the ammonia given off in the distillation. As each c.c. of tenth-normal acid is equivalent to 0.001401 gram of nitrogen, we multiply this factor by the number of c.c. of acid neutralized by the ammonia and obtain the amount of total nitrogen in 5 c.c. of urine. This result multiplied by 20 yields, of course, the percentage of total nitrogen, which may be changed into the actual total amount of nitrogen by multiply- ing it by the number of hundreds of c.c. in the 24-hour specimen. It goes without saying, that the reagents used in this determination must be ammonia-free or, at least, that their ammonia content be known. (6) Urea (NH2)2CO. A discussion of the various factors which have to do with urea excretion cannot be taken up at this time. The recent work in the laboratory of Hof- meister^ as well as that of Chittenden and of Folin has shown us conclusively that it is no longer possible to consider the rate of urea formation as a direct measure of protein metabolism. It is without question true, as Leathes' has said, that the nitrogen, or a great part of it, may be removed from the protein, converted into urea, and expelled with the urine before the oxidation of the rest of the protein molecule has been started upon; and the fact that we can trace in the urine excreted in a given time all or the greater part of the nitro- gen of the protein taken at a meal, tells us nothing whatever about the fate of that part of the protein which contains, it may be, as much as 80 or 90 per cent, of the total energy of the protein. Further, urea is not a measure of the ' See Dakin and Dudley, Jour. Biol. Chem., 1914, XVII, 275. ' Lang, Beitrage zur chem. Phys. und Path., 1904, V, 340. 3 Problems in Animal ^^^md^lVlimhoft® 2 20 DIAGNOSTIC METHODS true protein catabolism, because a great part of it is formed from nitrogen that has never been beyond the liver; and it is not the measure of the protein energy because it is largely derived from protein by reactions which leave the energy value of the molecules from which it originates but little altered. The importance of the denitrifying and desamidizat on reactions of the tissues must be much more considered in the future than they have been in the past.' As usually stated in text-books, urea constitutes from 80 to 85 per cent, of the total nitrogen output. It has been customary to figure directly the amount of tissue protein which must have been decomposed in order to yield this amount of urea. These figures can hardly be taken as conclusive of such decomposition. Folin has shown that a definite exogenous as well as an en- dogenous protein metabolism occurs. With a patient upon a nitrogen-free diet, the urea constitutes only about 60 per cent, of the total nitrogen. This would represent the true endogenous urea formation. A diet which requires the patient to eliminate much higher percentages of urea is, therefore, causing increased systemic activity, but is not increasing the direct tissue decomposi- tion, as this excess never becomes a part of the system. One of the most important laws of protein metabolism is that the amount of nitrogen in the body is not increased by, or not in proportion to, an increase in the nitrogen intake. The amount of urea excreted on an average diet varies from 15 to 40 grams. ^ Folin finds this excretion to be, on a diet of 119 grams of protein yielding 18.9 grams of nitrogen, 298. grams. On a nitrogen-free diet the amount of urea is 2.2 grams. This excretion will, of course, vary, depending upon the diet. Von Jaksch states that the excretion of urea bears a definite relation to the total nitrogen excretion, so that for clinical purposes direct urea determinations may well be dispensed with, as the correct urea-content of tlie urine may be found by multiplying the simple nitrogen of the urine by the factor 2. This statement should not be regarded seriously by the practitioner as the rule, as Folin shows, would require that 93.3 per cent, of the total nitro- gen in the urine be in the form of urea. Pathologic Variations. The pathologic increase in the amount of urea is observed under the same conditions as those mentioned under an increase of total nitrogen. Thus in febrile conditions, in diabetes mellitus and insipidus,' after the resorption of an exudate, in malignant conditions, and in exophthalmic goiter, the urea may be markedly increased. ; In conditions associated with destruction of hepatic parenchyma, or with a diminished rate of blood-flow through the liver, the urea excretion may be very considerably diminished.^ Thus we find in acute yellow atrophy, ^ See Henriques and Anderson, Ztschr. f. physiol. Chem., 1913, LXXXVIII, 357; Ibid., 1914, XCII, 21; Davis, Bull. Johns Hopkins Hosp., 1915, XXVI, 154. 2 See McLean (Jour. Exper. Med., 1915, XXII, 212 and 366) for a discussion of the law governing the rate of urea excretion. ' See Bassler, Jour. Am. Med. Assn., 1914, LXII, 282. ^ See Fiske and ^^^^^^^, ^^flM S^mcfd^M^' 399- THE URINE 221 carcinoma, cirrhosis, and phosphorus poisoning that the normal urea of the urine is replaced by other nitrogenous constituents. The normal function of the Uver in converting ammonium compounds and amino acids into urea is so markedly interfered with that the urea may completely disappear from the urine in such cases. It should be mentioned that the liver is not wholly responsible for the conversion of the ammonia and amino acids into urea, as the kidney unquestionably plays some role in this process.^ In acute nephritis there may or may not be a diminution in the excretion of urea, depending upon the extent of the renal insufficiency. In the chronic types of nephritis we find that the urea excretion fluctuates to a great extent, periods of increase varying with those of decrease. In the early stages, even though large amounts of albumin and casts be present, the urea may be normal, while in the later stages it is often greatly diminished. A diminished excretion of urea is observed in melancholia and in the ad- vanced stages of general paresis, while in epilepsy and hysteria an increase or a decrease may be observed. In some cases of diabetes melUtus Hirschfeld has shown that the urea output may be diminished as the result of delayed absorption from the intestine. In these cases of diabetes the ammonia of the urine may be markedly increased owing to its combination with acid bodies and consequent withdrawal from hepatic activity. While urea is a very important substance both clinically and chemically, it is very rarely tested by qualitative methods in medical work. The writer feels, therefore, that a description of the properties of this substance would best be learned by consulting works on physiologic chemistry. Deteiminatioii of Urea. The methods for the determination of urea are numerous. Many of them are inaccurate although giving results chnically of importance. In the selec- tion of a method for the determination of urea one must be governed en- tirely by the importance of the urea determination in any specific case. It should be said in advance that a urea estimation is absolutely useless unless taken in conjunction with the total nitrogen. The general practitioner usually insists on knowing both the percentage and total amount of urea ex- creted with utter disregard both for the nitrogen of the intake and of the output. If the urea is of any value at all it should be determined with these points in view. The prevailing idea is that a percentage output of two is approximately normal and he therefore bases his conclusion upon an increase or decrease with this as a standard. Not infrequently single voidings of urine are examined for the urea output. Such examinations are worse than useless and may even be harmful. The methods for the determination of urea are distinctly separable into those useful for purely clinical purposes and those for the more exact metabolic work. The general practitioner, believing as he does in the importance of urea as an indicator of systemic activity and excretion, must have a rapid and ' See Folin, Jour. Biol. Chem , 1912, XI, 87; Van Slyke, Ibid., 1913, XVI, 213; Marshall and Davis, Ibid., 1914, XVIII, 53; Fiske and Sumner, Ibid., 285; Jansen, Ibid., 1915, XXI, SS7. Palacios, Am. Jour. Med. Sc, 1915, CXLIX, 267. Digitized by Microsoft® 222 BIAGNOSTIC METHODS easy method for estimation of urea. It is to be said, however, that the more exact methods would better be applied if reliable conclusions are to be drawn. Knop-Hufner Method. The principle of this method is the decomposition of urea by means of sodium hypobromite and the measurement of the nitrogen evolved. Sodium hypobromite acts upon urea according to the following equation: CO(NH2),+3NaOBr = 3NaBr+2N+C02+2H,0. The carbon-dioxide evolved in this reaction is absorbed by the excess of alkali used, so that all that is necessary is to measure the amount of nitrogen evolved. This can be done by direct measurement or by collection in tubes which are so calibrated that each c.c. of nitrogen represents a certain percentage of urea. Various forms of apparatus, which are termed ureometers, have been advised for the estimation of urea by this method. The form introduced by Knop and Hiifner is probably the most accurate, but is too complicated for general clinical purposes. As this method has absolutely no claim to accuracy it is use- less in scientific investigations of the nitrogen partition of the urine. For clinical purposes, however, it serves as a rough approximation of the urea output and is, for this reason, largely used by the practitioner.^ This method, applied as in the following discus- ission, given merely because of the fact that it is almost the only method of estimating urea which can be carried out by the general practitioner. Fig. 73. — Doremus ureometer. Doremus Ureometer. This instrument is seen in the accompanying cut. The graduations of the tube are such that the number of mg. of urea in the i c.c. of urine used in the test are directly read off instead of the number of c.c. of nitrogen formed in the reaction. Aso.oi gram of urea in one c.c. represents i gram per 100 c.c, this tube will furnish directly the percentage values of urea. The tube is filled with a solution of sodium hypobromite made by adding I c.c. of bromin to 40 c.c. of 20 per cent, cold sodium hydrate solution. This hypobromite solution decomposes after standing for a few days so that it is never wise to attempt to keep such a solution for any length of time. In the writer's laboratory a stock solution of 20 per cent, sodium hydrate is prepared and the bromin added to it only as occasion requires for preparing fresh solu- tions. In this way one will always have the material at hand and need have no fear of his stock solution decomposing. After filling the tube with this 1 See Krogh, Ztschr. f. physiol. Chem., 1913, LXXXIV, 379; Robinsoa and Mueller, Jour. Am. Med. Assn., i9i4,^^fegy.Jby Microsoft® THE URINE 223 hypobromite solution, i c.c. of urine is added by means of the curved pipet accompanying the instrument. In injecting the urine into the solution, the curved end of the pipet should be passed well under the curve of the bulb, the tube tilted slightly forward, and the urine forced into the hypobromite solu- tion with a slow steady pressure. An evolution of gas will be observed at once and will cease in a short time. The carbon dioxid given off by the re- action of the urea upon the sodium hypobromite is absorbed by the excess of alkaU and the nitrogen collects in the upper portion of the tube. As soon as the evolution of gas has ceased (5 to 10 minutes), the amount of urea is read off directly from the calibrations of the tube as previously described. In this determination the urine should, theoretically, be free from both albumin and sugar. Practically, however, these sub- stances are never removed as the method yields comparative rather than accurate results. Indeed, a very delicate reaction for albumin is given by this test. One may be practically certain of the presence of al- bumin, if the urine, on being injected into the solution, forms rather characteristic heavy bubbles and if a dense froth collects in the upper portion of the tube. This froth settles slowly so that the percentage reading requires some time. The forms of this apparatus which substitute for the glass foot a wooden base are much to be preferred as they are not so easily broken. The modification of this instrument, as in- troduced by Hinds, is seen in the accom- panying cut. In this form the urine is allowed to run in from the smaller gradu- ated tube by opening the stop-cock. This modification is an advantage, but does not jdeld any more accurate results than does Fig. 74.— Doremus-Hinds ureometer. the preceding. (Hawk.) Folin'si Method. The principle of this method is as follows: At a temperature of 160° C. crystallized magnesium chlorid (MgCl26H20) boils in its water of crys- tallization. If urea be present it is decomposed by this boiling solution into ammonia and carbon dioxid. If the conversion be carried out in acid solu- tion, the ammonia formed will combine with the acid and may then be lib- erated by alkalinizing the mixture. The ammonia is distilled into a stand- ard acid solution and may then be determined as given under the total nitrogen. In this process the preformed ammonia as also the trace present 1 Ztschr. f . physiol. Chem£)^affe^pPpIJWH5?zD^6#®i903> XXXVII, 548. 224 DIAGNOSTIC METHODS in the magnesium chlorid will also be determined so that a separate estima- tion of these factors must be made and subtracted from the total amount. Technic. Five c.c. of urine are measured into an Erlenmeyer flask of about 200 c.c. capacity, 5 c.c. of concentrated hydrochloric acid, 20 grams of crystallized magnesium chlorid, a piece of paraffin about the size of a hazelnut and two or three drops of a i per cent, aqueous solution of alizarin-red are added. An especially constructed safety-tube (see cut) is then inserted and the mix- ture boiled until the drops flowing back from the safety-tube produce a very perceptible bump or hissing sound on coming in contact with the solution (10 to 15 minutes). The temperature^ is then somewhat reduced and the heating continued for one hour. It is important in this process ,v__-|in.— Jl ^^I^K. that the reaction must not remain alkaline, '^ ^e^£— «% and, therefore, as soon as the material m the flask turns red a very few drops of the acid distillate in the safety-tube are shaken back into the flask. ^ At the end of the hour the contents of the flask are washed into a liter Kjeldahl flask with about 700 c.c. of water. Twenty c.c. of 10 per cent, sodium hydrate are then added and the ammonia distilled, as described under total nitrogen, into a stand- ard tenth-normal solution of sulphuric acid. This distillation should be continued until the contents of the liter flask are nearly dry or till the distillate shows no trace of ammonia with litmus paper. This will require about one hour. The distillate is then boiled to drive off the carbonic acid, is then cooled, and titrated with tenth-nornial sodium hydrate to determine the amount of acid which com- bined with the ammonia formed. Alizarin- red or methyl red are used as indicators. One c.c. of tenth-normal sulphuric acid is equivalent to 0.001704 gram of ammonia (NH3), or 0.001401 gram of nitrogen. If the nitrogen value be multiplied by 2.143 the result will be the amount of urea in the 5 c.c. of urine taken. From the total c.c. of n/io sulphuric acid neutralized must be subtracted the n/io sulphuric acid values for the pre- formed ammonia as well as for the ammonia which may be present as an impurity in the 20 grams of magnesium chlorid used. 1 Folin advises (Handb. d. Biochem. Arbeitsmeth., 1911, V, 286) the use of small glass bulbs containing solid HgClI as an indicator of the proper temperature of this reaction. This substance melts at iS3°C., which is the optimum temperature to insure complete de- composition of the urea. ' See Folin, Jour. Biol. Chem., 1912. XI, 507. Digitized by Microsoft® Fig. 75. — Folin's urea apparatus {Hawk.) THE URINE 225 It has been found by Schoorl that when carbohydrates and urea are heated together they form very stable condensation products (ureids). For this reason this method of Fohn does not give accurate results with saccharin urine. A combination of this method with that of Morner, which will be described later, will give absolutely accurate results. In the determination of the ammonia values of the preformed ammonia and of the magnesium chlorid, the later methods of Folin must be used. Momer-Sjoqvist Method. This method^ is an extremely accurate one, but no more so than that of Folin, except in saccharin urines. If albumin be present it must, however, be removed by heat and acetic acid and the original volume of the urine re- stored. If the urine contains large amounts of hippuric acid this method may not give accurate results, as Salaskin and Zaleski have shown. Technic. Five c.c. of urine are placed in a flask with 5 c.c. of baryta mixture con- sisting of a saturated barium chlorid solution containing 5 per cent, of barium hydrate. One hundred c.c. of a mixture of two parts of 97 per cent, alcohol and one part of anhydrous ether are then added and the mixture allowed to stand in a closed flask overnight. It is then filtered and the residue washed with fresh alcohol and ether mixture and the combined filtrates evaporated at a low temperature (6o°C.). Urea will be practically the only nitrogenous body left in solution, with the exception of traces of ammonia. When the evaporated filtrate has been reduced to about 25 c.c. in volume, a few c.c. of water and a small amount of calcined magnesium oxid are then added, the mixture stirred, and heated to drive off the ammonia; or the residue may be treated by Folin 's method as previously described. This heating is con- tinued until the vapor shows no alkalinity when tested with moistened litmus paper, a result usually obtained when about 10 to 1 5 c.c. of the mixture remain. The fluid and the residue are then washed into a Kjeldahl flask and treated with concentrated sulphuric acid, copper sulphate, and potassium sulphate as in the determination of total nitrogen. One part of nitrogen is equivalent to 2.143 grams of urea. If much hippuric acid be present, Braunstein^ advises oxidation of the urea by heating the evaporated residue with 10 grams of solid phosphoric acid (in an air-bath) to 140 to I4S°C. for five hours. Alkalinize and distill as previously described. Benedict's' newer method also yields accurate results. Urease Method. Marshall has introduced a method for the determination of urea in various body fluids, which is based on an entirely different principle from the others advocated and which is accurate and extremely simple.* He takes advantage 1 Skand. Arch. f. Physiol., 1891, II, 438; Ibid., 1903, XIV, 247. 2 Ztschr. f. physiol. Chem., 1901, XXXI, 381. ' Jour. Biol. Chem., 1910, VIII, 405. See, also, Milroy, Biochem. Jour., 1913, VII, 399. * Takeuchi, Jour. College Agriculture, Tokio, 1909, 1, i; Chem. Ztg., 1911, XXXV, 408; Marshall, Jour. Biol. Chem., 1913, XIV, 283; Ibid., 1913, XV, 495; Hahn and Saphra, Deutsch. med. Wchnschr., 1914, XL, 430; Van Slyke and Cullen, Jour. Am. Med. Assn., IS Digitized by Microsoft® 226 DIAGNOSTIC METHODS of a fact, first discovered by Takeuchi, that an aqueous extract of soy bean {glycine hispida) contains a ferment which converts urea into ammonium carbonate and which neither acts upon anything else nor is interfered with by anything ordinarily found in the various body fluids. In his method Mar- shall employs this aqueous extract, but this has the disadvantage of requiring many hours to react quantitatively and, further, of losing its activity on standing for a few days, the loss being more rapid the higher the temperature at which it is kept. For these reasons the writer prefers to adopt the modifica- tion of Van Slyke and Cullen. These workers have produced a permanent preparation of this ferment by the following method: Extract soy bean meal with 5 parts of water and pour this extract into lo volumes of acetone. Filter off the precipitate and dry it.^ This urease dissolves readily in water, forming an opalescent solution. "Under given conditions, a given amount of it will decompose a definite amount of urea per minute and no more, regardless of how much excess urea may be present. The preparation is most active at a temperature approxi- mately S5°C. (i3i°F.) and in a perfectly neutral solution. Between lo and So°C. (so and i22°F.), increasing the temperature over any io° interval doubles the rate at which the preparation acts. Heavy metals, or acid in a relatively slight concentration, destroy the activity. The urease is not quite so sensitive toward an alkaline reaction, but its activity is decreased by even weak bases. Ammonium carbonate depresses the activity to about one-eighth the optimum, so that the action of the enzyme is self-retarding. The retard- ing effect of the ammonium carbonate formed can be neutralized and conditions for optimum activity obtained by the addition of potassium di- hydrogen phosphate and dipotassium hydrogen phosphate in equimolecular proportions. This phosphate mixture also acts as a stabilizer to solu- tions of enzyme." Preparation of Enzyme Solution. Two grams of the enzyme preparation, 0.6 gram of dipotassium hydrogen phosphate and 0.4 gram of potassium dihydrogen phosphate are stirred up with a rod in 10 c.c. of water. The preparation dissolves quickly, forming an opalescent solution. If this solution be covered with toluol it will hold its activ- ity for a week or so, but it is better technic to employ relatively fresh solutions. Technic. This method employs a portion of the technic originally introduced for determination of ammonia by Folin (see page 230). Dilute 5 c.c. of urine with water to 50 c.c. Mix this thoroughly and transfer, by means of a pipet, 5 c.c. of this diluted urine to a clean, fairly heavy test-tube of about 50 c.c. capacity. Add i c.c. of the enzyme solution and i drop of caprylic alcohol or a few drops of benzol to prevent foaming during the later aeration. Close 1914, LXII, 1558; Jour. Biol. Chem., 1914, XIX, 141 and 211; Van Slyke, Zacharias and Cullen, Deutsch. med. Wchnschr., 1914, XL, 1219; Plimmer and Skelton, Biochem. Jour., 1914, VIII, 70; Hahn, Deutsch. med. Wchnschr., 1915, XLI, 134; Eigenberger Ztschr.f. physiol. Chem., 1915, XCIII, 370; Jacoby and Sugga, Biochem. Ztschr., 1915, LXIX, 116. ' If one does not wish to prepare this urease for himself, he may obtain it from the Ar- lington Chemical Company of Yonkers, N. Y. Digitized by Microsoft® THE URINE 227 this tube (A) with a stopper and allow it to stand 1 5 minutes in order to per- mit the enzyme to act upon the urea present. Into a second test-tube (B) measure with a buret 25 c.c. of fiftieth normal (N/50) hydrochloric or sulphuric acid and add i drop of a i per cent, solution of alizarin as an indicator and i drop of caprylic alcohol or benzol. By means of glass and rubber tubing connect these two tubes in such a way that air may be forced through tube A into tube B (see page 230). After tube A has stood for 15 minutes, pass the air current for about }^ minute in order to carry over into the acid any ammonia which may have passed into the upper portion of tube A. Now shut off the air, open tube A and pour into it 4 or 5 grams of dry potassium carbonate. Close tube A at once and pass the air current till all the ammonia is driven into tube B. This time depends on the rate at which the air is passed. With the ordinary apparatus this varies from 15 to 30 minutes. When this process is finished, titrate the excess of acid in tube B with fiftieth normal (N/so) sodium hydrate, titrating to the appearance of the first permanent red point. Subtract the number of c.c. of standard alkali used from 25 (the number of c.c. of standard acid originally measured into tube B) and multiply this result by 0.056 to obtain the grams of urea nitrogen plus ammonia nitrogen in 100 c.c. of urine. The ammonia nitrogen, which is not referable to the decomposition of urea, is obtained by the same method as the above, using 5 c.c. of undiluted urine and adding no urease solution. In this latter case multiply the number of c.c. of excess acid by 0.0056 instead of 0.056 to determine the percentage of ammonia nitrogen originally present in the urine. Subtract this latter figure from the figure for the combined nitrogen of urea and ammonia and obtain the true .figure for the ammonia nitrogen derived from the action of the urease. If this nitrogen figure be multiplied by 2.143, one obtains the percentage of urea present as such.^ (c) Ammonia (NH3). This substance, although chemically belonging in the class of inorganic compounds, is so closely related to the nitrogen metabolism that it is more properly discussed under the heading of Nitrogenous Bodies. Ammonia is one of the most important products of protein metabolism. It is constantly present in small amounts in normal urine averaging about 0.85 gram of NH3 in 24 hours, representing from 4 to 5 per cent, of the total nitrogen. It is present in combination with various acids and may repre- sent largely a portion of the nitrogen which has not been transformed into urea, but has been used to combine with acid substances formed in the protein metabolism of the body. Any increase in the production of acid in the system or any increased intake of noncarbonate-forming acids will lead to an increased excretion of ammonium salts. This is an important factor in the metabolism of conditions associated with an acidosis. ' Instead of using the method of aeration, one may titrate directly the ammonium car- bonate formed by the action of the urease on the urea, using somewhat larger amounts of urine (5 c.c.) and titrating with N/io hydrochloric acid, with methyl-orange as an indicator. This was the original method of Marshall, but it does not yield as accurate results as the technic described. Digitized by Microsoft® 228 DIAGNOSTIC METHODS The total output of ammonia will vary under normal conditions with the diet or, in other words, with the intake of total nitrogen. ' While the increase of the total nitrogen of the urine or increased nitrogen intake is largely in the form of urea, yet a small increase in the absolute amount of ammonia must occur. Likewise we observe a diminished intake of nitrogen reducing the absolute value of ammonia, but largely increasing its relative value. Thus Folin finds with a total excretion of i6 grams of nitrogen, an ammonia output of 0.85 gram (4.3 per cent.); while on a nitrogen-free diet a total nitrogen output of 3.6 grams was observed with an ammonia elimination of 0.51 gram (11.3 per cent.). We therefore conclude with Folin as follows: "With pronounced diminution in the protein metabolism (as shown by the total nitrogen in the urine), there is usually, but not always, and therefore not necessarily, a decrease in the absolute quantity of ammonia eliminated. A pronounced reduction of the total nitrogen is, however, always accompanied by a relative increase in the ammonia nitrogen, provided that the food is not such as to yield an alkaline ash." Although the ammonium salts of many organic acids are converted into urea in the system we find the ammonium salts of the sulphuric and phos- phoric acids formed in the decomposition of protein material are excreted as such. Moreover, we observe that an increased consumption of fat, either taken in as food or derived from the tissue, is associated with a combination of ammonia with the fatty acids. This provision of metabolism, by which the system is protected against the deleterious effects of increased acidity by neutralization of acid compounds with ammonia, is of the greatest impor- tance, as the fixed alkalies of the tissues are thereby maintained in their usual concentration unless the pathologic processes be extreme. Pathologic Variations. An increased output of ammonia is observed in cases of diminished oxida- tive powers of the system, in febrile diseases, in hepatic disturbances such as carcinoma and acute yellow atrophy, in uremia, in acid intoxication, in dyspnea from any cause, in the toxib, vomiting of pregnancy, in delayed chloro- form poisoning, and especially in diabetes mellitus. In this latter condition the degree of acidosis may be conveniently followed by watching the ammonia output. A reduction in the amount of ammonia is observed in many cases of nephritis and in some cases of carcinoma of the stomach, although there is at the same time a diminished excretion of hydrochloric acid in the gastric contents. Edsall reports a reduction in cases of periodic insanity preceding the attack, while a rise is observed as the attack proceeds. Administration of large doses of the fixed alkalies will usually diminish the ammonia output.^ Quantitative Determination of Ammonia. Like the methods given under Urea, many have been advanced for the determination of the urinary ammonia. One of these, though inaccurate, has been so long used and even to-day is so rehed upon in many quarters that 1 See Tanji, Deutsch. Arch. f. klin. Med., 1914, CXVI, 92. ' See Janney, Ztschr. f. phgid^Che^^jj^XXXV^g. THE URINE 229 the writer includes it with the understanding that he advocates only the ac- curate methods. If the ammonia is worth determining, definite results should be sought and hence the most accurate methods are the ones to be used. They are no more complicated, not as time-consuming, and give more reliable results. Method of Schlosing. This method is the one most commonly used, but is open to the objection that it does not yield accurate results and is time-consuming, but has the advantage of simplicity. It is argued by many that the element of time is of no importance, as clinically one would not wait for an ammonia determination before instituting vigorous treatment. On the other hand, in metabolic work it is of a great advantage to get the work out of the way as quickly as is possible and consistent with accurate results. Technic. Twenty-five c.c. of urine are placed in the vessel B (preferably a Petri dish) (see cut). Above this is placed a glass triangle upon which rests a dish Fig. 76. — Schlosing's ammonia apparatus. (C) containing 20. c.c. of tenth-normal sulphuric acid. Twenty c.c. of milk of lime are then poured into the dish containing the urine and the whole cov- ered with a bell-jar, the borders of which have been well greased to make an air- tight union when the jar is placed upon the glass plate. This apparatus is then allowed to stand at room temperature from four to five days, during which time the ammonia, liberated by the action of the milk of hme upon the ammonium salts of the urine, will be taken up by the sulphuric acid in the vessel C. At the end of this time the bell-jar is removed, the acid titrated with tenth-normal sodium hydrate, and the number of c.c. of remaining acid determined. One c.c. of tenth-normal sulphuric acid neutralized by the evolved ammonia represents 0.001704 gram of ammonia. This figure is Digitized by Microsoft® 230 DIAGNOSTIC METHODS multiplied by 4 to obtain the percentage ammonia value. If any moisture is present on the inside of the bell-jar it should be washed into the sulphuric acid before titration. This method, as previously stated, does not give accurate results owing to the fact that ammonia may be split off from urea and thus give figures which are somewhat high. It has been found that if the apparatus be kept at 38°C., the time necessary for this reaction may be reduced to 48 hours. If we add to the urine instead of the milk of Ume, 0.5 gram of sodium carbon- ate and about 10 grams of sodium chlorid, no ammonia will be split off from the urea and no decomposition of the urine will occur (Schaffer). Fig. 77. — Folin's ammonia apparatus. {Hawk.) Folin's Method. The ammonia in this method"^ is set free by the addition of a weak alkali (sodium carbonate), is then removed from the urine at ordinary room tem- perature by means of a strong air-current, is collected in tenth-normal sul- phuric acid and then titrated. Technic Twenty-five c.c. of urine are poured into an aerometer cylinder (30 to 40 cm. high), 15 grams of potassium oxalate,^ i gram of dry Na^COsand some crude petroleum (to prevent foaming) are added. The upper end of the cylinder is then closed by means of a doubly perforated rubber stopper, through which pass two glass tubes, only one of which is long enough to reach below the surface of the liquid.' The shorter tube (about 10 cm.) in length is connected iZtschr. f. physiol. Chem., 1902, XXXVII, 161; Ibid., 1903, XXXIX, 477. 2 Jour. Biol. Chem., 1910, VIII, 497. ' Folin and MacaUum (Jour. Biol. Chem., 191 2, XI, 523) have originated a micro-chem- ical method, which permits of much quicker estimation, i to 5 c.c. of urine, depending on the amount suspected, are placed in a large Jena test-tube by means of an Ostwald pipet. Add to the urine a few drops of a solution containing 10 per cent, potassium carbonate and I s per cent, potassium oxalate and follow this with a few drops of kerosene or benzol to pre- vent foaming. Pass the strong air current for 10 minutes, collecting the ammonia in a 100 c.c. volumetric flask containing about 20 c.c. of water and 2 c.c. of N/io hydrochloric acid. Nesslerize this solution against a standard ammonium sulphate solution as described in section on Blood or titrate ^^ fff^j§^ff>lf^f^iffy^(fff^y^-'^^i as an indicator. THE URINE 231 ■with a calcium chlorid tube filled with cotton, which in turn is conjiected with a glass tube extending to the bottom of a wide-mouthed bottle (capacity about 500 c.c.) which contains 20 c.c. of tenth-normal sulphuric acid, 200 c.c. of water and a few drops of an indicator (alizarin-red). The complete ab- sorption of the ammonia by the sulphuric acid is most easily insured by the use of a simple absorption tube which compels a very intimate contact of the air coming from the cylinder with the acid and water in the absorption bottle. This absorption bulb consists of a glass tube, measuring about 8 mm., in diameter, one extremity of which has been blown into a small bulb. By means of a heated platinum wire, 10 to 1 2 holes, each about i mm. in diameter, are made in this bulb. The absorption bottle is then attached by means of a glass tube and rubber connection to a filter pump which will draw 700 liters of air per hour.^ The air passing through the alkaline urine will draw the ammonia into the standard acid in from one and one-half to two hours. In order to exclude any error due to the presence of ammonia in the aspirated air, a similar absorption apparatus is attached to the distal side of the evolution flask. The amount is then determined by titra- tion of the standard acid with tenth-normal sodium hydrate, using methyl-red as an indicator. Steel, Gies and others advise the use of NaOH instead of NaaCOs in this method on the ground that the latter will not liberate NH3 from ammonium magnesium phosphate. This would not seem to militate against the method as the triple phosphate is rarely, if ever, present in the urine in appreciable amounts except when the urine is decomposed. Under such circumstances the ammonia factor would be useless from the metabolic standpoint. Formalin Method. This method, originated by Ronchese^ and Malfatti,' de- pends on the fact that a solution of an ammonium salt, treated with formaldehyd, decomposes with the formation of hexa- methylen-tetramin, the acid combined with the ammonia being liberated. This can then be determined by titration. pj^, g Dilute 10 c.c. of urine with 50 c.c. of water, add 2 or 3 Folin's absorp- drops of a I per cent, alcoholic solution of phenolphthalein and /g'g^^ ) neutralize with N/io NaOH. Five c.c. of formalin, previously neutralized with N/io NaOH, are added to the neutralized urine and the mix- ture is again titrated with N/io NaOH to the appearance of a faint, perma- nent pink color, the amount of alkali used being noted. It is evident that i c.c. of this N/io NaOH is equivalent to i c.c. of N/io NH3 or, in other words, ^ In laboratories in which compressed air is available, the ammonia may be forced from the generating cylinder into the cylinder containing the standard acid. In this case, a cyUnder containing 100 c.c. of 10 per cent, sulphuric acid is interposed between the air blast and the generating cylinder in order to catch any ammonia which may be present in the air. ''Jour. Pharm. et Chim., 1907, XXV, 611; Bull. Soc. Chim. de France, 1907, I, 900. ^Ztschr. f. anal. Chem., 1908, XL VII, 273. Digitized by Microsoft® 232 DIAGNOSTIC METHODS represents 0.001704 gram of NH3. Multiply the number of c.c. of N/io NaOH used by this factor and obtain the NH3 in 10 c.c. of urine. {d). Uric Acid, C6H4N4O3. In times past uric acid has been credited with much more clinical im- portance than is to-day ascribed to it. It is not, as many believe, a product of protein decomposition, as such, but can be derived only by the splitting of the nucleo-proteins. These nuclein bodies are compounds of protein with nucleic acid, the latter constituent splitting up into thymic acid and deriva- tives of "purin," among which we find uric acid, xanthin, hypoxanthin, etc. Uric acid, as well as the purin bases to be discussed later, are derivatives of Fischer's hypothetical purin nucleus, with the formula C5H4N4 and the following graphic structure. The figures in brackets represent the number of the atom or group of atoms: (i) N= =CH (S) (2) HC C- (6) (7) -NH N- (4) -C- (9)/' — N CH (8) (3) Each of the purin derivatives is formed by the replacement of one or more of the hydrogen atoms in this nucleus by various atoms or groups of atoms. Thus we "find that uric acid is a derivative in which 3 atoms of oxygen have been substituted for the hydrogens in positions 2, 6, and 8. The hydrogen atoms are not replaced, but shift in the direction of the double bonds, passing to the nitrogen atoms i, 3, and 9. The structure of uric acid with the formula C5H4N4O3 is HN- -CO OC HN- -NH -NH CO Its chemical name is, therefore, 2, 6, 8, tri-oxy purin. Uric acid is derived from the nucleins of the food, as well as from the nucleins of the tissues.^ We can conceive, consequently, that we may have a uric acid excretion which may be, in part, referable to each of these factors. This is the basis of the theory of endogenous and of exogenous purin metabo- lism, the former representing the uric acid formation from the tissue nucleins, the latter that from the food directly. It is self-evident that the exogenous ^See Thannhauser, Ztschr. f. physiol. Chem., 1914, XCI, 320; Thannhauser and Boomes, Ibid., 336; Bass, Arch. f. exper. Path. u. Pharmakol., 1914, LXXVI, 40; Taylor and Rose, Jour. Biol. Chem., 1914, XVIII, 519. Digitized by Microsoft® THE URINE 233 purin metabolism, being directly dependent upon the diet, will vary more than will the endogenous form. This latter type is dependent not only upon the metabolism of the various cellular elements of the body, but also upon the degree of direct synthesis of uric ^cid in the system, as well as upon the extent of conversion of uric acid into urea. This latter statement must not be inter- preted as meaning that uric acid is simply a stage in the conversion of all protein into urea. This idea, which held sway for so long, has fortunately been abandoned. However, we do know, from the work of Frerichs and Wohler that the system does transform a certain amount of uric acid into urea. Burian and Schur point out that the uric acid eliminated by man on a purin-free diet (endogenous \lric acid metabolism) is for each individual a constant quantity and entirely independent of the total amount of nitrogen eliminated. This fact has so firmly fixed itself in the minds of the profes- sion that the important results of Folin have been largely unnoticed. This latter worker finds that "when the total amount of protein metabolism is greatly reduced, the absolute quantity of uric acid is diminished, but not nearly in proportion to the diminution in the total nitrogen, and the per cent, of the uric acid nitrogen in terms of the total nitrogen is, therefore, much in- creased." It would seem, therefore, that this point is still very debatable. Quoting again from Folin "if the endogenous uric acid is to be considered as derived from the cell nucleins exclusively, it would, indeed, seem highly plaus- ible that the quantity should tend to remain constant, even with very great variations in diet. Rigid proof that the endogenous uric acid elimination is for each individual a constant quantity would be strong evidence in favor of such a theory. Burian and Schur support the view that the endogenous uric acid is derived from the cell nucleins, but they contend that in man about one-half of the uric acid so derived is destroyed inside the organism and that only the other half is eliminated. With the introduction of this important modification of the nuclein theory, there is no longer any reason why the uric acid elimination should not be a decidedly variable factor which might well be susceptible to change under the influence of many different changes in the conditions, among others, changes in diet." It will be seen, therefore, that one must be on his guard in drawing conclusions from the amount of uric acid in the urine, as to the degree of nuclein decomposition within the system.^ Physiologic Variations. The output of luric acid varies, depending upon the diet, from 0.2 to 2 grams in 24 hours. On a diet of 119 grams of protein with a total urinary nitrogen output of 16 grams the uric acid eliminated was 0.37 gram (0.8 per cent, of total N); while on a nitrogen-free diet with a urinary nitrogen of 3.6 grams the output of uric acid was 0.09 gram (2.5 per cent, of total N). It is increased physiologically by increase in the nucleins of the diet, sweet-breads, liver, kidneys, and brain yielding very large amounts of uric acid. A meat diet will lead to a larger excretion of uric acid than will a vegetable diet, the ' See Siven, Arch. f. d. ges. Physiol., 191 2, XCLV, 283; Ibid., 1914, CLVII, 582; Faustka, Ibid., 1914, CLV, 523; Raiziss, Dubin and Ringer, Jour. Biol. Chem., 1914, XIX, 473; Daniels and McCrudden, Ar^^^j^^ jj^^^^Y»/?(g^^- 234 DIAGNOSTIC METHODS maximum output being observed about five hours after a hearty protein meal. According to Horbaczewski, this increase is dependent upon the leucolysis which occurs at the time of the disappearance of the digestive leucocytosis. The amount of exercise will also influence the output in the urine; while an intake of a large amount of water will increase the normal uric acid value. A certain relation between the amount of urea and of uric acid excreted seems to obtain. As a rule, it may be said that the nitrogen of the urea is to the nitrogen of the uric acid as 50 or 60 to i. It is probable that variations in this relation have at present much less clinical value than in times past. It does not seem to the writer consistent to assume a special " uric acid diathesis" if this normal relation be disturbed in the sense that the uric acid is increased. Pathologic Variations. A pathologic increase of uric acid is observed whenever we have increased protein catabolism. Thus, in fever, the output of uric acid runs parallel to that of urea. In cases associated with marked leucocytosis a large increase in the uric acid output may be noted, which is referable to the constant leucoly- sis. This increase is especially marked in leukemia, an output of more than 12 grams in 24 hours have been observed by Magnus Levy. In cases of pneu- monia, associated with a high leucocyte count, an increase in the uric acid out- put is seen, being especially marked after the crisis, but may even precede the crisis. Gout has so long been associated, in the minds of the profession, with increased uric acid formation that such a relationship is generally accepted. It is true that during the acute attack the blood may contain an increased amount of uric acid, but never in such a large amount as in cases of leukemia, for instance. The mere excess of uric, acid in the blood can, therefore, not be the determining factor. As our knowledge of the true etiology of gout is so ob- scure the writer will not attempt a discussion of uric acid in such relations. According to Futcher the uric acid is below the normal standard preceding the acute attack, rises to much increased values during the attack, and again falls below the normal limit after the subsidence of the acute symptoms. In acute articular rheumatism an increased elimination is noted during the febrile period, a decrease being observed as convalescence approaches. In diabetes mellitus an increase or a decrease may be observed, the uric acid excretion varying inversely as that of the sugar, giving rise to the term dia- betes alternans. In cases showing much degeneration of hepatic tissue, as in cirrhosis an acute yellow atrophy, uric acid may be largely increased. A diminished excretion of uric acid is usually observed in the ordinary forms of anemia while in pernicious anemia an increase may be noted. Chronic interstitial nephritis, chronic lead-poisoning, purpura hemorrhagica, and some cases of epilepsy, are associated with a diminished output of uric acid. Large doses of quinin or of opium may diminish the uric acid, while salicylic acid, chinic acid, colchicin, urotropin, piperazin, nucleinic acid, etc., may increase the amount. The chemical properties of uric acid may be found in works on physio- logic chemistry. Qualit|Jjg^/|^^(jarjj^j^g^gg5^ desirable for the detection THE URINE I 235 of the nature of certain deposits, as, for instance, renal infarcts. These will be discussed in a later section in association with the description of the various crystalline forms which uric acid may assume in a urinary deposit. Quantitative Determination. Folin's Method. This modification'^ of Hopkins' method depends upon the precipitation of uric acid, as ammonium urate, by addition of ammonium sulphate. The urate is decomposed with sulphuric acid and the liberated uric acid is deter- mined by titration with a standard solution of potassium permanganate. Technic. Two hundred c.c. of urine are treated with 50 c.c. of a reagent consisting of 500 grams of ammonium sulphate, 5 grams of uranium acetate, and 60 c.c. of 10 per cent, acetic acid, dissolved in 650 c.c. of water. The mixture is allowed to stand without stirring for about one-half hour. The precipitate of uranium phosphate has then settled and the clear supernatant [liquid is removed by siphonage or by decantation. One hundred and twenty-five c.c. of this clear fluid, representing 100 c.c. of urine, are measured into a beaker, 5 c.c. of strong ammonia are added, and the mixture set aside until the following day. The precipitate of ammonium urate, which is produced by alkalinizing the solu- tion saturated with ammonium sulphate, is then filtered off, and washed with 10 per cent, ammonium sulphate solution until the filtrate is practically free from chlorids. The filter is removed from the funnel, opened, and the precipi- tate rinsed back into the beaker, enough water to make about 100 c.c. being used. The'precipitate is now dissolved by adding 1 5 c.c. of concentrated sul- phuric acid. The solution is then titrated with a twentieth-normal solution of potassium permanganate, until the fijst pink coloration is observed extend- ing 'through the entire liquid after the addition of two drops of the perman- ganate solution. Each c.c. of potassium permanganate used corresponds to 3.75 mg. of uric acid. Owing to the solubility of the ammonium urate, a cor- rection of 3 mg. per 100 c.c. of urine must be made. The corrected result gives the percentage of uric acid.^ A method of making a twentieth-normal potassium permanganate solution may be found in any work in general chemistry. The student is to be cau- tioned that a twentieth-normal solution of potassium permanganate, as used in this connection, has reference to one of such a concentration that one liter would contain 0.05 gram of available oxygen for oxidizing purposes. This solution is obtained by dissolving 1.581 grams of pure KMn04 in one liter of water. As this weighing may not be sufi&cient, owing to slight impurities in the permanganate, it is wise to titrate this solution against a known twentieth- normal solution of oxalic acid. If the solutions correspond, i c.c. of each should be the equivalents. The titration of the oxalic solution is made by measuring out 10 c.c. of oxalic acid solution, diluting to approximately 100 c.c. with distilkd water, and adding 15 c.c. of concentrated sulphuric acid. The 'Ztschr. f. physiol. Chem., 1901, XXXII, 552. 2 Skorczewski und Sohn (Wien. klin. Wchnschr., 1911, XXIV, 1700) show that the urine of patients taking atophan may reduce the permanganate and, hence, introduce an error. Digitized by Microsoft® 236 DIAGNOSTIC METHODS permanganate solution is then added drop by drop until a faint permanent red color is observed throughout the liquid, which does not disappear on stir- ring but persists for at least 30 seconds. If these solutions do not correspond the permanganate solution must be diluted according to the formula pre- viously given under the determination of the chlorids in the urine. SalkowsM-Ludwig Method. This method is not as convenient as that of Folin, but is inserted as it serves as a combined method of determining uric acid and thepurinbases at the same time. The principle is as follows: Uric acid is precipitated, in the form of a double urate of silver and magnesium, by an ammoniacal silver solution in the presence of magnesium salts. The silver is removed by hydrogen sulphid and the uric acid precipitated by hydrochloric acid, after which it may be esti- mated by direct weighing or by determination of the nitrogen. Technic. Two hundred and forty c.c. of urine are treated with 60 c.c. of magnesium mixture, which is made up as follows: One hundred grams of crystallized magnesium chlorid and 200 grams of ammonium chlorid are dissolved in about 500 c.c. of water, ammonia is added until the mixture smells strongly of this substance and the whole is then made up to one liter. The above mixture is well stirred and immediately filtered through a dry filter-paper into a beaker. Two hundred and fifty c.c. of filtrate (representing 200 c.c. of urine) are then measured off and treated with 10 to 15 c.c. of an ammoniacal silver nitrate solution (one liter of which contains 26 grams of AgNOs and enough ammonia to dissolve the precipitate formed). The precipitate should be of a flocculent gelatinous nature and of a yellowish color. If the precipitate be white, too much silver chlorid is present and more ammonia must be added. The cjear solution above the precipitate should contain an excess of silver chlorid which may be shown by adding a little nitric acid to a few drops of the clear super- natant liquid. This mixture is then filtered, any particles adhering to the beaker being transferred to the filter by means of water and a rubber- tipped glass rod. The residue on the filter is washed with distilled water until the wash fluid shows no trace of silver or of chlorids. The funnel is now placed in the neck of a liter flask, the tip of the filter-paper is perforated with a glass rod, and the precipitate washed into the flask and thoroughly mixed with the water. This solution is made faintly acid by the addition of two or three drops of hydrochloric acid. Three to four c.c. of 10 per cent, copper sulphate solution are then added and the mixture boiled, after which hydro- gen sulphid is passed through the hot mixture to precipitate the silver salts. This gas should be passed until the solution is saturated with it, after which the solution is boiled and filtered. The precipitate is washed with hot water and the filtrate, which must be clear and colorless, is evaporated to a small bulk (10 to 15 c.c). Ten to fifteen drops of hydrochloric acid are then added, the mixture stirred and allowed to stand for a few hours, prefer- ably over night. This addition of hydrochloric acid precipitates the uric acid and leaves the purin bases in solution. The crystals of uric acid are filtered Digitized by Microsoft® THE URINE 237 off on a small weighed filter, are washed with water slightly acidified with HCl to such an extent that the total wash-water and filtrate should not be more than 50 to 60 c.c. The precipitate is washed with alcohol, carbon disulphid, and ether, and is then dried and weighed. The difference be- tween the original weight of the filter-paper and that of the paper and pre- cipitate is the amount of uric acid in the 200 c.c. of urine. Owing to the slight solubility of uric acid in acidulated water, a correction of 0.00048 gram must be added for every 10 c.c. of the filtrate and wash-water. Instead of weighing the uric acid, the filter-paper and contents may be placed in a Kjeldahl flask and a nitrogen determination made as previously described. The nitrogen value multiplied by 3 will give the weight of uric acid in 200 c.c. of urine. The filtrate and wash-water from the uric acid precipitation contains the purin bases. This filtrate is alkalinized with ammonia and again precipitated with the ammoniacal silver solution. This precipitate is collected on a small filter, washed with water, dried and carefully incinerated. The ash is dis- solved in nitric acid and the silver chlorid estimated by titration with potas- sium sulphocyanate as described under the determination of chlorids. One c.c. of the potassium sulphocyanate Solution is equivalent to 0.00734 gram of silver. One part of silver, in the form of the silver compounds of the purin bases, represents 0.277 gram of nitrogen, or 0.7381 gram of the purin bases. It is evident, therefore, that i c.c. of the potassium sulphocyanate solution will represent 0.002 gram of nitrogen and 0.00542 gram of purin bases accord- ing to the following proportion: I : 0.00734 : : 0.277 : x X = 0.002 By multiplying the number of c.c. of potassium sulphocyanate solution used to precipitate the chlorids by 0.00542 we obtain the number of grams of purin bases in the 200 c.c. of urine originally used. It is wise in determining the amount of purin bases to start with a larger quantity of urine, as, for instance, 600 to 700 c.c, as the amount present in 200 c.c. would be very small. This method is apt to give slightly high values for uric acid, as the purin bases may not be entirely soluble in the acid solution used to separate them from the uric acid. It requires much more equipment than is usually at the disposal of the practitioner so that it can hardly be recommended for routine use in general work. Method of Rudisch and Kleeberg. This method^ is quite as accurate as is the preceding and has the advan- tage that it can be carried out in from 20 to 30 minutes. The principle is as follows: The total purin bodies are precipitated by an excess of silver nitrate, and the excess of silver determined volumetrically^ by potassium iodid, using a mixture of nitrous and sulphuric acid with starch solution as an indicator. As the silver compounds of the purin bases are soluble in strong ammonia solution, it is possible to make a determination of the uric acid and then esti- 1 Am. Jour. Med. Sci., 1904, CXXVIII, 899. 2 See Kretschmer, Biochem. Ztschr., 1913, L, 223. Digitized by Microsoft® 238 DIAGNOSTIC METHODS mate the purin bases by subtraction of this value from the total purin compounds. Technic. One hundred and ten c.c. of urine are treated with 55 c.c. of fiftieth-normal AgNOa solution and diluted with strong ammonia to 220 c.c. (The fiftieth- normal solution of silver nitrate is made by dissolving 3.3932 grams of AgNOa, which has been heated for 10 minutes with a small amount of water to i2o°C., in 500 c.c. of water, adding 75 c.c. of strong ammonia and 10 grams of ammo- nium chlorid and making the whole up to one liter.) The addition of the ammonia dissolves the purin bases, leaving the uric acid in the precipitate. The original mixture is now. filtered in such a way that two 100 c.c. portions are obtained, each portion representing 50 c.c. of urine. With the first of these portions an approximate estimation of the uric acid is made, while with the second the accurate one is carried out. To one of these portions, fiftieth-normal potassium iodid solution is added from a buret, a few drops being removed from time to time and added to a solution of nitrous-sulphuric acid mixed with a little starch paste. (The fiftieth-normal potassium iodid solution should contain 3.32 grams of KI in one liter. The nitrous-sulphuric acid mixture is made by mixing 25 c.c. of H2SO4 with 75 c.c. of H2O and i c.c. of fuming HNO3). The addition of the drops of the solution to the indicator is for the purpose of determining the point at which an excess of potassium iodid occurs. This will be shown by the appearance of a distinctly blue contact ring. When the solution shows such a reaction, on adding a few drops of the mixture to the indicator, the number of c.c. of KI is read off, and we are then prepared for the more accurate control. It is wise to keep the indicator cold by immersion in ice-water, as otherwise a violent reaction may occur. The second 100 c.c. portion is then carefully titrated with the fiftieth-nor- mal KI solution by running directly about i c.c. less of this solution into the urine than was required in the first titration. After this point is reached the KI solution should be added drop by drop and a test made after the addition of each five drops. In this way an accurate end-point may be reached. As each 100 c.c. portion represents 50 c.c. of urine it will contain 25 c.c. of fiftieth- normal KI used and the number of c.c. of silver nitrate which combined with the uric acid of the urine is obtained. One c.c. of fiftieth-normal AgNOs solution represents 0.00336 gram of uric acid. By multiplying this factor by the number of c.c. of silver nitrate used, we obtain the amount of uric acid in 50 c.c. of urine. The second part of this method consists in the determination of the total purins. One hundred and ten c.c. of urine are treated with 55 c.c. of fiftieth- normal AgNOs solution and diluted with water to 220 c.c. The remainder of the process is exactly as outlined above. The number of c.c. of fiftieth- ' normal silver nitrate used will represent the values for the total purins. If the number of c.c. of silver nitrate solution used in the previous deter- mination of uric acid be subtracted from the number used in the determina- tion of total purin, the result wilLbe the. number of c.c. referable to the purin bases. One c.c. of fiftietn-fform^ A-gNOs smution represents 0.00152 gram of THE URINE 239 lodin, Potassium iodid, Absolute alcohol, Glycerin, Distilled water, q.s.. 0.50 gram 1.25 grams 7.50 grams 5.00 grams 100.00 grams purin bases calculated as xanthin. By multiplying this factor by the number of c.c. of AgNOs used we obtain the amount of xanthin in 50 ex. of urine. The writer has used this method frequently and has found its results very similar to those of the Salkowski-Ludwig method, although the amount of uric acid is somewhat less in this method than in the latter one. It is very simple, is quick and accurate and may, there- fore, be recommended for general work. A somewhat similar method is advocated by Bartley.^ The writer finds this latter method fairly reliable and simple. Riihemann's Method. This method is a very convenient clinical one, although its j results are by no means as accurate as those of the preceding 1 methods.^ What the general practitioner desires, as a rule, | is to know whether the uric acid is increased or diminished i and does not care as to the absolute value. Such results, ' giving the total purins, may be obtained for clinical purposes by this method. It consists in the use of a specially graduated tube, the uricometer, in which are placed the reagents and the urine to be tested (see cut) . The calibrations of the tube are such as to represent directly the amount of uric acid in parts per 1000. The principle of the method is the decolorization of an iodin solution by the uric acid of the urine, and the meas- urement of the amount of urine which must be added to a definite amount of iodin solution to effect this decolorization. Technic. Carbon disulphid is placed in the tube up to the mark S, in such a way that the lower meniscus of this reagent rests upon the mark. A solution of iodin in potassium iodid is then added so that the upper portion of the meniscus coin- cides with the mark J. This iodin solution has the follow- ing composition. mnia 0.175 0.1 7S 0.181 0.184 0,187 0.190 0.193 0.19S 0.19» 0.202 0,»)S O.SOS 0,211 0,215 0.21S 0.221 0.225 0.22S .0,231 .0,235 Fig. 7g.— Ruhemann's uricometer. The urine is added slowly by means of a pipet until the lowest calibration is reached. The glass stopper is inserted and the contents of the tube mixed by repeated inversion for about 15 seconds. The carbon disulphid absorbs the iodin, taking on a distinct purple coloration. If this amount of urine does not completely decolorize the iodin, ' shown by the porcelain-like color of the carbon disulphid solution, more urine is added and the tube ' Medical Chemistry, FhHial^^J^^MicrOSOft® ' See Bradley and Bunta, Jour. Am. Med. Assn., 1913, LX, 44. 240 DIAGNOSTIC METHODS again inverted for 15 seconds. This process is continued until repeated shaking of the tube causes the carbon disulphid to assume a pale pink color. The reaction is practically ended at this point, as by a little more shaking of the contents the indicator will assume the characteristic porcelain-white ap- pearance. This process requires from 6 to 15 minutes. The amount of uric acid is then read off directly from the tube in parts per liter. Should the urine contain less uric acid than can be read off from the cali- brations, a second test is made adding the iodin solution to the mark midway between S and J, the amount indicated on the tube being of course divided by 2. Conversely, should the urine contain more uric acid than is represented by the lower calibration, one adds the iodin solution to the point above J and multiplies his reading by 1.5, or adds the iodin solution to the second mark above J and multiplies the reading by 2. With this method the urine must be acid in reaction. If the urine con- tains a sediment of the urates, it should be thoroughly shaken before being added, so that the urates may be in suspension. Any free uric acid which may have separated in the sediment is not determined in this method. Strongly colored urines have no influence upon the decolorization. The presence of sugar does not interfere with the results, but if albumin be present in large amounts it should be removed by acidifying with dilute acetic acid, boiling, and filtering. The writer has used this method very frequently and finds it very useful for rough estimation of the uric acid outputs. The puri- nometer, introduced by Hall,^ seems to be a much more reliable and useful instrument, as it employs the reagents required by the Salkowski-Ludwig method.^ (e) Purin Bases. These bodies have been called purin bases, alloxur bases, xanthin bases, and nuclein bases. They are of extreme importance from the standpoint of physiologic chemistry, but are, clinically, of less value than is uric acid. The following bodies have been isolated from the urine in various conditions: adenin, guanin, epiguanin,. carnin, episarkin, xanthin, hypoxanthin, heter- oxanthin, paraxanthin, and methylxanthin. Certain methylated xanthin compounds are found in tea and coffee and are, therefore, introduced into the system as caffein, theobromin, and theophyllin, being excreted either as xan- thin or hypoxanthin. Xanthin (C6H4N4O2) is 2, 6, dioxypurin. These nuclein bases of the urine arise either from the nuclein of the food or from the increased nuclein metabolism of the system. Very little is known at the present time regarding the absolute variations in the excretion of these purin bases in the urine. Salkowski finds an excretion ranging between 0.0286 and 0.0561 gram (calculated as xanthin)., while Camerer regards an ' The Purin Bodies, Phila., 1904. '^ Folin and Macallum (Jour. Biol. Chem., 1912, XIII, 363) have devised a new method for uric acid based upon the action of phosphotungstic acid in alkaline solution upon uric acid. The depth of color of the blue solution formed is then compared with that of a stand- ard solution. Their "uric acid reagent" is prepared by boiling 100 grams of sodium tung- state with 80 c.c. of 85 per cent, phosphoric acid and 750 c.c. of water for a couple of hours and then diluting to i liter. See, also, Folin and Denis, Jour. Biol. Chem., 1913, XIV, 95; Autenrieth and Funk, Munch. Med. Wchnschr., 1914,1X1, 457; Host, Ztschr. f. klin Med ' 1914, LXXXI, 113; Benedict^^-^^^l^-gyi£,5g/^phem., 1915, XX, 619. THE URINE 241 average output as 0.087 gram in 24 hours. It is interesting to note that a vegetable diet appears to increase this output more than a meat diet, 0.044 gram being excreted, according to Camerer, on a meat diet and o.iii gram on a vegetable regime. This finding is the reverse of that for uric acid. As a general rule, it may be said that the output of purin bases is from 8 to 10 per cent, of that of uric acid, varying from 16 to 60 mg. per diem. It is evident that foods containing these substances should be absolutely interdicted in conditions which may be traceable to disturbances in the nucleia metabolism. An increase in the amount of uric acid is usually associated with an in- crease of the xanthin bases, but at times no such relations obtain, a decrease in these bodies being observed. In leukemia an excretion of 0.321 gram has been reported by Magnus-Levy.^ In certain cases of tuberculosis, nephr'tis, epilepsy, migraine, and pneumonia the output may be much increased. Edsall finds the urinary output increased as a result of X-ray treatment. Xanthin is occasionally found as a constituent of the urinary sediment and may form calcuh.^ (/) Creatinin (C4H7N3O). The older ideas regarding the excretion of this nitrogenous body have suffered a severe shock from the work principally of Folin, Shaffer, Hoogen- huyze and Verploegh, Mellanby, and Klercker. Acid urines were supposed to contain creatinin and little or no creatin; while alkaline urines were thought to show creatin instead of creatinin. It has been proven, however, that " nor- mal fresh urine, whether acid or alkaline, contains creatinin, and if the normal subject has not taken creatin in his food during the preceding days his urine will not contain creatin, whatever its reaction. There is no normal excretion of endogenous creatin,' as this, when ingested, is largely retained in the body unless the food contains a large amount of protein." Thus Folin has shown that the excretion of creatinin on a diet yielding a urinary nitrogen value of 16 grams, was 1.55 grams or 3.6 per cent, of total nitrogen; while on a nitrogen- free diet with a urinary nitrogen of 3.6 grams, the creatinin output was 0.6 gram of 17.2 per cent, of total N. He showed, further, that while the actual amount excreted varied with diftrent individuals, yet for the same person the output was practically constant, under the same conditions of health and mus- cular activity. He believes, therefore, that creatinin is by far the most re- Hable index as to the amount of a certain kind of protein metabolism occurring daily in any given individual. He bases his conclusions upon the facts that " the absolute quantity of creatinin eliminated in the urine on a meat-free diet is a constant quantity, differing for different individuals, but wholly independ- ent of quantitative changes in the total amount of nitrogen eliminated." Shaffer* believes that creatinin is not an index of the total endogenous protein catabolism, as patients in whom the endogenous catabolism is much in- > Loc. cii. " See Hefter, Deutscli.KArch. f. klin. Med., 1913, CIX,322; also, Koch, Jour. Biol. Chem., 1913, XV, 43. ' This statement does not apply to growing children who show a normal excretion of a relatively large amount of creatm. See Mendel and Rose, Jour. Biol. Chem., 1911, X, 213, 25s and 26s; also FoUn and Denis, Ibid., 1912, XI, 253. ' Am. Jour. Physiol, iQo^y^^Jgj, MicrOSOft® 242 DIAGNOSTIC METHODS creased may excrete very little creatinin. Folin finds that the chief factor determining the amount of creatinin eliminated on ^any special diet is the weight of the patient. Fat or corpulent persons yield less creatinin per unit of body-weight than do lean. ones. It is, therefore, necessary in metabolic work to consider not only the body-weight, but, also, the excess of fat in increasing thfe weight. According to Shaffer, the normal excretion of creatinin varies between 7 and 1 1 mg. of creatinin-nitrogen per kilo of body-weight. In pathologic subr jects it is low, varying from the normal to 2 mg. per kilo of body- weight in 24 hours. He calls the creatinin-nitrogen excretion per kilo of weight the "creatinin coefficient." He thinks that creatinin is an index of some special process of normal metabolism taking place largely, if not wholly, in the mus- cles. Upon the intensity of this process appears to depend the muscular efficiency of the individual. In acute febrile conditions, in which an in- creased destruction of muscle tissue occurs, an increase is seen in the creatinin output during the active febrile period, while in the period of convalescence a diminished excretion will be observed. This excretion in fever does not run parallel to the muscular efficiency of the individual (Shaffer). Simon has shown that a diminished excretion occurs in anemia, marasmus, myositis ossificans, chlorosis, phthisis, chronic parenchymatous nephritis, progressive muscular atrophy, and pseudohypertrophic paralysis. According to Shaffer, creatin may be excreted by subjects of acute fevers, in the acute stages of exophthalmic goiter, in other conditions in which there ■ is a rapid loss of muscle protein, and by women during the postpartum reso- lution of the uterus. Krause^ has reported the constant presence of creatin in diabetes mellitus referable, probably, to a deprivation of carbohydrates. Although administration of carbohydrate usually diminishes the creatin ex- cretion, Folin's theory would indicate that an increased excretion of creatin may follow a carbohydrate-rich diet, if the state of nutrition of the body is high, as a greater fraction of the creatin of the food would be eliminated. The study of the excretion of creatinin and creatin in various conditions is • fast becoming of the greatest importance.^ • t ' Quart. Jour. Exper. Physiol., igio, III, 289. 2 See Mendel, Science, 1909, XXIX, 584; Ditman and Welker, New York Med. Jour., 1909, LXXXIX, 1000, 1046 and 1091; Ellis, Jour. Am. Med. Assn., 1911, LVI, 1870; Skutetzky, Deutsch. Arch. f. klin. Med., 1911, CIII, 423; McCrudden, Jour. Exper. Med., 1912, XV, 457; Sedgwick, Am. Jour. Bis. Child., 1912, III, 209; Kraus, Arch. Int. Med., 1913, XI, 613; Scaffidi, Internat. Beitr. z. Ernahrungsstor., 1913, IV, 401, Orioli, Ibid., 421; Mellanby, Proc. Roy. Soc, 1913, LXXXVI, 88; Myers and Volovic, Jour. Biol. Chem., 1913, XIV, 489; Myers and Fine, Ibid., 1913, XV, 283 and 305; Ibid., 1913, XVI, 169; Thomas and Goerne, Ztschr. f. physiol. Chem., 1914, XCII, 163; Lampert, Ztschr. f. klin. Med., 1914, LXXX, 498; Folin and Morris, Jour. Biol. Chem., 1914, XVII, 469; Folin and Denis, Ibid., 493; Benedict, Ibid., 1914, XVIII, 183 and 191; Benedict and Osterberg.Ibid., 195; Shaffer, Ibid., 525; Tracy and Clark, Ibid., 1914, XIX, 115; Palmer, Means and Gamble, Ibid., 239; Ringer and Raiziss, Ibid., 487; Hull, Jour. Am. Chem. Soc, xgi4t XXXVI, 2146; Morris, Jour. Biol. Chem., 1915, XXI, 201; Myers and Fine, Ibid., 377, 383 and 389; Janney and Blatherwick, Ibid., 567; Myers and Fine, Ibid., 583; Taylor, Ibid., 663; Baumann and Marker, Ibid., 1915, XXII, 49; MacAdam, Biochem. Jour., 1915, IX, 229; Practitioner, 1914, XCII, 540; Hutchison, Indian Jour. Med. Research, 1915, II, 814; THE URINE 243 Qualitative Tests for Creatinin. Creatinin has the formula, C4H7N3O, with the graphic structure HN C = O HN = C H3C-N CH2 The chemical reactions which serve for the detection of creatinin depend upon the formation of different colored compounds. It forms a distinct crys- talline compound with zinc chlorid, which may be used in the quantitative estimation by Salkowski's method. For this process see works on physio- logic chemistry. A point to be remembered regarding creatinin is that it re- duces copper solutions and may be mistaken for sugar unless care be exercised. Weyl's Test. To the urine to be tested are added a few drops of a very dilute aqueous so- lutin of freshly dissolved sodium nitroprussid and a few drops of dilute sodium hydrate solution. In the presence of creatinin a ruby-red color appears which changes, after a short time, to an intense yellow. ' If this solution be heated with a little glacial acetic acid the color will change to green and finally blue. Acetone gives a similar reaction, but on the addition of acetic acid changes to a purplish-red instead of green. If the urine be heated previous to the appli- cation of this test, the acetone may be driven off. This test for creatinin is sensitive to 6 parts in 10,000. Jaffe's Test. To the urine to be tested are added a few drops of a saturated solution of picric acid and a few drops of dilute sodium hydrate solution. If creatinin be present a red color appears immediately, which increases in intensity and remains permanent for a long time. If glacial acetic acid be added the color becomes yellow. Acetone gives a reddish-yellow color of less intensity than that produced by creatinin. Glucose, if present, may give a red color if the mixture be warmed. This test indicates one part of creatinin in 5,000. Quantitative Determination. Folin's Older Method. The principle upon which this determination^ is based is the comparison of the color produced by Jaffe's reaction with that of a standard solution of potassium bichromate. A high-grade colorimeter is necessary for this com- parison. Folin recommends the use of the Duboscq instrument, while the writer finds one made by Sargent & Co., of Chicago, very satisfactory. This latter has the advantage of being much less expensive. The reagents necessary are (i) a half-normal potassium bichromate solu- tion containing 24.55 grams per liter, (2) a saturated picric acid solution contain- ing about 1 2 • grams per liter, and (3) a 10 per cent, solution of sodium hydrate. ' Am. Jour. Physiol., 1905, XIII, 45. Digitized by Microsoft® 244 DIAGNOSTIC METHODS Technic. Ten c.c. of urine are measured into a 500 c.c. volumetric flask, 15 c.c. of the picric acid solution and 5 c.c. of the sodium hydrate solution are then added, and the mixture allowed to stand for five or six minutes. This interval is used to pour a little of the standard bichromate solution into each of the two cylinders of the colorimeter. The depth of the solution in one of the cyUnders is then accurately adjusted to the 8 mm. mark. With the solution in the other cylinder a few preliminary colorimetric readings are made simply for the sake of insuring greater accuracy in the subsequent readings of the unknown solu- tion. The two bichromate solutions must, of course, be equal in color, and in taking their readings no two should differ more than o.i mm. or 0.2 mm. from the true value (8 mm.), leaving out of consideration the very first reading made, which is sometimes less accurate. Four or more readings should be made in each case, and an average taken of aU but the first. After a while one becomes sure of the true point, and can take the average of the first two readings. At the end of five minutes the contents in the 500 c.c. flask are di- luted up to the 500 c.c. mark. The bichromate solution is thoroughly rinsed out of one of the cylinders by means of the unknown solution and several colorimetric readings are then made at once. The calculation of the results is very simple. It is based on the experimentally determined fact that 10 mg. of perfectly pure creati- nin give, under the conditions of the determination, 500 c.c. of a solution, 8.1 mm. of which have exactly the same colorimetric value as 8 mm. of a half -normal bichromate solution.^ If, for example, it is found that it takes 9.5 mm. of the unknown urine- picrate solution to equal the 8 mm. of the bichromate, then the 10 c.c. of urine contain 10 X ^ = 8.4-I- mg. of creatinin. 9-S ^ 1 This relationship applies to the Duboscq instrument, which Folin advises. If one uses the Sargent or other colorimeter, he must, of course, make his adjustment according to his special instrument. It matters little what point is used for the standard solution, provid- ing one has tested his bichromate solution against solutions of creatinin treated as above and has obtained his relative values. Digitized by Microsoft® Fig. 80. — Sargent's colorimeter. (Courtesy of E. H. Sargent & Co.) THE TJRINE 245 If the 10 c.c. of urine used in the test are found to contain more than 15 mg. or less than 5 mg. of creatinin, the determination should be repeated with a correspondingly different amount of urine, because outside of these limits the determination is much less accurate. The color of the urine does not materially affect the results on account of the great dilutioii. Sugar and albu- min do not interfere with the determination, while acetone and diacetic acid do unless one allows a few minutes before reading. * Folin's Newer Method. In order to enlarge the scope of the colorimetric method for the determina- tion of creatinin and creatin, Folin advises the replacing of the bichromate solution with a standard creatinin solution for the color comparison. While creatinin may be obtained on the market it is usually far from pure, so that one should prepare his own creatinin according to the method of FoUn^ or of Benedict.' The method is as follows: One c.c. of a standard solution of creatinin (i gram per Uter, hence i c.c. containing i mg.) is measured into a 100 c.c. volu- metric flask and i c.c. of the urine into a similar flask by means of the accurate Ostwald pipets (care being taken to allow the pipet to drain against the neck of the flask for a few seconds and then to blow out the pipet clean). Twenty c.c. of saturated picric acid solution (measured with a cylinder) are added to each flask and then 1.5 c.c. of 10 per cent, solution of sodium hydrate are in- troduced from a buret. At the end of 10 minutes the flasks are filled up to the mark with tap water, are mixed thoroughly and the relative color of the two solutions determined in the colorimeter. It makes little difference whether the standard solution is set at 10, 15 or 20 mm., as the comparisons are equally exact at any one of these points. This standard reading is divided by the read- ing of the unknown to obtain the milligrams of creatinin present in the volume of urine (i c.c.) taken. If the urine reads less than two-thirds or more than one and one-half that of the standard the determination should, be repeated with more or less urine as the case may be. Creatin. The determination of creatin, as weU as of creatinin, is of much importance in various conditions. Many methods have been given for such work but the following seems to offer the best results with the simplest conditions. Folin's Method.^ Enough urine to give 0.7 to 1.5 mg. of creatinin is measured into a weighed Erlenmeyer Jena flask, (capacity 200 c.c). Twenty c.c. of a saturated picric solution, about 130 c.c. of water and a few very small beads to promote even boiling are added and the mixture is gently boiled, preferably over a micro- burner, for about i hour. At the end of this time increase the heat and boil down the solution to about 20 c.c. Transfer the flask to the scales iSee Greenwald, Jour. Biol. Chem., 1913, XIV, 87; also, Thompson, Wallace and Clotworthy, Biochem. Jour., 1913, VII, 445. 2 Jour. Biol. Chem., 1914, XVII, 463 and 46Q. 'Jour. Biol. Chem., 1914, XVIII, 183. *Joui. Biol. Chem., 1914, XVII, 469. See, also, Benedict, Ibid., 1914, XVIII, 191. Digitized by Microsoft® 246 DIAGNOSTIC METHODS and add enough water to make the total solution equal to 20 to 25 grams. Cool the solution in running water, add 1.5 c.c. of 10 per cent, sodium hydrate solution and determine the total creatinin as in the above method, using i mg. of pure creatinin solution as a standard. This method gives both the preformed creatinin and that derived from the creatin. Subtract the preformed (as determined by the direct method) from the total creatinin and obtain the creatinin derived from creatin. Multiply this latter figure by 1.16 to determine the actual amount of creatin as such, (g) Undetermined Nitrogen. By the undetermined nitrogen of the urine is meant the nitrogen remaining after that attributable to urea, uric acid, xanthin bases, ammonia, and creati- nin has been subtracted from the total nitrogenous output. This factor is made up of many substances present in variable amount and determined- with more or less difficulty. As the variations in the other nitrogenous constituents have been shown to depend to a great extent upon the nature of the diet, so we would expect the values for the undetermined nitrogen to show similar fluctuations. Folin ' finds on a diet yielding 16 grams of total nitrogen in the urine an undetermined nitrogen output of 0.6 gram or 3.75 per cent, of total nitrogen; while on a nitrogen-free diet with a urinary nitrogen value of 3.6 grams the undetermined nitrogen is 0.27 gram or 7.3 per cent. He is led, therefore, to make the follow- ing generahzation: "The absolute quantity of undetermined nitrogen de- creases under the influence of the starch and cream diet, but in per cent, of the total nitrogen there is always an increase." The separation of the various factors included in this undetermined nitrogen has clinically little value at the present time. (i) Amino Acids. Theoretically, we should find in the urine, whenever hepatic metaboHsm is disturbed, both mono- and di-amino acids, as these substances are normal products of protein hydrolysis in the intestine and are directly converted into urea in the normaLliver. It has long been known that acute yellow atrophy and phosphorus poisoning were associated with a diminished urea output and the presence of the two mono-amino acids, leucin and tyrosin, in the urine. Recent work^ with improved methods has shown that the urine contains these and other amino acids in any condition in which hepatic activity is impaired, so that our old-time diagnostic point of acute yellow atrophy must fall by the wayside. Although these substances are found in much larger quantities under pathologic conditions, traces of them are present in normal urine, especially when the nitrogen intake has been large. Thus von Noorden re- gards glycocoll (amino-acetic acid) as a normal urinary constituent, its excre- ' See Signorelli, Biochem. Ztschr., 1912, XXXIX, 36; Ibid., 1912, XL VII, 482; Galam- bos and Tausz, Ztschr. f. klin. Med., 1913, LXXVII, 14; Damask, Ztschr. f. klin. Med., 1913, LXXVII, 333; Hugounenq, Presse m6d., 1913, XXI, 825; Van Slyke and Meyer, Jour. BioL Chem., 1913, XVI, 213; and LoiHer, Ztschr. f. klin. Med., 1913, LXXVIII, 483; Benedict and Murlin, Jour. Biol. Chem., 1913, XVI, 385; Labb6 and Bith, Rev. de. mad., 1914, XXXIV, 89; Udaondo and Casteigts, Sem. med., 1914, XXI, 741; Geake and Nieren- stein, Ztschr. f. physiol. Chem., 1914, XCII, 149; Folin, Jour. Am. Med. Assn., 1914, LXiii, 823. Digitized by Microsoft® THE URINE 247 tion averaging i gram daily. These acids, are also found in cases of gout, pneumonia, especially during the absorption of the exudate, in diabetes, and in leukemia.^ A large number of these mon-amino acids have been isolated from the direct products of protein hydrolysis, but the number found in the urine has not been as large owing to the uncertain methods of examination. The intro- duction by Fischer of the esterification method has added much to our know- ledge of these bodies. By the di-amino acids we mean the bodies lysin,^ argi- nin and histidin, which are collectively known as the hexone bases. Benedict and Murlin's Method. This method^ is not as accurate as the gasometric one of Van Slyke,* but is much more suitable for clinical work. Measure into a 500 c.c. Erlenmeyer flask 200 c.c. of a 24-hour specimen of urine, which has been diluted to 2,000 c.c. Add an equal quantity of a 10 per cent, solution of Merck's phosphotungstic acid in 2 per cent, hydrochloric acid. Let the mixture stand at least 3 hours, preferably over night. Pour off 250 c.c. of the clear supernatant fluid, add i c.c. of a 0.5 per cent, alcoholic solution of phenolphthalein and then barium hydrate in substance until the whole fluid turns decidedly pink (this latter substance should be added a little at a time to avoid an excess) . Let this mixture stand for i hour. Now filter off two 100 c.c. samples, each representing 50 c.c,. of urine. Neutralize these specimens to litmus paper by means of N/s hydrochloric acid and add at once, to each, 10 to 20 c.c. of formalin which has been previously neutralized with N/io sodium hydrate solution. Titrate the mixture cautiously with N/io NaOH to a deep red color, i.e., until the drop of alkali produces no additional color. Make the duplicate determination in the same way and correct the results of deduction of the amount of N/io NaOH necessary to produce the same depth of color in an equal quantity of COa-free water with the same quantity of neutral forma- lin added. Each c.c. of N/io NaOH used in the titration, corrected as above, represents 0.0014 gram of nitrogen referable to the amino acids. Multiply the nitrogen value by 2 to obtain the percentage, and this latter amount by 20 to derive the total amino-acid nitrogen in the 24-hour specimen. 1 Warfield (Jour. Am. Med. Assn., 1914, LXII, 436) has attempted to base a diagnostic test for pregnancy on the presence in the urine of dialyzable products reacting to ninhydrin. This test, however, has no value as the urine, normal or abnormal, always contains dialy- zable substances, especially amino acids and certain ammonium compounds, which under proper conditions will react to ninhydrin. In this connection see Abderhalden's test in the section on Blood; also, Jamison, Jour. Am. Med. Assn., 1914, LXII, 1084; Fischer, Ibid., 950 and iS7S; Falls and Welker, Ibid., 1800; Berman, Am. Jour. Obs., 1914, LXX, 192; Holmes, Lancet-Clinic, 19 14, CXI, 400. Malone (Jour. Am. Med. Assn., 1915, LXIV, i6si),.using a modification of the technic of Kiutsi (Urine Diagnosis by Means of Filtration Process, Sapporo, Japan, 1914), removes the biuret-reacting urinary substances by means of the addition of 0.3 gram of kaoUn to 15 c.c. of urine, filters this mixture and neutraUzes the filtrate. This is then submitted to the usual Abderhalden technic, employing 10 c.c. of the biuret-free filtrate and 0.2 gram of dried placenta. This latter test may prove to be of some diagnostic value. 2 See Ackermann and Kutscher (Ztschr. f. Biol., 1911, LVII, 355) who report the pres- ence of lysin in a patient with cystinuria. Kocher (Jour. Biol. Chem., 1915, XXII, 295) finds these diamino acids largely increased in malignant tissues. « Jour. Biol. Chem., 1913, XVI, 385. *Ibid., 121 and 125. See, also, Chodat and Krummer, Biochem. Ztschr., 1914, LXV, 392;.Harding and MacLean, Tour. Biol. Chem.j 191S) XX, 217. Digitized by Microsoft® 248 DIAGNOSTIC METHODS (2) HippuricAcidCCgHgNOs). This acid is a normal constituent of urine and varies between o.i and I gram in 24 hours. It is derived to a large extent from foods containing benzoic acid, such as prunes, cranberries, green-gages, bilberries and many- other fruits, and is also formed from the metabolism of tissue and food protein. This substance is directly synthesized in the system by the combination of benzoic acid with glycocoll, but the exact place of synthesis is uncertain. A pathologic increase in the excretion of hippuric acid has been observed in acute febrile diseases, marked intestinal putrefaction, hepatic disturbances, and in diabetes mellitus, while in cases of acute diffuse and chronic paren- chymatous nephritis as well as in amyloid kidneys hippuric acid is practi- cally absent from, the urine. "■ (3) Oxyproteic and Alloxyproteic Acids. These acids have been isolated from the urine and seem to be constant constituents, derived from protein catabolism. Both of them contain sul- phur and have been credited with forming a large percentage of the neutral sulphur output as well as contributing 2 to 3 per cent, of the totalN excretion.^ Bondzynski believes the oxyproteic acid accountable for Ehrlich's diazo- reaction (page 334). Salkowski' has shown that a- certain amount (3 to 4 per cent.) of the mrinary nitrogen is precipi table by absolute alcohol and is non-dialyzable. This is the so-called " Colloidal N " and is largely referable to oxyproteic acid and N-containing carbohydrates. As this factor is increased in many cases of carcinoma up to 9 or 10 per cent, of total N, he believes this may be of value in diagnosis. However, numerous workers have demon- strated that this increase does not always obtain in cases of carcinoma and is found in pregnancy, diabetes, some hepatic disorders and in pulmonary tuber- culosis. Salmon and Saxl have outlined a method, for the determination of the sulphur factor of this oxyproteic acid as related to total N, and believe that an increase in this factor is indicative of carcinoma.* (4) Allantoin (C4H6N4O3). This substance is a product of oxidation of uric acid. It is somewhat variable in amount, usually being found only in traces, but after a large ' Dakin, Jour. Biol. Chem., 1910, VII, 103; Steenbock, Ibid., 1912, XI, 201; FoUn and Flanders, Ibid., 257; Hryntschak, Biochem. Ztschr., 1912, XLIII, 315; Raiziss and Dubin, Jour. Biol. Chem., 1914, XX, 125. "See Browinski and Dombrowski, Ztschr. f. physiol. Chem., 1912, LXXVII, 92; Domansky, Monatssdir. f. Psycli. u. Neurol., 191 2, XXXI, 53; Erben, Prager med. Wchn- schr., 1912, XXXVII, 4.27. ' Berl. klin. Wchnschr., 1905, XLII, 1581 and 1618; Ibid., 1910, XL VII, 533, 1746 and 2297. ■■ Marriott and Wolf, Am. Jour. Med. Sc, 1907, CXXXIII, 404; Mancini, Deutsch. Arch, f. klin. Med., 1911, CIII, 288; Caforio, Berl. klin. Wchnschr., 1911, XLVIII, 1843; Kojo, Ztschr. f. physiol. Chem., 1911, LXXIII, 416; Salomon and Sad, Wien. klin. Wchnschr., 1911, XXIV, 449; Riforma Med., 1911, XXVII, 421; Deutsch. med. Wchnschr., 1912, XXXVIII, 58; Neuberg, Der Harn, 1911, 788; Semenow, Folia Urol., 1912, VII, 215; Berl. klin. Wchnschr., 1913, L, 1436; Greenwald, Arch. Int. Med., 1913, XII, 283; Pribram, Miinch. med. Wchnschr., 1913, LX, 2047; Dozzi, Gazz. d. osp., 1913, XXXIV,, ;ioo7; Marenduzzo, Riforma Med., 1913, XXIX, 1149; Thar and Beneslawski, Biochem. Ztschr., 1913, LII, 43s; Lehmann, Deutsch. Arch. f. klin. Med., 1913, CXII, 376; StadtmuUer and Rosenbloom, Arch. Int. Med., 1913, XII, 276; Sassa, Biochem. Ztschr., 1914, LXIV, 19s; de Bloeme, Swart and Terwen, Miinch. med. Wchnschr., 1914, LXI, 1718; Biochem. Ztsehr., 1914, LXV, 34S; von Fiirth, Ibid., joij^LXIX 448: Gopdridge and Kahn, Biochem. Bull., 19x5, IV, 118; Damask, Vfiei^lym^wimikWJpt^^QiStVIII, 499. THE TJRINE 249 intake of meat especially rich in nuclein the amount may be quite percep- tible. It is found in fairly large quantities in the urine of the new-born, in the amniotic fluid, in ascitic fluid, in traces in the urine of pregnancy, and in leukemic blood. Pathologic variations have been little studied. The best method for the determination of this substance is that of Wiechowski.^ A point to be remembered in connection with allantoin is that it may be present in the urine in sufficient quantities to reduce copper solu- tions and must, therefore, not be mistaken for sugar. (2) Fatty Acids. Traces of volatile fatty acids are present in all normal urines. The most important of these are formic,^ acetic, propionic, and butyric acids. They are doubtless formed in the intestinal tract by bacterial action upon the carbo- hydrates of the food, and may, therefore, be to some extent an index of the degree of carbohydrate fermentation. Their normal amount does not much exceed o.oi gram in 24 hours, although BlumenthaP gives the figures under an ordinary diet as being equivalent to from 50 to 80 c.c. of tenth-normal sul- phiuric acid (0.25 to 0.39 gram). These acids are increased in febrile conditions, the amount running parallel to the rise in temperature. This increase in the fatty acids of the urine is known as lipaciduria. According to Rosenfeld, this increase in febrile states is observed only in those cases in which absorption of decomposing al- buminous material occurs, as in all suppurative processes within the system. In the convalescent stage of pneumonia these acids may be excreted in in- creased amount, while a diminished output is usually observed preceding the crisis. In hyperacidity of the gastric contents the fatty acids of the urine are increased, while in hypoacidity they are diminished. In contradistinction to the increase of fatty acids in cases of fever associated with suppurative con- ditions, we find a diminution in the amount of fatty acid in scarlet-fever, erysipelas, measles, and diphtheria. In cases of acute rheumatism formic acid is said to be excreted in large amounts. In cyclic vomiting lactic acid* may be increased. A simple method of determining the fatty acids of the urine is to acidify from 250 to 500 c.c. of urine with 50 to 75 c.c. of dilute sulphuric acid and distill. The distillate is then titrated with tenth-normal sodium hydrate solution using phenolphthalein as an indicator. The results are expressed in terms of the corresponding number of c.c. of tenth-normal acid. 1 Beitr. z. chem. Physiol, u. Path., igo8, XI, 129; Biochem. Ztschr., 1909, XIX, 378; Ibid., 1910, XXV, 431. See, also, Plimmer and Skelton, Biochem. Jour., 1914, VIII, 641; Givens, Jour. Biol. Chem., 1914, XVIII, 417. ^ See Dakin and Wakeman, Jour. Biol. Chem., 1911, IX, 329; Steppuhn and Schellback, Ztschr. f. physiol. Chem., 1912, LXXX, 274; Dakin, Janney and Wakeman; Jour. Biol. Chem., 1913, XIV, 341; and Strisower, Biochem. Ztschr., 1913, LIV, 189, for a discussion of formic acid as an intermediary metabolic product of both carbohydrates and protein. ' Pathologic des Harnes, Berlin, 1903. * For methods of determination see von Furth and Charnass, Biochem. Ztschr., 1910, XXVI, 199; Mondschein, Ibid., 1912, XLII, 91 and 105; also, Ishihara, Ibid., 1913, L, 468; Levene and Meyer (Jour. Biol. Chem., 1913, XV, 65) have shown that lactic acid is a prod- uct of the intermediary metabolism of carbohydrates. See, also, Yoshikawa, Ztschr. f. physiol. Chem., i9i3,LXXXVII, 382; von Furth, Biochem. Ztschr., 1914, XLIV, 131; Ibid., 156; Wien. klin. Wchnschr., 1914, XXVII, 877; Underhill and Steele, Am. Jour. Dis. Child., 1914, VIII, 127; Sch^^^r^Bgiop^em^.Ztgc^h5^^iS,LXX, 294. 250 DIAGNOSTIC METHODS (3) Oxalic Acid. The amount of oxalic acid eliminated in the urine in 24 hours varies from 10 to 20 milligrams. A portion of this excretion is undoubtedly derived from the diet, but some of it is produced in the metabolism of the tissues, especially of those containing nuclein substances. Although the carbohydrates do not take part in the production of oxalic acid normally, we find, in conditions asso- ciated with fermentation of the carbohydrates in the stomach, a large increase in the output of oxalic acid. As oxalic acid is more or less readily formed by oxidation of uric acid, passing through the intermediate stage of oxalunc acid, we can readily see that increase of nuclein metabolism may increase the output of this substance. Strangely enough, it has been found that the in- take either of pure nuclein or of nuclein-containing foods was not associated with a corresponding increase in the urinary oxalic acid, so that we must assume that a large portion of this intake escapes in the feces in the form of calcium oxalate. It has, moreover, been found that the administration of oxalates by the mouth is not associated with increased excretion either in the urine or feces, so that we must assume a decomposition of the oxalic acid into carbon dioxid and water somewhere in the system. This conversion appears to take place in the intestinal canal under the influence of bacterial action. Crystals of calcium oxalate are frequently found in the urine in cases showing a marked increase in the output of ethereal sulphates, so that we must either assume an increased formation from the carbohydrates of the food, an in- creased intake and absorption of oxalates of the food, or an increase in the nuclein metabohsm as a result of absorption of the toxic products of intestinal putrefaction. 1 Among the foods which are known to contain relatively large amounts of oxahc acid we find spinach, rhubarb, tomatoes, carrots, celery, string-beans, green peas, potato, figs, plums, strawberries, pepper, cocoa, tea, and coffee. Those which contain very little are meat, milk, eggs, butter, cereals, rice, asparagus, cucumbers, mushrooms, lettuce, cauliflower, cabbage, pears, peaches, grapes, and melons. The increased elimination of oxalic acid may or may not be associated with a deposition of crystals of calcium oxalate. An increased ehmination is observed in cases associated with irregular activity of the gastro-intestinal tract. These cases usually show many types of nervous disorder, especially neurasthenia, and are characterized by the large deposit of oxalate crystals in the urine. To this condition has been given the name oxaluria, which can hardly^be dignified as a definite pathologic entity. No inference can actually be drawn regarding the degree of elimination of oxahc acid from the ap- pearance of a deposit in the urine, as it has been shown by Fiirbringer that the urine may contain a large amount of oxahc acid without a sediment of calcium oxalate crystals being formed.^ Such cases, however, should be 1 See Wegrzynowski, Ztschr. f. physiol. Chem., 1913, LXXXIII, 112; also, Aron and Franz, Monatsschr. f. Kinderh., 1914, XII, 645. 2 Williams and Williams (Arch. Diagnosis, 1913, VI, 263), who show the relationship of obscure pain and hematuria to the excretion of small crystals of calcium oxalate. FittiDaldi, Rif. Med., 191S, XXX, 981. ^ Digitized by Microsoft® THE XTEINE 251 ■watched more or less closely as separation of calcium oxalate may occur within the pelvis of the kidney and lead to the formation of a calculus. In cases of jaundice a marked oxaluria may be observed which is directly ref- erable to the associated cholemia. In occasional cases of diabetes mellitus. the elimination of oxalic acid may be much increased. Quantitative Detennination. Baldwin's Method.* Five hundred c.c. of a mixed 24-hour specimen of urine are treated with 150 c.c. of 95 per cent, alcohol and the mixture set aside for 48 hours to allow the calcium oxalate to precipitate. It is then filtered, each particle of the pre- cipitate being transferred to the filter by means of hot water and a rubber- tipped glass rod. This precipitate is then washed with hot water and later with I per cent, acetic acid. The precipitate is then washed from the fil- ter-paper by a stream of dilute hydrochloric acid from a wash-bottle until every trace of the precipitate is removed from the filtet and dissolved. The filter is then washed with hot water until the washing.^ are no longer acid in reaction. The hydrochloric acid solution and the washings are evaporated to about 20 c.c, a little calcium chlorid solution is added, the solution is neu- tralized with ammonia, is then rendered slightly acid with acetic acid, and 95 per cent, alcohol added in an amount equal to one-half the volume of the liquid. The mixture is then set aside for 48 hours, after which the precipitate of calcium oxalate is collected on an ash-free filter, washed with cold water and dilute acetic acid until free from chlorids, and the filter with its contents is incinerated in a weighed platinum crucible. This latter process is first carried out over a Bunsen burner and later over a blast-lamp. The crucible is then dried in a desiccator and weighed. The difference in weight represents the amount of calcium oxid obtained from 500 c.c. of urine. Each gram of this oxid represents 1.6 grams of oxalic acid. (4) Ferments. Several ferments^ have been demonstrated in the normal and pathologic nurie, but do not seem to have any great clinical importance at the present time. Pepsin. This ferment is present in practically every specimen of urine. It has been found by Grober, Gehrig, Griitzner, Mathes, Stadelmann, and others. It seems to be absent or very much diminished in cases of typhoid fever, gastric carcinoma, and hypoacidity.' In cases of pneumonia, Lenobel and Kun and Lochbihler have observed the presence of a ferment, which seemed to be pepsin in increased amounts. Scola reports a diminution of the normal pepsin content in severe diseases of the nervous system. Diastase. Traces of this ferment are found in normal urine. It is increased by a carbohydrate-rich diet and diminished by a carbohydrate-poor regime. ' Jour. Exper. Med., 1903, V, 27. " See Lindemann, Ztschr. f. klin. Med., 191 2, LXXV, 58. ' See von Kozawa, Internal. Beitr. z. Path. u. Ther., d. Ernahrungsstor., 1912, IV, 44; Fuld and Hirayama, Ztschr. L.ejsp.. Path. u^Ther,, 1012, X, 248; also, Tachau, Ztschr. f. klin. Med., 1912, LXXVI, iQigaiS&a,b$imll€J'aSMt® FakaMt, Tokio, 1914, XI, 293. 252 DIAGNOSTIC METHODS Wohlgemuth^ beheves an increase is associated with a disturbance of pan- creatic function. It is lessened in nephritis. In diabetes the diastase varies with the sugar output. Lipase. This ferment is present normally only in minute traces. It is found, however, in cases of hemorrhagic pancreatitis, jaundice, and in diabetes melli- tus. It may be detected by the method of Kastle and Loevenhart which is as follows: In each of three flasks are placed 5 c.c. of urine. One of the flasks is boiled to destroy the ferment which may be present. To a second flask are added a few drops of phenolphthalein solution and the acidity determined by titration with tehth-normal sodium hydrate solution. The amount of alkali necessary to neutralize the 5 c.c. of urine is then added to each of the other flasks. To these flasks are then added 0.25 c.c. of ethyl-butyrate and o.i c.c. of toluol, the flasks being then placed in the incubator at 37°C. for 24 hours. To each of these flasks there is then added }^ c.c. more tenth-normal hydro-chloric acid than the amount of tenth-normal alkali previously added. The mixture is then shaken out with 50 c.c. of ether and 25 c.c. of alcohol to remove the butyric acid. This is then titrated with tenth-normal sodium hydrate, each c.c. of tenth-normal sodium hydrate representing 0.0088 gram of butyric acid.^ (5) Mucin-like Substances. {a) Mucin. True mucin is present in traces in practically all urine. It is found both as an insoluble portion which forms the nubecula and as a soluble portion which is much smaller in amount than the insoluble form, is precipitated by acetic acid, but is easily soluble in a slight excess of the acid. This form of protein is derived from the urinary passages and has practically no pathologic importance, although it may be much increased in catarrhal conditions of the urinary tract in which it may appear as a gelatinous ropy material, or in rare cases in the form of casts of the uterer or urethra from i to 10 cm. long and 3 to 4 mm. thick. These cases are rare and have been reported by von Jaksch under the name of "ureteritis membranacea" and by Frank under the name of "pyelitis productiva." Mucin is precipitated by the ordinary reagents for albumin (to be dis- ' cussed later), but is soluble in an excess of these reagents, so that it need not be mistaken for albumin. It is frequently confused with the " nucleo-albu- mins," but may be distinguished, chemically, by the fact that it contains no phosphorus and gives on heating with acids a substance which reduces copper solutions. (6) Nucleo-albumin. The largfe majority of specimens of urine contain a substance (other than 1 Biochem. Ztschr., igog, XXI, 432; Berl. klin. Wchnschr., igio, XL VII, 92 and 1444. See, also, Corbett, Jour. Obst. and Gynec. Brit. Emp., 1913, XXIII, 227; Neumann, Deutsch. Arch. f. klin. Med., 1913, CXI, 164; and Geyelin, Arch. Int. Med., 1914, XIII, 96; Marino, Semana Med., 1914, XXI, 907; Lister, Brit. Med. Jour., 1914, II, 584; Brown and Smith, Bull. Johns Hopkms Hosp., i9i4,XXV, 213; King. Am. Jour. Physiol., 1914, XXXV, 301; Kahn and Brim, Am. Jour. Obs., 1915, LXXI, 39. 2 See Quinan, Jour. Med. f ' THE URINE 253 true mucin) which is precipitated on the addition of cold acetic acid. Dilute acetic acid does not dissolve the precipitate, so that true mucin is excluded, as the latter is usually dissolved even by dilutions of acetic acid which do not precipitate the other bodies. The reaction with acetic acid is somewhat intensified if the urine be diluted. Practically every normal urine contains traces of such substances, which will give a precipitate with acetic acid, especially if the salts be removed by dialpsis. In all probability this body, found in normal urines, is either euglo- bulin or a mixture of this protein with fibrinogen. It has been called " nucleo- albumin," but this is in all probability a misnomer. It is probably true that real nucleo-albumin is never a normal consti-uent of the urine. In this con- nection it must be stated that much confusion exists regarding the nature of true nucleo-albumin.^ This substance has been considered to be identical with nucleo-protein, but with absolutely no basis of chemical facts. Nucleo- protein (true nuclein) is a combination of protein with the prosthetic group, nucleinic acid, which splits up into phosphoric acid and purin bodies; while nucleo-albumin (pseudo-nuclein) is a combination of protein with paranu- cleinic acid, which is rich in phosphorus, but does not yield purin bases tn hydrolysis. Morner^ beheves that most of the so-called "nucleo-albumin" is a com- pound of true serum-albumin with an albumin-precipitating body formed on addition of acetic acid. He showed that there were three such precipitating bodies present in the urine; chondroitin-sulphuric acid' was practically al- ways present, nucleinic acid occasionally present, and tauro-cholic acid, which may be normally present in traces, but in certain pathologic conditions is much increased. He believes that these precipitating bodies are normally present in excess and, therefore, any increase of a precipitate on addition of the acetic acid would mean an increased excretion of albumin. The more these precipitating bodies predominate the more the precipitate resem- bles "nucleo-albumin." From the pathologic standpoint what has been called true "nucleo- albumin" appears in conditions destroying the integrity of the epithelium of the uriniferous tubules or of the bladder as well as in conditions associated with the excretion of pus in the urine. Thus we would expect to find such a body in acute nephritis, whether the result of bacterial or of exogenous toxins, in the acute febrile diseases, in renal hyperemia, in leukemia, in acute yellow atrophy, and in obstructive jaundice, in which case this body is derived doubtless from the bile. In cases of nephritis this body, precipi' able by acetic acid in dilute solution, may precede and follow the true albuminuria. In amyloid kidney this body seems to be the chief type of protein present. In orthostatic albuminuria this substance may be the only protein present and may persist after the others have cleared up. In cases in which the urine 1 See Jones, "Nucleic Acids," Longmans Green & Co., New York, 1914. 2 Skand. Arch. f. Physiol., 1895, VI, 332. ' See Levene and La Forge, Jour. Biol. Chem., 1913, XV, 69 and 155; also, Pollitzer, Deutsch. Med. Wchnschr., 1912, XXXVIII, 1538; Med. Klin., 1913, IX, 2101; Levene and La Forge, Jour. Biol. Chem., 1914, XVIII, 12^; Ibid., 1915, XX, 433. Digitized by Microsoft® 254 DIAGNOSTIC METHODS contains a large number of epithelial cells, casts, and pus-cells, Matsumo to finds a substance precipitable by acetic acid, but only in very small amounts. This finding would seem to indicate that nucleo-albumin, if such ever occurs, is at least not an indication of cellular origin from increased epithelial desquamation. This body may be found in general catarrhal conditions of the urinary tract, as in cystitis or pyelitis, but in such cases we are more apt to obtain true mucin. In the above discussion the writer has not attempted to differentiate these bodies, as the reports in the literature have httle importance beyond the fact that a body precipitable by acetic acid was obtained. Each worker has named this body as he understood it and in many cases has had no definite basis for such conclusion. To remove it from the urine, add a solution of lead acetate and filter; the precipitated phosphates and chlorids carry down this protein^ , (6) Pigments and Chromogens. (a) Urochrome. This pigment is the chief coloring matter of normal urine, imparting a yellow, orange, or a brownish color to the urine, depending upon its concen- tration. It is closely related to urobilin, especially the so-called normal uipobilin of MacMunn, as this latter body may be readily converted into xiro- chrome by evaporation of its aqueous ethereal solution. It is, in all prob- ability, a mixture of one or more pigments, contains about 4 per cent, of nitrogen, and is free from iron. It is readily soluble in .water and alcohol; sparingly soluble in acetic ether, amyl alcohol, and acetone; insoluble in ether, chloroform, and benzol. An increase of urochromogen, the precursor of this pigment, seems to be of some clinical importance. Weisz^ has introduced the following test for its detection. Place the suspected urine in a test-tube and dilute with three times its volume of water. Mix well and add 3 drops of a i to 1000 aqueous solution of potassium permanganate. Shake well. A yellow tint or an increase in the yellow tone of the mixture indicates a positive reaction. This reaction is an indication of a toxic metabolic disturbance and is given in cases with high fever, especially in pulmonary tuberculosis and typhoid fever. It is never seen normally and, rarely, in carcinoma. A positive reaction is a contra-indication to the use of tuberculin in tubercular cases. (&) TJroerythrin. This pigment is a constituent of a large majority of normal urines, al- though it is probable that it indicates a slight deviation from the normal. It has been called rosacic acid by Prout and purpurin by Golding-Bird. To this pigment is due the salmon or brick-red color which the urinary sediments iMed. Klin., igio, VI, 1661; Biochem. Ztschr., 1911, XXX, 333;'Wien. klin.Wchnschr., 1912, XXV, 1183 _ See, also, Heflebower, Am. Jour. Med. Sc, 1912, CXLIII, 221; Paranhos and Giolito, Brazil-med., 1913, XXVII, loi; Vitry, Rev. internal, de la tuberc, 191^, XXIII, 325; Skorczewski, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 113; Salustri, Policlinico, 1913, XX, 1729; Nicola, Gazz. d. osp., 1913, XXXIV, 1465; Keim and Vigot, Presse m€d., 1914, XXII, 153; Martelli and Pizzetti, Policlinico, 1914, XXI, 182; GuUbring, Hygiea, 1914, LXXVI, 599; Metzger and Watson, Jour. Am. Med. Assn., 1914, LXII, 1886; Schaffle, Ibid., 1914, LXIII, 1294; Pignacca, Gazz. d. osp., 1914, XXXV, 353; Tuliato, Ibid., 665; Balduzzi and Ballero, Ibid., 2053; Rhein, Miinch. med. Wchnschr., 1914, LXI, 23SS; Bruni, Gazz. d. osp., 1915, XXXVI, 401; Ferrannini, Riforma Med., 1915, XXXI, lofi^j^P^S'S S." "^"^S^^hfMmM&^- '"^d. Wchnschr., 1915, LXII, THE URINE 255 take in highly concentrated febrile urines. Normally, it may not be present in sufficient amount to color the urine, but under pathologic conditions it may impart a deep orange tint to this fluid. It is soluble in amyl alcohol, slightly solubleinacetic ether and absolute alcohol and very difficultly soluble in water. This pigment appears to be increased on a meat diet, after severe exercise, profuse perspiration, or by irregular digestion. Pathologically, it is observed especially in cases of hepatic insufficiency, in chronic cardiac and pulmonary disease, in acute articular rheumatism, in malarial fever, and in general acute febrile diseases. In typhoid fever one does not find this pigment as fre- quently as in most other acute febrile conditions. (c) Urobilin. This substance appears in the urine not as a free pigment, but in the form of the chromogen urobilinogen, which is decomposed into urobilin through the influence of the light. It is claimed that various types of urobilin are found, as for instance the normal urobilin of MacMunn and the pathologic urobilin of Jaffe. Whether these are really different bodies is at present an un- settled question. Urobilin appears to be identical with the stercobilin of the feces and is not the same as the normal fecal hydrobilirubin. Much discussion has centered around the origin of this pigment. Passing through the stages of the hepatogenous, hematogenous, nephrogenous and histogene- tic urobilinuria, the general concensus of opinion seems at present to be that most of the urinary urobilin is of enterogenous origin.^ According to this theory, the pathogenesis of urobilinuria may be presented as foUows: "The liver-cell, both in normal and abnormal conditions, fojms only bilirubin from the blood pigment. Providing there is no marked obstruction to the passage of bile into the intestine, the bilirubin is acted upon by bacteria which reduce it so completely to urobilin that only traces of bilirubin appear in the feces. A part of the urobilin is absorbed and is excreted in the urinej while traces appear in the bile and in pathologic transudates. When bacterial action is excluded, as in the new-born, no urobilin is found in the urine. Further, when bile is not present in the intestine, as in cases of absolute oc- clusion of the ductus choledochus, urobilinuria dose not occur. It is sparingly excreted when the production of biliary pigment is diminished, as in hunger, while the amount is small or at most normal in cases of incomplete exclusion of bile from the intestine. On the other hand, the amount excreted may reach abnormal limits if a preceding obstruction, accompanied by stasis, has been overcome, and bile flows freely into the intestine. Likewise the quantity may be abnormally large if the production of biliary pigment from the red blood-corpuscles increases as a result of infection and intoxication, or of he- patic lesions, such as cirrhosis and cyanotic induration. In these cases the bile is tenacious and the condition may give rise to jaundice although it is seldom that the stasis is so great that the bile is completely shut off from the ' intestine. Indeed, in most cases, owing to the excretion of excessive pigments in the bile (pleiochromia), a more than normal amount of pigment passes into ' See Wilbur and Addis, Jour. Am. Med. Assn., 1912, LIX, 929; also, Fromholdt and Nersessoflf, Ztschr. f. exper. Path. u. Therap., 1912, XI, 400; Wilbur and Addis, Arch. Int. Med., 1914, XIII, 235; FeldnQ/gZikedb&if.M/(6Jaa£P®d. Med. u. Chir., 1915, XIX, 163. 256 DIAGNOSTIC METHODS the intestine, and a result of this there arises a marked urobilinuriawithamild degree of biliary stasis. In many cases the stagnation of bile is great enough to cause a passage of biliary pigment from the blood into the urine, leading to a marked urobilinuria, a mild degree of bilirubinuria, and a yellow-tinting of the tissues. In other cases the absorption of bile is so slight that only yellowing of the tissues results, yet the concentration of pigment in the blood does not suffice to permit of its excretion by the kidneys, so that marked urobilinuria and yellowing of the tissues without bilirubinuria ensues" (Weintraud). Urobilin is found in febrile conditions, chronic passive congestion, lead- poisoning, cases in which extravasation of blood into the tissues occurs, in any condition associated with marked hemolysis, hepatic cirrhosis, and in the cases of jaundice outlined above by Weintraud. It has been noticed in increased amounts in Addison's disease, extrauterine pregnancy, hemophilia, and in secondary syphilis.^ The presence of an increased amount of urobilin usually causes a dark- yellow color of the urine, the foam in such cases being colored, more perhaps due to the presence of other pigments than to urobilin itself. UrobiHn is soluble in ethyl alcohol, amyl alcohol, -and chloroform, and slightly soluble in ether, acetic ether, and in water. If an acid solution of urobilin be examined with a spectroscope, it shows a broad absorption band to the right of E, the left-border of which reaches nearly to b, while the right border encloses F. If the solution be alkaline, the spectrum shows a less broad absorption band between E and F enclosing b. This solution is best made as follows: Ten to 20 c.c. of the urine are acidulated with a few drops of hydrochloric acid and shaken out with from 6 to 10 c.c. of amyl alcohol, which then shows the characteristic speclrum of acid urobilin. If to a small portion of this amyl alcohol solution be added a few drops of i per cent, solution of zinc chlorid, which has been strongly alkalinized with am- monia, abeatifulgreenfluorescenceappears. WithEhrlich's dimethyl-amido- benzaldehyde reaction (p. 336) this pigment gives a distinct red color. {d) Indican.2 In the decomposition of protein occurring in the intestinal canal indol (CsHtN) and skatol (C9H9N) are found among the products of this bacterial cleavage. These substances are absorbed and oxidized in the blood to indoxyl (CsHtNO) and skatoxyl (C9H9NO). These bodies are then conjugated with sulphuric acid forming indoxyl and skatoxyl sulphuric acids, after which they are excreted in the form of the potassium salt, indoxyl potassium sul- phate (CsHeNO — SO2 — OK) and skatoxyl potassium sulphate (CgHsNO — SO2 — OK) . To the former of these is given the name indican. This chromo- gen is present in much larger amounts than is the skatoxyl compound so that our discussion will include principally the former. The absolute amount of indican occurring in the urine depends upon the amount of decomposition occurring in the intestine. The largest amounts are naturally observed in health following a meat diet, while the urine may be ' See Simon, Med. Klin., 1913, IX, 1164; Hildebrandt, Arch. f. Verdauungskr., 1913, XIX, 442; Kamsarakau, MedfObozr., igi^.tXXIX/^ft&fj Molnar, Virchow's Arch. f. path. Anat., 1913, CCXIII, 433; Hauimann, Zcbclir. f. cxpeKr'ath. u. Therap., 1913, XIII, 373. 5 See Baar, Die Indicanurie, Wien, 191 2; New York Med. Jour., 1914, XCIX, 669. THE URINE 257 practically free from indican on a vegetable or a milk d et. The pathologic variations may be summed up as follows. An increased elimination of indican is observed in all diseases which are associated with an increased intestinal decomposition. "^ This decomposition usually occurs in the large intestine, but may at times take place in the small bowel, in which cases the degree of in- dicanuria may be greater owing to the increased absorptive power of the small bowel. Although many writers state that indicanuria is not seen in cases of simple constipation, the writer must take exceptioris to these state- ments as some of his most intense and persistent cases of indicanuria have been found in uncomplicated constipation. Secondly, an increased excretion of indican is observed in cases showing diminished peristalsis, as, for instance, in ileus and peritonitis. These cases frequently show an intense reaction.^ If the obstruction leading to ileus be in the large bowel, indican is either absent or appears much later than is the case if the small bowel be involved. Lastly, in any condition associated with protein decomposition anywhere in the system, as, for instance, in empyema, putrid bronchitis, abscess formation, etc., indican may be much increased. The color of the urine is usually normal when voided, although a large amount of indican may be- present. In some cases oxidation of this chro- mogen has occurred within the system and the urine appears greenish or even blue when voided. If the urine be allowed to stand until decomposition occurs, a reddish or bluish metallic-like scum may be observed, due to the •conversion of the indican into indigo-blue. ,Most of the tests for the presence of indican in the urine depend upon the oxidation of the indican, according to the following equation (this body having been previously decomposed by concentrated HCl into indoxyl and sulphuric acid). 2C8H7NO + 20 = C16H10N2O2 + 2H2O. Tests for Indican. Jafife's Test. A few c.c. of urine are treated with an equal volume of concentrated hydrochloric acid, two or three drops of a strong solution of calcium hypo- chlorite (bleaching powder or "chlorid of lime") are added and the contents mixed. Two c.c. of chloroform are then added and the tube inverted several times. In this process the indican is oxidized to indigo-blue which is taken up by the chloroform. The depth of the blue coloration of the chloroform will serve as an approximate estimate of the amount of indican present. In absolutely normal urine no blue coloration or, at most, a faint bluish tinge is observed. Care must be taken in this test to avoid an excess of the hypo- chlorite, as this will convert the indigo-blue into isatin which is distinctly yellow, according to the following equation: C16H10N2O2 + 02= 2C8H6NO2. Obermayer's Test. A few c.c. of urine are mixed with an equal volume of Obermayer's reagent 1 See Morgan, Am. Jour. Med. Sc, 1912, CXLIV, 827; also, Benedict, Arch. Diagnosis, 1912, V, 159. " Although there appears to be no definite relation of indicanuria and albuminuria, the writer has rarely failed to oh^fg^l^ssf ftyi^j^^gfegflf^in urines showing marked indican reactions. 258 DIAGNOSTIC METHODS (a 0.2 per cent, solution of ferric chlorid in concentrated hydrochloric acid) and the solutions mixed by repeatedly inverting the tube. A few c.c.of chloroform are then added and the tube inverted as before. The presence of any appreciable amount of indican is indicated by a dark brown to black coloration of the urine and the later absorption of this color by the chloro- form, which becomes a more or less deep shade of blue. If the urine be very dark in color or if bile pigment be present, the pigments may be removed by adding a solution of lead subacetate and filtering. If potassium iodid be present this test yields a red color which will disappear on addition of sodium thiosulphate solution. Sahcyluric acid gives a violet coloration. If formalin be used as a preservative, indican is incapable of detection by any of the tests given. Thymol-containing urines give a violet coloration to the chloroform extract. This color is destroyed by sodium hydroxide or thiosulphate.' Jolles^ has made this fact the basis of a new test for indican in the urine. Instead of using the Obermayer reagent as such, the urine may be mixed with the hydrochloric acid and one or two drops of a 10 per cent, solution of ferric chlorid may then be added to the mixture. This test is a much more reliable one than is the Jaffe test, as the latter is very prone to carry the oxidation to the isatin stage rather than to the indigo-blue phase.' Skatoxyl-potassium Sulphate. A certain amount of skatoxyl-potassium sulphate is formed along with the indican. In some cases for reasons not well understood, this pigment or one closely allied to it appears in the urine in excess of the indican, so that the above test may give distinct red colorations of the chloroform instead of the usual blue. Just exactly what pigment causes these red variations is doubtful. Some of this pigment is always present along with the indican and may be extracted either with hot water or with a mixture of alcohol, ether and water. To this red pigment has been given the name indigo-red, skatoxyl-red, urorubin, urorhodin, and several others. If the urine be heated on applying Jaffe's test, a dark red coloration is observed, while if Obermayer's test be used the coloration will be of a reddish-violet. Rosenbach's Test. A few c.c. of urine are boiled and concentrated nitric acid added drop by drop during the boiling. The urine takes a deep red color and the foam ap- pears bluish-red. If the nitric acid be much in excess the urine will assume a yellowish-red color and the foam a distinctly yellow tint. If sodium hydrate or ammonia be now added drop by drop, a bluish-red precipitate is observed which is soluble in an excess of the alkali with a brownish-red coloration. This test is due to the presence of indigo-red. Its clinical significance is the same as that of indican. This pigment may be obtained directly from the urine by neutralizing it with sodium hydrate and shaking out with ether, when the ether will take a distinct red color. ' Rosenbloom, New York Med. Jour„ 1913, XCVIII, 814. 2 Ztschr. f. physiol. Chem., 1913, LXXXVII, 310. See, also, Stanford, Ibid., 188 and Ibid., 1913, LXXXVIII, 47; also, Edes, Arch. Diagnosis, 1913, VI, 152; Rossi, Policlinico, 1914, XXI, 297; CanteUi, Ga.2^j^je^^^,|^fj^^^^^^Jo\l^s, Ztschr. f. physiol. Chem., ^ See Natonek, Zentralbl. f. inn. Med., 1913, XXXIV, 1124. THE URINE 259 Quantitative Determination. Wang's Method. The principle of this method is the decompostion of indican and its oxidation to indigo-blue. This compound is then transformed into indigo- sulphuric acid, which is directly determined by titration with potassium per- manganate solution. Technic. A preliminary determination of the relative amount of indican is made with Obermayer's reagent. If a strong reaction is obtained, from25 to loo ex. of urine are used, while if the reaction be slight 200 to 500 c.c. are necessary. The urine should be acidified with acetic acid, unless its reactionbe already acid. Fifty c.c. of urine, or a larger amount if the conditions mentioned above obtain, are treated with 5 c.c. of a 20 per cent, solution of lead acetate, or one-tenth the volume of urine in case a larger amount of urine be taken. The urine is then filtered and a large and accurately measured portion of the filtrate is treated, in a separatory funnel, with an equal volume of Obermayer's reagent. This mixture is then shaken out with chloroform, using 30 c.c. of this menstruum and shaking for one minute. At least four such chloroform extractions should be made, more if the chloro- form still extracts indigo-blue from the mixture. The chloroform extract is placed in a small flask and the chloroform distilled. The residue in the flask is dried for a few minutes on the water-bath to remove the last traces of the chloroform, and is washed either with hot water or with a mixture of equal parts of alcohol, ether, and water. These solvents remove the red coloring matter, leaving the indigo-blue undissolved. The extract is filtered through a smaU filter and the indigo-blue completely transferred to the filter, after which it is thoroughly washed with hot water. This indigo- blue is dissolved on the filter with boiUng chloroform, the filtrate being allowed to run into the original flask. The chloroform is again distilled and the residue dried on the water-bath. This purified indigo-blue is dis- solved in 10 c.c. of concentrated sulphuric acid and the solution diluted to 100 c.c. with water. This solution of indigo-sulphuric acid is then titrated with standard potas- sium permanganate solution of such a strength that i c.c. will represent approximately 0.0062 gram of indigo-blue. This solution of potassium per- manganate contains about 3 grams of potassium permanganate to the Uter. Its titer is determined before use by titrating against a solution of pure indigo-blue in sulphuric acid. In making this titration the concentrated solu- tion is not used, but a dilute one made by diluting 5 c.c. of the stronger permanganate solution to 200 c.c. (each c.c. of which wiU represent 0.000155 gram of indigo-blue). In this titration the blue color of the indigo-sul- phuric acid does not change to any extent on the addition of the first drops of the permanganate solution, but giradually turns greenish and then becomes yellowish or entirely colorless. The amount of indigo-blue in the urine used is readily ascertained by multiplying the number of c.c. of permanganate solution used by the amount of indigo-blue represented by the titer of the Digitized by Microsoft® 26o DIAGNOSTIC METHODS solution (in the writer's laboratory i c.c. of the diluted solution equals 0.000155 gram of indigo-blue). It has been found by Ellinger that only about 87 per cent, of the theoret- ical yield is obtained by this method, owing, probably, to the simultaneous for- mation of isatin from the indigo-blue. The average daily excretion of indi- can, as revealed by this test, ranges between 5 and 20 mg. Folin's Method. This method has not, as yet, been extended so as to give absolutely quan- titative results. Its principle is the comparison of the color given when urine is treated with Obermayer's reagent with that of Fehling's solution as a stand- ard. To this standard has been given the arbitrary value of 100. Exactly one one-hundredth of the 24-hour specimen of urine is taken for each determination and treated with an equal volume of Obermayer's re- agent. The indigo-blue is then extracted with 5 c.c. of chloroform until all of the pigment has been dissolved. With the chloroform solutions are then made colorimetric comparisons with Fehling's solution, using either the Du- boscq or Sargent instrument. Folin finds the average indican excretion, on this basis, from a diet of 119 grams of protein to be 77, while on a nitrogen- free diet no indican is found, (e) Uroroseinogen. This chromogen, shown by Herter^ to be indol-acetic acid, is converted by oxidation into the pigment urorosein, which is soluble in ethyl and amyl alco- hol but insoluble in chloroform, ether and benzol. If the urine stands for some time bacterial action produces this pigment, although an alkaline reac- tion may destroy the coloration. Its amyl-alcoholic solution shows a sharp narrow absorption band between D and E.^ Although this pigment appears in normal urine only in traces, it may be markedly increased by a strict vege- table diet. Pathologically, it appears in cases of severe gastric and intestinal disturbances, tuberculosis, pernicious anemia, nephritis, severe chlorosis, diabetes mellitus, carcinoma, osteomalacia, typhoid fever and dementia praecox.' It has, therefore, little differential value although it is, often, as significant as indican of excessive protein decomposition in the bowel. Test for Urorosein {indol-acetic acid) : If 5 c.c. of urine be mixed with 5 c.c. of chemically pure HCl and a few drops of a i per cent, solution of sodium nitrite a characteristic rose-red coloration develops, which is due to oxidizing rather than nitrifying action. This pigment also gives a red color- ation with Ehrlich's dimethyl-amidobenzaldehyd reaction. {B) Abnormal Compositioii. (i) Proteins. There has been much discussion as to whether a true physiologic pro- teinuria occurs. Without going into a discussion of the subject the writer wishes to state his belief that a true physiologic proteinuria occurs, but that such may never be detected by the usual clinical methods of examination. For^dur purposes, therefore, the presence of protein of one type or another in 1 Jour. Biol. Chem., 1908, IV, 107, 239 and 253. ife ^i^r^u^i>>^mWMm^^® THE TJRINK "^^ 261 amounts amenable to detection by clinical methods must be considered pathological. (a) Serum Albumin. From the pathologic standpoint, serum albumin is the most important protein found in the urine. The amount excreted in 24 hours is variable and does not necessarily have any relation to the severity of the kidney lesion should one really exist. From 5 to 10 grams of this protein per diem may be regarded as a moderate pathologic excretion, while a lesser would be of little significance and a greater would be regarded as excessive. As large amounts as 40 grams have been observed, but such findings are exceptional. Morner considers an excretion of albumin varying from 25 to 75 mg. per liter as a nor- mal output, which amount would, however, escape the usual clinical tests. As some of the more delicate tests for albumin, as for instance Spiegler's reagent, will show traces of albumin in practically every specimen of urine examined, we should limit our conception of albuminuria to those cases which react with the ordinary tests rather than with the most delicate. According to Hofmeister, the standard upon which one bases a judgment as to the pres- ence of a pathologic albuminuria is the formation of a distinct albumin ring within three minutes after the urine and nitric acid are in contact in Heller's test (see below). Moreover, the term albuminuria should be limited to those cases in which there is some disturbance of the renal epithelium, especially of the glomeruli. This does not exclude those cases of purely functional albu- minuria in which no distinct lesion of the kidney exists, as these cases are all associated with some abnormality of the excreting organ. It not infrequently happens that albumin, as found in the urine, is derived from some portion of the urinary tract below the kidney, as when inflamma- tory exudates, blood, lymph, spermatic or prostatic fluids, pus and other ex- traneous material are mixed with the urine after it is excreted by the kidney.^ To such cases is given the name/afae or accidental albuminuria, in contra-dis- tinction to the true albuminuria in which the albumin is present when ex- creted by this organ. Before reporting a finding as one of true albuminuria, it is absolutely imperative that extraneous albumin be excluded. Ftmctional Albuminuria. Not infrequently do we find in perfectly healthy individuals a urine which is normal in every way with the exception of the presence of albumin, which is easily detected by our ordinary methods. This type of albuminuria is observed following severe muscular exercise beyond the point to which the subject is accustomed. Thus, in raw recruits under the severe forced marches during the early days of their service we find this type very prevalent. Leube states that 59 per cent, of such soldiers show a temporary albuminuria, which disappears after the subject becomes accustomed to the increased exercise.^ It is a common occurrence to find such a functional albuminuria in football players, bicycle riders, crew men, and general athletes after every period of ' See Belfield (Jour. Am. Med. Assn., igiSj LXIV, 2040) for a discussion of vesicular albuminuria due to presence of both globulin and albumin derived from the seminal vesicles. 2 See von Hecker, Berl. klin. Wchnschr., 1913, L, 1848; Bugge, Norsk Mag. f. Laegevi- densk., 1913, LXXIV, i6oi;^g^£^gdji^^^j:pggji^]. f. Schweiz. Acrzte, 1915, XLV, 262 DIAGNOSTIC METHODS increased exertion. This question may be summed up by the statement that the excretion of albumin is simply dependent, under these conditions, upon the limit of endurance of the subject, practically everyone being able to pro- duce an albuminuria if he overdoes in any way. Moreover, cold baths, excess- ive mental labor, and severe emotion may lead to quite an extensive albumin- uria, especially in an individual with somewhat lessened resistance. Ac- cording to Rem-Picci albuminuria is a constant finding after cold baths, diff- erent subjects reacting differently to the same stimuli. This albuminxiria never lasts over 24 hours and may be associated with the appearance of casts and blood, as may also the type following increased exercise. The colder the bath and the longer the immersion, the more rapid the appearance of albumin. Another type of functional albuminuria is that following the intake of a heavy protein meal. This is known as " alimentary albuminuria." It has been supposed to be due to filtration from the blood, the foreign protein being absorbed and excreted without intervening hydrolytic cleavage. Uhlen- huth,i Inouye,2 and Croftan,^ working with the precipitin test, have found egg albumin in the blood and urine in these cases. Ascoli* later demon- strated both serum and egg albumin by this test. Wilson^ using the fixation of complement test, found the main element to be of human origin. Recently Wells,* applying the anaphylaxis reaction,' has shown that sensitized animals do not react to egg albumin but show marked reactions to human protein. Owing to this disagreement of the anaphylaxis and precipitin tests, it would seem probable that the excreted albumin is derived from the blood rather than directly from the food, little if any unaltered food protein ever reaching the urine.* The albuminuria observed in the new-born for the first few days of life, aside from the influence of feeding upon foreign proteins, is probably a further example of a true functional albuminuria. Likewise, the albuminuria ob- served in pregnancy is usually distinctly functional. About 50 per cent, of pregnant women show this albuminuria, little difference being observed between primiparse and multiparae. The kidney undoubtedly shows some functional disturbance in its attempt to eliminate the toxic products absorbed from the fetus.' According to Senator'" such albuminurias as outlined above should be con- sidered functional when slight in degree, transitory in character, occurring ^ Deutsch. med. Wchnschr., 1900, XXVI, 731;. ^ Deutsch. Arch. f. klin. Med., 1903, LXXV, 378. ' New York Med. Jour., 1909, LXXXIX, 474. * Miinch. med. Wchnschr., 1903, L, 201 and 1761; Ztschr. f. physiol. Chem., 1903, XXIX, 283. ' Jour. Path, and Bacteriol., 1909, XIII, 484. ^ Jour. Am. Med. Assn., igog, LIII, 863. ' See Schittenhelm and Weichardt, Ztschr. f. exper. Path. u. Therap., 1912, XI, 69; Pearce, Jour. Exper. Med., 1912, XVI, 349; also, Schloss, Am. Jour. Dis. Child., 1912,111, 341- ' See Van Alstyne and Grant, Jour. Med. Research, 1911, XXV, 399; also, Van Alstyne, Arch. Int. Med., 1913, XII, 372; Salus, Biochem. Ztschr., 1914, LX, i; Cameron and Wells, Arch. Int. Med., 1915, XV, 746; Bronfenbrenner, Andrews and Scott, Jour. Am. Med. Assn., igis, LXIV, 1306. " See Bondi, J. and S., Arch. f. Gynak., 1914, CII, 89. " Erkrankungen der ^^^-fn^iMhJ-MicrOSOft® THE URINE 263 after unusual strain, either physical or mental, the subjects showing a further negative history after the removal of the direct stimulus. Whether this type is to be considered physiologic rests entirely upon the conception of the nor- mal or abnormal nature of the stimuli leading to the excretion of albumin. It would seem to the writer that the term "physiologic albuminuria" would better be given up or at least used with great caution, as it is difl&cult to believe that the severe strain put upon the system and kidneys, in particular, can be distinctly physiologic.^ He is inclined to reserve this term for the excretion of the minimal amounts of albumin, which cannot be detected by the simpler clinical tests, and to regard the types of albuminuria, as discussed above, as "functional" (Pavy), or "constitutional" (Martins). A second group of cases showing albuminuria is observed in which the excretion of albumin may persist for a period varying from days to months, may disappear for a variable space and then return for another indefinite length of time. To this type is given the name "intermittent albuminuria." This is purely functional, no lesion of the kidney being manifest. The cases usually show a history of an acute infection or of an antecedent nephritis as the result of such infection. The most frequent cause, however, is an uncom- pensated heart lesion which may or may not be associated with a direct renal lesion. Not infrequently we find an hereditary intermittent albuminuria in those with a distinct neurotic family history. The functional albuminuria may, at times, follow a definite course, disap- pearing and reappearing with such regularity that it has been styled "cyclic albuminuria." In this form the albumin usually disappears from the urine at night and when the patient is flat on his back, but reappears during the day or when the subject is erect. The terms " orthostatic, orthotic, or postural albumin- uria" would, therefore, seem to be more appropriate than the former appella- tion. This type is dependent, according to Erlanger and Hooker, upon a low- ering of the pulse pressure which constantly occurs when the individual changes from the recumbent to the erect position. Jehle and, more recently, Nothmann believe that this type is due to a lordosis, and style it, therefore, lordotic albuminuria.^ Certain pathologic cases, as beginning nephritis, may show a cyclic albuminuria which may extend well into the period of recovery. In true cyclic cases the negative physical findings would lead us to class the condition among the functional albuminurias. If a case of this type becomes persistent and casts are frequently found, subsequent cardiovascular changes will usually appear, changing the case into one of true nephritis. The albu- min in such cases usually appears in the urine after rising and reaches a maxi- mum from noon to 3 or 4 o'clock, then gradually declines, disappearing from ' See Wolf, Arch. Diagnosis, 1912, V, 260. ^ See Hamburger, Wien. klin. Wchnschr., 1912, XXV, 262; also Frankel (Deutsch. med. Wchnschr., 1912, XXXVIII, 1985), who finds the urinary acidity increased in these cases and states that, possibly, the albuminuria is due to abnormal production of acid as Fischer's theory assumes. See, also, GomoUtsky, Ztschr. f. klin. Med., 1913, LXXXVII, g6; Reyher (Monatsschr. f. Kinderhkde., 1913, XII, 82), Dietl (Wien. klin. Wchnschr., 1913, XXVI, 258) and von Jagic (Ibid., 1556) believe that tuberculosis is the important factor in this condition. See, also, Wendenburg, Arch. f. Kinderhkde., 1913, LXII, 34.; Jehle, Die Albuminuric, Berlin, 1914; Reyher, Pediatria, 1914, VI, i; Steensma, Nederl. Tijdschr. v. Geneesk., 1914, LVIII, 248; Groenendijk, Ibid., 885; Barlocco, Gazz. d. osp., 1914, XXXV, 2021; Fischl and Popper, Jalg^^^^^g^^^j^XXI. 264 DIAGNOSTIC METHODS 8 to 10 P. M. If the subject changes his habits of life, the cycle of albuminuria will also change. Along with the variations in the excretion of albumin, the other urinary constituents fluctuate in the same manner, the sequence being, according to Teissier, increase in pigments, albumin, uric acid, and urea. A peculiarity of this type of albuminuria is that it may be even diminished by exercise and is less, therefore, after a hard day's work, differing from the albuminuria in nephritis or in the cardiac types. While there has been much discussion as to whether such cases are not truly pathological, most of them at any rate must have a certain insufficiency of the renal epithelium. This may be due to changes in the circulation follow- ing change in posture, but it is rather surprising that increased exercise would not work more strongly in this way. Krehl regards these conditions as rela- tively harmless as they do not usually show any subsequent history of neph- ritis; Broadbent does not believe such cases ever develop actual renal disease; while Senator insists that most of them are cases of nephritis. The patients showing this type of albuminuria are weak anemic individuals about the age of puberty, subject to fainting spells, with a heart showing intermittent at- tacks of dilatation and palpitation, and with a probable congenital weakness of the kidney (37.5 per cent, having a movable kidney). The adults are neurasthenics with distinct vasomotor paralysis. A type of albuminuria has been observed in some patients with enlarged spleen, in which albumin is present when the patient is flat on his back but is absent when he is erect. This has been called " hypostatic albuminuria." Its pathogenesis is uncertain, but it can have only an indirect relation to the en- larged spleen; as many cases of splenic tumor do not show this albuminuria. A still further type of functional albuminuria is known as " albuminuria of adolescence." This occurs between the ages of 14 and 16, and then disappears. It is different from the cyclic type, although this latter occiurs in young people. The children are usually anemic, have a neurotic family his- tory, have an unstable vasomotor system and, possibly, a congenital weakness of the kidney. In this class should be included the albuminuria shown by masturbating children or after sexual excess at this period. The kidney does not keep pace with the physical growth and activity of the system, so that the association with the unstable vasomotor system may account for the albumine uria (Emerson) . According to Sutherland, movable kidney may be accountable for some of these cases, as he finds it present in one- third of his cases. Febrile Albuminuria. During the course of any acute fever, an albuminuria may be observed which is not associated with distinct changes in the renal parenchyma and dis- appears with the fall of temperature. The amount of albumin excreted may be small or great, depending upon the severity of the toxic action of the bac- terial products. 1 In ordiiiary cases there is practically no inflammatory con- dition present in the kidney, the albuminuria being due to an ischemia and a later hyperemia. In some of the infectious fevers the influence of the toxins is so great that a true nephritis originates, as especially noted in scarlet fever and diphtheria. Any f^fAlf/^UM^Wfl^f^K^y^a^ss into a true nephritis, so 1 See Grafe, Deutsch. Arch. f. klin. Med., 1914, CXVI, 328. THE URINE 265 that the case must be closely watched for the appearance of symptoms indi- cating such a complication. In some cases, an increase ifi the albuminuria is observed during convalescence, when only traces were previously noted. This is known as "colliquative albuminuria." Tratunatic Albuminuria. A transitory albuminuria may be observed following injuries to the kidneys or even after bimanual palpation of this organ. The albumin and casts may persist for a variable period with no other signs of renal involve- ment. In cases of movable kidney, especially during Dietl's crises, an albuminuria may be noted due to obstruction in the renal circulation. In cases of ureteral stenosis, an albuminuria may be observed as the result of the impeded outflow of urine. This same type of albuminuria may be seen after blocking of the ureter by a calculus, pressure from a tumor, or twisting of the ureter.^ Hematogenous Albuminuria. By this type, of albuminuria we have in mind one in which the albumin is excreted as a result of some alteration in the quality and quantity of the normal protein of the blood. On the other hand a distinctly hematogenous albuminuria may be the result of the excretion of an abnormal protein. This type is observed in purpura, scurvy, pernicious anemia, chronic lead or mercury poisoning, syphilis, leukemia, jaundice, cachexia, after the inhalation of anesthetics, and in diabetes. Toxic Albuminuria. This type is directly referable to the influence of various toxic agents upon the kidneys. The changes in the kidney may be either of a degenera- tive order, leading to a distinct nephritis, or may be purely circulatory. Among the substances causing such an albuminuria we find ether, chloro- form, mustard, cantharides, mercury, lead, arsenic and antimony compounds, all of turpentine, potassium nitrate and chlorate, phosphorus, carbolic acid, salicylic acid, tar compounds (aniline derivatives), petroleum and urotropin.^ Neurotic Albuminuria. A sUght transitory albuminuria may be observed in epilepsy (in which condition it may not always be found but is present invariably when marked cyanosis is seen during the attack), in apoplexy, tetanus, progressive paralysis, exophthalmic goiter, mania, delirium tremens, migraine, brain tumor, injuries to the head especially affecting the floor of the fourth ventricle, neurasthenia, and various psychoses. Not infrequently we find neurotic patients showing an albuminuria as the direct result of perverted metabolism and not as the consequence of pathologic changes in the nervous system. Albuminuria with Definite Renal Lesions. In acute nephritis an intense albuminuria is a constant and important symptom. The more acute the case the larger will be the amount of albumin, the elimination being generally proportionate to the severity of the disease, ' See Evans, Wynne and Whipple (Bull. Johns Hopkins Hosp., 1912, XXIII, 311), who have shown that a reflex albuminuria results from irritation of the urinary bladder. 2 See Cuntz, Munch, med. Wchnschr., 1013, LX, 1656. Digitized by Microsoft® 266 DIAGNOSTIC METHODS although some acute cases may show no albuminuria (Herringham^). The percentage of albulnin varies inversely as the amount of urine, as a rule, so that it is much better to excrete a larger amount of urine with a low per- centage of albumin than a diminished amount of urine with an increased percentage of albumin. The absoliite quantity of albumin excreted varies from 0.2 to I per cent. It may reach as high as 5 per cent, or higher, in one case of Senator being 8 per cent., but this is rare. The total excretion in 24 hours is rarely over 25 grams. Nephritis of syphilitic origin appears to be associated with the largest outputs of albumin. In cases of active renal congestion from exposure to cold or through the action of drugs, or in chronic passive congestion due to cardiac, pulmonary, or hepatic lesions an albuminuria may be observed without any trace of an active renal lesion. The albumin, in these cases, is small in amount and runs parallel to the quantity of urine, thus differing from the excretion in true nephritis. In chronic parenchymatous nephritis the elimination may be relatively large, exceeding, in some cases, that of the acute form.^ In the chronic inter- stitial type the albuminuria is very slight, rarely amounting to more than 5 grams. In this type of nephritis the albumin may be absent at various ex- aminations, so that frequent investigations of the urine must be made. In amyloid kidney, the urine closely resembles that of the interstitial type of nephritis, a total absence of albumin being, however, less frequently observed. The serum globuUn in this type of kidney disease is relatively more increased than in any other type of renal disorder, so that the albumin-globulin quotient may be of some importance in diagnosis. Tests for Senun Albumin. This protein is soluble in distilled water and is coagulated by heat, if the solution be acid, at a temperature varying between 56 and 8i°C. The tem- perature at which any protein coagulates on heating will depend upon the amount of salts present. This protein is precipitated by absolute alcohol and by salts of the heavy metals, as well as by the ordinary alkaloidal pre- cipitants. It is levo-gyrate, its degree being represented by the following formula (a)D = — 62.6°. It is precipitated by concentrated mineral acids, but is dissolved by somewhat large excess. With acetic acid the precipitate first formed is readily soluble in a slight excess of the acid. With con- centrated alkali serum albumin forms an alkali-albuminate which is less soluble in water than is albumin, but which is soluble in an excess of the alkali. This fact accounts for the spontaneous precipitation of albumin in a concentrated urine which is alkaline in reaction. Numerous tests have been given for the detection of albumin in the urinel The writer cannot attempt to describe all of these, but must select, therefore, those which he has found most useful. Before any test for albumin may be made, the urine must be absolutely 1 Trans. Clin. Soc. London, 1901, XXXIV, 34. 2 See von Hosslin, Deutsch. Arch. f. klin. Med., 1912, CV, 147; Cook, Jour. Am. Med. Assn., 1914, LXII, 684; Williams, 111. Med. Jour., 1915, XXVIII, 186; Macris, Gr6cem^d, 191S, XVII, 17. Digitized by Microsoft® THE URINE 267 clear. It is advisable always to use fresh specimens, but if these are not at hand methods must be adopted to clear up the urine. In the majority of cases filtration through several folds of filter-paper will usually accomplish this. If this does not succeed, as it practically never does if the urine be cloudy from the presence of bacteria, recourse must be had to percipitating agents which will carry down the suspension of bacteria.^ Such agents are powdered magnesium oxid or carbonate, silicic acid, or saw-dust. These substances are thoroughly mixed with the urine and the mixture then filtered through double folds of filter-paper or plugs of asbestos fiber. The addition of lead acetate or any of the salts of the heavy metals is inadvisable, as the precipitates formed will include a large part of the albumin, the other precipi- tants not affecting the albumin directly. Occasionally the urine may be cleared by centrifugation. It has been found that the tests for albumin are rendered more distinct if the urine be somewhat diluted. Hallauer has shown that the excess of urea and phosphates in a concentrated urine interfere to some extent with the delicacy of the reactions. As a rule, a 24-hour specimen is examined or a specimen of the urine voided in the morning and that voided at night. The variations of the voidings of the different periods of the day are occasionally quite marked, the morning specimen frequently showing no albumin while the evening specimen may show quite appreciable amounts. Heat Test. This test is based upon the principle that serum albuinin is coagulated by heat especially in the presence of acid. One may use either acetic acid or nitric acid, but the conditions of this addition are different in each case. If acetic acid be added one must be careful lest he add an excess, as the albumin precipitate is soluble in a very slight excess of acetic acid; with nitric acid the condition is the reverse, care being taken not to add too little else the albumin will not be precipitated by the acid. A few drops of dilute acetic acid are all that is required while with nitric acid between one-twentieth and one-tenth of the volume of the urine must be added (one to two drops of 25 per cent, nitric acid per c.c. of urine). Technic. A test-tube is filled about three-quarters full of the clear neutral or faintly acid urine and heated by directing the flame upon the upper portion of the tube, the. lower portion being held in the hand. If the fluid remains clear and the reaction is acid, no albumin is present. If a cloud is noticed it may be rendered more distinct by holding the tube against a black back-ground when the upper portion wiU appear more turbid than the lower. This cloud may be due to albumin or calcium phosphate, rarely to calcium carbonate. To determine which is the cause, acidulate with a few drops of 5' per cent. acetic acid. If the urine becomes clear, the precipitate first noticed is calcium phosphate; if it remains turbid and even increases in intensity of turbidity ' Vaughan (Jour. Lab. and Clin. Med., 1915, 1, 55) calls attention the erroneous practice of heating these bacteria-containing urines with alkali and filtering. As the alkali dissolves the bacterial proteins, later tests wiU show the presence of albumin, which was not present in the urine as voided. ^. ... ,, ... „^ Digitized by Microsoft® 268 DIAGNOSTIC METHODS the precipitate is albumin; while if due to carbonates an effervescence will be observed. It is wise to boil the urine after the addition of each drop of acid, so that the danger of getting an excess of acid may be more easily avoided. It is to be remembered that, if the protein be very slight in amount, and especially if the urine be originally alkaline, the protein will remain in solu- tion owing to the formation of acid albumin. If not enough acid is added, the precipitate of phosphates may not dissolve, while if too much be added the albumin will dissolve. For these reasons it is better to add the acid after boiling. In some cases the fresh urine is already too acid to permit of coagulation, so that alkali may be added to diminish the acidity. The presence of " nucleo-albumin " may lead to a wrong interpretation in this test. This substance is precipitateid in the cold by acetic acid and may thus be differentiated. The resinous acids, which are excreted in the urine after the intake of such drugs as copaiba, cubebs, and benzoin, are not so apt to interfere with this test unless a large excess of acetic acid be used, which is never admissible. If nitric acid be used in place of acetic acid the urine is boiled as above and concentrated nitric acid added to a strongly acid reaction. The nitric acid should never be added before boiling the urine nor should the urine be boiled after the nitric acid is added, a? traces of albumin will be dissolved by the hot nitric acid. A fllocculent precipitate is indicative of albumin. The phosphates and carbonates do not confuse in this reaction as they are readily dissolved. The "nucleo-albumin" is also eliminated by this test as it is readily soluble in the excess of acid. In this test the urine should be set aside and allowed to cool after the boiling is complete, as albumoses, if present, will separate out on cooling as a distinct white flocculent precipitate. A precipitate of uric acid may also form on cooling, but this is more granular and is usually colored, while the albumin precipitate is white unless an admixture of blood be present. In Case the urine be poor in salts, the tests are improved by the addition of a saturated solution of sodiuni chlorid. The salts hinder to a great extent the solution of the albumin by the acids. In such cases, therefore, it is wise to acidify strongly the urine with acetic acid and then add one-sixth its volume of a saturated solution of sodium chlorid as recommended by Purdy.^ The urine is now boiled as above when a precipitate on heating will indicate albumin. The nucleo-albumin reaction is slight, the albumoses appear only on cooling, while the resinous acids may be precipitated but are soluble in alcohol, while the albumin is rendered more compact by this reagent. Heller's Nitric Acid Test. This test is, perhaps, more frequently employed than any other of the tests for albumin in the urine. It has a very wide field of usefulness, although it is not as delicate as some of those to be mentioned.^ 1 Ulrich recommends the overlaying of the acidulated salt solution with urine as a con- tact test. Roberts uses a saturated solution of magnesium sulphate. 2 See Cavazanni, Policlinico, 1914, XXI, 557; Cronquist, Nord'. med. Archiv, 1914, XLVII, i; Morner, Ibid., rg.^i^YSB'by Microsoft® THE URINE 269 A few ex. of concentrated nitric acid are placed in a test-tube and the urine to be tested is allowed to run slowly down the side of the tube in such a way as to form a distinct layer of urine above the acid. Some workers advise the addition of the acid after the urine, but if this is done it is much better practice to allow the nitric acid to flow from a pipet introduced to the bottom of the tube. The writer is always accustomed to allow the urine to flow upon the nitric acid from a long pipet so that the urine does not perceptibly mix with the acid, the tube being held at an angle of 45 degrees. Albumin, if present, is precipitated at the zone of contact in the form of a white opaque cloud or ring. This precipitate is acid-albumin which is insoluble in a slight excess of acid. A red or reddish-violet transparent ring is always obtained with normal urine owing to the reaction of the urinary pigments with the nitric acid. If the urine contains abnormal coloring matters this colored ring may assume various tints. Thus if bile be present a play of colors from red to yellow through blue or green takes place, the green being the Fig. 81. — Conical test-glass. Fio. 82. — -Horismascope. characteristic coloration; if indican be in excess this colored ring may be bluish or even black; while pigments due to drugs will give colors ranging from deep red to violet. This colored ring is usually below the white opaque ring due to albumin and tends to extend down into the acid instead of up into the urine. If much nitrous acid be present in the nitric acid efferves- cence may be observed to such an extent that the ring of albumin may be lost. This albumin ring is usually sharply defined and separated both from the uririe and acid as a white opaque ring, whose breadth will depend upon the amount of albumin in the urine. If the tube be allowed to stand for some time the ring will lose its distinct outline, a more or less diffuse cloudiness rising throughout the urine. Albumin is, however, not the only substance to be precipitated by nitric acid under the conditions of this test. Thus we find globulin, albumoses, and resins precipitated exactly at the line of contact of urine and acids. If the urine be heated the albumoses will dissolve while the albumin becomes more compact. If this precipitate be due to resins the precipitate will dissolve Digitized by Microsoft® 270 DIAGNOSTIC METHODS in alcohol or ether while the albumin will remain unchanged. It is sometimes wise to shake out the urine with ether before applying this test. The globulin ring may be differentiated from that of albumin only by separating these proteins by the method to be discussed under Serum Globulin. GlobuUn is usually associated with albumin, is practically never found by itself, and has practically the same clinical significance, so that for clinical purposes the differentiation of these two bodies is unnecessary. Weinberger^ has re- cently shown that the addition of thymol, as a preservative of the urine, leads to the formation of a grayish-white ring just at the junction of the nitric acid and urine when this test is applied. " Below the ring there is a greenish zone extending somewhat into the acid, above it a reddish somewhat smaller zone." • If this substance is suspected, the urine should be extracted by agitation with an equal volume of petrolic ether. A somewhat similar reac- tion has been reported by Kenney^ in cases in which several drops of formalin were added as a preservative to a small amount of urine. Besides these rings at the zone of contact, a further white or yellowish ring may be observed at this point. This ring is found in urines which are especially rich in urea and appears as a distinctly crystalline ring due to the formation of urea nitrate. If the urine be previously diluted this ring does not appear. If an excess of uric acid be present in the urine, we observe, on allowing the tube to stand for a few minutes, a distinct white ring in the urine about I to 2 cm. above the point of contact of the acid and urine. If the mucin-lihe bodies previously discussed are present in slight excess, a diffuse cloud appears throughout the urine if the fluids have been slightly shaken or a distinct ring i to 2 cm. above the albumin ring may be observed if the urine is carefully added to the acid. This ring is seen in practically all urines, is never at the point of contact, and does not appear to have any clinical significance. Instead of performing this test in a test-tube as described above, it may be done in a conical wine-glass, as recommended by Simon and Ogden, or may be employed as recommended by Boston. This latter worker uses a fiat- pointed pipet into which is drawn from i to 2 inches of the urine to be tested. The exterior of the tube is then wiped perfectly dry and the pipet, with its upper end closed with the finger, is introduced to the bottom of a bottle con- taining pure nitric acid. By lessening the pressure of the' finger the acid gradually flows up into the pipet forming a distinct line of contact between it and the urine. The same points mentioned above obtain with this test. This modification is very simple and may be recommended for general use. A further method of performing this test is the use of the horismascope (see cut). The urine is placed in the larger tube C and the nitric acid allowed to fiow through the capillary tube A so that a distinct Hne of contact is observed. The use of this instrument frequently brings out much more clearly the albumin ring than do the other modifications. This test is frequently employed in combination with heat. Two methods 'Jour. Am. Med. Assn., igog, LII, 1310. 2 New York Med. Jour., ibQA,. LXXX, 453. ,,^ DigitizedWMicrosoft® THE tTRINE 271 are available, either one of which may be used, although the results are some- what different in the two cases. Some workers advise heating the nitric acid previous to the addition of the urine. This does not seem wise to the writer, as traces of the acid albumin are undoubtedly dissolved by the hot acid and may thus escape detection. The writer is accustomed to heat only the upper portion of the tube which contains the somewhat diluted urine. In this way the urates and albumose's are thrown out of the field of action while there is much less danger of traces of albumin being dissolved. If the various points included by this test are remembered and each interfering substance removed by proper differentiation, this test is practically the most useful and reliable one for the detection of albumin in the urine. Potassium Ferrocyanid Test. A few c.c. of urine are strongly acidified with acetic acid and a few drops of a 10 per cent, solution of potassium ferrocyanid are added drop by drop. In the presence of albumin a faint turbidity or a flocculent precipitate will be observed, depending upon the amount of albumin present. The slow addition of the ferrocyanid is necessary, as an excess of this reagent wiU dis- solve the precipitate first formed. In this test albumoses and nucleo-albu- min are both precipitated. The former is dissolved on heating the mixture, while the latter is detected by the precipitate on the addition of the acetic acid unless the acid be added in excess. The urates do not interfere if the urine is diluted previous to making the test. This test is recommended by many as a much more delicate one for albu- min than those previously described. It reacts with a smaller amount of albumin, but it does not, in the writer's opinion, give as much general infor- mation about the urine as does Heller's test. Sulpho-salicylic Acid Test. This substance may be used either in the form of a 20 per cent, solution or in the solid state. If the solution be used it is added to the acidified urine in such a way that a distinct Une of contact is formed. Albumin will be shown by a distinct white ring at the point of contact. It is preferable, how- ever, to add to the urine a small fragniient of this substance, when in the pres- ence of albumin a turbidity or a white flocculent precipitate will be observed, depending upon the amount of albumin. The albumoses are also precipitated by this reagent, but dissolve on heating. Neither uric acid nor resins react with this substance, while the mucin-hke substances are not appreciably affected. The test, known as Roch's test, is, perhaps, the most convenient one for the use of the general practitioner, as the substance may be readily carried in the medicine case and added to the urine at the bedside. ^ Spiegler's Test. As the original reagent of Spiegler was found to be of little value in many cases, Jolles^ has modified it with the following composition: 10 grams of mer- curic chlorid, 20 grams of citric acid, and 20 grams of sodium chlorid dis- solved in 500 c.c. of water. 'Folin and Denis (Jour. Biol. Chem., 1914, XVIII, 273) advocate this substance as a quantitative precipitant of albumin. ^Ztschr. f. physiol. ChemD^'fAgacKB^KM/WBSO/?® 272 DIAGNOSTIC METHODS The urine is acidified with acetic acid to precipitate the nucleo-albumin if present. This substance is filtered off and the filtrate superimposed by means of a pipet upon a few c.c. of the above reagent. In the presence of al- bumin a distinct white ring appears at the zone of contact. This reagent precipitates the albumoses which are soluble on heating. Nucleo-albumin, if present, should be removed before applying the test. In case iodids are present, mercuric iodid will be precipitated but may be removed by alcohol. This test is the most delicate test for albumin. It shows one part of albu- min in 350,000 parts of urine. This is almost too delicate for clinical work as it will show albumin in practically every specimen of urine. The writer has found this test extremely valuable in cases which showed only a faint reaction with Heller's test. If such an urine be treated with Spiegler's reagent a mucji more distinct albumin ring will be observed so that all doubt isthus cleared up. Many other tests have been advocated, but the writer does not feel that they have any advantages over those outlined above. As far as delicacy of reaction is concerned Spiegler's reagent is the most dehcate, the sulphosalicylic acid, heat and acid, ferrocyanid, and Heller's test following in the order named. It will thus be seen that Heller's test is the least delicate of any of the ones spoken of above, but the writer is accustomed to use it in general work, re- lying upon Spiegler's reagent to settle mooted points of delicacy of reaction. As a general working rule it should be said that no one of the less delicate tests should be relied upon without being confirmed by some one of the others. Quantitative Determinatioii of Albumin. Scherer's Method. Fifty c.c. of urine are placed in a beaker and heated upon a water-bath. Two or three drops of dilute acetic acid are then added and the mixture boiled. A flocculent precipitate of albumin should separate out; if not, a drop or two more of acid is added until such a precipitate is obtained. The solution is then filtered through an ash-free filter which has been previously dried and weighed. The filtrate should be tested by Spiegler's reagent to see if any albumin has been dissolved. If this shows no albumin, the precipitate may then be dried on the filter-paper after being washed with water, alcohol, and ether. If the filtrate shows albumin, another test with a fresh 50 c.c. of urine must be made. Much time is saved if a larger quantity of urine be originally taken, treated as above, and smaU portions filtered off and tested for albu- min. The addition of a small amount of a saturated solution of sodium chlorid will facihtate the precipitation of the albumin. The precipitate ©n the filter-paper is dried and weighed, the difference in weight between the original dried paper and that with albumin representing the amount of albumin in the 50 c.c. of urine used. The total amount is determined by a simple calculation. Esbach's Method. This test is carried out in a standard graduated glass tube, known as an albuminometer (see cut). This tube is filled with acidified urine to the point U and the Esbach reagent added to the mark R. This reagent consists of a solution of 10 grams of V^nifS^^j^^mbmi^^ citric acid in one liter of THE URINE 273 distilled water. The tube is now closed with the rubber stopper and inverted several times in order to mix thoroughly the contents. It is then allowed to stand in a test-tube rack for 24 hours, after which the amount of albumin is read off, the graduations on the tube representing the number of grams of albumin per liter of urine. This test has many fallacies and can give only a very approximate deter- mination of albumin. The precipitate does not settle evenly. If the albu- min reads more than 4 grams per liter, the urine must be diluted to a specific gravity between 1006 and 1008 and must be kept at constant acidity. If the urine contains less than ]/2 gram of albumin per Uter the test is absolutely useless. Further the room temperature must be kept constant at about i5°C. or errors as high as 100 per cent, may occur. Moreover, albumoses, uric acid, creatinin, resinous acids, etc., are precipitated by this reagent. Tsuchiya's Method. This method"^ is a modification of the above, the reagent be- ing a solution of phosphotungstic acid in acidulated alcohol with the following composition: Phosphotungstic acid, 1.5 grams Concentrated HCl, 5.0 c.c. 95 per cent, alcohol, q. s. ad., loo.o c.c. This reagent is used with the Esbach tube as in the above test or in the Purdy centrifuge tube discussed later, giving far more accurate results than the Esbach test. Normal urine gives only a faint unreadable precipitate, while small aniounts of albumin as well as large ones are completely precipitated by it.^ This albuminous precipitate settles regularly and quickly, foaming or floating of the precipitate being rarely seen. Variations in tem- perature have little influence on this precipitation. It is to be remembered, however, that other urinary albuminous substances will be also precipitated. Mattice' has shown that this method yields fairly accurate results for comparative purposes, the fig- ures agreeing closely with those of the gravimetric methods. He has also demonstrated that the modification of this test, as introduced by Goodman and Stern,* is neither accurate nor satis- factory, largely because the point at which albumin is shown by the first turbidity with this reagent (and hence the amount ?'^°; , ^^'T of albumin reacting) varies markedly with the dilution of the buminometer. urine. The writer advises, therefore, that this method of Tsuchiya be used for iZentralbl. f. inn. Med., 1908, XXIX, 105. ^ If large amounts of albumin are present, the urine must be diluted, as complete pre- cipitation may not occur with the amount of reagent used. The supernatant fluid must be water clear before the precipitation can be regarded as complete. ^Arch. Int. Med., 1910, V, 313. See, also, Pfeiffer, Berl. klin. Wchnschr., 1913, XLIX, 114; Semionov, Presse med., 1914, XXII, 579; Kahn and Silberman, New York Med. Jour., 1914, C, 667. * Jour. Am. Med. Assn., ■s^<)%,JAj 18 ^/g/f/z^ Jl^y Microsoft® 274 DIAGNOSTIC METHODS reliable comparative clinical results, the gravimetric or Kjeldahl methods being applied for scientific purposes. In case the latter method be used, multiply the nitrogen values of the precipitated and washed albumin by 6.3 to obtain the albumin values. It is perhaps unnecessary to state at this point that in speaking of per cent, of albumin, one should have reference only to the number of grams of albumin by weight in 100 c.c. of urine. The urine very rarely contains more than 5 per cent, of albumin, although Salkowski has reported a case in which 8 per cent, was observed, the albumin separating out as a white amorphous precipitate on standing. This fact should be remembered, as the appearance of such a precipitate in the untreated urine might be very misleading. Purdy's Centrifugal Method. To 10 c.c. of the urine placed in a centrifuge tube, 3 c.c. of a 10 per cent, solution of potassium ferrocyanid and 2 c.c. of 50 per cent, acetic acid are added. The reagents and urine are then mixed by placing the thumb over the end of the tube and inverting it several times, after which the tube is allowed to stand for 10 minutes. It is then placed in a centrifuge the radius of which, with its tubes extended, must be 6^ inches. The tubes are re- volved for exactly three minutes at a uniform speed of 1,500 revolutions per minute. The amount of albumin is then read off in bulk percentage, each division of the tube representing i per cent., as only 10 c.c. of urine are used and the divisions represent tenths of a c.c. One per cent, by bulk represents 0.021 per cent, by weight of albumin. This method is very satisfactory, although not absolutely accurate. It is difficult to keep a centrifuge running uniformly at the above rate, unless a speed indicator be watched during the entire period. The method is more exact and more expeditious than the Esbach method and is to be recom- mended, therefore, for clinical estimations of the amount of albumin.^ Removal of Albumin. It is advisable in many of the general quantitative tests applied to the urine that the albumin should be removed if it is present in more than traces. Usually this may be done by acidifying with acetic acid and boiling until the precipitate is flocculent. The filtrate in such cases will usually be clear and contain no albumin. As this test does not eliminate the albumoses and fur- ther as the boiling acid may hydrolyze a small amount of the albumin into albumoses, Hofmeister recommends the following method. Ten c.c. of a 40 per cent, solution of sodium acetate and the same amount of 10 per cent, ferric chlorid are added to the urine when it will be colored a bright red. The urine is neutralized or rendered very faintly acid and is then boiled. The al- bumin separates out along with the basic ferric acetate and is filtered off. This method is not applicable if glucose is present. ^ (&) Serum Globulin. Serum globulin is associated in the blood with serum albumin. This 'See Strzyzowski, Ztschr. f. physiol. Chem., 1913, LXXXVIII, 25. 2 See Tracy and Welker (Jour. Biol. Chem., 1915, XXII, 55) for the use of aluminium hydroxide cream in removing^^^g.^ ^^ Microsoft® THE URINE 275 protein is not a single body, but is probably a mixture of two forms known as euglobulin and pseudo-globulin. These two fractions differ in their precipita- tion and solubility constants and must be looked for in works on physiologic chemistry. The former of these probably occurs in most urines, constituting a large part of what has been called "nucleo-albumin" (see above). Serum globulin, in the specific sense, is usually found in the urine in every case in which serum albumin is observed. Cases are reported in which each one of these protein bodies has appeared separately, but this is not the normal finding. Its excretion as compared with that of serum albumin varies from 10 to 75 per cent, of the total protein. The relation of the albumin to the globuUn of the blood is as 1.5 is to i. This relation, known as the "albumin quotient," is by no means the same in the urine. In cases of amyloid degen- eration of the kidney the globulin may be very much increased beyond that observed in other chronic affections of the kidney, the albumin quotient being usually lower than one. Senator considers this an important point in the diagnosis of this condition. In the various types of nephritis we find the globulin being greater the more acute the condition, so that in acute diffuse nephritis the albumin quotient may be very low, while in chronic parenchy- matous nephritis the quotient ranges from two and five-tenths to five and five-tenths. As the nephritis improves the relative amount of globulin diminishes, but increases with each acute exacerbation.' Globulin is insoluble in water, but soluble in dilute solutions of sodium chlorid, dilute acids, or alkalies, unless these be exceedingly dilute and their action not prolonged. If, therefore, urine containing globulins be highly diluted with water the globulin will be precipitated in the form of a distinct cloud. The precipitation constants with various inorganic salts must be learned from text-books on physiologic chemistry. Qualitative Test for Globulin. The urine is rendered alkaline by the addition of a few drops of ammonium hydrate and the precipitated phosphates filtered off. To the filtrate is then added an equal volume of a saturated solution of ammonium sulphate, the mixture is allowed to stand one hour and is then filtered. The albumoses and nucleo-albumin may also be precipitated in this way. Ammonium urate does not usually separate out in the hour. The precipitate on the filter is then washed with a half-saturated solution of ammonium sulphate until the filtrate is albumin-free. A distinct precipitate is usually evidence of the presence of globulin, as the albumin is not precipitated until the urine is completely satu- rated with the ammonium sulphate. In order to eliminate the other factors the precipitate is dissolved in water and heated on a water-bath to coagulate the proteins. The solution is filtered, the precipitate washed with water and heated on a water-bath with a i per cent, solution of sodium carbonate. It is then filtered and neutralized with acetic acid. If a precipitate occurs it is globulin, as the albumoses and nucleo-albumin would not be precipitated by such treatment. 'Belfield (Jour. Am. Med. Assn., 1915, LXIV, 2040) calls attention to the presence of globulin, with or without aUjuimp, in, conditions associated with leakage of the seminal vesicles. Digitized by Microsoft® 276 DIAGNOSTIC METHODS Paton advises the use of a contact method in. detecting globulin. The phosphates are removed as mentioned above and the filtered urine allowed to run down the side of a test-tube containing a few c.c. of a saturated solu- tion of sodium sulphate. A white ring will indicate the presence of globulin. Quantitative Determination. The phosphates are removed as above and 100 c.c. of the clear filtered urine are treated with an equal volume of a saturated solution of ammonium sulphate or directly saturated with magnesium sulphate. The precipitated globulin is collected on a dried and weighed filter and washed with a half- saturated solution of ammonium -sulphate in the former instance or with a saturated solution of magnesium sulphate in the latter case. The final wash- ings should show no trace of a reaction for albumin. The funnel with the filter-paper and its contents are then dried at iio°C. The ammonium sul- phate is washed from the precipitate with hot water and the precipitate is then dried with alcohol, ether, and finally at iio°C., until the weight becomes con- stant. The difference between the weight of the filter-paper and the filter- paper plus the globulin gives the amount of globulin in 100 c.c. of urine. (c) Proteoses. These are intermediate products of the digestion of protein by ferments, by acids, or' by bacteria. In the normal digestion we find the products pass- ing through the following stages: protein, acid albumin, primary proteoses (of which there are two, namely, protalbumose and heteroalbumose), secondary proteoses (the only well-established representative being deuteroalbumose), peptone, amino-acids and hexone bases. The proteoses and peptones are very soluble diffusible bodies which are not coagulated by heating. The primary proteoses are precipitated by half-saturation with ammonium sul- phate, the secondary proteoses are precipitated only after complete saturation with this salt, while the peptones are not precipitable in either one of these ways. The primary proteoses are precipitated by nitric acid, thus differing from the secondary types which are not so precipitated. In the urine we find representatives of both types of proteoses, while more or less doubt exists as to whether true peptone has ever been isolated from the urine. Primary Proteoses. Bence-Jones' Protein. This body was first discovered by Bence-Jones' and was regarded as a heteroalbumose. Later work by Magnus-Levy^ has shown that it is in all probability a true albumin, as its digestion products include protalbumose which could hardly be derived from true heteroalbumose. As the exact chemical nature of this body has not been definitely settled, it will be dis- cussed under the above heading, although it probably does not properly be- long there. This body differs from all other types of protein material which occur in the urine in its property of precipitating when heated to as low a tem- perature as 4o°C. and of practically completely dissolving on boiling, to ap- pear again on cooling. A second characteristic of this body is the readiness 1 Med. and Chir. Trans., 1850, XXXIII; Phil. Trans. Royal Soc, 1848, 1, 55. 2Ztschr, f. physiol. Ch.em.[yi^z'§^^yMicrOSOft® THE URINE 277 with which it dissolves in dilute ammonia after it has been precipitated with alcohol. The excretion of this body in the urine has been called "heteroal- bumosuria," "myelopathic albumosuria of Bradshaw," "Kahler's disease" and " Bence- Jones' albumosuria." The amount of this protein body excreted is somewhat variable. In Bence- Jones' original case an output of 6.7 per cent, or a total amount of 70 grams in the 24 hours was observed, while Coriat' reports a case in which none was found in the urine, although 4 per cent, was present in the pleuritic effu- sion. Between these limits we find the majority of cases showing an output usually not over i per cent. The literature contains about 35 cases showing the excretion of this body. The output of this body appears to be constant during the day and not affected in any way by the diet. Little is known re- garding the direct origin of this body.^ It undoubtedly has some associa- tion with the bone-marrow, but just what is not clear. "We may imagine, however, that through the agency of the cells of the abnormal tissue, that is their products of metabolism, the normal transformation of the ingested albumin into tissue-albumin is impeded, resulting in the production of the substance in question, which is then ehminated as foreign matter" (Simon). This body is excreted in cases associated with the occurrence of multiple myelomata of the bones, especially when these affect the thoracic skeleton. It has been observed in a very few cases of leukemia and in a case associated with metastatic carcinoma.^ However, these findings are so rare that the presence of this urinary protein may be regarded as practically pathogno- monic of multiple myelomata. In some cases of this disease the urine does not show the Bence- Jones' body, so that a negative finding does not neces- sarily preclude the condition. EUinger has shown that this disease may take its course without the occurrence of local bone symptoms, but may be asso- ciated with a marked anemia. It is, therefore, wise in cases of obscure anemia to test the urine repeatedly for this body. Tests for Bence-Jones' Body. The specific reaction for this protein is observed on heating the acidified urine very slowly. At a temperature varying from 50° to 60° a slight cloud changing to a marked turbidity and then into a dense cloud will be observed. This may be so intense that the urine appears distinctly milky. This tur- bidity may change into a heavy sticky precipitate or coagulum as the tem- perature approaches the boiling-point. When the boiling-point is reached the precipitate entirely or partially dissolves, especially if the boiling be continued from one to three minutes. The precipitate may not absolutely all dissolve on boiling, as variations in the acidity of the urine and the amount of mineral salts present may affect this process. If the tube be allowed to 1 Am. Jour. Med. Sc, 1903, CXXVI, 631. ^Rosenbloom (Arch. Int. Med., 1912, IX, 236 and 255) advances the possibility of osseo- albumoid as a precursor of this protein. See, also, Cathcart and Henderson, Jour. Path, and Bacteriol., 1912, XVII, 238. ^ Boggs and Guthrie, Am. Jour. Med. Sc, 1912, CXLIV, 803; Bull. Johns Hopkins Hosp., 1912, XXIII, 3S3; Ibid., 1913, XXIV, 368; Folin and Denis, Jour. Biol. Chem., 1914, XVIII, 277; Schiitz, Deutsch. Arch. f. klin. Med., 1914, CXIII, 441; Sexsmith and Klein, Med. Record, 1915, LXXXVIII, 600; Schumm and Kimmerle, Ztschr. f. physiol. Chem., 1914 xcii, I. Digitized by Microsoft® 278 DIAGNOSTIC METHODS stand after being boiled the precipitate returns as the fluid cools. None of the other protein bodies give this sequence of precipitation, dissolving, and reprecipitation. Hugounenq suggests the name "thermolytic albuminuria" for the excretion of this body in the urine. Upon the addition of concentrated nitric acid, drop by drop, a temporary turbidity develops which disappears on shaking, but persists if more acid be added. If the mixture be heated the precipitate will dissolve and reappear on cooling. This same reaction may be observed on applying any of the tests for serum-albumin outUned above. This protein is precipitated from its solution by the addition of two volumes of a saturated solution of sodium chlorid to urine which has been previously acidified with acetic acid. The addition of two volumes of satu- rated solution of ammonium sulphate hkewise causes its complete precipitation. It may be then washed with alcohol and ether and dried over sulphuric acid. Boston' has proposed the following test for this body. Fifteen to twenty c.c. of filtered urine are placed in a test-tube and mixed with an equal volume of a saturated solution of sodium chlorid, the tube being shaken to insure a thorough mixing of the fluids. Two or three c.c. of a 30 per cent, solution of sodium hydrate are added and the mixture vigorously shaken. The upper one-fourth of the mixture is then gradually heated to the boiling-point and a solution of 10 per cent, lead acetate added drop by drop, the heating being continued after each addition. When the drop of lead solution comes in contact with the liquid a copious pearly or creamy cloud appears at the sur- face, becoming less dense as the boihng-point is neared; and when ebuUition is prolonged for from one-half to one minute the upper portion of the liquid shows slight browning, which deepens to a dull black color. Standing in- tensifies the reaction, and if this be prolonged for several hours the black precipitate falls through the clear stratum of Hquid, collecting in the bottom of the tube as a coarsely granular pigment. This reaction is based upon the fact that this body contains a large proportion of loosely bound sulphur. Lindemann^ finds that this body really contains no more such sulphur than does serum albumin, while Wood could obtain no more blackening than with other proteins. This test would seem to have Httle value as it is in no way distinctive for the Bence- Jones' protein. The quantitative determination of this body may be made by precipita- tion with two volumes of saturated solution of ammonium sulphate and the washing and drying of this precipitate as previously mentioned. The Tsuchiya method is useful as an approximate estimation in the absence of albumin. Secondary Proteoses. Deuteroalbumose is probably the body which has been found in the urine in cases in which peptone was reported. This secondary proteose differs in its reactions from the primary proteoses and the Bence- Jones' protein. It is precipitated only on complete saturation of the urine with ammonium sul- phate. In performing this test the urine should be made albumin-free, 'Clinical Diagnosis, Philadelphia, 1905. ^Deutsch. Archiv £. Win. ^fgftf^o^if^^Sm® THE URINE 279 preferably by the Hofmeister method previously discussed. Nucleo-albumin may be precipitated by basic lead acetate. Urine containing deuteroalbu- mose does not become cloudy on boiling; does not regularly give Heller's test, but does react with the ferrocyanid test when the neutral salts are present in fairly large quantities, and reacts in the cold with sulpho-salicylic acid and with Spiegler's reagent, but the precipitate dissolves on heating to reappear again on cooling. This reaction with the latter reagents in the cold may be distinguished from that of albumin by boihng the mixture and filtering while hot. The albumin remains on the filter while the albumose is present in the filtrate, appearing as a distinct precipitate as the solution cools. Tests for Albumoses. To a few c.c. of urine, from which albumin and nucleo-albumin have been removed as outlined above, add one-fifth its volume of concentrated acetic acid. A 10 per cent, solution of phosphotungstic acid is then added, when the urine remains clear on standing if albumoses are absent; while a milky turbidity is observed in 5 to 10 minutes if these substances are present. If this precipitate be filtered off (warming will faciUtate the clumping of the precipitate), washed with distilled water and then dissolved on the filter with a very dilute solution of sodium hydrate, the solution will have a distinctly blue color. This solution is warmed until it becomes clear, more sodium hydrate being added -if necessary. It is then cooled and the biuret test ap- plied by adding a few drops of strong sodium hydrate and a few drops of dilute (2 per cent.) copper sulphate solution. On warming this mixture a beautiful red color will be observed. FUtipaldf advises that 10 c.c. of urine be treated with 60 c.c. of absolute alcohol and allowed to stand until the next day. The alcohol is carefully poured off, the precipitate is dissolved in the least possible amount of 30 per cent. NaOH and this alkaline solution is treated with a few drops of a freshly prepared ammoniacal nickel solution.* In the presence of albumose or peptone an orange-red coloration appears. This test seems reliable and does not react with albumin. Bang's Method. Ten c.c. of urine are heated in a test-tube with 8 grams of finely powdered ammonium sulphate until the salt has been dissolved. The mixture is boiled for a few seconds and is then centrifugalized for one-half to one minute. The supernatant fluid is then poured oS and the precipitate extracted with alcohol to remove urobihn. After pouring off the alcohol the residue is dis- solved in a little water, the solution is boiled to remove albumin and filtered. The filtrate is shaken out with chloroform to remove any traces of urobilin which may have escaped previously. The watery solution is then poured off from the chloroform and tested as above for the biuret reaction. Clinical Significance. Deuteroalbumose may occur in the urine either alone or associated with albumin. It is observed in a great variety of conditions, so that distinct types of albumosuria may be noted. 1 Deutsch. med. Wchnschr., 1911, XXXVII, 1890. Gazz. d. osp., 1911, XXXII, 515. ' A ? jjer cent, solution of nickej-sulnhate rnixed with an equal volume of ammonia water. ^ Digifizeaby IWicrosort®^ 28o DIAGNOSTIC METHODS Large accumulations of pus anywhere in the system lead to the excretion of deuteroalbumose as a result of the breaking down of the pus cells and the later absorption of the hydrolyzed material. This form, known as pyogenic albumosuria, is observed in pneumonia during the stage of resolution, in gangrenous processes anywhere in the system, in empyema, bronchiectasis, abscess formation, and in epidemic cerebrospinal meningitis. In this latter condition the differential diagnosis from a tubercular meningitis often rests on the appearance of albumose in the urine. A hepatogenous form of albumosuria occurs in any condition associated with marked disturbance of hepatic function, as for instance in acute yellow atrophy, phosphorus poisoning, cirrhosis, carcinoma, and catarrhal jaundice. Little is known of the origin of the albumose in this condition. An enterogenous type is observed in cases of gastric or intestinal ulcer, whether the latter be due to typhoid fever or dysentery, while intestinal tuber- culosis is less frequently associated with the appearance of albumose in the urine. In these cases the breaking down of the tissues may be responsible for increased absorption both of the products of hydrolysis of the tissues and of the food. An albumosuria of hematogenous origin has been observed, in cases of scurvy, leukemia, purpura, dermatitis, poisoning with hemolytic agents, preg- nancy, especially after the death of the fetus, and in various psychoses, as well as in carcinoma affecting any part of the system. The albumosuria in these cases is probably referable to the increased lysis of the cells under the influence of the exogenous or endogenous toxins. A febrile type is observed in practically all fevers, more especially the infectious types, such as measles, scarlet fever, diphtheria, acute articular rheumatism, smallpox, and mumps. This is referable both to the influence of the toxins of the disease in producing increased protein disintegration as well as to the associated septic conditions. A large number of other conditions are associated with the appearance of albumose in the urine. Such a state is due to the breaking down either of tissue or of an exudate, and may, therefore, appear in almost any type of dis- ease. In some cases albumose may appear in the urine, following the inges- tion of a large amount of albumose. This is the digestive or alimentary albumosuria and appears to be indicative of an ulcerative condition some- where along the intestinal tract. Pollak^ shows that this albumosuria may be due, in part at least, to pathological conditions of the kidney, and suggests the name renal albumosuria for such cases. As previously stated, albumose may be associated in the urine w;th albumin, constituting the mixed albuminuria of Senator. In these cases the albumosuria may precede the albuminuria, may alternate with it, or continue after it has disappeared. This condition is particularly prominent in cases of nephritis, especially of the syphilitic type, and should be watched with care. In any case of albumosuria it is necessary to exclude contaminations with foreign material, especially with spermatic or prostatic secretions. ' Ztschr. f. d. ges. exper. WtgitiZ^JJ^ 6/3^rOSOft® THE URINE ' 281 (d) Peptone. True peptone rarely if ever appears in the urine. Peptones are the last hydrolytic products of protein which give the biuret reaction. This body is not precipitated on saturation with ammonium sulphate as are the other types of protein material. Ito reports the finding of true peptones in the urine in cases of croupous pneumonia, pulmonary tuberculosis, ulcer of the stomach, and in women during the puerperal period. In these cases deutero- albumose was also present, so that there is a possibility that the peptone isolatedby Ito was derived from the urinary albumose. Many reports of pep- tonuria are found in the literature, but the substance dealt with in practically all of these cases was probably some type of albumose and not true peptone. (e) Hemoglobin. This body is the normal coloring matter of the blood and is to be regarded from the chemical standpoint as a chromoprotein. In the normal metabolism disintegration of red blood-corpuscles is constantly occurring, but this is not sufficient to lead to a hemoglobinemia and a resulting hemoglobinuria. These two conditions must go hand in hand, the latter being impossible without the former. When the destruction of the red cell becomes so extensive that the accu- mulation of the blood pigment in the blood-current is so great that the liver is unable to convert it into bilirubin, hemoglobinemia and hemoglobinuria must result.^ While the distinct limit of destruction of cells necessary to produce this condition is not definitely settled, it may in general be said to occur when approximately one-sixtieth of the hemoglobin of the corpuscles is set free. The protein really excreted in the urine is not hemoglobin, but methemoglobin, so that the term methemoglobinuria would be better used, although a direct hemoglobinemia does obtain. From what has been said above it is evident that the excretion of this protein will inevitably occur after the use of the so-called hemolytic poisons. Among these we find ether, chloroform, snake-venom, arseniuretted hydro- gen, phosphorus, hydrogen sulphid, toluylendiamin, mushrooms, anilin, lacto- phenin, bile salts, chlorates, pyrogallic acid, naphthol, carbolic acid, carbon monoxid, and tuberculin. Hemoglobin will appear in the urine in cases of poisoning with the above substances only when the hemoglobinemia is ex- tensive. In mild cases, the liver will be-called upon to form increased biliary pigment and the urine will, therefore, contain bile pigments instead of blood pigments. Likewise, we find a hemoglobinuria following transfusion of the blood of animals into man, after severe burns, exposure to cold, in the course of any of the specific infectious diseases, and in malaria^ and syphilis. The so-called "black- water fever" is more probably a malarial hematuria than a hemoglobinuria. A paroxysmal type of hemoglobinuria has been occasionally reported in the literature. This occurs in typical paroxysmal forms after exposure to cold or exertion, and is often preceded by a typical "infectious" onset, such 'See Wilbur and Addis, Arch. Int. Med., 1914, XIII, 235; Addis, Ibid., 1915, XV, 413. " Urriola (Interstate Med. Jour., 1Q12, XIX, 74) claims that the black urinary pigment of the urine is pathognomonic ^j^f^j^ by Microsoft® 282 DIAGNOSTIC METHODS as chill, fever, and malaise, along with pain in the lumbar region. The hemo- globin may be excreted for several days and then disappear with no untoward symptoms. This condition is very rare and its cause uncertain.^ An epi- demic hemoglobinuria occurs at times in the new-born and is associated with a distinct hemoglobinemia, jaundice, and cyanosis. The urine, in cases of hemoglobinuria, may be clear, but is generally turbid, and varies in color from a bright red to almost a black. The turbidity gives the appearance of a peculiar smoky or hazy urine. The urine must be ex- amined when freshly voided, as blood-corpuscles soon disintegrate in the urine, giving it the same appearance as noted in hemoglobinuria. The clinical significance of hematuria and hemoglobinuria are much different and should not be confounded. If the urine be centrif uged the supernatant fluid will be a clear blood-colored liquid and the sediment will show none or very few red cells. The tests for the presence of blood pigments must be applied in order to differentiate this protein from the other types. Naturally, anyspecimen of . urine containing hemoglobin will react to the albumin tests previously given so that it may be very difficult to determine whether a true albuminuria is coexistent. As a rule, in such conditions there is an associated nephritis so that all the findings of this latter condition may obtain. The chemical tests indicate the presence of hemoglobin or of any of its derivatives and do not differentiate a hemoglobinuria from a hematuria. Microscopic examination for the presence of red blood-cells is the only possible way of clearing up such a diagnosis. A spectroscopic examination of the urine will differentiate the types of blood pigments (see Blood). Tests for Hemoglobin and Derivatives. Heller's Test. A few c.c. of urine are strongly alkalinized with sodium hydrate and heated. Either at once or on standing a brownish-red precipitate of the phosphates and the carbonates of the alkaline-earths is formed, the color being due to the hematin carried down by the phosphates if blood is present. If the urine contains a large amount of foreign pigments, this red coloration may not be easily noted. In this case filter off the precipitate and dissolve it in acetic acid, when the solution becomes red if blood pigment is present, the color gradually fading upon exposure to air. If this test be controlled by the spectroscopic tests for hematin in alkaline solution it becomes quite reUable and very delicate. It indicates one part of oxyhemoglobin in 4,000 of urine. Donogany's Test. Ten c.c. of urine are treated with i c.c. of ammonium sulphid solution and I c.c. of pyridin. If blood be present, the urine will assume a more or less intense orange color, which may be more evident on looking through the test-tube lengthwise. In this case the hemoglobin has been converted 1 According to Cooke (Am. Jour. Med. Sc, 191 2, CXLIV, 203), syphilis is the most impor- tant, if not the only, etiologic factor in paroxysmal hemoglobinuria!. See, however, Lind- bom, Hygiea, 1913, LXXV, 885; Ztschr. f. klin. Med., 1913, LXXIX, 147; Young, Jour. Am. Med. Assn., i9i4,LXII, 356; Widal, Abrami and Brissaud, Sem. M6d., 1914, XXXIII, 613; Forges and Strisower, Deutsch. Arch. f. klin. Med., 1914, CXVII, 13; Dennie and Bob- ertson. Arch. Int. Med., iQiSj^^^^o^-j^j, Microsoft® THE URINE 283 into hemoGhromogen, which may be recognized by the spectroscopic test. This test is more delicate than the previous, showing one part of blood to 8,000 of urine. Instead of the above tests, which are more directly applied in urine, the guaiac and aloin tests as discussed under Feces may be applied. The spectrum of the various blood pigments will be discussed later, so that the writer need only refer to the section on Blood for this. If the urine con- tains fresh blood the spectrum is that of oxyhemoglobin, while in cases of hemoglobinuria or of hematuria of renal origin the spectrum is that of met- hemoglobin. The^urine to be tested spectroscopically should be sUghtly acid and perfectly clear. If a large amount of blood pigment be present, the spec- trum will be much clearer if the urine be diluted. This dilution should not be carried too far, otherwise the absorption lines wiU not appear. In testing for methemoglobin the spectrum of neutral as well as of alkaUne methemoglobin should be looked for. (/) Fibrin. The occurrence of this protein in the urine is very rare. As fibrin is de- rived from fibrinogen through the action of the fibrin ferment, the presence of the former body presupposes that the latter two substances have been present somewhere along the genitourinary tract. This substance is an elastic, grayish, stringy material insoluble in water and alcohol. Chemically, fibrin belongs to the group of globulins; it is soluble with difficulty in dilute saline solutions, is coagulated by heat, and precipitated either by great dilution with water or by saturation with magnesium sulphate. This protein may occur in the urine either in the coagulated form or in solution. It is found in any condition in which large amounts of blood are present in the urine, whether the blood comes from the kidneys or points be- low. It may coagulate immediately after voiding or may occur as preformed clots which are formed either in severe inflammations of the pelvis of the kid- ney, of the ureter, bladder, or urethra. It occurs also in cases of chyluria and rarely in direct nephritis. In some cases the fibrin is in solution, especially in urines containing no blood. This fibrin separates out in the form of a coagulum on standing or may change the urine into a distinctly gelatinous mass. This so-called "spontaneously coagulable urine" is seen more frequently in cases of chy- luria, but may be observed in rare cases of nephritis. Test for Fibrin. The clotted material is filtered off from the urine, is thoroughly washed with water and boiled in a i per cent, solution of sodium carbonate. On cooling, this solution may be tested as outhned under Serum Albumin. (2) Carbohydrates. Normally the urine contains traces (o.oi to 0.03 per cent.) of carbo- hydrates which are incapable of detection by the ordinary chnical tests. Besides these true carbohydrates the urine contains other substances which react, especially toward copper solutions, as do the monosaccharides. These latter reducing bodies are uric acid, creatiniri, conjugated glycuronic acids, Digitized by IVHcrosoft® 284 DIAGNOSTIC METHODS and various pigments, either normal ones excreted in unusual amounts or abnormal ones excreted in usual amounts. The total output of the reduc- ing bodies of the normal urine varies between 2 and 3 grams in 24 hours, while the normal true carbohydrates of ^the urine vary from, 0.2 to i gram per diem. (a) Glucose (d-GIucose) (CH2OH— (CHOH)4— CHO). The normal blood contains about one part per thousand of glucose. Whether this sugar is in the free state or in combination with other molecules, as for instance as the so-called jecorin of Drechsel, is at present an unsettled question. According to Claude Bernard, sugar will appear in the urine when- ever more than three parts per thousand are present in the circulating blood. This figure is, in view of the recent work of von Noorden, Stern, and Lief- mann, much too high, as the average finding in their cases was 0.85 part per thousand. The excretion of sugar in the urine is known as glycosuria and presupposes an excess of sugar in the blood (hyperglycemia). In only one form of glycosuria do we find absence of this hyperglycemia; that is, in con- ditions in which the kidneys become less impervious than normally to the sugar circulating in the blood. This type of glycosuria is most frequently observed in cases of poisoning with phloridzin and, also, after intravenous in- jection of large quantities of physiologic salt solution (the so-called "salt glycosuria"), and has led to the assumption of the clinical entity "renal dia- betes mellitus." The pathology of sucli a condition is little understood, so that we may for the present disregard this type and limit our discussion to the glycosuria which inevitably follows a hyperglycemia. ^ Glycosuria. The normal metabolism is such that any excess of carbohydrate food is converted, up to a certain point, into glycogen and stored up in the liver. Should this ingestion of carbohydrates exceed the functional power of the liver to convert it into glycogen, the excess will pass through the hepatic filter into the circulating blood, thus causing directly a hyperglycemia. Unless in- creased muscular activity is suflScient to utilize this excess, the kidneys will excrete sugar until the normal relations again obtain. This is the purely aU- mentary type of glycosuria and ceases as soon as the intake is diminished. The type of food ingested has much to do with the extent of the glycosuria. Under normal conditions it matters relatively little how much carbohydrate is ingested in the form of starch, as the products of hydrolysis are gradually ab- sorbed and do not lead to overactivity of the liver with a resulting hypergly- cemia and glycosuria. On the other hand, a certain limit, different for each individual, is observed in the amount of sugar which may be ingested without causing a glycosuria. For this reason Naunyn has regarded alimentary gly- cosuria as of two distinct types: (i) that following the ingestion of starch, which he styles glycosuria ex amylo, and (2) that following the ingestion of an excess of sugar, glycosuria e sacckaro. The amount of starchy or of saccharine 'See deLangen, Nederl. Tijdschr. v. Geneesk., 1913, II, 1454; Ferannini, Riforma Med., 1914, XXX, -232; Landau, Rev. de Med., 1914, XXXIV, 145; Strouse and Beifeld, Jour. Am. Med. Assn., 1914, LXII, 1301; Salomon, Deutsch. med. Wchnschr., 1914, XL, 217; Galam- bos. Ibid., 1301; Gram., noi&f^^d^^Mf&^OM) THE URINE 285 food which a person may ingest without a glycosuria is known as the assimila- tion limit or degree of tolerance for such food. This factor varies for each indi- vidual under normal conditions and under pathologic influences is dependent upon the state of the intestines, liver, pancreas, muscles, and kidney. A nor- mal person may stand an intake of from 200 to 300 grams of glucose without excreting more than traces in the urine, but in the majority of persons this fig- ure would, perhaps, be found to be more nearly 150 grams than 300. It has been found that the administration of as small amounts as 50 grams of galac- tose and lactose was followed by an excretion of these sugars in the urine, while maltose, dextrose, levulose, and saccharose required much larger intakes. The excreted sugar, following the increased ingestion, is in most cases similar to that taken in, although Moritz has shown that some of the polysaccharides may be partially hydrolyzed into their monosaccharide components. It has been found by Worm-Miiller that a large intake of cane-sugar is not followed by a maximum excretion of this sugar, even though the assimilation limit has been greatly exceeded. Thus he observes, after an intake of 50 grams of cane-sugar, an excretion of o.i gram, while after an intake of 150 grams the excretion was only 0.85 gram. "Alimentary glycosuria occurs in a healthy person only by saturating the organism with soluble carbohydrates. Therefore it is absent after admin- istering starch, as in this case no more sugar will be absorbed than can be metabolized in the body. It is also scanty, or quite absent, if sugar solutions be given on a full instead of on an empty stomach. Naunyn has observed, regarding alimentary glycosuria, that there is excreted in the urine only that sugar which, according to Ginsberg, reaches the general circulation through the thoracic duct, thus avoiding the liver. Still other external influences may come into play, such as altered capacity of the tissues, especially those of the importantly concerned liver. The result of this may be that excessive doses of glucose are stored up as glycogen or fat in a given time. For saccharosuria and lactosuria the relationships are somewhat different. Here it is very obvious that these double sugars, if given in excessive quantities, are not com- pletely split up in the intestine or during their passage through the intestinal wall, but enter the general circulation as such. The organism, like most of the yeasts, cannot decompose these sugars to any extent, so that they leave the body with the molecules unaffected" (Magnus-Levy). In testing for the pathologic type of alimentary glycosuria, it is customary to give 100 grams of cane-sugar or glucose either in the morning on an empty stomach or 2 hours after a very light breakfast. The urine of healthy persons should remain free from sugar, while a pathologically lowered limit of tolerance will be observed by a more or less extensive glycosuria beginning in about i hour, reaching a maximum within 2 to 4 hours, and lasting about 8 hours. If this glycosuria follows the administration of starchy foods, the condition is probably a pure diabetic one, while a glycosuria following 100 grams of grape-sugar does not necessarily indicate a diabetes. It is probable that cases showing a somewhat low assimilation limit for sugar belong to the type of mild diabetes.. althou^ ^-^ classJhem in the general category 286 ' DIAGNOSTIC METHODS of "hepatic insufficiency." That the liver is incapable in such conditions of polymerizing the sugar into glycogen cannot be disputed, but the question at issue is, as von Noorden shows, upon what does the insufficiency of the liver depend? I quote from the article in his Handbuch der Pathologic des Stoff- wechsels as follows: "The cause cannot be overfilhng of the glycogen reser- voir, by which we explain the alimentary glycosuria of the healthy. In most cases the subjects are in ill health and their previous diet anything but excess- ive, so that there is little reason to suppose that their glycogen repository was already filled to overflowing. There is no more reason for supposing that they have a diminished power of utilizing sugar. ^ In none of the affections in question are the processes of oxidation and of energy production decreased; rather is there a great increase in oxidation and especially in the combustion of carbohydrate in certain of the diseases, such as Graves' disease and high fever, which predispose to alimentary glycosuria. Either the liver cells are unable to polymerize all the sugar reaching them or else the glycogen formed is too quickly converted into sugar, through some increase in the diastatic process. The latter would imply that the automatic regulation between sugar combustion and sugar forination is no longer so evenly balanced as in health. Physiologically the diastatic process is dependent only on the sugar requirements of the body; here it would also be controlled by the sugar supply." The idea of hepatic insufficiency as a distinct clinical entity responsible for the alimentary glycosuria can hardly hold. One should not be satisfied with a mere statement that such a condition exists, but should attempt to ex- plain why it does obtain. In many conditions of the liver in which an un- doubted insufficiency is present, no glycosuria can be caused by administra- tion of a fairly large amount of glucose. On the other hand, administration of levulose in these conditions produces a distinct alimentary levulosuria. To the classes of cases which give this levulosuria should be, according to Strauss, applied the term "hepatic insufficiency" rather than to those showing an aU- mentary glycosuria. (See p. 305.) It would lead me too far afield to discuss the various factors which influ- ence the appearance of sugar in the urine. ^ In many cases a restriction of the diet within the assimilation hmit will rid the urine of sugar, while in others a constant production of sugar within the system occurs. This endogenous production of sugar may again be of several types. Thus we find in nervous conditions, especially in affections in the region of the fourth ventricle, an ex- cretion of sugar which continues only so long as the glycogen of the body is not used up. If no carbohydrates be taken in the food, glycosuria will soon disappear. This condition is due to interference with the normal nervous control of the glycogenic function of the liver. This t}^e of glycosuria, to ' Minkowski, Luck, Murlin and, more recently, Roily and David assert that this is not true, but that the primary factor in human diabetes is an inadequate utilization of carbo- hydrates rather than the overproduction of sugar as von Noorden's theory would demand. 2 See Barrenscheen, Biochem. Ztschr., 1913, LVIII, 277; Woodyatt, Jour. Biol. Chem., 1913, XIV, 38; Greer, Witzemann and Woodyatt, Ibid., 1913, XVI, 455; Ringer, Ibid., 1914, XVII, 107; Roily and David, Munch, med. Wchnschr., 1914, LXI, 169; Macleod, Jour. Am. Med. Assn., 1914, LXII, 1222; Sansum and Woodyatt Jour. Biol. Chem., 1914, XVII, 521; THE URINE 287 which the name " neurohepatogenous glycosuria" has been appUed, is found in a variety of conditions, such as progressive paralysis, multiple sclerosis, cere- bral tumors, peripheral neuritis, traumatic neuroses, mania, melanchoUa, and hysteria. In this type we find the administration of carbohydrates in the food being followed by a glycosuria, because the diastatic processes are con- tinually urged to increased activity, to such an extent that the hepatic artery becomes loaded with sugar. In other words, we have, in this type, an increased rate of saccharification rather than a diminished formation of glycogen.^ In other cases a continuous glycosuria is observed even after Complete exclusion of carbohydrates from the diet. In this type there is a constant formation of carbohydrates from the protein and fat of the food. The hver appears to be capable of storing up glycogen in the usual amount, but the system in general is practically unable to utilize the sugar brought to it. In consequence of this the liver-cells are repeatedly called upon to convert the glycogen reserve into glucose, and as a result the blood becomes laden with sugar. Even under these circumstances the hyperglycemia would not neces- sarily cause a glycosuria, providing the normal ferments of the blood arising from the internal secretion of the pancreas were present. This type of glyco- suria is in reality typical diabetes mellitus and is so dependent upon such a large number of factors that the writer must refer to other works for its discus- sion. Recently Pfliiger has shown that a very close relationship exists be- tween the nervous influences of the duodenum and pancreas. His work ap- parently indicates that what is generally known as pancreatic diabetes is de- pendent to some extent upon disturbance of the proper correlation between the duodenum and pancreas. A peculiarity in the true diabetic glycosuria is that other types of sugar, especially levulose, appear to be well tolerated by the diabetic individual without leading to a glycosuria. Besides, the admin- istration of the primary hydroly tic products of glucose, as for instance gluconic and saccharic acids, apparently diminishes an already existing glycosuria. This would seem to indicate that the system is primarily unable to bring about the initial cleavage of the glucose molecule. As it has been shown that many diabetic cases are associated with sclerosis of the islands of Langerhans in the pancreas, this organ is usually regarded as the principal seat of the pathologic changes in true diabetes mellitus. On the other hand, many cases which are clinically indistinguishable from those of pancreatic diabetes show no lesion in this organ postmortem nor any characteristic lesion in other organs.^ Occasionally, cases are seen in which a diplomellituria exists. This is the contemporaneous or alternate occurrence of diabetic and non-diabetic glycosuria in the same individual (Stern'). The clearing up of one type usually reveals the other. A transient glycosuria may obtain in conditions in which the oxygen supply is reduced, as in suffocation, poisoning with carbon- monoxid, curare and amyl nitrite, or after administration of such drugs as 'See Novak, Porges and Strisower, Ztschr. f. klin. Med., 1913, LXXVIII, 413; also, Mann, Ibid., 488; Labbe and Bouchage, Lancet, 1914, I, 13. 'See Homans, Jour. Med. Research, 1914, XXX, 49; Major, Ibid., 1914, XXXI, 313; 288 DIAGNOSTIC METHODS strychnine, cocaine, caffeine and adrenalin.^ Further, a transient glycosuria is not infrequently seen following the use of alcohol, especially in the form of beer or champagne. A post-anesthetic glycosuria is observed after the use of chloroform and ether, probably as a result of the action of these agents in stimulating the transformation of glycogen into dextrose (Hawk^). Qualitative Tests for Glucose. The quahtative tests for sugar depend for the most part upon the chemical structure of its molecule. The hexoses belong either to the class of aldehyds or ketones, and as such will reduce metallic oxids to lower forms. The CHO and CO groups of the aldoses and ketoses, respectively, are the reacting points in all of the reduction tests, such as those with copper and bismuth solutions, as well as in the tests showing the formation of the characteristic osazones. Moreover, these carbohydrates show the peculiarity of fermenting, in the presence of yeast, into alcohol, carbonic acid, and other products. This fermentation test, especially with the saccharomyces cerevisiae, is given only by the sugars having three or a multiple of three carbon atoms in the molecule. Fischer's work has shown that only those sugars may be fer- mented by a specific ferment in which the ferment and sugar stand to one another in such a relation that a chemical union is possible between them or as he expresses it, only when the ferment fits into the sugar molecule like a key in a lock. This explains why only certain types of the hexoses will ferment in the presence of yeast. Before any qualitative or quantitative test may be made for the pres- ence of sugar in the urine, albumin must be removed, especially if present in more than traces.' Trommer's Test. To a few c.c. of urine in a test-tube are added one-third its volume of a lo per cent, solution of sodium hydrate and then, drop by drop, a lo per cent, solution of copper sulphate. This copper sulphate should be added with con- stant shaking until a slight excess of the precipitated cupric hydrate (Cu (0H)2) remains undissolved and is visible, on shaking the tube, as a distinctly green- ish-blue flocculent precipitate. The upper layer of the urine is then warmed, when a yellow or red precipitate appears in the heated urine if sugar be pres- ent. As a rule, it is unnecessary and even unwise to -boil the solution, as otherwise substances other than sugar may produce the reaction. It is true that the reaction is not as sensitive unless the solution be heated to boiling, but other substances do not so readily interfere with the reaction at a lower ' See Emerson (Jour. Am. Med. Assn., 191 2, LIX, 2245) for a discussion of glycosuria in the insane; also, Herbert, Biochem. Ztschr., 1913, XL VIII, 120; Landau, Ztschr. f. klin. Med., 1913, LXXIX, 201; Folin, Denis and Smillie, Jour. Biol. Chem., 1914, XVII, 519; Luzzatto, Ztschr. f. exp. Path. u. Ther., 1914, XVI, 18; Loewy and Rosenberg, Biochem. Ztschr., 1914, LXI, 189; Eustis, Am. Jour. Med. Sc, 1914, CXLVII, 830; Anders and Jameson, Ibid., 1914, CXLVIII, 323; Pozzo, Policlinico, 1915, XXII, 417. 2 Arch. Int. Med., 1911, VIII, 39. See also. King, Moyle and Haupt, Jour. Exper. Med., 1912, XVI, 178; Greenwald, Jour. Biol. Chem., 1913, XVI, 375; Ross and Hawk, Arch. Int. Med., 1914, XIV, 779. 3 See Meinertz, Med. Klin., 1913, IX, 1771; Glaserfeld, Med. Klin., 1914, X, 1388, 1413 and. 1452; Tirard, Lancet, 1914, II, 1133; Geelmuyden, Norsk Mag. f. Laegevidensk., 1915, Lxxvi, 985. Digitized by Microsoft® THE URINE 289 temperature. The yellow or red precipitate wUl gradually form through- out the mixture, from above downward, and will finally settle out, leav- ing a colorless or yellow fluid above. This reaction is due to the reduction of the cupric hydrate, which is formed by the action of the sodium hydrate upon the copper sulphate, into cuprous hydrate, which becomes dehydrated, on heating, into cuprous oxid. If no sugar be present in the urine and other reducing substances are not ex- cessive in amount, the cupric hydrate will settle out on warming as a black precipitate of cupric oxid. The equations showing these points are as follows : CUSO4 + 2NaOH = NaaSO* + Cu(0H)2 Cu(0H)2 + heat = CuO + H2O 2CuO + glucose (CHO - (CH0H)4 - CH2OH) = CuaO -|- gluconic acid (COOH - (CH0H)4 - CH2OH). Whether a yellow Or a red precipitate forms will depend upon the alka- linity of the solution, the stronger the alkalinity the more pronounced is the red color due to cuprous oxid, while in less strong alkaUne solutions the yellow color of cuprous hydrate will predominate. Certain substances normally present in the urine as well as some which may be added to it have the property of holding in solution the cupric hydrate first formed. This property is shown by the deep blue color which the solution assumes. This color is very intense in the presence of sugar, but it is unwise to assume the presence of sugar from this fact alone. Among the substances which dissolve cupric hydrate and which may be present in the urine in varying amounts we find ammonium compounds, albumin, uric acid, creatinin, aUantoin, mucin, glucose, lactose, maltose, pyrocatechin, hydroquinon, alkapton acids, bile pigments, and glycuronic acid. On warming the solution, which may not contain sufl&cient amounts of glucose to give a typical reaction, a slight reduc- .tion of the copper solution will occur leading to the formation of a dirty yellow solution. If these bodies be present in excess a distinct precipitate may occur, and as a result marked confusion may arise regarding the presence of sugar. 1 It has been shown that uric acid and creatinin do not readily reduce at as low a temperature as does sugar, so that slight warming is much better than boiling. The presence of these bodies very frequently leads to a change in color from the bright blue to the greenish-yellow which may be due to the presence of sugar in small amounts, but should never be regarded as indicative of a pathological glycosuria. It should be a working rule, therefore, that mere decolorization of the fluid should not be regarded as due to sugar. Glucose reduces so much more markedly than the other bodies mentioned that a distinct granular pre- cipitate either of cuprous oxid or hydrate forms and settles out, leaving a supernatant fluid partially or completely decolorized. Normal urine or urine containing an excess of uric acid, creatinin, or ammonium compounds practically never produces an immediate precipitate unless the solution be boiled for some time. This is due to the fact that these substances hold in ' See Schulz, Ztschr. f. physiol. Chem., 1912, LXXVII, 121; also, Salkowski, Ibid., 1912, LXXIX, 164. '«> Digitized by Microsoft® 290 DIAGNOSTIC METHODS solution the amount of cuprous oxid which is formed by their reducing action. If such solutions be allowed to stand for a time a reddish-yellow precipitate may occur, but this should not confuse as the typical sugar reaction occurs im- mediately on warming unless too much copper solution be added. In case only traces of sugar be present, we may obtain Uttle or no positive reaction because the sugar holds in solution the traces of cuprous oxid formed just as do the other substances above mentioned. As a rule, however, sugar in patho- logic amounts shows a reducing action over and beyond its dissolving action, so that a red or yellow precipitate must settle out. The limit of this test for sugar is about 0.2 per cent., in which case the reduction wiU occur as in normal urine without the separation of the characteristic copper precipitate. Even in this case the yellow color is somewhat more intense and is clearer than the dirty yellow color produced in the presence of excessive uric acid or creatinin. If the urine contain an excess of the conjugated glycuronates, the test may be quite as distinctive as that for sugar. To differentiate such reactions, one should resort either to the phenylhydrazin or the fermentation tests. Benedict's Test. This qualitative test' is, in the writer's opinion, the best modification of Trommer's test. The reducing action of glucose in alkaUne solution is dimin- ished by strong alkalies such as the hydrates. This property may prevent such well-known solutions as Fehling's and Haines' (discussed below) from demonstrating small quantities of sugar. Further, the urine contains many compounds which interfere with the detection of glucose by the strongly alkaline solutions, the loss of delicacy in the other tests being probably due to the fact that normal reduction of the solution is inhibited for a period long enough to allow the strong alkali to decompose the reducing substance. However, in solutions of the alkali carbonates the reduction develops more slowly and is not destroyed by this weak alkali. The addition of sodium citrate, instead of the Rochelle salt as used by FehUng, makes the solution very stable, although Uttle more than does the glycerin of Haines' solution. For the reasons given, this solution of Benedict is more sensitive (about ten times) to urinary sugar than are the other copper solutions. Moreover, it is not appreciably reduced by creatinin and uric acid, nor by such preserva- tives as chloroform, chloral or formaldehyd, which introduce errors into the other tests. It is, however, promptly reduced by the alkapton acids and the conjugated glycuronic acids. The formula of the reagent is as follows: Copper sulphate (C. P. crystallized), 17.3 grams Sodium or potassium citrate, i73-o grams Sodium carbonate (crystallized),- 200.0 grams Distilled water, q. s. ad., looo.o c.c. Dissolve the citrate and carbonate (with aid of heat).in about 700 c.c. of water and filter if necessary. Dissolve the CuSOi in about 100 c.c. of water and pour into the alkaline solution. Cool and make up to i liter. 1 Jour. Am. Med. Assn., 1911, LVII, 1193. See, also, Kleiner, Ibid., 1914, LXII, 1307; Sheaflf, Ibid., 1915, LXV, 1181; Weinberger, Am. Jour. Med. Sc, 1914, CXLVII, 407. ' Or one-half the amount of the anhydrous salt may be used. Digitizecfby Microsoft® THE URINE 291 The technic is as follows : 5 c.c. of the reagent are placed in a test-tube and not more than 8 or 10 drops of urine are added. The mixture is then heated to vigorous boiling for one to two minutes and allowed to cool spontaneously. In the presence of glucose, the entire body of the solution will be filled with a precipitate which may be red, yellow or greenish in tinge. If the quantity of glucose be low (under 0.3 per cent.) the precipitate forms only on cooUng. If no sugar is present, the solution either remains perfectly clear or shows a faint turbidity, which is blue in color and consists of precipitated urates. This test may be especially recommended for all qualitative work. Fehling's Test. This is, perhaps, the best-known test for sugar but it is inconvenient and frequently inaccurate for the reasons above mentioned. The formulae for the solutions are as follows: Solution A. Solution B. , Copper sulphate, 34-64 gm. RocheUe salt, 173 gm. Distilled water, q. s., ad., 500 c.c. Sodium hydrate 50 gm. Distilled water, q. s., ad., 500 c.c. In performing the test with Fehling's solution equal parts of solutions A and B are taken and the mixture brought to a boil. The urine is then added, drop by drop, when a reduction of the copper solution will appear in the presence of sugar. 1 The amount of urine added should rarely exceed 10 drops, at the outside 20, if a reduction is to be taken as typical and the solu- tion should not be boiled for more than a few seconds after adding the urine. The addition of larger volumes of urine will usually introduce errors from the factors mentioned under Trommer's test. This test has the same points of interest as has Trommer's test, so that it is wise to dilute the urine before making the test. It is more frequently used than is Trommer's test, but it is not as convenient. Haines' Test. Haines has introduced a modification of Trommer's test by adding glycerin, instead of Rochelle salt, to increase the amount of copper in solution. This test is much more convenient than FehUng's, the solution having the advantage of keeping almost indefinitely. It is, however, far less dehcate than is Benedict's and is reduced by preservatives as well as by excess of many normal urinary constituents, especially by uric acid and creatinin.^ The composition of Haines' qualitative solution is as follows: Copper sulphate, 12 grams Potassium hydrate,' 45 grams Glycerin, 90 c.c. Water, q. s., ad., 1000 c.c. 'See Crameri Biochem. Jour., 1915, IX, 71. ' The creatinin may be almost entirely removed from the urine, if desired, by adding to 10 c.c. of urine about 2 grams of picric acid and about 2 grams of Merck's blood charcoal, shak- ing iqv a few minutes and filtering (see Folin, Jour. Biol. Chem., 1915, XXII, 327). ' Or 32 grams of sodium hydrate. Digitized by Microsoft® 292 DIAGNOSTIC METHODS A perfectly clear, transparent, dark-blue liquid results which throws down a very slight reddish deposit of cuprous oxid on standing a week or more. This does not affect the value of the solution, as the clear blue solution is simply decanted as required. Four or five c.c. of this solution are placed in a test-tube and gently boiled. Six drops of the suspected urine are added and the upper portion of the mix- ture brought to a boil and immediately removed from the flame. If sugar be present an abundant yellow or yellowish-red precipitate is thrown down; if no such precipitate occurs sugar is absent. The precautions to be observed in using this test are never to add at the outside more than 10 drops of urine and not to boil the mixture for more than one or two seconds after the addition of urine. Many other modifications of the copper test have been introduced, both for qualitative and quantitative purposes, but they do not have any ad- vantages over those mentioned especially over Benedict's test. This latter is used daily in the writer's laboratory and has been found very reUable and serviceable as it does not react with the usual preservatives which are added to urine. When the other tests are used an error may creep into the report which can be corrected only by resort to fermentation and other confirmatory methods. Almen-Nylander's Test. This test is a modification of the original Bottger test and is a distinct improvement. The reagent is prepared as foUows: Four grams of RocheUe salt are dissolved in 100 c.c. of 10 per cent, sodium hydrate solution with gentle heat, and as much bismuth subnitrate is added as will dissolve (about 2 grams). After the mixture is cooled the undissolved bismuth subnitrate is filtered off and the filtrate kept in a dark bottle, where it will remain permanent for a long period. To a few c.c. of urine in a test-tube is added one-tenth of the volume of this reagent and the mixture boiled for a few minutes. If glucose is present, the fluid will darken and a black precipitate of metalhc bismuthseparate out. This black precipitate must occur while the solution is being warmed and not after it has cooled. If only a small amount of sugar be present the phosphates precipitated by the alkali may be slightly gray in color instead of the usual white. This test is somewhat more delicate than is the copper test, as it will show about J^o per cent, of sugar. This reagent is not reduced by uric acid, creatinin, p3nrocatechin, hydro- quinon or homogentisic acid; but it is reduced by the conjugated glycuronic acid, excess of urinary pigment and pentoses. It is particularly necessary in this test that albumin be removed, as bismuth sulphid forms in the presence of albumin, and may precipitate either in the form of a reddish or a distinctly brownish, even black precipitate. The reduction observed after the adminis- tration of medicaments, such as rhubarb, senna, antipyrin, camphor, salicyUc acid, salol, sulphonal, trional, quinin, eucalyptus, oil of turpentine, and chloral hydrate, is usually a brown rather than a black unless the solution stands for some time. The adminkteatraiof sa^harin u^ally results in a reduction of THE URINE 293 this test, while the copper tests are not affected by it. Sulphur bodies (methyl mercaptan) excreted after the patient has eaten asparagus give a distinct precipitate which may be more or less confusing. If the urine be ammoniacal the reaction may not appear in a characteristic way owing to the fact that the free ammonia is evolved and the alkalinity of the solution is reduced by the combination of the sodium ion with the acid radical formerly bound to the ammonium ion. This test is used by many as a routine test for sugar, as it is practically always negative with normal urine. The reducing action of the glycuronates and pentoses must, however, be remembered.' Fennentation Test. As previously stated, the reduction tests do not absolutely prove the pres- ence of sugar. All that one can say is that a reducing substance is present and if the reaction be typical the probability is that the reducing substance is sugar. The fermentation test is, perhaps, the most certain of all the tests for glucose and depends upon the fact that only those sugars which contain three or a multiple of three carbon atoms are fermentable with yeast. ^ Not all members of these groups of sugars will ferment with yeast, so that for absolutely scientific purposes fermentation will- not differentiate them. Fischer's work along this Une should be carefully read by anyone interested in the biologic properties of the various types of sugar. As the hexoses, which occur in the urine are practically limited to glucose and levulose, we are safe in saying that any sugar fermenting with yeast is one or the other of these monosaccharides, which may be differentiated by tests to be outlined later. The test is performed as follows: Ten c.c. of urine are placed in a test- tube and a piece of compressed yeast, which should be perfectly fresh, about the size of a pea is added and the urine gently shaken until the yeast is finely divided. This mixture is then poured into a fermentation tube, which is allowed to stand in the incubator for a few hours. Two control tests, using in one normal urine and yeast and in the other normal urine, yeast, and a trace of dextrose, are then made and placed in the incubator along with the suspected urine. The presence of sugar is indicated by gas (CO2) in the upper portion of the fermentation tube. The rapidity of formation of this gas de- pends upon the amount of yeast as well as upon the age of the yeast. The test indicates from o.i to 0.05 per cent, of sugar, especially if the urine be sterilized by previous boiling. The compressed yeast as usually purchased develops a certain minimal amount of gas in normal urine, so that the control test is necessary both to show whether "self -fermentation" is excessive and also whether the yeast is at all active in producing CO2 from glucose when it has been added. It is necessary, moreover, that decomposition of the urine be prevented, either by previously boihng the urine, addition of a trace of sodium fluorid, or tartaric acid. This test has the great advantage that the other substances, which reduce copper and bismuth solutions, do not ferment. If precautions are observed ^See Mende, Munch. Med. Wchnschr., 1914, LXI, 1120. *Boros (Med. Klin., 1913, IX, 874) shows that yeast cells may be so numerous in the urine, as voided, to destroy tl^)^g^f ^^»ty\/yjf^^j^^f^n as masked or occult glycosuria. 294 DIAGNOSTIC METHODS to add the right amount of yeast, not to shake the yeast and urine violently enough to include much air, and to prevent bacterial decomposition, this test will positively show the presence or absence of glucose or levulose in the urine and no other substances. It is always wise to use this test either as confirmatory or decisive in conjunction with the previous reduction tests. Phenylhydrazin Test. This test is much more delicate than any of the previous tests mentioned. Theoretically it will show sugar in the amount present in normal urine, but practically no definite reaction is observed. The principle of the reaction is the decomposition which occurs between the aldehyd or ketone group of the sugar molecule and the amino group of the phenylhydrazin. In this reac- tion, characteristic bodies known as hydrazones are first formed, which are converted, in the presence of dilute acetic acid and an excess of phenylhydta- zin, into crystalline bodies known as osazones. These latter bodies are char- acteristic of the sugar group, usually differing from one another, depending upon the original sugar from which they were formed. The osazones crystal- lize in definite forms, the purified crystals showing rather sharp melting points. It is, therefore, essential not only that a crystalline body be obtained in this reaction, but that the crystalline form and the melting-point of the crystal be that characteristic of the sugar suspected. The success of the test will depend largely on the relation of the sugar to the reagents, the best proportions being approximately one of sugar, two of phenylhydrazin, and three of sodium acetate. All of the members of the hexose and pentose groups show this reaction, as do many of those of the polysaccharide series. As will be seen from the reaction given below, those sugars which differ only in the space- relations of the atoms attached to the first two carbon atoms can possibly give the same osazone. For this reason we find glucose, levulose, and man- nose as well as glucosamine giving exactly the same osazone (phenylglucosa- zone), showing a characteristic yellow needle-shaped crystalline deposit, with a melting-point of 204 to 205°C. The reactions leading to the formation of this body are shown in the following equations: CH2OH - (CH0H)4 - CHO + NH2 - NHCeHs = CH2OH - (CHOH)4 - CH = N - NHC6H5 -I- H2O CH2OH - (CH0H)3 - CHOH - CH = N - NHCeHs + NH2 - NHCeHs = CH2OH - (CH0H)3 - C - CH + 2H2O II II CeHsNH - N N - NHCeHs Twenty-five c.c. of urine are treated with a few drops of a solution of lead acetate and filtered to remove the albumin if present. To the filtrate, which must be acidified with acetic acid if it is not already acid, is then added phenylhydrazin hydrochlorate (>^ to i gram) and about 2 grams of sodium acetate. The tube is then shaken thoroughly to mix the contents and is placed in a boihng water-bath for from one to two hours. Some workers recommend a shorter period, such as 20 minutes, but the writer has never been able to get as good jr^^f^gfi^p A!fy&A?fe?J)^^eating. At the end of this PLATE IX. OsAzONS. {Hawk) Upper form, dextrosazon; central form maltosazon; lower form, lactosazon. Digitized by Microsoft® Digitized by Microsoft® THE URINE 29s time the material in the tube is filtered while hot and the filtrate allowed to cool slowly rather than as some advise to cool suddenly by immersion in cold water. If glucose be present a yellow crystalline deposit will appear, which under the microscope will show the characteristic needle-shaped crystals arranged in bundles or sheaves. This microscopic examination does not absolutely prove the crystals to be phenylglucosazone so that a melting- point determination must be made before definite proof is forthcoming. For this purpose the crystals must be purified by dissolving in a hot 60 per cent, alcohol and recrystallizing by adding water and evaporating the alcohol. A few of these crystals are then placed in a perfectly dry capillary tube and the melting-point determined by methods previously learned in organic chemistry. Neumann has applied Fischer's method of using this test to the urine. Five c.c. of urine are treated in a test-tube with 2 c.c. of a 50 per cent, solution of acetic acid saturated with sodium acetate and two drops of pure phenyl- hydrazin. This mixture is then evaporated by boiling to 3 c.c, after which it is cooled quickly and is then rewarmed and allowed to cool slowly. If glucose be present to the amount of 0.02 per cent., pure crystals of phenyl- glucosazone separate out in 5 to 10 minutes. If the urine has a high specific gravity and a low sugar content the crystals do not form so quickly. This test is much to be preferred in well-equipped laboratories, but is not as con- venient as the use of the crystalline phenylhydrazin hydrochlorate for the gen- eral worker, as the fluid phenylhydrazin is quite irritating and is not as easy to work with. This test cannot be used for quantitative purposes as the yield is never complete. For qualitative purposes, however, it is to be especially recom- mended. When properly applied it is the most delicate and one of the most reliable tests at our disposal. If the melting-point of the crystals be deter- mined the only confusing substances will be levulose, glucosamin, and man- nose. The osazones formed from the other carbohydrates and the glycuronic acid compounds crystallize in somewhat similar form, but do not show the characteristic melting-point of 204° to 20S°C. of the phenylglucosazone. It is true that the impure crystals melt at somewhat lower temperature, but if care- fully purified from 60 per cent, alcohol they will melt at approximately 204°. Quantitative Methods. Fehling's Method. The solutions to be used in this test have been given under the qualitative test for sugar. The principle of the test is the determination of the exact amount of sugar necessary to decolorize a mixture of 5 c.c. each of solutions A and B. Providing chemically pure copper sulphate has been used in pre- paring solution A, the mixture is reduced by an amount of urine which con- tains 0.05 gram of glucose. If there is any question regarding the purity of the copper sulphate, the strength of the solution must be determined by titrat- ing against a known solution of chemically pure glucose, or of cane-sugar which has been inverted by heating with dilute acid (see Sutton's Volumetric Analysis). ^. ... ,, .,. „^ Digitized by Microsoft® 296 DIAGNOSTIC METHODS Technic. ' Five c.c. each of solutions A and B (p. 291) are carefully measured from a buret or pipet into an Erlenmeyer flask of about 250 c.c. capacity. Forty c.c. of distilled water are then added and the mixture boiled. The urine is now added in small quantities from a buret to this boiling copper solution until the blue color has entirely disappeared. It is advisable to add the urine rather rapidly as a preliminary test, it being practically impossible with a single titration to obtain accurate results. In doing this one adds a c.c. of urine at a time and observes whether the color still remains. If so, the titra- tion is continued, adding from 1/2 to i c.c. at each period, until the color has disappeared. The number of c.c. used is then observed and a second test made as follows: To the boiling copper solution is added directly i c.c. less of urine than was used for complete decolorization in the preliminary test. The urine is then added two drops at a time, noting after each addition whether the color remains. In this way an accurate end-point may be reached. As the amount of urine used to decolorize the 10 c.c. of copper solution is equivalent to 0.05 gram of glucose, a simple calculation yields the percentage and total values for glucose in the urine. As this test is not accurate with high concentrations of the urine, it is necessary that the sugar content should range between 1/2 and i per cent. Dilution may, therefore, be necessary. A good working rule is to dilute a urine of a specific gravity of 1030 five times (by adding 4 volumes of water) and ten times if the specific gravity be above 1035. There is little danger of getting the dilution too great to show the characteristic reaction for sugar, as this test shows the presence of sugar when present to the amount of 0.08 per cent. In the use of this method, as well as of most reduction tests, albumin must first be removed if present in more than traces. Moreover, the presence of preservatives as well as excess of uric acid or creatinin will increase the reducing power of the urine as determined by this test, a considerable error being occasionally introduced unless overcome by dilution or removal of the interfering substances. Bang's Method. This method^ appears to the writer to be the best one at our disposal for the quantitative estimation of sugar in the urine. Like the solution of Bene- dict previously mentioned, it contains alkali carbonates instead of hydrates. The principle of this method is as follows: The urine is treated with an excess of standard alkahne copper solution and boiled to bring out the reduction due to sugar. The amount of copper remaining in excess is then determined by titration with a solution of hydrdxylamin sulphate (instead of with potassium sulphocyanate as used by Citron) and from this the amount of sugat is calculated. The solutions necessary in this test are as follows : (a) One hundred grams of potassium bicarbonate are dissolved in about 1300 c.c. of distilled water contained in a 2-liter flask. To this solution are 1 Biochem. Ztschr., 1907, II, 271 ; igo8, XI, 538; 1911, XXXII, 443. Bang (Ibid., 1913, XLIX, i) has devised a zga^^thgi^whj^ Js^che^^ and equally exact. THE URINE 297 added 500 grams of potassium carbonate and 400 grams of potassium sulpho- cyanate. Exactly 25 grams of pure copper sulphate (CUSO4 SH2O) are then dissolved in about 150 c.c. of warm distilled water. After cooling this solu- tion is added quantitatively to the carbonate solution. Add water up to the BANG'S TABLE OF REDUCTION EQUIVALENTS Cubic centimeters of Milligrams of Cubic centimeters of Milligrams of hydroxylamin solution sugar hydroxylamin solution sugar 0-7S 60.0 25 -SO 23-S 1. 00 59-4 26.00 22.9 I. SO S8.4 26.50 22.3 2.00 S7-3 27.00 21.8 2.50 56.2 27.50 21.2 3.00 SS-o 28.00 20.7 3-So S4-3 28.50 20.1 4.00 S3-4 29.00 19.6 4-So 52.6 29.50 19. 1 S-oo 51.6 30.00 18.6 S-So SO. 7 30.50 18.0 6.00 49-8 31 00 17-S 6.50 48.9 31 -SO 17.0 7.00 48.0 32.00 16.5 7. SO • 47.2 312-50 15-9 8.00 46.3 3'3-oo 15-4 8.50 4S-S 33-50 14-9 9.00 44.7 34.00 14.4 9 -SO 44.0 34 -so 13-9 10.00 43-3 35 -00 13-4 10.50 42-S 3S-SO 12.9 11.00 41.8 36.00 12.4 11.50 41. 1 36.50 II. 9 12.00 40.4 37.00 II. 4 12.50 39-7 37-50 10.9 13-00 39-0 38.00 10.4 13 SO 38.3 38.50 9-9 14.00 37.7 39.00 9-4 I4.S0 37-1 39-50 9-0 IS- 00 36.4 40.00 8-S IS SO 35-8 40.50 8.1 16.00 3S-I 41.00 7-6 16.50 34-S 41 -SO 7-2 17.00 33-9 42.00 6.7 I7-SO 33-3 42 -so 6.3 18.00 32.6 43-00 5-8 18.50 320 43-50 S-4 19.00 31-4 44.00 4-9 19.50 30.8 44-50 4-S 20.00 30.2 45.00 4-1 20.50 29.6 45-50 3-7 21.00 29.0 46.00 3-3 21.50 28.3 46-50 2-9 22.00 27-7 47.00 2-5 22.50 27.1 47-50 2.1 23.00 26.5 48.00 1-7 23 -SO 25-8 48 -so 1-3 24.00 25.2 49.00 0-9 24.50 24.6 49-50 0.5 25.00 24.1 50.00 0.0 For every i/io c.c. hydroxylamin solution used more than is given in the table subtract o.i mg. if the reading be betwp^n^4^_and W^'^l?^UiUiS(bf ''^een 15 and i subtract 0.2 mg 298 DIAGNOSTIC METHODS 2-liter mark, allow to stand for 24 hours and filter. This solution is stable for about three months.^ (b). Two hundred grams of potassium sulphocyanate are dissolved in about 1500 c.c. of distilled water in a 2-liter flask. 6.55 grams of hydroxy- lamin sulphate are dissolved in water and added quantitatively to the sulpho- cyanate solution. Add water up to the 2-liter mark and preserve the mix- ture in dark colored bottles. This solution is very permanent. Technic. Ten c.c. of urine (5 or 2 c.c. diluted with water to 10 c.c. if more than 0.6 per cent, sugar is present) are measured into a 200 c.c. Jena Erlenmeyer flask and treated with 50 c.c. of the copper solution (a). Heat on a wire gauze to boiling for exactly three minutes. Cool quickly to room temperature by im- mersion of the flask in cold water. Now titrate with the hydroxylamin solu- tion (b) until a colorless mixture results. From the number of c.c. of hydrox- ylamin sulphate solution used, calculate the sugar in milligrams by means of the accompanying table. A simple calculation yields the percentage and total amounts. It has been found difl&cult to get a sharp end-point in this reaction if the urine be rather highly colored. Bang and Bohmansson^ have shown that the urine may be cleared by the following process. Twenty c.C. of urine are treated with 5 c.c. of 25 per cent. HCl and 2 grams of blood charcoal. Shake a few times during five minutes and filter through a dry filter into a dry beaker. This filtrate is then used for the test as described above. Woodyatt and Helmholz' and Andersen* have called attention to the necessity of testing the charcoal by control experiments before using the above method of clearing. As some of the blood charcoals give very erroneous results, they recommend the use of Merck's preparation. A further advantage of using this clearing method is that the "auto-reduction" of the urine is reduced to a low limit owing to the almost complete removal of urochrom, uric acid, creatinin and glycuronic acid derivatives.^ Purdy's Method. It has been found that the addition of strong ammonia to the mixed copper and tartrate solutions makes the end-point much more distinct. Pavy, Sahli, Kumagawa and Suto, Kinoshita, and others have modified the original Fehling's solution in this way. It was shown, however, by Lowe that the substitution of glycerin for the Rochelle salt made a much more stable solution and one which could be originally mixed and kept for indefinite lengths of time. Purdy has succeeded in obtaining a copper solution which, in the writer's experience, is much to be preferred for general laboratory purposes over most of the other modifications of Fehling's solution. His formula is as follows : ^ Hatta (Biochem. Ztschr., 1913, LII, i) shows that the self-reduction of this solution may give as high an error as 9% on the positive side. '^ Ztschr. f. physiol. Chem., igog, LXIII, 443; Biochem. Ztschr., 1909, XIX, 281. 3 Arch. Int. Med, igii.'VII, 598. "Biochem. Ztschr., 1911, XXXVII, 262. '■ Bang (Biochem. Ztschr., 1,912, XXXVIII, 168) has recently advised the substitution of 2 c.c. of 95% alcohol for t]0jgjtizeil ^WlicrOSOft® THE URINE 299 Chemically pure copper sulphate, 4-752 grams Potassium hydrate/ 23.500 grams Strong C. P. ammonia (sp. g. 0.88), 350.000 c.c. Glycerin, 38.000 c.c. Distilled water, q. s. ad., 1,000.000 c.c. This solution is prepared by dissolving the copper sulphate and glycerin in 200 c.c. of distilled water, heating gently if necessary. The potassium hydrate is dissolved in a second 200 c.c. of water and mixed with the copper solution. When the mixture has cooled add the ammonia and bring the total volume up to i liter with distilled water. Thirty-five c.c. of this solution are decolorized by 0.02 gram of glucose. Haines has slightly modified this original solution of Purdy so that 10 c.c. of the solution are decolorized by o.oi gram of glucose. This titer is some- what more convenient than that of Purdy as the calculation is distinctly simplified. The formula for his modification is as follows : Pure copper sulphate, 8.314 grams Pure potassium hydrate,^ 25.000 grams Glycerin, 40.000 c.c. Ammonia, 350.000 c.c. Distilled water, q. s. ad., 1,000.000 c.c. The principle of this test depends upon the fact that, in the reduction of cupric oxid in solutions of definite strength by glucose, the blue coloration disappears on the addition of a definite amount of glucose without any attend- ant precipitate, the reduced solution remaining transparent and colorless. Technic. Thirty-five c.c. of Purdy's test solution or 10 c.c. of Haines' solution are measured into an Erlenmeyer flask and 50 c.c. of water added. The flask is then closed with a doubly perforated rubber stopper, through one hole of which passes the stem of a buret containing the suspected urine and through the other a bent glass tube to conduct the fumes of ammonia away from the observer. The object of closing the flask with the stopper is to exclude the air and thus prevent reoxidation of the cuprous oxid. The con- tents of the flask are now brought to a gentle boil and the urine added, i c.c. at a time, shaking the flask after each addition until the fluid is completely de- colorized. The number of c.c. used is then noted and a second test made, adding at once i c.c. less of urine than the total number of c.c. used in the first experiment. The last portions of the urine are added two drops at a time, allowing from three to five seconds to elapse between the addition of these separate portions. When the urine is completely decolorized the number of c.c. used is noted. As the amount of urine used is equivalent to 0.02 gram of glucose with Purdy's solution or o. 01 gram withHaines' solution, the percent- age may be obtained as in the previous method. It is necessary with this method that'the urine be diluted for the same reasons as previously mentioned. In all of the copper tests for glucose the influence of preservative agents ' Or 16.8 grams of sodium hydrate. 2 Or 18 grams of sodium ^^f^ed by MicrOSOft® 300 DIAGNOSTIC METHODS must be remembered. Thus chloroform, chloral hydrate, and formaUn will all reduce copper solutions so that an error may be introduced unless these substances are removed. In selecting a method for the quantitative determination of sugar in the urine, the general worker should remember that the method used should be both simple and exact. With these points in view, the writer has little hesi- tancy in recommending Bang's method. If the urine be cleared as pre- viously described, the results are all that could be desired, the error being extremely small. Many other modifications of the copper tests have been advocated, but the writer does not feel that they have any advantage from either the scientific or clinical standpoint. The method of Allihn, in which a copper solution is reduced and the precipitated cuprous oxid either weighed as such or further reduced to metalhc copper in a stream of hydrogen, is not clinically available. The modifications of Rudisch, Rudisch and Celler, and of Gerrard and Allan do not give as accurate results as does the Bang method in the experience of the writer. The recent method of Benedict"^ is apparently quite reliable. Polariscopic Method. In this test the urine must be absolutely clear and must contain no albu- min. Moreover, it is essential that such substances as glycuronic acid and i3-oxybutyric acid be removed before polarization as they will introduce con- FiG. 84. — One form of Laurent Polariscope. {Hawk.) B, Microscope for reading the scale; C, a vernier; E, position of the analyzing Nicol prism; H, polarizing Nicol prism in the tube below this point. siderable error in the reading. The best clearing agent is that, previously mentioned under Bang's test, of shaking the urine with HCl and blood char- coal and filtering. Instead of this method the urine, acidified with acetic acid, may be treated with a solution of normal lead acetate, which will precipitate the albuniin and remove excess of pigment. The mixture is filtered and the clear filtrate is used for the test. It must be remembered that a correction must be made in the percentage of sugar in the filtrate, if the urine be cleared with a solution of lead acetate. This may be done by adding 25 c.c. of a 10 per cent, solution of lead acetate to 75 c.c. of urine. The sugar in the filtrate ' Jour. Biol. Chem., igii.BJ^tizeSt^tiy MrcMs^W^n., 1911, LVII, 1193. THE URINE 301 will represent only three-fourths of that of the original urine. Basic lead acetate should be used with caution, as it precipitates various sugars. If the urine con- tain no albumin, magnesium oxid or silicic acid may be used asclearing agents. Various types of polarimeters have been suggested and most of them are quite satisfactory for the determination of sugar. One of these with its description is seen in the accompanying cut and legend. The principle upon which their use depends is the fact that optically active substances when in solution have the power of turning the plane of polarized light either to the right or the left. The zero point of the instrument is determined by observing the point at which the halves of the optical field have exactly the same degree of illumination, when the light passes through a tube either empty or containing an optically negative fluid. The point at which the graduated scale of the instrument and the vernier correspond is regarded as the zero-point. In the use of the polariscope any deviation of the plane of light passing through the polarizing Nicol prism will be noticed by a darken- ing of one portion of the field, so that the compensating or analyzing Nicol must be rotated until both parts of the field are equally illuminated. In this way one readily determines how much the plane of polarized light has been deviated, by observing the degree of rotation necessary to bring the two portions of the field into equal illumination when the light passes through an /\7 BDI e f g Fig. 85. — Diagrammatic representation of the course of light through the Laurent polariscope. (Direction reversed from that of previous figure.) (Hawk.) a, Bichromate plate to purify the light; b, the polarizing Nicol prism; c, a thin quartz plate covering one-half the field and essential in producing a second polarized plane; d, tube to contain the liquid under examination; e, the analyzing Nicol prism;/ and g, ocular lenses. optically active fluid. The most rehable instruments for general work are known as the " half -shadow " types, but they rarely find a place in the equip- ment of the general worker. The examination is usually made in a dark room, the light passing through the tube of the polariscope from a sodium flame. It must be remembered that the urine may contain substances other than glucose which rotate the plane of polarized light. The normal urine is slightly levorotatory, the degree varying between 0.05 and 0.18. Albumin is also levorotatory and will interfere markedly with the degree of rotation refer- able to glucose, unless it be very slight in amount or be entirely removed. Levulose, /3-oxybutyric acid, and the conjugated glycuronates are also levo- rotatory, the second being especially prone to interfere with the glucose rota- tion as it so frequently is associated with glucose in diabetic conditions. If the urine has been previously heated with acid before being examined in the polariscope, glycuronic acid will also interfere, as it shows distinct dextrorota- tion. Cane-sugar as well as lactose are occasionally found in the urine and may lead to confusing results as both are dextrorotatory substances. It is, therefore, necessary befpr.e .any rehaMe results may be obtained with the 302 DIAGNOSTIC METHODS polariscope to remove the interfering substances, either as preliminary to the determination of glucose or subsequently, a correction being then made for the rotation of the interfering substance. It is to be said that the polariscope is more fitted for a clinical laboratory than for the general practitioner. The rotation of light when passing through optically active solutions is dependent upon several factors, among which we find the temperature at which the observation is made, the length of tube through which the light passes and the concentration of the active solution. By specific rotation of a fluid is meant the rotation observed when light passes through a solution containing i gram of the active substance per c.c. of fluid and placed in a tube one dcm. in length. Thus we find the specific rotation of glucose in such a tube being (a)^ = +52.74. It is, therefore, evident that the per- centage of sugar in an unknown solution contained in a tube i dcm. in length may be obtained by dividing the degree of rotation by 0.5274. Some of the tubes for clinical purpose are constructed of such a length (188.6 mm.) that one degree of rotation equals i per cent, of glucose. For a full discussion of the sub- ject of optical activity of fluids the writer would refer to the work of Landolt.^ Technic. Having determined the zero-point of the instrument, the tube, which should be thoroughly cleaned arid dry, is filled with the fluid to be examined. In filling this tube precautions must be taken not to include any bubbles of air, which is best done by filUng the tube until a convex meniscus is observed and then sliding the glass disk over the end in such a way that the excess of fluid is shoved off. The metal cap is then screwed on tightly, but in such a way that undue compression is not exerted upon the glass disk. The tube is placed in position, the field distinctly focussed and the degree of rotation determined by revolving the analyzing Nicol until the two portions of the optical field have exactly the same intensity of illumination. This will require considerable experience, as the accuracy of the determination will depend not only upon the clearness of the fluid, the degree of focussing, the sensitiveness of the instrument, and the brightness of the Hght, but also upon the sensitiveness of the observer's eyes. Several readings should be made, turning the prisms from both directions and observing the degree at which the fields are isochromatic, the average being taken as the final result. As the eyes soon become fatigued, they should be used for only a few seconds at a time. Having determined the degree of rotation, the percentage of sugar is then calculated as previously mentioned. This method, if carefully used and interfering substances avoided, is the most accurate method for the determina- '■ Das optische Drehungs-" Fig. 86.^ — -Einliorn sac- charometer. THE URINE 303 tion of glucose. It is, however, difficult, requires much experience, and is not as sensitive as the other methods, rarely detecting the presence of less than 0.2 per cent, of glucose. Theoretically, the percentage of sugar as de- termined by the polariscope should exactly agree with that obtained by titra- tion of copper solutions, but in general work such is never the case, as inter- fering substances may be present or have not been completely removed. The writer must refer elsewhere for methods of correcting the readings of the polariscope when interfering substances are present, as he does not think it wise to recommend the general worker to waste his time with this instrument when sufficiently accurate clinical results may be obtained by methods which are for him much easier and less liable to error. Fermentation Methods. The principle of this method has been previously outlined. It has been found that, if the precautions mentioned are observed, carbon dioxid is evolved quantitatively from glucose by the action of yeast. The most con- venient method of applying the test is to use an Einhorn fermentation tube which is so graduated that the amount of CO 2 evolved is directly read off in terms of per cent, of glucose. For this purpose the urine must contain less than I per cent, of glucose, the urine being diluted as previously described to bring the amount within this figure, multiplying the result, of course, by the degree of dilution. The urine, which should be acid in reaction, is shaken with a piece of compressed yeast about the size of a small pea, aU precautions being observed as mentioned above. The mixture is then poured into the fermentation tube in such a way that no air-bubbles collect at the upper end of the tube. Controls as previously described are then made and the three tubes placed in the incubator at 37°C. In a few hours bubbles of gas (CO2) will collect at the top of the tube, the fermentation being practically complete overnight. The percentage of sugar is then read direct from the calibration (1/4-1 per cent.) on the tube (the figures from one to five representing c.c. of gas and not per cent, of sugar). Lohnstein's Saccharometer. This apparatus is seen in the accompanying cut. Twelve c.c. of mercury are placed in the bulb of the apparatus. One-half c.c. of the urine to be tested is then floated upon the mercury and treated with a thick paste of com- pressed yeast dilute two or three times with water. The stopper is then carefully greased with vaseline and inserted so that the two apertures corre- spond. By tipping the apparatus a trifle the column of mercury in the long tube is then adjusted to the zero point of the scale. When this is done the stopper is turned so that the holes on longer correspond and the weight is placed on the stopper to prevent leakage from the increased pressure of the gas liberated in the fermentation. The apparatus is then placed in the incu- bator and the extent of fermentation read off by noticing the height to which the column of mercury rises in the long arm of the instrument. The fermen- tation is usually complete within six hours. After removing the apparatus from the incubator it is allowed to stand in the air for a few minutes to adjust itself to room temperatufj^^^^g^y^^g^^ted in this way. 304 DIAGNOSTIC METHODS This method gives results which correspond very closely to those of titra- tion and is to be recommended for the quantitative determination by fermentation methods. Lohnstein has also introduced a saccharometer which may be used with dilute urine. It is seen in the accompanying cut. It does not, in the writer's opinion, have any advantage over the above-mentioned apparatus as the principle is the same, although the urine must be diluted. Robert's Method. This method has been recommended for the quantitative determination of sugar and is based upon the fact that the specific gravity of the urine is changed in a quantitative way when the sugar of the urine is fermented. Fig. 87. — Lohn- stein's fermenta- tion tube for undi- luted urine. Fig. 88. — ^Lohnstein's fermentation tube for diluted urine. The urine must be acid before applying this test. A piece of yeast about the size of a bean is added to the urine which is allowed to ferment at incu- bator temperature until no further qualitative test for sugar is obtained. This will usually require from 24 to 48 hours, so that a trace of sodium fluorid should be added to the urine to prevent bacterial action. The specific gravity of the urine before it is subjected to fermentation is very carefully taken either with a very accurately standardized hydrometer or, preferably, with the pycnometer. After fermentation is complete the specific gravity of the fermented urine is determined in the same way. The difference in the specific gravity of th^tw^^^gm^s^s^th^g^multipUed by 234 to obtain THE URINE 305 the percentage of sugar. Or, according to Purdy, each degree of specific gravity lost in fermentation corresponds to i grain of sugar per fluidounce. If the specific gravity be accurately determined the results are correct within 0.1 per cent. As this method requires, however, the use of a pycnom- eter and a very accurate chemical balance, it can hardly be recommended to the general practitioner for his work. (b) Levulose (d-fructose), CH2OH— (CH0H)3— CO— CH2OH. Levulose is found very widely distributed throughout the vegetable king- dom, especially in fruits. Honey is almost a pure levulose. It may be found in the urine, transudates, or exudates, after a large intake of levulose-con- taining food or may occur spontaneously, when the subject has taken little such food. The levulosuria, reported by Zimmer, Ventzke, Czapek, Worm- Miiller, Seegen, Mauthner, Cotton, Roehmann, Personne, Henninger, Marie, and Robinson, must be accepted with reserve, as the incompleteness of the methods then in use afforded no certain means of recognizing levulose (Neu- berg'). The most authentic cases of true levulosuria are those of May, Schlesinger, Rosin and Laband, Lepine and Boulud, and Neubauer. This pure levulosuria occurs in both sexes and at all ages, the amount of sugar excreted being subject to variations from 2.7 grams per diem (Schlesinger) to 24 grams (Lepine). More common than pure levulosuria is its association with a glycosuria. This combination appears in all forms of diabetes, in the severe types levulose being practically never missed, according to Umber, especially when no restriction is placed on the carbohydrate intake. Neubauer's observations on this point are interesting. He finds that withdrawal of carbohydrates causes both levulose and dextrose to disappear from the urine. When levulose was given it was utihzed, but when glucose was taken it was less completely assimilated, being excreted in part as levulose. The tolerance for levulose is, as a rule, less than that for glucose, so that we are not surprised to find that the administration of 100 grams of levulose to normal individuals is followed by a levulosuria in about 10 per cent, of cases, while no such effect may be observed in diabetes mellitus. Here the question of individual tolerance must be considered, as Umber finds 25 grams of levulose excreted following an intake of 100 grams, while the writer has observed an excretion of 75 grams on the same intake. Strauss^ finds that an alimentary levulosuria occurs, after an intake of 100 grams of levulose, in 90 per cent, of cases of functional hepatic disturbance. This test would seem, therefore, to be a valuable indication of hepatic insufficiency, although it is not pathognomonic. Also alimentary galaciosuria, following the adminis- tration of 40 grams of galactose to the fasting stomach, as originated by Bauer,' has been shown by Reiss and Jehn and, also by Roubitschek to be ' Handbuch der Pathologie des Stoffwechsels, Berlin, 1907, 716; also, Cammidge and Howard, Lancet, ipij, I, 320. 'Deutsch. med. Wchnschr., 1901, XXVII, 757 and 786; -Ibid., igra, XXXIX, 1780. See, also Oszacki and Wagner, Med. Klin., 1913, IX, 1549; Draudt, Arch. f. exper. Path. u. Pharmakol., 1913, LXXII, 457; Hohlweg, Miinch. med. Wchnschr., 1913, LX, 2271; and Schirokauer, Ztschr. f. klin. Med., 1913, LXXVIII, 462. ' Deutsch. med. Wchnschr., rgoS, XXXIV, 1505. 20 Digitized by Microsoft® 3o6 DIAGNOSTIC METHODS inconstant. A decidedly positive excretion of galactose is observed only in cases in which the entire liver parenchyma is presumably involved, as in the acute infections and intoxications characterized by catarrhal jaundice. Cases of cirrhosis, carcinoma, cholelithiasis and phosphorus poisoning react either negatively or very slightly.^ Thd chemical reactions of levulose are very similar to those of dextrose. Owing to the presence of the ketone group in its molecule, it shows the same reducing actions as does the aldehyd group of the glucose. Like glucose it ferments, but not quite so readily. It is levorotatory, its specific rotation being (a)i, = —91 degrees. Leo has reported the finding of a levogyrate carbo- hydrate in diabetic urines, which he believes to be laiose. This difiers from lev- ulose in being unfermentable with yeast. Levulose forms exactly the same phenylosazon as does glucose, so that it is a matter of great difficulty to differ- entiate these bodies by the tests given above. Lobry de Bruin and Alberda von Eckenstein have shown that glucose and levulose may change, the one into the other, by the action of traces of alkah, acids, and neutral salts, such as sodium acetate. Seliwanoff's Test. This test has been advanced as one characteristic for the ketoses in dis- tinction from the aldoses. Ten c.c. of urine are treated with a few crystals of resorcin and 5 c.c. of concentrated HCl. If the mixture be warmed a bril- liant red color appears in the presence of a ketone (levulose) while no colora- tion is observed with an aldehyd (glucose).^ Miiller has shown that gluco- samin gives this test, while R. and O. Adler find a reaction in the presence of nitrous acid. If the mixture be heated too strongly or too long, mannose and maltose may also give a positive test. Adler finds that the use of acetic acid with a trace of HCl gives better results than HCl alone. It will be seen, there- fore, that this test is not so characteristic as was believed, but it serves to distinguish levulose fsom glucose, which is the important point. If the red solution formed in this reaction be neutralized with sodium carbonate and extracted with amyl alcohol or, preferably, with acetic ether (Borchardt), the extract will have a yellow color with a faint green fluores- cence and becomes rose-red on the addition of alcohol. The spectrum of this solution shows a sharp line in the green between E and h, while if the solution be quite concentrated a second weaker line will be seen in the blue at F. Methyl-phenylhydrazin Test. Neuberg^ has definitely shown that fructose forms a characteristic osazone 1 Reiss and Jehn, Deutsch. Arcli. f. klin. Med., 1912, CVIII, 187; Roubitschek, Ibid., 226; Foster, Am. Jour.Med. Sc, 1912, CX5L,III, 830; Editorial, Jour. Am. Med. Assn., 1913, LX, 287; Bloomfield and Hurwitz, Biill. Johns Hopkins Hosp., 1913, XXIV, 375; Hurwitz and Bloomfield, Ibid., 380; Isaac, Ztschr. f. physiol. Chem., 1914, LXXXIX, 78; Wagner Ztschr. f. klin. Med., 1914, LXXX, 174; Hatiegan, Wien. klin. Wchnschr., 1914, XXVII, 358; Fleckseder, Ibid., 475; Arai, Deutsch. med. Wchnschr., 1914, XL, 792; Worner and Reiss, Ibid., 907; Maliwa, Med. klin., 1914, X, 762; Friedman and Strouse, Arch. Int. Med., 1914, XIV, 531; HofEmann, Ztschr. f. exper. Path. a. Therap., 1914, XVI, 337; Schede, Jahrb. f. Kinderhkde., 1915,'LXXXII, 45. ^ Konigsfeld (Biochem. Ztschr., 1912, XXXVIII, 310) shows that the HCl must not have a final concentration greater than 12.3 per cent, and that the heating should not be longer than 20 to 30 seconds. If dextrose, aso, be present in amount greater than 2 per cent., the possibility of its conversion into levulose mustjje remembered. See, also, Jolles, Ibid., 1912, XLi, 331. Digitized by Microsoft® 2 Ztschr. f. physiol. Chem., 1902, XXXVI, 227; Ber. d. d. chem. Ges., 1902, XXXV, 959; Ibid., 1904, XXXVII, 4616. THE tTRINE 307 with methylphenylhydrazin, while no such compound is obtained with glucose mannose, or glucosamine. The test is, therefore, the most reliable and scien- tific one for the presence of levulose, although it is not clinically so acceptable as the Seliwanoff reaction. The formation of this osazone occurs according to the following equation. CH2OH - (CH0H)3 - CO - CH2OH + 2NH2 - N(CH3) (CsHs) = CH2OH - (CH0H)3 - C CH + 2HO2 + 2H ■ II II (CH3) (C6H6)N - N N - N(CH3) (CeHs) Technic. The urine is acidified with acetic acid and boiled to remove albumin if present. The mixture is then filtered and the clear filtrate, which must be acid, is evaporated in a vacuum, at a temperature not over 40°, to a thin syrup. The reaction must remain acid during the evaporation so that a drop of acetic acid may be added if necessary. The residue is thoroughly extracted with 98 per cent, alcohol, using an amount of alcohol equal to one-half the original volume of urine. Filter and re-extract the residue with alcohol should any reducing action be observed in it. The alcoholic extracts are mixed and decolorized "with animal charcoal. A portion of the extract is ex- amined for its sugar-content by Fehling's test, all of the reduction being attributed to levulose. Methylphenylhydrazin is then added to the alcpholic solution, which should not measure over 30 c.c. The amount of the hydrazin to be added is in the proportion of 3 molecules for i of levulose or, in other words, for each gram of sugar a trifle over 2 grams of methylphenylhydrazin. The mixture is allowed to stand for a few hours in the cold and filtered if a precipitate forms. The filtrate is treated with 50 per cent, acetic acid, using the same amount of acid as of the methylphenylhydrazin, and sujficient alcohol is added to give a clear solution. The mixture is heated from three to five minutes or, preferably, allowed to stand at 40°C., for 24 hours in an incubator. Crystals will usually separate out at the end of this time, but if not they will appear on the addition of a few drops of water. The crude product is purified by recrystalhzation either from a mixture of chloroform and" petroleum ether or from hot water to which pyridin has been added. The yield by this method is 81 per cent, of the total sugar if pure solutions are used, while from urine it is but 50 per cent. The crystals of methylpkenylosazone are delicate yellow, long, fine, needles melting from 158 to i6o°C. This method can hardly find application in, the hands of the general practitioner. Pure levulosuria is recognized by the levorotation of the urine, which possesses reducing properties and is capable of fermenting with yeast. After fermentation, the urine loses its reducing and optical properties. The presence of a levulosuria is indicated by a considerable difference between the results obtained by titration and polarization, providing a glycosuria be coexistent. The Seliwanoff reaction should be used as a routine in every case which shows both fermentation and reduction. The general practitioner Digitized by Microsoft® 308 DIAGNOSTIC METHODS will rarely have access to a polariscope and even then may be misled if other interfering substances, such as albumin, glycuronic acid, and j3-oxybutyric acid be present. Do not presuppose that glucose is the only fermentable and reducing sugar of the urine, as the recognition of the true condition may make much difference in the treatment as well as in the prognosis. (c) Pentose. The pentose^ group of carbohydrates comprises eight possible stereo- isomers with the general molecular formula of CgHioOs. Three members of this group, rhamnose, fucose, and chinovose, are substituted pentoses, viz., methyl pentoses having the formula C6H12O6. These latter are the excep- tions to the general rule that carbohydrates contain the same number of car- bon atoms as of oxygen atoms, and have led to the more scientific method of classifying the carbohydrates according to the number of oxygen atoms rather than of carbon atoms. The pentoses are widely spread throughout the vege- table kingdom in the form of their anhydrids or in combination with other groups of atoms. They are also found as constituents of the nucleoproteins of animal tissues, being especially abundant in the pancreas. It has been found that the ingestion of large amounts of pentose-contain- ing food, such as apples, cherries, plums, beets, and the leguminous vege- tables, leads to the excretion of pentose in the urine. This alimentary type of pentosuria is characterized by the presence of optically active xylose or arabi- nose and appears after the ingestion of small amounts, in some cases follow- ing an intake of as low as 50 mg. of the pure carbohydrate. In true didlbetes mellitus the urine frequently contains 1-xylose which probably arises from the breaking down of the pancreatic nucleoprotein. Alfthan states that the pentoses are constantly present in diabetic urine, so that it is highly probable that these sugars might be found whenever search was made for them in diabetic conditions. It is not strange that these carbohydrates are not more frequently reported, as their reducing action leads to confusion unless con- trolled by fermentation methods. The excretion of pentoses in diabetic conditions is not necessarily increased in direct proportion to the intake, as their absorption may be so slow that accumulation is not possible before oxidation has occurred. Salkowski and Jastrowitz^ reported in 1892 the finding of a pentosuria which was not associated with intake of pentose food nor with diabetes mellitus. This type is known as idiopathic, essential, or intrinsic pentosuria, of which 24 cases were found by Janeway^ up to 1906. The pecuUar thing of this type of pentosuria is that the sugar excreted is r-arabinose, an optically inactive pentose. This is the single exception in which an optically inactive pentose is found in all nature. This fact characterizes this ;type of; pen- tosuria as an anomaly of metabolism sui generis. The origin of this urinary pentose is still unsettled. The source must be within the organism, as no inactive arabinose is taken as food, and if it be given in experimental cases it appears in the urine as d-arabinose. Moreover, it cannot be derived from ' See Bendix, Die Pentosuria, Stuttgart, 1903. ^ Centralbl. f. d. mad. Wissensch., 1802, 337. " Am. Jour. Med. Sc, igcBj^K^SWy^MlCrOSOft® THE URINE 309 the nucleoprotein as the pentose in these cases is l-xylose.^ Neuberg sug- gests that galactose might be considered the source of this pentose, but no proof of this has been forthcoming. In the true idiopathic pentosuria the assimilation of other carbohydrates is unchanged and does not influence in any way the excretion of r-arabinose, although the active types of this pentose may be excreted at the same time in the urine. It is interesting to find that the pentoses taken in as food are excreted in different proportions by the diabetic and nondiabetic subjects. Thus von Jaksch observes that diabetics excrete from 49 to 82 per cent, of arabinose of the food and nondiabetics i to 47 per cent., while nondiabetics excrete from 19 to SS per cent, of xylose and diabetics only a trace. The amount of pentose excreted in essential pentosuria has been reported as vary- ing between 0.08 and i per cent. Neuberg has recently shown that a certain amount of the r-arabinose is combined with urea in the form of a ureid, which does not reduce Fehling's solution until it undergoes hydrolysis with acid. For this reason he beUeves that the ainount of pentose reported is in practi- cally aU cases 100 per cent, too low. Luzzatto^ has reported the excretion of the optically active 1-arabinose entirely independent of the food intake. The pentoses reduce copper solutions as do other carbohydrates, but the reduction is much slower, appearing during the cooling of the fluid. Ten c.c. of Fehling's solution are reduced by 0.0542 gram of pentose. They do not ferment with yeast and do not give a typical reduction with the Alm6n- Nylander test, the color being a gray rather than a black. In the true idio- pathic pentosuria no reaction is observed with the polariscope, while in the alimentary type a slight dextrorotation is usually noted, although von Jaksch reports the excretion of an inactive arabinose after the ingestion of active pentoses. These pentoses form more or less typical osazones which melt between 157 and 160°, but the reaction is not so easily produced.' These pentosazones are readily soluble in warm water and show dextrorotation. The most characteristic chemical property of these types of carbohydrate is the formation of f urf urol(C 6H4O 2) when they are distilled in the presence'of acids . The color reactions given below are based upon the production of furfurol and the formation of distinct colorations on treatment with various reagents. ToUen's Test. A few c.c. of cdncentrated hydrochloric acid are saturated with phloro- glucin, care being taken to leave a small amount undissolved. This solution is then divided into two equal parts, to one of which is added 1/2 c.c. of the suspected urine and to the other 1/2 c.c. of normal urine. Both tubes are then placed in a boiling water-bath for a few minutes, when an intense red zone will appear in the upper portion of the tube if pentose be present. After a few moments this red color will gradually spread throughout the fluid, while the ' Elliott and Raper (Jour. Biol. Chem., 191 2, XI, 211; Ibid., 1913, XV, 481) suggest the possibility of this sugar being ribose. 2 Beitr. z. chem. Physiol, u. Path., 1905, VI, 87. ' Klercker, Deutsch. Arch. f. klin. Med., 1912, CVIII, 277; Levene and La Forge, Jour. Biol. Chem., 1913, XV, 481; Ibid., 1914, XVIII, 319; Rosenbloom, Jour. Am. Med. Assn., 191S, LXiv, S08. Digitized by Microsoft® 3IO DIAGNOSTIC METHODS control urine shows no marked change in color. It is advisable to remove the tubes from the water-bath as soon as the color appears, as the clearness of the reaction is interfered with by prolonged' heating. The coloring matter is then extracted by shaking with amyl alcohol, when spectroscopic examina- tion will show an absorption band between D and E. This test reacts in the same way with glycuronic acid so that it has little value in differentiating pentose from the former substance. As a rule, the free glycuronic acid is not so easily split from its conjugated compound as is furfurol from pentose, so that the test is at least suggestive of pentose. Orcin Test. For this test the urine should be decolorized by heating with animal char- coal and filtering. Five c.c. of urine are treated with an equal volume of con- centrated hydrochloric acid and a few crystals of orcin are added. The mix- ture is then warmed approximately to the boiling-point, when a dark green color appears in the presence of pentose or glycuronic acid. The formation of a greenish-blue precipitate is very strong evidence of pentose rather than glycuronic acid. The pigment is then extracted with amyl alcohol, when spectroscopic examination shows a characteristic absorption band between C and D. The presence of glucose may interfere with the reaction, so that it may be necessary to remove it by fermentation. Bial has modified this test in such a way that glycuronic acid is less apt to be a disturbing factor. His reagent consists of 500 c.c. of 30 per cent. HCl to which are added i gram of orcin and 25 drops of 10 per cent, ferric chlorid solution. Four to five c.c. of this reagent are heated to boiling and removed from the flame. The suspected urine is then added drop by drop, not ex- ceeding I c.c. in all, when a green color should appear almost immediately if pentose be present. The heat employed is hardly sufficient to split off glycuronic acid. Here also glucose, if present, should be removed by fermen- tation with a pure culture of yeast rather than with compressed yeast, as the bacteria possibly present in yeast may break up the.pentoses at the same time. The osazone may be formed as previously given under Glucose. The glucosazone is separated from the pentosazone by digesting with water not over 60° in temperature, the pentosazones being dissolved. If the pentosa- zone be treated with 20 c.c. of water and 5 c.c. of concentrated hydrochloric acid and distilled, the distillate wiU give a beautiful test with Bial's reagent, which absolutely eliminates glycuronic acid and other interfering substances possibly present in diabetic urine. Quantitative Determination. Neuberg and Wohlgemuth^ have introduced a method by which the ara- binose of the urine may be accurately determined. A preliminary determina- tion of the sugar present is made by Purdy's solution, eliminating glucose by previous fermentation. If less than i per cent, of reducing sugar, which is assumed to be arabinose, is present, the urine must be concentrated in a vacuum so that the sugar content is slightly over i per cent. 1 Ztschr. f . physiol. OvtrrQi^iil^S^Wfpr&^ft® THE URINE 3" Technic. One hundred c.c. of urine are acidified with two drops of 30 per cent, acetic acid and evaporated on a water-bath to approximately 40 c.c. It is then treated with 40 c.c. of 96 per cent. a|cohol, the mixture is allowed to stand for two hours, and is then filtered from the separated urates and inorganic salts. The residue is carefully washed with 40 c.c. of 50 per cent, alcohol. To the filtrate 1.4 grams of pure diphenylhydrazin are added and the mix- ture heated on a boihng water-bath for one-half hour, the alcohol being re- placed as it evaporates. The mixture is allowed to stand for 24 hours and is filtered through a Gooch filter, using the mother liquor to transfer the precipi- tate. The crystals are then washed with 30 c.c. of 30 per cent, alcohol, and the Gooch with its contents dried at 8o°C. to constant weight. The amount of arabinose is obtained by multiplying the weight of the diphenylhydrazone by 0.4747 or by dividing by 2.107. Canunidge's Reaction. Cammidge^ has found that the urine in cases of pancreatic disease contains a substance which gives an osazone when treated with phenylhydrazin. In his earlier work he was led to believe that this substance was possibly glycerin or a derivative. He advanced two reactions, the first of which he found to be due to a mixture of glycuronic acid and a true carbohydrate, while the second was apparently due only to the glycuronic acid. In his later studies on this reaction he was led to believe that the mother substance of the osazone was a pentose (probably xylose) split off from a d&xtrin-like body by the hydrolysis with hydrochloric acid. This view has been somewhat substantiated by others. The careful work done in recent years by Kinney,^ Whippel and King,' Whipple, Chaffer and Fischer,* Swan and Gilbridge,^ Wilson,^ and Roper and Stillman^ has proven conclusively that, although this reaction is given in some cases of pancreatitis, it is not pathognomonic as it occurs in many other conditions and may not, even, be present in pancreatic disease.^ This test is not to be regarded, therefore, as of much diagnostic value. Technic. The writer gives only the improved method known as "Reaction C" referring to the original work of Cammidge for reactions A and B. The urine to be tested should be a portion of the 24-hour specimen and must be freed from glucose and albumin by methods previously outHned. Forty c.c. of clear, filtered acid urine are acidified with 2 c.c. of concen- trated HCl and boiled for 10 minutes. The mixture is then cooled and made 'Lancet, 1904, I, 782; Ibid., 1905, II, 14; Robson and Cammidge, Surgery of the Pan- creas, London, 1907. See Cammidge and Howard, Lancet, 1914, II, 791; Pekelhering and van Hoogenhuyze, Ztschr. f. physiol. Chem., 1914, XCI, 151; Lameris and van Hoogen- huyze, NederL Tydschr. v. Geneesk., 1914, II, 675. 2 Am. Jour. Med. Sc, 1910, CXL, 878. ' Bull. Johns Hopkins Hosp., 1910, XXI, 196. * Ibid., 1910, XXI, 339. ' New York Med. Jour., 1910, XCI, 781. " Trans. Assoc. Amer. Phys., 1910. ' Arch. Int. Med., 1911, VII, 252. ' See Mayesima, Mitt. a. d. Grenzgeb. d. Med. u. Chir., 1912, XXV, 403; also, Karas, Ztschr. f. kiin. Med., 1913, i&:§tYi^d^y Microsoft® 312 DIAGNOSTIC METHODS up to 40 c.c. with distilled water. The excess of acid is then neutralized by the addition of 8 grams of lead carbonate and the mixture cooled if neces- sary. Filter off the resulting precipitate and treat the filtrate with 8 grams of powdered tribasic lead acetate to remove the glycuronic acid. Filter, treat filtrate with 4 grams of powdered sodium sulphate, heat to the boihng- point, and allow to cool. The lead sulphate is removed by filtration. Ten c.c. of the clear filtrate are made up to 17 c.c. with distilled water, 0.8 gram of phenylhydrazin, 2 grams of sodium acetate, and i c.c. of 50 per cent, acetic acid are added and the mixture boiled for 10 minutes. Filter while hot and make the filtrate up to 15 c.c. with warm water. The mixture is allowed to cool, when yellow crystals arranged in sheaves and rosettes may be observed under the high-power lens. (d) Lactose (C12H22O11). Lactose is found in the urine of women during the period of lactation and may be found in patients who have been on an exclusive milk diet for a long period. A distinct type of alimentary lactosuria is observed on account of the low assimilation limit for milk-sugar. In breast-fed children with gastrointestinal disturbance lactose associated with galactose may be found in the urine. In this case Langstein and Steinitz^ have shown that the excre- tion is not due to failure of the normal enzyme, but to an unknown derange- ment of the activity of lactase, which renders it incapable of splitting up the whole of the lactose, the remainder being absorbed unchanged. From the portion which is split up in the bowel the resulting easily assimilable glucose is utilized by the organism, while galactose partly escapes by the kidneys, • on account of the much lower assimilation limit for this latter carbohydrate. Lactose in these cases is usually associated, therefore, with galactose (Neu- berg). According to the work of Voit, an increase of lactose in the diet of a diabetic is associated with an increased output of glucose. The usual form of lactosuria is that observed in the parturient female. It is ordinarily first seen a few days after delivery of the child, but occasion- ally appears during the latter days of gestation, as Ney, Lemaire, and Porcher have shown. The amount of lactose excreted by the nursing mother equals 2 to 3 per cent., according to Naunyn, while McCann places the average at 0.3s per cent, for the first few days of the puerperium. Lactose may continue in the urine for some time, the actual amount depending upon the quantity of milk as well as its quality.^ If nursing is interrupted for any reason, more lactose will be found than when nursing is regular. Lactose reduces copper solutions, although somewhat less actively than does glucose. It also shows a positive Almen-Nylander reaction. It has a strong rotatory power, its specific rotation being practically the same as that of glucose (-\- 52.5°). It does not ferment with yeast; although bacteria if present may hydrolyze it into its constituents, glucose and galactose, the former of which will show fermentation. It is, therefore, advisable when ^ Beitr. z. chem. Physiol, u. Path., 1906, VII, 575. ' See Gronvall, Biochem. Ztschr., 1912, XL, 14s; Rosenbloom (Jour. Am. Med. Assn., 1915 , LXIV, 508) reports the finding of lactose in the urine of an adult male after intake of milk. Digitized by Microsoft® THE URINE 313 applying the fermentation test not to judge of a reaction which has progressed longer than a few hours. With phenylhydrazin it forms a lactosazon which appears in the form of sheaves of delicate curved needles much resembUng bunches of yellow thread. These crystals melt at 2oo°C. The test is not easily obtained unless the urine be concentrated to a small bulk and the residue extracted with alcohol, when the alcohol is evaporated and this residue taken up with water and the phenylhydrazin test then appUed. Rubner's Test. Ten c.c. of urine are treated with an excess (3 grams) of lead acetate and boiled for a few minutes.^ The yellowish or brown solution is then filtered and ammonia added to the filtrate until a slight permanent precipitate remains. An intense brick-red fluid is obtained which later shows the depo- sition of a cherry-red precipitate with a colorless supernatant fluid. This test is not very deUcate as it shows lactose only when present in amounts varying from 0.3 to 0.5 per cent. Glucose gives with this test a red solution, but a more distinctly yeUow precipitate. Lactosuria is to be assumed when the urine possesses reducing properties and dextrorotation, but is incapable of fermenting with ordinary yeast within 12 hours. If the urine be boiled with 2 per cent, sulphuric acid and then neu- tralized, its optical activity will be increased and it will be capable of under- going fermentation. It is to be remembered in testing for the amount of lac- tose by the use of Fehling's solution that 10 c.c. of this solution are reduced by 0.0678 gram of lactose instead of by 0.05 as in the case of glucose or levulose. (e) Maltose (C12H22O11). Maltose has occasionally been reported in the urine, although many of the cases are questionable as the proper identification of the sugar was not thoroughly carried out. The most reliable cases appear to be those of Noble, von Ackeren, Rosenheim and Flatow, and especially that of Magnus-Levy. In this latter case the urine showed a considerable excess of rotation when compared with its reducing power. After inversion with dilute acid, by which each molecule of maltose was converted into two molecules of glucose, the rotation diminished apd the reduction increased, so that the polarimetric and titration methods gave concordant results. Tl\e urine underwent com- plete fermentation with synchronous loss of optical activity and of reducing power. Calculations founded on these determinations showed that 1.5 per cent, of maltose and 2 per cent, of glucose were present. This seems to be a case in which the amount of maltose excreted exceeds all records (Neuberg). The cases in which maltose appears in the urine seem to be those of disease of the pancreas, especially those with interstitial lesions. Maltose reduces copper solutions, but not as strongly as does glucose. Ten c.c. of Fehling's solution are completely reduced by 0.0807 gram of mal- tose. It is much more strongly dextrorotatory than glucose and forms an osazone which crystallizes in large prism-like needles arranged in sheaves, and melts at 207°C. This osazone is soluble in water and shows a dextrorotation, it being more distinctly identified by determination of the nitrogen content, 1 See Cole, Biochem. JouQ/gjft'fe^Igy fl^^jflteojipigerson, Ibid., 1915, IX, 243. 314 DIAGNOSTIC METHODS which should equal 10.6 per cent. Maltose ferments with yeast only after in- version by heating with acid, the splitting products being two molecules of glucose. Other carbohydrates, such as dextrin, isomaltose, and saccharose, have been reported in the urine. These are extremely rare and need httle comment in this place. In the case of cane-sugar the assimilation limit is so high that an alimentary saccharosuria could occur only after an enormous intake. Spontaneous excretion of cane-sugar has never been actually proven, but this sugar may be found in the urine of hysterical patients who have added it to deceive the physician. The so-called animal gum, first isolated by Land- wehr, seems to be a normal constituent of urine. Alfthan' finds it is present in practically every case of diabetes to the extent of i to 37 grams per diem. This substance is probably not a definite chemical body, but a mixture of several. Little is known of its chemistry. Inosite was regarded for a long time as a carbohydrate, but it is now known to be a hexaoxyhexahydrobenzol with the formula C6H6(OH)6. This substance, has, therefore, nothing to do with true carbohydrate metabolism, but is discussed at this point as it has so long been regarded in this connection. Inosite enters into the composition of almost all animal tissues and occurs both in the optically active and inactive forms. A physiologic excretion of inosite is not infrequent, according to Hoppe-Seyler. It may occur in neph- ritis, diabetes mellitus and insipidus, and after a large intake of animal food. if) Glycuronic Acid (CHO— (CH0H)4-C00H). Glycuronic acid is an intermediate product of the oxidation of carbo- hydrate, the CH2OH group being converted into CHO while the original CHO group is oxidized into COOH. This acid still retains the aldehyd group, in consequence of which it shows the same reducing action as does glucose. It seems to be characteristic of glycuronic acid that, when pro- duced naturally, it is never found in the free state, but only in the combined form as the conjugated glycuronic acid. It seems to be especially capable of combining with substances showing alcoholic or phenolic characteristics.^ The free glycuronic acid may be split off from its conjugated compounds by heating with acid and other hydrolyzing agents. The conjugated glycuro- nates are levorotatory while the free acid shows dextrorotation. Among the substances with which glycuronic acid combines we find chloral hydrate, butyl chloral hydrate, chloralamid, camphor, menthol, carbolic acid, resorcin, acetanihd, antipyrin, phenacetin, pyramidon, sandal-oil, morphin and cocain. The normal metabolism following intake of any of these substances is such that excretion of conjugated glycuronic acids will follow and may, therefore, lead to the assumption of sugar in the urine unless precautions are taken properly to differentiate these compounds. Besides conjugated gly- curonic acid of the above type we find a combination of vrea with glycuronic acid as well as a certain amount of indoxyl, skatoxyl, phenol, and cresol in combination with this acid. Most of the products of bacterial decomposition I'Ueber dextrinartige Substanzen in diabetischen Ham, Helsingfors, 1904. Cammidge THE UEINE 315 in the intestine are excreted in combination with sulphuric acid, but some is invariably present as a conjugated glycuronate. The origin and formation of glycuronic acid within the system is not entirely understood. It has been supposed to be derived from protein as especially advocated byLoewi, but Mayer has rather disproven Loewi's work, and shows that probably glycuronic acid is a direct derivative of glucose and that all carbohydrate oxidation must pass through the intermediate stage of glycuronic acid. The status of this question is very well summed up by Neuberg as follows: The formation of glycuronic acid out of protein is by no means excluded, nor yet from fat; but as it is difficult to eliminate the direct formation of gly- curonic acid or its secondary development from previously existing grape sugar, Mayer justly contends that the question of glycuronic acid forma- tion from these substances is practically included in the broader question of the formation of sugar from fat and protein. The exact point of conjugation of glycuronic acid is unsettled. It has been assumed by some to occur in the liver, while others find that the liver plays no part. It is probable that the synthesis takes place in various parts of the organism. It has been shown that the output of glycuronic acid may be increased in diabetes melUtus, in mild cases the unoxidized sugar being present largely in this form. Mayer^ advances the hypothesis of incomplete oxidation of sugar to explain its appearance in these cases.'' He shows that after the ad- ministration of glucose in amounts beyond the assimilation limit, an occa- sional excretion of glycuronic acid occurs with an equivalent diminution of the ethereal sulphates. It is possible that the substance conjugating with glycuronic acid is unknown and that we have the same results as though similar substances werfe introduced by mouth. As Mayer has advanced no direct proof of the correctness of his theory, the decision must be left for the future. Clinically, the question of the highest importance is whether the excretion of glycuronic acid is of any diagnostic value and whether it is of prognostic significance in diabetes. It does not seem wise to assume that a patient showing an occasional increase in the glycuronic acid excre- tion, which cannot be accounted for by intake or increased production of conjugating substances, will in the future show typical diabetes. Edsall does not beheve in the value of glycuronic acid in the diagnosis of a latent diabetes, nor does Neuberg regard an increased excretion of glycuronic acid as the origi- nal derangement which may determine other deviations from health.' Very few of the conjugated glycuronates show reducing action when treated with copper solutions using the precautions previously laid down. The chloral and camphor compounds are much more apt to produce tj^ical reduction, but even these require heating for somewhat longer periods than ' Ztschr. f. physiol. Chem., 1901, XXXII, 518; Berl. klin. Wchnschr., 1903, XL, 292 and 514. See Biberfeld, Biochem. Ztschr., 1914, LXV, 479; Roger and Chiray, Bull. I'Acad. de Med., 1915, LXXIII, 444. ^ See Conzen, Ztschr. f. klin. Med., 191 2, LXXV, 426. ' See Medigreceanu (Jour. Exper. Med., 1913, XVIII, 259) for a. discussion of the glycuronic acid excretion in ©jgjfibset^ MfSt^^ft® 3l6 DIAGNOSTIC METHODS does glucose. A diagnosis of a glycuronic acid excretion is based upon the following points: The fresh urine is levorotatory, but shows little or no reduc- ing properties and does not ferment. This same finding will be observed if /3-oxybutyric acid be present so that a diagnosis may not rest on these findings alone. After being boiled with dilute acid for a period varying from one- quarter to three-quarters of an hour, the levorotation is changed to dextroro- tation and the urine shows strong reducing powers. Such tests will not be given by j3-oxybutyric acid. In some cases after heating the urine with acid the action upon light may remain levorotatory or the solution may be optic- ally inactive, on account of the fact that the conjugating substance may be levorotatory or that complete hydrolysis has not been effected. On heating the urine for some time with Bial's modification of the orcin test a positive reaction appears with the liberated glycuronic acid. This acid crystallizes with phenylhydrazin forming distinct yellow needles which melt at 114 to iiS°C. This test is, however, not readily obtained so that it is difficult to identify glycuronic acid by its osazone. It will be seen, therefore, that gly- curonic acid is differentiated from the pentoses largely by its levorotation when in the conjugated state or dextrorotation when free. ToUens (Ber. d. d. chem. Ges., 1908, XLI, 1788) has advanced a test which permits of a clear differentiation between glycuronic acid and pentose. To . 5 c.c. of urine add }4, cc. of a i per cent, alcoholic solution of naphthoresorcin and 5 c.c. of concentrated HCl. Warm over the free flame to boiling or place in the boiling water-bath for 15 minutes. Allow to stand for 4 minutes and then cool under running water. Add an equal volume of ether and shake vig- orously. On separating, the ether layer shows, in the presence of glycuronic acid, a violet to blue coloration. Spectroscopic examination reveals a sharp absorption band at the D line. The test is not given by pentose. (See ToUens, Ztschr. f. physiol. Chem., i9o8,LVI, 115; JoUes, Ibid., i9i2,LXXXI, 203; also, Neuberg and Schewket, Biochem. Ztschr., 1912, XLIV, 502.) Goldschmiedf^ has introduced a test which Mayerhofer^ believes of great value to the pediatrician, as it indicates intestinal derangement better than indican. One-half to i c.c. of urine is treated with 2 drops of a 15 per cent, alcoholic solution of a-naphthol. Carefully overlay 3 to 4 c.c. of concentrated H2SO4 with this mixture. A violet ring is observed, which changes to a dis- tinct emerald green on standing. In this test the urine must be free from ni- trites and nitrates. Neuberg's Test.^ Five hundred c.c. of urine are treated with sufficient sulphuric or phos- phoric acid to make the acidity from i to 2 per cent. This acidified urine is then heated in an autoclave for two hours at a temperature of iis°C. The mixture is then cooled, neutraUzed with sodium carbonate, acidulated with acetic acid, and filtered. Two hundred and fifty c.c. of the filtrate are mixed with a hot aqueous solution of 5 grams of parabromphenylhydrazin hydro- 1 Ztsclir. f. physiol. Chem., 1910, LXV, 389; 1910, LXVII, 194. ^Ztschr. f. physiol. Chem., 1911, LXX, 391. 2 Ber. d. d. chem. Ges., i9D;£S?Sgan£)^^ Shaffer has recently introduced a method for the determination of the total acetone, which is closely related to his method for estimating /S-oxybu- tyric acid and wiU be discussed in a later section. (b) Diacetic acid (CH3-CO-CH2-GOOH). This substance is derived from j3-oxybutyric acid and is the precursor of acetone. As a rule, if acetone be present in large amounts, diacetic acid is also present and indicates a much graver condition than does the mere pres- ence of acetone. If Fohn's contentions are true, most of what we now call acetone, is, in realty, diacetic acid, so that this latter substance should be tested for in routine urinary work. The remarks previously made regarding the appearance of acetone in the urine also hold for diacetic acid.^ This substance is very volatile and disappears from the urine in a relatively short time so that the tests should be applied to perfectly fresh specimens. Gerhardt's Test. To 10 c.c. of urine are added a few drops of 10 per cent, ferric chlorid solution. This is best added as long as a precipitate of phosphates occurs, these latter bodies being then filtered off. To the filtrate are added a few drops more of ferric chlorid solution when the urine shows a Bordeaux-red color in the presence of diacetic acid. This color appears cherry-red by transmitted light and purplish-red by reflected light. Unfortunately, however, this test is not specific for diacetic acid.^ A red color is observed in the presence of cyanates, formates, acetates, phenol compounds, salicylates, the conjugated glycuronates of phenacetin, antipy- rin, thallin, kryofin, and kairin, as well as meconic acid which may be excreted after intake of opium. If the urine be heated, diacetic acid decomposes more or less completely into acetone and carbon dioxid, so that the red color due to diacetic acid wiU either disappear or become much weaker, while that with the other substances mentioned is not affected by heat. This same disap- pearance of color is noted if the mixture be allowed to stand from 24 to 48 hours. Schreiber recommends filtration of the urine through animal char- coal, using about 10 grams of charcoal to 100 c.c. of urine. Antipyrinphe- nacetin, and kryofin are retained while sufficient of the diacetic acid passes through to permit of detection. The urine may even be acidified with sulphuric acid and extracted with ether. This ethereal solution, which con- ' See Dakin and Dudley, Jour. Biol. Chem., 1913, XVI, 515- 2 See Steensma and Koopi©^;/ij|^[gf:fejyi3l^^®50/i®neesk., 1914, LVIII, 800; Licht- witz, Berl. klin. Wchnschr., 1915, LII, 399. THE URINE 325 tains the dacetic acid, is then shaken with water and ferric chlorid solution added. A deep red color will be seen in the watery layer in the presence of diacetic acid. If the red color, on addition of ferric chlorid, be due to the presence of meconic acid, it will disappear on the further addition of stannous chlorid or of alkali hypochlorites, while that due to diacetic acid is unaffected. This test is, perhaps, more frequently used than any other test for diacetic acid in the urine, but is by no means as serviceable as the following. ' Arnold's Test. For the performance of this test two reagents are employed: (i) a solution consisting of i gram of paraamidoacetophenon, 100 c.c. of distilled water, and 2 c.c. of concentrated hydrochloric acid; (2) i per cent, sodium nitrite solution. Fifteen c.c. of urine are treated with a mixture consisting of 10 c.c. of solution i and 5 c.c. of solution 2 and one drop of concentrated ammonia added. In practically all urine, whether it contains diacetic acid or not, a brownish-red coloration is observed which changes in the absence of diacetic acid into yellow if the mixture be treated with an excess of concen- trated hydrochloric acid, while if diacetic acid be present the color changes to a beautiful purple on the addition of acid. If the mixture be shaken the foam also shows a distinct violet coloration. As this test is somewhat difficult in the presence of small amounts of diacetic acid, Lipliawsky^ has modified it as follows: Six c.c. of solution i and 3 c.c. of solution 2 are treated with the same volume of urine, a drop of ammonia is added and the mixture shaken, when it assumes a brick-red color. According to the probable contents of the urine in diacetic acid 10 drops to 2 c.c. of this mixture are treated with 15 to 20 c.c. of concentrated hydrochloric acid, 3 c.c. of chloroform and two to four drops of ferric chlorid solution. The test-tube is then closed with a cork and gently shaken for one- half to one minute. In the presence of traces of diacetic acid the chloro- form assumes a characteristic violet coloration, while in the absence of this acid the color is yellow or light red. This test is positive for one part in 400,000 of water. Acetone and j3-oxybutyric acid do not react with this test nor do the drugspreviously mentioned under the Gerhardt Test interfere. If the urine is highly colored it is advisable to filter through animal charcoal. (c) |3-0xybutyric Acid^ (CH3-CHOH-CH3-COO3H). This acid, the mother substance of the acetone bodies, is found in the urine in extreme cases of the conditions described under Acetone. The amount excreted may vary from traces to as high as 100 grams. Kiilz has reported an excretion of 225 grams in 24 hours. The free acid is practically never found in the urine, being excreted either as the ammonium, sodium, or potassium salt. These salts as weU as the free acid are levorotatory and may be detected by the polariscope after previous fermentation of the carbohydrates. In the condition known as diabetic coma a specific intoxication with /3-oxybutric acid is assumed as the causative factor. While this is undoubt- 1 Deutsch. med. Wchnsckr., igoi, XXVII, 151. ' The more scientific name for this body is 6-hydroxybutyric acid. See Sassa, Biochem. Ztschr., 1914, LIX, 362; Kennaway, Biochem. Jour., 1914, VIII, 230 and 355. Digitized by Microsoft® 326 DIAGNOSTIC METHODS edly true to a large extent it cannot be regarded as the only factor in diabetic coma, as administration of |8-oxybutric acid in large quantities will not neces- sarily lead to such a syndrome unless largely retained. We do undoubtedly have an acidosis which acts by ultimately depriving the tissues of fixed alka- lies and may be regarded, therefore, as of great importance in following a diabetic case. Were this acid, per se, accountable for the entire symptoma- tology, we should be able, by administering alkalies, to overcome the effect of the acid intoxication. In some cases such therapy is extremely beneficial, while in others it is practically useless, as it cannot influence the formation of the toxic bodies and may not increase their elimination. As previously stated, the ammonia output of the urine is an invaluable guide in following the course of an acidosis, especially in diabetes. One gram of ammonia (NH3) is equivalent to 6.12 grams of /3-oxybutyric acid, especially when in excess of the amount directly due to the food. To detect |S-oxybutyric acid follow the method of Hart.^ To 20 c.c. of urine add 20 c.c. of water and a few drops of acetic acid and boil until the volume is reduced to about 10 c.c. (to remove acetone and diacetic acid). To the residue add sufficient water to make the volume 20 c.c. and place 10 c.c. in each of two test-tubes. To one of these add i c.c. of hydrogen peroxid, warm gently and allow to cool. Apply Lange's test for acetone (see p. 320) and allow the tubes to stand for a few hours. No reaction is observed in the tube which contains no hydrogen peroxid, while a distinct red zone is seen in the other. Quantitative Determination. A large number of methods have been advanced for the determination of |S-oxybutyric acid in the urine. Among these we find the methods of Kiilz, Tollens, Wolpe, Magnus-Levy, Bergell, Stadelmann, and Darmstadter. While some of these are accurate under certain conditions, so many precau- tions must be taken that widely warying results may be obtained. On the other hand, the method of Magnus-Levy, which is undoubtedly very exact, requires 24 hours, while that of Bergell depends to a large extent upon the condition of the powder which is extracted with ether. Black's Method. This method^ is a modification of those of Magnus-Levy and of Bergell, but in the writer's hands has given much more satisfactory results. One hundred c.c. of urine are faintly alkalinized with sodium carbonate and eva- porated in a porcelain dish to one-third or one-fourth of the original volume. The residue is then concentrated to about 10. c.c. on a water bath in order completely to remove the diacetic acid. This is then cooled, acidified with a few drops of hydrochloric acid, and made into a thick paste with plaster of Paris. This mixture soon begins to set, when it is stirred and broken up with a glass rod. The porous mass is then transferred to a Soxhlet apparatus and extracted with pure ether for two hours. The ether extract is evapor- ated, the residue taken up with water, decolorized with bone-black and fil- 1 Am. Jour. Med. Sc, 1909, CXXXVII, 869. ^ Jour. Biol. Chem., 1908, V, 207. Digitized by Microsoft® THE URINE 327 tered perfectly clear. The filtrate is then made up to 25 c.c. and its rotation determined with a polariscope. In determing the amount of ;3-oxy- butyric acid from its rotation we must make use of the following calcula- tion. The specific rotation of the free acid is — 24.12 in a decimeter tube. One divison of the scale in the case of glucose equals 2 per cent, so that we may find the percentage of (3-oxybutyric acid by the following proportion: 2 : X :: 24.12 : 52.7. X = 4.37 per cent. By a similar proportion it may be found that one divison of the scale corre- sponds to 7.1 per cent, of sodium |8-oxybutyrate, whose specific rotation is -I4-3S- This method is just as exact as those of Magnus-Levy and BergeU and has the advantages that it is simpler, more reliable, and may be performed in shorter time. The greatest difficulty with this method arises in the deter- mination of the exact point of the scale at which the two portions of the polarimetric field are equally illuminated. Magnus-Levy has shown about a difference of J'lo of 1° in reading the polariscope amounts to about three grams per liter of j3-oxybutyric acid, so that great care must be used with the polariscope in this method as well as in all others in which it is applied. Shaffer's Method. This method'^ seems to the writer to the most desirable one that has been advanced. Shaffer and Marriott^ show that this method yields uni- formly about 90 per cent, of theoretical values. The results obtained must, therefore, be corrected by the addition of 10 per cent, of the amount found. See, also, Pribram' in this connection. The principle of the method is the oxidation of /3-oxybutyric acid to ace- tone and carbon dioxid and the determination of the amount of acetone thus evolved. The acetone and diacetic acid already existing as such in the urine are previously determined by this method, so that it serves as a gen- eral one for the acetone bodies. From 25 to 250 c.c. of urine, depending upon the amount of |3-oxybutyric acid expected, are measured into a 500 c.c. volumetric flask and an excess of basic lead acetate and 10 c.c. of concentrated ammonia are added. In se- lecting the amount of urine to be taken one should use sufficient to yield from 25 to 50 mg. of acetone derived from j3-oxybutric acid. The solution in the flask is then diluted to the graduating mark, is thoroughly shaken, and filtered. Two hundred c.c. of the filtrate (representing 2,^ of the original volume of urine taken) are diluted with water to 500 to 600 c.c. 15 c.c. of concentrated sulphuric acid and a few grams of talcum are added, and the mixture distilled until 200 to 250 c.c. of distillate collects (distillate A). In this distillation, the distilling flask, which may be an 800 c.c. Kjeldahl flask, should be fitted with a dropping funnel and water run in to prevent the volume of fluid in the flask from becoming less than 400 c.c. This distillate (A) contains the performed acetone and that from the diacetic acid 'Jour. Biol. Chem., 1908, V, 211. ^ Jour. Biol. Chem., 1913, XVI, 265. ' Ztschr. f. exp. Path. u. Therap., 1912, X, 279 and 284. Digitized by Microsoft® 328 DIAGNOSTIC METHODS as well as volatile fatty acids which may be present in the urine. To remove the fatty acids, especially formic acid, the distillate A is redistilled after addilig 5 c.c. of 10 per cent, sodium hydrate solution. This distillate (Ai) is then titrated with standard tenth-normal iodin and thiosulphate solutions as described in the Huppert-Messinger method for determination of acetone. The residue of urine and sulphuric acid from which A was obtained is again distilled, dropping in 400 to 600 c.c. of o.i per cent, to 0.5 per cent, potassium bichromate^ solution. This bichromate must not be added faster than the distillate collects unless the boiling liquid turns a pure green color, indicating that the bichromate is being used up more rapidly. When about 500 c.c. of distillate (B) have collected, 20 c.c. of 3 per cent. H2O2 are added to the dis- tillate together with a few c.c. of sodium hydrate solution and this redistilled. This second distillate (B2) is then titrated with tenth-normal iodin and thio- sulphate solution. One mg. of acetone represents 1.794 mg. of |8-oxybutyric acid. (4) Abnormal Pigments. (a) Blood Pigments. The principal blood pigment appearing in the urine is hemoglobin, whidh has been previously discussed under the heading of Protein in the Urine. Certain derivatives of this pigment are found, however, in conditions in which hemoglobin does not appear (see hemoglobinuria). Hematoporph3rrin. This is an iron-free derivative of hemoglobin and appears to be present in minutes traces in normal urine. Pathologically, it has been found incases of rheumatism, phthisis, Addison's disease, pericarditis, paroxysmal hemoglo- binuria, cirrhosis of the liver, exophthalmic goiter, croupous pneumonia, lead poisoning, syphilis, and many acute infectious diseases. Long-continued use of certain hypnotics, such as sulphonal,^ trional, and tetr9nal, is fre- quently associated with the appearance of hematoporphyrinuria. Urine containing hematoporphyrin is usually dark-red in color, but may vary from a brownish-red or port-wine color to a distinct Bordeaux-red. Hammarsten has shown that this color is not entirely due to the hematopor- phyrin, but partially to other abnormal pigments whose identity is not certain. In examining urine for the presence of hematoporphyrin the spectro- scopic method is practically the only one available. If this pigment be present in large amounts the urine may be directly examined with the spectroscope, showing the four bands of alkali hematoporphyrin discussed in the section on Blood. As this method is not always certain and not always easy of application, it would seem preferable to treat 50 c.c. of uJrine with 10 c.c. of 10 per cent, sodium hydrate solution. The precipitated phosphates 1 Cooke and Gorslin (Jour. Biol. Chem., 191 1, X, 291) have wisely advised the following modification. The residue of urine and sulphuric acid is diluted to 600 c.c. and 5 c.c. of a 5 per cent, solution of potassium bichromate are added. Distill down to 300 c.c. and add water through the dropping tubeto 500 c.c. Distill until 500 c.c. have been obtained. Ad- ditional s per cent, bichromate is run in, a few cubic centimeters at a time, whenever the distilling solution shows a greenish tinge. ^See Pfortner, Deutsch. med. Wchnschr., 1914, XL, 1563; Fischer, Ztschr. f.physiol. Chem., 1915, XCV, 34. Digitized by Microsoft® THE tJRINE 329 carry down the pigments. This precipitate is then treated with 10 drops of concentrated hydrochloric acid and 15 c.c. of absolute alcohol. The solution is filtered if necessary and examined with the spectroscope when the two absorption bands of acid hematoporphyrin will be observed. (6) Biliary Pigments. Normally, bile pigments do not occur in the human urine. As was previously discussed in the section on Urobilin, biliary pigments may appear in the urine in conditions interfering with the passage of the bile into the intestine or when increased formation of biliary pigments from blood.pigments has occurred and an associated obstruction of the bUe-ducts is present. We see, therefore, that choluria occurs in every case in which there is obstruction to the outflow of bile into the intestine. Thus in catarrhal jaun- dice, biliary calculi in the common duct, carcinoma of the liver, and cirrhosis, bUirubinuria is frequent. Moreover, we may also find biliary pigments aris- ing from purely hematogenous conditions, such as pernicious anemia, malaria, typhoid fever, arsenical poisoning, and yellow fever. Whether this latter t37pe is not really hepatogenous in origin, as Stadelmann believes, is still un- settled, but it would seem more plausible to assume a primary breaking down of the red cells and a secondary insufficiency of the liver. The chief biliary pigment found in the urine is bilirubin,' which is inter- mediate between hemoglobin and urobiUn. On oxidation of this pigment, either in the system or in the methods of examination, various other pigments may arise. Thus we may find biUverdin, bilicyanin, bilifuscin, bihprasin, cholecyanin, and choleteljn. In the fresh urine bilirubin is the only pigment noted. A bile-containing urine may show various shadings of color, ranging from a greenish-yellow, through yellowish-brown, deep brown, or greenish-brown, to a pure green. If the urine be shaken, a yellowish or greenish-yellow foam is observed, while in normal urines the foam is practically colorless. The presence of an excess of urobilin may also give a brownish foam. Urine con- taining bile always shows the presence of nucleoalbumin along with slight traces of serum albumin, so that bile should be regarded as a source of of extraneous albumin. The sediment will usually be more or less colored by the biliary pigment, the casts in yellow fever, for instance, being usually distinctly bile-stained. Qualitative Tests. A large number of tests have been advanced for the detection of biliary pigments in the urine and many of them are distinctly unsatisfactory, if large amounts of bile be present, the urine, acidified with HCl, may be shaken out w;ith chloroform which dissolves the bilirubin. If the chloroform be evaporated, rhombic crystals with rounded edges or distinct needles of a brownish-red color will be observed. These crystals will give the color tests mentioned below. * See van den Bergh and Snapper, Nederl. Tijdschr. v. Geneesk., 1915, II, 469, Kuster, Ztschr. f. physiol. Chem., 19 15, XCIV, 136.,,. „_ ^ ' ^iJJgitized By Microsoft® 33° DIAGNOSTIC METHODS Smith's Test. This test has been described under other names, as those of Trousseau, Kathrein, Rosin, and Marechalt. A few c.c. of urine, acidified if necessary with acetic acid, are treated with a i per cent, alcoholic solution of iodin in such a way that the latter solution is superimposed upon the urine, forming a distinct line of contact. If bilirubin or other biliary pigments be present, a beautiful emerald-green color is observed at the point of contact. This test is especially recommended, but it is not very sensitive, indicating only one part of biliary pigment in 10,000 of urine. Certain drugs, especially antipyrin, may lead to the formation of a green color with this test. Thymol, if used as a preservative, may give rise to confusion with this and other bile tests. Gmelin's Test. One or two c.c. of nitric acid areplaced in a test-tube and the same amount of urine is allowed to flow from a pipet in such a way that a distinct contact line is formed. If the nitric acid contains a trace of nitrous acid and the urine biliary pigment, a distinct green ring will be observed at the line of contact. In some cases, as the nitric acid oxidizes the pigment, a play of colors may be seen from green, through a blue, violet, and red, to a yellow. The primary green is the characteristic color, other colorations being occasionally due to pigments other than biliary. This test is supposed to indicate i part of bilirubin in 80,000 of urine. If the urine contains an excess of indican a deep-blue ring may be observed or the combination of this blue with the yellow of the urine may give a green. In the presence of skatoxyl a violet-red ring may be noted, while various me- dicaments may give colorations ranging through the entire spectrum. In such cases it is always advisable to extract the acidified urine with chloroform and apply the test either to the, evaporated residue or to its aqueous solution. This test is, perhaps, more frequently used than any of the biliary tests, but requires considerable experience for its proper interpretation. If the urine be diluted the test is somewhat more distinctive. Many modifications of this test have been advocated, the most serviceable being the following: Rosenbach's Test. A large quantity of urine, which has been acidified with HCl, is filtered severa'l times through a thick filter-paper, which will hold back the bile- stained elements of the urine. It is sometimes advisable to add a little milk of lime to the urine before filtering, instead of the HCl, as this will throw down the phosphates which will carry with them the biliary pigment. If the filter- paper and contents be dried by pressing with a second dry filter-paper and a drop of yellow nitric acid allowed to fall upon it, distinct rings will be seen, which will be colored as in the previous test, the green one being external. Nakayama's Test. This is a modification of the older Huppert test.'- Five c.c. of acid urine are teated with an equal volume of 10 per cent, barium chlorid solution and the mixture centrifuged. The barium chlorid precipitates the phosphates ' See Rosenbloom, New ^S^M£^^'^Bym%ybMW' "9- THE URINE 331 and sulphates and carries down the bihary pigments. The supernatant fluid is then poured off and 2 c.c. of the following reagent are added to the precipitate. The reagent consists of 99 c.c. of 95 per cent, alcohol, i c.c. of concentrated HCl, and 0.4 gram of ferric chlorid. If this mixture of precipi- tate and reagent be heated to boiling, a bluish-green or a brilliant green solution is obtained, which becomes violet or red on the addition of nitric acid. This test is said to indicate one part of bilirubin in 1,200,000 parts of urine. Hammarsten's Test. As the reagent in this test we use a mixture of one part of 25 per cent. HNO3 and 19 parts of 25 per cent. HCl. Before use one part of this reagent is mixed with four parts of alcohol. A few drops of the urine are added to this mixture, when it assumes a green color in the presence of biliary pigments. If the urine be treated as in Nakayama's test and the precipitate mixed with I to 2 c.c. of the acid alcohol reagent and the whole centrifuged for a short time, a green solution is obtained if one part of bilirubin in i, 000,000 of urine be present. Bile Acids. The bile acids, taurocholic and glycocholic acids, are found in the urine in the form of their sodium salts. They may be found in small amounts in the same conditions in which the biliary pigments are present, but their amount is usually so small that they cannot be detected, as a rule, without being previously isolated. It seems to be fairly well established that the bile acids must be present to the extent of J^ per cent, before detection in the urine is possible. As the clinical significance of these acids is the same as that of the pigment and as their amount is so small that the methods of isolation require a fairly large volume of urine, the writer will refer to works on physiologic chemistry for such procedures. No absolutely reliable test is known for the detection of the bile acids in the untreated urine. There are, however, a few tests which are occasionally given providing the bile acids be present in sufficient amount. Hay's Test. This test depends upon the reduction of the surface tension of the urine in the presence of the bile acids. As advocated by Beddard and Pembrey, a pinch of powdered sulphur is sprinkled upon the surface of urine which should be preferably at a temperature not over i7°C. In normal urines the sulphur will float upon the surface, while if the urine contains bile acids the sulphur may sink at once indicating one part in 10,000 or may sink only after a few seconds to one minute, in this latter case indicating one part in 50,000. According to Sahli this test does not discriminate between biliary acids and biliary pigments, but clinically it is a matter of indiffer- ence which one is present. Phenol or aniline compounds lower the surface tension of the urine so that their presence may lead to wrong conclusions.* 1 See Allen (Jour. Biol. Chem., 1915, XXII, 505) for a method of measuring accurately the variation in surface tension ci.the urine,,. „^ UigitizecTby Microsoft® 332 DIAGNOSTIC METHODS Oliver's Test. This test is based upon the well-known property possessed by the bile acids of precipitating peptone when in acid solution. The reagent is as follows : Powdered peptone, 8.33 grains Salicylic acid, 1.12 grams Acetic acid, 2 drops Distilled water, i liter One to two c.c. of clear filtered urine are placed in a test-tube and treated with s c.c. of the reagent. In the presence of bile acids a decided milkiness appears at once, being the more intense the larger the amount of bile acids. Other tests, such as those of Pettenkofer and Udranzky, are only service- able in the testing of the isolated bile acids. (c) Melanin. In cases of melanotic tumors the urine not infrequently contains a chro- mogen (melanogen), which is converted into melanin on allowing the urine to stand or after adding alkalies or oxidizing agents. The urine when freshly voided is normal in color, but on exposure to air gradually darkens until it becomes distinctly black. This coloration is first noticed in the upper portion of the urine and gradually extends toward the bottom. If ferric chlorid be added to the urine the coloration may be somewhat intensified so that one may assume an excess of phenol derivatives. It is to be remembered that indican must first be split up with acid before giving the coloration with ferric chlorid; moreover, melanin is insoluble in chloroform while the indigo is readily soluble. (See Eppinger.^) The addition of the ferric chlorid may produce a black precipitate which is soluble in sodium carbonate solution, from which it may be reprecipitated by mineral acids. This pigment may also be found in some cases of chronic malaria^ so that it is not absolutely pathogonomonic of melanotic tumors. {d) Phenol Derivatives. As previously mentioned in the discussion of the variations in color of the urine, it may be dark colored either on voiding or after standing. While such colorations may be due to melanin, they are much more frequently traceable to the presence of sulphuric acid in conjugation with phenol,' paracresol, pyrocatechin, and hydroquinone. These substances are excreted especially in conditions associated either with increased intestinal putrefaction or with putrefactive processes elsewhere in the system. Aside from direct poisoning with these substances, they may be regarded as having much the same clinical significance as indican. Qualitative and quantitative tests for these substances are rarely of clin- 1 Biochem. Ztschr., 1910, XXVIII, 181. ^ Urriola (Interstate Med. Jour., 1912, XIX, 74) claims that a urinary excretion of blood pigment (probably hematin and not melanin) is invariably present as a pathognomonic sign of malaria. This may appear in the centrifuged specimen as a very abundant intense black pigment; a blue pigment, very constant but small in amount; or as an ochre pigment occasionally. 'See Hensel, Ztschr. f. physiol. Chem., 191 2, LXXVIII, 373; also, Folin and Denis, Jour. Biol. Chem., 1915, XXII, 305 and 309; Siegfried and Zimmermann, Biochem. Ztschr. 191S, LXX, 124. Digitized by Microsoft® THE TJRINE 333 ical importance. They may be roughly determined by estimating the amount of ethereal sulphates in the iirine, when a large increase may be as- sumed to be referable to these bodies unless indican is greatly in excess. (e) Alkapton. In certain conditions of disturbed protein metaboUsm, the urine contains pigments which cause it to turn dark on the addition of alkali or on standing. These urines are normal in color when voided and become black almost im- mediately on the addition of alkali, whence the name alkapton bodies and alkaptonuria for their excretion in the urine. The substances causing this change of color are hydroquinon-acetic acid (as it has been prepared syn- thetically from gentisic aldehyd, it has been called homogentisic acid with the formula C6H3(OH)2CH2(COOH) and uroleucic acid, whose structure has not been absolutely settled. '^ The former of these acids is present in all cases of alkaptonuria, while the latter may be present in all cases, but in such small amounts that it remains undetected. Urines containing these bodies strongly reduce copper and ammoniacal silver solutions, while bismuth solutions are little affected. The so-called glycosuric acid of Marshall is probably identical with homogentisic acid and is not a definite chemical entity. This condition is observed at various periods of life and seems to be of theoretical more than practical interest, as of the 48 cases reported none ap- peared to be much disturbed in health, the condition usually being detected accidentally.^ The source of these substances is still uncertain. Baumann and Wolkow believe that the influence of specific bacteria in the intestinal canal upon the tyrosin, formed in the hydrolytic cleavage of the proteins, leads to direct formation of homogentisic acid. While this accounts for some of the abnor- mal substance, it cannot account for the entire amount of 3 to 7 grams of homogentisic acid excreted in the 24 hours. From the work of Meyer, Falta, Langstein and Wohlgemuth it would appear that this condition depends not on an abnormal formation of homogentisic acid but on an incapacity for further oxidizing it when formed. Dakin' believes that both factors play a part. However this may be, alkaptonuria has very few iU-effects, although transitory symptoms have been observed in occasional cases of diabetes, cirrhosis of the liver, tuberculosis, pyonephrosis and gastritis. The browning of the cartilage occurring in ochronosis is supposed by Albrecht and Zdarek to have some relation to the alkapton bodies, although Langstein could not show the alkapton acids in the urine in such cases. QuaUtative and quantitative tests for these acids are rarely necessary. The characteristic change of color on alkalinizing the urine is, at least, suggest- ive of homogenistic acid. Like all other hydroxy derivatives of benzol, this substance shows the Schiff reaction with ferric chlorid, producing a transitory ' See Morner, Ztschr. f. physiol. Cham., 191 2, LXXVIII, 306; Oswald (Ztschr. f. physiol. Chem., 1914, XCIII, 307) believes this acid is identical with homogentisic acid. See, also, Gross, Biochem. Ztschr., 1914, LXI, 165. ^ See Poulsen, Munch, med. Wchnschr., 191 2, LIX, 364; also, Baldwin, Am. Jour. Med. Sc, 1913, CXLV, 123. 'Jour. Biol. Chem., 191 1, IX, 151- See, also, Pincussohn, Ergeb. d. inn. Med., u. d. Kinderheilkde., 1912, VII, 4S^yP|ggrgh, ^^^-q'^^^'^K iQ'S, XLVIII, i. 334 DIAGNOSTIC METHODS blue coloration when present in amounts of i to 4,000. The reduction of copper solutions should not be mistaken for sugar, as homogenistic acid neither ferments nor shows any optical activity. The urine will usually show a relatively high acidity. As this substance is practically harmless in the system we should expect to find, as we actually do, no increase in the conju- gated sulphates or glycuronates nor any increase in the ammonia content of the urine (Neuberg). (/■) Ehrlich's Diazo Reaction. Under certain pathologic conditions the urine has been found to contain a chromogen which gives a deep red color to the urine when treated with diazo compounds and ammonia. This is known as the diazo reaction, the urinary substance causing it being somewhat uncertain. According to Bond- zynski, the alloxyproteic acid is the causative factor, but this needs confirma- tion, as Clemens has apparently shown that the body producing the reaction is sulphur-free. In the performance of this test two reagents are used: (i) sulphanilic acid 5 grams, 50 c.c. of concentrated hydrochloric acid, and 1,000 c.c. of water; (2) 3^ per cent, aqueous sodium nitrite solution. To a mixture consisting of 50 parts of solution i and one part of solution 2 is added an equal volume of the urine. The mixture is then shaken and about one-tenth of the bulk of ammonia added quickly, and the mixture thoroughly shaken. The ammonia may be added in such a way that a line of contact forms, this latter method frequently bringing out a much more beautiful reaction. According to Greene, a mixture consisting of 100 parts of solution I to one of solution 2 renders the test more delicate. Instead of sul- phanilic acid, paraamidoacetophenon may be used, as suggestedby Friedenwald . If the test be positive the entire urine will assume an intense red coloration or a colored ring will be observed at the point of contact between the ammonia and the mixture. With normal urine a distinct orange color may be observed. On shaking the mixture the foam will be more or less brilliant red in color, which is more characteristic than the red coloration of the mixture. On al- lowing the mixture to stand for 24 hours a green precipitate may be observed at the bottom of the tube, which Ehrlich regards as especially characteristic of the true diazo reaction. This green precipitate is not always present and is not necessary for a positive reaction, the red coloration of the mixture and of the foam being more frequent. The administration of certain drugs may markedly affect this test.^ Thus we find that naphthahn, chrysarobin, opium, and phenol derivatives give a reaction which is very similar to the true diazo reaction, but may be distin- guished, according to Wood, by the fact that color is more permanent in alka- line solutions, does not fade to any extent on the addition of a strong mineral acid, the foam is more yellow than in the typhoid reaction, and the green pre- cipitate does not appear on standing. Burghart has found that, following the administration of tannic acid, gallic acid, tannigen, and tannalbin, the 1 Skorczewski und Sohn (Wien. klin. Wchnschr., igii, XXIV, 1700) show that the urine of patients taking atophan gJ5f^,fi|ff |,f ^/p^OSO/^® THE URINE 335 diazo reaction disappears from the urine, the inhibiting effect being perhaps exerted upon the reagents used rather than upon the unknown factor which usually causes the coloration. According to Ehrlich, if the urine contains an excess of biliary pigment, a dark cloudy discoloration may occur, which is changed on boiling to a distinct reddish violet. He also finds on applying the diazo test in some cases that the urine and foam become yellow before the addition of ammonia. After ammonia is added the color changes to a lighter yellow. This reaction is known as Ehrlich's "egg-yellow" reaction, and is supposed to be due to the presence of urobilinogen. He regards this latter test as especially important in predicting the crisis of pneumonia. This diazo reaction is never positive in health. It was formerly regarded as pathognomonic of typhoid fever, but it has been shown to occur in many other conditions. It is true that in typhoid fever it may be present as early as the third or fourth day and may persist for some time, reappearing if a relapse occurs, while the Widal test, as will be remembered, does not vary with a relapse. Moreover, the intensity of this reaction is somewhat parallel with the severity of the case, while the Widal reaction may not be present in the severest types of typhoid fever. A positive diazo reaction occurs fre- quently in measles, somewhat less frequently in pneumonia, miliary tubercu- losis, scarlet fever, ^ diphtheria, and erysipelas, while in rheumatism and meningitis it is rarely obtained. Michaells believes that the presence of a positive diazo reaction in pulmonary tuberculosis indicates a progressive condition with a grave prognosis.^ This reaction is, therefore, a valuable aid in diagnostic work. As it is present in about 80 per cent, of cases of typhoid fever^ a negative reaction would not necessarily exclude typhoid nor would a positive reaction prove its presence. Its appearance in a relapse is of value, as the Widal test would give no information under such conditions. It is to be said, however, that the diagnosis should by no means rest upon this test. (g) Russo'sTest. This test' has been recently advanced and seems to have somewhat more diagnostic importance than has the diazo reaction. The technic is very simple and is as follows: Four drops of a i to 1,000 aqueous solution of meth- ylene blue are added to 4 or 5 c.c. of suspected urine. If the reaction be posi- tive the mixture turns to an emerald or mint-green hue. A light-green or bluish-green tint shows a negativereaction. The positive reaction is not affected by boiling the urine or by the previous ingestion of such compounds as phenacetin, salol, quinin, and calomel. The difficulty in the application of the test comes in the ability to recognize the various tints of green which may be present. With a little practice, however, a positive reaction may be readily detected, especially if a control test be made with normal urine. This test is shown as early as the second day of typhoid fever and persists 1 See Umber, Med. Klin., igi2, VIII, 322; Woody and Kolmer, Arch. Pediat., 1913, XXIX, 18; Marcantoni, Gazz. d.. osp., 1914, XXXV, 193; Jahrb. f. Kinderh., 1915, LXXXl' 168. ' Riforma Medica, igos.'^iCt'.'-^-.'-ilbdfi&A'^W.'^m. Med. Assn., 1905, XLV, 363. ' See Heflebower, Am. j^q^l^t^^^^^^ 336 DIAGNOSTIC METHODS throughout its course. The mint-green hue is first observed, the emerald- green tint appearing as the disease progresses. If the course is favorable the color tone becomes more and more bluish, while if unfavorable the emerald tint persists. This test is also given in measles, smallpox, chronic and suppurative tuberculosis, but is negative in varioloid, varicella, scarlet fever, miliary tuberculosis, appendicitis, and malaria. This test is as simple and just as reUable as is the diazo-reaction, being especially valuable in differentiating a t3^hoid from a miUary tuberculosis. Rolph and Nelson' have pointed out that urines containing bilirubin react to this test, so that this fallacy must be borne in mind. {h) Dimethylaminobenzaldehyd Reaction. This test, advanced by Ehrhch, is as follows: Prepare a 2 per cent, solution of p-dimethylaminobenzaldehyd in equal parts of concentrated HCl and water. Add a few drops of this solution to 5 c.c. of fresh cold urine and allow to stand for a few minutes. A positive reaction is indicated by the appearance of a cherry-red color, which may be extracted with chloroform or epichlor- hydrin. Heating facilitates the re- action, but here normal urine may give a slight reddish coloration. In the cold normal urine gives a greenish-yellow color. Apparently this coloration is due to the presence of metabolic prod- ucts derived from blood pigments (urobilinogen compounds, see p. 256).^ It is to be expected, therefore, that this reaction would be distinct in diseases of the liver and bile passages, although it is not constant eyen here. It is, also, observed in tuberculosis, pneumonia, typhoid fever, malaria and scarlet fever.' It has little clinical value. IV. Microscopic Examination or the Urine. The microscopic examination of the urine is important in every case. So varied are the elements which appear in the microscopic field that con- siderable experience is necessary before absolute interpretation can be made. Not infrequently the character of the sediment will change a diagnosis, as, ^ Medical Record, 191 1, LXXX, 373. See, also, Neuman and Behrend, Arch. Int. Med., 1913, XI, 456; da Pozzo, Gazz. d. osp., 1914, XXXV, 865. 2 Fischer and Meyer-Betz (Ztschr. f. physiol. Chem., 191 1, LXXV, 232) show that the reaction depends on the presence of a dipyrrylphenylmethane pigment of the class of non- stable pyrrol derivatives, to which group the blood and biliary pigments, as well as urobilino- gen, belong. ' See Hesse, Med. Klin., 1913, IX, 294; also, Steensma, Nederl. Tijdschr. v. Geneesk., 1914, LVIII, 467; Rabinowitsch, Berl. klin. Wchnschr., 1914, LI, 1456; Berkowitz, Med. Record, 1914, LXXXVI, 1087; Eisner, Deutsch. Ztsc^. f. Chir., 1915, CXXXII, 589; Robertson, Calif. State JouVigiiSi^^^y, /K«Hp«§nr©tzenberg (Chicago Gynec. Soc, Apr. 16, 1915) reports an increase of urobilinogen in the toxemias of pregnancy. Fig. 89.- — Purdy electric centrifuge. THE URINE 337 for instance, when pus- or blood-cells are present in sufficiently large amounts to account for an albuminuria previously determined by chemical methods. It is, therefore, essential that the microscopic examination of urine form a part of the ordinary routine. In obtaining the sediment of the urine for microscopic examination, two methods are possible. In the first place, the urine may be allowed to stand in a conical glass for periods ranging from 12 to 24 hours. The sediment originally present in the urine as well as that formed by chemical changes taking place during the standing will collect in the lowest portion of the glass and may be removed by a glass-tube drawn out to a somewhat small point. It is to be remembered that a sediment at the end of 12 to 24 hours may be entirely different from that originally present in the freshly voided specimen. The changes in the reaction of the urine will, necessarily, lead to the dissolving of certain types of crystals and to the formation of other varieties. Moreover, casts, if originally present, may dissolve or disappear as a result of the reaction of the urine becoming alkahne. For these reasons it is absolutely essential, in the use of this gravity method, that preservatives be added to the urine. Among the preservatives which may be used to pre- vent bacterial action during the sedimentation, we find a small piece of cam- phor or a rather large crystal of thymol serving the purpose. Some workers add one-fifth the volume of a 4 per cent, solution of borax which is equally useful, but the addition of chloroform or for- malin, does not serve as well in these cases, as the former does not completely preserve the casts and the latter introduces a crystalhne compound of formalin and urea which may be confusing, as it is not unlike impure leucin. This method is not to be recommended for routine work, as it requires too long a period and as changes may occur which make it ab- solutely impossible to differentiate between a primary and a secondary sediment. Secondly, the sediment may be thrown down by the use of the centrifuge. This ap- paratus is seen in the accompanying cut. By the use of this method no preservative is needed, a deposit is obtained within three min- utes in a much more concentrated form, and changes in the sediment do not take place. The writer would recommend, therefore, the use preferably of the electric centrifuge or, at least, of the type run by hand. Whatever method may be adopted for obtaining the urinary sediment, the next steps in the process are the same. A pipet, consisting of a glass tube drawn out to a point about one-half the diameter of the tube, is introduced to the bottom of the vessel containing the 22 - Digitized by Microsoft® Fig. 90. — Sediment tube for Purdy cen- trifuge. Fig. 91. — Per- centage centri- fuge tube. 338 DIAGNOSTIC METHODS sediment, a finger being placed over the upper end to prevent fluid passing into the tube as it is introduced. When the tip of the pipet comes in contact with the deposit, the pressure of the finger on the upper end of the pipet is removed and the deposit allowed to flow up into the pipet. The finger is then placed tightly over the tube as it is withdrawn from the fluid. By placing the tip of the pipet in contact with a perfectly clean slide which is absolutely free from scratches and by gradually rotating the pipet, a small portion of the sediment is obtained. A cover-glass is then placed upon this drop avoiding any undue pressure, which might distort the organized elements of the sediment. Some workers dispense with the cover-glass and use somewhat larger amounts of sediment, but the writer does not find this method as acceptable owing to the fact that the lens of the objective may dip into the fluid and thus give indefinite microscopic pictures. Moreover, the focus cannot be as accurately adjusted without the use of the cover-glass. • In the examination of the microscopic specimen, prepared as above, the point of special importance to be observed is the proper adjustment of the light. The writer is accustomed to use the low-power objective in the pre- liminary examination. In this case it is essential that the light be shut off to a large extent, as the recognition of casts cannot be made in a briUiantly illuminated field. This examination with the low power has the advantages that larger visual fields are subject to inspection, casts are easily recognized and crystalline deposits, as well as morphological elements, are usually differ- entiated. After the preliminary examination with the low power, the final examination is made with the high-power dry lens. In this way elements which appear suspicious under the low power are more clearly brought out and differentiations rnade possible between various types of cellular elements. With the high-power lens it is, of course, essential that the field be somewhat more illuminated than when the low-power is used. While a mechanical stage'is, at times, advantageous in the microscopic examination of the urine, the writer has found that the fingers serve practically every purpose in the manipulation of the slide under examination. Where the urine is to be examined for the presence of bacteria, stained specimens must be made and examined with the oil-immersion lens. Urinary sediments are classified into two divisions: (a) chemical or nonorganized and (b) anatomical or organized sediments. The nonorganized sediments exist in solution in normal urine and appear as deposits under conditions of excessive formation, excessive excretion, or of alterations in the urine affecting its solvent properties. The chief chemical sediments are uric acid and its salts, calcium oxalate, phosphates, sulphates, cystin, leucin, tyrosin, xanthin, fat, and fatty acids. The organized sediments are usually foreign substances and are not met with in normal urine. They consist of epithelial cells, pus corpuscles, blood-cells, renal casts, spermatozoa, infusoria, bacteria, and tissue fragments. (4) Unorganized Sediments. (a) Those Appearing in Acid Urine. (i) Uric Acid (C^J^^t)zed by Microsoft® This occurs as a sediment in the urine under 3 conditions: (i) great THE URINE 339 concentration, (2) high acidity, and (3) low temperature. The deposit differs from others in possessing a deep yellow or orange-red color, although some of the smaller crystals are occasionally colorless. The primary form of the uric acid crystal is that of the rhombic prism. Modifications of this, in the form of square plates, cubes, ovoids, dumb-bells, or whetstone crystals are sometimes noticed. A rare type, especially of the colorless crystals, is a per- fect hexagon which resembles cystin so closely that chemical means of differ- entiation must be used. The crystals may be single or grouped in rosettes or fan-shaped masses. Occasionally typical needle-shaped crystals may be seen which are arranged in sheayes. The microscopic* peculiarities of uric acid are usually such as to permit of its easy recognition. In some cases, however, it is wise to confirm the Fig. 92. — Various forms of uric acid, i, Rhombic plates; 2, whetstone forms; 3, quad- rate forms; 4, s, prolonged into points; 6, 8, rosettes; 7, pointed bundles; 9, barrel forms precipitated by adding hydrochloric acid to urine. (Hawk.) microscopic finding by the murexid test as follows: Place a small quantity of the sediment in an evaporating dish and add a few drops of concentrated nitric acid. Evaporate on the water-bath to dryness, when a yellowish or reddish residue will remain. Allow the residue to cool and add a few drops of ammonium hydrate solution. In the presence of uric acid a distinct reddish purple color will appear. If water be added to this purple solution and the mixture evaporated to dryness the color disappears. This latter point is of importance as xanthin, which may resemble unusual types of uric acid in microscopic appearance, also gives the murexid test, but the color does not disappear on heating with water. (2) Sodium Acid Urate (CsHsNaNiOs). This salt of uric acid forms the bulk of the "brick-dust deposit" or "sedi- mentum lateritium" ^ou^^^g^ gn^^^^^^^d. In such cases the urine 340 DIAGNOSTIC METHODS first shows a milky appearance and the sediment soon settles on the sides, and bottom of the container. This deposit is usually in the form of irregular Fig. 93. — Acid sodium urate. {Hawk.) amorphous granules of a brownish or pink color. Occasionally the sediment may be distinctly crystalline, occurring as prismatic needle-like crystals which are grouped in star-shaped, fan-shaped or dumb-bell-like clusters. Fig. 94. — Xanthin. {Hawk.) (3) Potassium Acid Urate (C6H3KN4O3). This substance occurs only as a granular amorphous deposit. Owing to its greater solubility, it does not form as large an amount of the brick-dust deposit as does the sodium salt. These two latter sediments are occasionally associated with amorphous deposits of the calcium and magnesium acid urates. These are, however, rare and need not be separately considered. In detecting the presence of the urates in a deposit, a small portion of the turbid urine is poured into a test-tube and gently heated. If urates are present the sediment will com- pletely dissolve. These ©;^^;afei!^ fejvM&eSiEiflJiXid test. THE URINE 341 (4) Xanthin (C5H4N4O2). This substance which is chemically closely related to uric acid is rarely found as a sediment in the urine. Its chief chnical importance is found in its appearance as a urinary calculus. It crystallizes in whetstone-shaped colorless crystals which resemble those of uric acid, from which it is differen- tiated by its solubility on heating and in hydrochloric acid as well as ammonia. It may be chemically recognized by Weidel's reaction. Place a portion of the suspected crystalline deposit in an evaporating dish and dissolve by warm- ing with a few drops of bromine water. Evaporate to dryness and place the dish containing the residue under a large beaker, allow the fumes of ammonia to fill the inverted beaker when a red or purplish-violet color will be produced in the presence of Xanthin. (S) Calcium Oxalate (CaCaO^). This substance appears most frequently in acid urine, but may be found €8. ^ M * Fig. 95. — Calcium oxalate. {Hawk after Ogden.) after the urine has undergone alkaline fermentation. If it occurs in acid urine it is associated with uric acid; if in alkaline urine, with the triple phosphates. The deposit is a colorless crystalline one having two distinct forms: (i) octahe- dral crystals (four-sided pyramids lying base to base) ; viewed from the side, these appear as squares crossed by two sharp lines, giving the so-called "en- velope" crystal. (2) Dumb-bell crystals in the form of ovoid or circular disks with round margins depressed at the centers. These latter often pre- sent radial striations. Emerson has called attention to a rare type of cal- cium oxalate crystal which appears in the form of flat plates with parallel sides and rounded ends, looking like superimposed sheets of mica. It is characteristic of the crystallization of this substance, as of most crystalline urinary deposits, that the crystals are practically always of the same t3^e, variations rarely appearing in the same specimen of urine. These crystals are insoluble in acetic acid, but soluble in hydrochloric acid. This is a point of some importance, as it is occasionally difiicult to distinguish microscopically between calcium oxalate and some crystals of triple phosphate. This latter crystal is soluble in acetic acid. These crystals may be chemically identified by dissolving them in hydrochloric acid, alkali- nizing with ammonium hydrate, and precipitating with ammonium oxalate. Digitized by mcrosoft® 342 DIAGNOSTIC METHODS (6) Cystin (C3H6NS02)2. The appearance of cystin in the urine is known as cystinuria. This is a condition of perverted protein metabolism which is not well understood. It may make its appearance at any period of life and in either sex, perhaps somewhat more frequently in the male. It shows a remarkably frequent hereditary character, being observed in some cases through several genera- tions and in several children of the same parents. Many of these cases do not show any clinical characteristics, being present during their entire life without any apparent symptoms. In other cases, owing to the formation of calculi, frequent manifestations are noted and surgical intervention interposed. This condition is not very frequent, being reported only i8o times in the literature. 1 "Prior to the time when cystin was found to be a product of the disinte- gration of protein substances it had been conjectured, on the ground of its content in sulphur and nitrogen,that it might be a product of the intermediate protein metabolism. The explanation which von Udranzki and Baumann gave of its excretion corresponds in principle to that which is familiar to-day / Fig. 96. — Cystin. {Hawk after Ogden.) for the appearance of conjugation products of glycuronic acid and of glycocoll in urine. Like the latter, it should be normally further oxidized, and only in the presence of definite bodies should it be intercepted. These authors considered the binding substances to be the previously mentioned diamins, especially putrescin and cadaverin, which they had found in the urine and in the feces of a cystinuric patient. The formation of the diamins was sup- posed to be brought about by specific bacteria in the digestive tract by an ex- traordinary chronic intestinal mycosis. The resorbed part of the cadaverin and putrescin was supposed to protect the cystin from combustion, just as benzoic acid does glycocoll, by entering into a loose combination which de- composes again after passing through the kidneys. Serious difficulties, how- ever, have opposed themselves to this interpretation of cystinuria. First, numerous cases have been described without simultaneous diaminuria, and, conversely, diaminuria occurs in malaria, and in the conditions brought about by the cholera vibrios and the Finckler-Prior bacillus, without cystinuria having ever set in, any more than it does when diamins themselves are administered." 1 See Fromherz, Berl. klin. Wchnschr., 1913, L, 1618; Neumann, Deutsch. med, Wchn- schr., 1914, XL, .065. Digitized by Microsoft® THE URINE 343 "Further investigations have shown that cystinuria is really a disturbance of the amino-acid metabolism. Of the end-products of protein hydrolysis which arise in the system, the cystinuric cannot avail himself in the normal way of the cystin, and in part excretes it; the remaining products of protein hydrolysis undergo their ordinary fate. If free monomolecular a-amino acids appear in places which are at present not well known, or if they occur there even in unusual amounts, then, unlike the normal individual, the cystinuric is unable to burn them and they leave the organism unchanged just as cystin itself does. The basic diamino acids behave in practically the same way, except that the CO2 group is split off from them and we arrive at diaminuria" (Neuberg) . Crystals of cystin are rare in the urinary sediment. In some of the cases reported the cystin did not separate from the urine until this was acidified with acetic acid and allowed to stand for 12 hours. It crystallizes in two i Fig. 97. — Pure leucin. (Hawk.) forms: (i) six-sided tablets having an opalescent luster and sometimes traced with fine lines of secondary crystallization; (2) four-sided square prisms lying separately or in stellate forms. These crystals are soluble in hydrochloric acid, alkaline hydrates, and insoluble in acetic acid. These tests differentiate it from uric acid. If the urinary sediment suspected of containing cystin be treated with strong sodium hydrate solution and a few drops of benzoyl chlorid, and the mixture shaken, a voluminous precipitate of benzoyl cystin is obtained. (7) Leucin (CeHiaNOa). Chemically leucin is a a-aminoisobutylacetic acid. It occurs in the urine in conditions associated with more or less marked derangement of hepatic functions (see amino acids). As found in the urine, leucin appears in the form of yellowish, highly refractile spherules, with alternating light and dark concentric layers and with radial striations. In the pure state it crystallizes either in thin, white, hexagonal plates or as scales or rosettes of irregular shapes. Digitized by Microsoft® 344 DIAGNOSTIC METHODS Leucin is soluble in water, acids, and alkalies, and insoluble, to a more or less extent, in alcohol. Not always do we find crystals of leucin in the sedi- ment when the urine contains this substance. If it be suspected, the urine should be evaporated to a small bulk and alcohol added to the residue, which may then be examined for the characteristic crystals. This leucin may be identified by Scherer's test as follows: Some of the solid residue obtained by concentrating the urine to a small bulk is evaporated with concentrated nitric acid on a platinum crucible cover. With pure leucin the residue remains Fig. 98. — Impure leucin. {Hawk after Ogden.) colorless, but as usually applied to the urine a yellowish residue obtains. This is heated with a few drops of sodium hydrate solution, when a yellowish or brown color will be observed. If further heating be applied the leucin will collect into an oily drop which rolls around on the heated surface. As leucin does not stain with Sudan-Ill it should not be confused with fat. (8) Tyrosin (C9H11NO2). Chemically, tvrosin is p-oxyphenyl-a-amino-propionic acid. As found Fig. 99. — Tyrosin. (Hawk.) in the urine tyrosin crystallizes in the form of fine colorless needles, which may appear black and are arranged in sheaf -hke collections or rosettes. Like leucin, it may not crystallize out unless the urine be concentrated. Tyrosin Digitized by Microsoft® THE URINE 345 is soluble in water, acids, and alkalies, while it is slightly soluble in alcohol and insoluble in ether. As other crystals, which may appear in the urine closely resemble the tyrosin needles, it is advisable to confirm the microscopic findings by chemical tests.^ This may be done by evaporating the urine to a small bulk, removing the fluid and dissolving the residue in water. Morner's test may then be applied as foUows: To this aqueous solution is added i c.c. of a reagent consisting of i c.c. of formalin, 55 c.c. of concentrated sulphuric acid, and 45 c.c. of water. If the mixture be heated to boiling a beautiful green color will be observed in the presence of tyrosin. (9) Calcium Sulphate (CaSOiaHaO). This is a very rare sediment, appearing only when the urine is extremely acid. The crystals appear in the form of long, thin, rhombic plates or needles which may be single, but are more frequently observed in clusters. If the sediment be boiled with hydrochloric acid and barium chlorid added, a pre- cipitate of barium sulphate will point to the presence of calcium sulphate in the sediment. Fig. 100. — Calcium sulphate. (Hawk after Hensel and Weil.) Fig. ioi. — Bilirubin (Hsematoidin). (Hawk after Ogden.) (10) Bilirubin (CieHisNaOs). Bilirubin or its isomer hematoidin may appear in the urine in conditions previously discussed. The t3^e of crystal is either a brilliant yellow or ruby- red rhomb or a yellow needle. Rarely the deposit may be in the form of a yellow granular sediment. Not infrequently small curved needle-hke spines are observed projecting from the angles of the rhombic crystals. These crystals may be identified by extracting the acid urine with chloroform and applying the tests previously discussed under BiUary Pigments. (11) Hippuric Acid (C9H9NO3). This substance has been observed as a sediment, although rarely. It ap- pears in the form of semitransparent, colorless, four-sided prisms, or in long pointed rods or needles, occasionally in forms closely resembling those of the triple phosphates to be described later. These crystals are soluble in warm water, alcohol and ether and may be distinguished from uric acid by the fact that they do not give the murexid test. ' See Rosenbloom and Gardner, New York Med. Jour., 1914, C, 574. Digitized by Microsoft® 346 DIAGNOSTIC METHODS (12) Neutral Calcium Phosphate (CaHP042H20). This substance is found only in faintly acid or neutral urine. It is quite rare as a sediment, crystallizing in colorless needles or slender pyramids which group themselves together with their points in a common center to form rosettes or cross-shaped figures. These crystals are soluble in acetic acid and may be converted into calcium carbonate when treated with a strong solution of ammonium carbonate. (13) Fat. Under normal conditions the urine contains no free fat, but amounts varying from traces to rather large excretions may be found under pathologic conditions.^ The excretion of fat in the urine is known as lipuria. This is characterized by the presence of small or large strongly refractile globules which may be stained black with osmic acid or red with Sudan-Ill. These globules are soluble in ether and may, therefore, be extracted from the urine by shaking out with this solvent. It not infrequently happens that the urine is contaminated with fat which may have been used in obtaining catheterized specimens or with fat coming from the bottle containing the urine. This may lead to a diagnosis of lipuria unless care be taken to exclude such a source. True lipuria has been observed in various conditions. Thus we may find after a large intake of fat in the diet, or as a therapeutic agent the so-called ^'alimentary lipuria." Pathologically, it has been observed in various cachectic conditions, in crushing injuries, especially of the bones, in eclampsia, in chronic heart disease, fatty tumors, diabetes mellitus, tuberculosis, various affections of the pancreas and liver, nephritis, and after the use of various general protoplasmic poisons. In these cases the blood may also contain an excess of fat, although this has not been observed in all cases. In fatty degeneration along the genitourinary tract, fat droplets may be seen in the epithelial cells and in the casts. Free fat is rarely found in such conditions, but occasionally it may collect in droplets which float on the surface of the urine and then constitutes true Hpuria. Occasionally flat superimposed plates with notched corners (cholesterin) may be seen and may be so numer- ous as to justify the term "cholesterinuria." In conditions associated with infection by the filaria, large amounts of fat may be present, giving rise to the appearance of an emulsion. To this con- dition has been given the name chyluria. In this form the fat may be present occasionally in large masses resembling tallow, but more frequently is seen in finer clumps of globules. The appearance of the urine is much like that of skimmed milk, but may have a reddish tinge due to the presence of blood. On allowing the urine to stand, a cream-like mass of fat will rise to the sur- face. It is not unusual in such cases to find the ova or the parasite in the masses of coagulated material. The excretion of the fatty material at times runs a somewhat cyclic course, being present during the day and absent at night or vice versa. Occasionally the excretion varies with the position of the patient, being somewhat more frequent when he is erect, and may be mark- 1 See Sakaguchi, Biochem. Ztschr., 1913, XLVIII, i ; Lavyrynowicz, Ztschr. f. klin. Med., 1914, LXXX, 389. ^ ' Digitized by Microsoft® Digitized by Microsoft® PLATE X. Ammonium Urate, showing Spherules and Thorn-apple-shaped Crystals. (From Ogden, after Peyec.) Digitized by Microsoft® THE URINE 347 edly increased after severe exercise. This condition should be taken simply as a symptom of filariasis. A nonparasitic tj^e of chyluria has been observed, but its etiology is somewhat uncertain. It probably is closely related to the conditions above mentioned as causing true lipuria. It does have some relation to an increased fat diet and apparently is associated with exudation from the lymphatic vessels, as the cellular elements are largely lymphocytes.^ Fig. I02. — Cholesterin. {Hawk.) {b) Those Occurring in Alkaline Urine. (i) Ammonium Urate (C6H3(NH4)N403). This sediment occurs most frequently in combination with amorphous calcium phosphate'~and triple phosphate crystals. It is the only urate deposit found in alkaUne urine, but may occur in neutral urine. It appears as a crystalline deposit of dark brown spherical masses studded with fine spiculae, from which fact the name of "thorn-apple' crystals has been given to them. Occasionally these spheres may show concentric or radial striations. Not in- frequently one observes crystals having irr.egular shapes, such as those of a dumb-bell or a pear. Chemically, these crystals may be identified by dissolving in hydrochloric acid, when uric acid, which may be identified by the murexid test, will sepa- rate. If sodium hydrate be added to the dry sedime;it and heat applied, vapors of ammonia are given off. (2) Calcium Triphosphate (Ca3(P04)2). This compound is frequently found in alkaline urine, as a white amorphous flocculent deposit arranged in irregular patches. This is the iisual deposit which appears in the urine when it becomes alkaline after meals. In the so- called " phosphaturia" the urine is always turbid when voided so that the assumption was made that an excess of phosphoric acid was being excreted. Such is found not to be the case, as a deposition of the normal phosphates of ' See Stern, Arch. Diagnosis, 1913, VI, 114; Young, Jour. Trop. Med. and Hyg., 1914, ^^^^'^41- Digitized by Microsoft® 348 DIAGNOSTIC METHODS the urine must occur when the reaction becomes alkaline. It is to be said in this place that no conclusion whatever can be drawn from the separation of a substance in the sediment as regards an increase in its excretion. So many- factors influence the separation or nonseparation of a sediment that a finding should not be regarded as evidence of increased formation and excretion unless quantitative chemical examination points in this direction. Calcium phosphate is soluble in acetic acid without evolution of gas, which test may be used to show the presence of this substance in the deposits. It may be absolutely identified as a calcium compound by dissolving in acetic acid and precipitating with ammonium oxalate; the phosphoric acid radical may be proven by dissolving the sediment in nitric acid and precipitating with ammonium molybdate. ■ ^ (3) Magnesium Phosphate (Mg3(P04)2). Theoretically this compound appears along with calcium phosphate as an amorphous deposit in alkaline urine. Its amount is, however, usually less than the latter compound. It is observed in rare cases as large, long, rhombic plates with beveled edges which closely resemble the crystals of triple phosphates. These crystals are found in cases in which not sufficient ammonia is present to form the true triple phosphate, and may be considered as transition crystals. (4) Magnesium-ammonitxin Phosphate (Mg(NH4)P04). The appearance of this substance in the urine is essentially characteristic ^ \ Fig. 103. — Magnesium-ammonium phosphates. {Hawk after Ogden.) of ammoniacal urine. It may very rarely be seen in amphoteric urine when ammonium salts are present in large amounts. The crystals belong to the rhombic system, appearing most frequently as triangular prisms or "coffin- lid" crystals. These may be shortened in the form of squares or one or more corners may be rounded or beveled. By refracted light a greenish tone is observed when these crystals are present. A second type of the " triple phos- phate" is that of a star-shaped feathery crystal with points not unUke fern leaves. These crystals are easily soluble in acetic acid and may be identified by treating with ^°'^^^'^^^^^^^^i^^fg§Q§(^^^^ ammonia is evolved. THE URINE 349 (s) Calcium Carbonate (CaCOa). This substance frequently occurs in alkaline urine in association with the amorphous phosphates. It may appear as groups of amorphous material or may form large spheroidal masses with concentric radiations. Occasionally it may be observed in dumb-bell Uke masses which resemble somewhat the same type of calcium oxalate crystal, from which it may be differentiated by the fact that it is soluble in acetic acid with production of CO2, while calcium oxalate remains undissolved. Fig. 104. — Calcium carbonate. (Hawk.) (B) Organized Sediments, (i) Mucoid material. Mucus is a constituent of practically every specimen of urine, in the form of the "nubecula." This appears in the form of small threads which branch and interlace in such a way that the entire microscopic field may be practically taken up by this material. In the meshes of the nubecular threads are observed the so-called "mucous corpuscles," which are prac- tically identical with the ordinary leucocyte. Little signifiance is attached to this form of muc ousthreads unless a great increase is observed, when it indicates, as does mucin, a vesicle catarrh. (2) Epithelial Cells. Normally, the only epitheUal cells found in the urine are the irregular flat cells from the bladder and urethra or the large flat epitheUa seen in the urine! of women and arising from the vagina. The presence of large num- bers of others types of epithelial cells is always pathological and denotes, an inflammatory or destructive lesion somewhere along the genitourinary tract.* It is a matter of more or less diflSculty absolutely to identify, in all cases, the source of the epithehum found in the urine. According to Heitz- mann, the positive recognition is based largely upon the size of the cell, as the shape may vary from pathologic conditions as well as from the portion from ' See Schkarin, Russk. Vrg.h^.^1^ gf /l^gfeso/?® 350 DIAGNOSTIC METHODS which they are derived. As stratified epithelium is found in the pelvis of the kidney, the ureters, bladder, and urethra, it is to be expected that large flat cells, cuboidal cells, or columnar cells will appear depending upon the layer from which the cell is derived. As the simple epithelium exists in the urinif- eroas tubules, the prostate gland, seminal vesicles, and ejaculatory ducts, the recognition of such cells will limit their origin, the size being important in de- termining the exact point from which they are derived. It is to be remem- bered, therefore, that the shape of the cell is of far less importance thanisits size. The epithelial cells derived from the bladder are usually the large flat irregular cells commonly seen in all normal urine. They have a clear pro- toplasm and usually a small distinct central nucleus and are extremely granu- lar. These flat epithelial cells may be single, in groups, or if the irritation is marked may occur in large sheet-like masses. The large cuboidal cells of Fig. 105.- — Epithelium from different areas of urinary tract, a, leucocyte (for com- parison); b, renal cells; c, superficial pelvic cells; d, deep pelvic cells; e, cells from calices; /, cells from ureter; g, squamous epithelium from bladder; h, neck of bladder cells; i, epi- theliumjlfrom prostatic urethra; k, urethral cells; I, scaly epithelium; m, m'-, cells from seminal' passages; n, compound granule cells; 0, fatty renal cell. {Hawk after Ogden.) the bladder epithelium may be seen in acute cystitis in which they are asso- ciated with large numbers of the flat cells previously mentioned. If the conditions become chronic the flat cells may entirely disappear and be replaced by cuboidal and by a few columnar epithelial cells. These latter cells are especially observed in the severe inflammatory processes in the bladder. The large, flat, squamous epithelial cells derived from the vagina are more frequently arranged in stratified groups so that their recognition is usually simple. As these types of cells denote simple desquamation, being pathologic only when present in enormous numbers, an absolute differentia- tion is of little consequence clinically and, if it be so, the clinical symptoms of the case will usually clear up the decision. The urethral epithelfiipf/yetifJbylfliyfi^safi&Sibles that above described. THE URINE 351 The cells are large and irregular, being partly flat, partly cuboidal and partly columnar. The cylindrical types of urethral epithelium may occur in the form of longer, irregular, smaller types, than those of the bladder or vagina. This type constitutes the so-called "tailed" cells, which may be derived from the pelvis of the kidney and were at one time held to be indicative of a pyelitis. Sahli regards a preponderance of such tailed cells over the flatter and more regular types as distinct evidence of trouble in the renal pelves. As these cells may be derived from other portions of the urinary tracts it is unwise to make an absolute diagnosis on such a finding. The small polygonal cells as well as tailed cells may be derived also from the ureter so that our diagnosis would necessarily rest upon findings other than such epithelium. In the writer 's opinion, it is a practical impossibility to make a positive diagnosis of a lesion in any specific portion of the genitourinary tract based entirely upon the appearance of the urinary epithelium. The points to be remembered are that we may have any type of epithelium and may have many variations in shape as well as in size. Such variations may be present in any portion of the urinary system, although distinctly renal epithelial cells are more frequently in the form of round or cubical cells somewhat larger than the leucocyte and containing a large vesicular nucleus. These latter renal cells are the only ones which seem to the writer distinctly diagnostic. They are differentiated from the similar cells arising from the ureters and prostate gland by the fact that^the latter cells are about twice the size of the pus-cell, being consequently larger than the true renal cell. Degenerative changes are frequently observed in these epithelial cells, even when examined immediately after voiding. The usual type of this de- generation is the presence of fat granules or globules, especially in the small renal cells. If the sediment be treated with Sudan-Ill these granules will appear distinctly red. (3) Pus-cells. A few leucocytes may be observed in practically every specimen of urine, especially in those from women, in which case they may be in large numbers and derived from the vagina. A marked increase, as recognized by numerous, indistinct, small, circular or irregular, granular cells, should be regarded [as pathologic' To this condition the name pyuria has been given. The simple finding of a pyuria does not necessarily indicate the point from which these cells were derived. Severe inflammatory processes anywhere along the genitourinary tract or the rupture of an abscess into the urinary tract will be associated with a pyuria, so that other features must be relied ubon in deciding as to the source. As a rule, it may be said that the amount of pus is small in direct affections of the renal cortex, while disease of the urinary passages is associated with a larger number. If an abscess has ruptured into the pelvis of the kidney the number of cells may be enormous. If the pyuria be of renal origin, it will be associated with the presence of the small, round, renal epithelial cells as well as with tubular casts. Frequently leuco- ' See Posner, Arch. Diagnosis, 1912, V, 269; also, Pedersen, New York Med. Jour., 1913, xcviii, 1141. Digitized by Microsoft® 352 DIAGNOSTIC METHODS cytes in small numbers are found adherent to the casts or they may even be so closely grouped as to give the name pus cast to such formations. If large numbers of pus-cells appear in the course of a chronic nephritis, they indicate either an acute exacerbation of the condition or a complicating process in some other portion of the urinary tract. The sudden appearance of very large numbers of pus-cells is especially indicative of a ruptured abscess. In inflammatory processes in the pelvis of the kidney the amount of pus may vary within wide limits. In some cases the urine may be perfectly clear when voided, showing the presence of only a few pus-cells, while in others enormous numbers may appear. This paradoxical condition may be accounted for Fig. io6.— Pus corpuscles, i, Normal; 2, showing ameboid movements; 3, nuclei rendered distinct by acetic acid; 4, as observed in chronic pyelitis; s, swollen by ammonium carbonate. (Hawk after UUzmann.) by the possibility of obstruction of the ureter on the affected side and the later forcing out of the large numbers of pus-cells. In pyelitis the urine is usually acid, which may serve as a distinguishing point from cystitis in which the urine is almost always alkaline. In tuberculosis of the renal parenchyma pus-cells appear very early and vary in number from a few to many thousands. This pyuria is usually con- stant, and is frequently associated with hematuria. The pus-ceUs in tubercu- losis are usually of the monuclear type instead of the ordinary polymorphonu- clear form. This is not easily determined, as the degenerative processes make it somewhat difficult to distinguish the nuclear form. In such conditions the sediment should be frequently examined for the presence of tubercle baciUi and a portion inoculated into, a guinea-pig. This is the only certain method of making a diagnosis of tuberculosis of the kidney. It is, perhaps, neediess to add that for absolute differentiation a specimen obtained by ureteral catheterization must be examined. In cystitis the number of pus-cells appearing in the urine will vary ac- cording to the severity o^^^/fggcj^tiggj^^g^ijj^ severe the more pus-cells. THE URINJE 353 In this condition the urine is alkaline and may be, when voided, glairy and ropy. In chronic cases of cystitis the pus-cells, although present in the blad- der in large numbers, may be so degenerated by the alkaUnity of the contents that practically no cells are recognizable. Here we find the appearance of a large amount of mucus, the urine being in some cases distinctly jelly-like. In inflammatory processes of the urethra pus may be present in varying amounts. In the acute conditions the number of cells is much more numer- ous than in the chronic types. The recognition of the causative factor, in most cases the gonococcus, will be treated of in a later section. As the acute condition becomes subacute or chronic, the urine contains large numbers of the so-called gonorrheal threads which enclose numerous pus-cells.^ These will be treated in detail later. It is sometimes a matter of cUnical importance to distinguish between an anterior and a posterior urethritis. This is best done by the so-called "two-glass" test. If thefirstportionsof the urine be collected in a receiving vessel and the later portions in a second vessel, the urine in the first vessel will be cloudy while that in the second vessel is clear in the case of anterior urethritis; while in posterior urethritis associated with the anterior type the first portion will be cloudy and the second usually so, although at times it may be clear. The reaction of the urine in both vessels will be acid unless a complicating cystitis has arisen, when the urine in the second vessel will usually be alkaline. The appearance of the pus-cells will vary depending upon the reaction of the urine. In acid urine their structure is very well preserved, the addition of acetic acid rendering the nucleus somewhat more distinct. Their usual form is that of the polymorphonuclear neutrophile, their size varying from 7 to 12 microns. If stained the vesicular character of the nucleus of the renal epithelial cell will absolutely differentiate it from the irregular type of pus-cell. In alkaline urine the cells swell up, lose their shape and become opaque. The addition of acetic acid usually clears them in such a way that the nucleus becomes visible, but occasionally does not. If the urine remains long in con- tact with the alkaline material in the bladder it becomes slimy, stringy, and gelatinous, owing to its large content in mucus. Albumin is always present so that it may be difficult to decide whether or not a true albuminuria exists. As the pus-cells may undergo such marked change when in alkahne urine and be converted entirely into a gelatinous mass in which corpuscles cannot be detected, certain tests must be appUed for positive recognition of pus in such cases. VitaU's Test. Acidity the urine with acetic acid and filter. Treat the material on the filter with a few drops of tincture of guaiac, when a deep blue color will appear in the presence of pus. If the material is not filterable, as happens when the purulent material is extremely gelatinous, place a portion of this slimy urine in a test-tube and allow a few drops of tincture of guaiac to flow ' See Broadman, New York Med. Joiir., 1913, XCVIII, 28; also, Thomas, Am. Tour. Med. Sc, 1913, CXLVI, 696. 23 Digitized by Microsoft® 354 DIAGNOSTIC METHODS upon the surface. If pus be present a distinct blue line of contact will be observed. Donne's Test. A portion of the urinary sediment in a centrifuge tube is treated with a few drops of concentrated solution of sodium hydrate. If pus be present an extremely viscid gelatinous mass will be obtained. If this mixture be heated, it will dissolve, according to Miiller, with the formation of |8-nucleinic acid. If the pus-cells be treated under the microscope with a few drops of Lugol's solution, they will take a mahogany-brown color owing to the presence of glycogen. Enumeration of Pus-cells. Such a procedure is not a part of the ordinary routine examination of urine. It is, however, sometimes advisable, as it permits of a decision regard- ing the presence or absence of a true albuminuria. If the latter exists, casts and renal epithelial cells will usually be present so that a diagnosis is often possible without a count of the cells. Technic. A portion of the 24-hour specimen of urine is thoroughly shaken to bring the corpuscles into suspension. This turbid fluid is then drawn up to the upper (11) mark of the leucocytometer, a drop placed upon the glass sHde and the cells counted as described under Blood. If more than 30,000 per cmm. are present, it is advisable to dilute five times with 3 per cent, sodium chlorid solution. For each 100,000 leucocytes per cmm. of urine o.i per cent, of albumin is assumed to be present, according to Wunderlich. (4) Red Blood-cells. The presence of red blood-cells in the urine is known as hematuria. This condition should be sharply differentiated from hemoglobinuria as the cUnical significance is entirely distinct. Blood may be found in the urine in a variety of conditions. Thus in the more maUgnant t3rpes of the acute infectious fevers hematuria is frequently observed. Likewise, in scurvy, hemophilia, purpura, leukemia, and Werlhof's disease, the kidney may be so markedly affected that hematuria obtains. In the hematuria of purely renal origin we find both acute and chronic congestions as well as inflammatory processes in the kidney associated with this condition.^ In the more acute types of nephritis, hematuria is so com- mon that the name "hemorrhagic nephritis" is frequently applied. Such cases are especially observed after posioning with cantharides and phenol derivatives.^ The chronic parenchymatous type of nephritis is, according to Weigert, always hemorrhagic in type, the number of red corpuscles being an indication of the intensity of the process. In malignant growths of the kid- ney, tuberculosis, renal calculus and cystic degeneration of the kidney, 1 See Randall, Jour. Am. Med. Assn., 1913, LX, 10. * See Belkowski, Rev. de. med., 1913, XXXIII 663, for a discussion of hematuria caused by urotropin. Digitized by Microsoft® THE URINE 355 hematuria is especially common; while in infection with certain parasites, such as the filaria, echinococcus, and the distoma hematobium, hematuria is relatively frequent although few cases of these conditions are seen. Hematuria may also be observed as a result of lesions or disease of any portion of the urinary tract. Thus stone in the ureter or urethra, tumors, ulcers, and parasites of the bladder, urethritis, prostatitis or injury during catheterization may also be associated with the appearance of red cells in the urine. A further type of cases in which hematuria occurs is known as the func- tional or idiopathic hematuria. In this class of cases no definite lesion has been found to account for the condition. It has been called "Gull's renal epistaxis," "essential renal hematuria," "angioneurotic hematuria," "renal hemophilia" and "renal aneurysm." The lesion, whatever it may be, is usually unilateral and the attacks appear at variable intervals. Some of the cases recover without any treatment after one or two profuse hemorrhages, while others require extensive treatment of the nervous system.^ . In the diagnosis of a hematuria it is important to observe the appearance of the urine both with the naked eye and with the microscope. The urine is turbid, and varies from a light, hazy, "smoky" appearance to a bright-red or deep-brown color. The red cells appear in various stages of preservation. In some cases the normal yellow color of the cell will be quite distinct while in others the color will be entirely washed out. If the urine be particularly con- centrated many crenated forms will also be observed. The blood-cells may exist singly and scattered, or may be grouped in large masses forming distinct clots or adherent to tube casts forming the so-called blood casts. In true renal hematuria the blood is intimately mixed with the urine, the individual corpuscles usually appearing as pale shadows or " ghosts." In hemorrhage from the bladder the urine may show the presence of blood-clots of irregular form and size. If the two-glass test be applied, the second glass will contain the more blood, while in hematuria of renal origin both glasses will show equal amounts. In some cases clots of blood in dis- tinct casts are seen. In the chronic parenchymatous nephritis clots are rarely present, while in malignant disease of the kidney clots are relatively common. It is important in making a diagnosis from the presence of blood that extraneous sources of blood-cells be excluded. If the blood be of renal origin it will be associated with the presence of casts and epithelial cells while no such elements will be present from a hemorrhage lower down in the genito-urinary tract. Albumin will also be present in more or less amount. It has been stated that if the blood be derived from other than renal sources, the clear supernatant fluid in the centrifuge tube will be albumin-free. The writer has convinced himself that this is an error, as he practically always obtains faint albumin reactions in cases of hemorrhage other than renal. ■■ See Braasch, Jour. Am. Med. Assn., 193, LXI, 936; Kemble, Wash. Med. Ann., 1914, XIII, 150; Kretschmer, Interstate Med. Jour., 1914, XXI, 1256; WulfE, Hospitalstid. 1914, LVII, 1465. Digitized by Microsoft® 356 DIAGNOSTIC METHODS (5) Casts. True casts are moulds of the uriniferous tubules. Their mode of forma- tion is not entirely clear. Undoubtedly a colloid substance is thrown into the lumen of the tubule and later solidifies forming a distinct cast of that particu- lar tubule. In this process of hardening the material may enclose cells of different types which are, also, present in the tubule. Whether this coagula- ble material is derived from the blood as a transudate, whether it be a secre- tion of the epithelial cells which have become pathologic, or whether it be material arising from degeneration of renal cells is not at present settled, the latter source being the more probable.'- In the urine we find true casts of the renal tubules, as well as pseudo casts which have nothing in common with the true type of these pathologic formations. True Casts. Hyaline Casts. The true hyaline casts are pale, transparent, homogeneous cylinders with rounded ends. Their size may vary from a very small fragment to one several mm. in length. In diameter they may be narrow or broad. . •; As a rule, little Fig. 107. — Hyaline casts. One cast is impregnated with four renal cells. Hawk.) difference is cHnically made between these various types of hyaline casts, but the broader types seem to the writer to be somewhat more significant than do the narrower ones. The characteristics of the true cast are their cylindrical appearance, their sides being parallel and usually straight, although they may at times be observed in typical tortuous forms. They are never tapering at the ends, but may show an irregular' outline at one or both ends, but the length and the parallel sides will usually differentiate them. ' See Erdman, Jour. Am. ^ffg^lW-fo^M/C^^b^- THE URINE 357 The pure hyaline casts are perfectly homogeneous and free from granules. Such types are, however, not frequently observed as very fine granules may almost always be detected embedded in the surrounding homogeneous ma- terial. There may be even inclusions of epithelial, renal, blood-, or pus-cells, so that the gradations between the pure t3^e of hyaline casts and many of the other varieties are outlined with difficulty. It should be stated at this point that a distinction exists between hyaline casts with enclosures of cells to such an extent that the cast is named from the variety of cell present and the type of pseudo cast in which groups of such cells are massed so as to form an apparent cast, but which do not have any definite matrix. The true hya- line cast is soluble in acetic acid and may be stained yellow withLugol's solution. These hyaline casts are not always easy to find in the sediment. In examining the urine for the presence of casts the light should be shut off as much as possible and a low-power lens used. With the use of the high power the field is limited and one is not so apt to observe the cast as with the low power. It is, however, always advisable to examine a cast, first seen with the low power, under the high power, so that the decision may be much more definite as to whether the cellular elements are real inclusions or simply material resting upon the true cast. The same is to tie said regarding the presence of granules in the hyaline cast. Significance. Regarding the significance of hyaline casts in the urine, it is to be said that they occur in any condition in which the kidney is altered by circulatory, toxic, of inflammatory disturbances. They are not pathognomonic of any one condition and may be found as a result of simple functional disturbance. A few hyaline casts may be found in practically every urine, providing suffi- cient search is made. Any undue strain, such as running for a car in one who is not used to such exertion, may be sufficient to add quite a number of hyaline casts to the urine. In many thousand urine examinations made in the writer's laboratory, it has been rather the unusual thing not to find an occa- sional hyaUne cast. It would seem, therefore, that no significance whatever should be attached to the presence of an occasional hyaline cast. When, however, these casts become very numerous they should then be interpreted as meaning a disturbance of the kidney, although the absence of other types would rather speak against a marked pathologic change. In diabetes melli- tus, "showers" of casts are especially observed preceding the appearance of coma (Kiilz). Granular Casts. These are modifications of the true hyaline cast in the sense that fine or coarse granules are found in the matrix of the hyaline cast. Several types of . granular casts are observed. The granules may be very fine, very coarse, or may be distinctly composed of denegerated epithelial cells. The fine as well as the coarse granules are undoubtedly derived from the renal epithelium, which has degenerated completely. The coarser the granules the more severe the inflammatory proces^/g/f/zed by Microsoft® 358 DIAGNOSTIC METHODS These granular casts vary in shape and in size, but are usually shorter than the hyaline type. To these granular casts may be attached various cells so that it is difficult to tell whether the cast is really a true granular or a cellular one. In some cases these cellular inclusions may undergo fatty degeneration giving a much higher refractility to the specimen. Not infre- quently one observes hyaline casts which are distinctly granular in one portion while the other is perfectly homogeneous. Fig. io8. — Granular casts, X 225. {Tyson.) The so-called hrown granular casts appear to be almost entirely degener- ated epithelial cells, although the coloring matter is probably hemoglobin. The fact that the hyaline matrix cannot be distinctly made out does not argue against this type being truly hyahne in character, although the matrix is completely saturated with the pigment. Waxy Casts. This type is very refractile, transparent,' and either perfectly colorless or showing a slight shade of yellow. Usually they are very long and broad and may be either straight or curved. The ends show a very distinct fracture while the cast itself may show a tendency to split transversely. Their ap- pearance is, therefore, i\Diigiit^ y j; . JD Digitized by Microsoft® 366 DIAGNOSTIC METHODS this organism in the case of gonorrheal cystitis. Under such condition the symptoms and the association with an existing gonorrhea would furnish the decisive clue. Bacilluria. By this condition is meant the presence in the freshly voided urine of so liiariy organisms that the urine is distinctly cloudy. These organisms are usually those associated with a mild cystitis or may be those of an existing general infection. ^ In cases of persistent bacilluria, we may find a true renal origin, which is largely associated with the presence of the typhoid and colon bacillus. As has been well established, the typhoid organism may be excreted for months after the patient is convalescent so that it becomes necessary to use strict measures to disinfect all urine of typhoid patients. The colon bacillus is at present assuming so much importance in clinical work that cases are being almost daily recognized which may be directly traceable to the colon bacillus and not to the typhoid as usually assumed. It is necessary, therefore, for the labora- tory worker especially, and, where possi- ^ V.^ ble, for the general practitioner to be able m^ to recognize each of these organisms when ^^ \ ' present either in the feces, urine, milk, or water-supply (see Feces). A second general class of cases associated with J^ath^rine M rll bacilluria are those of urethritis and pros- FiG. 113.— Scolex and booklets of tatitis. Usually there is a secondary taenia echinococcus in urine. ,-.• • • ^ 4.1. cystitis arising from the same organism or, at least, the resistance of the bladder has been so far reduced that these organisms which find their way into the bladder develop profusely therein. (9) Parasites. Various types of parasites are observed in the urine. Thus the tricho- monas vaginalis has been found by Klinstler, Miura, and Dock. Amebse have been found by Balz, Jiirgens, Wijchoff, and by Musgrave and Clegg. Various portions of hydatid cysts are frequently observed, among which we find the echinococcus booklets and fragments of membrane. Nematode worms, especially the filaria sanguinis hominis, are present in cases of chyluria, while the anguillula aceti or "vinegar-eel" has been reported, especially by Stiles, while Billings and Miller report its presence as a possible contamina- tion from the bottle in which the urine was collected.^ 1 Freifeld (Berl. klin. Wchnschr., 1913, L. 1761) reports the finding of diphtheria bacilli in urine. Rosenow (Jour. Infect. Dis., 1909, VI, 296) and Townsend (Jour. Am. Med. Assn., 1913, LXI, 1605) report the pseudo-diphtheria bacillus as a causative factor of cystitis. See, also, Curtis, Jour. Am. Med., Assn., 1915, LXIV, 270; Surg. Gyn. and Obs., 191S, XXI, 423. Barber and Draper, Jour. Am. Med. Assn., igis, LXIV, 205; McGowan, Ibid., 226; Cunningham, Ibid., 231; KoU, Ibid., 297; Greeley, New York Med. Jour., 1915, CI, 250; Hoover, Interstate Med. Jour., 1915, XXII, 163; Kretschmer, Ibid., 173. ^ King (Jour. Am. Med. Assn., 1914, LXIII, 2285) reports a case of Myiasis of the urinary passage due to the presence of the larva of the small "latrine fly" (fannia scalaris). Digitized by Microsoft® PLATE XIII. J« , / V 1^ I ' ^1 ;- /# ^w ICatharine Hill Cystitis due to Colon Bacillus. (Methylene Blue Stain.) Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® PLATE XIV, i 'M %> Katharine Hill Staphylococcus Cystitis. (Leishman Stain.) Digitized by Microsoft® THE URINK 367 Eggs of the schistosomum hsematobium are not infrequently observed together with large numbers of red cells in cases of bilharziasis. This worm as well as its ova wiU be discussed in the section on Blood. Stuertz has reported the findings of the egg of eustrongylus gigas in the urine in a case of chyluria. V. Calculi Concretions of a more or less hard and dense character are prone to form in the urinary passages. These bodies are termed, according to their size and location, sand, gravel, stone, and calculi. These formations consist of accretions of any of the various crystalline or amorphous sediments previously mentioned, the type of stone depending upon the reaction of the urine. These calculi are formed by the deposition of the crystalline material around a definite nucleus, which usually consists of organic material, such as fibrin, blood, desquamated epithelial cells, mucus, or even a crystal of uric acid or calcium oxalate. ^ It is very difficult to decide as to the reason for the deposition of this material in the form of a renal stone. The growth of the Fig. 1 14. — Ova and miracidium of schistosomum hematobium, X ioo: A, Ovum as seen in urine; B, the same after addition of water; C, miracidium. {Tyson after Railliet.) calculus takes place by accretion, the deposition of successive layers of mater- ial occurring around the original nucleus. The material of which the stone consists will usually be of one kind, so that we speak of uric acid or phosphate calculi, for instance, while, occasionally, mixed calculi may be formed by the deposition of two or more chemical combinations. The classification of urinary concretions is based on the chemical constit- uents of which they are composed. Before examining a calculus chemically a thorough optical examination should be made, as this may give a definite clue as to its composition. After this preliminary examination, the calculus 'See Posner, Ztschr. f. Urol., 1913, VII, 799; also, Lichtwitz, Ibid., 810; also, Williams, New York Med. Jour., i9i5,^J^6o^^ ^^ Microsoft® 368 DIAGNOSTIC METHODS is ground to a fine powder and examined according to the following table of Heller. Uric Acid Calculi. These are, perhaps, the most common renal stones. They are not always composed of pure uric acid, but are made up of a mixture of this substance with the urates. They are always colored, usually yellowish or brownish, but may at times appear distinctly red. They are fairly hard and usually show a rough irregular nodular surface, although at times this may be smooth. They fracture very easily and show, on cross section, a distinctly laminated structure, the layers frequently being of different colors, in some cases even being composed of deposits other than uric acid. If heated on a platinum foil they are combustible, burning without a flame. They give murexid test and do not liberate appreciable amounts of ammonia on treatment with sodium hydrate. Ammonium Urate Stones. The ammonium urate calculi occur rarely in the adult. They are small, yellow and very soft, being distinctly clay-like and easily powdered when dry. These stones give the murexid test and also give a strong reaction for ammonia on treatment with sodium hydrate. Calcium Oxalate Stones. Next to the uric acid calculus the oxalate stone is most frequently met.* The smaller t)rpes of these calculi are practically colorless and have a smooth surface, while the larger ones are grayish, brownish or even black in color and have a rough nodular surface with sharp projecting angles. These stones frequently cause severe hemorrhage and much irritation when passing through the ureter and urethra. From their appearance they have been called the mulberry calculi. They are, perhaps, the hardest of the urinary stones. These calculi are insoluble in acetic acid, but soluble in hydrochloric acid without effervescence unless the powder is previously heated. It is rare to find these stones perfectly pure, admixtures with various other sediments leading to distinct concentric arrangement as shown on fracture. Phosphatic Calculi. Stones composed of pure alkaline phosphates or triple phosphates are exceedingly rare. Usually the phosphatic calculi contain admixtures of ammonium urate, calcium carbonate, and calcium oxalate. The color of such stones may range from a white through yellow to some with distinct red- dish tones. They are frequently of very large size, especially when formed in the bladder, are of a chalky consistency, and show a rough surface. These calculi are soluble in hydrochloric or acetic acid, such solutions giving reactions both for phosphoric acid and the alkaline earths. ' See Rowlands, Biochem. Jour., 1908, III, 346; Kahn and Rosenbloom, Jour. Am. Med. Assn., 1912, LIX, 2252; also, Kahn, Arch. Int. Med., 1913, XI, 92; Kahn, Ztschr. f. exp. Path, u. Therap., 1915, XVII, 88; Rosenbloom, Jour. Am. Med. Assn., 1915, LXV, 161; Cabot, Surg. Gyn. and Obs., 1915, XXI, 403; Braasch and Moore, Jour. Am. Med. Assn., 191S, LXV, 1234. ^.g.^.^^^ ^y Microsoft® THE URINE 369 Pi < p o 12; o 1—1 H o ^ (U u « *w (J .s ^ ^ S '=i ° y ^ aj ^ >^ 3 i! oPh O 3 =d o 4> 3 3 |i^-d CO " p Ph ^ in 'g Sii J! g W. 4JtH>4)tnoa)tn !a 4) K X-d j3 H H^^ > 4) U5 U 4) yi a 3 'd p . a^ a 2 <: 3 « ^_) 41 t- .2 >^ ^a d.3 tS p 4) " '^ 4J fern p t; J=l,d ^■1 §§ --3 a1°s ^ ^ y „ L.- "i p Safe u o '■h'5 S . p ■" a SH T3 wi >> P '-<*0 w M P CS ^ is .2 S.5 a ^ a.B-a -3 cj ca a=d "1-J3 d 5: S b "' 3 -3 2H T3 ^.3 I g i 4J 4) . •B ■■<«"d 1 rt *j p . O (J d d *i O^ a 3 a a cs ca g d J3 P 4) dj< ,d dM-,^ && jd p a Digitized by Microsoft® 370 DIAGNOSTIC METHODS Calcium Carbonate Calculi. Such stones are exceedingly rare. They are small in size, are distinctly chalk-like in consistency and color, and have a smooth surface. If treated with acid, carbon dioxid is evolved. Cystin Calculi. These stones are white or pale yellow in color, have either a smooth or irregular surface, and are soft and waxy in consistency. They vary in size occasionally being found as large as a hen's egg, although those of true renal origin are about the size of a pea. The formation of such calculi and their passage through the ureter and urethra constitute practically all of the untoward symptoms shown by subjects affected with cystinuria.^ Such stones burn readily if heated on a platinum foil, giving off a pecuhar sharp odor. The powder is soluble in ammonia from which the characteristic hexagonal plates separate on allowing the ammonia to evaporate. Xanthin Calculi. These stones occur especially in children, although even here they are very rare. They are usually light brown in color, moderately hard, and vary in size from that of a pea to a tennis-ball. On cross section they appear amor- phous and if rubbed take a polish much resembling that of wax. The pow- der shows the typical reaction for xanthin previously outlined.^ Urostealith Calculi. These masses consist of fat, calcium and magnesium soaps, and choles- terin. They are usually soft and may be somewhat irregular in shape. This material burns with a pale yellow flame giving an odor of resin. The dry powder is soluble in alcohol and ether, from which rhombic notched plates of cholesterin separate on evaporation. VI. Functional Diagnosis It is usually of great importance, especially in cases in which surgical intervention is contemplated, to know just exactly what the functional capa- bilities of the kidney are. If one kidney is to be removed, the question arises as to whether the remaining kidney can sufficiently accommodate itself to the increased work which must be put upon it. As such a variation has been occasionally found between the results of the chemical and microscopical examination of the urine on the one hand and the pathologic condition shown in the kidneys postmortem on the other, an at- tempt has been made to find delicate tests by which a true renal lesion might be indubitably determined and thus permit of an absolute diagnosis even though the urinary findings were or were not conclusive. It is granted that a more or less severe lesion of the kidney may exist and yet its functional capacity be almost normal. If this functional capacity can be determined by ' See Frankenthal, Deutsch. Ztschr. f. Chir., 1914, CXXXI, 442; Neumann, Deutsch. . med. Wchnschr., 1914, XL, 2065. 2 See Rosenbloom, New York Med. Jour., 1915, CI, 120. Digitized by Microsoft® THE URINE 371 tests which are more or less simple, it is evident that such methods should form part of the daily routine of the practitioner.' Before discussing some of these tests, which have been advocated for estimating the functional activity of the kidney, the writer would say that few of them have proven entirely satisfactory. If we are to regard nephritis as a constitutional condition with secondary renal manifestations, it is self-evident that such tests can show only the manner in which a normal or pathologic kidney reacts toward certain substances and can have little reference to the primary etiologic factors. Cryoscopy. The method of determining the freezing-point of a solution is one of the most delicate of those of physical chemistry. As it is based upon the principle that substances in solution lower the freezing-point of the solvent in direct proportion to the molecular or ionic concentration of the solution, this method serves as a ready means of determining the molecular weight of a substance as well as the molecular concentration of a solution. For a successful outcome of a cryoscopic determination, the most assiduous attention to detail must be paid, so that this method certainly can find no place in the hands of the general practitioner or even in those of the laboratory worker, who has not been especially trained along these lines. Such being the case, it is absurd to expect that slight variations in the freezing-point (A) of such a complex mixture as the urine can yield any valuable information, especially when one remembers that fluctuations wider than those shown under pathological conditions may be noted as a result of not observing such slight details as that of constantly agitating the urine during the cooling and freezing. The normal freezing-point of the urine varies between — o.gand — 2°C.,anincrease being known as hypersthenuria and a decrease as hyposthenuria. As will be seen in the discussion of this subject in the section on Blood, the results obtained by this method have been far from satisfactory, as noth- ing of distinct diagnostic value has as yet been derived from comparative studies by various workers. It would seem to the writer, therefore, that for the present this method would better be left to the research worker than to be adopted by the general or special student, who should make use of methods which will yield results of more immediate value to him. For these reasons the writer must refer to other works for a detailed description of the method. Electric Conductivity. The remarks made later in the section on Blood regarding electric conduc- tivity are especially applicable to the urine. This test is altogether too deli- cate to be applied to such complex fluids as the urine with the hope that slight variations in the conductivity will show anything of importance. As the electric current is conducted only by dissociable compounds, this method '■ See Blum, Renal Diagnosis in Medicine and Surgery, New York, 1914; Stevens, Jour. Am. Med. Assn., 1914, LXII, 1544; Geraghty, New York Med. Jour., 1914, €,312; Piersol, Interstate Med. Jour., 1914, XXI, 1165; Thomas, Jour. Med. Assn., 1914, LXIII, 1909* Braasch and Thomas, Ibid., 1055. LXIV. 104: Geraghty. Bull. Johns Hopkins Hosp., loii;, XXVI, 155. Digifized by Microsoft® 372 DIAGNOSTIC METHODS can show absolutely nothing regarding the excretion of the nondissociable organic substances. Such being the case the writer can see no reason for re- sorting to such delicate procedures as the determination of the conductivity of the urine when no attempt is made, as may be observed in many of the ex- periments reported, to control the intake of the inorganic substances which would. affect the conductivity of the urine. A little more attention to ordi- nary methods of chemical examination, with especial regard to ascertaining the intake and output of the patient, would, in the writer's opinion, yield much more valuable information than could be obtained by the rather un- certain urinary manipulations with the method of Kohlrausch. Chlorid Excretion. By the determination of the chlorid excretion in a given case under the influence of a specific intake of sodium chlorid it was hoped that some informa- tion might be derived as to the "glomerular sufficiency" of thekidney. This test, known as alimentary chloruria, advanced by Claude and Mante,^ has like the above yielded little of value. The exceptions to the general rule of pathologic retention are too numerous to permit of absolute differentiation. While it has been shown by Widal that a retention of sodium chlorid does occur in nephritis and that a restriction of the salt in the diet frequently does alleviate the edema and albuminuria, yet many factors not well understood frequently give rise to opposite results (see Chlorids in Urine) . The general consensus of opinion regarding this test may be summed up by the statement that the chlorid excretion in cases of health and disease is of no distinct value in diagnosing the functional activity of the kidney. Methylene Blue Test. It was hoped by the introduction of such tests that the capability of the kidney to excrete substances present in the blood could be determined by the ease with which artificially introduced substances were eliminated. Achard and Castaigne administer by the mouth o.i gram of methylene blue in a capsule or, preferably, 0.05 gram subcutaneously. This substance is excreted by the kidneys in the form of a colorless chromogen in 15 to 30 minutes after injection. This chromogen may be converted into the colored pigment by heating with acetic acid. In about five minutes after the excretion of the chromogen, the urine becomes greenish-blue from the excretion of the pig- ment itself. Under normal conditions the excretion reaches a maximum from three to four hours and may last two to three days. If the pigment is not excreted within one hour after subcutaneous injection, pathologic conditions are supposed to exist. In some cases of chronic interstitial nephritis it as found that such injections were not allowed by an excretion of the pigment until one to three hours had elapsed. This would be a valuable diagnostic method had it not also been shown conclusively that many cases of advanced interstitial ne- phritis show the same excretion of this pigment as normal individuals. Such ^eing the case the test is of no more use than many clinical and routine urine methods. 1 Arch. gen. de.m6d., iqoQfgitiS^Qi^^y MicrOSOft® THE URINE 373 Phloridzin Test. This test is based on the assumption that phloridzin normally gives rise to a glycosuria through distinct alternations in the renal cells. In other words, a so-called "renal diabetes" is set up. The technic is as follows: i c.c. of a 1 :20o aqueous solution of phloridzin is injected subcutaneously. The urine is tested at intervals of 15 minutes for the appearance of sugar. Normally, sugar may be detected in one-half to one hour and may be present for as long as five hours. The quantity eliminated may vary from 0.5 to 3 grams of glu- cose. In nephritis the sugar is usually absent or below 0.5 gram. The test does not distinguish the various types of nephritis, yet it does usually indi- cate that renal activity is disturbed. It is, however, being rapidly displaced by the following test. Phenoisulphonephthalein Test. Rowntree and Geraghty^ have introduced a test for the functional activity of the kidney which is by far the most accurate at our disposal.^ The solu- tion used is prepared as follows: 0.6 gram of phenoisulphonephthalein and 0.84 c.c. of 2/N NaOH solution (8 per cent.) are added to sufficient 0.75 per cent. NaCl solution to make 100 c.c. Each c.c. of this solution contains, therefore, 6 mg. of the dye. As the mono-sodium salt, formed in this solu- tion, is slightly irritant locally when injected, 2 or 3 drops more "of the 2/N NaOH are added, when the solution changes to a beautiful Bordeaux red color and becomes non-irritant.' The technic is as follows: 20 to 30 minutes before applying the test, the patient is given 300 to 400 c.c. of water in order to insure a free urinary secre- tion, otherwise delayed time of appearance may be due to lack of secretion. Under aseptic precautions a catheter is introduced into the bladder and the bladder completely emptied. Noting the time, i c.c. of the above solution is administered subcutaneously in the upper arm by means of an accurately graduated syringe. The urine is allowed to drain through the catheter into a test-tube in which has been placed a drop of 25 per cent. NaOH solution and the time of the first pinkish tinge is noted. In patients without urinary obstruction, the catheter is withdrawn at the time of appearance of the drug in the urine and the patient is instructed to void into a receptacle at the end of one hour and into a second receiver at the end of the second hour. A rough estimate of the time of appearance may be made by having the patient void urine without the use of the catheter at frequent intervals. In prostate cases it is wise to have the catheter in place until the end of the observation. The catheter is corked at the time of appearance of the drug in the urine and the cork is removed at the end of the first and second hours, the bladder drained each time. ' Jour. Pharm. and Exper. Therap., 1910, I, 579; Arch. Int. Med., 1912, IX, 284. . ' See Conzen, Deutsch. Arch. f. klin. Med., 191 2, CVIII, 353; Smith,. Am. Jour. Dis. Child.,' 1913, V, 25; Pepper and Austin, Am. Jour. Med. Sc, 1913, CXLV, 254; also, Rown- tree, Fitz and Geraghty, Arch. Int. Med., 1913, XI, 121; Geraghty, Rowntree and Gary, Am. Surg., 1913, LVIII, 800; Dietsch, Ztschr. f. Exper. Pathol, u. Therap., 1913, XIV, 512, Rowntree, Geraghty and Marshall, Surg. Gynec. and Obs., 1914, XVIII, 196. ' This mono-sodium salt of phenoisulphonephthalein may be obtained from Hyhsoh' Wescott and Co. of Baltimore in the form of ampules, each c.c. of the solution containing 6 mg. Digitized by Microsoft® 374 DIAGNOSTIC METHODS Each sample of urine is measured and the specific gravity taken. Suffi- cient 25 per cent. NaOH is added to make the urine decidedly alkaline in order to elicit the maximum color (a brilliant purple-red). This solution is now •placed in a liter volumetric flask and distilled water added to the i L. mark. Thoroughly mix the solution and filter a small portion for comparison with the standard solution. This standard consists of 3 mg. of phenolsulphone tha- lein (or i c.c. of the solution used for injection) diluted to i liter and made alkaline with i or 2 drops of 25 per cent. KaOH solution. This solution will retain its intensity of color for weeks. The comparison of color is made in the Duboscq or the Rowntree and Geraghty modification of the Autenrieth- Konigsberger colorimeter. The known (standard) solution is adjusted to the 10 mm. mark and the intensity of the unknown solution is made to correspond by means of the usual manipulations. One may readily calculate the amount of drug eliminated as follows: If, for instance, the reading for the unknown solution is 20, it is evident that only 50 per cent, as much dye is present as is contained in the standard solution. That is, the excretion equals 50 per cent, of the 3 mg. of the standard or 25 per cent, of the 6 mg. injected. The read- ings may be then made on a second and third voiding until the drug is com- pletely eliminated. In normal cases it has been found that the time of appearance varied from 5 to 1 1 minutes and that 40 to 60 per cent, of the drug was excreted in the first hour and 20 to 25 per cent, in the second hour. The excretion of the drug does not run parallel to the excretion of water. The smaller the amount of urine in normal cases the greater the concentration of the drug. It is im- material, as far as the excretion of the drug is concerned, whether the urinary output is 50,200,400 or more c.c. In pathological cases it has been demonstrated that the permeability of the kidney for this drug is decreased in both chronic parenchymatous and chronic interstitial nephritis, the decrease being most marked in the latter and varying with the intensity of the disease. In cases with obstruction in the lower urinary tract, this test may show renal involvement to such an extent that operation should be deferred, especially if the time of appearance is de- layed beyond 25 minutes and the output of the drug is below 20 per cent, for the first hour. This point is of special value to the surgeon as it shows the danger of using surgical intervention until the kidney may become less in- sufficient. It would seem to be of value, also, in the study of the renal con- dition preceding uremia. To the obstetrician it may show the possible approach of eclampsia, as has been demonstrated in several cases. In uni- lateral and bilateral kidney diseases the absolute amount of work done by each kidney as well as the relative proportion can be determined by resort to ureteral catheterization followed by this test. The writer has no hesitancy in advising a careful study of this test in all cases with suspected or actual renal involvement. ^ 1 See Behrenroth and Frank, Ztschr. f. exper. Path. u. Therap., 1913, XIII, 72; Rowntree and Fitz, Arch. Int. Med., 1913, XI, 258; Geraghty, Jour. Am. Med., Assn., 1913, LX, 191; Goodman, Ibid., 1913, LXI, 184J Frothingham, Fitz, Folin and Denis, Arch. Int. Med., 1Q13, XII, 24?; Geraghty and Ro.wntree, Joun Am. Med. Assn., 1013, LXI, 919; Foster, Digitized by Microsoft® THE URINE 375 BIBLIOGRAPHY 1. Benedict. Influence of Inanition on Metabolism. Washington, 1907. 2. Blarez. L'Urine au point de vue clinique et medical. Paris, 1906. 3. Blumenthal. Pathologic des Harnes am Krankenbett. Berlin, 1903; Hand- buch der Speziellen Pathologie des Harns. Berlin, 1913. 4. Chittenden. Physiological Economy in Nutrition. New York, 1905. The Nutrition of Man. New York, 1907. 5. CoHNHEiM. Chemie der Eiweisskorper. Braunschweig, 1906. 6. Croftan. Clinical Urinology. Chicago, 1907. 7. Daiber. Mikroskopie der Harn-Sedimente. Wiesbaden, 1906. 8. Fischer. • Untersuchungen ueber Amiosauren. Berlin, 1906. 9. Gerard. Traite des Urines. Paris, 1906. 10. Gerard. L'analyse des urines consider6e comme un des 616ments de diagnostic. 2' ^d. Paris, 1913. 11. Hammarsten. Lehrbuch der physiologische Chemie. Wiesbaden, 1914. 12. Heitzmann. Urinary Analysis and Diagnosis. New York, 1915. 13. Hoppe-Seyler-Thierfelder. Handbuch der physiologisch- und pathologisch- chemischen Analyse. Berlin, 1909. 14. Krehl. Ueber die Storung chemischer Korrelationen im Organismus. Leipzig, 1906. Pathologische Physiologie. Leipzig, 1912. 15. Mann. The Physiology and Pathology of the Urine. London, 1913. 16. Naunyn. Der Diabetes Mellitus. Wien, 1907. 17. Neubauer UNO Huppert. Analyse des Harns. Wiesbaden, 1910 and 1913. 18. Neuberg. Der Harn. Berlin, 1911. 19. von Noorden. Die Zuckerkrankheit. Beriin, 1912. 20. Ogden. Clinical Examination of the Urine. Philadelphia, 1903. 21. Pavy. Carbohydrate Metabolism and Diabetes. London, 1906. 22. Purdy. Practical Urinalysis and Urinary Diagnosis. Philadelphia, 1900. 23. Rieder and Delephine. Urinary Sediments. London, 1899. 24. Saxe. Examination of the Urine. Philadelphia, 1906. 25. Scott. Clinical Examination of the Urine. Philadelphia, 1900. 26. Spaeth. Untersuchung des Harns. Leipzig, 1903. 27. Sutton. Volumetric Analysis. Philadelphia, 1904. 28. Tyson. Bright's Disease and Diabetes. Philadelphia, 1904 29. Wells. Chemical Pathology. Philadelphia, 1914. Arch. Int. Med., 1913, XII, 452; Fishbein, Jour. Am. Med. Assn., 1913, LXI, 1368; Roth, Berl. klin. Wchnschr., 1913, L, 1609; Marogna, Gazz. d. osp., 1914, XXXV, 172; Widal, Weill and Radot, Presse m6d., 1914, XXII, 565; Pezzana, Policlinico, 1914, XXI, 563; Mach- witz, Rosenberg and Tschirtkoff, Munch, med. Wchnschr., 1914, LXI, 1268; Hess, Ibid., 183s and 1874; Ware, New York Med. Jour., 1914, XCIX, 416; Robertson, Ibid., 972; Jones, Ibid., 1914, C, 518; Fanz, Ibid., 1214; Tracy, Surg., Gyn. and Obs., 1914, XIX, 734; Block, Jour. Am. Med. Assn., 1914, LXII, 1309; Griessmann, Deutsch. Arch. f. klin. Med., 1914, CXIV, 32; Deutsch and Schmuckler, Ibid., 61; Hedinger and Schlayer, Ibid., 120; Hessel, Ibid., 396; Lichtwitz and Stromeyer, Ibid., 1914, CXVI, 127; Rowntree, Am. Jour. Med. Sc, 1914, CXLVII, 352; Fitz, Ibid., 1914, CXLVIII, 330; Thayer and Snowden, Ibid., 781; Agnew, Arch. Int. Med., 1914, XIII, 485; Frothiugham and SmiUie, Ibid., 1914, XIV, 514; Quimby and Fitz, Ibid., 1915, XV, 303; Miller and Cabot, Ibid., 369; Rowntree, Marshall and Baetjer, Ibid., 543; Hopkins and Jones, Ibid., 964; Bauer and Habetin, Ztschr. f. Urol., 1914, VIII, 355; Bauer and Nyiri, Ibid., 1915, IX, 81; Tilestou and Comfort, Am. Jour. Dis. Child., 1915, X, 278; Hess, Bull. Johns Hopkins Hosp., 1915, XXVI, 52; Smith, Jour. Am. Med. Assn., 1915, LXIV, 223; Elliott, Ibid., 1885, Pedersen, New York Med. Jour., 1915, CI, 770; Siegel, Pub. Jefferson Med. Coll., igiic- VI, 78. Digitized by Microsoft® CHAPTER VII SECRETIONS OF THE GENITAL ORGANS I. Male Secretions General Considerations. The normal secretion of the male generative organs is known as semen and is a mixture of the secretions of the prostate gland, the glands of Cowper, the testicles, and the seminal vesicles. It is a practical impossibility from the clinical standpoint to separate the different elements of the semen, so that this must be discussed as a whole. The semen is a white or slightly yellowish, somewhat thick and viscid liquid with a peculiar odor, somewhat resembling fresh glue, and showing a neutral or faintly alkaline reaction, a nonhomogeneous milky appearance, and a specific gravity greater than that of water. It is composed of semisolid material in the form of white masses floating in a limpid liquid and holding in suspension specific elements, derived from the secretory glands of the geni- tal apparatus and from the desquamation of the various canals through which the semen passes. Semen contains about 6 per cent, of organic and 4 per cent, of inorganic matter. Its chief chemical characteristic is the presence of spermin (C2HS- N)2, which is at least isomeric, if not identical, with diethylen-diamin, accord- ing to Ladenburg and Abel. This spermin, which is derived largely from the prostate gland, combines with the phosphoric acid radical to form spermin phosphate, which crystallizes in the form of four-sided spindles or prisms which may appear as flattened needles. In some cases these crystals re- semble very closely the diamond-shaped double pyramids known as Charcot-Leyden crystals, which are found in the sputum. They are, how- ever, of a different crystalline group and are soluble in formalin, while those found in the sputum are insoluble in this menstruum. These spermin crystals are known as Bottcher's crystals. Miescher has studied the com- position of the heads of the spermatozoa and has been able to isolate certain bodies, known as protamins, which are the simplest type of protein material.^ Microscopic Examination. The most important and characteristic constituent of semen are the sper- matozoa.^ These sexual elements consist of an anterior broader portion or head and a narrow thread-like tail. The former is oval or egg-shaped and measures about 5 microns in length, 4 in breadth, and 2 in thickness. Just behind this pyriform head is a short cylindrical portion measuring 6 microns ^ See Steudel, Ztschr. f. physiol.Chem., 1913, LXXXIII, 72. ^ Koessler (Trans. Chic. Path. Soc, 1912, VIII, 280) has shown that the spermatozoa may act as direct carriers of infectious agents, among, these being the spirochseta pallida. Digitized by^crosoft® SECRETIONS OF THE GENITAL ORGANS 377 in length, which is known as the middle piece. This tapers somewhat to the point of union with the tail. This so-called tail is a thread-like posterior portion and is approximately 45 microns in length. In the freshly voided semen these tail portions show active undulatory whip-like motions, which persistfor 24 to 48 hours, and even longer under proper conditions, and enable the spermatozoa to progress from point to point. Alkalies seem to favor this niovement, while dilute acids inhibit it very rapidly. This movement of the spermatozoa is closely associated with their sexual activity, as the cells show- ing no movement when freshly voided may be regarded as possessing no Xathaii-'ine. Hill Fig. 115. — Normal semen. functional power. For a- discussion of spermatogenesis as well as of fertili- zation of the ovum the writer would refer to the admirable description of McMurrich.i Besides these characteristic portions of the semen, large numbers of lecithin globules are seen, which give the milky appearance to the fluid. Espe- cially to be noted among the cellular elements are the so-called corpora amylacea which resemble very closely starch granules, having concentric striations, a finely granular center and occasionally a nucleus. These cells take a distinct blue color on treatment with iodin solution. Moreover, various epithelial cells are observed, which are derived from the several glands contributing to the composition of the semen. Some of these cells are ' The Development of thi 1907. 378 DIAGNOSTIC METHODS distinctly granular, some contain fat globules and very closely resemble the colostrum corpuscles of the lacteal secretion, while some of the granules re- semble myelin. In rare cases cylindrical casts are seen which simulate the hyaline casts of the urine, but as a rule they are larger and longer. These are supposed to be derived from the prostate gland and seminal vesicles. If the semen be allowed to stand for a few minutes, several -types of crystal may be observed, especially the phosphate of spermin, ammonium-magne- sium phosphate, fatty acids, and oxalate of calcium. This last crystalline component is especially noted in the urine in cases of spermatorrhea. Pathologic Variations. Direct pathologic variations in the semen are limited to two conditions. Either spermatozoa are absent or those present are nonmotile. The deter- mination of the presence of spermatozoa in the semen or in suspected stains is a matter of simple microscopic examination. With the question of the mo- tility of such elements, when present, the conditions under which the exami- nation is made may markedly influence the findings. If possible, semen should be examined, with regard to the motility of the spermatozoa, as soon as ejected, but if such is not possible the fluid must be kept warm until exami- nation may be .made. It is absolutely out of the question to make a positive diagnosis of true nonmotility of spermatozoa from examination of specimens, which have been allowed to cool. In some cases, if the time has not been too extended, warming may bring back the motile power of these cells, but in many cases it does not. It is, therefore, unjust and unwise to brand a man as sterile without absolute proof that such a condition really exists. If no spermatozoa are present, especially after several examinations, sterility is absolute. This condition is known as azoospermatism. According to Kehrer, 40 per cent, of cases of conjugal sterility are due to the absence of spermatozoa in the semen. It is, therefore, necessary that the ordinary gynecological idea that women are the responsible factors in the family sterility should be, at least, partly borne by the man, as it is unjust to the woman to blame her for faults existing in the husband. The writer does not wish to be interpreted as stating that sterility does not frequently exist in women, but he does desire to emphasize the point that many more men are ster- ile than is generally supposed and that the sterile women are in this condition argely through theresults of gonorrheal infection through their husbands.' Spermatozoa may be absent from the semen during convalescence from acute febrile conditions, valvular heart disease, and in general conditions associated with lowered nutrition. On the other hand, the constant presence of spermatozoa in the urine, as well as in the semen, may be noted as a result of various pathological conditions as well as of venereal excesses or mastur- bation. To this condition is given the name of spermatorrhea. Medicolegal Aspects. Not infrequently the physician is called upon to decide whether certain stains are due to spermatic fluid or whether assault has been committed. If 1 See Reynolds, Jour. Am. Med. Assn., i9i3,LXI, 1363; Ibid., 1915, LXV, 1151; Dozzi, Gazz. d. osp., 1914, XXXV, f^f^jf,^^^ f^y Microsoft® SECRETIONS OF THE GENITAL ORGANS 379 the question is one of suspected rape, an examination of a drop of the vaginal fluid or of scrapings from the vulva or vagina will usually reveal the sperma- tozoa. Of course other signs, which are important from the medicolegal point of view, will be observed in the examination of the external organs. The stains usuajly subjected to medicolegal examinations for the presence of spermatic fluid have a grayish-yellow color, their size is somewhat variable, their contour usually irregular, and the linen upon which the stain is usually found is almost as stiff as if it had been starched. As spermatozoa are very resistant to the action of reagents as well as to putrefactive processes, they may be detected many years after the stain was made. It is, therefore, almost an impossibility to say by examination of a stain anything about the length of time the stain has been upon the cloth. A fragment of the linen, which shows the stain, is placed in a watchglass and allowed to soak for one hour in 30 per cent, alcohol or in faintly alkaline water. It is then lifted from the solvent, placed in another watchglass, and teased with needles in a solution of i per cent, eosin in glycerin. A few drops of this mixture are then placed upon a glass slide, covered with a cover-glass and examined with a high-power dry lens. Spermatozoa, if present, will practically always be seen by this method. The heads are stained a deep red while the tails, which are usually broken off by the teasing, show a light reddish tint, which distinguishes them from the unstained vegetable fibers. Florence's Test. It not infrequently happens that spermatozoa may not be found, although the stain be due to spermatic fluid. The principle of this test is that sper- matic fluid when treated with a solution of iodin in potassium iodid gives crystals which were supposed to be iodospermin. According to Bocarius, this substance is not iodospermin, but an iodin compound of cholin. This test would be given, therefore, by any substance containing cholin and cannot, for this reason, be distinctive for spermatic fluid. Such being the case, a negative result is of far greater importance than is a positive one. Technic. The reagent used consists of 1.65 grams of iodin and 2.54 grams of po- tassium iodid dissolved in 30 c.c. of distilled water. If a drop of spermatic fluid or of an aqueous extract of a suspected stain be treated with a drop of this solution and immediately examined under the low-power lens, long rhombic platelets of a dark brown color, fine needles, lance-shaped or ovoid bodies^ often grouped in rosettes may be observed. A positive reaction is seen many years after the formation of the stain so that a positive result is of value when other sources of cholin are excluded. Barberio's Test. Barberio has found that the treatment of a drop of spermatic fluid or an aqueous extract of a suspected stain with a saturated aqueous solution of 1 See Lecha-Marzo and Conejero, Semana Med., 1913, XX, 74; also Maestre and Lecha-Marzo, Ibid., 1914, ^^gffj^ed by MicrOSOft® 380 DIAGNOSTIC METHODS picric acid gives immediately a precipitate of sharply refractile, yellow, ovoid or needle-shaped crystals which gradually increase in size. This test was supposed to be of much greater diagnostic importance than that of Florence, but the recent work of Fraenkel and Mliller has shown that the crystals are not sufficiently characteristic to permit of an absolute diagnosis. They call attention to the fact that substances other than spermatic and prostatic fluids may give similar crystals, but that in such cases these crystals are iso- lated and form usually on the border of the drop, while with spermatic fluids the crystals are numerous and are formed throughout the specimen. These workers recommend this test for the recognition of prostatic secretions or for the condition of azoospermatism, but caution the worker against making an absolute diagnosis from its presence in medicolegal cases. A negative result does not necessarily exclude the presence of semen.'- It will be seen, therefore, that neither one of the micrbchemical tests given above should be regarded as absolutely indicative of the presence of semen. It is much better practice to make repeated search for spermatozoa than absolutely to identify a stain as semen by the microchemical method. II. Female Secretions (i) Vaginal Secretions. The normal secretion of the vagina is scanty, usually just sufficient to moisten the mucous membrane. It is clear or occasionally opalescent, semi- liquid in character, and is composed largely of mucus and epithelial masses. Its reaction appears variable. As a rule, it should be considered acid in the case' of virgins, while in those who have borne children it is usually alkaline. Little is known regarding the chemical properties of this secretion. From the clinical standpoint the normal vaginal secretion is of importance owing to the fact that it possesses marked bactericidal properties. According to Kronig, pus organisms introduced into the vagina of pregnant women dis- appear in from four to thirty-six hours. Whether this bactericidal power is due to the reaction of the secretion or to some unknown agent is unsettled. A remarkable fact seems to be that frequent irrigation of the vagina with water or antiseptic solutions decreases the bactericidal power. If this be true, it is questionable whether frequent douching is advisable.^ Microscopic Examination. Outside of the great number of large, irregular, stratified, squamous epithelial cells which are constantly found in the vaginal secretion, one observes mucous corpuscles, a few large mononuclear leucocytes, cellular debris, and numerous bacteria. The bacterial flora^ of the vagina is very extensive. These organisms are normally saprophytic and rarely take on pathologic functions, but they may occasionally do so. Among these bacteria we find the colon bacilli, streptococci, staphylococci, and bacilli which are not ^ See Lecha-Marzo, Arch, internal, de MM. leg., 1913, IV, 341. ^ See Zweifel, Monatsschr. f. Geburtsh. u. Gynak., 1914, XXXIX, 459. ' See Kiister, KoUe and Wassermann's Handbuch, 1913, VI, 458. Digitized by Microsoft® SECRETIONS OF THE GENITAL ORGANS 381 unlike true diphtheria bacilli. None of these organisms are particularly im- portant from the clinical standpoint and will be passed with mere mention. We do find, however, certain organisms which give rise to no particular clini- cal symptoms, but which are extremely confusing in the examination for the presence of the gonococcus. As will be learned from the later discussion of this organism of Neisser, it appears in the form of biscuit or coffee-berry shaped diplococci, which are both intra- and extracellular and do not stain by Gram's method. In the early cases the intracellular organisms are few in number. Saprophytic cocci often have the same morphology as the gonococ- cus, but are slightly larger, are variable to Gram's stain, and are rarely intracellular. They are, however, differentiated by the fact that they grow easily upon ordinary media, while the gonococcus requires special media. The more or less normal presence of these saprophytes should be constantly in mind and a diagnosis of gonorrhea made only when clinical symptoms are present to point to the gonococcus. It is wise in all doubtful cases to resort to cultivation, as one may very much regret having made a diagnosis of gonorrhea when such did not really exist. The practitioner should be cau- tioned to take his smear high up, in the vagina, preferably from the region of the cervix uteri. If this be done as a routine fewer specimens will be found showing these confusing saprophytes. Smears are frequently sent to labora- tories for examination which will not show the gonococcus even though pres- ent in large numbers in the vagina. It is not sufficient to take a specimen from simple purulent material which may be present in the lower portion of the vagina as the organisms are frequently absent in these locations. Pathology. Blennorrhea. Physiologically, an increased vaginal secretion (blennorrhea) is seen dur- ing sexual excitement, preceding menstruation, and during pregnancy, when a very profuse secretion may be observed. If this secretion contains a large number of epithelial cells and leucocytes, as seen in nonspecific inflammations, it becomes more or less creamy in color and is then called leucorrhea."^ This type of leucorrhea should be sharply differentiated from the true pus secre- tions observed in the blennorrhagia of gonorrhea, as the former is not neces- sarily associated with pus formation. In pregnancy a slight catarrhal vagini- tis is not infrequent, so that leucorrhea is more apt to appear at such times. If the inflammatory process becomes intense, large shreds of epithelium may be found and ulceration followed by vaginovesical or vaginorectal fistulae may be observed. Such pathologic findings are usually the result of gonor- rhea. In slight catarrhal conditions of the vagina yellowish-gray patches may be seen, which are due to infection with mycotic fungi. It is not infrequent to find the trichomonas vaginalis in the vaginal prepa- rations. This organism has been previously discussed in the section on Feces, to which the reader is referred. The oxyuris vermicularis as well as its ova 1 See Curtis, Surg. Gyn., and .pbs.i^iQ.14. XVIII, joo: Ibid., 1914, XIX, 25. ""■' i^^i^^'edW/h^^MkoW 382 DIAGNOSTIC METHODS have been reported in the vaginal discharge, but do not seem to have excited any pathologic changes. Purulent Secretions. True purulent secretions arising from the vagina are almost always due to the presence of the gonococcus.^ This organism is accountable for a large number of gynecological conditions, so that it is wise for the practitioner to be able to identify it both from its clinical manifestations as well as by its labora- tory detection. In doubtful cases cultures should be made and a portion of the pus dropped into the eye of a rabbit. The gonococcus itself may later be recovered from the conjunctival exudate. It is not to be assumed that the finding of the gonococcus in the vaginal discharge is necessarily evidence of a gonorrheal vaginitis or vulvovaginitis. It has been shown that the semen of the male as well as the urethral discharge may contain gonococci and that these may be introduced into a perfectly normal vagina without necessarily setting up gonorrhea. However, this is unusual. The gonococcus may arise from the urethra, the Bartholin glands, acute inflammatory processes of the uterus, or from a ruptured pyosalpinx. In any of these cases the gonococcus may be found in the vaginal discharge, so that the laboratory worker should be guarded in his diagnosis of a vaginitis. Further, suppurative processes which were originally due to the gonococcus may later take on a type of mixed infection or even become of the sterile type. This latter condition is especially observed in old Bartholinitis, metri- tis, and cystic salpingitis. The organisms usually associated with the gono- coccus in the mixed infection are the streptococcus, staphylococcus, colon bacillus and pseudodiphtheria bacillus. In chronic metritis or salpingitis it is not infrequent to find the tubercle bacillus as the causative agent. Fetid Secretions, In these conditions the pus is usually chocolate colored, has a fatty ap- pearance, is extremely repulsive, is frequently sanguinolent and contains numerous degenerated cells as a result of marked leucolysis. This condition is especially observed in puerperal infection and may be extremely severe. (2) Uterine Secretions. Normally, the .uterus has no secretion beyond a slight mucoid one which is recognizable clinically. In inflammatory conditions, during normal men- struation, or following abortion or parturition, certain types of discharges are observed which have some clinical importance. Menstruation. Under normal conditions the menstrual fluid is at first mucoid in char- acter, but within a short time red cells appear and later the discharge takes on almost the character of pure blood. This menstrual fluid should be bright red in color, should contain no clots and should be discharged without caus- ing active pain. This fluid contains red cells, leucocytes, and prismatic epithelial cells showing large areas of fatty degeneration. The duration of the ^ See Sharp, Jour. Infect. Dis., 1914, XV, 283. Digitized by Microsoft® SECRETIONS OF THE GENITAL ORGANS 383 menstrual period is variable, running between two and five days. The amount of blood lost under normal conditions averages about 200 grams, but may be much larger under pathologic conditions. Not infrequently do we find cases in which menstruation is associated with marked pain during more or less of the period of flow. This condition is known as dysmenorrhea and may be associated with the exfoliation of large shreds of mucous membrane, in some cases reported these shreds constituting almost a cast of the uterine cavity. To this latter condition is given the name membranous dysmenor- rhea. For the pathologic significance of these abnormal types of menstrua- tion as well as for a discussion of the condition associated with failure of menstruation (amenorrhea) the writer must refer to works on gynecology. The Lochia. By this term we have reference to the discharges from the uterine cavity during the puerperium. At first such discharges consist of blood, which may be in the form of clots, and decidual shreds along with epithelial cells which are probably of vaginal origin. This type is known as the lochia rubra or cruenta. During the next two or three days the discharges become paler and thinner, the red cells diminish and the leucocytes increase, while the decidual shreds may continue approximately the same. This type is known as the lochia serosa. After about a week the discharge assumes a grayish or yellow- ish color and a creamy consistency, the red cells diminishing rapidly and the white cells increasing markedly. Microscopic examination shows, besides the leucocytes and epithelial cells, numerous fat globules and cholesterin crystals. This discharge may continue during the remainder of the period of uterine involution and is known as the lochia alba. Under normal condi- tions the lochial discharge has a faint odor, but is never fetid. If a portion of the placenta or membranes have been retained, the lochia may assume a dirty brownish color and become extremely fetid. After the first two or three days numerous bacteria, such as staphylococci, streptococci and colon bacilli, may be present, but no untoward symptoms exist unless these, along with other saprophytes, give rise to a distinct puerperal infection or sapremia. Amniotic Fluid. This is a thin, whitish or pale-yellow fluid containing the constituents of ordinary transudates. The reaction is neutral or faintly alkaline, the specific gravity varies between 1002 and 1008, and the amount of solids rarely reaches 2 per cent. The albuminous bodies are principally vitellin, serum albumin, and traces of mucin, while glucose is absent. Urea and allantoin are present in traces, while creatinin has occasionally been reported. The amount of amniotic fluid varies between 700 and 1,000 c.c. Under pathologic conditions, however, this amount may be increased or decreased, giving rise on the one hand to polyhydramnios or dropsy of the amnion and on the other to oligohydramnios. For a discussion of these pathologic varia- tions as well as for a treatment of the subject of pathologic changes in the membranes, the writer will refer to works on obstetrics. Digitized by Microsoft® 384 DIAGNOSTIC METHODS Abortion. The recognition of abortion is usually made by examination of the material discharged from the uterine cavity. Usually one finds blood-clots in which the villi of the chorion are present. These usually appear as club-shaped masses with epithelial coverings, showing the characteristic capillary net- work. Moreover, decidual cells are usually present, and may be recognized by their large size, their round, polygonal, or spindle-shaped form, and their irregular and Urge nuclei with nucleoU.^ Fig. 1 1 6. — Chorionic villi. {McMurrich.) Vesicular Mole. This condition has been called dropsy of the villi of the chorion, hydatidi- form degeneration of the chorionic villi, cystic mole, and myxoma of the placenta. One of its most important symptoms is the expulsion through the vagina of the vesicles forming the degenerated mass. The mole is a mass of pedunculated vesicles which closely resemble a bunch of grapes or gooseberries. Each vesicle may vary in size from a millet seed to a large hazelnut and con- tains a fluid which is usually colorless and limpid, but may be reddish and somewhat dense. Macroscopic examination of the tissue shows the peculiar myxomatous degeneration of the chorionic villi. ^ Carcinoma. It is not infrequent to observe, in cases of severe hemorrhage through the vagina, the appearance of occasional shreds, which on microscopic examina- tion show the characteristic appearances of carcinoma of the cervix or body of the uterus. The diagnosis of carcinoma, however, would better be made upon sections removed by the surgeon rather than upon shreds found in the hemorrhagic fluids. Although a diagnosis may be at times possible, it should be somewhat guarded unless the clinical symptoms are distinctive. 1 See Lackner, Surg., Gyn. and Obs., 1915, XX, 537. 2 See Westermark, Hygiea, 1914, LXXVI, 801. Digitized by Microsoft® CHAPTER VIII THE BLOOD I. General Considerations The blood is perhaps the most important tissue in the body, inasmuch as it is at once the purifier and the nutritive source of the cell. Any normal or abnormal product of cellular activity finds its way ultimately into the blood, either to be taken up by the assimilatory organs or to be thrown out by the excretory ones. While but relatively few disease processes are as- sociated with diagnostic findings in this tissue, yet many are characterized by definite manifestations which are invaluable aids to the clinician. . It is, therefore, of the utmost importance that we should have a proper knowledge of the normal blood in order better to understand the various phases which characterize abnormal blood and which give to hematology such an interesting and important position in diagnosis. While it is true that some of the methods involved in hematological examinations require definite apparatus and a large experience for their proper interpretation, yet the results obtainable from the ordinary routine blood examinations are so invaluable, being in some cases pathognomonic, that no practitioner should consider himself fitted to give his patients the proper service without being equipped with a clear working knowledge of the methods of examination and the findings of normal and of abnormal blood. It is essential to remember that certain physiologic as well as pathologic conditions influence the quantity and quality of the blood. So great are the effects of digestion, exercise, nervous factors, massage, cold, heat, sweats, dysentery, "constitutional and specific diseases, that one does not wonder at the many conflicting reports of cases showing widely varying hematological findings. As Grawitz has pointed out, no conclusion should be drawn from an examination of the blood without taking into consideration the physiologic and pathologic condition of the patient. It is a pleasure to observe in these days the tendency toward a more rational and thorough study of the plasma, the so-called "intracellular fluid" of the blood. We have' forgotten, in our enthusiasm over the many valuable findings obtaine'd from histological investigations, that the relations of the fluid portions of the blood are, in some cases, of quite as much importance as are the variations in the cellular elements. It is necessary only to cite the work on lysins, precipitins, agglutinins, opsonins, etc., to show the value of a more extended study of the plasma or serum. Regarding the technic of blood examinations, the writer wiU have much to say later, but he wishes, to inipressimon his readers one point which has 386 DIAGNOSTIC METHODS been well expressed by Tiirk, namely, an indispensable basis for the proper utilization of any diagnostic, prognostic, or therapeutic method of clinical examination is a knowledge of the absolute limitations of the method. Reli- able results can, however, be obtained only by those who are thoroughly familiar with the principles as well as with the technic and the Uttle "knacks" of the method used. It will be found, when the attempts are made to apply the methods outUned, that quite as much depends on the exactitude with which the separate details are carried out as upon the selection of the method itself. It is not to be expected that a first trial will yield exact results or that a few determinations will perfect one in the methods of examination. Experi- ence is the only teacher that can equip one with the skill and power of inter- pretation necessary to cope with the many difficulties to be overcome in the hematological investigations. II. Physiology and Chemistry (i) Blood Formation and Blood-forming Organs. While it is impossible in a general work of this character to go into great detail regarding the formation of the blood, yet it seems to the writer that a brief discussion of this subject is extremely valuable both to the student and practitioner. '^ " Red Corpuscles. According to KoUiker, the first blood-corpuscles have their origin, in embryonal life, in the embryonic heart and blood-vessels. They appear as nucleated colorless cells, which later develop into colored corpuscles by the appearance of hemoglobin in some of the cells of the mesodermal cord, which cells go to form the first capillaries. Upon the formation of these vessels the cells lie within them as nucleated reds. At this time there are no true leucocytes and none appear until after the complete formation of the red cells, which is advanced as an argument against Pappenheim's theory of single origin of red and white cells. It will thus be noted that the vessel wall and the primitive erythrocyte have a common origin in^the meso- dermal cord, the peripheral cells going to form the endothelium of the vessel and the internal cells the corpuscles. Up to the end of the fourth or fifth week of embryonal life all of the red cells are nucleated; while from that time on the relation of the nonnucleated to the nucleated forms gradually increases until at birth few if any nucleated cells obtain. In later embryonal life (about the third month), the liver becomes the chief seat of blood formation. During the fifth month, the spleen^ and lymph- glands take up this work, and finally the bone-marrow becomes the seat of such activity. In extrauterine life, the bone-marrow is the chief point of formation of the red cells, but under pathological conditions the spleen and Uver may as- sume their embryonic functions. It appears that the formation of nucleated 1 See Gruner, The Biology of the Blood Cells, NewliYork:, 1914. 2 See Morris, Jour. Exper. Med., 1914, XX, 379; Hirschfeld and Weinert, Berl. klin. Wchnschr., 1914, LI, 1026. „. .^. ,, ,,. ^^ Digitized by Microsoft® THE BLOOD 387 reds in the adult is practically the same as in the embryo and that, at all . periods of life, the red cell is the product of several series of mitoses of a colorless mesoblastic cell. The difficulty of tracing this series, from the large nucleated red cell to the colorless mesoblastic "mother-cell" in the marrow, has given rise to the diverse opinions now held regarding the ulti- mate development of the red corpuscles. Leucocytes. The earliest indications of the formation of leucocytes are seen in the presence of primary wandering cells, of mesodermal origin, which are found principally in the loose connective tissues of the, early embryo. Though of mesodermal origin they are, from the first, quite distinct in morphology and, apparently, in function from the capillary endothelium and fixed con- nective tissue cells. Their development has been traced by Ziegler to masses of mesodermal cells surrounding the cords from which the capillaries are formed. It thus seems that originally the parent leucocytes lie outside the vessels, into which they make their way by virtue of ameboid powers. Most observers find that the primary wandering cells produce, by mitotic division, one or more generations of colorless cells which gradually approach, in morphology, the early basophilic leucocytes of the circulation. Denys, Lowit, Ziegler, von der Stricht, and others claim that red cells and white cells develop from separate series of cells, which have become differentiated from the primary mesodermal cells with the first appearance of blood and blood-vessels. Kostianecki, MiiUer, Schmidt, Saxer, Pappenheim, and others believe that the primary wandering cell persists in the blood-forming organs as the parent of both red and white cells. Before the leucocytes begin t3 appear in the circulation, mitotic figures are abundantly seen in the primary wandering cells in various situations. These are gathered in groups, first in the loose connective tissues of various regions, where lymph nodes subsequently develop; but the chief seat of the production of the leucocytes is found in the embryonal liver. In both situa- tions the wandering cells are found in lymph and blood capillaries, in the interstices of the connective tissues, and between the liver cells. In later embryonal life the process is gradually transferred from the liver to the lym- phoid and adenoid tissues, as indicated by the development of lymph nodes, spleen, marrow, and thymus. Under normal conditions, the reproduction of leucocytes, in the adult, is limited to the lymphoid structures both of the lymph-glands and bone-marrow. (2) Total Volume of Blood. The various methods which have been advanced for the estimation of the total quantity of blood in the body are subject to such wide variations that they have jdelded Uttle exact information regarding this subject. The procedures advocated by Valentine, Vierordt, Buntzen, and Thibault have an error sufficiently great to exceed the physiological and pathological varia- tions of the bloo(^. By these methods, the quantity of blood in the body has been estimated as egi^J;ied)65^VtffE*«^fe®f the body-weight. The so- 388 DIAGNOSTIC METHODS called clinical methods of Quincke or of TarchanofE are of purely the oretical interest, because certain factors, such as the appearance of the patient or the volume of the pulse, are taken into consideration in making a rather un- reliable guess as to the total quantity of blood in the body (Buckmaster). By the use of a reliable method, introduced by Haldane and Smith,' the total volume of blood may be fairly accurately estimated. This method is based on the following points. The capacity of hemoglobin for oxygen and for carbon monoxid is identical. On the assumption that none of this latter gas is oxidized in the body, and that no substance in the blood, other than hemoglobin, unites with it, the experimenter is able to determine the CO capacity and hence the O capacity of the blood. This method has, however, little clinical application and will be left with reference to the original work. In the cases studied by Haldane and Smith by this method, the average value was 3,240 c.c. or, on the basis of a specific gravity of 1,060, about 3,434 grams. This yields, according to Smith, a figure ranging between one-six- teenth and one- thirtieth of the body- weight.^ In obese persons the volume of blood is less, proportionately, than in the more normal specimens of mankind. The question of the volume of blood in the body is of great importance in the study of the changes taking place in this tissue. It must be remembered that the number of red or of white cells in a cmm. of blood will depend upon the total amount of blood present. If for any reason the volume is diminished ed or increased, corresponding changes, in the inverse sense, will be observed in the number of the cellular elements per cmm. It seems to the writer, there- fore, that certain factors, not ordinarily, taken into account in blood examina- tions, should be known before any definite report is made upon a blood count. It may be readily seen that a concentration of the blood, due to hemorrhage, diarrhea, sweating, etc., will lead to an apparent increase in the number of corpuscles. Certain physiologic and pathologic conditions lead to definite changes in the volume of the blood, as such, or of some of its constituents. As Plehn has recently shown, the volume remains quite constant or is adjusted through the activity of the capillary endothelium and through the influence of the nervous system. However, definite changes of a more or less transitory nature do occur and exert marked influences on the results of blood examinations as well as upon many pathologic conditions.^ Oligemia. By this term is meant a reduction in the total volume of blood, both as regards the liquid and the cellular portions. This condition is most fre- quently noticed after profuse hemorrhage and, may be so marked that death results. In other cases, in which the hemorrhage is less extensive, the loss of 1 Jour. Physiol., 1896, XX, 497; Ibid., 1900, XXV, 333. ^ Keith, Rowntree and Geraghty (Arch. Int. Med., 1915, XVI, 547) consider that the blood constitutes 8.8 per cent. (11/11.4) of the body-weight. Normal individuals have approximately 85 c.c. of blood per kilogram. '2 See Kammerer and Waldmann, Deutsch. Arch. f. klin. Med., 1913, CIX, 524; Miller, Keith and Rowntree, Jour. ^g^^cf?g>-yvfftM558^"9. THE BLOOD 389 blood is made up by osmosis from the lymph spaces into the capillaries and, later, by an increase of the cellular elements due to compensatory activity of the hematopoietic organs. Plethora. The opposite of the preceding condition is known as plethora, a state characterized by an increase in the total volume of blood. There has been much discussion as to whether a true plethora exists, but there can be little doubt that a transitory plethora vera may occur as a result of direct transfusion of blood, and also, according to Bergmann and Heissler, who have established the fact that there is a direct ratio between the volume of blood and the size of the heart, on the one hand, and the muscular development of the subject, on the other, as the result of increased muscular activity, provided the loss of fluid by perspiration is not excessive. Such a plethora disappears, of course, in a very short time. In this discussion we must distinguish between a serous and a cellular plethora. By the former is meant an increase in the volume of blood due to excessive quantities of its liquid and soluble constitutents; while by the latter we understand an increase in the number of corpuscular elements, that is a polycythemia. Serous plethora is most frequently observed in organic lesions of the kidneys and of the heart, in which a diminished elimination of water and inorganic constituents is noted. This condition is usually of a transient duration, as the volume of blood is soon restored to normal by intra- capillary transudation and by diffusion. Osmotic effects must also be taken into consideration here, inasmuch as the salts will tend to diffuse out from the blood and will consequently draw water after them, giving rise, under certain conditions, to dropsical effusions of a more or less transient duration. Hydremia. Another condition of the blood is frequently observed, in which an in- crease in the quantity of the liquid of constituents is observed. This is known as hydremia and is different from serous plethora, as the latter carries with it an increase in the saline as well as in the watery portion of the blood. In hydremia the specific gravity of the blood is reduced, while in serous plethora it is increased. As EngeP has shown, the estimation of the coefficient of refraction of the blood serum is a reliable method for the clinical study of the subject of the water content of the blood. Hydremia may be produced by any factor which changes the normal relationship of the blood constituents in such a way that the watery portion is relatively increased. It is in these more or less physiologic states that we are apt to observe the greatest variation in the blood counts. As the cellular elements are not simultaneously increased, the drop of blood under examina- tion contains relatively fewer cells than normally. The most common physiologic causes of hydremia are the ingestion of large quantities of fluids, saline transfusions, and vasomotor dilatations as a result of exercise or nervous influences. In severe anemias we find the watery portion of the blood rel- 1 Magyar Orvosi Arch., i "Bf^iiizS^^y Microsoft® 39° DIAGNOSTIC METHODS atively increased. In dropsical states, following cardiac or renal lesions, we often observe such a condition, whose duration will depend, of course, upon the etiological factors of the trouble. Anhydremia. This is a condition characterized by a diminution in the liquid constituents of the blood. There is no change in the cellular elements and hence a blood count will show an erroneous increase in the number of cells. In this condi- tion the specific gravity of the blood is naturally increased. Anhydremia follows any condition which results in the excessive loss of fluid from the body, as, for instance, that following profuse diarrhea, poly- uria, sweating, vomiting, and effusions into the various serous cavities of the body. According to Oliver, this state may be caused by influences which in- crease the arterial tension and hence bring about an increase in the passage of water from the vessels into the tissues. Thus, for instance, we may observe anhydremia following local and general exercise, massage, bathing, etc. (3) Volume Relations of Cells of Plasma. The study of the relationship between the cellular and the intracellular portions of the blood is a comparatively recent addition to the technic of blood examinations. This determination is based on the principle that the corpuscles may be thrown by centrifugal force to the distal end of a calibrated tube, while the plasma will collect in the proximal portion. If the tube be properly calibrated, the percentage relations of the cells and plasma may be readily ascertained. These ideas were used by Hedin in elaborating the earlier methods of Blix. Fig. 117. — Daland's hematocrit. Daland's Hematocrit. Daland'has introduced a modification of the clumsy model of Hedin and has succeeded in simplifying the technic to such an extent that this method is directly applicable to clinical use. His instrument is shown in the accom- panying cut. One of the calibrated glass tubes is fitted with the rubber tubing and is filled with blood from the ear or finger. The forefinger, smeared with a little vaselin, is then placed over the beveled end of the tube and the rubber tubing withdrawn. Insert the tube into one arm of the frame, the other arm of which should carry the second tube filled in the same manner, in order to balance the instrument and to control the reading. Rotate the spindle for three minutes at such a rate of speed as will insure 10,000 revolutions per minute (80 revolutions of handle). In this way the corpuscles are separated Digitized by Microsoft® THE BLOOD 39^ from the plasma and are distinguishable as a distinct column, which may be read off directly from the graduations of the tube. These divisions will give the percentage relations of the cells and plasma as the tube is divided into I GO equal portions, each division of the scale representing approximately 100,000 cells. This latter makes it possible to make a rather rough blood count with this instrument, but it is to be remembered that accurate results cannot follow, as we find such variations both in the size and elasticity of the cells in the different conditions in which the number of cells is most sought. Fig. 118. — Hematocrit tube. Volume Index. Capps^ has introduced the conception of volume index, that is the quotient of the volume per cent, as obtained with the hematocrit, and the blood count in terms of per cent. Sahli advises the use of the expression volume quotient or volume value for this factor. By means of the hematocrit the volume of the red cells, as compared with that of the whole blood, is taken. In normal cases this is about 50 per cent, which is reckoned as one. Hence the volume of the red cells may be obtained directly in percentage value. The red cells are then counted by the method to be later outlined, and the result is expressed in percentage by comparing this count with a so-called normal one of 5,000,000 red cells. By dividing the volume of red cells (in per cent.) by the per cent, of red cells, Capps obtains his volume index of the red cells. In normal conditions this quotient is one. According to Capps, an in- crease of this index is a constant factor in pernicious anemia. The color in- dex never exceeds the volume index in such cases, which fact shows that there is no supersaturation of the corpuscles with hemoglobin. In primary and also in secondary anemia this factor is diminished. Here we find the color index often falling below the volume index. This method may be used in detecting various pathological conditions of the blood. According to Emerson, it is used in the Johns Hopkins Hospital in ascertaining the presence of lipemia, cholemia, or hemoglobinemia. It would seem to the writer that it could be employed with advantage as a routine procedure, especially in hospital practice. The osmotic pressure of the plasma plays a great r61e in this determination, as the concentration of the blood may be such as to cause swelling or shrinkage of the cells. As Capps has found, normal red cells with a volume index of one have their dis- coplasm saturated with hemoglobin. Hence, if the hemoglobin index be- comes greater than one, an enlargement of the red cells is indicated. Upon the other hand, the color index may fall, regardless of a corresponding lower- ing of the volume index. It follows, therefore, that, if the color index is > Jour. Med. Research, 1903, X, 367. Digitized by Microsoft® 392 DIAGNOSTIC METHODS above normal, the volume index must also be increased; while, if the hemo- globin index is below normal, the volume index is not necessarily diminished (SahU). (4) Methods of Obtaining Blood. The method of obtaining blood for examination depends upon the amount desired and on the examination which is to be made. For ordinary routine work only a few drops are necessary, while for bacteriological investigations 2 to 20 ex. may be required. In obtaining the blood no set rule can be laid down as to the proper place from which to take the specimen. We should select the part which promises the best results, avoiding naturally the points which are cyanosed, eczema- tous, edematous, hyperemic, cold, unduly heated, or, in other words, any part which is not normal. The ear usually furnishes the best results, in the writer's opinion, because its puncture is relatively painless, it is easily acces- sible (which point is often of importance in attempting to obtain blood from those who are comatose or who refuse to submit their hand for puncture), and because the patient, who may be easily affected by the sight of blood, can not see the drop. If the lobe of the ear is thick it is usually pricked on the flat side, but if it be thin it is well to make the puncture on the edge and par- allel to the surface. Some workers prefer the palmar surface of the ball of the middle or second finger of the left hand and others advise pricking the arm over a small superficial vein. In cases in which repeated examinations of the blood are to be made the parts should be varied in order to prevent soreness and also to avoid anesthesias which occasionally follow repeated use of the same site of puncture. Quite a number of special forms of blood needles, as, for instance, those of Francke and of D aland, are on the market, and each of them has its ad- vocates. Many of them are in the form of special holders, which permit a puncture of a desired depth to be made without any danger of going too deep. These are to be recommended to those only who seem unable to control their stab. The writer does not find, that the results obtained by the student are any better, with such instruments than are those following the use of the ordinary Hagedorn surgical needle. In lieu of any better article, a clean steel pen -vi^ith one nib broken will yield admirable results. The one thing to bear in mind in selecting a needle for blood work is that the point have a cutting-edge and should not be round or sharp nor should it be too long or slender. Having decided upon the part from which the blood is to be taken, this surface is washed carefully with water and alcohol or ether and is then thor- oughly dried. Avoid any undue rubbing as this will cause hyperemia and will introduce an error into the work. It is, as a rule, unnecessary to sterilize the needle but if it seems advisable to do so on account of the patient's attitude, the sterilization is best done by heat, hydrogen peroxid or alcohol, allowing the needle to cool before making the puncture. In this latter part of the technic, much depends on the amount of blood desired as regards the use of a short quick stab or a slow steady puncture. The latter procedure usually Digitized by Microsoft® THE BLOOD 393 jaelds more blood but is more painful. It is much better to prick the patient too deeply, going even through the lobe of the ear, than it is to subject him to repeated punctures. The part pricked should not be squeezed nor held in a position which will cause an abnormal circulation. If the puncture is suc- cessful, the blood will come out in good-sized drops. The first of these are wiped away and subsequent ones used for the examination. As often hap- pens, the blood coagulates fairly quickly, so that the coagulum should be wiped off with a little alcohol followed by a dry cloth. It is important to remember that a patient with hemophilic tendencies or history may bleed very easily from a very slight puncture. Care should, therefore, be taken to question the patient regarding the ease with which blood flows from a wound and also regarding the history of "bleeders" in the family. If considerable blood is desired, resort must be made to venous puncture. Thesite of this operation is usually the median basilic vein at the bend of the elbow. This vein may be made more prominent by tying a tight bandage around the arm, but the band- age should be removed before the blood is withdrawn, except when serum reactions are to be studied. The site of puncture must be thoroughly cleansed, using the precautions observed before any surgical operation. Likewise, the needle and the aspirating instrument must be absolutely sterile before puncture is made. The instrument best adapted for this purpose is, in the writer's experience, the Llier syringe, which is made of glass with a tightly fitting glass plunger and adjustable platinuni needle. From 2 to 20 c.c. of blood are withdrawn and immediately placed in work. The question of the bacteriological examination and of serum reactions will be discussed in a later section. (s) Physical Properties. The blood must be regarded as a fluid tissue, consisting of a transparent liquid, the plasma or liquor sanguinis, in which are suspended the corpuscular elements, erythrocytes, and leuco- cytes. Besides these latter cells we find two constituents, the blood plates of Bizzozero and the hemoconien (blood dust), which Fig. up.— are hardly to be considered as true corpuscular entities. needle As it flows from the vessels, blood is a thick, viscid, red liquid, having a peculiar odor, a salty taste, and an alkaline reaction to litmus. If allowed to stand it shows, unless certain precautions are observed, the pecuhar phenomenon of coagulation. In this process the blood is separated into two portions, the cellular elements and the plasma, the latter changing, as the process goes on, into serum and the clot (fibrin), which holds the corpuscles in its meshes. In the diagram given below, adapted by Webster and Koch, may be seen the composition of the blood. Digitized by Microsoft® 394 Plasma. Blood' Serum DIAGNOSTIC METHODS Serum Albumin. Serum Globulin. Glucose, extractives, calcium salts, sodium and potassium chlorids, carbonates, phos- phates, etc. Fibrinogen (yields fibrin). Cellular Elements ■Red Corpuscles White Corpuscles - Blood Plates. Hemoconien. Oxy-hemoglobin. Lecithin. Salts. Fibrin Ferment. (A) Color. The color of the bleod is due to the presence in the erythrocytes of an iron- containing albuminous substance, hemoglobin, which has remarkable affinity for oxygen and other gases. This latter property enables this pigment to play one of the most important roles in the body economy. Arterial blood is bright red in color, while venous blood shows a purplish-blue tint. These variations are due entirely to the relative proportions of oxygen and of carbon dioxid in the two types of blood. Many different shadings are observed, physiologically and pathologically, in the coloration of the blood, and each is due to some combination of hemoglobin with normal or abnormal substances. The presence of large numbers of red corpuscles in the blood gives rise to a characteristic opacity of this tissue. If, for any reason, such as admixture of blood with water, dilute salt solutions, urea, ether, snake venom, extract of mushrooms, etc., the blood loses its opacity, the change is due to the dis- solving out of the hemoglobin from the red cells. This is the well-known phenomenon of " laking' ' or, better, of hemolysis, about which we will have something to say later. The normal color of the blood is often changed in pathological conditions. Thus, in anemia the blood is pale and watery; in leukemia it may be milky; in diabetes buff-colored; while in poisoning with potassium chlorate it is chocolate-colored and in that with carbon monoxid it is bright red.^ {B) Odor. The odor of the blood is peculiar and indescribable. This halitus san- 1 See GaisbSck, Med., Klinik, 191 2, VIII, 1906. Digitized by microsoft® THE BLOOD 395 guinis is due to the presence of certain volatile fatty acids and may be ren- dered more distinct by the addition of concentrated sulphuric acid, which increases the volatility of these acids (Barruel's test). (C) Reaction. If we are to accept the teachings of physical chemistry, that the alkalinity of a solution is due to the presence of free hydroxyl (OH) ions and that its acidity depends on the surplus of free hydrogen (H) ions, we must grant that the blood is a practically neutral fluid. If, however, we have in mind the acid- combining power of the blood, we must regard the reaction of this tissue as alkaline. It is certain that the blood shows both acid and alkali combining powers due to the presence of protein constituents as well as to both acid- and alkali-reacting substances, the measure of such powers being dependent on the indicator used in the estimation. As the combining power for acids is greater, in the case of blood, than it is for alkalies, the reaction, as judged by titrimetric methods, must be alkaUne. The normal free or diffusible alkalinity of the blood is due to the presence of disodium hydrogen phosphate (Na2HP04) , sodium bicarbonate (NaHCOs) , and sodium carbonate (Na2C03). This total diffusible alkali constitutes, according to Brandenburg, about 20 per cent, of the entire alkalinity and may be measured by dialyzing against known alkaline solutions and observing the concentration at which the strength of the known solution does not change. This factor represents the so-called alkaline tension of the blood and remains fairly constant, in normal cases, at about 60 mg of NaOH per 100 c.c. of blood, while in pathologic conditions, such as uremia, diabetes, etc., it is somewhat reduced. Besides this diffusible alkali, the blood contains nondiffusible alkali bound to the proteins. This portion represents normally about 80 per cent, of the total alkalinity, and is dependent largely on the cellular content of the blood, as the soluble protein constituents are not generally subject to wide varia- tions. The fluctuations in this nondiffusible alkali are no doubt accountable for the great differences in the figures given for the alkalinity of the blood. The subject of the reaction of the blood is one which should furnish, if properly'' studied by reliable methods, much valuable data upon subjects which are now very obscure. Unfortunately, however, most of the methods at our command are so unreliable and so inexact that the results have little more than a comparative value. As Moore and Wilson^ have pointed out, the titration methods do not give us the true neutrality of the blood, but rather the amount of alkali or of acid which may be added to it without rais- ing the hydroxyl or hydrogen concentration above certain low limiting values. The reaction of the blood depends on the indicator used for the determina- tion of the neutral point and cannot be definitely measured by any method which employs an indicator for such purposes. These writers have intro- duced the term "reactivity" to indicate the property, possessed by the ' Biochem. Jour., 1906, I, 297. See, however, Sellards, Bull. Johns Hopkins Hosp., 1914, XXV, loi. Digitized by Microsoft® 396 DIAGNOSTIC METHODS blood, of combining with both alkaUes and acids in such a way as not to raise its ionic composition. In selecting the methods for the quantitative estimation of the so-called alkalinity of the blood, I limit myself to those of Dare' and of Salkowski,'' as neither depends upon the titration of the blood. The figures given by the older titration methods have such a wide range, being as low as 162 mg. and as high as 800 mg. of NaOH per 100 c.c. of blood, that the writer feels com- pelled to give them up for methods which seem to yield more exact and ac- ceptable results. Method of Dare. ^ -y. This method is based on the fact that the absorption B jl bands of the spectrum of oxyhemoglobin disappear at the H ^£^^ point of exact neutralization of the blood. Although it I ^^HE. has not been conclusively proven that oxyhemoglobin is f ^UP not destroyed before the point of complete neutralization ill ^« is reached, yet the results with this method are rarely so variable as are those with the titration methods. The apparatus (hemoalkalimeter) may be seen in the accompanying illustration. It consists of a special grad- uated tube (A), a blood pipet (B) which holds 20 cmm. (15 mg. by weight) of blood, a reagent pipet (D), and a hand spectroscope (E). The method of using this instrument is as follows: The tube (A), into which fits the pipet (B), is held hori- zontally in such a manner that the pipet may be filled by capillary attraction with blood from the finger or ear. With a medicine dropper, containing distilled water, the pipet is washed free of blood, using sufiicient water to Fig. 120. — Dare's bring the diluted blood to the zero-point of the tube A. hemoalkalimeter: A, 'pjjjg diluted blood is then thoroughly mixed by shaking alkahmeter tube; B, , . . , , . ,.,....„ , automatic blood pi- and invertmg the tube, after which agitation it is allowed pet; C, air hole; D, (-q settle. In this process care should be taken to close the opening (C) with the thumb to prevent loss of fluid. The reagent pipet (D) is then filled with the following test solution (N/200 • tartaric acid, each c.c. of which is equivalent to .0002 gram of NaOH) and is connected with the end of the blood pipet by means of rubber tubing. M Tartaric acid, Alcohol (95 per cent.), Distilled water, q. s. ad.. 0.37s gram. 100.000 c.c. 1,000.000 c.c. By compression of the bulb of the pipet the reagent is forced through the blood pipet into the tube A. Mix the fluids by inversion, care being taken to avoid loss of fluid. Adjust the spectroscope to the tube below the zero point after each addition of reagent and observe the spectrum for the presence of the ' Phila. Med. Jour., 1903, XI, 137. 2 Centralbl. f. d. med. Wissensch., 1898, XXXVI, 012. Digitized by Microsoft® THE BLOOD 397 two bands of oxyhemoglobin. This is best done by holding the tube a con- stant distance from an artificial light. This part of the technic is very tedious, but is necessary until the observer learns to associate the change in color of the mixture with the point of neutralization. If the spectroscopic bahds of oxyhemoglobin still persist, more reagent is added and the mixture shaken as before, until these bands can no longer be seen. This is the point of neutralization and the end-point of the test. Read off, from the gradua- tions on the tube, the number of c.c. of reagent used. The following table, representing the graduations on the tube, gives the equivalent of NaOH for every fraction of a c.c. of reagent used. It is calculated on the basis of 15 mg. of blood for every 2 c.c. of the N/200 tartaric acid. Dare considers the nomal figure to be 2 c.c. of acid, which represents 266 mg. of NaOH per 100 c.c. of blood. C.C. of Milligrams of NaOH reagent. per 100 c.c. of blood. 3 433. ,, ... „^ 26 Digitized by Microsoft® 402 DIAGNOSTIC METHODS place under the influence of a ferment, thrombase, which is present in the leucocytes in the form of prothrombase. This latter zymogen, through the influence of calcium compounds, is changed into thrombase, the active agent in bringing about coagulation. It is occasionally of clinical importance to know the time of coagulation of the blood under certain conditions, as one of the normal processes of this tissue, when outside the vessels, is coagulation. This process takes place normally in from two to eight minutes, depending on several factors, among which are the length of time the blood is in contact with the tissues, the depth of the incision, the pressure with which the blood is expelled, the nature of the vessel into which the blood flows, and the temperature at which coagulation takes place. It has been found that certain variations in the normal coagulability are present at different hours of the day and are observed when blood is drawn from different parts of the body.^ Dorrance's Method. This method^ is simple and reliable. The instrument consists of an 8- ounce thermos bottle without the silver lining, an aluminium stand and two rubber stoppers, one of which has one hole for the passage of a thermometer, while the others bears four holes each of which is lined with a brass flange. Into each of these holes fits a glass rod 4M inches long and 8 mm. in diame- ter. The lower end of each rod is cone-shaped, the flat tip being 4 mm. in diameter. The other end is slightly bulbous to hold the rod in place. Fill the bottle with water at 98° F. to within an inch of the top. Insert the stopper with thermometer. Scrub the glass rods with soap and water and cleanse them with alcohol and ether. Substitute the stopper with rods for the other, so as to warm them to 98" F. Puncture the ear or finger and wipe away the first two or three drops of blood. Remove the stopper with rods, wipe the latter dry and touch the drop of blood with the tip of each rod in turn, getting as nearly as possible the same amount of blood on each. Note the time and replace the stopper in bottle. The end-point is determined in three ways: (i) At the end of two and one- half minutes push rod i down into the water. All the blood normally falls off the end and breaks up into a fine cloud. At intervals of one minute the other rods in turn are pushed into the water. Some blood falls off but the particles become coarser, the amount remaining on the tip becoming greater until with rod 4 most of the blood remains, if coagulation has occurred. If no '■ See Cohen, Arch. Int. Med., 191 1, VIII, 684 and 820; Howell, Am. Jour. Physiol., 191 d XXIX, 187; Therap. Gaz., 191 2, XXXVI, 95; Bordet and Delange, Ann. del'Inst. Pasteur. 1912, XXVI, 657; Berl. klin. Wchnschr., 1914, LI, 497; Barratt, Jour. Path, and Bacteriol.i 1913, XVII, 303; Whipple, Arch. Int. Med., 1913, XII, 637; Cannon and Gray, Am. Jour. Physiol., 1914, XXXIV, 232; Cannon and Mendenhall, Ibid., 225, 243 and 251; Gray and Lunt, Ibid., 332; Howell, Ibid., 1914, XXXV, 143 and 474; Pepper and Krumbhaar, Jour. Infect. Dis., 1914, XIV, 476; Fingerhut and Wintz, Miinch. med. Wchnschr., 1914, LXI, 363; Stuber and Heim, Ibid., 1661; Fonio, Mitt. a. d. Grenzgeb. d. Med. u. Chir., 1914, XXVII, 642; Ibid., 1914, XXVIII, 313; Lee and Vincent, Arch. Int. Med., 1914, XIII, 398; Ibid., 1915, XVI, 59; Hurwitz and Drinker, Jour. Exper. Med., 1915, XXI, 401; Drinker, K. R. and C. K., Am. Jour. Physiol., 1915, XXXVI, 305; Mendenhall, Ibid., 1915, XXXVIII, 33; Denny and Minot, Ibid., 233; Bloch, Arch. d. Mai. du Coeur, i9is,VIII, 249. 2 Am. Jour. Med. Sc, 1915, CXLVI, 562. Digitized by Microsoft® THE BLOOD 4°3 coagulation obtains, the test is repeated with larger intervals between the immersions in water. (2) Remove stopper with rods and hold up to the light. The tips will show more or less of a red color depending on the degree of coagulation, rod i being clear while rod 4 is almost covered with a red clot. (3) Touch the tip of each rod to filter paper. The depth of color of the blot will indicate the degree of clotting. The average coagulation-time by this method is three and one-half to five and one-half minutes. Rudolf's Method. This"^ is a modification of the older one of Sabrazes^ and applies some of the points of the method of Kottmann.^ It is so simple and clinically reliable that the writer advocates it for general use. Thin glass tubes 1.5 mm. in diameter and about 18 cm. (7 inches) in length are used. A pint Thermos bottle is employed, its ordinary cork being replaced by a triply perforated rubber stopper. In these perforations rest two brass tubes 7 inches long and just large enough in caliber to hold easily the glass blood-tubes. The third perforation contains a thermometer. The apparatus rests on its side in such a way as to prevent its rolling about. The bottle is filled with water at 20°C. and the stopper is inserted. The finger or lobe of the ear is punctured, the exact time of puncture being noted. Two glass tubes are partially filled from the same drop, the blood being made to run nearly to the far end of each tube, which is placed, as filled, in a brass tube of the thermostat. The protruding ends, the ones at which the blood entered the tubes, are then sealed with a spirit lamp. In about five minutes the first tube filled is drawn out of its holder by the left hand (covered with a glove to diminish the effect of heat of the fingers), is touched with a sharp file, broken across and the broken ends slowly separated. The tube is at once replaced in the thermostat. This technic is carried out with the first tube at intervals of 15 to 30 seconds, until a thread of fibrin appears between the broken ends. The time between the puncture and the appearance of the fibrin is the "coagulation time" of the blood. Tube No. 2 may then be used as a control. Normally the coagulation time averages eight and one-half minutes by this method. Method of Boggs. This is a modification of the older method of Russell and Brodie and gives a lower coagulation time than does the preceding method. It is based on the fact that the corpuscles, set in motion by a current of air in a moist chamber, move freely and independently of one another at first but, as coagulation pro- ceeds, clumping of the cells occurs, and finally an elastic radial motion of these cells obtains. This is observed under the low power of the microscope. The writer refers elsewhere for the technic (see cut) . ? Normally, the coagulation time, as evidenced by the formation of fibrin, is between two and nine minutes.* Anything above nine minutes means 1 Am. Jour. Med. Sc, 1910, CXL, 807; 191 1, CXLII, 481. 2 Folia Haemat., 1904, I, 394. 3 Ztschr. f. klin. Med., 1910, LXIX, 415. * Carpenter and Gittings (Am. Jour. Dis. Child., 1913, V, i) show that the average time for children is slightly longer than for adults. Digitized by Microsoft® 404 DIAGNOSTIC METHODS delayed coagulation. The importance of testing this function of the blood is observed more particularly in cases of suspected hemorrhagic diathesis, in which the period of coagulation is remarkably increased. In some cases of hemophilia it requires fifty minutes, while in certain of the purpuras from lo to 15 or more (Emerson). Likewise, it is customary to test this factor in cases of long-standing jaundice, in which surgical intervention for obstructive lesions of the biliary passages is to be undertaken, as here also the time is much increased. Moreover, we find the coagulation time delayed or imper- fect in cases of hemoglobinemia, asphyxia, and general dropsy.^ Poisoning from the bite of certain serpents, such as the cobra, is characterized by a greatly delayed coagulation of the blood as well as by hemolysis. On the other hand, we notice a quicker coagulation in conditions associated with stasis, repeated hemorrhages, transfusion, hunger, and also under the therapeutic use of calcium chlorid and of gelatin. Xdlharme Mill Fig. 12 2.^ — Boggs' coagulometer: a, moist chamber; h, tip of tube through which air passes; c, cover which fits over moist chamber and which holds glass cone; d, pin-hole for escape of air; e f, cross section of cover c; g, tip of glass cone upon which is placed the drop of blood. The formation of fibrin, as the end-product of coagulation, is increased in some cases and diminished in others.^ An increase in the amount of fibrin in the blood (hyperinosis) is observed in acute inflammatory processes and in most infectious diseases. Hayem states that the density of the fibrin network, observed when blood is allowed to dry in thick smears on a glass slide, indi- cates the degree of resisting power of the individual against disease. The largest amounts of fibrin are observed in such conditions as pneumonia^ and acute articular rheumatism, but are seen to a lesser extent in parenchymatous inflammation, in inflammations of the mucous membranes and the skin, in the febrile stages of chronic suppurations, in hepatitis, influenza, diphtheria, acute gout, and erysipelas. A decrease in the amount of fibrin (hypinosis) may be observed in malignant growths, malarial fever, pernicious anemia, leukemia, and purpura. In parenchymatous nephritis the amount of fibrin ' See EJreiss, Inaug. Dissert., Heidelberg, 1912; Nel, Inaug. Dissert., Berlin, i9i2;Lands- berg, Biochem. Ztschr., 1913, L, 245; Lee and White, Am. Jour. Med. Sc, 1913, CXLV, 495; Jaff6, Folia Ha^mat., 1913, XV, 167; Steiger, Wien. klin. Wchnschr., 1913, XXVI, 1749. ^ See Bosworth, Jour. Biol. Chem., 1915, XX, 91. ' Dochez (Jour. Exper. Med., 191 2, XVI, 693) finds the coagulation time lengthened in the acute stage of pneumonia. Digitized by Microsoft® THE BLOOD 405 is but slightly if at all increased, while in interstitial nephritis the increase may be notable (Da Costa). (G) Osmotic Pressure and Cryoscopy. The cryoscopic examination of the blood may be of some importance in the diagnosis of certain conditions, particularly in reference to the sufficiency or insufficiency of the kidneys as regards the elimination of the urinary solids. While this subject has not yielded as much information as was expected of it, a brief discussion seems essential. For a theoretical discussion of osmotic pressure and cryoscopy, I must refer the reader to works on physical chemistry. By cryoscopy from the Greek kryos, frost, and skopeo, to see, is meant the determination of the freezing-point of a solution and the referring of this figure to the freezing-point of the solvent, which is regarded as o. Substances in solution lower the freezing-point of the solvent in direct proportion to their molecular (dissociated or nondissociated) concentration. In determining the osmotic pres- sure of a solution, and cryoscopy is one of such methods, it is important to remember that the pro- teins have practically no osmotic value. We have, therefore, in this cryoscopic method a means of ascertaining directly the molecular concentration of the body fluids. The determination of the freezing-point of the blood is best made by means of the Beckmann ap- paratus, which may be found in works on physical chemistry. As the osmotic pressure of the serum is equal to that of the plasma or of the whole blood, the serum is generally used for this determination. Withdraw from a vein, preferably the median bas- ilic, about 30 c.c. of blood and allow it to coagulate in a clean closed vessel. Place the serum in the freez- ing tube of the Beckmann apparatus, adjust the thermometer and stirring rods, and proceed as directed elsewhere. The freezing-point of the blood is usually desig- nated as the small Greek delta (5), while that of the urine is given the sign A. Normally, blood freezes at — o.56°C., distilled water freezing at o. This point is subject to more or less physiologic variation depending upon the changes which occur in the blood after meals, exercise, baths, etc. This physiologic variation is transient and slight because the kidneys soon regulate the osmotic pressure (molecular concentration) of the blood by withdrawal of the constituents which have caused the abnormal tension. This close relationship between the kidneys Digitized by Microsoft® ^ Fig. 123. — Beckmann apparatus. {Long.) 4°6 DIAGNOSTIC METHODS and the blood is of great importance in the study of renal insuflSciency, as many products of metabolic activity remain in the blood in cases of renal disturbance. This is more particularly true of the inorganic constitutents of the blood, as the organic elements do not materially affect its osmotic pro- portions. It is in those cases of renal insufficiency with a tendency to uremia that the most is to be expected from the cryoscopic examination of the bloody' yet we find that Schoenborn/ in the study of 88 cases, observed practically normal figures for the cryoscopic point of the blood. Engelmann,^ on the other hand, reported a series of 36 cases in which the freezing-point averaged — o.664°C. It is true that in certain surgical conditions of the kidneys a parallelism is noticed between the lowering of the freezing-point and the development of the uremic symptoms. In cyanotic conditions of any origin whatever we find the cryoscopic point lower than the normal, owing to the fact that the CO2 is increased in amount in such cases. If the CO2 be driven off, the freezing-point becomes normal. Hence we see why, in certain cases of uncompensated cardiac disease, the freezing-point has frequently been re- ported much lower than the normal standard. There is practically no disease of the blood itself about which a cryoscopic study will furnish any information.' From these considerations, as well as from many others, which I have not enumerated, the study of the cryoscopy of the blood does not seem to the writer of sufficient importance to warrant its adoption as a routine method of blood examination. It is, certainly, of no value whatever to the general practitioner who does not have access to a thoroughly equipped laboratory. The time consumed in such examinations would be much better spent in a more thorough study of the fresh and of the stained specimens. While cryoscopy may give some idea of the osmotic activity of the kidneys, it adds to blood examinations a slightly enlarged laboratory record, whose interpre- tation is a matter of more or less difficulty and whose results rarely ever give any information, beyond that which may be more easily and more readily learned by other methods. It is a purely theoretical method of following the metabolic activity of the system and reveals no signs of an oncoming uremia nor does it show us anything regarding the results of therapeutic measures. Naturally, variations in the osmotic pressure of the blood are of import- ance in the study of conditions associated with effusions into the various serous cavities, as such exudations are markedly influenced by the molecular concentration of the blood. For a discussion of such phases of the subject I must refer the reader to works on pathology. {H) Electric Conductivity. In the effort to enlarge the scope of blood examinations, the clinician and' more particularly, the laboratory worker have taken advantage of every- thing offered for the furtherance of their aim. In so doing they have often over-stepped themselves and complicated the examinations by useless ad- ditions to their technic. ' Wiesbaden, 1904. ? Mitt., a. d. Grenzg. d. Med. u. Chir., iqo^j XII, 3.96. Digitized by Iwicroson® THE BLOOD 4°? By electric conductivity is meant the reciprocal of the resistance, which a certain amount of solution offers to the passage of an electric current of known strength between two platinum electrodes of given size and a given distance. apart. This method is merely a measure of the number of elec- trolytes (both dissociated and nondissociated) in solution and is not affected by the nonelectrolytic organic substances of the blood, although, according to Hardy, ^ the proteins move to one or the other pole of the battery depending on the reaction of the fluid. The method usually adopted for estimating this factor is that of Kohlrausch, the resistance being balanced on a Wheat- stone bridge against a rheostat, the point of equilibrium being determined by means of a telephone attachment. As this determination can give us no information regarding the retention of organic products in the system, it is of comparatively little value in the study of metabolic or of blood diseases. It is in those conditions which are associated with retention of inorganic constituents that this method should be of value, and yet we find, according to the work of various authors, that in advanced nephritis, in which the retention of chlorids has been assumed, the conductivity of the serum is not increased to any extent, although the freezing- point is lower than the normal standards. This proves, its seems to the writer, that in such cases the substances capable of increasing the conduc- tivity — that is, the chlorids — are either not retained in the blood or are in such a combination as not to influence the conductivity of this tissue. We must, therefore, assume that the lowering of the freezing-point ob- served in such conditions is due more to the organic than to the inorganic constituents of the blood. As this determination is, in the writer's opin- ion, of so little practical value in blood work, a detailed description of the methods and results of observations will riot be taken up. (6) Chemical Properties. The chemical examination of the blood resolves itself into a consideration of the composition of the whole blood, of the plasma, of the serum, and of the various cellular elements. Is is evident that the coiiiposition of the whole blood ■will depend upon the relations in which the single constituents of the blood stand to one another.^ Naturally, physiologic influences are of great importance in the consideration of the composition of the fluid portion, while the cellular elements are less affected by these changes. In view of the recent work on opsonins and immunity, we must conclude that the chem- ical examination of the fluid elements of the blood may yield much impor- tant information as soon as proper methods of research are evolved. Normally, the composition of the cellular elements remains almost con- stant and is but sUghtly affected by the varying composition of the fluid por- tions. As is well known, the corpuscles act more or less like semipermeable membranes, although Kahlenberg would have us believe that no such a con- dition is possible. At any rate, the membrane surrounding the erythrocyte ' Proc. Royal Soc, igoo, LXVI, 95. '^ See Bonninger, Ztschr. f. exp. Path. u. Therap., 1912, XI, i; also, Veil, Deutsch. Arch, f. klin. Med., 1914, CXIII, 226; Lowy, Deutsch. Arch. f. klin. Med., 1915, CXVII, 79. Digitized by Microsoft® 4o8 DIAGNOSTIC METHODS allows the passage of certain inorganic and organic constituents of the plasma into the cells and permits the back-passage of certain constituents of the cells into the plasma. This question is intimately related to the subject of hem- olysis, which will be discussed in detail later. It is true that a many-sided exchange exists between the red cells and the hypertonic plasma in which they float, such an exchange accounting very often for the alterations in the volume of the corpuscles and frequently being the cause of abnormally developed cells. This exchange is subject to certain restrictions. Thus we find that the corpuscles show a very high content in potassium salts and a very low sodium content, while the reverse conditions obtain in the serum. If free exchange took place between the cells and serum no such conditions could exist. It is known that potassium salts are closely associated with phenomena of growth and hence it might be possible to prove that cells, which are morphologically the older, show a less potassium content than do the newly developing ones. It is evident that a quantitative analysis of the blood can be of only com- parative value as far as the blood as a whole is concerned. We should ascer- tain on one side the relationship of the plasma and blood-corpuscles to each other, and on the other side the composition of each of these two chief con- stituents. As there are many difficulties in the way of such determinations, we will not go into detail regarding the chemical composition of the different portions of the blood, but will discuss the blood as a whole. According to C. Schmidt, the composition of the blood is as outlined in the following table. This table, while giving the composition of the whole blood, may readily serve as one from which the composition of the corpuscles and serum may be ob- tained by reducing the constituents to parts per i,ooo of either corpuscles or serum. It is beyond the scope of this work to discuss the chemical properties of the blood or of its constituents in detail, but certain points which have clinical interest must be taken up. Man's. Corpuscles . 513.02 Water Solids . . . Hemoglobin, proteins, and other organic bodies Inorganic bodies . K2O NasO CaO I MgO. . . . 0.320 0.48s FeaOs . J CI 0.898 0.362 P2O6 . 0-695 0.643 Digitized by Microsoft® Woman's. 396.24 349 ■ 690 272.560 163.330 123.680 159-590 120.130 3-740 .' 3-55° 1.586 1. 412 0.241 0.648 THE BLOOD 409 Man's. Woman's. Serum . . ... 486.98 603.76 Water .... Solids ... Protein and other organic bodies 43 . 820 46 . 700 Inorganic bodies .... K2O . NaaO CaO . MgO. CI . 1.722 0.144 PaOs . . 0.071 2.202 439.020 551-990 47.960 51-770 4.140 5 070 0.153 0.200 I. 661 I. 916 0.533 0.608 The large excess of chlorin in the serum of man as compared with that of woman and the excess of phosphoric acid in the serum of woman as com- pared with that of man is noteworthy. These variations may later be shown to have a great influence upon sexual differences as regards metabolism. (A)- Total SoUds. The determination of the total solids and, hence, of the water content of the blood is often of importance, especially in cases of anemia. ^ The method is as follows: Allow about i c.c. of blood to flow onto one of two previously weighed matched watch-glasses. Cover this with its mate and weigh them while in the moist condition. Separate the glasses and place them in a des- iccator over CaCU or H2SO4 for twenty-four hours, at the end of which time weigh them as before. The blood should have dried to a hard glassy mass in this time. The loss of weight will represent the water of the blood taken, from which amount the percentage may be calculated. Gumprecht and Stintzing and Biernacki advise the use of higher than room temperature, the former usine 67°C. in an incubator, while the latter employs a heat of 100 to i2o°C. Drying by these latter methods is often associated with loss; hence the writer is accustomed to use room temperature (about 2o°C.). Working in this way, many have found the dry residue (total solids) of the blood to be from 21 to 22.5 per cent., the water content being 77.5 to 79 per cent. This figure shows marked variation under the influence of such processes as diarrhea, excessive sweating, and exudation into serous cavities. In anemia the solids are much reduced, while in leukemia they are increased. (B). Blood Pigments. Hemoglobin. The normal color of the blood is due to the presence of the pigments, hemoglobin and oxyhemoglobin. Usually the former is not large in amount, while the latter is the predominant factor in the color. In blood after as- phyxia we find a mixture of hemoglobin, pseudohemoglobin, and parhemo- globin; in arterial blood large amounts of oxyhemoglobin, and in venous blood a mixture of hemoglobin and oxyhemoglobin. 1 See Busch, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 335. Digitized by Microsoft® 4IO DIAGNOSTIC METHODS Hemoglobin belongs to the class of bodies known as "chromoproteins" and, owing to its power of combining with various gases and thus aiding in the gaseous exchange of the body, may be styled a "respiratory protein." It is easily decomposed into a protein, globin (about 96 per cent.), and a pig- ment, hemochromogen (4 per cent.), which contains iron and is easily changed in the presence of oxygen into hematin. The iron content of hemoglobin is the portion which enables this pigment to exert its peculiar vital power of oxygen transference. The amount of hemoglobin in normal blood is variable, depending on the age of the subject examined. Normally, 100 c.c. of adult human blood contain from 13 to 14 grams of hemoglobin (the average being about 13.77), which amount is not a constant factor at aU ages. From the following table of Leichtenstern it will be seen that a definite age curve exists for this substance. Age. Grains per 100 c.c. of blood. I to 4 days, 19-329 to 21.160 8 to 14 days, 17.869 to 16.124 8 to 20 weeks, 15.362 to 12.928 6 months to 5 years, 10.971 to 11.373 5 to 15 years, n-iSi to 11.796 15 to 25 years, i3-o34 to 13.870 25 to 45 years, 14-727 to 15.013 45 to 60 years, 12.484 to 13.150 Over 60 years, i4-790 Under certain conditions the hemoglobin is dissolved out from the red cells, leaving only their stromata behind. This gives rise to the condition known as hemoglobinemia which will be discussed in a later section. Accord- ing as hemoglobin is free or combined with certain gases, we have in the blood various derivatives of this pigment, each one of which has certain character- FiG. 124. — Direct- vision spectroscope. istics and a definite clinical importance. These derivatives are recognized by qualitative tests, especially by spectroscopic methods, and will be presented here; while the important tests used for the recognition of hemoglobin, either in the fresh state or as dried stains, will be taken up under the medicolegal discussion. "^ Hemoglobin also called reduced hemoglobin, is much more soluble than oxyhemoglobin, its solution in water being more violet or purphsh than one of oxyhemoglobin of the same concentration. Such solutions of hemoglobin absorb the blue and violet rays of the spectrum to a less marked degree than ' See Maestre and Lecha-Marzo, Arch, intemat. de m6d. leg., 1914, V, 49. Digitized by Microsoft® Absorption Spectra. PLATE XV. ■M ^ Oxyhaemoglobln. Haemoglobin. Carboxy- haemoglobln. Neutral Met- haemoglobln. Alkaline Met- haemoglobln. Alkali Haematln. (from hawks "PHrSIOLOGICAL CH£MISTf>r") Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Absorption Spectra. Ji % Reduced Alkali Haematln or Haemochromogen. Acid Haematln in ethereal solution. Acid Haemato- porphyrin. Alkaline Haematopor- phyrln. Urobilin or Hydro- bilirubln in acid solution. Urobilin or Hydro- blllrubin-ln alkaline solution after the addition of zinc chloride solution. Bllicyanin or Cholecyanin in alkaline solution. f/^ROM HAWKS "PH^'SIOLOG/CAL CH£MISTRr"J Digitized by Microsoft® THE BLOOD 4" do those of oxyhemoglobin, but they strongly absorb the rays lying between C and D. In proper dilution a solution of hemoglobin shows a spectrum with one broad not clearly defined band between D and E, lying toward the red end of the spectrum a little over the Fraunhofer line D (see plate). Pseudohemoglobin. According to Ludwig and Siegfried, blood, reduced by h5^osulphites or by a stream of hydrogen to such an extent that the spectrum of oxyhemo- globin disappears, yields large amounts of oxygen when exposed to a vacuum. This loose combination of hemoglobin and oxygen, which gives the spectrum of hemoglobin, is called psuedohemoglobin. Hammarsten considers it an intermediate body between hemoglobin and oxyhemoglobin. Oxyhemoglobin. Oxyhemoglobin, also called hematoglobulin, is a molecular combination of hemoglobin and oxygen. The ability of hemoglobin to take up oxygen is a function of its iron content. When this factor is calculated as 0.33-0.40 per cent., I atom of iron in the hemoglobin molecule corresponds to two atoms (one molecule) of oxygen. This combination is a loose one and hence the quantity taken up will depend upon the partial pressure of the oyxgen. This oxygen is set free when the oxygen pressure is reduced, thus giving rise to the characteristic property of oxygen transference. As Pfliiger has shown, oxy- hemoglobin may, when it is gradually oxidized, act as an "ozone exciter" by the decomposition of neutral oxygen into the atomic form. It may also act as an "ozone transmitter" as in certain tests to be outlined later. ^ A dilute solution of oxyhemoglobin or of arterial blood shows a spectrum with two absorption bands between the Fraunhofer lines D and E. One band, a, is narrower, but darker and sharper and Ues on the line D, the other, ff, is broader, less defined and less dark and lies at E. As the dilution becomes' weaker the band /S first disappears. By increased concentration the two bands become broader, the space between them smaller, and the blue and violet parts of the spectrum darkened. Other substances may give this same absorption spectrum, but oxyhemoglobin may be differentiated by its be- havior toward reducing agents, such as ammonium sulphid or Stokes' solution of ammoniacal ferro tartrate (see f late). Methemoglobin. This pigment is closely related to oxyhemoglobin, as it contains the same amount of oxygen and is isomeric with it. The oxygen is, however, not' in loose combination and cannot, therefore, be utilized by the system. This coloring matter is formed by the spontaneous decomposition of blood, as observed in hemorrhagic transudates and cystic fluids, and occurs also in cases of posioning with potassium permanganate, potassium ferricyanid, chlorates, nitrites, nitrobenzol, acetanilid, antipyrin, turpentine, sulphonal,' and arsenic, and in cases of cyanosis with diarrhea. ' See McClendon, Jour. Biol. Chem., 1915. XXI, 27?. Digitized by Microsoft® 412 DIAGNOSTIC METHODS According to Jaderholm and Bertin-Sans, the absorption spectrum of methemoglobin, in aqueous or slightly acidified solution, is similar to that of acid hematin (see below), but is easily distinguished from the latter by the readiness with which it turns into that of hemoglobin on treatment with alkali and a reducing substance. Hematin, under the same conditions, gives the spectrum of an alkaline solution of hemochromogen (Hammarstein). Met- hemoglobin, in alkaline solution shows two absorption bands, which are like those of oxyhemoglobin, but differ from them in that the band nearer E is stronger than the one at D. A third fainter band may be observed,' according to Hammarsten, lying between C and D. Carbon-monoxid Hemoglobin. This pigment is a molecular combination of one molecule of hemoglobin with one molecule of CO. This combination is stronger than those of hemo- globin and oxygen. The oxygen of hemoglobin is easily replaced, therefore, by CO and, in consequence, the tissues suffer for want of oxygen. This pig- ment imparts to the blood a bright cherry-red color both in the venous and arterial circulation. The most common cause of formation of the pigment is the inhalation of coal gas or of illuminating gas. The characteristic color in these cases may disappear after a few hours or it may persist for days, depending on the sever- ity of the case, being frequently found in the blood after death. Such blood shows an absorption spectrum similar to that of oxyhemoglobin, but the two bands lie more toward the violet end of the spectrum than in the case of oxy- hemoglobin. On the addition of a reducing agent to blood showing this spectrum, the absorption bands of carbon-monoxid hemoglobin are una- fected, while those of oxyhemoglobin are changed to those of hemoglobin. This spectroscopic test is not delicate as less than 20 per cent, of carbon- monoxid hemoglobin in the blood is difficultly, if at all, detected. Carbon-dioxid Hemoglobin. According to Bohr, hemoglobin forms three molecular combinations with CO2, in which products, a, j3, and 7, one gram of hemoglobin combines with 1.5, 3, and 6 c.c. of CO2. The spectrum of these compounds is similar to that of reduced hemoglobin. If a large excess of CO2 be present the hemoglo- bin is decomposed and globin is precipitated. The absorption band under these conditions is probably referable to the presence of free hemochromogen. Sulph-hemoglobin. This pigment, formed by the action of hydrogen sulphid upon hemoglobin, is found in the corpuscles and not free in the plasma. Sulph-hemoglobin- emia has been reported by several, especially by van der Bergh^ whose name is given to the condition known as "idopathic enterogenous cyanosis" and characterized by cyanosis, headache and marked constipation. Clarke^ has reported the first case in the United States. Spectroscopic examination of 1 Deutsch. Arch, f.klin. Med., igos,LXXXIII, 86; Berl. klin. Wchnschr., igofi.XLIII, 7. 2 Medical Record, 1909, LXXVI, 143; 1910, LXXVIII, 987. See, also, Wallis, Quart. Jour. Med., 1913, VII, 73. Digitized by Microsoft® THE BLOOD 413 the diluted blood is essential to show the presence of this pigment. The spectrum shows 3 absorption bands, two similar to those of oxyhemoglobin and a third in the red not so hear the C line as is the third band of methemo- globin. Addition of dilute ammonium sulphid destroys the third methemo- globin band, while it does not effect that due to sulph-hemoglobin. Decomposition Products of Blood Pigments. As stated previously, hemoglobin and oxyhemoglobin are proteins, which are converted under the action of different physical and chemical means into a globulin-like protein, globin, and an iron-containing pigment, hemochromo- gen. Fron; the table below, adapted from Webster and Koch,^ may be seen the relations of hemoglobin to its various derivatives and also to the bile pigments and to chlorophyll. Hemin .11 (C32H5oN,FeO.,HCl) Hematin (C32H3,03N4Fe)-^Hematoporphyrin (C,jH,jOiN,) . / II in/ Hemoi Phylloporphyrin (C,jH,8NjO) t Hemoglobin/ Hemochromogen Hemopyrrol (CjHijN)— Phyllocyanint-Chlorophyfl Methemoglobin % Urobilin (CjjH.oOjNj Bilirubin (C,„H,gNjOj)i :5.Hemat6idtft y Biliverdin (e.eHijN^O,) Hematin. This decomposition product is found in any situation in which oxyhemo- globin is destroyed; thus, in the digestive tract where it is formed by the ac- tion of gastric and pancreatic juice on oxyhemoglobin, in old extravsations, in the stools after hemorrhage, in urine after poisoning with arsenical compounds, and in the blood of persons poisoned with nitrobenzol and 1 Laboratory Manual of Physiological Chemistry, Chicago, 1903 . '^siological Lhemistry, Lhici Digitized by Microsoft® 414 DIAGNOSTIC METHODS acetanilid.^ Hematin is a dark brown or blue-black amorphous powder, insoluble in water, dilute acids, alcohol, ether, and chloroform, but readily soluble in acidified alcohol or ether and in dilute alkalies. Arnold has shown that there are three modifications of this pigment, a neutral, an alkaline, and an acid hematin, each showing spectroscopic differ- ences. The neutral hematin has little importance clinically and will be passed in this discussion. Acid-hematin solutions show four absorption bands, one between C and D, a second broad but not clearly defined band between D and F, which divides under certain conditions into two narrower bands, a fourth band between D and E which is nearer D and is very weak. Usually only the band between C and D and the broad band between D and F can be seen (see plate) ; Solutions of alkaline hematin show one absorption band between C and D which reaches out to some extent between D and E. If the alkaline hema- tin solution be reduced with ammonium sulphid, the spectrum of hemo- chromogen is observed, which shows two characteristic absorption bands, one very sharp and dark between D and E and a second paler and broader band covering the £ line. Hematin forms very characteristic compounds with hydrochloric, hydro- bromic, or hydriodic acids. With HCl hematin crystallizes with one mole- cule to form the compound hemin. The crystals are light or dark brown rhombic forms and are called, after their discoverer, Teichmann's crystals. Their forniation is specific for blood, but the kind of blood cannot be deter- mined by their presence. As their detection is largely a medicolegal question, I will refer a discussion of the technic to the later section. Hematoporph3nrin. When hematin is treated with concentrated sulphuric acid in presence of air,- iron is split off leaving the pigment hematoporphyrin. If air be excluded the product yielded by such treatment is hematolin. This pigment is insoluble in water, but dissolves in alcohol, strong and weak alkalies, and in acids. It is isomeric with bilirubin, with which it is associated in the liver cells. In acid solution hematoporphyrin shows two absorption bands, one fainter and narrower between C and D and nearer D, the other darker, sharper and broader in the middle between Z) and E. In dilute alkaline solutions this pigment shows four absorption bands. A band between C and D, a second broader band surrounding D with the broadest part between D and E, a third between D and E nearly at E, and a fourth broad and dark band between E and F. Hematoporphyrin is of great impo tance from the medicolegal standpoint as certain suspected stains may be identified only by its spectrum. For this phase see the later sections. Hematoidin. This ruby-red or reddish-yellow pigment is derived from blood coloring matter and like hematoporphyrin is iron free. It is found in old blood-clots, in hemorrhagic exudates, in sputum, and in feces. It is more abundant ' Schumm (Ztschr. f. physiol. Chem., 1912, LXXX, i) reports hematinemia in a case of acute chromium poisoning. Digitized by Microsoft® THE BLOOD 4^5 when the blood pigment is not much exposed to the action of living cells, as in the center of large extravasations and in hemorrhages into preformed cavi- ties of the body (Ziegler,). Hematoidin is identical with bilirubin and shows no absorption bands, but only a strong absorption of the violet to the green portion of the spectrum. Hemosiderin. This yellow, orange, or brown pigment is a derivative of hemoglobin and contains iron. Unlike hematoidin, it is found more particularly in extrava- sated blood which has been subjected to the action of living cells. After a time this pigment changes into one (probably hematoidin) which contains no iron. Malarial Pigment. The older view of Ewing' and others that this yellowish-brown or black pigment was melanin seems to have been disproven by the later work of Brown^ who has shown that it is, probably, identical with hematin and is formed by the action of the malarial parasite (possibly of a proteolytic enzyme) upon hemoglobin. On spectroscopic examination of an alkaline alcoholic solution of this pigment, a single broad band is seen starting sharply at D and extending to the left, gradually shading into the red between C and D. The melanins, observed in the leucocytes in relapsing fever, melanotic sarcoma and Addison's disease, are quite distinct from this malarial pigment as shown by decolorizing and solubility reactions. Estimation of Hemoglobin. A very large number of methods have been introduced for the determina- tion of the blood coloring matter. Some of these are extremely accurate, but are too complicated for general clinical purposes; others are less accurate, but are more applicable to our work. It may be said that, as a rule, the clinical instruments at our command will give comparative figures, providing their construction and their standardization is accurate. However, we find so much inaccuracy or eveii lack of proper standardization in some of these in- struments, that no absolute comparison may be attempted between the re- sults obtained by various workers using the same or different methods. Normal adult blood contains about 13.77 grams of hemoglobin per 100 c.c. of blood, this figure being subject, however, to variations at different periods of life, so that these instruments which are standardized against nor- mal adult blood and which furnish the amount of hemoglobin in terms of percentage of such blood cannot be absolutely accurate no matter how per- fectly constructed or how accurately standardized, when they are used for the estimation of hemoglobin in the blood of a child or of an elderly patient. It is, therefore, much better practice to obtain the values in terms of the actual amount per 100 c.c. of blood. It is well to recognize, as Tlirk points out, that we are working with possible errors and can obtain comparative results only when we use the same exactitude and care in each of our estimations. 1 Jour. Exper. Med., 1905, VI, 119. i^Arch. Int. Med., 191 1, 3aiI,,2go., , „,. „^ Digitized by Microsoft® 4l6 DIAGNOSTIC METHODS The errors are essentially constant, but we must remember that the arbitrary color standards change as time goes on, necessitating a restandardization of our instruments if accurate results are to follow. The writer has seen errors of 50 per cent, arise from the use of an old von Fleischl instrument and one of 25 per cent, from a faulty standardization of Sahli's solution. It must be insisted upon that we must not assume that a patient, who is pale and "anemic looking," is in reality anemic. Pallor depends not only upon the amount of pigment in the blood but also upon the delicacy and transparency of the skin and upon the superficial distribution and size of the blood-vessels. By remembering these points we may often save ourselves unnecessary chagrin on finding that the results of our determinations do not accord with the "anemic" expression of the patient. A simple puncture of the ear, allowing the blood to drop upon a clean linen towel (so-called " towel- test"), will many times set us right and prevent a diagnosis of anemia without further examination of the blood. Such examinationless diagnoses are unwarranted and inexcusable. Direct Methods of Estimation. These methods are, owing to their complexity, not applicable to clinical work. The spectrophotometer of Hiifner is undoubtedly the very best and most accurate method, but I must refer to the original article for its descrip- tion. Likewise the colorimetric double pipet of Hoppe-Seyler and the meth- ods of Nebelthau and of Zangemeister must be neglected. Indirect Colorimetric Methods. These indirect methods employ a comparison of the blood solution with a second medium which approximates the blood in color. Two principles are possible in such methods. We may either have a fixed unchangeable medium of comparison, with which the blood is matched by constant dilution, or we may use a fixed blood solution of known strength and compare there- with the standard graduated color scale. Both of these principles have been used in such determinations, the variations being shown in the fact that in- struments for this purpose have been introduced by Gowers, Sahli, Hayem, Malassez, Henocque, Bizzozero, von Fleischl, Miescher, Haldane, Griitzner, Gartner, Dare, Oliver, and Tallqvist. It is evident, a priori, that these indirect methods must have greater errors than the direct. Two things are essential for any exactitude whatever. First, the medium of comparison must agree in the most Complete manner with the various color tones which the blood shows at different percentage, values of hemoglobin. Second, the standardization of the medium of com- parison must be extremely accurate. We must have no illusions regarding the exactness of the hemoglobin values obtained by these methods, as it is very difiicult to construct two instruments of the same kind that will agree exactly. Certainly no two instruments of different make will agree, but comparative results suflSciently accurate for clinical purposes are obtained by the use of the more reliable instruments. The personal equation in reading the color comparisons must be remembered, as some individuals show abnormal Digitized by Microsoft® THE BLOOD 417 sensitiveness or lack of sensitiveness to shadings of red. It is an impossibility in this book to give in detail all of the methods advanced for the estimation of hemoglobin. I select, therefore, those that have proven most reliable in my hands. Hemometer of Fleischl-Miescher. Up to recent times the most frequently used of the instruments for the estimation of hemoglobin was the old von Fleischl instrument. With the introduction of the Miescher modification, this original form has been or should be less often employed. We avoid, therefore, a discussion of the older instrument, referring to other works which have included it. This new apparatus, made by Reichert, under the direction of Miescher, Fig. 125. — Hemometer of Fleischl-Miescher; R, Stage; T, milled head, which moves the color scale; m, opening in stage through which the instrument is read; M, mixing cell- D', cover glass; D, cap; PS, gypsum mirror from which light is reflected; mel, diluting' pipet. is similar in general appearance to the old von Fleischl. It has the same stand and the same scale principle, although this latter is standardized differ- ently and graduated on a different basis. It differs, materially, in the method of measuring and diluting the blood, in the form of the comparison chamber and in the meaning of the graduation of the scale (see cut and legend fo/its description). The Diluting Pipet. This is similar in construction to the pipet of the Thoma-Zeiss hemo- cytometer, its calibrations, however, being different. The marks are J^ ?^ and I. Above and below each of these main divisions are two marks each corresponding to Koo °hBfizW^hf)^i8foMt^^^^^^^^ ^"^^- ^his device 4l8 DIAGNOSTIC METHODS enables the worker to measure accurately the column of blood taken, in case he gets too little or too much blood in the tube. The relation of the capillary to the ampulla is such that blood, drawn to the mark i and diluted to the mark above the ampulla, receives a dilution of 200; if drawn to the mark ^ the dilution is 300; whUe the line M furnishes a dilution of 400. The diluent used is Ho per cent, sodium carbonate solution. This dissolves the stromata of the red cells furnishing a clear solution. Occasionally the diluent becomes turbid after standing some time, and should be freshly made and should con- tain no bicarbonate. The taking of the blood and the mixing with a diluent is done with the same precautions mentioned under the method of making the blood count. In choosing the proper dilution, one should select that which will enable him to use the central portions of the graduated scale. A dilution of 400 is usually applicable except in cases in which marked anemia is suspected when dilutions of 300, 200, or even 100 should be made. In making this last dilution the erythrocytometer may be used. In doing accurate work, it is well to make a preliminary determination of hemoglobin in order to tell, the better, just what dilution would be advisable. This may be done by the Tallqvist method described later. The Comparison Cells. With this instrument two cells for holding the blood and diluent are fur- nished. One of these has a depth of 15 mm., the other one of 12 mm. The former is the standard cell, the latter the control, giving ^ the value of the larger cell. Their external appearance is similar to the cell of the old Fleischl, but their capacity is less owing to their greater thickness. The dividing partition between the halves of the cells projects about J^ mm. above the borders, thus preventing any mixing of the fluids in the two portions. A grooved cover-glass is slid over the compartments without fear of mixing the fluids. If fluid is lost no error is introduced, as the dilution is uniform in the pipet and the depth of the chamber is definite. A diaphragm, with an opening 4 mm. wide, is placed over the cover-jglass after this latter has been adjusted, the opening being so placed that its long axis is perpendicu- lar to that of the vertical partition. The field of comparison is thus limited to 4 mm., which corresponds to about 3° of the scale, affording a comparison of a single tint of the scale with the color of the diluted blood. Graduation of the Scale. The slide for comparison of color is tinted with Cassius' golden-purple, as in the old Fleischl instrument. The graduations of this color scale are made by comparison with standard solutions of hemoglobin and not with the ar- bitrary standard of blood of so-called normal individuals. The scale shows the same divisions as that of the Fleischl instrument, but their significance is different in the Miescher modification. Here one does not read directly the percentage of hemoglobin, but must obtain the corresponding value by reference to a " table of £)^fej^jpg^,'A^JgJ3S(^#®ompanies each instrument. THE BLOOD 4^9 While the figure thus obtained is in terms of actual percentage (grams per loo CO.), it is quite different from the percentage figure given by the Fleischl instrument. Method. After testing the chamber by placing in one compartment the diluting fluid in order to see that none runs into the other, the blood is drawn into the pipet to the desired point and diluted to the mark, all precautions mentioned under Blood Counting being observed. Thoroughly mix the blood and dilu- ent by shaking and blow out the unmixed contents of the capillary tube. Fill one compartment of the 15 mm. cell with the diluted blood so that a con- vex meniscus appears above the border of the chamber, the other compart- ment being fiUed with the diluent or with distilled water. Adjust the grooved slide and cap, place the cell in the central opening of the stand, and adjust the light. This latter portion of the technic is of considerable moment, as this instrument gives the best results when used in a dark room illuminated only by a small candle flame which is placed about 18 inches from the stand and to the side. In the absence of a dark room, a light-proof shield for the eyes ma,y serve. This may readily be obtained by the use of a tube of stiff, dark paper, which fits over the comparison cell and shuts off the light of the candle from the field of vision. The observer should stand in such a position that he looks into the cell from the side and not from the front or back, the eyes being about one foot above the cell. In the comparison of the color tones the variations are much better seen by quick movements of the slide rather than by slow gradual changes. The eyes should be rested at short intervals to prevent fatigue and lack of sensitiveness to the different shadings of color. When the color is matched take the reading of the instrument, by observing what mark of the scale coincides with the notch on the edge of the opening above the scale, and control this reading by several duplicates. Remove the blood from the 15 mm. cell by means of the pipet and transfer it to one compartment of the 12 mm. chamber, tested as was the larger one. Adjust this chamber and make readings in the same way. These values should be only four-fifths of those obtained with the 15 mm. cell. This modification has the advantage of using different portions of the color scale and should give comparative figures. If there should be any variation, which should never be over i per cent., a correction may be made. Calculation of Results. This is possible only with the use of the "table of calibrations," which con- tains the series of scale divisions and the absolute amount of hemoglobin in mg. per 1,000 c.c. of blood, corresponding to each division of the scale when the 15 mm. chamber is used. Thus, if the scale shows 56, we find the value corresponding to this to be. 447 mg. of hemoglobin in 1,000 c.c. of diluted blood. As the dilution may have been 400, we would have, in 1,000 c.c. of undiluted blood, 400 x 447 = 178.8 grams. As we wish to know the amount in^ioo c.c. we have merely to divide by 10, thus obtaining 17.88 grams of Digitized by Microsoft® 420 DIAGNOSTIC METHODS hemoglobin. If we wish to get the percentage figures, as read on the old Fleischl, we divide the figure obtained with the Miescher, in this case 17.88, by the amount of hemoglobin corresponding to the 100 division of the scale of the instrument used, in this case 14, and we obtain 127.7 P^^^ cent. This Miescher modification is our very best clinical apparatus for estima- tion of hemoglobin, giving results which are accurate within 0.2 to 0.5 per cent. It is open to the objections that it is bulky, expensive, and requires more time and practice for its use than is at the control of the busy practi- tioner. For hospital use, however, it is the instrument par excellence and should never be substituted by others. It has one disadvantage, that each instrument is standardized, not against a known hemoglobin solution, but against a so-called "normal" instru- ment, which has been properly stand- ardized. It is to be hoped that each instrument will, in the future, be properly calibrated and thus insure us against the possibility of errors arising from any change in the standardization of the "normal" instrument. Hemoglobinometer of Dare. The instrument, introduced by Dare, has the advantage of using un- diluted blood, and avoids any error consequent upon dilution. The prin- ciple of this instrument is as follows : E^ Fig. 126. — Hemoglobinometer of Dare: R, milled wheel; s, case inclosing the color disk; T, movable wing, which is swung out- ward; XT, telescoping camera; v, aperture The color of undiluted blood is COm- v'itachlKnd"; hoS^rTl KhZg P^^^ by artificial light with that of a which the percentage of hemoglobin is read, graduated glass scale colored with golden-purple, the loo-point of which is standardized against a solution of 13.77 grams of hemoglobin in 100 c.c. of serum. For a description of the apparatus see cut. Method. Swing outward the movable screen, which serves as a cover for the case, adjust the camera tube, and fit the candle attachment in its place opposite the camera tube. The candle should be so adjusted that its upper end is flush with the top of the clips which hold it. If the wick be curved, it should be so turned that the intensity of the light is midway between the two aper- tures. See that the pipet, composed of the rectangular glass plates, is thoroughly cleansed and dry. The space between the plates is filled by applying the edge of the pipet to the side of a fairly large drop of blood. Adjust this pipet in its place and rotate the colored scale, by means of the milled screw, until the colors match. Hold the instrument steady to prevent the flickering of the flame as much as possible. No dark room is necessary, but it is advisable to point the instrument at some dark object and to avoid Digitized by Microsoft® THE BLOOD 42 1 direct sunlight, as the shadings of color are not so easily matched by direct daylight. As soon as the colors are matched make the readings and check the results with several duplicates. This reading is observed on the left side of the case in the small open space, the line which coincides with the beveled edge of the opening representing the percentage of hemoglobin, on the basis of a value of 13.77 grams of hemoglobin per 100 c.c. as 100 per cent. It is, there- fore, easy to calculate the direct amount of hemoglobin in the blood examined. This instrument has the advantages that undiluted blood is used, that the scale of comparison is usually very accurately standardized, that it is con- venient, easy of manipulation, and rapid in giving results. Coagulation of the blood does not occur sufficiently soon to introduce an error, providing the reading is taken within a reasonable time. It is more convenient for general Fig. 127. — Method of filling the Dare blood pipet. {Da Costa.) use than is the Miescher, is less expensive, can be used in a light room, and gives results second only to those of the Miescher. The disadvantages of this instrument are that an occasional faulty standardization may introduce errors, it costs much more than some of the instruments to be described, and it is not a long-lived instrument unless care is taken in handling it. In the writer's laboratory this instrument has given great satisfaction and can be recom- mended for general use on the ground of its convenience and ready application to clinical work. Hemometer of Sahli. This instrument is a new modification of the older hemoglobinometer of Gowers, and has so many advantages over the older instrument that this latter will be passed over. A modification of the Gowers instrument intro- duced by Haldane is simple and reliable, but has the disadvantage that coal gas is used in converting the hemoglobin into CO hemoglobin, and consequently, is not of easy application for bedside work. As Sahli has pointed out, a colored fluid under examination should liot be compared with a different substance similar in color, but with a solution of known strength of the same coloring matter. His modification of the Gowers method employs an instrument constructed on exactly the same lines, but using a different standard of comparison. This standard of comparison is an acid hematin solution in a concentration corresponding to a i per cent. Digitized by Microsoft® 422 DIAGNOSTIC METHODS solution of normal blood. This solution is somewhat dark, as it is standard- ized against blood showing high hemoglobin value. As Sahli states, the stand- ard fluid, as furnished with his hemometer, corresponds to a blood which shows with the Miescher instrument at a dilution of 200 a reading of 109, or an ab- solute quantity of 17.2 grams of hemoglobin per 100 c.c. of blood. The fine particles of hemin, which are in suspension, may adhere to the glass, especially if the instrument lies unused for some time. This process changes the color of the standard to some extent, so that it is supposed that deterioration has occurred. This may be remedied by completely inverting the tube, without violent shaking, thus allowing the precipitate to dif- fuse uniformly. Occasionally one of these instru- ments is found to bfe improperly standardized, but this is rare. Method. This consists in diluting the blood with 10 times its volume of N/io normal hydrochloric acid. After a few seconds the fluid becomes dark brown from the forpiation of hematin hydrochlorid (hemin), which substance is not in solution, but in fine suspension. The blood is taken with the 20 cmm. pipet and blown into the graduated tube, which contains N/io normal HCl up to the mark 10. This acid may be accurately enough made by diluting 15 c.c. of concen- trated chemically pure HCl to 1,000 c.c. Shake the mixture of blood and acid and dilute with ordinary water as soon as a clear dark brown color is visible. Fig. 128. — Hemometer of Add water until the shade of the mixture corresponds Sahli. (Greene.) exactly with that of the standard solution, when the percentage of hemoglobin may be read off. The water should be added very carefully from a dropping pipet, as the accuracy of the method depends upon adding the exact amount of fluid necessary. The comparison of colors may be made in any light, as the two solutions, being the same, will be similarly affected. This instrument is very conveniently gotten up, being mounted in a case, carrying a white-glass plate to reflect the light to better advantage. It is simple, inexpensive, and is accurate within 2 per cent. The author would recommend this instrument to the general worker above all others, with the possible exception, of the Dare, whose advantages have been mentioned. Hemoglobinometer of Oliver. In this method the color of the blood in a definite dilution is compared, by light reflected from a white surface, with a series of tinted glass standards. Such a method has the advantage that the color of the diluted blood is com- pared with one single ^^^f^f^M^J^^Mj^hpff^^^^^^ glass disks correspond THE BLOOD 423 accurately, as determined by the tintometer, to the specific color curve of progressive dilutions of normal blood. Two sets of standards are furnished, one for use in daylight, the other by candlelight, the latter being preferable. Each set of 12 disks is mounted on plaster of Paris and enclosed in two Fig. 129. — Hemoglobinometer of Oliver. {Coplin.) wooden frames, six disks in each frame. They represent the color of solu- tions of hemoglobin with percentages from 10 to 120. To obtain interme- diate values, colored riders are used, each representing 2.5 and 5 per cent, of hemoglobin, when used with the disks from 70 to 120, but twice tliese values if used with the djigj^^/^^ggg ^ Wli^soft® 424 DIAGNOSTIC METHODS The blood is taken in a capillary pipet similar to that of von Fleischl, holding 5 cmm. of blood. The diluting chamber has a white background of plaster of Paris and, when filled with distilled water in which one pipetf ul of blood has been dissolved, yields a solution of i per cent. When filled the cell is covered with a blue-glass cover. Method. The pipet is filled with blood drawn by capillarity from a puncture of the ear or finger. Wash the blood from the pipet into the diluting chamber by means of water from a medicine dropper, fill the chamber with water, stir well with the handle of the pipet, and adjust the cover-glass. A small bubble of air should be included as an evidence that the chamber was not overfilled. Compare the color of this solution with that of the standard disks, using the light of a candle placed 3 to 4 inches away in such a manner that the light strikes the two cham- bers alike. It is well to use the camera tube to shield the eyes and enable one to compare the tints more accurately. If ' the color matches that of any of the disks, the percent- age is read directly from the case. If, how- ever, the color is intermediate between two disks, the riders must be superimposed on the disk of lower percentage, and a second com- parison made by using a corresponding plate of unstained glass as a rider on the diluting chamber. The adjustment of tints and riders must be continued until the colors match ex- actly. Naturally the variation wiU equal 2 per cent, as the ordinary riders are equivalent to 2)^ per cent, of hemoglobin. It is not necessary for this determination that the room be absolutely dark. This method is accurate within the limit mentioned (2.5 to 5 per cent.), but it is trying, time consuming, and does not equal in accuracy the methods of Dare and of Sahli. The instrument is inexpensive and the disks are prone to deteriorate. Tallqvist's Hemoglobinometer. Tallqvist has introduced a method for the estimation of hemoglobin which is so simple that certain writers have been led to recommend it above other methods. The principle of this method is essentially the same as Oliver's method, although the application is entirely different. Tallqvist compares the color of the undiluted blood with that of a series of lithographed standard tints, which range by differences of 10° from 10 to 100 per cent. These stand- ard tints were prepared by matching in water colors the tint of the blood of various patients (whose J^^l^sL?!^^/ )W^W Zeiss D, Leitz 6 or 7, and Reichert^3^. As the student becomes accustomed to cell-counting he may use a lower objec- tive. This has the advantage of bringing the entire field of one sq. mm. into focus. A mechanical stage is of some convenience in making the count, but the fingers answer quite as well after some practice in manipulating the sUde. The unit of counting surface is a matter of individual preference. Sahli recommends a unit of four small squares, Grawitz and Simon use sixteen of these squares, while Cabot advises the use of 36. In common with Tiirk, Ewing, Da Costa, Wood, and Emerson, the writer prefers the units of 25 small squares, as this is the unit of the ruling and the calculation is much simpler than with the other units. In order to simplify the process of counting, some routine method must be used. These methods depend on the worker, but the usual procedure in the writer's laboratory is as follows: Adjust the slide upon the table of the microscope so that the upper left-hand corner of the central ruled area of 16 large squares of the Tiirk chamber is brought into the field in such a manner that one may count the cells in the small squares from left to right. If the low-power objective is used, the complete group of 16 units will lie within the field of observation and may be easily examined. With the higher power, which the student may be forced to use in his earlier work, the optical field is necessarily limited. Count the total number of cells lying within the 25 small squares of the single unit. In doing this count the upper row of the unit from left to right, drop down a row, and count the cells from right to left and so on until the cells in the whole unit have been counted. The accom- panying cut will indicate the method to be followed. In making this count, cells which touch the right hand or lower boundaries of the unit are disre- garded, while those which touch the upper and left-hand hne are included in the count of that square. After counting the cells in the upper left-hand unit, count those in the remaining fifteen units, thus covering a field of 16 units of 25 small squares each, making a total of 400 small squares counted. Tiirk recommends the counting of eight units as a minimum, Emerson advises the counting of the four corner units in each of two separate preparations, while Da Costa and others count the cells in four groups of units from above downward and repeat with four units not adjacent. It seems to the writer that the error is less^the larger the area covered, and he, therefore, advises the beginner to make the total count of the sixteen units, although it is not supposed that one will try to cover up defects in technic by this larger count- ing area. A variation of more than 25 cells in the counts of the various units should be taken as evidence that the distribution is not perfect. In such cases the count would better be rejected on the ground of inaccuracy. After the technic is mastered and the worker has discovered just exactly wherein his error lies, a count of 8 or 4 units will sujEce. It is advisable, Digitized by Microsoft® 464 DIAGNOSTIC METHODS where accurate scientific results are indispensable, to clean the sHde and make a second count with a fresh drop of blood so that one may have a check on •his work. For clinical purposes, however, the count of four units will ordi- narily be sufficient. In ordinary counting it is not necessary to differentiate the red cells from the leucocytes, as the error thus introduced is small and may be disregarded. In cases, however, which show large leucocyte values, this error will be guite appreciable and must be overcome by the following method. All of the cells observed may be counted as erythrocytes and the reduction made for the num- ber of leucocytes as obtained by the special leucocyte count to be described later. In the use of Hayem's diluting fluid, the leucocytes are not colored, while the erythrocytes retain their normal yellow color. The leucocytes ap- FiG. 135. — Plan of Counting the Cells. {DaCosta.) The small squares are examined in the order indicated by the arrow. pear, by good illumination of the field, of a bluish tone, are somewhat larger than the red cells, and are characterized by a sharper border. These facts will enable one after some experience to distinguish the white from the red cells. Toisson's fluid stains the leucocytes blue, and may, therefore, be used to outline these cells. As a rule, it is better to learn to recognize the leucocytes by differences in refraction than to rely on the staining quahties of these cells. In some cases the methyl violet of the diluent colors some of the red cells so that they cannot be easily distinguished from the white ones. The number of cells in the blood is invariably reported as the number contained in a cubic millimeter of blood. In making the calculation of this number it is necessary to know the number of units counted, the number of cells in these units, the area of each small square of the unit, and the degree of dilution of the blood. Thus, if 16 units, each of 25 squares, have been counted, the total number of small squares is 400, each having a cubic area of M, cmm. The total volume of the units counted is, therefore, H cmm. It is evident that the number, of cells in i cmmL of blood is 10 times that in THE BLOOD 4^5 « the area counted over if the blood were undiluted. But the count is always made with diluted blood and we must, therefore, take this factor into account. With a dilution of loo multiply the number of cells in i cmm. by loo, and with a dilution of 200 multiply by this factor. Thus, if 2,500 ceUs were counted in the 400 small squares and the cubic contents of the units gone over was 3d!o cmm. then i cmm. of diluted blood would contain 25,000 cells. As the blood was diluted 200 times, the total number of cells in one cmm. of undiluted blood is 5,000,000.^ A very simple way of remembering this calcu- lation is to multiply the number of cells counted in the total area of 400 small squares by 1,000 if the dilution was 100, and by 2,000 if the dilution was 200. If the total area of 16 units be not counted, the method of calculation is the same but the factors are variable. This method goes as follows: Multi- ply the number of cells counted by the degree of dilution and this result by the cubic contents of each small square (4,000). Divide this result by the num- ber of small, squares counted. Thus, to calculate the number of cells in a cmm. of blood when 100 small squares (4 units) were counted at a dilution of .1 1 • ^ „ , • • /• n 625 X 200 X 4000 200, the count bemg 625 cells, the equation is as follows: = 5,000,000. Leucocytes. In counting the leucocytes much depends on the sort of ruled slide at the disposal of the worker. With the old Thoma chamber at least five different drops must be examined in order that a sufficiently large number of leucocytes may be counted, while with the Tiirk cell a counting area of 9 sq. mm. is afforded for each drop. The more leucocytes counted so much the less is the error. It is usually sufficient to count the white cells in a single drop, using the Tiirk chamber, but for scientific purposes three or even four drops would better be examined. If it is desired to count the leucocytes in the same specimen as the red cells the procedure is as follows: Prepare the drop of blood exactly as described for counting the red cells, using Toisson's fluid as the diluent and the erythrocy- tometer as the diluting pipet. After the reds have been counted, enumerate the whites in the entire ruled area of this chamber. In this process the leuco- cytes will be found to be stained a faint blue. It is often advisable, in case of low leucocyte counts, to repeat this process with a second drop of blood. The calculation by this method is very simple. As the entire ruled area of the Tiirk chamber covers a surface of 9 sq. mm., each equal to the central area used in counting the red cells, we have the equivalent of 3,600 small squares in the ruled surface. Multiply this figure by the number of drops used to obtain the total number of small squares covered by the count. Thus, if 54 leucocytes were observed in two drops (7,200 small squares) and the dilution 1 , 1 • S4 X 200 X 4000 was 200, then we have the equation, = 6,000 cells. ^ 7200 ' A second method of calculating the number is to consider each sq. mm. of the surface of the Tiirk chamber as a unit. If then, the number of cells 30 Digitized by Microsoft® 466 DIAGNOSTIC METHODS counted in two drops (i8 units) be 54, we divide this number by the number of units counted, 18, and multiply the result by 10 (the cubic contents of each unit) and then by the dilution. Thus, 3 = 6,000. It is usually preferable in counting the leucocytes to use the special leuco- cytometer previously described, as this gives a smaller dilution, and conse- quently a larger number of leucocytes to the counting surface. In using this pipet, the blood is drawn to the mark i and the diluent, i per cent, acetic acid, added to the mark 11. This diluent destroys the red, cells and brings out the leucocytes clearly. The dilution of the blood will thus be 10. If a large increase in the number of leucocytes is anticipated, it is better to use a dilution of 20, drawing the blood to the mark 0.5, as a routine the dilution of 10 being, however, preferred. In counting the white cells with a dilution of 10, using the Tiirk chamber, it is not as a rule necessary to go over the entire counting field, but more ac- curate results will obtain if this be done. A count of at least 250 cells is ad- visable, while one of 1,000 is more to be preferred in scientific work. The method of calculation is the same as given above. Thus if 540 cells were counted in the 9 sq. mm. of the ruled surface, the dilution being 10, we have 1:40 X 10 X 10 , -rr 1 -11 , , , r- = 6,000. If we have not used the total 9 sq. mm., but have used 4 or any other number which may be considered sufficient by the worker, the divisor in this division will be the number of units counted. If only a Thoma chamber be at hand for counting the cells, two methods are available. By the first, several drops may be gone over counting the total number of cells in the ruled sq. mm. of the cell. In the second method we find the cubical contents of each visual field and then count the leucocytes in many of such fields. The method of computing these factors is given by StengeP to whose work the author refers the reader who may not have access to a Zappert or a Tiirk chamber. In making this leucocyte count great care must be exercised to have the diluent fresh and free from yeast spores, which so freely develop in such mixtures. If this factor be observed, all the cells seen may be counted as leucocytes, but occasionally nucleated red cells may be confusing, especially if these be present in large numbers. The physiological condition of the patient should always be considered in making a report on a leucocyte count, as such factors as digestion and exercise influence this count to a great extent. The normal error in making a leucocyte count, with a count of 200 and more leucocytes, is about 5 per cent., while in the case of a red count it should not be over 3 per cent. Careful work with special attention to all the details mentioned will often reduce this error to a lower figure. Naturally, the error in counting the leucocytes will be much reduced by using the Tiirk chamber and giving the blood a dilution of 1 ■. There are certain errors due to faulty construction both of the pipet and of the counting chamber which remain constant in the same apparatus Hence it is wise to procure the very best ^ Twentieth Century Practice of Medicine, New York, i8g6, VII, 271. Digitized by l^icrosoft<^ ^ ' ' ^ THE BLOOD 467 equipment possible and to test the different portions of the pipet for such errors. In this way a very appreciable difference may be obviated. Not all of these blood counters show such variations, but some do, and it is, therefore, a matter of moment to know your tools. (5) Cleaning the Apparatus. This is the last step in the technic of making a blood count. While perhaps not as important as some of the other steps, yet if not properly carried but it will introduce errors which may prove very annoying. It is readily seen that an unclean pipet or counting chamber will interfere with the proper manipulations as described above. After the cover-glass has been removed from the slide, wash out the chamber with distilled water and dry thoroughly by means of a clean piece of linen. Place the slide in its proper receptacle, so that it may be conveniently found when desired. This may seem a small point, but the author has seen Fig. 136. — Cross-section of Durham's blood pipet. {Da Costa.) T, Glass' tube; n, rubber nipple; p, lateral perforation in nipple; c, cork in which a capillary pipet is fitted. too many slides lost by carelessness in putting them away. The cover-glass is rubbed clean and dry and put in the case with the slide. Wash out the pipet with water until all of the blood is gone. In some cases fine clots will be observed sticking to the side of the tube. Under such conditions remove the adherent blood by means of a fine wire or, if this is not effective, draw a little strong potassium hydrate solution into the capil- lary and allow it to act on the clot. After the tube is apparently clean, wash it out with alcohol and ether. Blow a stream of air through the tube by means of compressed air or the suction pump. Be certain that the apparatus is perfectly dry and Clean, and that the glass bead in the ampulla is freely movable as the tube is shaken, before putting the pipet in its case. Durham's Hemocytometer. Recently Durham has introduced a modification of the older instruments for blood-counting. This embodies the principles of the various methods, but substitutes a self -measuring capillary pipet for the. suction pipet of the Thoma apparatus, and special mixing vessels for the dilution of the blood. This device makes it possible for one inexperienced in blood-counting accu- rately to measure the blood and diluting fluid and thus eliminates the error possible with the older pipet. The direct count is made in the counting chamber of Thoma. This capillary of Durham count is more easily cleaned than that of Thoma, thus giving an advantage in cases where several blood examinations are to be made in a limited time. While this method of diluting the blood has the advantages above mentioned, the writer has never been able to convince himself that the use of this apparatus gives the student any Digitized by IVIicroson® 468 DIAGNOSTIC METHODS better results than he obtains with the Thoma instrument. After some experience, and this is needed in any method, the dilution of the blood with the Thoma pipet is quite as easily made as with the Durham modification, and the results of the count give quite as close checks as do those with the newer method. Oliver's Hemocytometer. This instrument was intended to furnish a more accurate method of count- ing the red cells than was given by the older instruments. The method is based on a principle entirely different from that of the older instruments and does not afford an actual count of the cells. If blood be diluted with a fluid, which preserves the corpuscles, in a rectangular test vessel composed of longitudinally stri- ated glass, each striation of the glass will act as a lens projecting an image of a candle flame viewed through the suspension of opaque particles of the blood, providing the suspension is of a sufl&cient dilution to permit of the almost unobstructed passage of the rays of light. At the proper dilution, these images of the candle flame will IE* \~JJ\ form a bright streak horizontally across the tube. Ex- I "" M^A periments have shown that the development of this bright Kssss^s^S^sli line, on dilution of the blood with Hayem 's solution, is an Fig. 137. — Oliver's accurate measure of the percentage of red cells in the (Greene.) specimen examined. The dilution of the blood is made in a rectangular glass cylinder by means of a capillary pipet, which is washed out with Hayem 's solution. The cylinder is graduated into divisions from 10 to 120, each division representing 50,000 red corpuscles. As this instrument has little clinical value, owing to the fact that the error is very great in cases in which the blood is diseased, I refer the reader to other works for a description of the method in detail. In the study of the physiologic variations of the red cells, this method affords very accurate re- sults, giving figures which would be lost with the Thoma instrument. It must be remembered that, in this method, as in many others the personal equation plays a large r61e and may account for serious error which is not found with the Thoma instrument.. Emerson has shown that the variations in the re- sults may be as much as 2,000,000 cells when this instrument is compared with the Thoma in counting the blood in primary anemia. Baumgarten has proven that variations in the size of the cell as well as deformities of the cell will introduce a serious error into this method. Ellermann and Erlandsen* as well as Dunger^ have recently introduced new methods, for making ac- curate counts of the white cells, which may prove very advantageous. Counting of the Blood-platelets. The technic of counting these cellular elements has been imperfect and the results variable. The methods advanced are both direct and indirect. The > Deutsch. Arch. f. klin. Med., 1910, XCVIII, 245. ^ Munch, med. Wchnschr.. 1911, LVIII, 1131. Digitized by Microsoft® THE BLOOD 4^9 technic of Determann and of Brodie and Russell belong to the latter class, while the method of Helber is a direct one. Recently Wright and Kinnicutt^ have introduced a method which is simple, exact and reliable. The technic is as follows: The blood is diluted i : looby means of the pipet used for count- ing the red cells and the counting is done in the Thoma-Zeiss counting-cham- ber, using all the precautions previously discussed. The specially thin cover-glass of Zeiss, with central excavation, is used to render the platelets clearly visible. The diluting fluid consists of 2 parts of a i : 300 aqueous solu- tion of "brilliant cresyl blue" and 3 parts of a i : 1,400 aqueous solution of potassium cyanid. These two solutions should be fairly fresh, kept separate, mixed and filtered just before taking the blood. After the counting chamber is filled, it is left, at rest for 10 to 15 minutes in order to allow the blood- platelets to settle to the bottom of the chamber and be more easily and accurately counted. The platelets appear as sharply outlined, round, oval or elongated lilac-colored bodies, some of which form a part of small spheres or globules of hyaline substance. The red cells are decolorized and appear as "shadows," while the nuclei of the leucocytes are stained a dark blue and their protoplasm light blue. This method shows a normal platelet count of 225,- 000 to 350,000 per cu. mm. No constant relations seem to obtain between the variations in the number of platelets and of the leucocytes. According to Determann, the ratio between the red cells and the blood plates is, on the average, 22 :i. The pathologic variations in the number of these cellular elements will be discussed in detail in a later section. III. Morphology of the Blood Before any examination which is concerned with the study of the morpho- logical characteristics of the blood can be made, it is essential that all of the glassware which comes in contact with the blood should be absolutely clean and dry. The glass slides, as they come from the shops, are often coated with substances which are removable with diflSculty. Moreover, these slides are not in all cases perfectly level on both surfaces. It will need but one experience with an uneven slide to convince the. worker that it is a loss of time to attempt the use of such slightly convex or concave slides. The cover- glasses should be of the very best quality of glass, should be as thin as possible (number o), and three-fourths inch square. The seven-eighths inch square covers as also the larger rectangular ones are not as desirable for blood work, especially in the examination of fresh specimens. The sUdes and covers should be cleaned with soap and water followed by water and alcohol. In some cases it is necessary to soak them in concentrated hydrochloric acid for some hours and then wash with water, alcohol, and ether, or the ordinary acid-alcohol may be used. After being cleaned they should be kept either in 95 per cent, alcohol or, preferably, polished with a clean linen cloth or a piece of tissue-paper and kept in dust-proof receptacles. It is a wise precaution invariably to polish the slides and covers before use 1 Jour. Am. Med. Assn,. 1911, LVI, 1457. See, also, Fonio, Deutsch. Ztschr. f. Chir., 1912, cxvii, 176. Digitized by Microsoft® 47° DIAGNOSTIC METHODS as dust particles are prone to collect even under the best conditions. As a rule, it is better to use only new cover-glasses and not attempt to clean them after use. The slides may, however, be cleaned by boiling with a strong alkali solution, washing with hot water acidified with hydrochloric acid, then with hot water, alcohol, and ether. After the cover-glasses have been polished it is the best practice to handle them only with forceps, as moisture is almost certain to collect on them if the fingers be used. This is not only better technic, but more rapid work may be done with their use. Two kinds of forceps are necessary in such work. The first is one for holding the cover firmly, being found as the locking forceps of Ehrlich or the cross-point forceps, while the second is the ordinary pinch forceps with which the second cover-glass is handled in mg,king smears, (i) Examination of Fresh Blood. The examination of fresh blood is a very important part of hematological work and should be a routine procedure in every case possible. If the blood cannot be examined for several hours after being taken, it is wise not to attempt the study of a fresh specimen, as so many changes will occur in such slides that no certain findings obtain. The information obtainable from such examination of fresh blood often supplements that which one may derive from a study of the stained specimens, as some peculiarities, such as the ameboid movement of the leucocytes or the motility of the malarial parasite, may be studied only in this way. Technic. Assuming that the slides and cover-glasses are clean and dry, the ear is punctured as previously described. Wipe away the first few drops of blood and touch the center of a cover-glass, held with the pinch forceps, to the top of the next drop, which should be about the size of a small black-headed pin. If this drop be too large the layer of blood will be too thick to permit of proper examination. Care should be taken that the cover-glass does not touch the skin. Drop this cover onto a slide, which has been warmed by rubbing or by passing through a flame. If the glassware be clean, the drop will spread evenly in a thin circular layer, not quite to the edge of the cover-glass. Under no circumstances should pressure be used to thin the layer or to readjust the cover after it has settled on the slide, as artefacts may be easily introduced in this way. The slides thus prepared are examined first with a low-power lens to obtain an idea of the even distribution of the cells over the entire area. The detailed study is carried out with the 3d! 2 oil immersion lens, but it should be remembered that a smaller magnification may give a better general survey. These preparations will keep long enough for the purposes of exami- nation, but if one wishes to preserve the blood fresh and uncoagulated for a longer period it is well to enclose the cover-glass with vaselin or paraffin or to use the ordinary hanging-drop chamber as suggested by Rosin and Bibergeil. It is sometimes desirable, especially in the study of malarial parasites, to use a warm stage or a warm chamber. If, however, the specimen is examined soon after its preparation, no such precaution is necessary, provided the room is not Cold. Digitized by Microsoft® PLATE XVII. KaTha ^-^'' Fresh Normal Blood. (Zeiss Ocular 4, Objective DD.) Digitized by Microsoft® Digitized by Microsoft® THE BLOOD 471 In order to judge of the changes which abnormal blood may show in the fresh state,' one must be thoroughly famiUar with the appearance of normal blood. This latter knowledge can by obtained only by frequent study of fresh normal specimens and not from any text-book description. To at- tempt to learn without microscopic study the size, shape, color, and refraction of the red and white cells, the relation of the blood-plates to fibrin formation, the number of the various cells and their ratio to one another, would be absolute idiocy. An examination of the fresh blood as described above gives information regarding the presence or absence of the malarial parasite, the spirochete of relapsing fever, the filaria, and trypanosomes. It affords evidence of in- creased or decreased rouleaux formation, number, deformities, and degenera- tions, as well as of the amount of hemoglobin of the red cells; the presence of a leucocytosis or of a leucopenia and of ameboid movement of the leucocytes. However, care must be taken to avoid premature conclusions from such study and to institute further examinations of the stained specimen to clear up doubtful points. The observer must be on his guard lest he mistake the nor- mal Brownian movement in the protoplasm of the cells for ameboid or parasitic movement. Curious phenomena are observed in the fresh specimen as the blood dries and should not be misinterpreted. The various characteristics of fresh blood will be taken up in detail later. The introduction of the ultra- condenser or dark-field illuminator has opened up a field of great possibilities in the examination of specimens of fresh blood, especially when malarial parasites or spirochaetas pallidse are suspected. Fig. 138. — Preparation of smears with two glass slides. {Da Costa.) (2) Preparation of Smears. To prepare blood smears, which are to be later examined in the stained condition, one may spread the blood in capillary layers on slides or between cover-glasses. The former method is the one used in the writer's laboratory and has given excellent and satisfactory results. A fair-sized drop of blood is collected on one end of a clean dry slide, held between the thumb and second and third finger of the left hand. A second slide is held in the same manner by the right hand, but at an angle of 45 degrees to the first one and touching the drop of blood. Allow the blood to spread out by capillarity along the edge of the second slide. As soon as this occurs, draw the drop of blood along the first sUde with a clean sweep, exerting little pressure with the second slide and maintaining the angle of 45° between the two slides, allowing the second slide to rest rather upon the blood than upon the slide (see cut). In the proc- ess, as recommended by some writers, the second slide is gradually drawn Digitized by IVIicrosoft® 472 DIAGNOSTIC METHODS into a position perpendicular to the first one. This procedure does not yield, in the writer's hands, as good results as the former method, as it is more dif&cult to maintain equal pressure, the smear being as a result too thick or too thin in places. Instead of a slide, a cigarette paper may be used as a spreader and gives good results. This method of making blood smears has the advantage of ofEering a large surface for examination, of making smears which are fairly uniform after some practice, of dispensing with the necessity of mounting the specimen, and of permitting the fixation of the smear in the Fig. 139. — Preparation of blood smear with cigarette paper. {Da Costa.) free flame. It is less expensive than the method to be described later and permits of the cleaning and later use of the slides. The beginner may find, on examining his early specimens made by this method, that the leucocytes collect at the distal end of the smear and that the general surface contains few white cells. ' This is due to the use of undue pressure in making the smear, and may be avoided by proper attention to this detail. A second method which has many advocates is the use of two cover- glasses. One clean, dry cover-glass, which should not be too large (prefer- ably three-fourths inch square or the larger rectangular slips) , is held by the Ehrlich or cross-bladed forceps or, as some advise, between the thumb and first finger of the left hand. The other cover, held in the pinch forceps or between the thumb and first finger of the right hand, is touched to the drop of Fig. 140. — Ehrlicii forceps. blood as it wells from the puncture in the ear. This second cover is then dropped at once upon the first in such away that the corners of the two glasses do not coincide. If the glasses are clean the blood spreads out evenly in a thin capillary layer between them. As soon as the spreading is complete, the two covers are drawn apart, in a line parallel to the plane of their surfaces, by a steady, quick motion, being sure to avoid lifting them apart. This manipu- lation can be learned only by practice and never from any description. If the fingers are dry and if care be taken to touch only the corners of the covers the forceps need not be used in separating the covers, but it must be remem- bered that moisture will cause changes in the specimen. It is, therefore • advisable to use the forceps in this part of the technic unless the rectangular shps be used. As soon as the covers are separated, they are allowed to dry in the air or by waving thei^gj^^(;J;]g5^9j^j^§(J^@nd fro. They should be'at THE BLOOD 473 once placed in a clean, closed receptacle, such as a Petri dish, until ready for the later complete fixation and staining, as dust will collect upon them or flies may attack them if left in the open air. It is rarely necessary to fix these smears at once, but with some stains such treatment is advisable. This second method of making the blood smears is more difficult than the first, is not so reliable, does not give as great a surface for examination, and always shows the lower cover-glass better and more uniformly spread than the upper. Smears made by either of these methods should be uniform throughout with the exception of the edges, which should never be used as they are too thick for allowing definite conclusions to be drawn. The red cells should lie on their broad surface, should not be in rouleaux except at the edges, and should not show deformities due to errors in technic. The leucocytes fre- quently collect at the edges of the specimen if too great pressure be used in Fig. 141. — Pinch forceps. making the smear, while the platelets always collect at the point first touched by the second slide or cover-glass. The preparation of thin even smears is necessary for the proper carrying out of the later technic. Those specimens which are irregular or are too thick would better be discarded, as the time consumed in studying such specimens will not be compensated for by the results obtained. It is much better to make several smears than to be content with a few bad ones. A little experience with poor smears will convince the worker that it is advisable to use great care in preparing them, especially if a differential count is to be made or if a study of the degenerations and deformities of the cells is to be undertaken.^ (3) Fixation of Smears. Before any staining of the cellular elements of the smear takes place, the protein constituents of the blood must be coagulated by exposing the air- dried film to the action of a high degree of heat or to that of various chemical reagents. The selectioji of the method of fixation will depend to a great ex- tent upon the stain to be used later. Fixation is always essential if aqueous stains are used, while it is not so necessary if strong alcoholic solutions are employed. In the use of the different modifications of the Romanowsky stain, the fixation is done by the methyl alcohol employed as a solvent for the various stains. As a general rule, fixation by heat is preferable to that by chemicals, as artefacts are less prone to appear, providing the degree of heat is carefully regulated. Fixation by Heat. This method, which is the most difficult to use and which is at the same time the best, is the only one which is reliable when Ehrlich's triple stain is to 1 See Schilling, Deutsch. i&i^itfi^^TjfrMioicQsMl^lX, 1985. 474 BIAGNOSTIC METH0D3 be employed. The principle is as follows: The air-dried specimen is sub- jected to the action of a temperature of iio° to 150°, for a more or less varying length of time, depending on the experience of the special worker. The lower the temperature the longer must its action be exerted. The apparatus most frequently employed is the copper plate introduced by Ehrlich. This is an unpolished triangular plate of copper about 3 mm. thick, 30 to 50 cm. long, and 10 cm. wide, which is held in position by vertical standards. It is heated by an alcohol lamp or a Bunsen burner placed under the narrower end until the temperature of the plate becomes constant, the parts nearer the flame being naturally warmer than those more remote. The temperature of the different portions of the plate may be readily found by determining the points at which water (100), toluol (no), xylol (140), or of turpentine (150) boil. The slides or cover-glasses are then placed, smeared side up, at the desired point (the outer margin of the glass being three-fourths inch from the boiling-point and toward the flame). Just how long a period is necessary, at the temperature selected, to^ive a perfect fixation will depend upon the age of the specimen and upon the condition to be studied. It is good practice to place several slides at the point desired for a period of one hour and then remove a slide at intervals of 15 minutes thereafter. One of the specimens is sure to be good by this method and the remaining ones may, there- fore, be properly heated. Freshly made specimens require longer heating than the old ones, while normal blood requires a longer exposure than abnormal specimens. As a rule, the specimens require, when the triple stain is to be used (and this is the one riiost frequently employed with heat fixation), from one to one and one-half hours at a tem- FiG. 142.— OverTfor fixing blood perature of no, although some workers use films. {Da Costa.) only a few minutes' (one to three) exposure to such temperatures. The higher the tempera- ture the less time is essential. Rubinstein uses a point at which a drop of water does not boil, but assumes the spheroidal state (so-called Leidenfrost phenomenon) and places the slides, with the smeared side down, upon the plate at this point for one-half to three-fourths minute. Some workers, as Pappenheim, use this point, but place the smeared side upward. Instead of the copper plate, one may use the ordinary drying oven or a Victor Meyer heater. It is the writer's custom to use a copper drying oven heated by a gas flame regulated by a thermostat. The slides, with the smeared side downward, are placed on a glass plate whose temperature is measured by an accurate thermometer. The temperature is allowed to in- crease gradually to about 80°, from which point it is more quickly raised until the desired stage is appro©^afte^Tfeljlf^jld»©k)«l^g must continue slowly, the THE BLOOD 475 final temperature being maintained for 15 minutes. The best temperature for fixation, in the writer's experience, is 110° to 120° for 15 minutes when the staining is to be done with eosin-methylene blue or eosin-hemotoxylin, while for the tri-acid stain a temperature of 120° to 125° for one and one-half hours should be maintained. Some workers heat to 160° rapidly and then allow the films to cool to 30°, when fixation is complete in about 15 minutes. Engel and Cabot recommend, in the absence of other equipment, the passing of the smear through the flame several times. Such treatment often yields excellent results, but is uncertain and requires much experience. It is to be remembered that too rapid changes of temperature are to be avoided, as shrinking or splitting of the cells wiU occur under such conditions. So much depends upon proper fixation that a little more time spent in obtaining good specimens will shorten the time necessary for the future examination. Proper staining depends on proper fixation, especially with the tri-acid stain, in which cases the over- or under-heating is evident in the color tone of the erythrocytes. Chemical Fixation. (i) Absolute Alcohol. Allow the alcohol to act for five minutes to one hour, or boiling alcohol for one minute. The slide is simply covered with the fijjative and left for the desired period. Much depends upon the stain to be used as to the time neces- sary for good fixation. If an alcoholic stain, five minutes is sufficient; if a watery or alkaline one, a longer time is essential. After fixation drain off the alcohol and aUow the residue to evaporate in the air or wash with water and dry between sheets of filter-paper. If alcoholic stains are to be used the moist specimen may be directly passed through the flame. This fixative is unre- liable if a study of the neutrophile granules is to be made, but it brings out the nuclear structures fairly well. (2) Nikiforoff's Method. Cover the smear with equal parts of absolute alcohol and ether and allow the fixative to act from one-half to two hours. After the fixation allow the fixative to evaporate or wash with water and dry. Some workers advocate short fixation especially where the malarial organism is to be studied. This fixative brings out the degenerations of the red cells in good shape. (3) Methyl Alcohol. This fixative is used absolutely pure for three to five minutes. It is the most generally applicable chemical fixative and gives beautiful specimens. If used in combination with stains, as in the Romanowsky methods, it gives as perfect preparations as absolute alcohol in one-half hour and brings out the neutrophile granulations in better outline. A longer fixation than three to five minutes does no harm; and a shorter one, especially if the fixative is the solvent for the stain, will give fair results, the outline of the cells being sharper the longer the fixative is allowed to act. (4) Formalin. This fixative may be used as a i per cent, solution in 95 per cent, alcohol and allowed to act for oBej;mmiteA,aaiE£najin^vocates, but the writer has 476 DIAGNOSTIC METHODS had much better success with the 0.25 per cent, solution in 95 per cent, alco- hol for one minute, as Futcher and Lazear suggest. Allow the fixative to act for one minute, wash in water, and dry between filter-paper. Some writers advocate the direct treatment of the fixed smear with the stain without an intermediate washing and drying, but I have never found the results as good by this method. Instead of the formalin solutions, the vapors may be used by placing the specimen under a bell jar with a few drops of 40 per cent, for- malin and allowing the fixative to act for one to five minutes. The normal staining of the cells is not always as clear as could be desired after the use of such a fixative. A large number of inorganic fixatives have been advised, among them being mercuric chlorid, chromic acid and osmic acid, but these fixatives are much more apt to produce artefacts than are the others mentioned above. While these latter fixatives give good demonstrations of the nuclear structures and of mitotic figures, the granules are very imperfect; but chromic acid has many advantages as a fixative in the study of the chromatin elements. (4) Staining Methods. {A) General Considerations. Since the work of Witt, we have recognized that the color of an organic substance is due to the presence of two definite atomic complexes in the molecule of the colored substance, The first of these, the chromophore group, is responsible for the chromogenic properties of the substance, while its in- fluence as a dye is increased by the presence of the second or auxochromic group. The color of the compound is the more intense the more of these groups are present. Groups or atoms which intensify the color of the sub- stance are called, by Schiitze, bathochromes, while those which reduce the color are called hjrpsochromes. While the dyes, the so-called anilin dyes, used in hematological work are all aromatic derivatives, it must not be assumed that such are alone characterized by staining qualities, as many simple aliphatic derivatives show a definite color and exert some staining property, depending on the presence of the two groups above mentioned. The chief chromophore groups are the CO (carbonyl) group, the CS group, CN, HCN, the — N = N — (azo group) the I yO (azoxy group), NO (nitroso group), — N^' | (nitro group), — N^ ^O and the — N = SO group. The most important auxochrome groups are the NH2 and the OH groups, the former being a much more powerful one than the latter. These auxochrome or haptophore groups make possible the union of the stain with the tissue as a direct salt formation. Substances, which possess a chromophore group and are colored or intensified by the presence of the auxochrome group, are called chromogens. It is, therefore, evident that the effect of an auxochrome depends not only upon its own na- ture, but also upon that of the chromogen. Through the introduction of H by means of hydrocarbogj^a^j^jjjjijp^P^yjg^omes are produced and the THE BLOOD 477 color becomes intensified, the effect being so much greater the higher the molecular weight of the substituting hydrocarbon radical. As a large number of colored substances have a tendency to form tauto- meric compounds, the salt formed by combination with the auxochrome groups may have a different constitution from that of the free base or acid. Many chromophore groups are also capable of forming salt, but only with strong acids or bases. In this formation of salts, by union with the auxo- chrome or chromophore groups, we must remember that halochromia may be seen. By this is meant that uncolored or weakly colored substances may combine with acids to form salts without the color being due to the presence of a chromophore group. It is essential that the chromophore group possess a double bond of union, as oscillation in one portion of the molecule is thus possible. As the chromophoric as well as the auxochromic radicals may have acid or basic tendencies, it is manifest that the reaction of the substitution product will depend upon the interrelations of the acid and the basic radicals. The best dyes are, therefore, obtained by substitution in one direction, that is, by accumulating either basic or acid groups separately. It is thus seen that we 'have two general classes of dyes, the acid and basic, depending upon the preponderance of the total acid or basic groups in the molecule of the dye. It has been shown by Ehrlich that these acid and basic dyes, may be so combined that a third one results, showing neither acid nor basic properties. This class of dyes, known as neutral dyes, is of the greatest importance in hematological work. In them we have not only the staining properties due to the original chromophore and auxochrome groups of both the acid and basic dyes, but also those due to the union of the compo- nent groups in the neutral dye. Such dyes are hence called polychrome dyes and are usually soluble in an excess of one of the component mother dyes, generally the acid one. It must be understood that in speaking of a dye as acid, basic, or neutral, we do not refer so much in our staining work to the chemical reaction of the dye, but to the portion of the dye to which the stain- ing is due — that is, to the specific auxochrome and chromophore groups. Among the basic stains we find methyl green, methylene blue, amethyst violet, neutral red, dahlia, pyronin, thionin, fuchsin, methyl violet, Bismarck brown, alum hematoxylin, and safranin. Most of these stains, depending on the strength of their component groups, color the nuclear substance of both red and white cells, as also the cytoplasm and certain granules of abnormal red and of some normal white cells, the staining being influenced, as Matthews has shown, by the alkaline reaction of the tissues. The most important acid stains are eosin, acid fuchsin, orange-G, indulin, nigrosin, aurantia, and salts of picric acid. These dyes color the red cells and the eosinophile (oxyphile) granulations of the leucocytes. The neutral portion of the dyes color the so- called neutrophile granules of the leucocytes. In the process of staining, it is a question whether we have to do with purely chemical processes as Knecht 's theory assumes or whether the solid- solution theory of Witt or the mechanical theory of surface attraction are accountable for the phenomena observed. It is probable that the salts of the Digitized by Microsoft® 478 DIAGNOSTIC METHODS dyes are decomposed by the cells and that new compounds result from the union of the acid and basic stains and the various chemical entities of the cells. Yet we have instances in which the dye is simply stored in the cell without any chemical union taking place. We must, however, account for the elective character of certain stains by a purely chemical activity, as, for instance, chromatin, which undoubtedly consists of nucleinic acid, alvyays takes a basic stain, even though a neutral compound is used as the staining agent. It is to be remarked that a neutral stain does not color all of the acido- phile or basophile substances of the cells of the same tint. Thus, eosinophil granules are differently colored from the oxyphile hemoglobin of the red cells. The neutral portion of the stain has nothing to do with the staining process beyond the coloring of theneutrophile substances in the protoplasm of certain leucocytes. In the selection of a stain for general hematological work, it is necessary to use a compound stain or, at least, two simple stains, one after the other. A single simple stain colors only a few of the elements and affords no general idea of the blood picture. Indeed, to obtain definite conceptions of the finer structure of the blood, it is necessary to study several slides stained by differ- ent methods. It is usual to select for routine work a stain which will reveal the greatest amount of information regarding the histological characteristics of the blood. This is the so-called panoptic staining and is to-day generally accompUshed by the use of the various forms of neXitral stains. In making the stains for one 's self or in buying them in the market, one must be certain that only chemically pure pigments are used and that the solution is made according to the formula with the purest solvent obtainable. It will need only one experience with a poorly made stain to convince the worker of the importance of this detail. (B) Methods of Staining. In his work the author uses, for routine purposes, the Wright's stain, the eosin-methylene blue, and eosin-hematoxyhn stains. These stains have the advantage of giving clear-cut pictures of practically all of the important blood elements along with simplicity of technic. As it is not always posi sible to secure or even to make a Wright or an Ehrlich triple stain which will give reliable results under all circumstances, the writer feels that the general worker would better use the eosin-methylene blue stain for his daily work. The many modifications of the Romanowsky stain have their advocates, and it must be said that they yield reliable results when they are working properly, but no one can say when they will go wrong or how to make them work right when once they do give poor results. Eosin-Methylene Blue. A number of methods of using these two simple stains have been advo- cated by such workers as Chenzinsky, Ehrlich and Lazarusj von Willebrand, Plehn, Aldehoff, and Gabritschewsky, but the writer has found the method advocated by Miillern^ to be the most generally applicable and if properly, 1 Grundriss der klinischen^^^^untejs^chjj^^^Wi^igog. THE BLOOD 479 applied, the most reliable modification. By it we are able to stain all of the blood elements, including the neutrophile granules and obtain, thus, a panoptic picture whose findings are not excelled by those of the various modi- fications of the Romanowsky stain. Technic. (a) Fixation of the smear in pure methyl alcohol for three minutes, {b) Preliminary staining in J^ per cent, alcoholic (70 per cent.) solution of Griibler's "french pure" eosin from three to five minutes, (c) Wash in dis- tilled water and dry between filter-paper, {d) Lay the slide in a carefully measured and weU-mixed solution of 20 drops of 3^ per cent, aqueous solution of methylene blue (B. pat.) and ten drops of the above eosin solution for one- half to one minute, (e) Wash quickly and briefly with distilled water and dry at once between filter-paper or over the flame, (f) Mount if desired in Canada balsam or examine directly with a high-power lens. This stain shows the red cells and the eosinophile granules of the leuco- cytes of a bright red tone, the neutrophile granules pink to bright red (distin- guished from the eosinophiles by their smaller size), the nuclei, mast-cell granules, bodies of the lymphocytes, platelets, malarial organisms, trypano- somes, and filaria varying shades of blue. The preliminary staining with eosin serves the purpose of bringing out the neutrophile granules more clearly while the basophile granulations are of course unaffected by such treatment. It is probable that the later staining with the eosin-methylene blue mixture has the same characteristics as the neutral stains to be mentioned later. While this stain has the advantage of simplicity, reliability, and panoptic power of staining, it is somewhat inferior to other stains in bringing out some forms of the malarial organism, owing to its lack of chromatin staining qualities. The writer has found that the eosin and methylene blue above mentioned are the best to use in this process and that Turk's advice, regarding the use of fairly fresh eosin solutions and of old methylene-blue solutions, is warranted. The blood preparations should not be over a few days old to show the best pictures, as those over a week old may show a diffuse plasma staining and the neutro- phile granules not clearly differentiated. If the nuclei do not stain well with the methylene blue as many writers claim, make a second preparation fixing somewhat longer in methyl alcohol. By following out these precautions the worker will be rewarded with beautiful specimens. This method is to be recommended for all routine examinations. Eosin Hematoxylin. This stain is especially important in cases in which the nuclear structures are to be studied.^ It stains the nuclei bea^utifully, showing their finer structure, karyokinetic figures, and pycnotic qualities, as well as the basophile granules of both red and white cells. The solutions required are: (i) J^ per cent. Griibler 's blood eosin in 70 per cent, alcohol. We may use, with equally good results, the eosin mentioned in the previous method. (2) Delafield's hematoxylin, the formula of which is 1 See Williams (Jour. Am. Med. Assn., 1913, LXI, 1627), who describes a modification of this stain which he calls ")^0^^i,ly Microsoft® 480 DIAGNOSTIC METHODS Hematoxylin crystals, 4 grams. Alcohol (absolute), 25 c.c. Ammonium-alum crystals, C. P., 52 grams. Distilled water, 400 c.c. Glycerin, C. P., 100 c.c. Methyl alcohol, C. P., 100 c.c. Rub up the hematoxylin crystals with the alcohol until they are dissolved and place the solution in a loosely corked glass bottle, allowing it to stand exposed to the light for four days. Dissolve the ammonium-alum in the water and allow it to stand exposed in the same way for four days. At the end of this time mix the two solutions, shake thoroughly, and filter at the end of three hours. Add the glycerin and methyl alcohol to the filtrate and allow this to stand overnight. Filter the mixture, place it in a clear bottle, and allow it to ripen, exposed to the light for six weeks, when it is ready for use. Technic. Stain the specimen with the eosin solution for one-half minute, and wash in water. Without drying place the slide in the hematoxylin solution for one to three minutes, the time varying with the particular stain and with the experience of the worker. Wash with water, dry, and mount. This stain does not give as good results as does the former method, but is to be espe- cially recommended when the nuclear structures are to be studied. Ehrlich's Triple Stain. In the literature of hematology we find the expressions "triacid" and "triple stain" used synonymously. The triacid stain, as originated by Ehrlich, was a mixture of equal parts of saturated solutions of indulin, nigrosin, and aurantia, and was used to differentiate the eosinophile granules. When used synonymously in these days, the triple stain is always meant. The composition of this latter stain is as follows, made up of two add and one basic stain: Saturated watery solution of orange-G, 13-14 c.c. Saturated watery solution of acid fuschin, 6-7 c.c. Distilled water, 15 c.c. Alcohol (absolute), 15 c.c. Saturated watery solution of methyl green 00, 12.5 c.c. Alcohol (absolute), 10 c.c. Glycerin, 10 c.c. The pigments must be chemically pure and the solutions must be added in the order named, the methyl-green solution being added drop by drop with constant stirring. This stain is difficultly prepared, being usually a failure. The writer would, therefore, advise the worker to obtain it already made; and even then it may not prove satisfactory. The correct stain should have a russet-brown color and should not show any trace of a precipitate. It improves up to a Digitized by Microsoft® certain point with age but, after a time, precipitates invariably occur, making it useless. It should never be filtered or shaken, the solution to be used being taken from the center of the bottle with a dripping pipet. Technic. The smear should be ficxed by heat as previously described. After proper fixation, cover the smear with the stain and allow it to act for one to ten min- utes as the experience of the worker with the particular stain may indicate. Wash with distilled water, dry, and mount. One advantage of this stain is that it cannot overstain, those films appearing so being underheated, while those understained are overheated. The stain shows the red cells of a buff or orange color, without the slightest shade of red (a reddish tint is given with underfixed specimens while a yellow tone is shown by those overheated), the nuclei of the leucocytes a dark green, those of the normoblasts black, the neutrophile granules a lilac color (though some occasionally show a reddish tinge), and the eosinophile granules of a crimson tone. This stain was introduced as specific for the neutrophile granules, but its disadvantages are too numerous to warrant its recommendation as a routine stain. It is a poor nuclear stain, does not show the structure of the normal mononuclear leucocytes, does not stain the basophile granules , nor the malarial or other parasites. For a reliable preparation, showing those features for which it is especially adapted, a proper fixation is an absolute essential. On account of the lack of nuclear staining with the triple stain, Pappen- heim has substituted methylene blue or methylene azure for the methyl green, and eosin for the acid fuchsin. The writer has had no experience with this modification and cannot, therefore, speak regarding its value. Polychrome Methylene-blue-eosin Stains. These stains are very numerous, each having its advocates. They are easy to use, contain a reliable fixative, and give satisfactory results, but are not always obtainable or easily prepared. They are the stains which give, perhaps, the best panoptic results and are especially serviceable in the study of the malarial organism and other parasites as many of them contain chroma- tin-staining elements. The granulations of the leucocytes are not as well marked in all cases as they .should be, so that for a complete study of the various types of granules in the cells several stains would better be used. Romanowsky had found that the addition of a watery eosin solution to an aqueous methylene-blue solution, until an insoluble precipitate began to form, gave rise to new staining properties of the solutions, in the sense that the chromatin substance of malarial organisms was stained a beautiful red. The specific staining properties of this mixture were later found to be due not to a combination of eosin and methylene blue, per se, but rather to the formation of a new compound between eosin and an impurity or decomposi- tion product in the methylene blue, namely, methylene azure. Jenner in his stain makes use of a methyl-alcohol solution of the isolated precipitate, the 31 Digitized by Microsoft® 482 DIAGNOSTIC METHODS eosinate of methylene blue, which lacks the red chromatin staining element. The same may be said of the May-Griinwald stain. In making up these polychrome stains it is not general to use the pure methylene azure and eosin, but rather solutions of methylene blue containing a variable amount of the methylene azure to which eosin is added. Recently Wilson,^ in a careful study of the methylene-blue-eosin combinations, has shown that very little methylene azure and methylene violet exist in the stains as commonly employed. He finds evidences of at least four staining bodies in such mixtures, namely, the eosiiiate of methylene blue, eosinate of methylene violet, eosinate of methylene azure, and eosinate of thionin. Wright's Stain. This stain is a much improved .modification of the Leishman stain,'' as its method of preparation requires but a few hours and as the resulting dye is somewhat more panoptic than is that made by the Leishman method. Preparation. To a 0.5 per cent, aqueous solution of sodium bicarbonate add methylene blue (B. X. or "medicinally pure") in the proportion of i gm. of the dye to each 100 c.c. of the solution. Heat the mixture in a steam sterilizer at ioo°C. for one full hour, counting the time after the sterilizer has become thoroughly heated. The mixture is to be contained in a flask, or flasks, of such a size and shape that it forms a layer not more than 6 cm. deep. After heating, allow the mixture to cool, placing the flask in cold water if desired, and then filter it to remove the precipitate which has formed in it. It should, when cold, have a deep purple-red color when viewed in a thin layer by transmitted yellowish artificial light. It does not show this color while it is warm. To each 100 c.c. of the filtered mixture add 500 c.c. of a o.i per cent, aqueous solution of "yellowish, water-soluble" eosin and mix thoroughly. Collect the abundant precipitate, which immediately appears, on a filter. When the precipitate is dry, dissolve it in methyl alcohol (Merck's "reagent") in the proportion of o.i gm. to 60 c.c. of the alcohol. In order to facilitate solution, the precipitate is to be rubbed up with the alcohol in a porcelain dish or mor- tar with a spatula or pestle. This alcoholic solution of the precipitate is the staining fluid. It should be kept in a well-stoppered bottle because of the volatility of the alcohol.^ Technic. Cover the film with a noted quantity of the staining fluid by means of a medicine dropper. After one minute add to the staining fluid on the film the ' Jour. Exper. Med., 1907, IX, 645. - Leishman Stain. Prepare a i per cent, aqueous solution of medicinal methylene blue (Griibler) and add 0.5 per cent, sodium carbonate; heat for 12 hours at 6s°C. and keep for 10 days at room temperature. Prepare a. 0.1 per cent, aqueous solution of "yeUosvish, water-soluble " eosin. Mix equal volumes of the two solutions and leave for 10 to 20 hours, shaking the mixture at frequent intervals. Filter, wash the precipitate with distilled water until the washings are a pale blue, dry on the filter and powder the dried residue. The staining solution is prepared by dissolving 0.2 gram of this powder in 10 c.c. of methyl alcohol. This stain is used precisely as the Wright stain, the blood-picture being very similar. ' Jour. Am. Med. Assn., 1910, LV, 1979. Digitized by Microsoft® THE BLOOD 483 same quantity of distilled water by means of the medicine dropper aiid allow the mixture to remain for two or three minutes, according to the intensity of the staining desired. A longer period of staining may produce a precipitate. Eosinophilic granules are best brought out by a short period of staining. The quantity of the diluted fluid on the preparation should not be so large that some of it runs off. Wash the preparation in water for thirty seconds or until the thinner portions of the film become yellow or pink in color. The experi- ence of the worker may be such as to demand longer decolorization with any particular specimen. When the desired degree of differentiation is reached, dry the specimen quickly between filter-paper, mount if desired, and examine first with the low-power lens to observe the staining effects, and then with the high-power lens for the more minute study. When searching for malarial parasites, the decolorization would better be of short duration, as the chroma- tin suffers to a great extent in this process. This stain shows the red cells colored either orange or pink (depending on the time of decolorization), nuclei of leucocytes blue or dark lilac, neutro- phile granules lilac, eosinophile granules red or pink, fine basophile granules deep blue, large mast-cell granules purple, protoplasm of the lymphotytes robin 's-egg blue, blood-plates deep blue or purple, bacteria blue, malarial and other parasites blue, the chromatin element varying from lilac to ruby- red to black. Polychromatophilia and granular degenerations are well shown, the granules being blue. This stain is very useful in studying lympho- cytes, mast cells, blood-plaques, and the finer structure of the malarial organ- ism; but the leucocytic granules, at times, are not suflBiciently differentiated. Giemsa Stain. Giemsa has shown that the complicated methods of preparing the dyes may be dispensed with by the use of the pure staining substance extracted from the polychrome methylene blue, namely, the methylene azure. If this pure pigment be used the stain becomes a pure chromatin one. It is advis- able, therefore, to use Griibler's azure II, a combination of equal parts of methylene azure and medicinal methylene blue. The formula of the Giemsa- stain is as follows : Azur II-Eosin, 3 . o grams. Azur II, 0.8 gram. Glycerin (C. P.), 125.0 grams. Methyl alcohol (C. P.), 375 -o grams. Grind up the dyes in the alcohol and then add the glycerin. Technic. Fix the films in methyl alcohol, and stain for fifteen minutes in a mixture of 15 drops of the stain to 10 c.c. of distilled water. If necessary, a trace of sodium carbonate may be added to the water to intensify the basic stains. Wash in water, dry, and mount. It is to be remembered that oil of cedar will bleach these specimens quite rapidly, so that it is advisable, if the films are to be kept, to stain them upon the slides so that mounting becomes unneces- sary. Strong light should not be allowed to act on these stained films for any Digitized by Microsoft® 4»4 DIAGNOSTIC METHODS length of time as the chromogen stain fades rather quickly under these condi- tions. The various elements are stained as with the Wright stain, but the neutrophile granules are often not well defined. Pappenheim's Stain. Pappenheim (Technik der klin. Blutuntersuchungen, Berlin, 1911; Folia Hsemat., 1912, XIII, 339) advocates as the best and most serviceable stain the combination of the Giemsa stain as follows: Fix the blood film by covering it with May-Griinwald stain (Wright's answers as well) for 3 minutes, add to this stain on the slide an equal amount of distilled water and allow it to act for I minute. Pour off the fluid but do not wash. Now cover the stained film with dilute Giemsa solution (15 drops of stain to 10 c.c. of distilled water) and allow to act for 15 minutes. Wash thoroughly and dry, but not over the flame. Mount, if desired, in neutral Canada balsam. The result is extremely pan- optic. The nuclei stain violet. The lymphocytes, monocytes and undiffer- entiated lymphoidocytes have a delicate blue cytoplasm. The mast-cell granulations are ultramarine blue. Eosinophiles have brick-red granulations and the neutrophiles a rose-colored cytoplasm, while their granules are indefinite. Specific Stains for Malarial Organisms. While the above stains all give good results with the malarial organisms, enabling, one to make a diagnosis from an examination of the smear, yet certain other stains are often of advantage in that little else is stained and thus the confusion arising from indefinite staining is obviated. Thionin Stain (Futcher and Lazear). Add to 100 c.c. of 2 per cent, carbolic acid 20 c.c. of a saturated solution of thionin (Lauth's violet) in 50 per cent, alcohol, and allow this mixture to ripen for a few days. Fix the specimen by the formaUn method given above and stain the smear for 10 to 15 seconds. Wash in water, dry, and mount. These preparations do not keep indefinitely, usually fading within a year. The Plasmodia are shown as deep purple, irregular masses enclosed in the faint green red cells. The hyaline forms show as reddish- violet ring-like bodies. The basophile granules and the nuclei are the only other elements showing any particular staining qualities. Nocht's Stain. This is essentially a Romanowsky stain and is made as follows: Add two or three drops of i per cent, aqueous eosin solution to 2 c.c. of water. To this diluted eosin solution polychrome methylene blue is added, drop by drop, until the red color of the eosin is only faintly visible (the polychrome methylene blue is a i per cent, solution of methylene blue chemically changed by heating with 3^ per cent, sodium carbonate solution for two days at S5°C.). Technic. Fix the film in methyl or ethyl alcohol and lay it face downward in the above mixture for five to ten minutes. Wash with water, dry and examine. The picture is the same given by Wright 's stain, except that the nuclear trans- Digitized by Microsoft® THE BLOOD 4^5 formations and the chromatin substance are better differentiated, being stained a bright red color. The many other stains for the examination of fixed specimens will be passed over, as the writer finds the above stains applicable to practically all routine work. Many writers prefer different stains from those mentioned, but these stains do not seem to have any advantages which would warrant their use to the exclusion of those given above. Special stains used for bringing out certain granules and deformities will be discussed under later headings. Vital Staining. Although the examination of stained specimens of the blood is usually made with the dried and fixed smear, excellent results obtain when the fresh blood is stained without previous fixation. It is true that a " vital" staining of the blood-cells does not actually take place, as the dyes are decolorized by the reducing and oxidizing action of the living cells. However, a "post vital" staining, that is the staining of whole cells or portions of the cells, after their removal from the circulation and before the death of the cell results, may be accomplished in several ways. We may either add to the fresh drop of blood a few crystals of the stain, as advised by Arnold, and note the staining of certain leucocytic granules and nuclei and recticular structure of many erythrocytes; or we may first dry a staining solution upon the slide, cover this dry stain with a drop of fresh blood, adjust the cover-glass, and seal this to the slide with wax. Some workers prefer to use the hanging-drop slide in the preparation of these fresh specimens. The stains which may be used for vital staining of the nuclei, granules, and plates are methylene blue, toluidin blue, thionin, neutral violet, Capri blue, Nile blue, brilliant-cresyl blue, Janus green, and paraphenyl blue. Of the protoplasmic stains we have fuchsin, acridin red, pyronin, safranin, and neutral red. While the results derived from this method of staining are not as numerous as those of the more usual method, yet it affords much valuable information regarding the vital properties of the cells and regarding the normal structure and the circulatory changes of the cells. ^ This method has been used exten- sively by Ito, Rosin and Bibergeil, Levaditi, Cesaris-Demel, and Pappenheim. Their results lead us to assume that further study will give us much valuable information concerning details of structure both of the cell and of the nucleus. It may be possible by this method to differentiate between certain forms of degeneration of the cell which are now known only indefinitely under the names of metachromatic and polychromatic staining.^ 1 See Luzzatto and Ravenna (Folia Haemat., 1912, XIII, 102) for a discussion of the numerical relations of the granular and non-granular erythrocytes as shown by this method. 2 See Ross (Induced Cell-reproduction and Cancer, Philadelphia, 1911, and London, 1 9 1 2) for the use of agar films containing stains and activating salts (auxetics) in this connec- tion. See, also, Anitschkow, Med. Klin., 1914, X, 46s; Barbaro, Gazz. d. osp., 1914, XXXV, 68j; von Mollendorff, Deutsch. med. Wchnschr., 1914, XL, 1839; Hober, Biochem. Ztschr., 1914, LXVII, 420; Lindborn, Nord. Med. Arch., 1914, XLVII, i ; Evans and Schule- mann, Science, 1914, XXXIX, 443; Folia Hsemat., i9iiiXIX (I), 207; Rubino, Rif. Med., igrs, XXXI, S33; Traube, H!Dy^aiBectfej*M;«6®§;aH5ex, 309. 486. DIAGNOSTIC METHODS (5) Erythrocytes. (A) Appearance and Structure. In fresh, normal blood the red cells, or erythrocytes, appear as thin, flattened, homogeneous, biconcave, nonriucleated, discoid bodies with a sharply defined regular outline and a clear semitransparent center. In some cases they may apppar distinctly cup- or bell-shaped. The cells show, when examined singly, a pale greenish-yellow color but, when more thickly grouped, exhibit a reddish tint. The degree of color in these cells depends up on their hemoglobin content, the clear central area becoming larger and the entire cell becoming paler as the hemoglobin decreases. The loss of hemoglobin may be so great as to lead to the formation of the so-called "pessary" form, in which only the periphery of the cell is apparent. This central pale area varies much in individual cells and is not at all evident in those which are flattened out. In dry specimens, if thinly spread, the cells are circular, the normal bicon- FiG. 143. — Normal blood showing rouleaux formation and fibrin network. (Da Costa.) eavity is obliterated, and a uniform stain is observed. If the smear be thick the central pale area is observed. - In chlorosis and secondary anemias, the color of the cells is usually uni- formly paler than normally, while in pernicious anemias the color may be even deepei" than normal. These are the usual but not invariable pathologic findings. In malarial conditions, discolored cells are often observed in the fresh specimens, the bronzed or '-'brassy" tone often drawing attention to the presence of a parasite of the estivoautumnal type or of the quartan form. The red cells show a marked tendency, to cohere to one another in more or less regularly arranged piles, forming long rows (rouleaux), like rolls of coil piled up face to face. The exact cause of this phenomenon is unknown, but' it rhay b^ depehdentO^;|fe©gi5!p^fl>pg/^S^J]ff(^tty membrane surrounding THE BLOOD 487 the cell, as Peskind's findings show that the red cells are enveloped by a layer composed of lecithin, cholesterin, andanucleoprotein.^ In certain pathologic conditions this normal rouleaux formation is increased and in certain ones is decreased. The diminished rouleaux formation is observed in conditions associated with increased viscosity of the cells, as observed in most inflam- matory diseases and in the anemias due to malignant disease. This hyper- viscosity of the cells has not at present much clinical significance, but may be shown to be of importance when our knowledge concerning the normal viscosity of the blood becomes more extensive. The structure of the erythrocytes is still an unsettled point in hematology. Neither membrane nor stroma have been fully demonstrated yet after the hemoglobin has been removed from the cells by hemolysis, a stroma may be definitely seen which could hardly be called an artefact. Schafer assumes that the hemoglobin is held in firm combinations by chemical union with other albuminous constituents of the cell and supported by a stroma similar to that of Peskind's outer membrane. That some sort of an outer membrane does exist would seem to be proven by the experiments on hemolysis which have shown that many substances penetrate the cell producing hemolysis, while others in the same concentration have no such effect. It is hard to believe that a selective vital activity is at work here, as the results follow too closely the laws of physical chemistry as apphed to diffusion and osmosis. The cor- puscles are very elastic and contractile so that rapid and marked temporary distortions of shape are possible under the influence of variations in the com- position of the circulating plasma. As the blood dries various changes in the appearance of the red cells are observed. These changes, known as crenation, are due to the evaporation of water and depend upon the quantity of air which comes in contact with the specimen as well as upon the length of time this influence acts. " The develop- ment of one or more small, bright, highly refractile spots in the body of the cell and a slight indentation of the periphery of the cell are the most conspicu- ous indications of beginning crenation. As the process goes on, more and more of these hyahne points develop, until finally the whole surface of the corpuscle becomes thickly studded with glistening bead-like spines. As the stroma becomes drier and drier, its typical biconcavity and sharply cut out- line are lost, contracting strands of the stroma are seen to extend from point to point among the beaded projections, the periphery of the cell changes to a cogged rim, and finally the cell becomes shrunken and shriveled up into a small, many-starred asterisk. Some of the erythrocytes become fragmented and small bits of their stroma are observed to break off and float through the plasma. Others become progressively paler and paler, as the hemoglobin is dissolved out, until complete decoloration occurs. Still others become dis- torted into designs of every conceivable shape so that their resemblance to the normal cell becomes more remote " (Da Costa) . These changes must not be confused with those occurring as a result of pathological changes. Crenation is often induced more rapidly than in normal blood, in the blood of persons ' See Wiltshire, Jour. Pathijaod-JBaote 488 DIAGNOSTIC METHODS suffering from acute infection and from chronic diseases. True ameboid move- ment of the red cells is sometimes seen as a result of a high-grade anemia.^ (B) Size and Shape. The average diameter of these normal human erythrocytes (normo- cytes) is 7.5 ft, the normal variations being between 6 and 9 m (a micromilli- meter, }iooo oi a, millimeter). The size varies depending on the method of preparing the specimen and also upon the osmotic tension of the plasma. Although dwarf and giant cells may occur to a sHght degree at all ages, the normal infant blood shows these variations more markedly (3.3 to 10.3 /i, ac- cording to Hayem). According to Hamburger, the cells are slightly larger in the venous than in arterial blood. Gram states that the size of these cells varies with climatic conditions, being greater in those of the northern cooler countries than in those of the southern warmer climates. The varia- tions in the size of the red cells is very slight in the different sexes. Pathologically, variation in the size of these cells is a common and important feature. Generally speaking, variations in the cellular size indi- cate a severe and chronic anemia, while in the more mild acute forms of ane- mia such variations are unusual. The average size is said to be increased in jaundice, cholera, lead-poisoning, leukemia, congenital heart disease, and cretinism. Tsrpes of Pathological Erythrocytes. Variation in the normal size of the red cells is indicated by the term anisocytosis. This term does not include the misshappen red cells which appear in the blood as a result of degeneration or of mechanical injury and to which are given the name of poikilocytes. Microcytes. These are cells under the normal size, the variations being between i /x and 6 /jt, the usual representatives being about 3.5 fi. It cannot be stated at present whether these cells are mere schistocytes (fragments of larger cells) or are prefect cells of degenerative origin. They occur normally in the blood of embryos and infants, but are rare in that of the adult except in patho- logical conditions. It is certainly true that these undersized cells may arise from purely physical causes, as a result of increased osmotic pressure of the plasma as well as from division of undersized mother cells. This latter phase is made possible by the appearance, especially in pernicious anemia, of nucleated reds of corresponding size. Pathologically, these smaller cells are observed in all severe anemias. As a rule, they stain deeply and evenly, but in some cases of pernicious anemia these cells are deficient in hemoglobin and show irregular staining, yet many of them may have an increased hemoglobin content. Occasionally, but not invariably, we find these undersized cells in chlorosis, in which the hemoglobin is deficient. At times these cells may show polychromatophilia, but this is not the rule. According to Tallqvist, an increase in the number of these microcytes (microcytosis) is an indication of rapid destruction of blood. 1 See Krizenecky, Ztschr. ©/S^Zf efy^^'§f§,SS^, i. PLATE XVin. Q; U 8. 14: o *. S. a (l0r ^ la. IS. tr IS. #1 ^5 ^"*v. 22. Z3. ^ Ka+haWne fliil Types of Red Cells. (Weight's Stain. Zeiss Ocular 4, Oil Immersion Objective.) I — Normal Cells. 2-3-4 — Normal Cells as seen with different Focus. 5 — So-called "Pessary Form." 6-7-8 — Polychromatophilic Cells. 9 — Macrocyte. 10-12-13 — Poikilocytes. 1 1 — ^Poikiloblast. 14-15 — Punctate Basophilia in Red Cells. 16-1 7 — Normoblasts. 18 — Normoblast ^vlTH Pycnotic Nucleus. 19 — Punctated Normoblast. 20-23 — Megaloblasts. 24 — GiGANTOBLAST. Digitized by Microsoft® Digitized by Microsoft® THE BLOOD 4^9 Macocytes. These are cells above the normal size, the variations being between lo and 20 II. Those cells from 9 to 12 /* are called macrocytes; those between 12 and 16 ju are known as megalocytes; while those above 16 /* are termed gigantocytes. These cells are of regular shape, of even staining quaUties, and generally without a well defined central clear area. The larger size of these cells may be partly due to the swelling incident to a lowered osmotic tension of the blood, but more probably this increase in size is traceable to the origin of the macrocytes from the large nucleated reds of the bone-marrow. These cells may show an excess or deficiency in hemoglobin, the former characteristic being observed in the primary pernicious anemias, while the latter is evident in the secondary forms. Morris and Thayer^ report ameboid movements in these cells. Pathologically, the presence of these various forms of macrocytes, giving rise to the condition of macrocytosis, indicates a severe and unusually chronic anemia. They are most frequeptly seen in pernicious anemia, in which the largest cells are sometimes the darkest and some of the microcytes are exceed- ingly pale. These large cells do occur, however, in lexikemia, cholemia, and chlorosis, being frequently pale or "chlorotic" and "dropsical." These " drop- sical" cells are i;ot sufficiently numerous, however, to change the volume index, as Capps has shown, of a secondary anemia to one shown in true pernicious anemia. Poikilocytes. These are misshapen red cells of large or of small size, the varieties of such deformities being numerous. The presence of poikilocytes in the blood is known as poikilocytosis (first described by Damon) and is closely related to crenation as in both cases the cells may be similarly misshapen. The former is a pathologic process demonstrable the moment the blood is taken, while the latter is a physiological process appearing only after the blood has been in contact with air for some time. Poikilocytes arise in several ways. First, faulty technic, especially pressure on the cover-glass of the fresh specimen, will give rise to fragmenta- tion of some of the corpuscles into small spherical masses, dagger-shaped bodies, and small elongated rods (pseudo bacilli of Hayem). Such fragmen- tation is indicative of lowered vitality and feeble powers of resistance of the cells. If the cover-glass be moved after the cells have spread, a large number of them will be distorted into oval or pear-shaped forms, the long axes of which usually point in the same direction. Secondly, true poikilocytes, or cells misshapen while in the circulation, are probably due to ameboid motion of a portion or the whole of a cell or to alterations in the plasma. These mis- shapen cells are usually pear-shaped with a budded projection at one or more poles. Cells may be seen which resemble a tennis-racket, a kidney, tomahawk-blade, dumb-bell, or anvil, while oval forms are especially observed in pernicious anemia ^"(^r^y^ggsW^^l^^^g^^t of diagnostic importance. > Arch. Int. Med., 1911, Vin, 581. 49° DIAGNOSTIC METHODS Poikilocytosis is an indication of severe anemia with degenerative changes in the red cells; although it is not characteristic of any single disease, it is found more frequently in pernicious anemia and leukemia. (C) Nucleation. Nucleated red cells may be considered pathological at any period of extrauterine life, although they are usually found in the blood of the child during the first few days of life. These nucleated reds are always found in the bone-marrow, the normal and large forms being quite distinctive. The large form is the oldest and gives rise by cell division to the smaller cell or the normoblast. It is probably true that the nonnucleated red cells are derived from the nucleated form, but the denucleation takes place before the normal cells reach the blood. Just how we are to explain the disappearance of all trace of the nucleus from the normal erythrocyte is a question, but the general concensus of opinion seems to be that the nuclear material gradually fades within the cell, although some slight evidence of extrusion of the nucleus can be advanced: Although our ordinary methods show no such evidence, King^ has recently introduced a method by which, he demonstrates stainable rem- nants of a nucleus in practically every red cell in the normal adult blood. Nonnoblasts (Trachyochromatic Erythroblasts). These are nucleated red cells similar in size, shape, and color to the normocytes. They do not usually show a biconcave form and do not unite in rouleaux. The protoplasm of the cell is usually regular in outline, stains more intensely than does that of the normocyte and frequently shows evidence of polychromatophilia, although it is normally orthochromatic. In myeloid leukemia, cells are frequently observed in which the protoplasm presents a ragged outline and may even-, in some cases, be so degenerated as to show only a small fragment attached to the nucleus. These latter cells are prac- tically always polychromatophilic. This type of small cell might be called a microblast, which is the rarest form of erythroblast and corresponds in size to the microcyte. Moreover, we may find such cells or fragments of cells attached to a nucleus, such cells coirrfesponding in size and shape to the poikilocytes and being termed poikiloblasts. The nucleus of the mature normoblast (Howell's mature nucleated red) has a diameter of about one-third that of the cell, is densely stained, homo- geneous, sharply defined, spheroidal in shape, and without any decided chromatin network (the so-called pycnotic nucleus). It is situated irather toward the periphery than in the central portion of the cell and is surrounded by a clear zone shading off into the cellular protoplasm. Occasionally the nucleus is observed resting upon a margin of the red cell or even extruded entirely from it probably as a result of degeneration of the surrounding pro- toplasm (Pappenheim). These nuclei often show amitotic figures, being subdivided into two -or more lobes or fragments which may be connected by strands of chromatin. These mature forms may show all gradations in which the chromatin network becomes more and more evident, until we reach 1 Jour. Med. Research, igEiigHMB% bV- Microsoft® THE BLOOD 491 the very immature forms (Howell's immature nucleated reds). These latter cells are somewhat larger than the mature forms and are somewhat lighter in color; the nucleus is relatively larger and is composed of delicate faintly basic chromatin fibers radially arranged, and frequently showing mitotic figures. These two types of cell are the forerunners of tne normocyte and their appearance in the adult blood is indicative of increased activity of the hemato- poietic organs, especially of the bone-marrow, either as a result of a poor condition of the blood itself or as a direct disease of the blood-forming organ. These cells are most commonly seen in the milder forms of anemia, chlorosis, and acute anemia from hemorrhage, inanition, or organic disease. In the severe types of anemia they are constantly met with and are usually associated with the larger megaloblasts. During the course of severe anemia, especially in chlorosis, there obtains a periodic increase of normoblasts and of leucocytes lasting several- days. This is followed by a marked increase in the normal number of red cells, giving rise to the condition described by von Noorden as a " blood crisis." The normoblasts disappear, the blood count falls off, and a second crisis may obtain. This condition is considered as a transitory at- tempt on the part of the bone-marrow to regenerate the blood. Such a crisis is not always a sign of improvement, but it does demonstrate that the type of blood formation is becoming physiological and that recovery may follow. Megaloblasts (Amblyochromatic Erythroblasts). These are cells of larger than normal dimensions, corresponding in size to the macrocytes and varying from 9 to 20 microns in diameter, if exceeding 20 microns thay are known as the gigantoblasts of Ehrlich. Occasionally some cells are seen which are no larger than a normoblast, but in which the nucleus shows definite characteristics which should enable one to classify the cell as a megaloblast (Pappenheim). The protoplasm of the megaloblast often appears swollen and enlarged (dropsical). The cell is usually circular or oval, but it is easily deformed, giving rise to irregular-shaped bodies. Although it usually contains an excess of hemoglobin, it may show a deficiency. It is usually polychromatophilic, the shade of cellular staining varying from yellow to purple. These, various color tones may not be regular, but may be varied by tintings of alinost any shade in the same cell. The nucleus of the megaloblast is very large, varying between 6 and 10 microns. This may be situated either centrally or somewhat peripherally. It shows a great variety of forms appearing as a vesicular body with intra- nuclear network and nodal thickening, but without nucleoli. It may rarely be pycnotic, may show mitoses or many stages or karyorrhexis with frag- mentation, vacuolation, fading of the segments of, the nuclei, as well as minute subdivisions into fine basic staining particles widely scattered jn the cell (Ewing). It is frequently poorly defined and shows feeble basic staining qualities. It may be sharply differentiated from the, body of the cell by a distinct white margin which is thrown into relief by the. deeper staining of the nuclear and cell-substances. Occasionally the nucleus is over- looked owing to the pol^g^(Hfifift&^Mf e/g!fi^/^ies of the cell, which do not 492 DIAGNOSTIC METHODS always permit of clear differentiation of nucleus and cell substance. Careful examination of the nuclear structure with its wide-open meshwork, should, however, prevent the mistake of classifying this cell as a large lymphocyte. The clinical significance of the megaloblast is more or less in doubt. As such cells are foreign to the blood of an adult and as they are not present be- yond IS per cent, of the total nucleated reds in the marrow, according to Emerson, it must be assumed that their presence in considerable number is indicative of a reversion to an embryonal type of blood formation, or at least denotes an arrested development of normal cells and in consequence an in- creased production of these abnormal types. Megaloblasts are, therefore, evidences of degeneration of blood-forming organs, while normoblasts are sig- nificant of regeneration of blood. A few of these cells, along with a larger num- ber of normoblasts, has no special significance in cases of severe anemia, being found in small numbers in any variety of anemia. If, however, the majority of the nucleated red cells be megaloblasts, especially if gigantoblasts be pres- ent and unequal mitoses be observed, a diagnosis of primary pernicious ane- mia seems justified. It may be considered that the number of erythroblasts has no special significance as regards the severity of a particular case, but that i1 indicates merely the effort on the part of the bone-marrow to overcome the effects of blood destruction. The appearance of megaloblasts may, therefore, be regarded as an evidence of incomplete formation of the younger elements and, not necessarily, as an unfavorable sign. In the anemia following infection with bothriocephalus latus, the specific toxins produce a megaloblastic de- generation of the bone-marrow, so that the blood picture may assimie the characteristics of a severe pernicious anemia. Ehrlich and Lindenthal have reported a case of nitrobenzol poisoning, in the later stages of which the megaloblasts out- numbered the erythroblasts. Thayer^ is the first to report the finding of ameboid movements in a megaloblast in a case of Addisonian anemia. {D) Number of Red Cells. The normal number of red cells is generally regarded as 5,000,000 per cmm. in the blood of the adult male, while the normal value for the female is 4,500,000. These figures are purely arbitrary, but they serve as an approxi- mate basis upon which one may form his opinion as to the probable normality of a specific specimen of blood. We frequently observe marked variations in the red count under the influence of both physiologic and pathologic conditions. These variations may be due to an actual increase or decrease in the number of cells or to a change in the volume of the plasma as discussed under Total Volume of Blood on page 388. It is not uncommon to find much higher counts than these normal ones on healthy individuals, especially in those living the "simple life," so that any special count must be a law unto itself and must be considered normal or abnormal only after considering all the factors which may influence the number of cells. Physiologic Variations. (i) Sex. The variations due to^ex are not so. markecLas those from other causes. While the adult woman almost invariably snows a lower blood count than 1 Arch. Int. Med., 1911, VII, 223; Ibid., 1911, VIII, 581. THE BLOOD 493 does the adult man, yet we occasionally find the girl showing, before puberty, a somewhat higher count than the boy. of corresponding age, and also the woman, after the menopause, showing somewhat higher values than does her brother of similar years. It can hardly be doubted that menstruation, as well as pregnancy and lactation, have some influence in lowering a count in woman at certain periods of life, yet these conditions are simply transient and would not seem to have as much bearing on the question as does the somewhat more hydremic state of the normal plasma of woman. . (2) Age. The number of the red cells varies more or less with the age of the subject examined. The highest values are usually observed at birth, when the count ma/ run as high as 7,500,000, as in a case observed by the writer, the hemo- globin being practically always over 100 per cent, at this time; but, as a rule, a lower count is noted, averaging about 6,000,000. These high figures are due, to some extent, to the concentration of the plasma at birth through the loss of body fluid before a compensatory intake. The count gradually falls during the first few days and becomes fairly constant about the tenth day, when it is rare to find nucleated red cells. The number of cells is then stated to be somewhat reduced until the age of puberty, when a gradual increase occurs until about 40 years of age, after which a slight decrease may be ob- served in man. These variations, observed at different ages, are to be regarded as slight and influenced, to some extent, by the many modifications of the plasma as the result of growth and development as well as to the gradual decrease of functional activity after the middle periods of life. (3) Altitude. An increase in the number of cells has been observed under the influence of a higher altitude. The count has been shown to increase at the rate of approximately 50,000 cells per 1,000 feet of ascent and to diminish, within 36 hours, at a corresponding rate, this increase or decrease being more marked the more sudden the ascent or descent. Just what factors are at work in causing these changes is undecided.^ The rise is too rapid to be entirely accounted for by new formation of blood-cells under the influence of diminished oxygen tension, and the fall is not accompanied by signs of destruction of the reds. Weinzirl considers the increased count of high altitudes due to the lowered temperature at these elevations. This factor would seem to have some influence, as counts are frequently observed in which a variation is noted at places showing the same elevation. Change of residence from warm to a cold or from cold to a warm climate may lead to an increase or decrease,^ as the case may be, in the number of cells varying from 500,000 to 2,500,000. (4) Nutrition. The general nutritive condition of the subject has more or less influence ' See CohrLheim, Kreglinger, Tobler, and Weber, Ztschr. f. physiol. Chem., 1912, LXXVIII, 62; Schneider and Havens, Am. Jour. Physiol., 1915, XXXVI, 380. 2 Chamberlain has, however, shown (Philippine Jour. Sc, Sec. B., 1911, VI, 467) that the average number of red cells in the tropics is little different from that in temperate zones. See Laquer, Deutsch. Arch. f. klin. Med., 1913, CX, 189; Cohnheim and Weber Ibid., 22s; and Lope, Seman^^^^^^^j^ ^^jg^@ 494 DIAGNOSTIC METHODS upon the number of red cells per cmm. This statement must not be inter- preted to mean that the obese person shows a higher blood count than does his sparer brother. It is well known that obesity is an indication of poor as- similation of food; hence we should expect to find, as we really do, that the muscular person has a somewhat higher count than the obese subject and well-develope.d robust individuals a larger value than do the poorly nourished patients. Diet has, of course, much to do with the general nutrition, so we find the meat eaters averaging somewhat higher in their percentage of red cells than do vegetarians. Immediately following a hearty meal we may observe a temporary decrease in the number of red cells, but this soon returns to normal, owing to the rapid adjustment of the water content of the blood. 1 (s) Exercise. Active muscular exercise produces a transient increase in the red count due both to the increased blood-pressure and to the concentration of the blood through the loss of water by perspiration. Physical exercise, taken to the point of producing fatigue, may produce a marked diminution in the number of red cells, due probably to the fact that regeneration of new cells cannot keep up with the destruction of the old cells. Passive exercise, in the form of massage, has a transient influence in producing an increase in the number of the red cells, owing to its increasing the general circulatory tone ^ (6) Baths. Careful investigation of the influences of both cold and hot baths has shown that an increase in the number of reds occurs under the action of these two conditions. The increase as a result of a cold bath may be as great as 2,000,000 cells, due to capillary stasis as a result of vasomotor constriction. Likewise, a hot bath will increase the number of cells by causing dilatation of the peripheral vessels and a consequent increase in the amount of blood at the point from which the specimen is taken. When marked perspiration follows either a hot or cold bath the blood becomes concentrated and, as a result, an increase in the number of red cells will obtain. (7) Therapeutic Measures. Any drugs which cause rapid loss of fluid from the body, as for instance emetics, purgatives, diuretics, and diaphoretics, will cause concentration of the blood and hence a coincident increase in the number of cells, providing the change is sufficiently rapid and the blood examination is inade before compensation occurs. Among the drugs which increase the number of red cells we find iron and arsenic, both of which are particularly valuable in anemic conditions, the former in chlorosis and the latter in pernicious anemia. The compounds of mercury and of lead, on the other hand, have a destructive action upon the red cells, so that we find these drugs causing a diminution in the number of cells. ' See Hopmann, Arch. f. Verdauungskr., igi3, XIX, 456. ^ See Hawk, Am. Jour. Physiol., 1904, X, 384; Schneider and Havens, Ibid., 1015, XXXVI, 239. Digitized by Microsoft® THE BLOOD 495 Pathologic Variations. (i) Oligocythemia. This is a condition characterized by a diminution in the number of red cells. It is usually associated with a decrease in the percentage of hemoglobin and with a slight reduction in the total volume of blood, although this latter factor is not invariably present. This condition is found, to a more or less degree, in all forms of anemia and may be temporary or permanent. The extent of the decrease in the number of red cells varies from 500,000 cells to a reduction of 4,000,000. This diminution in the number of cells is usually an indication of the severity of the anemia, the most marked decrease being observed in the pernicious types. Osier reporting probably the lowest count recorded, namely, 100,000 red cells. While this oligocythemia is usually associated with oligochromemia, yet we find in chlorosis that the diminution of red cells is not as marked as is the reduction in the amount of hemoglobin, the color index in this condition being usually low. In some cases of chlorosis, however, the oligochromemia may keep pace with the oligocythemia. In pernicious anemia, on the other hand, we have a marked reduction in the number of cells and coincidently a large decrease in the amount of hemoglobin, the result being that we have a very high color index as the usual sign of this condition. Cases showing the loss of a large amount of blood, as the result of hemorrhage have naturally an oligocythemia, but the percentage of hemoglobin may not necessarily be reduced to any great extent. This gives rise to the condition of secondary anemia, in which the color index may be high. In these cases of hemorrhage a sudden reduction to the point of 1,000,000 or less cells is usually followed by a fatal result, although even here recovery has been made possible by rapid infusion of salt solution and compensatory activity of the blood-form- ing organs. In leukemia we find that the red cells are not usually diminished to a very great extent, the oligocythemia being generally more marked in the lymphatic than in the myeloid variety. Occasionally, however, we do find very low counts, in both varieties of leukemia. These same statements apply to splenic anemia, although the count here very rarely reaches a lower point than 2,500,000 cells. A large number of conditions, aside from direct blood diseases, cause a diminution in the number of these red cells. Thus we find that the toxins of certain of the specific fevers, such as typhoid and pneumonia, may cause a marked anemia, but this is not necessarily the rule. Acute infection with pus organisms is frequently observed to cause an extreme and rapidly pro- gressive anemia, the destruction of the red cells being in some cases very extensive. In mahgnant disease we usually find, especially if cachexia is present, a very extensive anemia which may lead to a diagnosis of the per- nicious type. It is to be remembered, however, that a secondary anemia may assume all of the characteristics of the primary pernicious type. (2) Polycytheniia. This is a condition characterized by an increase in the number of red cells and is sometimes called 9^^^iM-*^"^fi&^ this increase is actual and ' Also erythremia. 496 DIAGNOSTIC METHODS permanent or whether it be simply apparent, due to concentration of the blood or to unequal distribution of blood in the peripheral vessels, is still an unsettled question. It is true that many physiological causes previously enu- merated do bring on a polycythemia, so that these should be remembered when- ever a blood count is to be made. From the pathologic standpoint we find an increase in the number of red cells occasionally following active blood regeneration after hemorrhage. Likewise, we observe an increase in the number of cells in phosphorus poisoning, acute yellow atrophy of the liver, and in certain cases of general hepatic insufficiency. Why such an increase of cells occurs in these conditions is very hard to say. It has been found that the solids of the plasma are not increased, so that we are not warranted in assuming a concentration of the fluid elements. A polycythemia of uncer- tain origin is also seen in cases of poisoning with carbon monoxid and illumi- nating gas.^ In a condition called by Osier autotoxic enterogenous cyanosis, which is characterized by a marked increase in the number of red cells and also by enlargement of the spleen, we have reason to assume that a direct real increase in the number of red cells occurs, but the pathogenesis of the condition is uncertain. It has been traced to improper aeration of the blood and occurs also in congenital heart disease, mitral lesions in the adult, and in pneumonia and acute miliary tuberculosis. While this high blood count must be definitely admitted, its importance from a clinical standpoint is still unsettled. It is uncertain whether the rapid increase in the number of reds is due to an attempt on the part of the blood-forming organs to overcome the influences of the toxic substances upon proper oxidation in the system; yet it is certain that relief very frequently follows such an increase in the number of red cells, although in Osier's disease or, as it is called by some, Vaquez's disease, death frequently follows without any relief from the condition.^ (£) Staining Properties of the Red Cells. The normal red cell, like all living cells, is incapable of being stained with anilin dyes; that is, it is achromatophilic. Previous to staining, fixation of the cell must take place, the protoplasm being killed in this process. The normal fixed red cell has a marked affinity for various dyes of the acid type, such as eosin, orange-G, acid fuchsin, and indulin, and is therefore caUed acidophilic or oxyphilic. This cell normally takes up but one color from a mixture of dyes, and is called, therefore, monochromatophilic. Polychromatophilia. Under various pathologic conditions we find red cells which show a tend- ency to take up the basic stains, and for this reason are called basophilic or polychromatophilic cells. The tint of such abnormal red cells varies from that of a light indefinite shade of the basic stain to a dark distinct tone. Just 1 See Karasek, Trans. Chic. Pathol. Soc, 1911, VIII, 173. « See White, Lancet, 1912, XCII, 7; Lucas, Arch. Int. Med., 1912, X, 597; Cahn, Inaug. Dissert., Berlin, 1912; Chauffard and Troisier, Presse m6d., 1913, XXI, 653; Friedman Med. Record, 1913, LXXIV, 701; and Moewes., Deutsch. Arch. f. klin. Med.', CXI 281' Hertz and Ehrlich, Deutsch. Arch. f. klin. Med., 1014, CXVI, 43; Mosse Ztschr f' klin' Med., 1914, LXXIX, 431; ^^S)^yiV^g^-j^5'^^rOSO^^''P''- ^^^'^P- ^9^5, VII, i69'. THE BLOOD 497 what factors are at the bottom of this change, which is called polychromato- philia or polychromasia, is difficult to say. The normal acidophilic tenden- cies of the cell are due to the presence of hemoglobin and, in consequence, the normal staining properties will depend upon the relative richness of the cell in this pigment. As hemoglobin is always acidophilic, we cannot assume that the polychromatophUic properties of the abnormal red cells are due to variations in the hemoglobin, but must content ourselves with the belief that these changes have something to do with the protoplasm of the cell. This condition, which has been termed by Ehrlich anemic degeneration, is characterized by a diffuse basic staining property of the red ceUs. These basophiUc cells are somewhat larger than the normal ones, show less bicon- cavity, and often are abnormal in shape. The megaloblasts are practically always polychromatophUic and the normal erythroblasts of the bone-marrow usually show this degeneration. This fact would seem to point to the prob- ability of this condition being a sign of regeneration of the blood as the ery- throblasts are more prone to be polychromatophUic the younger they are. Two distinct forms of polychromasia have been found. The first of these, known as the polychromatophUic degeneration ofGabritschewsky, is the diffuse basophilic staining of the cells which is found in various forms of anemia and extensively in the cells of the normal bone-marrow. The second of these, the polychromasia of Maragliano, is shown in severe anemias and in toxemias and appears as a more punctate basophilia, being closely related to the basophilic degeneration of Grawitz, which will be discussed later. While the poly- chromatophilia of the diffuse type is considered by Ehrlich as evidence of a degenerative process, that is as a coagulation necrosis of the discoplasm in consequence of which this takes up the albuminous principles of the plasma while it loses its power of retaining hemoglobin, yet there are evidences show- ing that this may be a regenerative process.^ Polychromatophilia is often seen in cells which are undergoing degeneration, especially those following malignant disease, eruptive fevers, malaria, and after various poisons, such as that of snake venom. The forms of partial polychromatophilia, which have been described under various names, such as vacuolization, pseudonucleation, globular decolorization, and more commonly IVTaragliano's endoglobular degeneration, are seen in normal blood in from 30 to 70 minutes after the specimen is made. They are usually found in the center of the cell, but may be near the periphery. There may be several such areas in a single cell, but the more common form is the single area of degeneration, which is usually round, may be elliptical, and may resemble a vacuole. These are shown quite distinctly in the unstained specimen and in this condition very frequently are seen in rapid motion. This motion is not of the true ameboid type, but is due more to the gradually progressing coagulation and consequent constriction of the protoplasm. These areas, whether in the fresh or stained specimens, may be mistaken for malarial parasites, so that the worker should be on his guard lest he make a wrong diagnosis without suflicient evidence. They differ in size from the 1 See Pfuhl, Ztschr. f. kUip^-^gggj, y^^^^ff^i. 498 DIAGNOSTIC METHODS parasites, and on focusing the specimen are characterized, usually, by a change froni a smaller to a larger form, which variation is not evident with the malarial organism. The longer one searches in the unstained specimen for malarial organisms the more apt a mistake in diagnosis is to be made, as these " Maraglianos " develop quite rapidly. In the stained specimen these areas of coagulation necrosis will show a basophilic staining quality, while the. chromatin element of the malarial organism will differentiate this body, from the more usual degenerative area. (F) Degenerations. While the preceding conditions of polychromatophilia are to a certain extent true degenerations, yet they are more properly grouped under the heading of atypical staining reactions of the red cell. Basophilic Degeneration of the Red Cells. This condition is distinct from the polychromatophiha above described and is known as the punctate basophilia of Grawitz. It is characterized by the appearance, in the body of the red cell, of granules of varying size, which stain with the basic dyes. The cell may be dotted throughout with these granules or may show this degeneration only in parts. These granules are not observed in the fresh unstained specimen and are not increased, as are the Maragliano areas, by allowing the blood to stand. These cells are observed in pernicious anemias, leukemia, in the toxemia of malignant states and especially in cases of lead-poisoning. The size of the granules may vary from small dots to large granules, showing a diameter of one or more microns. Their origin is much in doubt, but they are generally considered to be areas of coagulation necrosis associated with either degeneration or regeneration of the red cells. They are quite distinct from the granular basic "stippling" observed in malarial conditions, but are probably evidences of direct toxic effects upon the protoplasm of the red cells. Their greatest clinical importance is probably in lead-poisoning, where they may be the only signs of abnormality in the blood picture. They may vary in number from day to day, showing in some cases five or six in one microscopic field, but, as a rule, they are present in much fewer numbers, as one observes them only after examining several visual fields. As a rule, they appear very early in cases of lead-poisoning, in one case observed by the writer within three days, and they may be present in the blood of a lead worker for several years after his exposure to the effects of the lead. They are usually the first sign of anemic change and usually persist longer than do the other abnormalities of the blood. ^ Ring Bodies. Occasionally one observes in the red cells curious ring-like bodies which are in shape very much like the hyaline malarial ring with a circular refractive center. They change their shape in a peculiar way, much resembling the undulatory movements of the hyaline body. They do not increase in number or grow larger on standing as do the Maraglianos and they are observed in 1 See Petry, Biochem. Ztschr., 191 2, XXXVIII, 02: Anders, Jour. Am. Med. Assn., 1914, LXII, 1164; Lmenthal/3g»teecf96ygW0»t(B®p/TOUtsch. Arch. f. klin. Med., 1915, CXVII, 127. PLATE XIX. ^eiffltilW/cKOS©rei), 1915, XXXI. Srj and 873. THE BLOOD 50I when a gradual increase occurs again until middle life, after which a steady decrease is observed. It is to be expected that variations in the color tones (hemoglobin content) of these cells will vary normally with the age of the subject, being very high in the early days of life, gradually diminishing as puberty approaches, increasing until the middle periods of life, and ste;adily declining as the age increases. Aside from the number of cells, thii blood in childhood shows little variation in the characteristics pf there^ cell'^V^iit the variations in the plasma are more or less marked, as jthe.sypthaiifc.and retro- grade products of tissue metabolism are much more evideiit.'ia tbe period of development than during the middle periods of life. As the subject becomes older the blood is laden with products of retrograde tissue change and the blood-forming organs become less active. It must be remembered that the various organs of the body contribute certain factors to the blood and may, therefore, all be considered as blood-forming organs in a definite sense. The ductless glands,in particular, pour into the blood certain substances which markedly influence the proper correlation of these various organs. Just what these substances are is not well established, so that the introduction of the word "hormone" by Starling does not at present have a specific meaning. As old age approaches variations become evident in the plasma, and through such influences changes in the morphology of the red cells are more or less frequent. Besides these plasma changes, gradual atrophic degeneration of the blood-forming organs takes place, so that we may find red cells showing the various types of anemic degeneration and may even see nucleated red cells as an evidence of the attempt on the part of the bone-marrow to overcome the gradual destruction of cells. The more nota- ble variations in the red cells are observed in their size, owing to the hydre- mic state which is almost always characteristic of the plasma in advanced periods of life, but associated diseased conditions will, of course, give their characteristic changes. (/) Functions of the Red Blood-cells. The only well-recognized function of these cells is their oxygen-carrying power, which is dependent upon their hemoglobin content. Other oxygen catalysts, such as oxidase, peroxidase, and catalase, are found more largely in the leucocytes and play a significant r61e in the function of the whole blood. These will be discussed later. As is well known, the fluid portions of the blood are much more influenced by the various organs of the blood than are the cellular elements, although the red cells will naturally be affected by variations in the hematopoietic organs, such as the bone-marrow, liver, and spleen. Our knowledge regarding the variation in the plasma is so uncertain that we have been led to interpret the changes in the histological appearance of the blood as significant of certain general pathological conditions. It is true that in some cases we do have direct disease of the blood-forming organs and, in consequence, variations in the cellular structures are of diagnostic importance, yet in the large majority of cases the preliminary changes must be resident in the plasma, those in the cellular structure being simply inci- dental. It may be, therM^^zgg(.^^2(!^^s^@ng ^ functional activity on S02 DIAGNOSTIC METHODS the part of the red cells to overcome biologic changes in the plasma and if so we must attempt to discover just what plasma changes we are to regard as significant of the various conditions shown by the red cells in each true blood disease, (6) Leucocytes. (A) Appearance. In the fresh specimen the leucocytes appear as colorless, high refractive bodies, somewhat larger than the red cells, and showing a definite nucleus. These white cells show distinct ameboid movement in the fresh specimen by virtue of which they are able to surround a foreign body and enclose it within their own protoplasm. This is the well-known property of phagocytosis which is such an important factor in Metschnikoff's theory of immunity. In contradistinction to the red cells, these white cells show many variations both in size and in shape. As the relations between the nucleus and the cellular protoplasm is quite distinct in many of these forms, some writers have been led to classify the white cells according to the peculiarities of the nucleus. Other writers classify them into granular and nongranular forms, as many of these cells show distinct granulations which are more clearly differentiated in the stained specimens. As more or less confusion exists regarding these different varations, it seems more rational at present still to classify these white cells with Ehrlich, who combines both the nuclear and granular char- acteristics in his classification. "^ (B) Leucocytes in Normal Blood. (i) Lymphocytes. The cells of this class which occur normally in the blood are of two kinds, (a) the small cell, about s to 8 microns in diameter and (b) large cells showing a diameter of from 8 to lo microns. Both of these cells have a very large nucleus which is usually centrally located but may have an eccentric posi- tion. It may be compact or coarsely reticulated and is not always as strongly stained as is the surrounding cytoplasm. Nor is it as deeply stained as is the nucleus of the normoblast, although it is very rich in chromatin. The nucleus of the larger cell may show very irregular staining properties. The nuclei of both of these cells are usually circular in outline, but may show an oval or a kidney-shaped structure, in which at times may be seen a distinct nucleolus. The protoplasm of these cells is usually in the form of a narrow rim surrounding the nucleus and showing a strongly basophilic homogeneous cytoplasm. The older cells may at times show a difficult staining property, may even be acidophilic, and occasionally exhibit a net-like structure in which are observed a few granules scattered throughout the protoplasm of the cell. This granulation, which occurs in about one-third of the ceUs seems to have little clinical significance, except as a probable sign of age of the cell, this granular stage being regarded as the end-point of their development. These granules are probably not representatives of true granulation, but seem to be more referable to nodal points of the reticular structure of the protoplasm. 1 See Pappenheim, Die Ereebnjsse derinn JVted. u^i Kinderlieilkde. 1013, VIII, 183; also, Hoxie, Interstate Med. fmfl^^PX.WG^i^^® Digitized by Microsoft® DESCRIPTION OF PLATE XX. {Triacid Stain.) 1, 2, 3, 4. Small Lymphocytes. Contrast the faintly colored protoplasm of these cells in the triple stained specimen with their intensely basic protoplasm in th« film stained with eosin and methylene- blue, 17 and i8. The cell body of i is invisible. Note the kidney-shaped nucleus in 4. 5, 6. Large Lymphocytes. With this stain the nucleus reacts more strongly than the protoplasm; with eosin and methylene-blue (19, 20), on the contrary, the protoplasm is so deeply stained that the nucleus appears pale by contrast. This peculiarity is also observed in the smaller forms of lymphocytes. 7, 8. Transitional Forms. Note the moderately basic and indented nucleus, and the almost hyaline non- granular protoplasm. Compare 8 with the myelocyte, 7. Plate IV. these cells dif- fering chiefly in that the myelocyte contains neutrophile granules. 9, 10, 11, Polynuclear Neutrophiles. These cells are characterized by a polymorphous or polynuclear nucleus, sur- rounded by a cell body filled with fine neutrophile granules. In 11 the nuclear structure is obviously separated into four parts; in 9 it is moderately, and in lo markedly, polymorphous. 12, 13. Eosinophiles. The nuclei are not unlike those of the polynuclear neutrophile, except that they are somewhat less convoluted, and poorer in chromatin, staining less intensely. The protoplasm is filled with coarse eosinophile granules, the characteristics of which are clearly illustrated by 13, a " fractured " eosinophile. 14. Eosinophilic Myelocyte. Compare with 15. 15. 16. Myelocytes. {Neutrophilic.) These cells are morphologically similar to 14, except that they contain neutrophile instead of eosinophile granules. Note that the granules of the myelocyte are identical with those of the polynuclear neutrophile. A dwarf form of myelocyte is represented by 16. {Eosin and Methylene-blue.') 17,18. Small Lymphocytes. Note the narrow rim of pseudo-granular basic protoplasm surrounding the nucleus and the pale appearance of the latter. 19,20. Large Lymphocytes. Budding of the basic zone of protoplasm is represented by 20, Both of these cells belong to the same type as 5 and 6. 21, 22. Large Mononuclear Leucocytes, Compared with 19 and 20, these cells have a decidedly less basic, protoplasm, but a somewhat more basic nucleus. In the triple stained film these differences cannot be detected, so that they must be classed as large lymphocytes. 23. Transitional Form. The distinction between this cell and 24 is not marked; the nucleus of the latter simply being somewhat more basic and convoluted. 24, 25, 26, 27. Polynuclear Neutrophiles. With this stain these cells show a feebly acid protoplasm, and lack granules. Note that the more twisted the nucleus the deeper it is stained. Compare with g, 10, and II. 28, 29. Eosinophiles. Compare with 12 and 13. 30, Eosinophilic Myelocyte. Compare with 14. 31, Basophile. {Finely granular.) This cell is characterized by the presence of exceedingly fine 5-granules, staining the^wre color of the basic dye. The nucleus is markedly convoluted and deficient in chromatin. The cell here shown was found in normal blood. 32, 33. 34, 35, 36. Mast Cells, The granules take a modified h^sxo. color, as shown by their royal-purple tint in this illustration. Note their unusually large size and ovoid shade in 35, their peculiar distribution in 35 and jfi, and their irregularity in size in 32 and 36. With the triacid mixture these granules, as well as those of the finely granular basophile, 31. remain unstained, showing as dull-white stippled areas in the cell body. The nuclear chro- matin of the mast cell is so delicate and so freely stained that it is barely visible. These cells were found in the blood of a case of spleno-medullary leukemia. Digitized by Microsoft© PLATE XX. o '•.-. ■.!.?■• 'l-jf-'' -13 20 17 18 ooQ 24 ^ € t^ ^ 27 ® '».*j>»» The Leucocytes. (l-l6, Triacid Stain; l^-i6, Eosin and Methylene-blue.) (E. F. FABER,/ec.) (From Do Costa's "Clinical Hematology.") Digitized by Microsoft® Digitized by Microsoft® THE BLOOD 5°3 These cells usually constitute from 20 to 25 per cent, of the leucocytes, their absolute numbers being from 1,200 to 2,000 per cmm., but the relative pioportion of the small and large form being unsettled. The larger tjrpes of these cells are more closely associated with increased functional activity of the lymphoid tissues and should probably be considered the older ceU of . the two. According to Pappenheim, the large lymphocyte represents the mother cell from which all other leucocytes as well as erythrocytes are indirectly de- veloped as a result of heteroplastic differentiation. Variations in technic of preparing the slides will often lead to differences in size of these cells, the thinner preparations showing these cells as larger forms, while the thicker preparations present the smaller type of these cells. These cells are beautifully colored by the hematoxylin-eosin stain and by the various modifications of the Romanowsky stain, although the boundary between the nucleus and the protoplasm is not always clearly outlined by these latter stains. The granules, which are occasionally found in these cells and which are in size between the a and the e granules of Ehrlich, show most clearly in preparations stained by the Giemsa stain and appear distinctly azurophilic. These granules do not appear at all with the triple stain. These cells are frequently increased and frequently diminished, the former condition being spoken of as lymphocytosis, the latter as lymphopenia. A relative increase of the number of these lymphocytes is regarded as more or less characteristic of typhoid fever, especially when associated with a diminu- tion in the total number of white cells. In lymphatic leukemia these cells are present in large numbers and are indicative of marked disturbance in the lyinphatic structures. (2) Large Mononuclear Leucocytes. These cells, which are supposed to be derived from the spleen and are called, therefore, splenocytes, are as a rule two or three times as large as a red cell (12 to 15 microns), and show a' nucleus which may be large and round, but is more frequently oval in shape or may be indented, forming the so- called kidney type of nucleus. It is to be remembered that we may have both large and small types of this large mononuclear leucocyte, the smaller forms being distingiushed from the lymphocyte by the relation of the protoplasni to the nucleus. These nuclei are usually eccentric in position and are not always sharply outlined, they are poor in chromatin, but are strongly baso- philic, although less so than are the nuclei of the lymphocytes. The proto- plasm of these cells is very abundant in relation to the size of the nucleus and is very clear, hyaUne, and nongranular in appearance. This protoplasm is much less basophilic than is the nucleus and shows a very fine reticulum with nodal thickenings which are somewhat more strongly basophilic and may give the appearance of granulation. ^ These cells should not, however, be regarded as granular cells. Occasionally we find cells, derived from the above, which have been called by Ehrlich " transition forms," which are the largest of all the white cells. 1 Bergel (Deutsch. Arch. f. klin. Med., 1912, CVI, 47) has shown that these cells play a large r61e in the liemoj^^^^g^^dy^y^^^l^^^r injection of cells and lipoids. 504 DIAGNOSTIC METHODS The nucleus is pale, often deeply notched giving it usually the appearance of a saddle-bag, and shows the characteristics of the large mononuclear tj^e, being distinguished from the polymorphous nucleus of the neutrophile cells by its greater thickness and by its diminished intensity of staining. The extensive granulation of the polymorphonuclear leucocyte should make any mistake in diagnosis impossible The protoplasm of these transitional cells is very abundant and shows faint basophilic properties and may even have a few fine granules, which are neutrophilic in character, in the neighborhood of the nucleus. These two forms of cell constitute between 3 and 5 per cent, of the leucocytes, their absolute number varying between 200 and 400 per cmm. Pappenheim considers that these large mononuclear cells develop directly from the large lymphocytes and then pass into the transition forms, which -are the final developmental types. (3) Polymorphonuclear Cells. (a) Polymorphonuclear Neutrophiles. These cells, sometimes called the finely granular cells of Schultze, are from two to three times the size of the red cell (10 to 12 microns) in diameter, the size depending upon the extent to which the cell is flattened by pressure. These cells are always smaller than the large mononuclear tjrpe and quite small specimens are frequently found in cases of myeloid leukemia. They are the most sharply characterized cells of the blood, their protoplasm being relatively great and showing slightly acidophilic staining properties. The re- ticular portion of the protoplasm is very slightly basophilic, showing occasion- ally nodal thickening or granules when stained with the methylene blue dyes. Throughout this protoplasm are scattered fine dust-like granules, the € granules of Ehrlich, which are not all of the same size and which stain with the neutral principle of the dyes. In some cases these granules may gradu- ally diminish so that they may be apparently absent or at least undetected. The triple stain is the characteristic stain for these granules, showing them of a distinct lilac color; but various other acid dyes, such as eosin, give various tints to them, so that they are recognizable as distinctly reddish granules in specimens stained by any of the Romanowsky modifications. The nuclei are elongated and constricted and may appear in the form of a bent rod or a mass of interwoven fine fibers, showing a thin chromatin-rich structure, or a distinctly S-shaped formation with oval thickenings. Fre- quently these nuclear masses appear as if separated into two or more distinct nuclei, but as a rule these masses are cormected by fine bands and should, therefore, be considered rather as polymorphous than as polynuclear. The nucleus shows a reticular structure with nodal thickenings and is very baso- phiHc. These forms constitute from 65 to 75 per cent, of the total number of cells, their absolute number averaging 5,000 per cmm., the percentage rang- ing from 20 to 40 per cent, in the first years of life when the lymphocytes are increased. When outside of the blood-vessels they form the usual pus-cells and are extremely active phagocytes. These cells are derived from the mono- nuclear neutrophiHc myelocytes which are normal habitants of the bone- marrow. They run theP'&^§fl^sM!S''SSS^^ not transformed into other THE BLOOD $0$ cells, their granules diminishing in pathological conditions or with age, such change being associated with degeneration in the nucleus. Perinuclear Granulation. Sometime ago Neusser reported .a finding of basophilic granules in certain leucocytes, especially the mononuclear and polynuclear type. These granules surrounded the nucleus and even appeared attached to it, showing a variable size and being more or less refractive. He regarded these perinu- clear graunulations as characteristic of the uric acid diathesis, but the work of Futcher and of Simon show that these granules are undoubtedly artefacts which can be produced by heating and by variation in the staining. Ameth's Classification. Arneth^ in his study of the blood in health and disease has been led to classify the polymorphonuclear neutrophiles into five classes depending upon the number of nuclear lobes. His divisions seem to be fairly constant in health, but show great variation, especially in infectious conditions. His first subdivision, known as Class i, is divided into (a) M cells, which are mononuclear forms identical with Ehrlich's myelocyte, (6) W cells, represent- ing forms which show only a slightly indented nucleus, the indentation never extending beyond the middle of the nucleus; this cell forms what is called the metamyelocyte, (c) T cells, in which the indentation of the nucleus is deeper than in the W cell, but there is no distinct separation into isolated loops, this form constituting the true polymorphonuclear tj^e. The first two varieties of Class I are usually seen only under abnormal conditions, although the W cell may be found to the extent of 0.2 per cent, in healthy conditions. The cells of Class I are usually present, according to Arneth, to the extent of 5 per cent., Simon giving this percentage as from 4 to 9. The second class of Arneth embraces cells with two distinct nuclear fragmentations and shows three subdivisions: (a) 2 K cells, neutrophiles whose nucleus consists of two round nuclear portions; (6) 2 S cells,. neutrophiles whose nucleus consists of two distinct S-shaped forms; (c) i K i S cells, neutrophiles whose nucleus consists of one round nuclear portion and one S-shaped division. These cells of Class 2 constitute, according to Arneth, 35 per cent, of the total neutro- philes, while Simon's figures are 21 to 47 per cent. In Arneth 's figures we find the 2 S cells forming about 23 per cent, of the total of this class. The third class has three nuclear divisions and is subdivided into four parts, as follows: (a) 3 K cells, (6) 3 S cells, (c) 2 K i S cells, (i) 2 S i K cells. Arneth gives the percentage of Class 3 as 41, the 2 K i S and the 2 S i K subdivisions, each representing approximately 16 per cent, of the total of this class. Sim- on's figures for this class range from 55 to 48 per cent. The fourth class comprises cells with four nuclear divisions showing five subgroups, as follows: (a) 4 K cells, (b) 4 S cells, (c) 3 K i S cells, (i) 3 S i K and (e) 2 K 2 S cells. This class, from Arneth 's figures, shows a percentage of 17, the 4 K, 3 K i S and 2 K 2 S types being largely in excess of the other forms. The fifth class comprises cells with five or more nuclear subdivisions and may be arranged 1 Jena, 1904. Digitized by Microsoft® So6 DIAGNOSTIC METHODS into five groups, this class representing about 2 per cent, of the total neutro- philes. It is probable that these various classes represent the gradual develop- ment of the polymorphonuclear neutrophile, the older the cell the greater the tendency to reach Class 5, while in conditions associated with new formation of cells, as in infectious conditions, we find the percentages of the earlier classesbeing increased, that of the later ones diminished. Minor and Ringer^ have recently emphasized the prognostic value of this method in pulmonary tuberculosis. (b) Polymorphonuclear Eosinophiles. These cells, sometimes called the coarsely granular cells of Schultze, are somewhat smaller than the preceding, varying in size from that of a lymphocyte to that of the neutrophile. Their protoplasm is usually some- what less in amount than is that of the neutrophile and may not be distinct. It is filled with coarse, round, or slightly oval granules about one micron in diameter, which are very refractile and appear in the fresh specimen dis- tinctly black, while in the stained smear they take up the acid portion of the dye and are called, therefore, acidophilic, oxyphilic, or eosinophilic cells. ^ These granules are the a granules of Ehrlich, and are sometimes associated with the |3 granules of Ehrlich which are about the same size or a little smaller than the a granules and take both the acid and the basic stains, although ■ with the ordinary staining solutions they appear, like the a forms, stained with eosin. The nuclei of these cells are coarsely reticulated, are larger and thicker than those of the neutrophils, are usually bilobed, and more fre- quently show distirict separation of these lobes. These nuclei do not stain very deeply with the nuclear dyes, so that they may be rather indistinct. These cells are probably derived from the mononuclear eosinophile myelo- cytes of the bone-marrow and constitute from two to four per cent, of the total number of leucocytes, their average number being from 100 to 200 cells. (c) Polymorphonuclear Basophiles. These cells, frequently called mast cells, resemble the neutrophile cells in the fresh specimen, but show quite distinct characteristics in the stained form. Their protoplasm is much the same as that of the neutrophile and shows the same relation toward the nucleus which does not, however, so frequently form distinct lobes as does the nucleus of the neutrophile. The size of these cells averages about 10 microns, but shows a marked variation, being very small in myeloid leukemia. These cells are characterized by their granules, the 7 granulation of Ehrlich, which are more irregular in size than are the neutrophile granules and are not so extensively scattered through the protoplasm. These granules show the peculiar property of metachro- masia, being colored red with violet dyes and with the blue dyes violet, although lAm. Jour. Med. Sc, 1911, CXLI, 638. See, also, Ringer, Ibid., 1912, CXLIV, 561; Kramer, New York Med. Jour., 1913, XCVII, 1241; Cummings, Calif. State Jour. Med., 1913, XI, 286; Cooke, Brit. Jour. Tubprc, 1914, VIII, 211. ^ See MuUer (Wien. klin. Wchnschr., 1913, XXVI, 1025) for a discussion of the chemical properties of these granules. Acton and Knowles (Indian Jour. Med. Research, 1914, I, 523) believe the KurloflE bodies of the bone-marrow to be forerunners of the eosinophile granules. See, also, Pappenhdm..Folk HKinaL^^ 224; Downey, Ibid., 1915, THE BLOOD SO 7 with absolutely pure methylene blue they take a blue shading. In cases of myeloid leukemia these granules are particularly soluble in water and may, therefore, not be seen if aqueous stains are used, while in normal blood the granules are usually water-fast. These cells stain best with Ehrlich's dahlia stain, or Tiirk's iodin solution, and take a tone which is not strictly baso- philic, but resembles more that of mucin, on which account they have been called mucinophiles. Whether these granules are all of the same significance is questionable. The true mast cell granules are know as the 7 granules, while other basophilic granulations have been found by Ehrlich and are known as the delta (5) granules.^ These latter granules are found in the large mononu- clear cells, especially in the lymphocytes, and do not stain with Ehrlich's dahlia stain. Whether these latter bodies are true granules or nodal thick- enings is at present uncertain. True mast cells probably originate in bone- marrow from a granular mononuclear type corresponding to other types of myelocytes (Pappenheim). These cells constitute about J^ per cent, of the total leucocytes, averaging between o and 50 per cmm. Ehrlich's dahlia stain for these mast cells is made as follows: Distilled water, 100 c.c. Saturated absolute alcohol solution of dahlia, 50 c.c. Glacial acetic acid, 10 c.c. The specimens are heated or fixed by alcohol and are then stained in the above stain for 5 to ib minutes, the mast cells appearing of a distinctly violet tone. Tiirk's Iodin Method. The specimens are fixed with heat and are then first stained in a i per cent, alcoholic methylene blue solution, warming the slide very carefully until it steams. It is then allowed to cool, washed quickly in water, and dried between filter-paper. The slide is then covered with a solution of iodin in potassium iodid of the strength i : 300. Allow this solution to act not longer than 3^ minute, pour off and mount in the following syrup: Iodin, I gram. Potassium iodid, 3 grams. Distilled water, 100 grams. Gum arable q.s. to make a syrup. The mast-cell granules appear, when treated in this manner, very dis- tinctly outlined and colored black. The nuclei are brownish in color, the erythrocytes yellowish-green, and the polychromatophilic erythrocytes dark green, the neutrophile and eosinophile granules faintly yellow. (C) Leucocytes in Pathological Blood. (i) Myelocytes. Under this heading we must regard any cell of the bone-marrow as a myelocyte, but for diagnostic purposes we have reference more to mononuclear iHuguenin (Zentralbl. f. allg. Path. u. path. Anat., 1912, XXIII, 725) reports the presence of sudanophile granges i^^th^ W'M^^O^i^O^S^^'^^'^^ '^^^®' 5o8 DIAGNOSTIC METHODS cells which are distinctly granular. While the granulations of these cells are usually either neutrophilic or eosinophilic, we may rarely find, especially in myelogenous leukemia, cells which show basophUe granulations. The size of these myelocytes varies from that of the red blood-corpuscle to that of a large mononuclear cell. {a) Neutrophile Myelocsrtes. This myelocyte may be either large or small, in the first case being known as Cornil's myelocyte (amblyochromatic typt), a cell which is much larger than the polymorphonuclear leucocyte (at least 15 microns in diameter), and showing a round, pale, eccentric nucleus which stains feebly but not diffusely. This cell shows many distinct neutrophile granulations and has a narrow zone of basophilic protoplasm surrounding the nucleus. It is found almost en- tirely in myelogenous leukemia and has been called by Pappenheim the heteroplastic promyelocyte. The second type of neutrophile myelocyte is known as the Ehrlich myelocyte (trachyochromatic type of Pappenheim) which is a medium-sized cell with a pale central nucleus which stains deeply but not diffusely. This cell shows extensive neutrophilic granulation of the protoplasm, which is faintly ox3TDhilic, and has a nucleus which is either per- fectly round, oval, or indented, but is never lobed nor pycnotic. A distinc- tion between the myelocyte and the polymorphonuclear leucocyte should be based entirely upon the structure of the nucleus, all those cells with round, oval, or kidney-shaped nuclei which occupy .at least one-half of the cell and show neutrophile granulations but no diffuse staining of the nucleus must be called myelocytes. It is to be remembered, however, that this t}^e develops into the polymorphonuclear neutrophile, so that in abnormal blood we may have all gradations between these two types. {b) Eosinophile Myelocytes. These cells are exactly analogous to the preceding, with the exception that the granules of the more mature form show distinct eosinophilic tendencies. The younger forms of these granules may show a purplish-violet or even blue color, owing, as Simon states, to the fact that the young eosinophilic granule is physically cyanophilic and chemically amphophilic, whereas the mature granule is physically erythrophilic, but chemically absolutely oxyphilic. The size of these cells is more or less variable, so that it is probable that we have the two types of eosinophile myelocytes, corresponding to the large and small neutrophile myelocytes. These cells occur more frequently in leuke- mia, in association with tumors of the bone-marrow^ and in the psuedoleuke- mic anemia of children. (c) Basophile Myelocytes. These myelocytes may be of variable size, but are characterized by the large centrally located nucleus, which is not clearly defined from that of the surrounding slightly basophilic protoplasm. The granules are distinctly basophilic and in some cases are very numerous, while in others they may be widely scattered through the protoplasm. These cells are practically never 1 von Roznowski (Ztschr.^/^^^^y^SV&liSJ?©. 377) believes that the presence of myelocytes in large numbers m cases of cachexia points to metastasis in the bone-marrow. Digitized by Microsoft® PLATE XXI. 'fi^-'-i it^ A'-K '-'J 4'?.'" .«■ *f lODOPHILIA. Digitized by Microsoft® THE BLOOD SOQ seen except in cases of severe splenomyelogenous leukemia, in which they may reach as high as 47 per cent, with an absolute count of 140,000 cells (Taylor). (2) Irritation Forms. These cells vary in size from a lymphocyte to a large mononuclear cell, resembling more nearly the former. They are mononuclear, nongranular cells, thus differing from the myelocyte which is always granular. The nucleus is round and eccentrically placed, showing a very slight chromatin network and staining with the triple stain a bluish-green color while while with the Romanowsky dyes the color is a pale blue. The protoplasm is stained a deep brown with the triple stain and is thus differentiated from other forms of cells. With the methylene blue dyes the protoplasm appears more deeply stained than does the nucleus . These cells were first des- cribed by Turk and would seem to have the same significance as do the myelo- cytes, namely, an indication of marked activity of the bone-marrow. Pappen- heim regards them as plasma cells and largely derived from the lymphoctyes. (3) Degenerated Forms. Occasionally we find in normal blood degenerated leucocytes which stain poorly and show no granules. These may be even so much degenerated that they show as the so-called basket cells or "shadows." This condition is very frequently seen in severe infectious diseases, while in the acute leucocy- tpses which occur under many influences diminution in the number of neutro- philic granules as well as swelling and fragmentation of the bodies of the leucocytes is very common. The changes in the staining qualities of the nucleus seem to be the most significant of the lesions in the acute type of degeneration of the leucocytes. In chronic degeneration of the leucocytes we find hydropic degeneration, which is frequent in the blood of chlorosis and, when the nuclei are involved in this degeneration, seems to be limited to certain cases of leukemia. Besides such changes we find fatty degeneration as well as glycogenic degeneration. This fatty degeneration is characterized by the appearance of fat globules in the leucocytes which stain with osmic acid and with Sudan-Ill. For this latter reason they have been styled sudanophiles, and have been very carefully studied by Buttini and Comesatti, as well as by Cesaris-Demel. lodophilia. In many pathologic conditions, especially in acute infectious diseases and in those associated with all types of sepsis, a so-called glycogen reaction or iodophilia may be demonstrated in the bodies of the leucocytes, as well as in certain extracellular granules. The technic of this method is as follows: An unfixed dry blood smear is exposed to the vapor of solid iodin until it is stained a brownish color. After the specimen is stained it is mounted in the syrup described on page 507 and is examined with an oU-immersion lens. The blood of normal individuals, stained by this method, shows the proto- plasm of the leucocytes of a bright yellow, while the nucleus takes on a much lighter tint. In pathologic blood two types of reaction can be noted. The intracellular one, which is of greater clinical importance, shows a more or less Digitized by Microsoft® 5IO DIAGNOSTIC METHODS marked diffuse brown color of the entire protoplasm of the leucocyte, or the protoplasm contains reddish-brown granules which may be more or less dis- tinct. The extracellular reaction is evident in the blood plates, while the intracellular type is more particularly confined to the neutrophiles, although the mononuclear leucocytes may occasionally be tinged brown. Much difference of opinion exists as to the nature of this brown-staining substance, Ehrlich regarding it as glycogen, while Czerny considers it as an antecedent of amyloid, and Goldberger and Weiss regard it as peptone. Kaminer regards this reaction as a degenerative change and not as an evidence of regeneration. While this reaction has little value in differentiating infectious or septic condi- tions one from the other, it is sometimes of importance in making a diagnosis between purulent and nonpurulent affections, being present in the former and absent in the latter. These granules have been found by Hofbauer in pernicious anemia, secondary anemia, and leukemia, but not in chlorosis or pseudoleukemia. This reaction is observed in pneumonia, but is seldom seen in tuberculosis, typhoid fever and diphtheria. It is, however not to be re- garded as dependent upon infection, as it occasionally obtains in non-infec- tious conditions. {D) Differential Counting of the Leucocytes. By a differential counting of the leucocytes is meant the counting of the different varieties of the leucocytes found in the stained smear and the calcula- tion of each type in terms of percentage. The technic of this method is that previously outlined and consists in making an even smear upon a glass slide and staining it with any of the stains previously mentioned, noting that the triple stain does not bring out the granulations of the leucocytes with the exception of those of the neutrophiles.^ It is self-evident that the larger the number of leucocytes counted the greater will be the possibility of arriv- ing at true percentage relations. It is wise, therefore, to count at least 250 of these cells, and in many cases, to extend this to 500. If the smear is even and the leucocytes well distributed throughout, 100 cells will frequently suffice. For a differential count a satisfactory classification is an absolute essen- tial. As none of the systems at present advanced are entirely adequate, we still use the classification of Ehrlich, which is as follows: Small mononuclears, large mononuclears (including the transitional), polymorphonuclear neutro- philes, eosinophiles, basophiles (mast cells), and myelocytes. The character- istics of these cells have been previously given, but may be summed up in this connection. By a small mononuclear is meant any nongranular mononu- cleated cell smaller than a polymorphonuclear neutrophile. A large mononu- clear is any nongranular cell with a round or oval nucleus and larger than a polymorphonuclear neutrophile. A cell of the same description and size but with an indented nucleus is a transitional form. The polymorphonuclears a,re cells which are about 10 microns in size and show a diffuse granulation, which may be either neutrophile, eosinophile, or basophile. It should be remembered that the mast cells appear as nongranular forms when the triple stain is used, so that the characteristics of the nucleus in its relation to the 1 See Arneth, Deutsch. Med. Wchnschr., 1913, XXXIX, 2560; also, Dunzelt, Munch. Med. Wchnschr., 1913, LX, Digitized by Microsoft® Percentage. Number per cmm. 20-25 1200-2000 3-5 200-400 65-75 5000 2-4 100-200 c^K 5-50 THE BLOOD SII protoplasm must be borne in mind. The percentage relations of these cells in normal blood are as follows:^ Small mononuclears, Large mononuclears, Polymorphonuclear neutrophiles, Polymorphonuclear eosinophiles. Polymorphonuclear basophiles. In the writer's laboratory differential staining is usually carried out with the use of the Wright or Giemsa stain as he has found the usual triple stain- quite unreliable. Although the granular differentiation is not as distinct as could be desired, yet one soon becomes accustomed to the staining of various cells so that it is not a matter of great difficulty to distinguish the various types. In many cases it is not the easiest matter to distinguish myelocytes from the small mononuclear types, but careful study will usually clear up any obscurities which exist. (£) Number of Leucocytes. The normal number of leucocytes in a cmm. of blood has been given vari- ous figures. As a rule, it may be said that anything above 10,000 leucocytes per cmm. should be considered pathological, the normal variation running from s,ooo to g,ooo cells. In estimating the normal number of white cells, both in health find in disease, a large, number of factors which influence these cells must be taken into consideration. Thus vasomotor phenoniena, varia- tions in the volume of the plasma, inflammatory processes, state of digestion, age, variations in different parts of the circulatory system, and many different disease processes usually bring about an increase in this number, while many pathological conditions are associated with a reduction. An increase in the number of white cells is usually spoken of as a leucocytosis, but it must be remembered that such an increase may be purely physiological and should be sharply differentiated from a pathological increase which is clinically the more important. This increase is usually referable to the increase in the number of the polymorphonuclear neutrophiles, while an increase in the other varieties of cells is spoken of as a lymphocytosis, myelocytosis, esoinophilia or an eosinophilocytosis, or a mixed leucocytosis. A diminution of the number of polynuclear neutrophiles is designated as a leucopenia, which is the more usual form in which reduction of the cells occurs. It is to be remembered that these conditions may be transitory and symptomatic pointing to a purely physiological process, while a more per- manent and more marked increase or decrease in the number should be con- sidered pathological. It is rare that we find the increase limited absolutely to one variety of cell, the increase in the others being less marked. The ^ See Bunting, Am. Jour. Med. Sc, 1911, CXLII, 698; Galambos, Folia Hsemat., 1912, XIII, 153; von Torday, Virchow's Arch. f. path. Anat., 1913, CCXIII, 529; Mehrtens (Arch. Int. Med., 1913, XII, 198) calls attention to the frequency of relatively low poly- nuclear leucocyte counts with high lymphocyte counts in the routine clinical work. Warfield (Jour. Am. Med. Assn., 1915, LXIV, 1296) advises subdivisions of these groups so that we have seven classes. Digitized by Microsoft® 512 DIAGNOSTIC METHODS absolute number of these cells is much more to be regarded than their per- centage relation, as with an increased number of leucocytes the actual number of some of these varieties may be increased, although the percentage may be diminished; while with a low leucocyte count the percentage may be increased and the absolute number diminished. It is wise, therefore, to report not only the percentage relations of the leucocytes, but also the actual number of these cells per cmm. This will correct mistaken ideas as to an apparent in- crease or decrease in any particular variety of cell. Thus a differential leucocyte count may show a percentage of 50 for the neutrophiles and at the same time an actual number of 10,000 per cmm., giving rise to confusion as to the actual relation of these important leucocytes. Leucoc3rtosis (Polymorphonuclear Neutrophilosis). As stated above, anything above 10,000 leucocytes per cmm. should be regarded as a leucocytosis. This is, however, relative and should not be con- sidered pathological without taking into consideration all of the physiological and pathological influences. Regarding the theoretical cause of leucocytosis, little is definitely known. The influence of infectious processes is such as usually to increase the number of leucocytes in the blood, as an attempt on the part of the system to overcome by phagocytosis the action of the bacteria of the various diseases. Yet we find in some of these infectious processes, notably in typhoid fever, a marked reduction in the number of the white cells, although the bacillus typhosus is present in large numbers in the blood at the same time. We must, therefore, assume some specific influence upon phago" cytosis and chemotaxis, as a general infection is not necessarily associated with an increase in the number of white cells, but is dependent more upon the specific nature of the infection. The work upon opsonins and vaccine therapy may open up an entirely new field in our study of this subject. The leucocytosis shown in noninfectious conditions is still a matter of much dispute and has probably more to do with variations in the plasma than with direct increase in the number of white cells. Classification. According to Limbeck,^ the following classification of the various leuco- cytoses is the most comprehensive: (i) Physiological leucocytosis. (a) Leucocytosis of digestion. (b) Leucocytosis of pregnancy. (c) Leucocytosis of the new-born. (2) Pathological leucocytosis. (a) Inflammatory leucocytosis. (b) Leucocytosis associated with malignant tumors (cachectic leucocytosis). (c) Posthemorrhagic leucocytosis. (d) Agonal or antemortem leucocytosis. (3) Leucocytosis following medicinal and therapeutic measures. (4) Leucocytosis from other causes. ' Jena, 1896. Digitized by Microsoft® PLATE XXII. POLYNUCLEAR LeuCOCYTOSIS. (WkIGHt's StAIN.) Digitized by Microsoft® Digitized by Microsoft® THE BLOOD 513 (i) Physiological Leucocytosis. (a) Leucocytosis of Digestion. Normally a leucocytosis of the polynuclear type will be observed beginning about one hour after a meal rich in proteins and will reach its maximum in from three to five hours. ^ The actual figure reached varies in different per- sons, being usually an increase of about one-third, the maximum often reach- ing 15,000 cells, but more usually not much over 10,000. In this increase the small mononuclear cells may be absolutely as well as relatively increased. In some persons this leucocytosis of digestion does not appear, which fact may be referable to marked torpidity of the intestines, to a prolongation of the process of digestion, or to a large absorption of fluids. It has been found that a highly albiiminous diet has a much more marked influence upon this leucocytosis than does a diet of vegetables and fat. The rapidity of ab- sorption and of digestion must be taken into consideration as factors which influence the appearance or nonappearance of the leucocytosis. In children the increase in cells is much more marked than in the adult, due probably to the increased digestive and absorptive powers, providing the food taken is, as it should be, easily digestible and absorbable. Various pathologic conditions influence this digestion leucocytosis. Thus Miiller has found that in cases of carcinoma of the stomach a digestion leucocytosis is rarely observed after heavy meals. There are, however, a few ■cases of gastric cancer in which a slight increase has been observed. This failure of leucocytosis is probably due, as Schneyer shows, to lessened ab- ;Sorption as a result of involvement of the lymphatics rather than to a malig- nant stenosis of the pylorus, as the benign stenoses are usually associated with a leucocytosis. That the lack of leucocytosis is due more to diminished ab- sorption than to lack of digestive power is shown by the usual occurrence of a digestion leucocytosis in ulcer of the stomach, chronic gastritis, and in dyspep- tic conditions. Just why carcinoma, with or without stenosis, should be .associated with a normal or subnormal number of leucocytes after digestion is hard to explain, in view of the fact that these other conditions are associated -with a leucocytosis. Practically all absorption of food material occurs from the bowel and we would naturally expect to find disorders of the intestinal -canal leading more frequently to a normal leucocyte count than to a leucocyto- sis. Very little detailed work has been done upon the influence of enteric troubles upon the leucocyte count, but the writer has seen several cases in which a digestion leucocytosis did not occur and in which the findings both .ante- and postmortem were entirely related to the bowels. The examination of the blood may, however, clear up a diagnosis of carcinoma, but will not always permit of a differential diagnosis between this condition and pernicious anemia. It is to be remembered that a leucocyte count must be frequently made in order that this test may be of any value whatever and that a consider- able rise only is to be taken as evidence of a leucocytosis. Patients with cancer either of the stomach or of other viscera frequently show a leucocytosis 1 See Brasch, Ztschr. f. exp. Path. u. Therap., 1912, X, 3&1; also, Mitchell, Am. Jour .Dis. Child., 191S, IX, 358. Digifi^ecl by Microsoft® 514 DIAGNOSTIC METHODS as the result of cachexia and this should be remembered in the interpretation of a leucocyte count following digestion. (b) Leucocytosis of Pregnancy. It has been shown that from 5 to 75 per cent, of cases of pregnancy are associated with a leucocytosis, averaging about 15000 cells per cmm. This is especially true of primiparae, but is often shown in the multiparas. Just what cause can be given for this rise is uncertain. It is more than likely that a condition of slight intoxication is present due to the overloading of the blood of the patient with the products of metabohsm of the fetus. These sub- stances are not normal to the blood of the woman and in consequence act as foreign bodies which may attract, by their chemotactic influence, the white cells and bring about an increase. As these cases of pregnancy show an ab- sence of a digestion leucocytosis it has been assumed that the increase in leucocytes is due to a prolonged digestion leucocytosis, but this does not seem probable. The changes in the breasts and in the uterus during this period have suggested these organs as the factors influencing this leucocytosis, but such inferences do not seem to be directly warrantable. The leucocytosis of pregnancy is a mixed leucocytosis, all of the various types of leucocytes, with the exception of the eosinophiles being increased. After the birth of the child the leucocytes gradually diminish in number and normally reach their usual values in from four to fourteen days after delivery. Should complications, such as postpartum hemorrhage or septic fever arise, the leucocyte count may remain high until these complications have subsided. In multiparas these changes in the number of leucocytes are not so marked as in the case of the primiparae but a slight rise always occurs. This fact has been attributed to the lessened reactivity of the organisms to the influence of the toxic substances thrown into the blood from the cells of the fetus. (c) Leucocytosis of the New-bom. As Askanazy has found, the blood of the fetus shows a diminution in the number of leucocytes, owing to the fact that there is no function as yet estab- lished for these cells in utero. In the blood of the new-born, however, a leucocytosis running from 15,000 to 20,000 cells may be observed, these fig- ures going as high as 40,000 under the influence of the first feeding. As the weight of the child begins to diminish these cells are markedly reduced in number to about 8,000, and are subsequently increased to 10,000 as the child begins to gain in weight. The high leucocyte count at birth is more probably due to the rapid blood formation than to a concentration of the blood, al- though this latter factor as well as that of the influence of digestion must be taken into consideration. The increase in the number of cells is chiefly limited to that of the small mononuclear cell, the differential count of the leucocytes showing during the early periods of life from 40 to 60 per cent, of the total number. This leucocyte count, if taken at the moment of birth, will not, however, vary much from that of the adult, the change becoming more marked in the early penc§^^.t,y Microsoft® THE BLOOD 51$ (2) Pathological Leucocytosis. (a) Inflammatory Leucocytosis. In most cases of inflammatory nature, of acute infections and general febrile diseases, there is observed an absolute increase of the polymorphonu- clear neutrophiles, which runs more or less parallel to the temperature. This increase may run from 10,000 to 50,000 cells and diminishes as the influence of the inflammatory process is diminished. In general it may be said that leucocytosis represents the reaction of the individual to the disease. A high count may mean a vigorous reaction to the infection; a low cent may mean either a poor reaction and hence an unfavorable condition of the patient, or it may indicate a very mild degree of infection with a normal reactivity of the patient. It must be said that a.11 diseases of an infectious nature are not necessarily associated with a leucocytosis. For instance, pneumonia shows a leucocytosis which runs parallel to the degree of viru- lence; measles, influenza, malaria, and tuberculosis are rarely if ever associ- ated with a leucocytosis unless complications arise or the conditions become very severe. In typhoid fever we usually find a leucopenia which, however, is associated with a relative lymphocytosis; if complications such as perfora- tion arise a leucocytosis may appear. Among the conditions causing leucocy- tosis are acute lobar pneumonia, the count running between 20,000 and 100,000, depending upon the severity of the infection and the degree of resist- ance against the infection. Acute articular rheumatism, diphtheria, acute cerebrospinal meningitis, follicular and suppurative tonsillitis, scarlet fever, mumps, rabies, erysipelas, ulcerative endocarditis, small-pox, cholera, general pus infections of the serous membranes and of the mucous membranes,^ acute bronchitis, and many other conditions cause a leucocytosis of the polymor- phonuclear type. The variations in these many conditions will be discussed under the Pathology of the Blood. The exact cause of this leucocytosis following inflammatory processes is still a matter of much discussion. The bone-marrow has been shown to be markedly increased as regards cellxilar proliferation in the early stages of the inflammation and may be so markedly changed as to cause permanent de- rangement in the functions of this blood-forming organ. That leucocytosis has much to do with immunity both from the standpoint of phagocytosis and from that of Ehrlich's side-chain theory cannot be questioned.^ A leucocy- tosis must represent the attempt on the part of nature to rid the blood and the system of the bacterial and toxic products of the disease. Whether phagocytosis as such or under the influence of various opsonins is the direct cause of immunity and of recovery from any specific infection must be left to a later chapter. (b) Cachectic Leucocytosis. This variety of leucocytosis is the le;ast uncertain. The cases of carci- noma and of sarcoma show a leucocytosis which is not definite for the particu- ' See Smith, Surg. Gynec. and Obst., 1913, XVI, 403. ' Gay and Claypole (Arch. Int. Med., 1914, XIV, 662) call attention to the specific hyperleucocytosis following i]S0^^ j^l^j^^^lyg^yy^cines. 5l6 DIAGNOSTIC METHODS lar kind of cancer, but is more usual with the latter than with the former type. The leucocytosis is usually one of the polymorphonuclear tj^e, but is frequently associated with an increase in the number of mononuclear cells. This leucocytosis has no direct relation with the situation of the tumor and is not always present in all cases of cancer. Whether this leucocytosis be due. to an intercurrent infection is a question which must be left for more detailed work, but it would seem wise to accept Ewing's statement that a marked leucocytosis in the course of a cachexia, from tertiary syphilis, tuber- culosis, nephritis, and in the majority of cases of carcinoma, should suggest a search for a complicating infection, while in the sarcomata a leucocytosis is much more common as a direct result of the disease. (c) Posthemorrhagic Leucocytosis. A well-marked leucocytosis, which may begin in from ten to fifteen minutes and may reach as high as 20,000 cells within an hour, has been of ten observed following extensive acute hemorrhages. The leucocytosis in these cases bears a general relation to the extent and rapidity of the loss of blood and usually disappears or diminishes long before regeneration of the blood has occurred.' This leucocytosis is of the polymorphonuclear type and is referable rather to the sudden outflow of lymph which occurs as a compensation for the loss of fluid than to a new production of cells, as this latter process does not take place for some time. In hemorrhages which are slight and long-continued as in cases of gastric or intestinal ulcer, the duration of the leucocytosis as, well as its extent is very brief. Stassano and Billou assume from their work that a hypoleucocytosis follows a severe hemorrhage while a true leucocytosis is observed after the loss of small qauntities of blood. These findings seem to be rather doubtful, except where the hemorrhage has been so extensive as to cause death. {d). Antemortem Leucocytosis. This form of leucocytosis has been questioned, especially by Arneth, but there seems to be little doubt that such a leucocytosis may occur if death does not take place too rapidly. In some diseases the leucocytes do not fall in number, but in some a distinct rise is noted which has been attributed by Ehrlich to the accumulation of white cells along the periphery of the blood- vessels as a result of slowing or stasis of the circulation. This type of leuco- cytosis is usually of the polymorphonuclear variety and may support the view of Limbeck that antemortem leucocytosis, when it does occur, is the result of a terminal infection, although it cannot explain those cases which show a lymphocytosis rather than the ordinary leucocytosis. The character of the antemortem leucocytosis must depend largely upon the precedent condition and will be associated with antemortem dissemination of bacteria, antemor- tem hyperpyrexia, vasomotor paralysis, serous exudation, etc. (3) Leucocjrtosis Following Therapeutic Measures. {a) Drugs. It has been found following administration of tonic drugs, ethereal oils, myrrh, turpentine, cs.m^s^iJ^&W^^f'oi&f^' ^"^ °^^^^ ^"^^Ss, that a 1 See Levison, Jour. Am. MeS. Assn., 1915, LXIV, 1294. THE BLOOD S^? leucocytosis of a more or less extent occurs, which is probably referable to the same cause as is digestion leucocytosis. Many of these drugs if applied locally for the purpose of counterirritation were shown to have the same effect. Extracts of tissues, especially those containing large amounts of nucleinic acid and of purin substances, produce an extensive leucocytosis, which fact has been taken advantage of in the administration of the nuclein substances as therapeutic remedies. In the case of those drugs which de^ stroy the red cells, such as the coal-tar antipyretics, chlorates, and illuminat- ing gas, a normal number of white cells is usually observed, although Simon wrongly states that a hyperleucocytosis follows the use of such drugs. After the prolonged use of chloroform or ether a polynuclear leucocytosis is gener- ally observed which is usually of short duration. In this connection it is well to remember that an increase in the number of leucocytes of 10,000 to 15,000 above the normal value of the individual should be regarded as evidence of infection if this increase is sustained for more than a few hours. (6) Baths. After a cold bath the leucocytes of the polynuclear variety have been shown to be increased from 100 to 300 per cent.^ This is true only if the bath is of moderate duration, a prolonged cold bath taken to the point of exhaus- tion will diminish rather than raise the number of white cells. Experiments on the result of hot baths have shown just the reverse condition, namely, a hot bath of short duration produces a decrease, while one of long duration causes an increase in the number of white cells. Massage in itself following either a hot or cold bath tends to increase the number of leucocytes, (c) Exercise. Prolonged muscular exercise, as taken in the form of gymnasium work, as applied in the various therapeutic movement treatments, and also as given n the more violent athletic contests produces a rise in the number of leuco- cytes which is temporary and is characterized more by an increase in the num- ber of polynuclear cells than of any other variety of the leucocytes. (4) Leucocytosis from Other Causes. Cyanosis, passive hyperemia obtained by the method of Bier, shock whether physical or mental, injection of various toxins, such as Koch's tuber- culin, the various autovaccines, and injection of various organic principles, such as peptone, pus, organ extracts, etc., have all been shown to produce a polynuclear hyperleucocytosis, either by causing stasis of the circulation or by increasing the chemo tactic power of the serum. ^ The exact explanation of chemotaxis is still in doubt and we find that some substances are positively and others negatively chemotactic, thus accounting for the lack of phagocy- tosis in certain conditions, such as pneumonia, although in these same condi- tions we may have a leucocytosis as a direct result of an infection. From the work of Rosenow we learn that the phagocytic action of the leucocytes in 1 See Rovighi and Secchi (Rif. Med., igi4, XXX, 617; Miinch. Med. Wchnschr., 1914, LXI, 1721) for a discussion of leucocytosis following exposure to cold. ^ See Veraguth and Seyderheim (Munch, med. Wchnschr., 1914, LXI, 301) for the leucocytosis following use of electricity. Digitized by Microsoft® 5l8 DIAGNOSTIC METHODS pneumonia is increased by the addition of an extract of the active pneumo- cocci; whether this extract increases the chemo tactic power of the bacteria in the blood or whether certain restraining influences are overcome is still unsettled. Future work upon phagocytosis and upon the factors governing increased opsonic power in the various infections may show us what causes are effective in such conditions. In the previous discussion of leucocytosis we have had reference more to an increase in the number of polynuclear cells, this type being more prop- erly called polynuclear hyperleucocytosis. It is very rare that this condition exists in the absolute pure state. Mixed Leucocytosis. By this is meant an increase of both granular and of nongranular cells of various types, its more common interpretation, however, being a leucocy- tosis characterized by an increase in the number of neutrophile myelocytes. This neutrophilic myelocytosis is best seen in leukemia in which the absolute number of myelocytes may reach 150,000, the increase in no other condition, according to Ehrlich, rising above 1,000 cells. The increase in the myelo- cytes in leukemia is not limited to those of the neutrophile variety, but is com- monly associated with a marked increase in the number of eosinophile types as well as with an increase in the number of mast cells. In pernicious anemia we may also find a large increase in the number of these myelo- cytes. These cells when increased in number are clinically significant of ex- haustion of the bone-marrow, but should never be interpreted in this way unless they remain while the leucocyte count is falling; in other words, they are significant only when constituting a large percentage of the leucocytes. Lymphocytosis. A relative or absolute increase in the number of lymphocytes is of fre- quent occurrence and has occasionally some significance. In judging of this condition the actual number of cells present as well as their numerical rela- tions to the other varieties of leucocytes must be considered. The term lymphocytosis must always be applied to an absolute increase in the number of these cells, the normal number being between 1,200 and 1,500 per cmm. Physiologically we find this condition in infants and during a digestive leucocy- tosis in the adult. i The child shows at birth from 50 to 65 per cent, of these cells which percentage gradually diminishes, reaching the normal percentage (20-25) about the age of puberty. Pathologically this condition is observed in poorly nourished children, ^ those showing the "constitutio lymphatica," rachitis, whooping-cough, gastro-intestirial disturbances in the child, cervical adenitis, splenic tumors, in most infectious diseases of children,^ and especially in lymphatic leukemia, in which as high as 90 per cent, of a largely increased leucocyte count is ref- erable to the small lymphocytes. In the splenomyelogenous form of leuke- ' Kaufmann, Jour. Am. Med. Assn., 1914, LXIII, 1104; Staines, James and Rosenberg (Arch. Int. Med., 1914, XIV, 376) report a lymphocytosis due to increased altitude. ' See von Hoesslin, Munch, med. Wchnschr., 1913, LX, 1129 and 1206. ' See Marchand, Deutsch. Arch. f. klin. Med., 1913, GX, 359. Digitized by Microsoft® THE BLOOD SIQ mia and following removal of the spleen we observe a steady increase in the number of lymphocytes continuing during the first year. It must be remem- bered that a leucocyte count may be low and yet a lymphocytosis exist. This is shown in typhoid fever, amebic dysentery, chlorosis, pernicious anemia, scurvy, and other conditions in which the leucocyte count is low, the granular cells being diminished in number, but the lymphocytes being increased.^ In the congenital and also secondary acquired syphilis we find a lymphocytosis which must be referred to involvement of the lymph-glands. The worker must not be led into a mistaken interpretation of his blood-findings, as en- larged lymph-glands may give the same picture of lymphocytosis as is seen in Hodgkin's disease, chronic and acute lymphatic leukemia. Eosinophilia. By this is meant an absolute increase in the number of eosinophilic cells. The average number of these cells is between one and two hundred per cmm., hence the term eosinophilia should be limited to those cases showing counts something above 250. We find a physiological eosinophilia during child- hood, the average increase being about i to 2 per cent, above the normal adult finding. No physiological relations have been established between eosin- ophilia and sex, pregnancy, menstruation, digestion, or old age; however racial distinctions are sometimes shown by eosinophilia, the natives of southern China showing between 15 and 20 per cent, of the leucocytes as eosinophiles. Pathological Eosinophilia. We observe variations in the number of eosinophiles in various affections of the bone-marrow. Thus in splenomyelogenous leukemia, we may find these cells increased as high as 30,000 per cmm. According to Ehrlich, a diagnosis of this form of leukemia is warranted only when we have an increase in the number of eosinophiles, but it cannot be doubted that cases are rela- tively frequent in which these cells are not increased. In sarcoma of the- bone-marrow as well as in osteomyelitis and osteomalacia, we find these cells " usually increased in number. In chlorotic conditions we may find the eosino- philes moderately increased, while in secondary anemias especially those following infection with parasites a marked eosinophilia may be observed. After extirpation of the spleen as well as in cases of chronic splenic tumor we find an eosinophilia which may run from 10 to 40 per cent, higher than the normal, constituting as high as 40 per cent, of the total number of leucocytes. Whether or not a pure disease of the lymph-glands is associated with the eosinophiha is unsettled, but if metastases have extended to the bone, an eosinophilia of marked degree is usual. True bronchial asthma is always associated with an increase in the num- ber of eosinophiles, one case of Billings showing 54 per cent, of the total leucocytes as eosinophiles. This relationship in asthma is of importance 1 Borchardt (Deutsch. Arch. f. klin. Med., 1912, CVI, 182) shows that a lymphocytosis obtains in a large majority of cases of disease of the thyroid, hypophysis and suprarenal gland. This is oftentimes sissociated with a leucopenia. Cabot (Am. Jour. Med. Sc, 1913, CXLV, 33s) calls attention to the occasional occurrence of a lymphocytosis in infections usually associated with a polynucleosis. See, also, Huhle, Deutsch. Arch. f. kiln Med 1914, CXIII, 455- Digitized by Microsoft® 520 DIAGNOSTIC METHODS from a diagnostic standpoint as asthmatic attacks from other causes are not associated with an eosinophilia. It is true that in emphysema a marked eosinophilia does occur so that we may find, in cases in which this condition complicates a tuberculosis, that an eosinophilia obtains. Tuberculosis of the lungs or of other tissues does not show an eosinophilia unless complicated by emphysema, cachexia, or secondary infection. ^ A large number of skin diseases are associated with eosinophilia, the high- est count being reported by Zappert in a case of pemphigus showing 4,800 cells per cmm. The occurrence of this condition in skin diseases depends not so much upon the nature of the lesion as upon its extent, intensity and lack of healing tendency. Many skin lesions are known to be produced by toxic agents which have special chemotactic influences over the eosinophile cells; this fact may account for the eosinophilia in such conditions. The most usual skin diseases showing this increase are pemphigus, eczema, psoriasis, urticaria, purpura, scleroderma, lupus, leprosy, herpes zoster, and general gouty affections.^ It is a general rule that the infectious fevers are not associated with eosino- philia, but with the acute polynuclear leucocytosis.' In scarlet fever, how- ever, we find that eosinophiles are frequently markedly increased, while in acute rheumatism and in malaria these cells are usually present in more or less increased numbers.* In gonorrheal infections the eosinophile leucocytes are very frequently marked in the discharge in the early days of infection, diminishing in number as the number of neutrophiles increases, and increasing again as the discharge clears up. It is generally believed that the eosino- phile cells in the blood are increased coincidently with their increase in the gonorrheal discharge. It has been shown that the eosinophiles are usually increased in all forms of ovarian disease with the exception of cancer. In syphilis a uniform increase of the eosinophiles has been observed, but some cases do not show such a regular finding. Outside of the splenomyelogenous form of leukemia and the true bron- chial asthma, infection with various parasites is accompanied by the most pronounced eosinophilia. Any parasite from the harmless pin-worm to the most malignant uncinaria may cause an eosinophilia (Emerson). This eosinophilia is not necessarily constant nor does its extent bear any relation to the severity of the infection. A differential diagnosis between typhoid i See Arneth (Deutsch. Arch. f. klin. Med., 191 2, CVIII, 323), for the relations of the eosinophiles in pneumonia. Chuprin (Med. Obozr., I9I3,LXXIX, 17) believes that eosino- philes gradually increase in the blood in favorable cases of tuberculosis; also, Brosamlen, Deutsch. Arch. f. klin. Med., .1914, CXV, 146; Peppard, Journal-Lancet, 1915, XXXV, 478. ^Lifschutz,, MonatsEchr. f. Kinderhkde., 1914, XII, 603; Aschenheim, Ztschr., f. Kin- derhkde., 1914, X, 503; Putdg, Ibid., 505; Engman and Davis, Tour. Cutan. Dis:, 1915, XXXIII, 73. 2 Schlecht and Schwenker (Deutsch. Arch. f. klin. Med., 191 2, CVIII, 405) show the relationship of eosinophilia to anaphylaxis. See Herrick, Arch. Int. Med., 1913, XI, 165; also, Ahl and Schittenhelm, Ztschr. f. d. ges. exper. Med., 1913, 1, iii; Steiger and Strobel, Zentralbl. f. inn. Med., 1913, XXXIV, 1073. Leopold (New York Med. Jour., 1914, C, 225) finds an eosinophilia in chorea. Besancon and Moreau, Ann. de Med., 1914, II, 85; Johns, New Orleans, Med. and Surg. Jour., 1914, LXVII, 453; Weinberg and Sfiguin, Ann. de rinst. Pasteur, 1914, XXIII, 470; Herrick, Arch. Int. Med., 1914, XIII, 794. * See Ebbell (Norsk Mag. f. Laegevidensk., 1912, LXXIII, 617) for a very naive theory of the relation of eosinophilia to the parasitic diseases. Digitized by Microsoft® THE BLOOD 52 1 fever and trichinosis is frequently possible on the basis of the marked eosino- philia in this latter condition/ although eosinophilia does not always obtair\ here. Mast-cell Leucocytosis. In myelogenous leukemia we find these cells increased to a very large extent, often outnumbering the eosinophile cells. An increase of these cells is generally regarded as the sole isolated pathognomonic sign of this disease. Their increase may be as high as 20 per cent, of the leucocytes. These cells have been found in a few cases of cancer, tuberculosis, syphilis, and other lesions of the skin, whUe in bone disease complicated by septic infection a.few reports of positive findings have been made. Leucopenia. This is a condition characterized by a reduction either in the total number of leucocytes or in one or more groups of leucocytes. The most usual con- dition showing a leucopenia is typhoid fever in which we find the polynuclear cells markedly diminished, thus lowering the leucocyte count, while the large mononuclear cells are relatively increased. Anything below 5,000 cells is regarded as a leucopenia. It must be considered in this connection that the typhoid leucopenia remains only when the disease is limited to the intestinal canal; when other organs become involved a leucocytosis supervenes. The count in typhoid fever may run as low as 2,000 cells, while in tuberculosis of the lymph-glands it may reach 500 cells. In cases of starvation or malnu- trition the leucocyte count is always low and. in chronic intoxication with the heavy metals, morphin, alcohol, and cocain, we observe a very low count, as a rule. A general rule to be observed in typhoid fever is that an increase in the number of white cells following a low leucocyte count is evidence of com- plications or of a mistaken diagnosis. It must be remembered that a relapse in typhoid may bring on a hyperleucocytosis, but this must occur during the afebrile period, otherwise a leucopenia will remain.^ In measles, also, we observe a leucopenia following the eruption and a leucocytosis preceding the eruption. This leucopenia affects the polynuclear neutrophiles while the lymphocytes are relatively increased, with disappearance of the eosinophiles. In cases of uncomplicated influenza we usually find a diminution in the num- ber of leucocytes, although a normal number may obtain. This enables one to differentiate influenza from pneumonia, in which latter condition a marked leucocytosis is practically always present.' In pernicious anemia and in splenic anemia we find a marked leucopenia during the active periods of the disease, the count running, as a rule, between two and three thousand cells. (F) Variations in Infancy and Childhood. In the first few days after birth the leucocytes may reach as high as 20,000, while in the nursing period the cells average 13,000. In this connection the 1 Cooper (Wisconsin Med. Jour., 1914, XII, 365) reports a case of trichinosis with 72 per cent, of eosinophiles. "See Schneider (Deutsch. Med. Wchnschr., 19 15, XLI, 393) for a discussion of leu- copenia following typhoid inoculation. ' Camp and Baumgartner (Jour. Exper. Med., 1915, XXII, 174) show that severe inflammatory processes may he associated with a leucopenia. 522 DIAGNOSTIC ACETHODS influence of a digestion leucocytosis must be remembered as the cells may reach as high as 25,000 of which the lymphocytes constitute about 60 per cent. As the age of the child increases, the number of leucocytes gradually diminishes until the age of puberty, when the average number is about 8,000. As the development of the child progresses, the number of lymphocytes is gradually diminished and that of the neutrophiles correspondingly increased. The relations between the- lymphocytes and the neutrophiles must be con- stantly remembered in making a blood count, as the percentage of lympho- cytes is high at first and then gradually diminishes -until the age of pu- berty; the neutrophiles during the same period are at first low, reaching the normal 65 to 70 per cent, about the age of 15. There are no marked differ- ences in the morphology of the granular and of the nongranular cells of the child as compared with those of the adult. The characteristic changes in the leucocytes of the blood of the growing child are more related to the changes consequent upon development and show no signs of degenerative changes. (G) Functions of the Leucocytes. The functions of the leucocytes are largely related to their powers of overcoming the effects of bacterial processes. These results are accomplished both through their ameboid powers and their characteristic property of phagocytosis. This latter property is largely influenced by chemotaxis as well as by the indefinite increase of so-called opsonins. Just what we are to regard as the basis of the opsonic index of the leucocyte is unsettled, but it is certain that the same leucocyte may show marked variations in its power of absorbing bacteria of different types. Whether this opsonic power is related in any way to the various assumptions embraced by Ehrlich's side-chain theory must be left for a later section. Besides the above important func- tions of the leucocytes, these cells show oxidizing, reducing, and fermentative powers, all of which are of more or less importance in the study of immunity and of reaction to bacterial infection. As stated (p. 501), the oxygen cata- lysts of the blood include oxidase, peroxidase, catalase and hemoglobin. To these ferments we owe many chemical tests for blood and pus, which are destroyed if the specimen be previously boiled, while hemoglobin is still capable of inducing certain oxidations. Kastle and Amoss^ have studied the peroxidase activity of the blood in health and disease, while Kastle^ has made an exhaustive study of the oxidases. Winternitz and Meloy,^ Winter- nitz and Pratt,^ and, especially, Winternitz, Henry and McPhedran* have turned their attention to catalase. The technic of these latter workers is as follows: 0.025 c.c. of blood is taken from a puncture of the ear by means of a specially graduated pipet. This is immediately diluted with 10 c.c. of dis- tilled water, giving a dilution of i : 400. Five c.c. of this diluted blood are placed in each of two 100 c.c. salt-mouth bottles, one being used for the test, 1 Bull. 31, Hyg. Lab. U. S. Pub. Health and Mar. Hosp. Serv., 1906. 2 Bull. 59, Hyg. Lab. U. S. Pub. Health and Mar. Hosp. Serv., 1909. See Schultze, Miinch. med. Wchnschr., 1909, LVI, 167; Dunn, Jour. Path, and Bacteriol., 1910, XV, 20; and Klopfer, Ztschr. f. exper. Path. u. Therap., 1912, XI, 467. 'Jour. Exper. Med., 1908, X, 759. ■i Jour. Exper. Med., 1910, XII, i and 115. xcV^fsifwSi^f nd' lMi?dMMm»:f: ^67 """' ""''■ '°"^' ''''' THE BLOOD 523 the Other being held as a check. In one is placed a small vial containing 5 c.c. of neutralized commercial hydrogen peroxid (3 per cent.). The large bottle is connected with a gas buret, to collect the gas formed by the action of the catalase upon the H2O2. The bottle is agitated for one minute, read- ings being taken every 15 seconds. Eighty per cent, of the normal cases studied show a liberation of 14 to 17 c.c. of oxygen in 15 seconds, this being constant for the same individual for a period of many months. The impor- tance of this test rests in the variations from day to day or in any change from the normal activity of the individual's blood. The amount of gas hberated is, therefore, a personal rather than a general factor. Tschernoruzkii has recently shown that i gram of leucocytes is capable of liberating 312.9 c.c. of molecular oxygen under the influence of catalast. The diagnostic value of this test in disease is as follows: In typhoid fever there is no change in the early stages, but toward the third week there is a gradual fall accompanying the anemia. During the course of lobar pneumo- nia a slight decline may be noted. Diabetes, mellitus shows no change. In diseases of the thyroid, the catalytic activity is not constant from day to day, in hyperthyreosis a tendency to increase being noted while in hypothyreosis a lower level than normal is observed. In renal disease we find no change as long as no marked renal insufficiency obtains, but the activity becomes irregu- lar from day to day, and, as uremia approaches, becomes much lower than normal. With retention of urine due to obstruction of the lower urinary tract a marked decline is observed. Little change is noted in cardiac cases even m the severe types. The toxemias of pregnancy are separable into two classes by this test; without renal involvement eclampsia shows no change but, when renal insufficiency obtains, a marked decline prevails. This test should be of much value in diagnosis. (7) Blood-plates. (a) Appearance. These bodies which have been called "third corpuscles" are probably not true cellular entities. Hayem has considered them as the direct forerunners of the erythrocytes and has styled them, therefore, "hematoblasts." These bodies have been variously called plaques by Osier, blood-plates by Bizzo- zero, and by Arnold fragments of cells. ^ These so-called corpuscles are small colorless bodies containing no hemoglobin. They are about 3 microns in diameter, round, oval, or rod-shaped without any biconcavity. They appear bluish, homogeneous or occasionally granular, and stained lightly by both basic and acid dyes. They do not contain any nucleus or membrane and become hyaline and glassy as soon as removed from the vessel, but on standing they become pale and unite to form granular masses. They are very sticky and adhere very extensively to one another, forming masses from which fibrinous threads radiate. This fact has lead to the belief that they have an important part in the formation of fibrin. (See Duke.') ^Ztschr. f. physiol. Chem., 1911, LXXV, 216. ^ See Downey, Folia Hsemat., 1913, XV, 25; Brown, Jour. Exper. Med., 1913, XVIII, 278; Winogradow, Folia Hsemat., 1914, XVIII, 207. 3 Jour. Am. Med. Assn., ^SfiQiMbd^'l9f^Mh&^- ^^'^■' ^9i=> X, 445- 524 DIAGNOSTIC METHODS Specimens of the platelets are best obtained by puncturing the tip of the finger or the ear through a 10 per cent, solution of sodium metaphosphate. In this way the blood becomes at once mixed with the fixing fluid and the drop may then be placed on a slide and covered with a cover-glass. It has been customary to call anything a platelet which is smaller than a red-blood cell and which does not contain hemoglobin. The term platelet, however, should be reserved more particularly for these bodies which have a peculiar bluish refractility, no nucleus, show marked cohesive properties, and soon disintegrate. (6) Size. The normal size of the blood-platelet averages about 3 microns, although Preisich and Hein have reported them as high as 7 microns. As a rule, their size varies inversely as their number. Some of these bodies show clear areas either in the center or on one side, or on the whole periphery; others become crescents, triangles, spindles and threads (Emerson). (c) Number. The normal number of platelets per cmm. is approximately 250,000, this number varying in the same person at different times of day. The physio- logical factors influencing the number of these cells are not well understood and in pathological conditions we may find large or small numbers of these cells. It is generally accepted that they are increased in anemias from any cause and may be related to the red blood-cells as i to 10. They are usually normal in chlorosis, increased in severe secondary anemia and decreased in pernicious anemia. In splenomyelogenous leukemia we find a large increase of. these bodies, while in the lymphatic leukemia these cells are diminished. They are increased in chronic diseases associated with cachexia and malnutri- tion, being more marked in cancer, nephritis and tuberculosis^ than in anemia, due to other causes. The more acute, more severe, more threatening the dis- ease, the fewer the number of platelets^ so that we have a direct relationship between severity of disease and number of these bodies. The method of counting these cells has been given in a previous section to which the reader is referred. Just exactly what significance is to be attached to their increase or decrease is uncertain, but it must be remembered that there is a certain relationship between their number and the severity of general conditions. (d) Staining Properties. These bodies stain very much like nuclear material with a basic stain, but also take the acid dyes under certain conditions. With the Wright stain these cells are seen as distinctly blue bodies grouped in numbers from i to 10 and seem to be composed of nucleus and protoplasm. The apparent nucleus consist of rows of blue or reddish dots occasionally arranged in spherical masses, while the indefinite poorly-defined protoplasm-like substance seems ' See Webb, Gilbert and Havens, Arch. Int. Med., 1914, XIV, 743; Webb and Gilbert, Jour. Am. Med. Assn., 1914, LXIII, logS. 2 See Duke, Arch. Int. Med., 1913, XI, 100; also, Port and Akiyama, Deutsch. Arch. f. klin. Med., 1912, CVI, 362; Nob^court and Maillet, Bull. Soc. de Pediat., 1914, XVI, 285; Weltmann, Wien. klin. Wchnschr., 1914, XXVII, 1013; Dimond, Jour. Path, and Bac- terioi., 191S, XIX, S08. Digitized by Microsoft® THE BLOOD 525 swollen to almost the size of a red corpuscle. As a rule, however, these bodies appear as grouped bluish masses of indefinite structure and outline, (e) Function. The function of these cells is very indefinite. It is possible that they have much to do with the formation of fibrin and may be the source of the so-called fibrin ferment, thrombogen.^ If we are to regard these bodies as derived from the leucocytes this function of fibrin formation is acceptable, but if they are to be considered as derivatives of red cells or as true independent bodies such an hypothesis is untenable. No facts of any clinical value have so far been forthcoming from the study of the blood plates, and it may be possible that they are really artefacts as Lowit has claimed. (8) Hemoconien. By this term we have reference to the presence in normal blood of very fine granules which are actively motile, not truly ameboid, but with motion more of the Brownian type. These granules, also called blood-dust by Miiller, are small, round, colorless granules which vary in size from very fine dust- like particles to some as large as i micron in diameter Their exact chemical nature is uncertain as they do not show, according to Miiller, either the re- actions of fat or of albumin. The general idea prevails that these bodies are the extruded granules of the leucocytes, as they resemble in size and in stain- ing qualities those of various leucocytic types. The number of these gran- ules is uncertain, but apparently the relation of these granules to the red cells, as shown by the ultra condenser, is about 50 to i. Although no clinical significance is known to attach to these granules, the writer has observed marked variations in their number in various patholog- ical conditions, but cannot at present draw any conclusions from such observations. (g) Morphology of the Blood-forming Organs. As the bone-marrow is of such importance in the production of both red and white cells, it would seem advisable briefly to discuss the histology of this tissue. In it we find practically every cell which occurs in the blood both in normal and abnormal conditions, and also many transitional forms between various groups of cells. The following brief outline of the histology of the bone-marrow is taken from a valuable paper of Dickson. ^ Varieties of Bone-marrow. (i) Primitive or Embryonic Marrow. A delicate interlacing network of mucoid cells, which later in the process of development go to form the connective- tissue framework or adenoid reticu- lum of the tissue. (2) Red "Lymphoid" or Formative Bone-marrow Proper. This is found in the adult in the short and flat bones, sternum, ribs and vertebrae, and to a varying extent in the ends of the long bones. This is the 1 Bayne- Jones (Am. Jour. Physiol., igi2, XXX, 74) lias shown that the platelets aid in the clotting of blood in two ways: (i) by setting free prothrombin, which is later activated to thrombin, and (2) by liberating a thromboplastic substance which neutralizes the anti- thrombin normally present in the blood. 2 Jour. Path, and BacteriiSp^Kj^OilBr {i^fCrOSOft® 526 DIAGNOSTIC METHODS most important variety as in it are found the red and the majority of the white cells of the bloo^, and, according as one or the other of these series of cells predominates, the type of marrow may be classified as erythroblastic or leucoblastic, a varying admixture of these two t3npes being practically always found in any given case. (3) Fatty or Yellow Marrow. This is found mostly, as age advances, in the central part of the long bones and is formed by a process of physiological transformation or degradation of the connective-tissue elements, together with the gradual disappearance of most of the blood-forming cells of the red marrow. (4) Fibroid Marrow. This is found in old persons, especially if debilitated by long-standing dis- ease, and is characterized by the proliferation of the connective-tissue ele- ments and by the progressive sclerosis of the marrow, followed by the gradual disappearance of the hemopoietic cells of the tissue. (5) Gelatinous Marrow. This is essentially a retrogressive change in the tissue and is in no way identical with that type already described as primitive or embryonic marrow. This change has been described by a previous writer as a chronic condition only, but has been frequently found by Dickson as an acute change in many of the acute infectious fevers and allied diseases. Cjrtology of the Bone-marrow. In this discussion little more will be taken up than a brief enumeration of the various varieties of cells, as the more important ones of these have been treated of in other sections. (/) Blood-fonning Cells. (A) Leucocyte Series. (a) Nongranular Cells with Basophile Protoplasm. (i) Large. (2) Small. (a) Cells similar to but smaller han the large variety. (/3) Cells identical in ppearance and in staining reactions with the small lymphocyte of the blood. Of these there are also probably two varieties. (b) Granular Cells. (i) Neutrophile. (a) Myelocytes with large rounded or oval nuclei. These have been definitely traced by Dickson to nongranular or hyahne cells in which the granules may be seen gradually develop- ing. There are two types known as the larger and the smaller neutrophile myelocyte. (j3) Intermediate cells with indented or horse-shoe-shaped nuclei. These are developed from the myelocytes (a) and i^ turn^^^^igj, Microsoft® THE BLOOD 527 (7) Polymorphonuclear cells or adult leucocytes which pass out into the blood stream. (2) Eosinophile. The same types of these cells with eosinophile granulations are found in the marrow, as have been previously tabulated under the heading of neu- trophile cells. (3) Basophile. (a) Mast cells. The three types above dicussed are also observed in the basophile mast cells of the bone-marrow. (|3) Cells resembling the eosinophile myelocytes but with granu- lations staining with the basic dye. These cells are prob- ably altered eosinophiles. (B) Hemoglobin-holding Series. (i) Normoblasts Normocytes or ordinary red corpuscles. , . T,^ 1 , , ^ f Normoblasts, normocytes. (2) Megaloblasts < ,^ , , . , ,,.,., , , ^ [ Megalocytes (entirely pathological m the adult). (77) Giant Cells. (i) Mononucleated or megakaryocytes. (2) Multinucleated or polykaryocytes. (777) Cells of Connective-tissue Type. (i) Fat cells. (2) Cells of the reticulum. (3) Various forms of phagocytic cells. (4) Ordinary connective- tissue cells. (7F) EndotheUal Cells. (i) Found in their normal position in the vessel wall. (2) Found proliferating and taking on phagocytic functions. Reactions of the Bone-marrow in Disease. Many of these reactions are connected with the production of the so- called inflammatory leucocytosis and take place with great rapidity. Other varieties of change are intimately concerned with the production of the hemo- globin-holding series of cells. These reactions may, according to the tjrpe of cell involved, be summarized as follows: (7) Leucoblastic. (i) Neutrophile. (3) Basophile. (2) Eosinophile. (4) Hyaline or non-granular. (77) Erythroblastic. (i) Normoblastic. (2) Megaloblastic. The above brief outline will show the reader that we have in the marrow all possible types both of red and of white cells and that any variation in the normal activity of the marrow will result in the overloading of the blood with cells of a particular type depending upon the kind and extent of the affection. The histology of the other blood-forming organs, as the liver, spleen, and hemolymph nodes mQf&M^j^i^MfiOmiook on histology. 528 DIAGNOSTIC SIETHODS IV. Pathology of the Blood. (I) Special Pathology. Under the head of the special pathology of the blood we have to consider the conditions which are manifested directly by changes in the composition and cellular structure of this tissue. In very few of the blood diseases proper is the blood picture so characteristic that a definite diagnosis is always possi- ble, but in a few of them certain changes are more frequently found and more often lead to a presumptive diagnosis. The pathological conditions in the blood may be considered either primary or secondary, but it should be re- membered that severe secondary changes may so closely simulate those found in primary conditions that differentiation is almost impossible. It is probably true that all pathological changes of the blood are really secondary, but in a certain number of these states the etiologic factors are so obscure that we can do no more than interpret the blood findings as primary conditions. (A) Anemia. This is a condition characterized by a deterioration, both qualitatively and quantitatively, in one or in aU of the bloo'd constituents. It is usually char- acterized by a diminution in the percentage of hemoglobin (oligochromemia) and by a decrease in the number of red cells (oligocythemia), but we should regard as essential factors in the anemic condition a reduction in the total volume of blood (oligemia) as well as a reduction in the amount of protein (hypalbuminosis) . As much more attention has been paid to the first two factors than to the latter ones, anemia has come to mean a reduction in the amount of hemoglobin with a more or less extensive reduction in the number of red cells. Associated with these conditions we have, in the severer types of anemia, variations in form, size, and structure of the red cells as well as def- inite changes in the relationship of the different white cells. In the study of the anemic conditions we must differentiate the primary from the secondary form, by this we mean a differentiation between those forms which have no demonstrable cause from those types whose etiology is more or less secondary to other pathologic conditions. Primary Anemias. (1) Simple Primary Anemia. This form which has no demonstrable cause is difficultly separable from the secondary form as well as from certain other primary forms, such as pri- mary pernicious anemia. It will probably be shown to be a true secondary anemia and must he sharply differentiated from the pernicious type from the point of prognosis. These cases are only recognizable when they are typical in form and are frequently not amenable to any form of treatment which we may institute. The rule, however, is that these forms of anemia yield more or less promptly to proper dietetic and therapeutic treatment. It is probable that the question of prognosis in these cases must depend largely upon the amount of degeneration of the red cells which occurs.^ In these cases we find 1 See PoUitzer (Ztschr. f. klin. Med., 1912, LXXV, 367) for a discussion of the types of regeneration and degeneration in anemia; also, Wichern and Piotrowski, Deutsch. Arch f klin. Med., 1912, CVI, 533; 'Di^ze3>b^sM^Cfi0^ft(Bdm. Med., 1914, CXVI, 535. ' Digitized by Microsoft® PLATE XXm. \^ ^*^ 'bS'ih S,M ,^6^° o<"^;^ ^p^„^ v^ ^^e Q^ Chlorotic Anemia. (Wright's Stain.) Digitized by Microsoft® THE BLOOD 529 that the diminution in the number of red cells is usually parallel to the re- duction in the amount of hemoglobin, so that a high color index will usually obtain. More or less degeneration, as evidenced by the appearance of poiki- locytes, normoblasts, Maragliano's polychromatophilia, etc., will be observed depending upon the severity of the case. The leucocytes are usually normal in number and in differential relations, while the blood-plates are usually in- creased. The changes in the plasma in this primary anemia are not char- acteristic, although we do observe a diminution in the specific gravity which runs parallel to the oligochromemia. (2) Chlorosis. Chlorosis is a primary anemia occurring almost exclusively in girls about the age of puberty and characterized by a marked reduction in the amount of hemoglobin and a slight change in the number of red cells. Clinically, this state is evidenced by the appearance of a wax-like changing into a greenish tone of the skin and a sky-blue coloration of the cornea. Some cases may show a variety of colors of the skin. Certain changes are observed in the digestive and generative organs and certain general abnormalities are seen which are due to lessened production of blood-cells and to diminished oxida- tive and fermentative powers of the system. This form of primary anemia differs from all other forms in the absence of blood degeneration, as very rarely marked degenerative signs appear in the blood picture. The blood finding is not absolutely characteristic for this clinical entity, as it is stimu- lated by many anemias of the secondary type. Clinically, this disease is so sharp that a diagnosis is often possible without a blood examination. The chief characteristics of the blood in this condition are: (i) Reduction in the hemoglobin. This may run as low as 20 per cent., giving a color index of 0.5. Secondary anemias rarely reach such a low level, (i) Variations in the number and size of the red cells. The number of red cells is not reduced to a very great extent, the average being about 4,000,000, although counts as low as 1,000,000 have been reported. When these low counts do occur some complication should be suspected. Ordinarily the size of the cell is dimiinished, although we frequently find large "dropsical" cells which are due to absorption of fluid from the hypotonic plasma. These latter cells are usually few in number, the great majority of cells being smaller than the normal size. Poikilocytes and degenerated reds rarely occur except in the severer forms of this disease, while chromatophilia is usually regarded as a sign of active re- generation of the blood. When nucleated reds occur, which is a rare find- ing, they are practically always of the normoblastic type and rarely appear of the megaloblastic form. The leucocytes in this condition are usually normal both in size and number and degenerative forms are rarely seen. It has been stated that the eosinophile cells are much increased in this condition, but the writer has found that their ratio is very rarely above the upper limit of the normal figure for these cells. The platelets are usually about normal and are usually large in size. In chlorosis we find cer.tain variations in theDhysical and chemical prop- 530 DIAGNOSTIC METHODS erties of the blood. The specific gravity is usually reduced in proportion to the reduction of the hemoglobin and may fall as low as 1028. The plasma is usually much diluted so that a condition of oligemia may be considered more or less characteristic of chlorosis. Whether or not a hydremia, as indirectly manifested by a diminution in the amount of albumin, obtains, is debatable. Chlorosis belongs to the class of primary anemias and as such has no definite etiology. Various conditions such as hypoplasia of the arterial system, intestinal autointoxication, disturbances of the nervous system, such as a vasomotor neurosis, have been advanced to explain this condition, but none of them are tenable in all cases. A great importance must attach to hygienic conditions, poor food, and mental depression, especially about the age of puberty, as this disease is usually apparent under these conditions at this time of life. A further factor which must necessarily bear upon the etiology of this condition is the defective power of absorption of iron com- pounds. Patients afiiicted with chlorosis improve rapidly under the ad- ministration of iron, but not unless the digestive and absorptive powers im- prove at the same time. It does not concern us here as to the dynamics of the absorption and the effect of iron, but it should be accepted an as axiom that no therapeutic effects may be expected from iron unless the absorptive power is made better. Just exactly what the pathological conditions are which are accountable for the functional insuflS.ciency of the bone-marrow is uncertain, but it must be recalled that gross as well as microscopic athologic changes are not necessary to produce functional disturbance in any organ. It should be remembered that we may have various types of chlorosis which show different prognostic characteristics: (i) Those in which the red cells are very slightly reduced (about 4,000,000), a marked diminution in the amount of hemoglobin, a low color index, and no change in the size and shape of the cell. Such cases usually recover promptly without showing any re- lapse. (2) Cases in which the red cells are below 4,000,000, which show a marked diminution in the amount of hemoglobin with a very low color index and which give very slight evidences of degenerative changes in the red cells. These cases are usually characterized by marked prostration, but usually re- cover more or less promptly although relapses frequently occur. (3) Cases in which the red dells are reduced as low as 2,500,000, a reduction in the hemoglobin with a very low color index, marked changes in the shape and size of the red cells. These cases respond slowly to treatment and have a bad prognosis (Ewing). As these cases of chlorosis convalesce, we observe an increase in the number of red cells to a point somewhat above the normal and a later increase in the hemoglobin content of each individual cell. These changes are usually evident in from eight to ten days after institution of treatment, but as a rule a much longer time is necessary for any marked change to be observed in the number of the cells or in their hemoglobin content. The changes in the plasma are usually the first to appear and should be considered essential for the proper regeneration of the blood in chlorotic conditions. This disease belongs in the group of curable conditions and usually has a Digitized by Microsoft® Digitized by Microsoft® PLATE XXIV. Blood in Pernicious Anemia. (Weight's Stain.) Digitized by Microsoft® THE BLOOD 53 I good prognosis, but the susceptibility to intercurrent acute diseases during this period is much increased so that we should always bear in mind the probability of complications arising in the convalescent period. (3) Progressive Pernicious Anemia (Biermer's Anemia). This term is applied to a form of severe anemia which, in spite of all treatment, progresses steadily toward death. It results from defective hema- togenesis and excessive hematolysis and is characterized by definite changes, both numerically and morphologically, in the red cells and by characteristic changes in the bone-marrow (Ewing). This condition has frequently been described as the result of infection with certain intestinal parasites so that it cannot in all cases be considered a truly primary disease, although the larger majority -of cases show no known etiology.^' According to Herter, certain anerobic bacteria, found in the large intestine, produce a substance of marked hemolytic power, which penetrates the intestinal wall and enters the portal circulation.^ In some cases of idiopathic purpura hemorrhagica the blood picture is that of pernicious anemia, but may be distinguished by the absence of megaloblastic change and by the prominence of hemorrhage. The blood picture in this disease is not absolutely characteristic, as certain forms of secondary anemia may show similar findings. The chief characteristics of the blood in primary pernicious anemia, as also in those severe types of secondary anemia which simulate this form, are (i) signs of rapid blood de- struction, such as degenerated reds, endoglobular degenerations, polychroma- tophilia, increased iron compounds in the serum and corpuscles, an increase of iron in the liver and spleen (2) poikilocytosis; (3) a high color index result- ing from a marked diminution in the number of red cells and a correspondingly less degree of diminution in the amount of hemoglobin; (4) megaloblastic blood formation. This latter indicates a direct reversion to the embryonal type of blood formation in which the presence of megaloblasts as direct pre- cursors of megalocytes is observed.^ The red blood-cells in this condition are few in number being reduced to as low as 1,000,000 cells, counts of 500,000 having been observed without the patient suffering any marked in- convenience. Naegeli reports a case showing 138,000 reds. This fact should be taken as evidence that the oligocythemia in pernicious anemia is not alone accountable for the symptomatology. This count may remain stationary, may show slight decrease, but usually progresses slowly until death ensues. The average diameter of the red cells is somewhat increased in pernicious anemia. While many of them may be normal in size and many very small, the cells average from 4 to 13 microns in diameter. A pernicious anemia is a distinct large cell anemia, A macrocy tosis is much more character- istic of this disease than of any other, 70 per cent, of the cells in these cases being of this type.^ Microcy tosis is rare but may occur to such an extent iSee Friedstein, Inaug. Dissert, Berlin, i9i2;Ludke and Fejes, Deutsch. Arch. f. klin. Med., 1913. CIX, 433; also Pilcher, Am. Jour. Med. Sc, 1913, CXLVI, 226; Ragosa, Folia Hiemat., 191S, XIX, 269. 2 See Banti, Semaine m6d., 1913, XXXIII, 313; also, Robertson, Jour. Biol. Chem., 1913, XVI, 652. 3 See Brosamlen, Deutsch. Arch. f. klin. Med., 1913, CXII, 83; Vogel and McCurdy, n:e°DuS,B\ri:k^S'«».li»a#^'' '^^^ ^^^^^^ '''■ 532 DIAGNOSTIC METHODS that the average size of the red cell may be about normal. Poikilocytes are very common and often show extreme shapes and are frequently numerous in number. Polychromatophilic degeneration is very extensive in this form of anemia. While both normoblasts and megaloblasts occur in large numbers in per- nicious anemia, the megaloblasts usually outnumber the normoblasts. This megaloblastic increase may be considered pathognomonic of this anemia, as a preponderance of these large nucleated red cells does not occur, except in rare cases, in the other varieties of anemia. The hemoglobin may be markedly reduced showing values rarely above 50 per cent, and often as low as 10 per cent. The color index is always high; being more frequently above one than below. A low index is found, according to Ewing, in the chronic c^ses, while the acute forms are more frequently associated with a high index. If im- provement occurs the index is always lowered, an increasing index denoting a bad prognosis. In this pernicious form of anemia we find the leucocytes practically al- ways diminished in number, averaging about 4,000, a condition which almost never obtains in a secondary anemia. Their number usually runs parallel to that of the red cells, a leucocytosis pointing probably to a complication. As the case improves the neutrophile cells increase in number, the low leucocyte count being, as a rule, due to their diminution. The percentage of the non- granular mononuclear cells varies inversely to that of the granular form. As the disease progresses the percentage of the nongranular cells increases, while the granular cells are diminished. This disease shows, therefore, a high lym- phocyte count which is relative and not real, being due to the diminution in number of the polymorphonuclear cells. The lymphocytes may constitute as high as 50 per cent, of the leucocytes, the eosinophiles may reach as high as 10 per cent., the myelocytes 2 per cent., while the mast cells may run as high as 3 per cent. Degenerations of all kinds are observed in the leucocytes, but nothing characteristic of pernicious anemia is found in the white cells. ^ The blood-platelets are largely decreased and may be as low as one- twenty-fifth of the normal number. Von Limbeck and Sahli claim that these cells are increased in number, but the usual finding is one of diminution. (4) Splenic Anemia. This is a form of chronic anemia characterized by idiopathic enlargement of the spleen without any involvement of the lymph nodes. A large number of conditions may be responsible for the anemia of the splenic type, so that a direct etiologic factor should be looked for in all cases. Among the conditions which may give rise to this type of anemia, we find gummata of the spleen, large round-cell sarcoma of the spleen, chronic splenitis of the malarial type, ' See Nicol, Deutsch. Arch. f. klin. Med., 1913, CXI, 417; Sisto, Policlinico, 1913, XX, 509; Ibid., 1914, XXI, 34, 91 and 115; "Wolff, Deutsch. Med. Wchnschr., 1914, XL, 643; Cederberg, Berl. klin. Wchnschr., 1914, LI, 585; Mikhailoff, Russk. Vrach., 1914, XIII, 837; Cavaglieri, Gazz. d. osp., 1914, XXXV, 1169; Schmidt, Am. Jour. Med. Sc, 1914, CXLVIII, 313; Briggs, Ibid., 413; Moffitt, Ibid., 817; Krumbhaar, Ibid., 1915, CL, 227; , Drinker and Hurwitz, Arch. Int. Med. 191S1XV, 733. Digitized by microsoft® THE BLOOD 533 and splenomegaly associated with cirrhosis of the liver. This latter con- dition is known as Banti's disease and its etiology is uncertain.^ While it is true that the splenic lesions do not differ essentially from certain stages of the lesions in ordinary pseudoleukemia, yet we do not find in this condition any involvement of the lymph-glands, although the spleen may be enormously enlarged. The blood picture in this condition is character- ized by a relatively high red cell count, a marked reduction in the percentage of hemoglobin, and a consequent low color index. The white cells are rarely increased, a leucopenia being rather the rule. When we do find an increase in the number of white cells a relative lymphocytosisoccurs usually associated with an increase in the number of basophiles. Poikilocytes and nucleated red cells are very uncommon while degeneration may occasionally be observed. Whether or not we have an increase or a decrease in the number of leucocytes, a relative lymphocytosis associated with enlarged spleen and no involve- ment of the lymph nodes must be considered characteristic of splenic anemia. In Banti's disease we find an enormous increase in the size of the spleen and associated with this an extensive cirrhosis of the liver. Along with these factors we find a high-grade toxogenic protein decomposition, which is, associated with very high values for the total nitrogen of the urine and of the output of purin bases. The number of erythrocytes diminishes correspond- ing to the degree of anemia, while the hemoglobin percentage is more markedly reduced. The leucocytes are either normal or more frequently diminished in number, a relative lymphocytosis existing as in the pure type of splenic anemia. 2 (s) Anemia Infantum Pseudoleukemica. Von Jaksch has described a rare form of anemia seen in children which is characterized by enlargement of the spleen, liver, and lymph nodes. The most striking points in this condition are the great diminution in the number of red cells, one case showing only 820,000; numerous nucleated red cells; diminution of hemoglobin; the leucocytes always increased in number, being from 20,000 to 50,000, as a rule, and displaying a remarkable variety of form and frequently attaining to unusual size. The morphological changes in the blood resemble both those seen in leukemia and in pernicious anemia, the dis- ease passing either into one or the other of the previously mentioned con- ditions. The blood findings are not alone sufficient to warrant a diagnosis of infantile pseudoleukemia, but are significant when taken in conjunction with the clinical findings. ' See Banti, Berlin, klin. Wchnschr., 1911, XL VIII, 2328; Stein, Am. Jour. Med. Sc, 1912, CXLIV, 856; also, Umber, MUnch. med. Wchnschr., i9i2,LIX, 1478; Lacouture, Dup6ri6, and Charbonnel, Jour, de m6d. de Bordeaux, 1913, LXXXIV, 805; Caronia, Pediatria, 1914, XXII, 752; Sailer, Penn. Med. Jour., 1914, XVIII, 91; Krull, Mitt. a. d Grenzgeb. d. Med. u. Chir., 1914, XXVIII, 718. ^ Yaties, Bunting and Kristjanson (Jour. Am. Med. Assn., 1914, LXIII, 2225) report the isolation of pure cultures of Bacillus hodgkini (see Chap. XI) in two cases of Banti's disease. They believe there is an etiologic relationship here. See, also, Gibbons (Quart. Jour. Med.,. 1914, VII, 153), who reports ^^^gig^^y)^yg^gjj^x in six cases. 534 DIAGNOSTIC METHODS (6) Leukanemia. This condition has been classed by some with the leukeniias as the blood findings are occasionally more prominent among the leucocytes, while by others it is classed with pernicious anemia owing to the frequent changes in the red cells. Von Leube considers this anemia a mixed form of pernicious anemia and of leukemia. Luce^ regards this as a symptom of many conditions rather than an independent blood disease. In the majority of these cases the changes are more evident in the red cells, marked diminution in number (as low as 200,000 per cmm.) associated with extensive destruction of red cells with all irregular and unusual types of these cells being observed. The dim- inution in the amount of hemoglobin is great, but the color index is usually high just as we find it in pernicious anemia. The white cells show an exten- sive disturbance in the neutrophile and eosinophile blood picture with a large increase in the number of large lymphocytes. The number of white cells is usually increased, but not to as great an extent as in leukemia. The changes in the red cells and in the hemoglobin usually precede those in the white cells, so that the early stages may show us a typical picture of pernicious anemia, while later-^oaininations may lead to the diagnosis of the mixed condition. (7) Aplastic Ane^oia. Ehrlich has reported a rapidly progressing anemia accompanied by hemorrhages into the mucous membranes, associated with hyperplasia of the bone-marrow, and not showing the ordinary changes in the blood which are supposed to accompany pernicious anemia. This type has been called aplastic anemia and has not been frequently reported. The red cells are usually markedly reduced in number, being as low as 790,000 in a, case reported by Wood. The hemoglobin may fall as low as II per cent, as reported by Muir, while the leucocytes are usually normal in number but showing much reduced percentages of the polynuclear neutro- philes. In this condition we find an enormous increase in the number of lymphocytes, the percentage being in Lipowski's case 93, the remaining 7 per cent, being neutrophiles. No nucleated reds have been found in the blood and only a very few in the marrow itself, which is fatty, almost white and contains few neutrophiles and no eosinophiles.^ Secondary Anemia. By secondary anemia we mean one in which etiological factors seem sufficient to explain the variations observed in the blood. The principal variation seems to be more directly observed in the reduction of the hemo- globin, although the number of red cells is coincidently reduced, but not to the same degree as is the hemoglobin. In the mild cases the color of the blood is but slightly paler than normal, but in secondary anemia of a severe type the color may resemble the watery drop observed in cases of typical pernicious anemia. ^Deutsch. Archiv. f. klin. Med., 1900, LXXVII, 215. See, also, Martelli, Virchow's Arch., 1914, CCXVI, 224; Gazz. internal, di med., 1914, XVII, 553 and 577. 2 See Hirschfeld, Folia Haemat., 1911, XII, 347; Musser, Arch. Int. Med., 1914, XIV, 27s; Kleinschmidt, Jahrb. f. Wf^ti^^^^'Mcfd^§Jf®^- PLATE XXV. Blood in Leukanemia. (Wright's Stain.) Digitized by Microsoft® Digitized by Microsoft® THE BLOOD • 535 Cabot suggests a classification of the secondary anemias as follows: (i) Mild cases, showing a normal count of red cells, but having the hemo- globin diminished. (2) Moderate cases in which the count is normal but the cells show signs of moderate degeneration, abnormal staining qualities, and a diminished tendency to rouleaux formation. (3) Severe cases, in which the count is not much reduced but in which the hemoglobin is very much lessened and the cells show marked qualitative and quantitative changes. (4) Very severe cases with a slightly lessened blood count, a marked diminution of the hemoglobin, and evidences of degeneration and destruction of the cells as well as evidence of regeneration. In some of the severer types of secondary ane- mia the blood picture may so closely resemble that of pernicious anemia that a diagnosis is possible only through the careful investigation of the etiology of the condition. Blood Picture. The general points observed in secondary anemia are as follows : A vari- able decrease in the amount of hemoglobin, a reduction in the number of red cells less in degree than in the diminution of hemoglobin and a subnormal color index, which rarely reaches the low grade shown in chlorosis. The lowest color index is seen in those secondary anemias following cancer, severe hemorrhage, and gangrenous processes. The specific gravity of the blood is reduced corresponding to the degree of reduction of hemoglobin. The rapid- ity of coagulation is increased depending upon the grade of oligochromemia and of oligocythemia. The reduction in the number of red cells may be very marked as, for instance, in a case of von Limbeck the number was 306,000. The red cells show a lack of hemoglobin, frequently appearing as the pessary forms. Polychromatophilia is quite common, but bears no direct relation to the hemoglobin content of the cell. Only in the severer cases do we find poikilocytes, although anisocytes, especially microcytes, are frequently pres- ent, the larger "dropsical" cells being less common than in chlorosis. Nu- cleated erythrocytes are frequently seen in some cases, while in others even of severer grade of anemia they are absent. These nucleated reds are, as a rule, of the normoblastic variety, megaloblasts being exceedingly rare. The leucocytes vary in number depending on the cause of the anemia, from a leucopenia, which is rare, to a leukemic condition. The increase in the number of leucocytes is more frequently in the polynuclear neutrophiles, the lymphocytes being rarely if ever increased, while the eosinophiles, although not increased, are usually at the upper limit of the normal value for these cells. In some cases of the severe chronic types of secondary anemia we may find a lymphocytosis, but this is rare. The blood-platelets are usually increased in number, in some cases being two and one-half times the normal values. (i) Acute Hemorrhage. The character of the anemia following an acute hemorrhage will depend upon the type of the hemorrhage, that is whether the loss of blood occurred suddenly and at one period, or whether slowly and at intervals.^ The loss of 1 See Milne, Jour. Exper. Med., 1912, XVI, 325; Deutsch. Arch. f. klin. Med., igi-j CIX, 401. Digitized by Microsoft® 536 DIAGNOSTIC METHODS one-half of the total volume of blood at one time is usually fatal, as Panum has shown. If the loss be less following one large hemorrhage, regeneration takes place in from five to thirty days, depending upon the amount of blood lost. Regeneration is quickest in men between the ages of 20 and 40, slower in women, and slowest in children. Immediately following a hemorrhage the blood picture will be normal qualitatively as there has been no time for the morphologic changes to take place. Shortly following the hemorrhage lymph pours into the blood to re- store the volume and maintain the pressure, so that the blood count and the hemoglobin diminish usually to the same degree. As the formation of new cells goes on the color index decreases, as the new cells are, as a rule, deficient in hemoglobin. These cells are more or less easily degenerated, showing varia- tion both in their shape and staining qualities. The number of cells reaches the normal much sooner than does the percentage of hemoglobin, so that we may find for weeks evidences of marked anemia. The most frequent causes of acute hemorrhagic anemia are traumatism, ectopic pregnancy, abortion, gastric and duodenal ulcers, uterine tumors, pul- monary tuberculosis, and hemorrhagic pancreatitis. (2) Chronic Hemorrhage. By a chronic hemorrhage we mean one in which repeated hemorrhages follow one another so closely that the blood has no time to regenerate before a second loss of blood occurs. This rules out of consideration those cases in which repeated hemorrhages occur but at sufficiently long intervals to permit of regeneration of the blood. This latter condition, although a chronic hemorrhage, gives the picture described under Acute Hemorrhage. In the chronic hemorrhage we find the red cells markedly reduced, the hemoglobin very much diminished and usually a marked leucocytosis. The red cells are usually small and pale, show a low color index, and usually few nucleated forms, although the picture may rarely assume the perncious type. Regeneration in this form of anernia is slow, as the blood-forming organs seem to lose their power of regenerating the blood after repeated hemorrhages. The most frequent causes of chronic hemorrhage are scurvy, epistaxis, hemorrhoids, intestinal ulcers, gastric and other carcinomata, and intestinal parasites. (3) Inanition. In the discussion of the anemia of inanition, it must be remembered that other factors than starvation are necessary in its causation. That starvation alone will not cause anemia is shown from the examination of the blood of Cetti who fasted ten days. His blood showed about 6,000,000 red cells, a small diminution in the percentage of hemoglobin, and a leucocyte count of 4,200. Although the changes in the cells and the pigment of the blood are not marked following starvation, it is true that we have a loss of albumin of the plasma and a diminution in the total volume of blood. This is taken as evidence by Grawitz that a true anemia occurs. Such changes are not strik- ing if the days of fasting are alternated with days of slight nourishment. It is not so much the quantill^'gBf/ieflHbjirsMIcrQpjrf^ which is of importance in THE BLOOD 537 bringing about the anemic conditions. As is well known, the foods containing iron are the principal sources of the hemoglobin of the blood, and these are frequently, owing to disturbed gastric and intestinal functions, poorly digested and assimilated. Although the amount' of iron contained in the ordinary food is sufficient under the best conditions to maintain the hemoglobin con- tent of the blood, yet the methods of food preparation, as well as the abnor- mal methods of rapid eating, are important factors in the poor assimilation of the iron of the food. The lack of sunlight as well as impure air are contributory factors in causing anemia through their influence upon the general body functions. No tissue, whether animal or plaiit, can flourish in air which does not have sufficient oxygen to support the combustion processes of the system. This statement needs no retraction in the case of anaerobic bacteria, as it has been definitely shown that these organisms obtain the oxygen necessary for their development from the culture media upon which they grow, although they are ' incapable of developing in an atmosphere of pure oxygen. Overwork, espe- cially when associated with worry, has great influence upon all of the func- tions of the system. For this reason overwork has been credited with the power of producing anemia as well as many other serious systemic disturb- ances. However, it is rare to find an authentic case of anemia which can be traced directly to overwork without the mental influence of worry and the coincident nervous strain from this latter cause. ^ (4) Intestinal Parasites. The anemia caused by intestinal parasites may be of such a severe grade as to resemble very closely the type of pernicious anemia. In many cases it is an impossibility to make a differential diagnosis between these types with- out the finding of an intestinal parasite which will clear up the diagnosis. In these cases the blood picture returns more or less quickly to normal after re- moval of the parasite in question, while in the pernicious anemia of unknown origin the progress of the disease is always toward a fatal termination. It is probable that the cause of the severe secondary anemia due to the presence of the intestinal parasite is a result of the toxic condition set up by the ab- sorption of the hemolytic toxins elaborated by the parasite. A very severe anemia of the secondary type is frequently seen as a result of decomposition of the intestinal contents and in cases of chronic constipation in which the direct toxic agent is at present unlinown. The most common intestinal para- sites causing the severe types of Secondary anemia are (i) uncinaria duode- nale, (2} strongyloides intestinalis, and (3) bothriocephalus latus. The first of these causes an anemia which is very closely related to that shown by miners and tunnel diggers, and seems to be much more prevalent in the southern part of the United States, although it occurs in many different countries. The blood count may fall below one million red cells and the hemoglobin may be as low as 15 per cent., while all varieties of degenera- tive changes may be seen in the erythrocytes. In the anemia following infection with the bothriocephalus we find very marked similarity with the 1 See Ash, Arch. Int. MedOjpfe^lg^M/crOSO/?® 538 DIAGNOSTIC METHODS primary pernicious type. One-half to two-thirds of the nucleated reds in this variety may be of the megaloblastic type and yet may disappear within two to three weeks after the worm has been expelled. It is uncommon to find in secondary anemias caused by the parasites above mentioned any marked eosinophilia, which is so common in cases of infection with many of the other forms of intestinal parasites. The anemia of the severer type seems to prevent a chemo taxis toward eosinophile cells. (5) Fever. It is still very much of a question whether the blood in febrile cases shows the characteristics of a secondary anemia, as the result of the temperature increase. It has been shown that increased temperature, in itself, does not always produce anemia, although we do have marked destruction of the red cells and a coincident loss in the amount of hemoglobin of the remaining cells. So great is the influence of the toxin of the febrile condition that it is highly probable that the anemia so frequently observed in febrile cases is due to a combination of causes, rather than to a specific effect of the increased temperature. The changes in the white cells in febrile cases are not always of the same character, the variations being dependent upon the specific causative factor of the fever. As the blood changes in the acute infectious fevers are of more or less importance, they wiU be discussed later under separate headings. The anemias which are secondary to both the acute and chronic infections are probably directly due to the influences of the toxins upon the blood and blood-forming organs. The condition of general nutrition as well as the state of digestion, especially in the chronic states, such as tuberculosis, leprosy, and syphilis, must be regarded as important factors in the causa- tion of these secondary types. (6) Blood Poisons. There are a very large number of compounds which produce, when taken in toxic doses, very marked changes in the qualitative and quantitative com- position of the blood. As is well known, iron compounds in therapeutic doses increase the amount of hemoglobin in the red cells and also increase the num- ber of red cells up to a certain point. Many of the effects which are attrib- buted to iron compounds may be due to the improved hygienic and dietetic conditions which usually prevail during the administration of these sub- stances. Yet the therapeutic results following the administration of iron are such as to make it certain that a specific influence of this drug is present in anemic conditions, especially of the chlorotic type. Many compounds produce a very marked secondary anemia, the most important of these being alcohol, opium, lead compounds, cocain, and ace- tanilid. While others, such as arsenic, nitrobenzol, nitroglycerin, phenacetin, and poisonous mushrooms, cause dissolution of the red cells with marked hemoglobinemia. The anemia following the use of lead, either in toxic doses or after its slow absorption from constant contact with it in the arts, is of great practical importance. The causes of this lead anemia are rather com- plex. The lead compoui^s have^^dijj^a.acti^^on the red cells and on the THE BLOOD 539 blood-forming organs as well as upon the gastrointestinal tract and the elimi- native organs. While the anemia shows no especial characteristics as regards the number of red cells and the amount of hemoglobin^ yet the peculiar granu- lar degeneration and the polychromatrophilia are sufficient to differentiate this type from most of the other secondary anemias. The basophilic degen- erations of the reds is more marked in lead anemia than in almost any other condition and usually runs parallel to the severity of the clinical symptoms of the case. In arsenical poisoning also we occasionally find a slight amount of granular degeneration of the red cells, but the hemoglobinemia in this latter condition will differentiate it from lead anemia. (B) Leukemia. Although acute forms occur, leukemia may be regarded as an essentially chronic condition which is characterized on the one hand by definite changes in the lymphatic and myeloid tissues of the body, and, on the other hand, by certain peculiar changes in the number and relations of the various cellular constituents of the blood. These latter conditions must be regarded as purely symptomatic of the preceding states and not as the direct pathological condi- tion in themselves.^ Leukemia has been classed as a primary anemia, al- though the changes in the blood are here more directly related to variations in the white cells than to characteristic changes in the red corpuscles; yet we do find a diminished red count as well as a diminution in the amount of hemo- globin in this condition. It is a disease marked by the constant presence in the blood of granular mononuclear or polynuclear cells, or an increase of the nongranular cells with round nuclei. While the leucocyte count is almost invariably increased to a marked extent, we find cases in which the number of cells is normal, but we find great deviations from the normal relations of these white cells. While the tendency is becoming more and more general to regard this condition as a single entity, manifested by various blood pictures, yet we find the cells grouping themselves together in such definite ways that we are justi- fied in dividing leukemia into three general types, with transitions from one to the other form.^ These types are (i) splenomyelogenous leukemia or "myelemia," (2) lymphatic leukemia or "lymphemia," (3) mixed leukemia. Each of these types shows a distinct blood picture which permits of the classi- fication of the condition studied. (i) Splenomyelogenous Leukemia. This condition was formerly subdivided into two distinct types— the true splenic and the myeloid leukemia. However, practically none of the cases ' It is probable that leukemia, both of the chronic and acute types, is of infectious origin. Streptococci and the bacillus hodgkini have both been isolated from these cases. See Steele, Boston Med. and Surg. Jour., 1914, CLXX, 123; Simon and Judd, Jour. Am. Med. Assn., 1915, LXIV, 1630; Wilbur, Ibid., 1915, LXV, 1255. ^ It is probably true that a classification of these types on purely morphological bases is futile. See von Bqmbard, Ztschr. f. klin. Med., i9i4,LXXX, 506; Emden and Rothschild, Deutsch. Arch, f . klin. Med., 1914, CXV, 304; Panton, Tidy and Pearson, Quart. Jour. Med., 1914, VII, 304; Rummo, Rif. Med., 1914, XXX, 897 and 926; Castellino, Ferrata and Rummo, Gazz. d. osp., 1914, XXXV, 1813; Rotky, Zentralbl. f. inn. Med., 1914, XXXV, 953; Martelli, Rif. Med., ^9^Bi^^d1^'IW?)t0^^mW^ '324- 54° DIAGNOSTIC METHODS reported could be definitely classed under either one of these headings, as the blood picture was always referable to disturbance in both the spleen and marrow. This type of. leukemia is characterized by a marked increase in all of the granular cells, especially of the neutrophile, eosinophile, and basophile types, while the nongranular cells are not so characteristically increased. Gross Appearance. The gross appearance of the blood is normal even though the leucocytes are increased to an enormous extent. In extreme cases it may appear pale and opaque and does not flow from a puncture as readily as normal blood. In making smears of such blood, the preparations appear granular and are not readily spread so that the future examination is rendered somewhat difficult. Red Cells. As a rule, these cells are diminished in number, but the oligocythemia is of a mild degree, the average count being about 3,000,000 although it may run as low as 1,500,000. As a general rule, the red cells diminish in proportion to the increase in the number of white cells. Occasionally we find cases in which an oligocythemia persists with a normal or slightly increased leucocyte count. Such a condition might lead to the diagnosis of pernicious anemia, unless the differential count was carefully studied. The red cells are usually pale and of the chlorotic variety. Very little degeneration of the red cells is observed, microcytes and macrocytes are rare, but a few poikilocytes are seen in prac- tically all cases. Polychromatophilia is more or less common and cells showing basophilic granulations appear with more or less frequency. Normo- blasts are very common in this condition, yet their absence does not rule out the diagnosis of leukemia. Megaloblasts and gigantoblasts are frequently seen and are sometimes many in number, although they rarely if ever exceed the normoblasts in number. Hemoglobin. The hemoglobin is reduced to a somewhat greater extent than is the num- ber of red cells, the color index being about 0.6 and the average percentage of hemoglobin about 40. Leucoc3rtes. In this condition we find the leucocytes increased, as a rule, to a very marked degree, counts running as high as 750,000 having been seen by the writer. Osier gives as his average for the white count 298,700, while the average may vary to a slight extent at different periods of the day. Some of the cases show a uniformly high count, others a moderate count and a few others a low count of about 100,000 cells. It is this increase in the number of white cells which gives the blood its peculiar opacity in this condition and may make a diagnosis possible by mere inspection. In some cases of leuke- mia we find a normal count of white cells while in others the count may be similar to that of a simple leucocytosis. It is the differential count in com- bination with the large increase in cells which should be considered charac- teristic, rather than a ^^^^^tWbfMcPSlh® PLATE XX\'I. Blood in Spleno-myelogenous Leukemia. (Tri-acid Stain.) Digitized by Microsoft® Digitized by Microsoft® THE BLOOD 54^ Differential Count. Neutrophile Myelocytes. These cells are large mononuclear cells with neutrophile granules. They are present in large numbers, averaging about 35 per cent, of all the leucocytes present. A diagnosis of leukemia is almost always possible when we have such an extreme neutrophile myelocytosis along with an extreme leucocyto- sis. These myelocytes appear in two forms: (i) the large myelocytes of Cornil, which may be as large as 30 microns in diameter and have a large, pale, eccentric nucleus which is poor in chromatin. These cells are seen only in splenomyelogenous leukemia and in some of the secondary leukemias of children and must be regarded as practically pathognomonic of this con- dition.^ (2) Small myelocytes about the size of the normal polynuclear leucocytes with a centric, round nucleus staining deeply with the various aniline dyes. We find all gradations between these large and small mye- locytes, sometimes observing a few which are about the size of the red cell. The granulations of these cells are sometimes numerous, but may be entirely lacking so that they may be indistinguishable from the large lymphocytes unless the pale quality of their nuclei is remembered. The degenerative changes in these myelocytes are few in number and are limited, as a rule, to the hydropic form usually seen in chlorosis. Poljmuclear Neutrophile Leucocytes. These cells are relatively diminished, their average, according to Cabot, being about 46 per cent., although an absolute increase is present, amounting to as much as 60,000 to 75,000 cells. Marked variations in the size of these cells are common, some of them being very large, some very small and no defi- nite relation existing between the numbers of the large and small cells. These variations in size of the polymorphonuclear cells are rarely if ever seen in ordinary leucocytosis. It is very common to find cells with irregularly shaped nuclei and with more than one form of granule, which may vary in tint depending on the method of fixation. Marked degenerative changes in these cells are very common. Thus we find their stickiness is increased, their nuclei usually pale and frequently showing karyokinetic figures. All grades of variations in the granulations may be observed, the granular cytoplasm being occasionally replaced by a homogeneous highly refractive material. Eosinophiles. The eosinophile cells are usually much increased in the splenomyeloge- nous form of leukemia, but their percentage relations to the other leucocytic forms are practically normal. Their number may run from 3,000 to 100,000 the average absolute number being about 12,000, while their percentage is about five. The total number of these cells per cmm. greatly exceeds that found in any other condition so that we accept, with Ehrlich, such an increase as pathognomonic of splenomyelogenous leukemia. These cells appear in all modifications, some of them being very small, while some of them are very ' See Klein, Deutsch. med. Wchnschr., 1Q13, XXXIX, 2513. Digitized by Microsoft® 542 DIAGNOSTIC METHODS large. The eosinophile form of myelocyte occurs in large numbers, but never is as numerous as is the neutrophile myelocyte. We occasionally observe all forms of transition between the myelocyte and the eosinophile leucocyte, the eosinophile myelocytes occasionally forming the majority of the eosinophile cells. The granulations of these cells may be of uniform size and staining quality or there may be some basophile granulations among the eosinophiles while the granulations themselves may vary greatly in size. Ewing considers eosinophile myelocytes with granules of unequal size and density of stain as pathognomonic of myelocythemia. Basophiles. According to Ehrlich, we always find an increase in the number of mast cells in leukemia, there absolute increase being in some cases greater than that of the eosinophiles and is always proportionately higher. This in- crease is so marked as to constitute a very reliable diagnostic feature of the blood. The number of basophiles may run as high as 140,000 (Taylor), while the percentage may vary from 5 to 47. Lymphocytes. The number and proportions of lymphocytes in the splenomyelogenous leukemia vary in different cases and at different times in the same case. As a rule, their percentage is reduced averaging about 10, while an absolute in- crease is usually present, this increase having no uniform relationship to the stage or character of the disease. These cells vary much in size, the large cells usually outnumbering the small ones. Large mononuclear cells with very faint cytoreticulum and vesicular nucleus occur in large numbers in this form of leukemia and seem to have no special significance, although they may be mistaken for the large lymphocyte or for the myelocyte. De- generative changes are observed in both the small and large lymphocytes in leukemia; thus the nuclei of the small cells may become incurved and bilobed or even trilobed, while the cell body remains basophilic (Rieder) . Points in Diagnosis. An excessive leucocytosis, with a large proportion of neutrophile mye- locytes, the presence of a large number of eosinophile myelocytes and of basophile cells, the presence of atypical cells, both of the mononuclear and polynuclear variety, and large numbers of nucleated red cells are the chief characteristics. Any one of these points may fail for a time, but will usually be evident at some stage of the disease. The large size of the myelocyte is much more characteristic than the mere presence of these cells, so that we should confine our diagnosis to cases which show irregularity in size, stain- ing qualities, and degenerative reactions of these cells rather than to those showing merely an increase. The presence of the large number of eosino- phUes, especially those showing granules of irregular size and staining quali- ties, is a very important point to be remembered in the diagnosis of this condition.' 1 See Dieballa and Entz., Foiia Hsmat., 1913, XV, 59; also, Ghon and Roman, Ibid., 72; and Milne, Jour. Am. Med. Assn., 1913, LX, 822. Goodall (Boston Med. and Surg. Jour., 1914, CLXX, 789) discusses ffl©fSHiegb^M«Sfflfi(HS®in this condition. Digitized by Microsoft® PLATE XXVIT. Lymphatic Leukemia. (Tri-acid Stain.) Digitized by Microsoft® THE BLOOD 543 (2) Lymphatic Leukemia (Lymphemia). In this form of leukemia we observe a marked increase in the number of mononuclear nongranular cells in distinction from the previous form of leukemia in which the increase is rather in the number of the granular types. While a variety of the mononuclear nongranular cells are present, there is usually observed a predominance of one particular form and size, in some cases the small mononuclear cell with a narrow ragged rim of protoplasm, in others the cells of the large lymphocyte type, and in others large cells whose protoplasm is basophilic or in some cases distinctly acidophilic. ' Red Blood-cells. In this form of leukemia we find a much greater anemia than in the splenomyelogenous form, although we may observe a normal red count for some time. The number of cells varies between 1,500,000 and 4,000,000, while the average percentage of hemoglobin is about 37 per cent. Nucleated red cells are rare in this condition, yet in the severer cases we may find them as numerous as in the splenomyelogenous type. All forms of degeneration noted under the previous type of leukemia are occasionally seen in this latter form. Leucocytes. The leucocytes are, as a rule, increased, the average being about 145,000, according to Osier. In this form we may find aleukemic periods which may last for a considerable period of time, the count usually rising just before death. Differential Count. According to Grawitz the cases of lymphatic leukemia may be divided into (i) those in which the increase of leucocytes is especially in the small mononuclear variety, (2) those showing an increase in the medium-sized cells with basophilic homogeneous protoplasm, and (3) those in which the cells which predominate are very large and usually degenerated. All these forms may occur together and may vary in the same case at different times. These mononuclear cells may constitute as high as 99 per cent. (Osier) of the total number of leucocytes. These leucocytes show in a very large number of cases much degeneration either of the protoplasm or of the nucleus, very few of the cells showing mitosis which is so common in the splenomyelogenous form. In this type of leukemia polymorphonuclear cells are rare, eosino- philes usually absent, and myelocytes and basophiles rarely if ever present. This type of leukemia is not easily amenable to diagnosis, especially in differ- entiating it from some cases of sarcoma in which the blood may show a simi- lar picture. In some cases we find a lymphatic leukemia with a considerable number of myelocytes both of the eosinophile and neutrophile type. This has led to the differentiation of a "mixed leukemia," which does not seem to be ad- visable as we may find myelocytes in the pure lymphatic type of this disease. (3) Acute Leukemia. This form of leucemia is characterized by its brief course (from six to eight weeks), by the severity of its symptoms, the frequency of the hemor- ^ See Nakamura, Deutsch. Ztschr. f. Chir., 1914, CXXXII, 275; Jackson and Smith, Boston Med. and Surg. .Toiir.,/5^^,/^g^^]/l^e^OSO/?® 544 DIAGNOSTIC METHODS rhagic diathesis, rapidly developing cachexia, and death. This condition occurs chiefly in young people and is usually of the lymphatic type,* although a few cases of the myelogenous variety have been reported (Billings and Capps).^ In all cases the anemia is extreme, the red cells usually running below 1,000,000 and the hemoglobin as low as 10 per cent. There is no type of cell which is characteristic of this form, although the cells are much more uniform in size than in the chronic states of this disease. In some cases nearly all of the cells have a basophile protoplasm, while in others they show acidophilic properties. Nucleated reds are usually rare, although they may be present in fairly large numbers. The drop in the count of red cells is usually sudden and denotes rapid blood destruction. The leucocyte picture resembles closely that of acute infections. (C) Pseudoleukemia. Under the heading Pseudoleukemia have been grouped a great variety of diseases, which have in some cases the external appearances of the disease, such as the glandular swelling, splenic tumor and progressive cachexia without, however, showing the blood picture which is so characteristic of leukemia. On the other hand, we find conditions which have little in common with the clinical findings of leukemia and yet show a blood picture similar in some respects to that of leukemia. It is difiicult to group all of these cases under one heading, as the blood picture is not characteristic for any one of these conditions. (i) Hodgkin's Disease. This condition, first described by Hodgkin in 1823, is characterized by chronically progressing cachexia with enlargement of the lymph-glands and spleen. It has been called, synonymously, lymphatic pseudoleukemia, lym- phosarcoma, malignant lymphoma, lymphatic anemia and aleukemic mye- losis.^ It is probably infectious in nature, the etiologic factor being probably the Bacillus hodgkini (see Chap. XI). The blood characteristics in this disease are more particularly those of a true cachexia, the red cells showing a diminution in the number which may be as low as 2,200,000, but which is usually between 3,000,000 and 4,000,000. The more severe and pro- nounced the signs of anemia and cachexia the lower the number of cells, the number of these cases running as low as 1,500,000. Morphologically, the red cells show much less deviation from the normal than in other severe anemias, the average size of the cell being usually normal, degenerations of the red cells appearing only in the very severe conditions, microcytes and macrocytes, as well as nucleated erythrocytes, being very unusual except in the late stages. The hemoglobin content runs parallel to the number of red cells, being the lowest in those cases showing very low counts. The leuco- cytes are slightly increased, averaging about 12,000. This failure of a leuco- 1 See Strauch (Am. Jour. Dis. Child., 1913, V, 43) for a discussion of this condition in children. Also, Beltz, Deutsch. Arch. f. klin. Med., 1913, CXIII, 116; Ballagi, New York Med. Jour., 1914, XCIX, 68; Yamakawa, Mitt. a. d. Med. Fak. Tokyo, 1914, XI, 115; Citron, Berl. klin. Wchnschr., 1914, LI, 332; Deutsch. med. Wchnschr., 1914, XL, 629. 2 See Beifeld, Arch. Diag., 1915, VIII, 244. ^ See Hirschfeld, Ztschr. f. klin. Med., 1914, LXXX, 126; Ghedini, Gazz. d. osp., 1914, XXXV, 2037; Ibid., 191S, ^W^Szediby^1\4iee0S§^®i'^a, Sem. Med., 1915, xxi, 57. THE BLOOD 545 cythemia enables us to differentiate Hodgkin's disease from a true lymphemia. The differential count of the leucocytes may show a relative lymphocytosis, the relation of the lymphocytes to the polynuclear cells being as three to one instead of the normal one to three. ^ Ehrlich and Pinkus consider this relative increase of lymphocytes characteristic of true pseudoleukemia in contradis- tinction to sarcomatous and other lymphomatous conditions. According to Grawitz, an increase in the leucocytes is associated with an unsatisfactory course of the disease, while a diminution in the number is observed as the disease progresses toward convalescence. Cases have been reported which would seem to point to the transition of Hodgkin's disease into a true leukemia, so that we may find irregular prog- ress of a pseudoleukemia as an evidence of a transitional stage. (2) Tuberculosis of the Lymph-glands. Why this condition has been classed as a pseudoleukemia is questionable, as the blood picture shows nothing beyond a secondary anemia with cachexia or may show even a normal red and white count. A large increase in the number of leucocytes, which is so characteristic of leukemia, is rarely seen, although a true leukemia may arise in the course of a glandular tuberculosis. The differentiation of this condition should be based upon examination of an excised gland, which wiU show distinct tuberculous lesions and usually will contain the tubercle bacilli in demonstrable numbers.^ In other cases we may find a simple lymphoid hyperplasia without any distinct inflam- matory changes and without demonstrable bacilli. Such nodes should be tested by inoculation experiments are advised by Ewing. Heredity plays a great r61e in the diagnosis of these tubercular conditions, while a scrofulous child should always be looked upon with suspicion. A splenic tumor appear- ing coincidently with the glandular swelling would speak rather against tuberculosis and in favor of a lymphatic pseudoleucemia. The diagnosis of this condition by examination of the blood alone is a practical impossibility. It is this tj^e of case that is especially amenable to diagnosis by the use of the various tuberculin tests. (3) Lymphosarcoma. The lymphosarcomata usually run their course either as primary benign lymphomata or as the maUgnant sarcomata. The blood findings in these conditions show nothing beyond a sUght anemia with nothing characteristic in the appearance of the white cells. The diagnosis must be based entirely on the examination of the excised gland or tumor. ^ (4) Gummatous Lymphoma. An exact diagnosis of this syphilitic swelling of the lymphatic glands is at the present time a matter of more or less difficulty. The previous history, "■ Bunting (Bull. Johns Hopkins Hosp., 1911, XXII, 114 and 369) considers an incraese in the number of transitional cells together with increase of blood platelets as quite char- acteristic of true Hodgkin's disease. See, also. Bunting, Bull. Johns Hopkins Hosp., 1914 XXV, 173 and 177; Yates, Ibid., 180. * Resort should be made to the antiformin method, if ordinary staining processes are not conclusive. 'See Oliver, Jour. Med. Research, 1913, XXIX, 191; Steiger, Berl. klin. Wchnschr. i9i3,L,^2i29. Digitized by Microsoft® 546 DIAGNOSTIC METHODS as well as other manifestations of syphilis, must be studied and a careful search made for the presence of the spirochaeta pallida. It is to be said that these organisms have been frequently reported in lymphatic enlargements which seem to have no direct relationship to purely syphilitic conditions . The interpretation of one's findings is of the utmost import as artefacts, which commonly appear in preparations of broken-down glandular tissue, resemble very closely the spirochsete. The blood condition shows nothing character- istic and can have only incidental diagnostic importance. Application of the Wassermann serum test might throw much light on the diagnosis. (II) General Pathology. (a) Blood Changes Following Surgical Intervention. Under this heading the writer will not attempt to take up all of the siugical conditions, as the vast majority are not associated with any direct hemato- logical characteristics. As a rule, it may be said that pus formation anywhere in the system will cause a leucocytosis. The degree of this leucocytosis averages about twice the normal standard, but may greatly exceed this figure in individual cases. It mUst be said, however, that trivial as well as extensive pus formations may be accompanied by normal or even subnormal values for the leucocytes. This is due to the facts that small pus foci do not cause any systemic reaction and extensive pus formation may overcome the power of the system to react against the infection. If the pus cavity is well encapsulated, the absorption of the toxin from this focus is necessarily limited so that we may find no leuco- cytosis, even though a very large pus cavity is present. Thus we find in localized peritonitis following appendicitis that a leucocyte count may be normal, but may suddenly increase to a marked extent as an indication of the rupture of the cavity and an extension of the process.^ A general rule is that a distinct increase in the number of cells in excess of the figure originally obtained is indicative of the extension of pus formation and should put the surgeon on his guard as to operative interference. If the absorption of toxic material from a focus of pus formation is great enough to produce a systemic effect upon a patient as manifested by a high leucocyte count, we find an anemia characterized by a marked diminution in the hemoglobin and the number of red cells, which is parallel in intensity to the severity of the poisoning. Cases are frequently found in which a low leucocyte count prevails, al- though clinical evidence of severe sepsis is at hand. In these cases a differ- ential count of the leucocytes should be made in all cases, as the low leucocyte count may throw one off his guard unless this precaution be taken. Should the polynuclear neutrophiles form 80 to 95 per cent, of the total leucocytes, a severe infection is indicated, even though the leucocyte count may be subnormal. If a leucocyte count does not diminish or even return to normal within one to two days after operation, this should be taken as evidence that a rein- fection has occurred or that the pus cavity has not been properly drained. iSee Friedman, Am. Jou£)^,?^^(5to5?)^/(^2(/^ 54°. THE BLOOD 547 Recourse must, therefore, be had to measures to overcome the secondary infection. It has been found that administration of ether and chloroform causes a leucocytosis which usually lasts from 24 hours to 48 hours and which may interfere with the interpretation of a blood examination. This point must be borne in mind in the examination of blood of cases which have shown high leucocyte values prior to operative procedure. The differential count in such cases, however, will show only slight variations in the proportions of the differ- ent types of cells so that one may judge as to the cause of the secondary leucocytosis by determination of the polynuclear cells. The number of red cells as also the amount of hemoglobin are very markedly reduced in some cases following the administration of an anesthetic, so that a direct secondary anemia maybe the result. This fact has led to the refusal by many surgeons to. resort to operative procedure in cases which show as low as 30 per cent, hemoglobin prior to operation.^ As the blood changes in surgical conditions, which are not accompanied by pus formation, are not especially characteristic and are more especially associated with the diseases of the special organs, the writer will refer such discussions to other headings. (6) Blood in Constitutional Diseases. (i) Diabetes Mellitus. In diabetes mellitus the changes in the cellular content of the blood are not very marked. The leucocytes may be subnormal, normal, or slightly in- creased, usually a very slight leucocytosis being observed. The amount of hemoglobin is usually reduced to a very slight extent, while the number of red cells may be slightly increased. One of the most striking peculiarities of the blood in diabetes is the presence of an abscess of fat (lipemia). Microscopic examination usually reveals the presence of the extracellular globules, but in some cases fat is present in sufficient amount to permit of demonstration by macroscopic methods. Glycogen has been found both in the plasma and in the leucocytes of diabetic blood and shows the peculiar characteristics discussed in the section on lodophilia (p. 509). Certain peculiarities of the blood in diabetes have led to the introduction of tests supposed to be characteristic for such blood. These tests are occasionally of diagnostic value, although a diagnosis may, as a rule, be made even when these tests do or do not obtain. Bremer's Test. — This test is based upon the fact that diabetic blood does not stain to any appreciable extent when treated with certain aniline dyes. Thick smears of the blood are made upon slides and are fixed by dry heat. These smears are then covered with a i per cent, aqueous solution of Congo red and allowed to stain for a few minutes, after which they are rinsed in water and dried. Diabetic blood will be stained either a faint yellow or not at all, while normal blood will be colored a bright red. A i per cent, solution of Biebrich scarlet will stain the diabetic blood intensely while the normal blood is unstained. Bremer's original staining solution was made up as 1 See CuUen, Surg., GyneO/glffi2SKit£!yi^ip/305£!/?<^6. 548 DIAGNOSTIC METHODS follows: Satiirated watery solution of eosin and of methylene blue are mixed in equal proportion when a precipitate forms which is filtered, washed, dried and powdered. To 24 parts of this powder are added six of powdered methyl- ene blue and one of eosin. One-twentieth of a gm. of this mixed powder is dissolved in 10 c.c. of 33 per cent, alcohol and forms the staining solution in which the specimens are stained for four minutes. The diabetic blood stained by this solution has a greenish tint, while normal blood is reddish violet. Similar reactions have been found in normal blood, in leucemia, in exoph- thalmic goiter, in Hodgkin's disease, in multiple neuritis, and in some cachec- tic conditions, but the reaction in all these cases is very inconstant. Accord- ing to Bremer, cases of renal diabetes do not give this reaction, which is more characteristic of the pancreatic type of the disease. Williamson's Test. — This test is performed as follows: Two drops of blood (20 cmm.) are dissolved in four drops (40 cmm.) of water and to the solution is added i c.c. of a i to 6,000 aqueous methylene blue solution. To this is added four drops (40 cmm.) of 6 per cent, solution of liquor potassae and the test-tube placed in boiling water for four minutes. Diabetic blood will decolorize the solution, while normal blood leaves it a deep blue. The same effect is observed by using diabetic urine instead of the blood. (2) Gout. Little information is available as to the variations in the number of the red and white cells in the gouty condition. The recorded observations show that acute gout has little effect upon the number of red cells and upon the amount of hemoglobin, while chronic gout may be accompanied by an anemia which is more directly referable to causes other than the gouty condition itself. The leucocytes are usually increased in the acute attack, while in the chronic form the leucocytosis is of a more moderate grade. Neusser in working upon the blood of gouty patients found many polymorphonuclear leucocytes whose nuclei were surrounded by basophile granules — the so-caUed perinuclear baso- phile granules. These he considered diagnostic of the uric acid diathesis, but Futcher and Simon have found them in many other conditions, while Ehrlich regards them as artefacts. While the chemistry of the blood in gout has been the subject of much investigation for a long period, nothing of diagnostic importance has been found in the chemical properties of the blood. The excess of uric acid has been shown not to be pathognomonic of gout, as it is present in many other conditions which are clinically far removed from the gouty^state. (3) Addison's Disease. This disease is usually associated with a severe grade of anemia, the'num- ber of red cells being reported as low as 1,120,000 while the percentage of hemoglobin is coincidently reduced. The leucocytes are usually diminished, but may be slightly increased, while the relative proportions of the different cells are not markedly changed. As the disease progresses unfavorably, a relative lymphocytosis may be observed, but this is not always the case.^ 1 See Buettenmuller, Med. Klin., 1914, X, 1354; Engelsmann, Folia Haemat., 1915, XIX, 333; Scheltema, NederlD^jflgj^ /yy(^(fWgf^OffiiS, I, 1767- THE BLOOD 549 (4) Sickets. The state of the blood in rickets varies with the extent and severity of the primary disease and is markedly afEected by complications. Cases are re- ported in which the red cells are practically normal and in which the hemo- globin was only very slightly reduced. This disease is not associated with any special type of anemia, although the hj^jeremia of the bone-marrow might be expected to yield a large number of nucleated red cells. The usual condition of the blood in rachitic children is of the type of simple chlorotic anemia. A grave secondary anemia is seen in many cases in which there are serious complications. The leucocytes in practically all cases of rickets are increased, but may not exceed the normal limits for the child. As is usual in the blood of a child, the lymphocytes are increased whUe the eosinophile cells are often relatively numerous. "• Just exactly what the cause of the leucocytosis in rickets is must be left to the realm of hypothesis, as neither the gastroenteritis nor the hyperplastic splenitis are sufficient to explain all cases. (s) Myxedema. In this disease we usually find an anemia of the secondary chlorotic type along with a moderate leucocytosis. The number of cells is usually somewhat diminished, although their size is usually increased. The proportion of the different leucocytes does not vary, although a few myelocytes are sometimes seen, in the blood, which is numerically normal in other respects. Although many studies of the chemistry of the blood in myxedema have been made, little knowledge has been forthcoming as to the exact cause of this toxemia. It is highly probable that the changed activity of the thyroid gland in this disease influences other organs to such an extent that slight anemia is the result and we should, therefore, assume that this anemia is more a secondary one than a primary result of thyroid insufficiency.^ (c) Blood in Acute Infectious Diseases. In a study of the blood in acute infectious diseases, we must remember that there are certain general rules which apply to all of such diseases with a very few exceptions. The interrelation of the fever with the resistance of the system in general is so close that it is hard to say in any given case whether certain changes in the blood are or are not due to the increased temperature in itself. There can be little doubt that a high temperature working over a considerable period of time will destroy large numbers of red ceUs and will bring on various changes in the blood which might be misinterpreted. A rather extensive concentration of the blood along with a progressive loss of albumin is observed in practically aU conditions associated with fever. We should, therefore, expect to find the number of red cells increased at the out- set of such condition, owing to the concentration of the fluid portion, while a distinct anemic condition may become evident only after the lapse of some time. Fever in itself does not have a large influence upon the number of leuco- ^ See Ostrowski, Folia Haemat., 1912, XIII, 305. ^ See Salvatore (Riforma Med., 1913, XXIX, 1373) for a discussion of the blood changes in exophthalmic goW^fgjtJzed by Microsoft® 55° DIAGNOSTIC METHODS cytes, but it may be stated as a general rule that most infectious diseases (the exceptions being malaria, typhoid, tuberculosis, influenza, and measles) are associated with an increase in the number of white cells. This is not an invariable rule, as will be seen under the discussion of the various infectious types. The leucocytosis so commonly seen associated with infection is no doubt due to the action of the bacteria themselves and of their products upon the leucocytes. The positive chemo taxis which bacteria and their toxins exert upon the leucocytes is very marked. Moreover, as the blood becomes laden with these abnormal products, new leucocytes are thrown into the circulation to aid the old ones in their phagocytic action. Just what sub- stances are accountable for the increased opsonic power of the seruin in any specific infection must be left undecided for the present. In the following discussion of the various infectious diseases, the writer will not attempt to give more than a brief discussion of the blood changes in these separate conditions, leaving associated questions to other writers. (i) Pneumonia. This disease is, hematologically, one of the most definitely characterized of all the infectious diseases. While the physical findings of this condition are largely local, the systemic effects are so marked that definite changes are seen in the blood both in the early and in the later stages of the infection. While showing so many characteristic findings in the blood, it is, at the same time, one of the most obscure in its relations to opsonins and to phagocytosis. Just why the virulent pneumococci should be so little capable of phagocy- tosis and why the addition of attenuated cultures of pneumococci or of ex- tracts of virulent organisms should increase this phagocytic power of the leucocytes is at present very uncertain. We must, therefore, leave the dis- cussion of this phase of pneumonia as well as of other infectious diseases to the section on Bacteriology of the Blood. In pneumonia we find in the early stages that the blood is somewhat con- centrated owing to the action of the increased temperature, while this concen- tration gradually increases as the exudate forms. Such a condition can lead only to an increased count of both the red and the white cells. As the dis- ease progresses, the number of red cells shows a sUght but a steady decline, which points not only to a destruction of the red cells, but to a diminished formation. This decrease in the number of red cells is occasionally seen only at the time of crisis, while, in the cases in which the diminution is gradual the period of diminution does not usually exceed ten days. It will be seen, there- fore, that the red cells in pneumonia may be about normal in number and at the same time an anemia may be present which becomes evident only after the disease has progressed for some time. The red cells are, as a rule, nor- mal in appearance, but an occasional polychromatophilic cell may be seen, especially in the severe cases. Rarely normoblasts may be observed and very rarely megaloblasts. The hemoglobin usually shows a greater reduction than does the number of red cells, which decrease may become evident only after the fever has sub- Digitized by Microsoft® THE BLOOD 551 sided. A reduction in the hemoglobin below 60 per cent, is very unusual in pneumonia of the pure type. Pneumonia is one condition in which the leucocyte count may prove of great value. A leucocytosis appears in most of the cases, being absent in very mild cases as well as in those very severe ones which show very feeble resistance of the organism toward invasion by the pneumococci.^ Rieder's observations are very interesting on this subject. He has found that the leucocytosis of pneumonia is more a function of the intensity of the infection and the degree of resistance toward this infection than it is of the fever or of the extent of the exudate.^ A leucocytosis which may reach 12,000 to 20,000 appears very early in the course of this disease, and is usually evident at the time of the chill or immediately following. A steady increase is sometimes observed in the number of the white cells so that the maximum is usually reached just before the crisis. It is to be said that rapid extension of the dis- ease as well as continuous high temperature may cause much irregularity in the count, cases being reported in which the leucocytes are high at first and steadily diminish as the patients grow worse. Others may show a sudden in- crease in the number of cells as the time of crisis is approached.' According to Ewing, when the leucocytes increase slowly they usually diminish slowly and the disease defervesces by lysis. The degree of leucocytosis in pneu- monia may reach any stage between the normal figure and that of 115,000 as reported by Laehr. The increase in the number of white cells in pneumonia is largely referable to increase in the number of the polynuclear neutrophiles, these cells constitu- ting as high as 97 per cent, of the total number of white cells. Associated with this polynuclear leucocytosis we have a marked diminution of lymphocytes, while the large mononuclear ceUs usually persist in considerable numbers. The eosinophile cells are always much reduced at the height of the leucocyto- sis, so that we may not be able to find a single one after very prolonged search. Cabot has reported a case in which the lymphocytes constituted 66 per cent, of a total of 94,600 white cells, but such a finding is not the usual one follow- ing infection with the pneumococcus. As defervescence goes on the polynu- clear cells diminish very rapidly, while the lymphocytes increase and the large mononuclear leucocytes become very numerous, reaching as high as 16 per cent, in a case reported by Tiirk. The eosinophile cells usually appear about the time of crisis, but occasionally their appearance is postcritical. The degenerative changes seen in the leucocytes in pneumonia are in no way different from those observed in other infectious diseases. It will be noted, from the above remarks, that the blood changes in pneu- monia are those of a mild anemia associated with a high-grade polynuclear leucocytosis and a distinct lymphopenia. Too much reliance must not be placed on the blood finding in a case of pneumonia, owing to the fact that many abnormal cases are present and show results different from the above 1 See Hess, Am. Jour. Dis. ChUd., 1914, VII, i. 2 See Kline and Winternitz, Jour. Exper. Med., 1913, XVIII, 50. ' Dick (Jour. Infect. Dis., 191 2, X, 383) shows that proteolytic ferments develop in the blood about the time of crisis. , Digitized by Microsoft® 552 DIAGNOSTIC METHODS which can be interpreted only by a complete study of the complications in any special case. As a general rule, it is to be said that an absence of leucocytosis is strong negative evidence against pneumonia, while leucocytosis may serve to differentiate this condition from typhoid fever and malaria with which it might be confounded, especially where the systemic and cerebral symptoms are more pronounced than are the local pulmonary changes. (2) Typhoid Fever. This condition, like the preceding, is very often associated with such marked systemic disturbance that the local intestinal manifestations are obscured and the diagnosis rendered somewhat difficult. While typhoid fever is a purely infectious condition and subject to the ordinary laws governing such cases, yet we find for some reason that the invasion of the blood by the specific qrganism is not associated with a leucocytosis, although the febrile rise may be very marked. The study of the characteristic serum reaction for typhoid fever as well as of the bacteriology of the blood must be left for a later section, the discussion here being limited to the changes in the microscopic appearances of the blood. In this condition we find the total volume of blood very much diminished in the early stages, both as a result of the high temperature and the diarrhea and repeated hemorrhages which may occur at any stage of the disease. This concentration of the blood leads to an initial polycythemia which may last for two or even three weeks. However the characteristic change in the red cells is one of a slight and gradual decline, the number of these cells not usually falling below 4,000,000. One must be on his guard in an examination of the blood in any infectious disease lest he conclude from a slightly increased count that no anemia is present. It is a very safe precaution, although rarely followed, to determine the specific gravity of the blood so that one may com- pute the degree of concentration. In this way he may be able to show that the number of cells normally present in such a concentrated blood is much higher than in the case of suspected typhoid fever which he is examining. At any rate, it is wise to make frequent determinations of both the number of cells and of the hemoglobin, as a reduction in both of these elements takes place gradually as in the cases reported by Thayer and Da Costa. The reduction in the amount of hemoglobin is in some cases very marked, being as low as 50 per cent, in one case observed by the writer. The repeated hemorrhages which so often occur in typhoid fever may cause marked varia- tion in this value. The morphological changes which occur in the red cells are not very marked, as a rule, but may be very severe in case much blood is lost by frequent hemorrhages. Polychromatophilia is more or less frequent and irregularity in the size of the red cells is occasionally seen, whUe nuclea- tion of these cells along with formation of a few megaloblasts may occur in severe hemorrhagic cases. The leucocytes are usually normal in numbef in the early stages of uncom- plicated cases, but any complication may cause a polynuclear leucocytosis which may be confusing to the worker. The behavior of the leucocytes is THE BLOOD 553 very variable in the early stages, so that one should never rest his diagnosis of tj^hoid fever upon a negative leucocytosis. As the disease progresses, the leucocytes show a gradual reduction, especially in the number of the polynu- clear cells, which reduction continues until the disease has reached its highest point, after which they slowly increase. The more severe the action of the typhoid toxin the lower is the leucocyte count, the reduction not usually going below 2,500 cells, the majority of cases showing a count between 4,000 and 6,000. It is not an uncommon thing to observe a leucocytosis during the later course of typhoid fever and it is not always easy to explain such a condition. Marked hemorrhage, cold baths, severe diarrhea, and usually perforation may account for the increase in the number of cells, but we do not always find a leucocytosis after such conditions. In typhoid fever we find quite characteristic changes in the relations of the various types of leucocytes. During the first week the neutrophile cells do not, as a rule, increase, while the lymphocytes, especially of the medium- size variety, show a progressive rise. The lymphocytes are rarely below 25 per cent, of the total number of cells and may reach as high as 65 to 70 per cent. The eosinophile cells are usually low in number during the febrile period, but reappear about the time of defervescence. It will thus be seen that the characteristic changes in the blood of typhoid fever are a slight anemia, together with a leucopenia and a relative, and in some cases absolute, lymphocytosis. Such characteristics are the usual ones of typhoid fever, but it is to be remembered that suppurative processes do not always produce a leucocytosis, nor is a leucopenia always present in typhoid fever. (3) Scarlet Fever.* In this condition we find the usual effects of fever manifested in a slight concentration of the blood, leading in the early stages to a polycythemia. The usual change, however, in the red cells is one of gradual reduction in numbers to as low as 3,000,000 cells and occasionally much lower. The hemoglobin also suffers qviite a diminution, so that the anemia may reach quite a severe grade. The leucocytes in scarlet fever usually increase in number one or two days before the appearance of the rash and continue to increase until quite a marked leucocytosis, ranging from 10,000 to 50,000 cells, becomes evident at the time of the complete eruption. The degree of the leucocytosis cor- responds as a rule with the severity of the disease and in some cases is dimin- ished at the time of the eruption, but usually continues for several days and may even extend for weeks after the temperature has subsided. The increase in the number of leucocytes is largely referable to the poly- nuclear cells, these constituting from 85 to 99 per cent, of the total number. The lymphocytes diminish in the early stages of the disease, but later rise to normal or slightly above normal figures. The eosinophile cells are usually 1 See Koessler (Jour. Am. Med. Assn., 1912, LIX, 1528) for a discussion of the recent advances regarding scarlet fever. Digitized by Microsoft® 554 DIAGNOSTIC METHODS normal or even subnormal at first, but steadily increase as the disease pro- gresses and reach a degree of lo to 20 per cent, in the second or third weeks, after which they slowly decline. These rules are not invariable in scarlet fever, but a severe leucocytosis appearing prior to the period of eruption of an infectious fever is practically always suggestive of this disease. In some cases the polynuclear cells diminish about the end of the first week and the lymphocytes and eosinophiles rapidly increase, leading to a later sec- ondary leucocytosis.' (4) Measles. This condition shows in itself nothing particularly characteristic in the blood, but the absence of definite findings is of great importance in its dififer- entiation from scarlet fever with which it might be confounded. The red cells in this disease are not found to be greatly changed, although a slight reduc- tion in their number is usual. A loss of hemoglobin is practically always noticed. The leucocytes are usually normal or slightly reduced in number at the outset of the disease, being the lowest at the height of the eruption when the figure may reach as low as 2,500 cells, returning to the normal within a few days after subsidence of fever. A complicating bronchitis may cause a moderate leucocytosis of 8,000 to 16,000 cells, but this should not lead one to a mistaken diagnosis, as the clinical symptoms of both scarlet fever and of measles should be well-established at the time of the complicating bronchitis. An eruptive fever in the second or third day of its course should be considered scarlet fever, or at least scarlatina, if a leucocytosis is present, while if the disease be measles the number of leucocytes will be normal or even subnormal in the absence of extensive bronchitis. ^ ' DoMe (Centralbl. f. Bakteriol., 1911, LXI, 63; Ibid., 1912, LXV, S7_; Miinch. med. Wchnschr., 1912, LIX, 1688) has announced the almost constant presence in the polymor- phonuclear leucocytes of "inclusion bodies." These are observed near the margin of the cell as rod or coccus forms, which stain readily a faint bluish-green tint with the Giemsa or other blood stains. While these bodies'are observed in about 95 per cent, of scarlet fever cases in the early days of the disease, the number diminishes gradually. They are also found in many cases of diphtheria, pneumonia and tuberculosis, yet they are very infrequent in cases of serum sickness or of scarlatiniform rashes. Their presence is, therefore, not pathognomonic but their absence almost excludes scarlet fever. See Kretschmer, Berl. klin. Wchnschr., 1912, XLIX, 499; Deutsch. med. Wchnschr., 1912, XXXVIII, 2163; NicoU and Williams, Arch. Pediat., 1912, XXIV, 350; Ahmed, Berl. klin. Wchnschr., 1912, XLIX, 1232; Kolmer, Am. Jour. Dis. Child., 191 2, IV, i; Granger and Pole,|Brit. Jour. Child. Dis., 1913, X, 9;Lippmann and Hufschmidt., Zentralbl. f. inn. Med., 1913, XXXIV, 369; Schippers and de Lange, Berl. klin. Wchnschr., 1913, L, 544; Bongartz, Ibid., 544; Schwenke, Miinch. med. Wchnschr., 1913, LX, 752; Pappenheim, Folia Hasmat., 1913, XV, 379; Cummins, Jour. Med. Research, 1913, XXVII, 529; NicoU, Arch. Pediat., 1913, XXX, 346; and Brinchmann, Berl. klin. Wchnschr., 1913, L, 1248; Massini, Med. Klin., 1913, IX, 1729; MacEwen, Jour. Path, and Bacterid., 1914, XVIII, 456; Rosanoff, Arch, f. Kinderhkde., 1914, LXII, 321; Hill, Boston Med. and Surg. Jour., 1914, CLXX, 792; Isenschmid and Schemensky, Munch, med. Wchnschr., 1914, LXI, 1997; Kaiser and Lowy, Deutsch. Arch. f. klin. Med., 1914, CXVI, 82; Rehder, Ibid., 1914, CXVII, 37. ^ Schwaer (Miinch. med. Wchnschr., 1913, LX, 1203) believes that a diroinution or dis- appearance of eosinophiles at the height of the fever is characteristic of measles. See, also, Lucas, Am. Jour. Dis. Child., 1914, VII, 149; and Grumann, Miinch. med. Wchnschr., 1914, LXI, 132. Hess (Arch. Int. Med., 1914, XIII, 913) discusses the blood picture in German measles. Digitized by Microsoft® THE BLOOD , 555 (5) Variola. This condition is associated with more or less extensive destruction of the red cells. ^ In the early stages of the disease the red count may be slightly above normal, owing to the concentrating effect of the fever, but later the red cells will show a sudden reduction. This reduction is especially noticeable in the cases associated with extensive pustulation, when the septic process has such a marked influence in destroying the red cells. The hemoglobin is usually reduced in degree parallel of the diminution in red cells. In most of the cases of small-pox we find a distinct leucocytosis which may run from 10,000 to 20,000 as a rule, but has reached as high as 41,000 in the severe cases. This leucocytosis begins with the appearance of the vesicle, increases as the exudate becomes purulent, and reaches its height when sup- puration becomes extensive; that is, the degree of leucocytosis usually runs parallel to the severity of the septic process, the count returning gradually to normal as the suppuration subsides. The leucocytosis in small-pox is usually of the lymphocytic type, the number of these cells varying from 35 to 45 per cent, of the white cells. Associated with the increased lymphocyte value we find from 5 to 10 per cent, of the large mononuclear leucocytes and usually an average of 3 per cent, of neutrophile myelocytes, in some cases these latter running as high as 16 per cent. Eosinophiles and basophiles are occasionally observed, especially in the hemorrhagic form of this disease. It will thus be seen that small-pox causes a leucocytosis which may reach even the degrees given by scarlet fever, but the differential count as shown by the large percentage of lymphocytes in the former and the greatly increased number of polynuclear neutrophiles in the latter should make a mistake in diagnosis impossible. It is to be remembered that complications of true abscesses with the pustules of small-pox may increase the percentage of polynuclear cells in this disease, but never to such a degree as is shown in scarlet fever. An examination of the blood of children, who have been vaccinated with small-pox virus, shows a distinct leucocytosis of the polynuclear type, reaching as high as 20,000 cells. This leucocytosis usually begins on the third or fourth day after inoculation and gradually subsides until the end of the period of vaccination. The blood in cases of varicella^ seems to show the same characteristics as that of very mild cases of variola, or of vaccinia. The slight leucocytosis is usually of the polynuclear type, reaching a degree of 15,000. The large mononuclear cells which seem to play such an important rdlein the differential leucocyte picture of variola are for the most part absent in varicella, arid myelocytes are practically never found in this condition. 1 See Proescher (Interstate Med. Jour., 1915, XXII, 427) for a discussion of the cultiva- tion of the variola-vaccine virus. 2 See Mensi, Gazz. d. Osp., 1912, XXXIII, 1625; also, Erlenmeyer, Deutsch. med. Wchnschr., 1913, XXXIX, 21 ; Schatzmann, Cor.-BL f. schweiz, Aerzte, 1913, XLIII, 1515; Ztschr. f. klin. Med., 1914, LXXX, 333; Erlenmeyer and Jalkowski, Deutsch. med. Wchn- schr., 191 4, XL, 646. Digitized by Microsoft® 556 DIAGNOSTIC METHODS (6) Diphtheria. The number of red cells in diphtheria seems to be slightly increased, owing to the marked concentration of the blood in this condition. In practi- cally all cases of diphtheria the number ranges from a high normal value to as high as 7,800,000 reported by Cuffer. As the disease progresses, especially after the temperature has fallen, the number of red cells is diminished and a coincident decrease in the percentage of hemoglobin is observed. This slight anemia is not evident in the early stages of the disease due, no doubt, to the abnormal concentration of the blood. In this ' condition, like most infections, we find a leucocytosis ranging between 25,000 and 50,000, the higher the leucocytosis the more grave the prognosis. In one case reported by Felsenthal the leucocytes numbered 148,000, but this is very unusual, as the grade of leucocytosis is ordinarily proportional to the extent and depth of the membrane. The leucocytosis is usually of the polynuclear type, the lymphocytes being also slightly in- creased. In some cases a lymphocytosis of 60 per cent. (Ewing) has been observed, but this is not the usual finding. The eosinophile cells are reduced in number, but are relatively more numerous than in pneumonia. It is in diphtheria that we find quite marked degenerative changes in the leucocytes, "the leucocyte shadows" and increased acidophile tendency of the neutro- phile granules being especially worthy of mention. (7) Pertussis. The exact work of Barach* and others has so modified our ideas of the changes in the blood in pertussis that I can do no better than to give his summary upon these points : ^^ "In the early stages of this disease there is a leucocytosis with increase of all the forms; then a small-cell lymphocytosis becomes conspicuous and continues to increase when the other forms have reached their limit. The large lymphocytes follow the course of the small ones, but they reach their greatest numbers after the small cells have reached theirs. During the stage of active lymphocytosis, bilobed, small lymphocytes are frequently seen as well as numerous degenerated large lymphocytes, especially the basket forms. Then comes the simultaneous falling of the leucocytosis and lymphocytosis, while the polynuclears begin to resume their normal proportion. A little later the mast cells are observed more frequently, and an occasional myelocyte may be seen. While the leucocytosis and lymphocytosis continue to fall by lysis, an eosinophUia is noted; this continues for a variable time, after which the blood formula resumes its normal proportions. During this entire cycle the transitionals seem unaffected. If we were to speak of the first and second half of the blood cycle in this disease, we would say that in the first half the lymphocytes are the prominent factors and in the second half the polynuclears and the eosinophiles. Clinically, leucocytosis is present at about the time the child first coughs; as the coughing goes on, the leucocytosis increases and the lymphocytosis 1 Arch. Int. Med., 1908, I, 602. See, also, Schneider, Miinch. med. Wchnschr., 1914, LXI, 303. • Digitized by Microsoft® THE BLOOD , 5S7 becomes very marked. Churchill believes that ai lymphocytosis exists in the prespasmodic stage and is of extreme importance in early diagnosis. The height of the leucocytosis is reached in the spasmodic stage, sometimes early, and sometimes in the latter part, the sickest children showing the highest grade of leucocytosis. About the time that a marked improvement is noted in the child the leucocytosis has decreased, the polynuclears have increased and the eosinophilia is present." The degree of leucocytosis in this condition varies between 25,000 and 51,150- (8) Acute Bheumatism. In this disease we find that the red cells are quite markedly destroyed, causing, very frequently, a reduction of 2,500,000 cells. This reduction is not always evident in the early stages of the disease, as the blood becomes very much concentrated by the marked sweating which is such a prominent symptom of the disease. The hemoglobin suffers more than the red cells so that we may find the percentage of hemoglobin as low as 60 per cent, in this condition. This anemia is one of the characteristic signs of acute rheumatism and continues well on into convalescence, the hemoglobin not being as quickly restored as are the red cells. A largely increased formation of fibrin has been observed and may have some diagnostic importance. The leucocytes are increased in proportion to the severity and acuteness of the disease, the grade being usually moderate and the type being poly- morphonuclear. In the very mild cases we may find no leucocytosis and one reaching 20,000 or more is, according to Tiirk, always associated with com- plications. As the fever diminishes the leucocytes retvurn to normal and are not as much affected by the subsequent attack as by the previous initial one. The eosinophile cells are absent only in the early stages, while they are present in moderate amounts later in the disease and show a distinct increase after defervescence (Loeffler). It would be impossible in a work of this character to discuss in detail the blood changes in all diseases, whether infectious or non-infectious.^ The writer, therefore, has selected under the acute infectious diseases those which show the more characteristic changes in the blood and those' in which an examination of the blood is more frequently called upon to aid in diagnosis. Many phases of these diseases have been left to the chapters on bacteriology of the blood. {d) Blood in Chronic Infections. (i) Tuberculosis. The earlier studies of the blood of tubercular patients reveal the fact that the blood may show practically no changes which are comparable with the pallor of the skin and the degree of the emaciation of the subjects affected. An anemia is often seen of the very highest type, but usually one of moderate degree is present and even may not exist at all. The degree of anemia is independent of the localization of the disease, although pulmonary affections » See Barach (Arch. Int. 'Wi»ilSl3j^Pn«iu),pJ^i&od changes in mumps. Hess and Fish (Am. Jour. Dis. Child-.^PK^^lVP'sf' HlSttS'tK blood in infantUe scurvy. 5S8 DIAGNOSTIC METHODS are more frequently associated with high-grade anemia than are other tubercu- lar conditions. It is to be remembered that pulmonary tuberculosis is so frequently associated with extensive hemorrhage that one may not wonder at the severe anemia present, yet we find cases in which the regeneration is very rapid after severe hemoptysis. As a rule, a mild anemia of the chlorotic type prevails; that is, the count is practically normal with the hemoglobin somewhat reduced. Occasionally we find a slight lymphocytosis, especially of the smaller cells, and only when a secondary infection prevails does the leucocytosis take on the polynuclear type.' The lymphocytosis in tuberculo- sis is so common that we usually find even in the sputum an excess of the small mononuclear cells in the pure tubercular affection of the lungs. In tubercular infection of the meninges we practically always find a leucocytosis, but with this exception imcomplicated tuberculosis is not asso- ciated with an increase in the number of white cells. It is highly probable that the increased percentage of the mononuclear cells is more closely asso- ciated with the poor nutrition which the tubercular patient shows than it is with any specific effect of the bacillus tuberculosis. Occasionally we find the eosinophile cells somewhat increased, especially in pulmonary conditions with cavity formation, but it must be remembered that a sUght eosinophilia will obtain if tuberculin therapy is being used in such cases. (2) Syphilis. According to Becquerel and Rodier, a moderate grade of anemia is to be found in the majority of cases of syphilis, becoming more pronounced as the disease progresses. This anemia of syphilis is of the chlorotic type, but may increase untU the pernicious type becomes established. As a rule, the reduction in the number of red cells is moderate, being rarely below 3,000,000 cells. The hemoglobin is usually relatively more decreased than are the cells, and the application of mercury in the treatment of this condition frequently lowers this percentage still further, establishing an anemia which is directly referable to the mercury. This reduction in the number of red cells and in hemoglobin becomes more marked as secondary symptoms ap- pear, so that a diagnosis of an initial lesion becomes established by a later examination of the blood. The increase in the number of leucocytes is largely limited to the second- ary and tertiary stages of this disease, as the leucocytes usually remain normal up to the time of the eruptive stage. The increase in the secondary stage, which may reach as high as 20,000, is largely in the number of the small and large lymphocytes, but the eosinophile cells may be increased to as high as 5 per cent, in some cases. In very severe cases a progressive polynuclear leucocytosis is observed. As the tertiary stage comes on the leucocytosis usually persists, but- the lymphocytosis becomes less distinct and constant through the increase in the number of polynuclear cells. ^ 1 See Miller and Reid, Arch. Int. Med., 1912, IX, 609; Miller, Lupton and Brown, Am Tour. Med.'Sc, 1912, CXLIII, 683; Ringer, Ibid., 1912, CXLIV, 561; also, Rayevsty, New York Med. Jour., 1913, XCVII, 813; Wack,- Deutsch. Arch. f. klin. Med., 1914, CXV, 596. 2 See Hazen, Jour. CutanCi!^/zBiaf3fo^?9J5^cfe8#@i 739- THE BLOOD 559 Justus' Test. Justus has found, in studjdng the blood of patients suffering with florid syphilis, that injection or inunction of preparations of mercury cause a reduc- tion in the percentage of hemoglobin of from lo to 20 per cent, for a period of a few hours or days. After a certain time, varying with the general condition of the patient and the severity of the symptoms, the hemoglobin increases again. This test can hardly be considered diagnostic of syphilis, as the mer- cury salts all cause an anemia which may be directly traceable to their hemo- lytic action upon the red cells. In the blood of patients suffering with congenital S5T)hilis we always find a distinct anemia, associated with a slight leucocytosis, especially of the lympho- cytic type. The red cells in this condition show many changes, such as poly- chromatophiUa and nucleation, while the changes in the white cells often resemble the picture of a mild grade of leucemia. For a discussion of the causative factor, the spirochaeta pallida, see the section on Parasitology (p- 5 7 9) . (3) Leprosy. The blood in leprosy is quite different from that in either of the two pre- vious conditions. The usual rule is a very slight reduction in the number of red cells, although cases have been reported with a red count of i, 900,000 and a blood picture of pernicious anemia. The hemoglobin does not seem to be reduced to any extent, the percentage usually being relatively higher than the number of red cells, so that a high color index almost invariably obtains. The leucocytes are rarely increased in number, being usually subnormal, with a relative increase in the number of lymphocytes, their percentage reaching as high as 47 per cent., according to Winiarski. (4) Carcinoma. Although discussed under the heading of Chronic Infections, carcinoma has at present no etiological relation to such conditions. This is one of the most important causes of anemia, owing to the frequent hemorrhages and the mechanical effects of the growth as well as to the unknown toxin, which may produce severe constitutional symptoms, even though the growth may have become latent. The anemia of malignant disease usually runs parallel to the progressive cachexia. The grade of anemia may vary, depending upon the location of the tumor, from a very mild chlorotic anemia to one with a perfect picture of pernicious anemia. It is natural to suppose that the more malignant the disease the greater will the blood changes be, so that we should expect to find the rapidly growing cancers which form numerous metastases usually associated with the more extreme blood picture. That this is not always the case is shown by the following statement of Emerson: "Our cases with rapidly developing metastases) with large nodules, are those with a slight chlorotic anemia; those which simulate pernicious anemia are more often those with few objective signs of cancer, an insignificant-looking little nodule." It is possible that this paradox may be explained by the fact that the development of the cancer is so rapid that the toxin has not had suflBicient time to cause the blood dianges which .the raoie slowly developing growth may bring about. The raSfij^^iir i^CT^6i?lm are much more marked in the S6o DIAGNOSTIC METHODS slowly developing cancers than in the more rapidly growing ones, so that we might assume that the same rule applies to the blood changes as expressions of the general systemic disturbances. When the anemia of cancer develops it is usually more severe than in any other chronic disease. The chief changes are at first in the size, shape, weight, and degeneration of the red blood-cells; later, as the cachexia develops the red cells are often as low as 2,500,000 or even as low as in pernicious anemia, 1,000,000. The hemoglobin is always reduced in amount, but is rarely as low as in chlorosis, the average, according to Cabot, being about 58 per cent. The hemoglobin value seems to be lower in cases of visceral cancer than in those of peripheral type. In a majority of the cases a moderate leucocytosis obtains which is never seen in the benign tumors unless these be complicated by sup- puration. This leucocytosis depends largely upon the amount of hemorrhage from the tumor and upon the position of the cancer. We find in carcinoma of the stomach and uterus, in which hemorrhages are very frequent, quite an extensive leucocytosis, while in cancer of the esophagus a leucopenia may ob- tain. The larger and faster the tumor grows the greater will be the degree of leucocytosis, a condition which is the reverse of that usually found in the case of the red cells. The leucocytosis of cancer is usually of the polynuclear type, but this may not be over 45 per cent., in which cases the lymphocytes are relatively increased. The eosinophiles are rarely as much diminished as in other conditions, but they are not always increased. Myelocytes are, per- haps, more frequently found in cancer than in the other types of anemia, excepting pernicious anemia and leucemia. The degree of cachexia is often very extensive in cancer, but is not always so closely related as one would suppose to the changes in the blood. In those cases in which the cachexia is due to a combination of malnutrition with in- toxication by the malignant toxin the blood changes are naturally very severe, but, as previously stated, the more fulminating types of cancer are not associ- ated either with great cachexia or with severe changes in the blood. Cachexia, therefore, seems to be more a function of the chronicity of cancer than of its malignancy. The changes in the blood in the numerous specific diseases of various organs show nothing characteristic in themselves. It is to be expected that in all chronic diseases of whatever organ, a sHght anemia may be present owing to the effects of such disorders upon the general metabolism. These changes are, however, of the general type of simple anemia and are usually rapidly remedied by the application of the ordinary therapeutic agents. It is true that disease of the general organs causes changes in the composition of the fluid portions of the blood, which may bring about a secondary change in the cellular content. Ce) Effects of Splenectomy. The effects of splenectomy are usually the combined results of severe hemorrhage, a preexisting anemia, of the loss of functions of this organ, and of intravenous infusion^ which has been performed following the operation 1 See Ottenberg and Kali5Ei<^jil)edhlbi^MWfc/?temt)i. 15. Shrunken and fragmenting extracellular forms. 16. Vacuolation of an extracellular furm. Note. — The original water-color drawings were made from fresh blood specimens, ?-. Leitz i^g-inch oil-immersion objective and 4 ocular, with a Zeiss camera-lucida, being used. (E. F. Faber./^c) (From Da Costa's "Clinical Hematology.") Digitized by Microsoft© THE BLOOD 563 cellular organisms, and not to be content with a single examination in doubt- ful cases. 1 Examination of Fresh Blood. The technic of making a fresh specimen of suspected blood is the same as that previously outlined and consists in touching a perfectly clean cover-slip to a drop of blood and allowing the cover-slip to fall upon a clean glass slide. The quantity of blood should be rather small so that the red cells may be dis- tinctly separated from one another. The examination is best made by the use of a )^2 immersion lens. (a) The Tertian Organism (Hemameba vivax; Plasmodium vivax). The youngest form of the tertian parasite as it appears in the red cell resembles very closely the spore of the parent rosette.^ It is a small, compact, colorless, non-pigmented disk (hyaUne form) about 2 microns in diameter and shows an undulating outer rim of basophilic protoplasm which encloses a single large nuclear body which does not stain with methylene blue but shows a dis- tinct chromatin stain with any of the modifications of the Romanowsky stain. This nuclear body is usually surrounded by a clear space which does rot take the stain and which has been termed by Gautier "the milky zone." The parasite has a very rapid ameboid movement and shows a great number of changes in shape and position. It sometimes assumes a typical ring-like form which is usually a Uttle thicker at one point, from which the name "signet ring " has been given. Occasionally several of these rings may be seen within a single blood-cell. After about 12 hours the corpuscle increases slightly in size, becomes somewhat paler, but still has the sharp, smooth, round outline of the normal cell. At this stage the ameboid powers of the organism are very marked, so that many pseudopodia may be seen, connected to the .larger part of the organism by very thread-like pale and rather indistinct bands of union. This gives the appearance of disconnected globules of protoplasm, which is very slightly refractile. At this period (12 hours) pigment (hematin) appears in the parasite in the form of very fine light brown granules which have a very ' Bass has recently succeeded in cultivating the various types of malarial plasmodia. See Bass, Jour. Am. Med. Assn., 191 1, LVII, 1534; Bass and Johns, Jour. Exper. Med., 191 2, XVI, 567; Lavinder, Jour. Am. Med. Assn., 1913, LX, 42. The parasites are cul- tivated only in the red cells of human blood and are destroyed by the leucocytes as well as by the serum. See, also, Sinton, Ann. Trop. Med. and Parasitol., 1912, VI, 371; and Olpp, Munch, med. Wchnschr., 191 2, LIX, 2623. Thomson and Thomson (Proc. Roy. Soc. London, 1913, LXXXVII, 77, and Ann. Trop. Med. and Parasitol., 1913, VII, 153) show that cultures of benign tertian parasites dififer from those of the malignant tertian forms in that there is no tendency to clumping of the former types. This may explain why we do not find any but the young forms of the malignant tertian organism in the peripheral blood, as the clumping causes the larger older forms to collect in the internal organs. Ziemann, Centralbl. f. Bakteriol., i Abt., Orig., 1913, LXVII, 482; Sergent, Beguet and Plantier, Compt. rend. Soc. de biol., 1913, LXXV, 324; Bass, Am. Jour. Trop. Dis. and Prev. Med., 1914, 1, S39. MiUer (Jour. Am. Med. Assn., 1914, LXII, 1549) reports the cultivation and development of a second generation of parasites without the removal of the leucocytes. 2 Stephens (Proc. Roy. Soc. London (B), 1914, LXXXVII, 375, and Ann. Trop. Med. and Parasitol., 1914, VIII, 119) reports the discovery of a new malarial parasite, which he names Plasmodium ienue. Craig (Jour. Parasitol., 1914, I, 85) believes this to be nothing more than a rather atypical form of Plasmodium vivax in the unpigmented stage of development. Emin (Bull. Soc. Path. Exot., 1914, VII, 385) describes a further variety of the tertian organism to which he gives the name Plasmodium vivax variety minuta. Craig does not believe the evidience s"*^<='«^'^/A/f jg^J^f,^ff/5%pefiific rank. 564 DIAGNOSTIC METHODS rapid dancing motion and are clustered especially at the ends of the pseudo- podia. The organism continues to increase in size and, at the same time, the host becomes somewhat larger, paler, but still round in outline. At the end of 24 hours the organism fills about one-third of the cell, is still ameboid and shows increased pigmfent, which is somewhat darker in color and is less ac- tively motile, being distributed throughout the substance of the parasite. In this form one may occasionally see the nucleus as a globular body at the end of a pseudopod. In the last half of the cycle of development of the tertian organism the growth is much more rapid than in the first half, the parasite be- ing fully developed within 40 hours. The cell at this time is about one and a half times its normal size and is so little refractile that its outline can scarcely be seen. The organism is from 8 to 10 microns in diameter, is round, and is even less refractile than is the corpuscle. The pigment is much more abun- dant at this time and is still evenly distributed throughout the organism. The next stage in the development of the organism is known as the presegmenter stage. The cell becomes practically invisible, the pigment collects in one or more irregular clumps throughout the organism, the granules moving in irregu- lar lines. At this time the periphery of the organism shows slight crenation and refractive dots appear irregularly in the periphery of the organism. The line of demarcation between the presegmenter and the segmenter is very slight. The corpuscle is now practically eliminated and the organism be- comes more dense and highly refractile. The refractive dots which were visible in the presegmenter stage are now seen to be in the center of lines of separation which pass from the irregular crenated border down toward the center of the organism, thus marking off future segments, which are as a rule from 15 to 20 in number. As development proceeds the segments become more sharply defined until the clumps form into discrete circular masses with a distinctly refractile spot in the center. The pigment in these segment forms seems to be left in masses between the segments without any definite arrange- ment. Each segment now splits off from the mother segmenter cell and becomes free in the blood in the form of the original hyaline type which be- comes attached to a red cell and soon enters it to pass through the vari- ous stages discussed. It is to be remembered that the hyaline forms do not modify their host, either in shape, color, or size, such changes being observed only at the time when the pigment first becomes evident. The preceding is a concise description of the cycle of development within the cell (asexual generation or schizogone), but we occasionally find tertian forms other than hyaline which are extracellular. These seem to be of two types, the degeneration forms and the gamitocytes , cells capable of sexual development. The degeneration forms or, as they are sometimes called, ex- truded intracellulars, are sometimes the only ones seen in the specimen. These are parasites which have passed from the cell and have died, the organ- ism sometimes appearing as if it had passed out through a very fine hole. If it be entirely extruded, the hemoglobin leaves the cell with it and only a shadow of the red corpuscle remains behind. However, this does not always occur, so that we meet with .typical dumja-bell-sbaped organisms in the plasma. Digitized by Microsoft® PLATE XX rX. 6 r^} 8 9 10 fo^ The Quartan Parasite. 1. Normal erythrocyte. 2. Intracellular hyaline form. 3. Youn^ pigmented intracellular fo-rm. Note the coarseness, dark color, and scantiness of the pigment gtanules. 4. 5, 6, 7. Later developmental stages 0/3. Note the peripheral distribution of the pigment in all the parasites from 3 to 8. (Compare size and color of the erythrocytes in 5, 6, and 7 with 7, 8, and 9. Plate VI.) 8. Mature intracellular foryn. Note that the stroma of the erythrocyte is no longer demonstrable. 9, 10, II. Segmenting forms. In g are shown the characteristic radiating lines of pigment. (Compare with 10, 11, and 12, Plate VI, and with 10, 11. and 12, Plate VIII.) 12. Large swollen extracellular form. (Compare with 13, Plate VI.) 13. Flagellate fonn. (Compare with 14, Plate \l.) 14. Vacuolation 0/ an extracellular form. (E. F. FABER,y>cO (From Da Costa's "Clinical Hematology.") Digitized by Microsoft© THE BLOOD 565 If the blood be observed while this process of extrusion is going on, the pigment will still be extremely active but it gradually becomes quiet as the organism dies. The organism may break up into fragments forming several pigmented spherical masses or it may become deformed and vacuolated, con- stituting the so-caUed " sporulating" forms. The gametocytes are found at all times in the blood after the infection has been established for a few days. These are not in reality extracellular forms as one sees them in the stained specimens surrounded by the shell of the corpuscle. These gametocytes are of two forms, the macrogamete or female cell and the micro gamete, the male cell, which is one fiagellum of the micro gametocyte (parent male cell). The macro- gametes are large organisms, pale, indistinct, and three or four times as large as the red cell. Some of them show no trace of the corpuscle; their pigment is abundant and exhibits very active movements. Their nucleus is about three and one-hah" microns in diameter and is sometimes seen in the fresh specimen; either its outline is distinct or its size and shape may be recognized, as it is the only portion of the parasite which is not invaded by the pigment granules. The function of these macrogametes seems to be to continue the life of the organism within the mosquito after the organism has become fertil- ized by the male element or microgamete. The microgametocyte is smaller than the macrogamete, being eight to ten microns in diameter. Its pigment is very active, but soon forms a circle around the center and becomes sta- tionary. Occasionally this pigment may become even more active than be- fore, the margin of the cell may undulate and several flagella protrude. These flagella are the microgametes and are two or three times the length of a red blood-cell and often contain pigment granules which enable them to be followed when they break loose from the parent cell. After these flagella separate, the parent cell is seen as a small cell with central motionless pigment. This process of flagellation is not seen in the fresh specimen, but occurs 1 5 or 20 minutes after the blood has been drawn, which would point to the fact that such a process does not occur within the body. Normally, this change takes place only in the stomach of the mosquito. (&) The Quartan Organism (Hemameba malarias ; Plasmodium malarise) . This organism is much more rare than is the tertain. Its cycle of develop- ment requires 72 hours so that the normal -paroxysm occiurs every foiurth'day. If two groups are causing the infection there will be two days with paroxysms, one free day, and then two days of paroxysm following. If more than two groups are introduced v/e may have daily chills and fever, but only when the groups are large enough in number to cause a paroxysm. According to Ross, 250,000,000 organisms are necessary before a chill follows. The small hya- line forms of the quartan parasite are not distinguishable in the early stages from those of the tertian type, but are easily recognized at the time pigment appears, as the granules of the former are coarser, darker in color, and not so actively motile. As the parasite grows in size the corpuscle becomes smaller and stunted with an irregular crenated margin. The protoplasm of the organism is more ref ractile than that of the tertian organism and hence the outlines of the pseudo]g})f^fffz®"»J3y 5i 6. Intracellular hyaline /oryns. In 4 the parasite appears as an irregTilarly shaped disc with a thiiined-oul centra] area. In s and 6 its ameboid properties are obvious. 7. Voung pigmented intracellular forni. Note the extreme delicacy and small number of the pigment praTiuies. (Compaie with 6, Plate \'l, and with 3, Plate VII.) 8, 9. Later developmental stages 0/ y. 10, ir, 12. Segmenting fortns. 13, 14. Crescentic /o)ms at early stages 0/ their developm.ent. 15, 16, 17, i8, 19. Crescentic forms. In 15 and 19 a distinct " bib " of the er^'^throcyte is visible. Vacuolation of a crescent is shown in 18, and polar arrangement of the pigment in 17. 20. Oval form. 21. 22. Spherical foims. 23. J-lagellate Jorm. 24. Vacuolation and deformity of a spherical form. 2=;. Vacuolated leucocyte apparently enclosing a dwarfed and shrunken crescent. 26. Remains of a shrunken spherical for7n. (E. F. FABER,yVc.) (From Da Costa's "Clinical Hematology.") Digitized by Microsoft© THE BLOOD 567 becomes more and more continuous. The hyaline forms are similar to those of the tertian and the quartan types, but are slightly smaller and assume the "signet-ring" form much more commonly and maintain it longer. This early form of the estivo-autumnal parasite is distinguishable from the tertian by the shrinkage of the red blood-cell and from the quartan parasite by the smaller dimensions. In some cases the rings do not show the thickening of one segment, but remain of a uniform fine caliber throughout. These rings which do not show the distinct "signet-ring" type nearly always present two nuclear bodies lying at opposite poles or close together. Occasionally these rings appear as if unfolded and stretched across the cell like a thread, the nuclei appearing at irregular intervals. These rings may at times lose their refractility and become ameboid. As the parasite develops, a slight amount of pigment appears, usually seen as one or two granules which are motionless, as a rule, and are located at the periphery of the parasite or at the inner edge of the biconcavity. The cell is very commonly much shrunken, crenated, and brassy, even in the early stages. Some cells, which do not contain parasites, show the same injurious effects of the organism. The parasite at this time occupies about one-fifth of the cell. The infected cells now usually disappear from the circulation and continue their development in the lymph-glands, especially in the spleen. In some cases, however, the parasite does continue its development in the peripheral blood, but this is rare. In such blood or in that obtained from the spleen, the pigment appears much increased and seems to be rather coarse and dark in color, thus resembling very closely the quar- tan organism at this stage. It seems to be a general rule that the more malig- nant the type of estivo-autumnal malaria, the fewer older forms are seen in the peripheral blood, although numerous young parasites are present. In some cases the hemoglobin becomes concentrated around the parasite, leaving an almost colorless ring at the periphery of the cell. The cycle of development in the internal organs seems to take place within the macrophages, which are best studied in the fresh specimen. The parasite develops to about 5 microns in size, which is about half the size of the cell, and when fuU grown has its pigment all in the center, never diffusely scattered as in the tertian organism or peripherally located as in the quartan type. This form is rarely seen in the peripheral circulation. The segmenters vary in size from 2j^ to 5 microns in diameter, the process of segmentation being similar to that of the other organisms giving rise to the formation of 15 or 16 very small segments. Certain characteristic forms of this type of malaria appear in the pe- ripheral blood from about the seventh day of infection and in the internal organs as early as the fifth day. These forms are known as the crescents and the avoids. The crescents^ are slightly longer than the red blood-cells and show a distinctly crescentic shape with rounded ends, although irregular forms are at times observed. They are very refractile and usually show a fringe of the degenerated red blood-cell, which is more abundant in the con- cavity of the crescent and forms the so-called "bib." The pigment is large in amount and is masssed at the center of the crescent, occasionally in the form 568 DIAGNOSTIC METHODS of a sheaf or ring. The granules are usually coarse and rod-shaped. These crescents very frequently change their shapes, becoming oval, dumbbell- shaped, or circular and then may resume their original crescentic form. In the circular types no trace of the corpuscles is seen and the protoplasm is not as distinctly refractive as is that of the crescent. In this form of malaria we also find pigmented leucocytes, both the polynuclear neutrophiles and the large mononuclears assuming this function. In these phagocytic cells one may see masses of pigment or even parasites, especially the segmenting and flagellating forms. These pigmented cells are also seen in the other form of malaria, but only just after the chill, while in the estivo-autumnal form they may occur at any time during the infection. Examination of Stained Specimens. The technic of making preparations of malarial blood for staining is practically the same as that outlined previously. Precaution must be taken to make thin smears so that the parasite may be brought out more clearly. The stains to be used will depend largely on the experience of the worker, but the writer would recommend the thionin and Nocht stains above the others, although the Wright and Giemsa stains will frequently give beautiful pictures. Stains which have been kept for some time are not always reliable, so that it is well to have fresh specimens of the stain on hand for use. If the blood has been kept for some time before staining a diffuse plasma staining with methylene blue will be observed. The Tertain Parasite. The young hyaline form consists of a mass of blue protoplasm usually grouped in ring-form with a mass of reddish-violet stained chromatin, usually situated at the thinner portion of the ring and extending for a large part within the clear achromatic or vesicular part of the parasite. These hyalines are 2 to 3 microns in diameter. There is some discussion as to what portion of the parasite the name nucleus should be applied. Some give this term only to the chromatin staining part, while others include both the chromatin and achromatic portion. However this may be, it is necessary for the recognition of the tertian organism that the blue protoplas m and the red chromatin be both observed. There are frequently artefacts in the blood which resemble very closely these hyaline foms so that the worker must be constantly on his guard. Such artefacts are the Maragliano degenerations so commonly seen in red blood-cells and also the cases in which blood-platelets lie upon a red blood-cell. Any structure which lies upon a red blood-corpuscle ap- pears surrounded by a colorless zone, while the trU3 malarial ring is in direct contact with the hemoglobin of the cell. Moreover, such artefacts will not show the chromatin staining portion which is so characteristic of the hyaline ring. In the specimens examined at the end of 24 hours, one will observe that the achromatic area has become somewhat larger, while the chromatin porti6n seems to be grouped in more irregular masses, some ceUs appearing to have several nuclei. In the full-grown parasite the chromatin breaks up into a cluster of fine granules which are scattered diffusely through the cell in the form of ^^^^^^t^fyHf^cPff^Wcr^hWlB) chromatin clumps separate PLATE XXXI. Tertian Malarial Parasite. (Wright's Stain.) Digitized by Microsoft® Digitized by Microsoft® THE BLOOD 569 into from 15 to 20 dense round masses, around which the protoplasm collects with them as a center. The protoplasm at this stage is distinctly achromatic and is always so in the segmenting ceU. The distinct, achromatic, milky zone surrounds each segmentary chromatin clump, while the general proto- plasm shows a difiEuse faintly basic staining. The pigment which must not be confused with the chromatin is pushed toward the periphery and, after segmentation is complete, collects in masses near the center. It is to be re- called that at the time the pigment collects in th^ center in the fresh specimen there is no distinct evidence of segmentation, although this segmentation shows quite distinctly in the stained specimen. The sexual development of the parasite is easily followed in some cases in the stained specimen. According to Stephens and Christophers, the young gamete is characterized by the position of the chromatin as it lies in the center of the vacuole instead of at the edge. During the development the gamete is occasionally filled with basophile particles which are known as Plehn's karyochromatophilic granules or Schiigner's granules. The full- grown macrogamete contains an abundance of protoplasm which stains a deep blue and a small amount of chromatin in a compact mass, which is peripherally placed and surrounded by a thin vacuole-like area. The pig- ment of these female cells is uniformly distributed throughout the cell and the inclosing red blood-cell can be seen only with difficulty. The chromatin is much more voluminous in the microgametocytes, but is looser and centrally placed in a large achromatic zone arranged in the form of a band which stretches clear across the cell. The protoplasm is in the form of a ring around the nucleus and stains more of a grayish-green color than does the bluish protoplasm of the macrogamete. The Quartan Parasite. The structure of the quartan parasite resembles very closely that of the tertian form, but in the hyaline type the chromatin mass is less distinct and is in the form of an irregular clump of granules in the older forms, while in the younger a cluster of fine granules without any distinct achromatic zone is seen. As development proceeds the parasite generally takes a form extending across the cell and usually occupies the larger portion of the red cell which has be- come shrunken and irregular in shape. The segment forms are much more distinct in the quartan type than in the tertian and show much more regular and geometric lines of cleavage with the chromatin exactly in the center of the crenated surface. The pigment granules are coarser and much more dis- distinct than in the tertian form and are more peripherally located. The Estivo-autunulal Parasite. The hyaline forms in this type show the chromatin in two or more masses or filaments. The protoplasm is scantier than in the other forms and remains so throughout the cycle of development of this parasite. A very character- istic appearance of the hyaline rings of to be this tjqpe seem the thickenings of the protoplasmic layer opposite the chromatin mass. The gamete forms are distinctly spherical, being of the same thickness all the way round. Their nucleus forms a portion ©^^fgiaqgy)tfl-^,4^/?®t project as in the schizonts 57° DIAGNOSTIC METHODS (the asexual parasites). The red blood-cells in these sexual t3T)es usually show no coarse granular stippling . The crescent forms, which are character- istic of this type of malaria, show the chromatin in a loose network which occupies the larger portion of the cell, has little blue staining protoplasm, and has its pigment scattered throughout its body. This is the male form and is somewhat kidney-shi^^jed and is shorter and broader than the female type. The female crescent is longer and narrower, its chromatia more or less compact and centrally located, its pigment in a ring around the nucleus or in a clump at the center, while its protoplasm is more or less extensive and takes a distinct bluish tinge. We also find two types of the circular form. The microgametocyte is smaller than the red cell, distinctly spherical in shape, with its chromatin in the center in a large irregular mass, or in several dense masses near the periphery. These masses containing chromatin material are later extruded and form the flagella or microgametes. The macrogamete is two or three times the size of the microgametocyte, is often of a triangular shape, and has abundant blue-staining protoplasm. The chromatin is in a single mass at the periphery and is surrounded by a circle of pigment. The examination of the stained specimens does not give as great an oppor- tunity for study of the developmental cycle of these parasites as does the examination of a fresh specimen. The conditions found in the fresh blood resemble more nearly those found in the stomach of the mosquito than in the circulating blood, so that many pictures seen in the fresh specimen are practically never found in the stained slide. ^ It is further to be said that we do not always find malarial organisms either in the fresh or stained specimen, although the patient may be at the time suffering from malaria. It may be stated, as a rule, that in all well-marked initial attacks of malarial fever the parasite may be found in the blood if it be examined within i8 hours of the chill. The energetic use of quinin has so much influence upon the ameboid types of the parasites, that the blood may fail to show any of these organisms, although the patient may die from the effect of the infection. It is safe, how- ever, to state that there is practically no case of malaria in which parasites may not be found in the blood, if frequent and repeated examinations are made. Development of the Organism within the Mosquito (Sporogony). The cycle of development of the malarial organism has been more closely followed in the mosquito in the case of the estivo-autumnal parasite. For any development to occur within the body of the mosquito it is necessary that the macrogamete become fertilized, so that the course in the mosquito is one of sexual development. The microgametocytes throw out their flagella (microgametes) and the macrogamete ripens in the stomach of the mosquito by casting off karyosomes (polar bodies consisting of chromatin), and in so doing causes the formation of a slight mound at one portion of the organism through which the free flagellum enters. This process occurs in from one to one and a half hours after the mosquito has bitten a patient infected with malaria. The nuclear material of the macrogamete and microgamete then unite. The cell then forms a distinct motile spindle shape called the ver- 1 See Clark (Jour. ^^V^^- ^^izW^¥Mbr6'^^ ^ discussion of the value of the study of placental blood films iir tne diagnosis of malaria. PLATE XXXII. ESTIVO-AUTUMNAL PARASITE. (WrIGHt's StAIN.) Digitized by Microsoft® Digitized by Microsoft® THE BLOOD 571 c b Fig. 144. — -Cycles of the Malarial Parasite. (Deguy el Guillaumin.) I, Beginning of the development of the parasite as the ameboid intra-ceUular body. /, II, III, and IV represent the endoglobular cycle or schizogony. /, //, //', //", III", and ///' represent the extra-cellular cycle or sporogony. In //', there are two free gametes (g), one microgametocyte (ra), one microgamete (>»'), and the union of a macrogamete and a microgamete (»»")• HI', free gametes; ///", fertilized macrogamete, taken up by the mosquito, A. lnB,C, D and E it becomes encysted in the gastric musculature forming the [zygotes {z and z')- In E, the sporozoits are formed and in F they are thrown out by the saliva of the mosquito. In G, these are free to enter the cell forming the ameboid bodies. Digitized by Microsoft® 572 DIAGNOSTIC METHODS miculus or ookinet. The size of this fertilized macrogamete is f rom 20 microns up and may be found in about 48 hours after the blood has been ingested. This motile form is found only in the stomach of the mosquito. The ver- miculus then bores its way through the epithelial cells of the intestinal wall and becomes encysted between the intestinal epithelium and the elastic layer, which forms the membrane of the spore cyst {zygote, oocyst, sporoblast). This zygote increases rapidly in size and the nucleus divides rapidly. In its growth it bulges outward from the intestinal wall forming pendulous tumors into the body cavity which growth may vary from four and a half to ninety microns in diameter. This stage is associated with the appearance of much pigment and is called the medium zygote or medium sporoblast stage. The protoplasm gathers around the divided nuclei, forming daughter cysts which are con- nected by bridges of protoplasm forming the stage known as large zygote, large sporoblast, or large oocyst. In each of these divisions the nucleus divides many times, the daughter nuclei remaining on the surface of each daughter cyst. The protoplasm now collects around each daughter nucleus, the first forming spherical cells which then elongate into threads lying parallel over the remains of the sporoblasts. These threads are called sporo zoits and have an elongated nucleus. The final length of these sporozoits is about 14 microns and their width about i. Their protoplasm is thick, homogeneous, and very refractive. They are sometimes present to the number of 10,000 in some zygotes, but more frequently are not so numerous. As the oocyst becomes larger it bursts into the body cavity, the sporozoits of each cyst ripening at about the same time. These sporozoits wander at first free, but soon collect in the salivary gland of the mosquito. They are motile and move with a bending and gliding motion. When they are inoculated into the blood of man by the bite of the female mosquito they attach themselves to the red blood-corpuscle and finally penetrate it to form the initial hyaline type of the organism. The period of incubation after the bite of the mosquito is usually between the eighth and twelfth day, when the first chUl will appear, although the exact time of appearance of the initial symptoms will depend upon the number of sporozoits introduced into the circulation (Emer- son). The anopheles is the only type of mosquito which is at present known to be the host of the malarial organism and to give rise to the development of the gameto-schizonts (the sexual cells), which the bite of the female animal introduces into the blood cells which are known in their future development as schizogones. The sexual cycle within the mosquito is known as the sporo- gone, which Rowley-Lawson^ has recently shown may occur in the circulating blood of the human host. General Changes in the Blood in Malaria. There are few conditions which lead so rapidly to such an extreme reduc- tion in the red cells as does acute malaria. ^ An acute attack may reduce the ' Jour. Exper. Med., igii, XIII, 263; Ibid., 1913, XVII, 324. ^ Rowley-Lawson (Arch. Int. Med., 1912, IX, 420) believes that this severe anemia is due to the migration of the plasmodia from corpuscle to corpuscle. See James, Jour. Infect. Dis., 1913, XII, 277; Browft, Arch. Int. Med., 1913, XII, 315; Jour. Exper. Med., 1913, XVIII, 96; Rowley-Lawson (Ibid., 1914, XIX, 450 and 523; Ibid., 1915, XXI, 5 584) dis- cusses the extra-cellular lelai^n^i^th^ gar^tg^o^th^^d corpuscle. THE BLOOD 573 red cells to as low as 500,000 cells as reported by Kelsch. Frequently a re- duction of 1,000,000 is observed during the first day, with, a progressive reduc- tion as the time goes on. In the afebrile period of the disease a continuous fall is observed, but this is much less rapid. The regeneration of the cells is very active so that an increase in the number of cells has been observed directly after an attack in some cases. In cases of chronic malaria the red cells are commonly reduced to as low as 583,000 (Kelsch), while when attacks occur only at intervals and are promptly stopped by quihin no reduction in the red cells may follow (Marchiafava). In cases of moderate severity the usual changes of secondary anemia are present in the red cells. Polychro- matophilia and granular degeneration of the reds progresses steadily, while the hemoglobin content of the cells may be markedly reduced. Frequently cases are seen in which the anemia takes on the absolute pernicious type, so that the parasites seem to have been massed in the bone- marrow. As Ewing states, "there can be no doubt that the tendency of the estivo-autumnal parasite to be massed in the bone-marrow, in both ameboid and crescentic phases, and the excessive demand on red-cell production arising in the disease render pernicious malaria an extremely favorable condition for this dit turb- ance of the structure of the marrow and the development of specific megalo- blastic changes." Besides the changes which can be directly referred to anemia or toxemia, changes in the size of the cell are quite constant, the ter- tian parasite causing from the start swelling of the cell and progressive loss of hemoglobin, while the quartan and estivo-autumnal forms cause the red cell to shrink and take on a peculiar brassy tone. The leucocytes do not show very characteristic changes. In the acute malarial attacks of average severity the absence of leucocytosis is of consider- able corroborative value, although a slight leucocytosis amounting to about 10,000 with an increase in the percentage of polynuclear cells has been ob- served by Billings and others. Except during the thr^e or four hours im- mediately following a chill malarial blood usually shows a diminished number of leucocytes with a distinct relative lymphocytosis, which finding is that seen in typhoid fever. In the more severe estivo-autumnal attacks a definite leucocytosis has been distinctly observed, especially in the hemoglobinuric or black- water type of malarial infection. The extent of the leucocytosis varies between 10,000 and 35,000, although many attacks fail to cause any distinct increase. During the afebrile periods the eosinophile cells are usually in- creased and may be observed throughout the course of the attack. Neutro- phile myelocytes are occasionally present and rarely eosinophile myelocytes. Pigmented leucocytes are seen in the majority of cases, especially in the severe and fatal cases, the pigmented leucocytes being more closely related to the severity of the paroxysms than to the extent of the deposits in the various viscera. These pigmented or phagocytic cells include mononuclear and poly- nuclear leucocytes and a few endothelial cells. The large and smaU mono- nuclears usually contain pigment or rosettes, while many of the polynuclear leucocytes also contain the parasites. These phagocytes may contain, be- sides parasites and rnalarial pigment, hematoidin, hemosiderin, red blood- [arial pigment, nematoid cells, leucocytes, and occasioiffliy an unknown crystalline pigment 574 DIAGNOSTIC METHODS (2) Relapsing Fever (Famine Fever). The cause of this peculiar fever is the spirillum of Obermeier and is not a member of the class of bacteria but, more properly speaking, belongs to the class of spirochete. This organism is between 16 and 40 microns in length and about i micron in width, but is subject to considerable variation in size. It is thin, sharply curved, and appears to be structureless. It takes a deep chromatin stain and also stains with methylene blue in from two to five min- utes. It is seen in the blood only during the febrile period of the disease and at that time is actively motile with a rapid wavy motion, much resembling the movements of a coiled spring in its stretching and collapsing. It moves rather slowly among the corpuscles, but does not disturb them to any extent.' Cases have been reported in which these spirochete are present in the blood 24 hours before the chill, but they are usually to be found in larger numbers at the time of the rise in temperature, increasing rapidly from day to day. The fever, as a rule, continues about six days, at the end of which time these Fig. 145. — Spirillum of Obermeier. (Pitfield.) parasites leave the blood. Strangely enough these organisms have been found in varying numbers in different parts of the circulation, while there does not seem to be any strict parallelism between their number and the height of the fever. Loewenthal has applied the agglutination test to the blood of suspected cases and finds the reaction positive in 85 per cent, of the cases in the periods in which the parasites are absent. Nothing of a charac- teristic nature is observed in the general blood picture. Cases of relapsing fever are practically never found in the United States, unless imported through the medium of emigrants from Russia and especially India. The cases reported by Wellman show that relapsing fever, as found in West Africa, may arise from the bite of a tick transmitting the spirillum of Obermeier. 2 ' Noguchi^Qour. Exper. Med., 1912, XVI, 199) has succeeded in cultivating this organ- ism and shows that this pathogenicity is not thus diminished. 2 NicoUe, Blaizot and Conseil (Ann. de I'lnst. Pasteur, 1913, XXVII, 204) show that the bite of the louse does not transmit this disease as is commonly supposed. If the louse be crushed on the skin and there are abrasions on the skin from scratching, the parasites gain access to the system. „. ... ,, .,. „„ Digitized by Microsoft® THE BLOOD 575 (3) Sleeping Sickness. This very interesting condition which is so prevalent in Central and West Africa seems to be due to an actively motile fusiform flagellate known as the trypanosoma Gambiense, which can be found in the blood free in the plasma (never intracorpuscularly), moving with a screw-like motion among the red cells which it does|°not seem to disturb. This parasite doubtless has a sexual development, its host being the common fly, Glossina palpalis, while closely related trypanosomata are transmitted by the bites of various flies, especially one of the seven varieties of the tsetse fly. This organism is from two to three times as long as a red blood-corpuscle (18 to 25 microns) and 2 to 2}/^ microns wide, having a flagellum anteriorly and an undulating membrane ex- tending its entire length.^ In the fresh blood specimen these parasites should be looked for with only a medium magnification. These parasites vary much Fig. 146. — Trypanosoma gambiense. {Da Costa.) in number sometimes being absent for a long period and then suddenly re- , appearing in large numbers. Symptoms of the disease seem to bear little relation to the number of parasites in the peripheral blood, so that in some cases it may be necessary to examine the fluid in the edematous areas or even to puncture the cervical lymph-glands.^ When these parasites are stained with a polychrome dye they show a rather large red nucleus about the mid- dle, a centrosome staining intensely in a vacuole-like area near the blunt posterior end, and a line of chromatin taking a dense red stain running down the edge of the undulating membrane and terminating in the flagellum which is also stained red. The protoplasm of the body takes a distinct blue stain. The parasite contains no pigment and, therefore, obtains its nourishment from the plasma and not from the red cell. ' See Thomson and Sinton (Ann. Trop. Med. and Parasitol., 191 2, VI, 331), who have succeeded in cultivating this organism. * Wolbach and Binger (Jour. Med. Research, 191 2, XXVII, 83) show that the trypano- somes are not confined to tlie blood-vessels and lymphatics, but invade the tissues. See, also, Tuttle, Tour. Am. Med. Agsn.^ 1915, LXV, 240. „^ DigitizedriyMibrosoft® 576 DIAGNOSTIC METHODS This disease may take an acute course, but as a rule is exceedingly chronic, running for years, but becoming fatal as soon as the parasite reaches the cere- brospinal fluid. The true sleeping sickness appears only when the cerebro- spinal fluid is invaded and seems to be, according to the recent work of Koch, directly amenable to treatment with atoxyl. In examining the cerebrospinal fluid for these parasites it is best gently to centrifuge the fluid for five minutes, after which the sediment may be examined under a vaselined cover-glass. There are many other types of trypanosomata, but the Gambiense form is the more important. This is pathogenic toward man, but cannot be dis- tinguished from the trypanosoma of the tsetse fly which is so fatal to the horse and mule (trypanosoma Brucei), that of the surra disease^ (trypano- soma Evansi), or that of dourine (trypanosoma Equiperdum). (4) Kala-azar. Through the researches of Donovan, L'eishman, and Ross, parasites have been demonstrated in the blood which are probably directly associated with the condition known as kala-azar, tropical splenomegaly, piroplasmosis, cach- exial fever, and dum-dum fever. The organism has been called the Leishman- Donovan body, and is a small oval, round, or oat-shaped body from 2^ to 3 microns in diameter. These bodies have a definite cell outline and contain two chromatin masses, a larger one, a nucleus which is almost round or oval and stains faintly, and a smaller bacillus-shaped centrosome. which stains deeply and is directed almost at right angles to the axis of the nucleus. These two chromatin masses are both in the long axis of the cell, the larger one being at the periphery. Many of these forms are vacuolated and the outline of the cell cannot always be seen, although these two masses thus arranged are distinctive. They are easily stained with the various polychrome dyes and are best studied with the highest lenses. These bodies probably represent a stage in the development of a trypanosome as shown by the work of Leish- mann and Statham. They are not found in the circulating blood as a rule, but they have occasionally been reported in the form of intracellular bodies in fatal cases. They are easily seen in the blood obtained by splenic puncture and also in the granulation tissue taken from the ulcers.^ Many are found in the mesenteric lymph-glands, bone-marrow, and liver. Some of these bodies lie free, but most of them are intracellular, either in the leucocytes, endotheUal, or splenic cells, and frequently in large masses in the macrophages.' The changes in the blood are those of a moderate anemia, associated with a leucopenia with a relative and absolute increase in the number of the large mononuclears. The average leucocyte count is about 2,000. ' Mitzmain (Philippine Jour. Sc, B, 1913, VIII, 223) shows that this disease is trans- mitted by the tabanid fly {Tabanus Striatus Fabricus). * Bates (Jour. Am. Med. Assn., 1913, LX, 898) reports a case of Leishmaniosis of the nasal mucosa, the organisms being obtained from direct smears made from the ulcers. See, also, Patton Indian Jour. Med. Research, 1914, II, 492; Mackie, Ibid., 510. ' Darling (Jour. Exper. Med., 1909, XI, 515) has reported the finding of an intracellular parasite, the histoplasma capsulatum, as the cause of Histoplasmosis, an infectious disease of the Canal Zone much resembling kala-azar. The parasite is small, round or oval, i to 4fi in diameter, possesses a polymorphous chromatin nucleus, basophilic cytoplasm and achromatic spaces all enclosed within an achromatic refractile capsule. Digitized by Microsoft® THE BLOOD 577 (s) Filariasis. This is a condition associated with the presence of filariae in the blood {filaria sanguinis hominis). While many of these filariae' are known; the most common one is the filaria Bancrofii (filaria nocturna). These are from 270 to 340 microns (0.2 to 0.3 mm.) long and from 7 to 11 microns broad. They are enclosed in a sheath which is considerably longer than is the parasite and shows fine cross striations. The anterior end of the parasite is abruptly rounded and has a six-tipped prepuce and a sharp fang, while the posterior end tapers for about two-fifths of the length of the parasite. The median axis of the parasite is granular. The movement of these parasites is distinctly progressive at first as seen under the microscope, but they soon become motionless, appearing to attach themselves to the glass slide at their anterior end. Strangely enough these embryos appear in the circulation only toward 'tD Fig. 147. — Filaria Bancroffi. {Da Costa.) evening, their numbers gradually rising to a maximum about midnight and diminishing toward dawn. During the day they are found in the internal organs, especially the lungs. The forms appearing in the blood are prac- tically all embryos, as the adult types lie in the lymphatics where they ob- struct the lymph flow. The obstruction in the lymph-glands may also be brought about by the eggs, which are 25 to 38 microns long by 15 broad. The embryos reach the general circulation only through the thoracic duct. The female filaria is 85 to 150 mm. long with a distinct neck, a head with a simple, minute, terminal mouth, and a plain cylindrical body covered by a striated cuticle and tapering 1 Wellman and Johns (Jour. Am. Med. Assn., 1912, LIX, 1531) have succeeded in culti- vating the filaria immitis on artificial media. See FUlIeborn, in KoUe and Wassermann's Handbuch der pathogen. Mikroorg., 1913, VIII, 185; Johns and Querens, Am. Jour. Trop. Dis. and Prev. Med., 1914, I, 620; Smith and Rivas, Ibid., 1914, II, 357; Dutcher and Whitmarsh, Ibid., X91S, HI, ^f^^fS'hyWcmS^^h^"'''-' '"''' ''™' '^^ 578 DIAGNOSTIC METHODS toward the neck and tail. The tail ends bluntly and has a small depression surrounded by two lips. The male is about 80 mm. long, without a neck and having a tendril-like tail rolled into one or more spirals. Like the malarial organisms,, the filaria has an intermediate host in the mosquito, both of the culex and anopheles variety. The embryos, which are taken up by the bite of the mosquito, cast off their sheath in about one hour in the stomach of a mosquito. Some of these embryos die at this stage, but others bore actively through the intestinal wall to the muscle, where they remain. In the next two or three days the embryo becomes larger and its alimentary tract develops. On the seventh day the worm is about ij^ mm. long and is perfectly developed. It now travels toward the head and takes its position in the labium, whence it enters the blood of its new host during the biting by the insect. A large number of these adult forms is neces- sary to cause very severe cases and many years pass before any symptoms are manifest. In examining the blood for the filaria, it is best to take a specimen late, at night and to make a very thick, fresh specimen which should be examined with a low power. Besides the ordinary anemia which may develop in such cases, we find a very striking eosinophilia which may run from 4 to 17 per cent. A very characteristic finding in such cases is the condition of hematochy- luria followed by chyluria. This hematochlyuria seems to be due to rupture of the varicose lymph- vessels of the bladder, as these form a large part of the collateral circulation when the thoracic duct is occluded (Emerson). Such attacks may occur for years and be separated by long intervals. Their onset is spontaneous or following exertion and is usually associated with pain and fever. The urine shows the presence of blood, chyle (as high as 3.8 per cent, fat), and embryos. Many other forms of filariae are known, but this Bancroft type seems to be the more important. While this disease occurs endemically in the tropics there are undoubtedly many cases in this country. It is, therefore, wise in a case showing lymph tumor, elephantiasis, and hematochyluria, especially when pain and fever and enlarged spleen are present, to examine the blood for the filaria Bancrofti. (6) Syphilis. The search for the etiologic factor of S3fphilis has extended over a period of many years and various organisms have been described, from time to time. However, none of these former parasites have stood the test of criticism. Through the work of Schaudinn and Hoffmann in 1905, a parasite was dis- covered which was so constantly associated with syphilitic lesions, whether primary, secondary or tertiary, that it was almost uriiversally regarded as the causative agent in this disease. The cultivation of this organism in pure culture by Noguchi (see p. 581) and the demonstration of the pathogenic properties of this cultivated parasite have definitely established the etiologic relationship of this organism to syphilis. This organism was called the spirochete pallida or tre^pife®(Sig^}a(iiWfeposo/?® THE BLOOD 579 While it is now a relatively simple procedure and a matter of almost daily laboratory routine to demonstrate the presence of these organisms in the pri- mary and secondary syphilitic lesions or in the blood of affected patients, the detection of the spirochete in the tertiary stages or in .the parasyphilitic conditions is not by any means always successful. It is in the latter types of this disease that the Wassermann and luetin tests are so frequently called upon to settle the diagnosis, as the primary and secondary stages are more amenable to direct diagnosis. It has, of course, been known for some time that chronic nervous condi- tions, such as general paralysis and tabes dorsaliSj were closely associated with long-standing syphilitic infection, but this relationship remained to be clearly proven. Noguchi and Moore' have demonstrated the presence of the tre- ponema in the brain tissue of paretics and also in the posterior columns of the spinal cord in tabes. These results have been abundantly confirmed by others. It is true that the results show only about 25 per cent, of positive findings in these cases but this is not surprising in view of the latent character of the infection. Such a result indicates, merely, the association of these organisms with the pathologic lesions of the disease and does not prove their absolute etiologic relationship to syphUis in general. Later work by Noguchi and others has clearly shown that typical syphilitic lesions may be caused in rabbits by inoculation of an emulsion of the brain of a paretic individual. The results show about the same percentage of successful inoculations as do those obtained from direct histologic examination of the tissues.^ Graves' goes even a step further and believes that his experiments show that rabbits may be infected with syphilis directly from the blood of general paretics. The proof is, therefore, absolute that these diseases, general paralysis and tabes dorsalis, are the result of actual syphDitic infection of the brain and spinal cord. It is evident, therefore, that the Wassermann and luetin tests can be of inestimable service in such cases inthe way of clearing up a diagnosis. The spirocheta pallida^ (treponema pallidum) derives its name from its low refractive power and the difficulty with which it takes up anilin dyes. It has a very delicate structure, usually presenting 10 to 40 deep spiral incurva- tions in the larger specimens or only a few in the smaller ones. Its length varies between 4 to 10 microns and its width does not exceed J^ micron. The organism has been demonstrated in the circulating blood, in the scrapings obtained from the chancre, in the incised papules, in smears from the mucous patches, and in the fluid aspirated from the inguinal glands. It seems to be easily demonstrable in the blood from a splenic puncture, while in the congeni- tal forms it is found in the internal organs and in the peripheral blood. A ' Jour. Exper. Med., 1913, XVII, 232; Noguchi, Jour. Am. Med. Assn., 1913, LXI, 85; Munch med. Wchnschr., 1913, LX, 737; Jour. Cutan. Dis., 1913, XXXI, 543; Forster and Tomasczewski, Deutsch. med. Wchnschr., 1914, XL, 694. ^ See Hoche, Med. Klin., 1913, IX, 1065; Forster and Tomasczewski, Deutsch. med. Wchnschr., 1913, XXXIX, 1237; Wassermann, Ibid., i28i;Levaditi, Marie and Bankowski, Ann. de I'lnst. Pasteur, 1913, XXVII, 577; Nichols and Hough, Jour. Am. Med. Assn., 1913, LXI, 120; Wile, Ibid., 866. 'Jour. Am. Med. Assn., 1913, LXI, 1504. *See McDonagh, Brit. Jour. DermatoL, 1912, XXIV, 381; and Ross, Lancet, 1912, II, "°5- Digitized by Microsoft® S8o DIAGNOSTIC METHODS characteristic difference between this spirocheta and some other types (spirocheta buccalis), with which it might be confused, is that its ends lie above and below a longitudinal line drawn through the center of its curvatures, while in the other forms the ends lie on the projection of such a line. The organism moves in an oscillatory manner about its longitudinal axis, its. movements being winding, bending and whipping, while in the spirilla the longitudinal axis remains rigid. Schaudinn demonstrated the existence of a flagellum at each end, while the other spirochetas have an undulating membrane. Fig. 148. — Spirochetae pallidae and refringens. (Piifield.) The darker ones are the refringens. These organisms are seen only with great difficulty in the specimens of fresh blood, but thanks to the introduction of the ultra-condenser (the dark- field illuminator) we are in a position to see these organisms, both in the splenic and peripheral blood, although considerable practice is necessary prop- erly to adjust the light.' These organisms do not take anilin dyes readily, so special methods have been advanced for their demonstration in smears. A very good stain for them is the Goldhorn stain. The smears are fixed with pure methyl alcohol for 15 minutes and then covered with the stain (polychrome methylene blue) for three to five seconds, when the excess is drained off. The specimens are then slowly introduced into clean water with the film sides down. Keep the slide in this position for four to five seconds and then shake in the, water to remove the excess of the dye. The 1 See Sidwell and Smith, 111. Med. Tour., 1914, XXVI, 418. Digitized by microsoft® THE BLOOD S8i spirochetae appear of a violet color. This violet tint may be changed to a bluish-black by covering the specimen with Gram's iodin solution for 15 to 20 seconds, after which it is washed and dried as usual and the examination made with the immersion lens. The writer has also found the use of the Giemsa stain very reliable, especially when the staining is continued for 18 hours (see Exudates). Other stains, such as that of Levaditi, have been advo- cated, but they do not seem to give any better results and are more complicated. For staining the spirochetse in tissues the Levaditi stain is admirable. The examination of the blood is very often disappointing, owing to the fact that few spirochette may be present in the specimen. Better results t'lc. 14Q. — Ultra-condenser of Reichcrt. are olHained by examination of specimens from a curettage which has been carried sufficiently far to allow serum to appear. This serous fluid is then spread upon slides and treated in the usual manner (see Exudates). Cultivation of Treponema Pallidum. As previously stated, the causative factor of syphilis has been definitely settled only by fullrlling Koch's postulates regarding pure cultures and the production of the disease by means of these pure cultures. Schereschewsky,^ Aliihlens^ and Hoffmann^ were able to cultivate the pallidum but were unable to reproduce syphilitic lesions by means of their cultures. Bruckner and Galasesco^ and Sowada'^ reported the successful reproduction of the lesions by injection of their ''young impure cultures" but, as neither investi- gator was able to grow a second generation of these so-called cultures in ' Deutsch. mecl. Wchnschr., 1909, XXXV, 835, 1260 and 1652; Ibid., 1913, XXXIX, 1408. ■ Deutsch. med. Wchnschr., 1909, XXXV, 1261; Klin. Jahrb., 1910, XXIII, 339. ~ Ztschr. f. Hyg. u. Infcctkr., 1911, LXVIII, ' Compt. rend. Soc. de biol., ^Xiio^LyiVIIl, (j&a. „^. 5 Deutsch. med. Wchnschr., J^igif«&&yLlm>fpmS®lilin.. 1914, X, 161. 582 DIAGNOSTIC METHODS any medium, their results are questionable. Recently Noguchi' has suc- ceeded not only in producing many generations of pure cultures of the pallidum but, also, in reproducing the syphilitic lesions by use of these pure cultures. His work is of the greatest importance as it absolutely establishes the spirocheta pallida as the etiologic factor of syphilis. His technic is as follows: As the material for obtaining the cultures he uses the spirdchetse-containing testicular tissue of rabbits, which have been inoculated with human syphilitic material. From this first generation in rabbits any number of generations may be derived by transmitting the strain, at appropriate intervals (four to six weeks), from rabbit to rabbit. By this means the spirochetse are almost free from banal bacteria. The culture media is the following: 16 c.c. of serum- water (3 parts dis- tilled water and i part serum of sheep, horse or rabbit) are placed in tubes 20 cm. long and 1.5 cm. wide. These tubes are then sterilized by fractional sterilization at ioo°C. for 15 minutes on each of three successive days. Then a small piece of freshly removed sterile rabbit tissue (preferably kidney or testicle, although heart muscle may be used but not liver) is placed in each tube which is then incubated at 37° C. for two days and examined for sterility. To each tube a layer of sterile parafl5.n oil is added to shield the medium from contact with air and to prevent evaporation. The method of cultivation must be strictly anaerobic in obtaining the first generations of pallida. No- guchi employs a combination of hydrogen gas, vacuum and pyrogallic acid in an anaerobic apparatus, which is fully described in his article. He believes the following conditions essential in obtaining his first generation: (i) the presence of suitable fresh sterile tissue in serum-water, (2) strict anaerobiosis, (3) a slightly alkaline reaction as furnished by the serum and tissue, and (4) a temperature of about 35 to 37° C. When once adapted to the artificial serum-water tissue medium, the pallidum grows well under less strictly anaerobic conditions. In fact, certain strains even grow well in a medium containing agar, provided suitable fresh tissue is placed low down in a high column of medium (serum-water or serum agar), and is covered with paraffin oil. Out of ten strains of treponema pal- lidum, six have been cultivated in the above medium. The difficulty in this work seems to be to obtain the first generation. The culture may be purified by permitting it to grow through a Berkefeld filter, which the pallida pass about the fifth day. Certain strains grow together with bacteria along the stab canal in a serum-agar tissue medium. But while the bacteria do not grow out into the surrounding medium, the pallida grow out gradually in a pure state. The characteristics of this pure culture may be obtained from the literature.^ The morphology and motility are quite typical, difficulty being experi- enced in distinguishing these points from those of specimens taken from ' Jour. Exper. Med., 1911, XIV, 99; Ibid., 1912, XV, 90; Ibid., 201; Ibid., 1912, XVI, 211. See, also, Nichols, Ibid., 1914, XIX, 362; Hartwell (Jour. Am, Med. Assn., 1914, LXIII, 142) reports the isolation of this organism from the blood. " See Meirowsky ("Studien iiber die Fortpflanzung von Bakterien, Spirillen und Spiro- chaten," Berlin, 1914) for a discussion of the proper classification of this organism. Digitized by microsoft® THE BLOOD 583 human lesions. These pure cultures produce typical lesions when injected into animals, thus completing the chain of evidence. ^ (7) Yellow Fever. This infectious noncontagious febrile disease is caused by a specific organism not yet discovered. This etiologic factor is, however, blood-borne, as shown by the fact that injection of blood from a yellow-fever patient into a healthy subject causes the disease. Whatever the organism be it must be very minute, as the blood serum retains -its infecting power after passage through a Berkefeld filter. The various organisms which have been held responsible for this disease, among them the Bacillus X of Sternberg and the Bacillus icteroides of Sanarelli, have all been abandoned and even the myxo- coccidium stegomyiae of the Yellow Fever Commission is at present con- sidered as foreign to yellow fever. In 1 881 Finlay advanced the hypothesis that yellow fever was transmitted to man only through the bite of a mosquito of the Culex group, the stegomyia fasciata. The United States Commission, consisting of Reed, Carroll, Agra- monte, and Lazear, furnished the experimental proof that this hypothesis was valid and showed that the unknown organism of the disease required a period of 1 2 days' development in the body of the mosquito before it could be trans- mitted from the stegomyia as an infecting agent. A second U.S. Commission, consisting of Parker, Pothier, and Beyer with the help of Smith stated in 1903 that yellow fever was due to a parasite of the sporozoan type, the myxococ- cidium stegomyiae, which developed in the stegomyia. This organism has never been found in the human body, hence its schizogony (asexual develop- ment) is unknown. The French Commission, Marchoux, Salimbeni, and Simond, as well as Schaudinn and Carroll, do not believe that this organism has anything to do with yellow fever. ^ Seidelin' has reported the finding of a parasite, a protozoon, in the blood, which he believes the causative factor. As this disease is beyond question blood-borne, its hematological changes are of some interest. , Jones shows that anemia is infrequent, that fibrin formation is deficient, that the globucidal action of the serum is greatly increased, and that both cholemia and hemaglobinemia occur. The red cells show little variation in number, Pothier never finding them below 4,280,000. ^ In his later work Noguchi has cultivated the organism directly from the lesions in man, using a high cylindrical layer of solid medi^. consisting of two parts of 2 per cent, slightly alkaline agar and one part of ascitic or hydrocele fluid, at the bottom of which has been placed a fragment of sterile tissue. The small pieces of tissue taken from the lesion are inserted deep into this media and the growth carried on as outlined above. See, also, Baeslack, Jour. Infect. Dis., 1913, XII, 55. Noguchi has, also, cultivated various types of spirochetae, which closely resemble the pallida, and has thus shown their morphological variations (Jour. Exper. Med., 1912, XV, 466; 1912, XVI, 194, 199, 261 and 620; Ibid., 1913, XVn, 89). See also, Clark and Gates, Am. Jour. Dis. Child., 1915, IX, 126; Zinsser Hopkins and Gilbert, Jour. Exper. Med., 1915, XXI, 213. Zinsser and Hopkins (Ibid., 576) as, also, Kissmeyer (Deutsch. med. Wchnschr., 1915, XLI, 306) have shown the pres- ence of antibodies (agglutinins) for the treponema pallidum in tie blood of syphilitics. ^ See Yellow Fever, Senate Document No. 822, Washington, 191 1. ' Jour. Path, and Bacteriol., 1911, XV, 282 Otto, in KoUe and Wassermann's Handbuch der pathogen; Mikroorg., 1913, VIII, 623. See, also, Wenyon and Low, Jour. Trop. Med. and Hyg., 1914, XVII, 369; Ibid., 1915, XVIII, ss; Camerer, U. S. Naval Med. Bull., 191S, IX, 65. Digitized by Microsoft® 584 DIAGNOSTIC METHODS The hemoglobin suffers- considerable loss, being usually between 50 and 75 per cent. This loss is restored very slowly in convalescence. Albertoni draws attention to the lack of parallelism between the percentage of hemo- globin and the specific gravity of the blood, the latter falling much more than the former. Morphological changes in the reds are rare. An occasional normoblast may be seen. The leucocytes range between 4,660 and 20,000, the higher the count the more favorable the prog- nosis. In this leucocytosis the polynuclear neutrophiles are in higher proportion, only rarely be- ing normal. Eosinophiles are few and myelocytes only occasional. (8) Rocky Mountain Spotted Fever (Tick Fever). This disease is not to be con- fused with typhus fever or epidemic cerebro-spinal meningitis, to both of which the term " spotted fever" has been occasionally applied. Wilson and Chowning have reported that the blood of man affected with Rocky Mountain spotted fever shows the presence of an erythrocytic parasite which they call Piroplasma hominis. These parasites are ovoid in form, ^ , . , , . , have ameboid motility, and are Fig. 150. — Schistosomum hematobium; male . , ^, , , with female in gynecophoric groove. {Tyson unpigmented Ihree forms ot after Loos.) these intracellular ovoids were found: (i) a small, non-motile form, I to 2 microns in length by i micron in width; (2) a larger actively ameboid form, 3 to 5 microns in length by i to 1.5 microns in width, and showing a dark granular spot at one end; (3) a twin form, consisting of two pear-shaped bodies, lying with their tapered ends approaching and bear- ing a granulai^ .spot at each end. These bodies stain best with the polychrome dyes. Through the work of Ricketts and King it has been definitely established that the parasite of spotted fever finds its host in the wood tick (dermacentor venustus (Banks) or, as Stiles states, the dermacentor andersoni). There is apparently no cycle of development in the tick as an intermediate host. Ricketts advances much evidence against the piroplasma as the causative fac- tor, but is unable to say that such might not be the etiologic unit. After Digitized by Microsoft® THE BLOOD 585 much research he^ succeeded in finding an extra- and intra-cellular pleomor- phic polar staining bacillus, which is extremely small and is constantly present in the blood of infected animals as well as in the infecting tick. Whether this is the. true etiologic factor is still unsettled, but Ricketts believed it was.^ The red cells in this disease are reduced to about 4,000,000, while the hemoglobin content may be as low as 50, thus giving a low color index. Degenerations and atypical staining qualities are rare. The leucocytes are increased to 12,000 or more and show nothing differentially abnormal, except a slight increase of the large lymphocytes. (9) Distomiasis (Bilharziasis). This is la chronic parasitic disease due to the deposition in the tissues of the eggs of the worm, Sckistosomum hematobium, also called Bilharzia hema- tobia, gynecophorus, distomum hematobium, distoma capense, and Thecos- oma. It is a very common condition in Africa, but has been found but six times in America, according to O'Neil. Infection appears to be more com- monly induced by drinking unfiltered infected water, but occasionally it may come through the skin.' The adult parasites inhabit the blood of the portal vein and the vessels of the pelvis, rectum, and bladder. The male is smaller and thicker than the female, is 12 to 15 mm. long and i mm. broad, is flat and so folded as to form a gynecophoric canal which holds the female. The female is about 20 mm. long and 0.25 mm. thick and is the active agent in the infection. The eggs of the parasite are oval or spindle-shaped, measure about 0.16 mm. in length and 0.05 mm. in breadth, and have a distinct spine-like pro- jection from the posterior end or from one side. These ova are particularly frequent in the urine of such cases. Occasionally they may be found in the circulating blood. VI. Bactekiology of the Blood From the standpoint of differential diagnosis and treatment of a con- dition known or thought to be of infectious origin, it is frequently of especial importance carefully to examine the blood for the presence of various bacteria. Moreover, in many conditions, as for instance in typhoid fever, cultures made from the circulating blood often give positive findings long before other tests are obtainable. The bacteriological study of the blood requires not only a thorough working-knowledge of the principles underlying the bacteriological technic but, in many cases, special training, also, in the isolation and differ- entiation of the suspected organism.^ Technic. The success of this work depends partly upon the obtaining of a suffi- cient quantity of blood and partly upon the selection of the culture-media for ' Jour. Am. Med. Assn., igog, LII, 379. * See McClintic (Pub. Health Rep., 1912, XXVII, 732) for an excellent bibliography of the subject; also, Rucker, Ibid., 1912, XXVII, 1465. ^ SeeLooss, in KoUe and Wassermann's Handbuch d. pathogen. Mikroorg., 1913, VIII, i; Kartulis, Ibid., 23; Bovaird and Cecil (Am. Jour. Med. Sc, 1914, CXLVIII, 187) report two cases of infection with the closely related schistosomun japonicum. CI * See Cummer, Ohio State^e^^.Jo^.^i^^j X^|oi^|yntz, New York Med. Jour., 1915, S86 DIAGNOSTIC METHODS the future development of the organism present. The amount of blood re- quired varies from 5 to 20 c.c.,so that it must be obtained by venous puncture. The vein usually selected is the median basilic or the median cephalic at the bend of the elbow, but, if these be not available, the veins of the dorsum of the hand may be used, although the latter are less satisfactory. The site of puncture should be carefully cleansed, using all the precautions taken for any surgical procedure. As a rule, contaminations from the skin are rare pro- viding this cleansing has been adequate. In some cases, especially in very obese patients, it is necessary to expose the vein by incision of the skin and subcutaneous fat over the vein. The instrument best adapted for venous puncture is, in the writer's opin- ion the Liier syringe, which is made entirely of glass and has a tightly fitting platinum needle.^ Sterilization is best done by the use of dry heat to iso°C. for one hour, boiling in i per cent, sodium carbonate solution for 45 minutes, or using steam under pressure in an autoclave for 15 minutes. These instru- ments should never be sterilized with carbolic acid or bichlorid solution owing to the danger of inhibiting the growth of the suspected organisms. It is well, also, to sterilize a pair of forceps to use in fitting the needle on the s3T:inge. The properly sterilized outfits should be kept in long test-tubes, plugged with cotton, the point of the needle being protected by resting upon a pad of sterilized cotton. When the above precautions have been taken, an elastic band or a towel may be fastened about the arm above the point of puncture, to produce con- striction of the vessels and distention at the point of puncture. The skin may be anesthetized with a spray of ethyl chlorid, if the patient be very sus- ceptible to the slight pain caused by the puncture. The needle isplunged into the vein against the direction of the blood current, care being taken that the point of the needle is sharp as, otherwise, the vein may roll about beneath it. Very slight a'spiration is necessary to draw the blood into the syringe, as the blood tends to flow into the instrument through its own force. The usual amount withdrawn is 10 c.c. but occasionally 20 c.c. are preferable, especially if the patient is plethoric and can stand the loss easily. AVhen the desired amount is obtained, the bandage is removed before the needle is withdrawn. After withdrawal from the vein, the needle is quickly removed from the syringe and the blood divided among several tubes containing agar melted and cooled to 40°C., the tip of the syringe being passed through the flame before inoculating each tube. In deciding as to the amount of blood to add to the agar, a general rule is to increase the amount of blood when the feeble- growing organisms, such as the pneumococcus and gonococcus, are sus- pected. The usual proportions are 2 to 3 of blood to 5 of agar. It is, probably, preferable to add the blood to fluid media, such as bouillon or litmus-milk, using i to 2 c.c. of blood to 100 c.c. of media in order to over- come the normal bactericidal effects of the blood.'' The blood and fluid 1 See Judd and Simon (Jour. Am. Med. Assn., 1915, LXIV, 822) for the use of the Keidel tube charged with culture medium. ^ Hiss and Zinsser (Text-book of Bacteriology, p. 126) recommend the addition of a small piece of calcium carbonate to the liquid media in order to neutralize any free add formed in case the pneumoMccus-is^susneqtai See^^o, Gillespie (Jour. Exper. Med., 1913, XVIII, 584) for compyM¥&^Smtf^''Sm'im solid media. THE BLOOD 587 media are then directly incubated'at 37°C. In the case of the agar tubes, the blood and media are mixed by shaking as quickly as possible, poured into Petri dishes and placed in the incubator. It is advisable to use both solid and fluid media. Anaerobic cultures are prepared as usual, using, preferably, a combination of a vacuum with alkalinized pyrogallic acid solu- tion to insure absence of oxygen. If, after incubation for 24 hours, the plates show only a few surface col- onies, contamination is to be considered, while if the colonies be deep and are observed in several plates, contamination may be ruled out. This must not be interpreted to mean that any colonies will, necessarily, develop within 24 hours, as the plates may have to be kept for 5 to 10 days before a negative result may be certain. However, the contaminating organisms usually show up within 24 hours. The fluid media will become cloudy if any organisms are present. From the plate colonies and the fluid media sub-cultures are then made upon different media and every expedient possible used to isolate and identify the offending organism. Usually the infecting organism may be obtained in pure culture as a mixed infection in the blood is very uncommon. Organisms Found in the Blood. I. Bacillus Typhosus. Instead of transferring the blood, withdrawn from the vein, to bouillon or melted agar, one may add it directly to sterilized ox-bile in the propor- tion of I or 2 c.c. of blood to 5 of the bile. The blood may, if desired, be obtained in this case from a puncture of the ear (the puncture being prefer- ably made through a coating of collodion and the blood allowed to drop directly into the tube of bile). The mixture of bile and blood is allowed to incubate at 37°C.for 12 to 15 hours, when transfers of a fewloopsful are made , to the Drigalski-Conradi or modified Endo media discussed under Feces. The characteristic colonies are then sub-cultured and the agglutination tests made as described in the next section. According to KoUe and Hetsch,' this bacillus of. Eberth-Gaffky shows the following characteristics : it is an actively motile bacillus with numerous flagella, stains with the ordinary dyes but is Gram-negative, produces no indol in bouillon (colon bacillus does), produces no gas in glucose-bouillon (colon bacillus does), produces no acid nor any coagulation in litmus-milk (colon bacillus produces much acid, while the bacillus f aecalis alcaligenes is a strong alkali-former), produces no change in neutral red-agar (colon bacillus and paratyphoid bacillus B cause fluorescence of the solution and gas forma- tion, while the bacillus f aecalis alcaligenes and the dysentery bacillus produce no effects), produces dew-drop colonies on litmus-lactose-agar with no change in color of the media (colon bacUlus produces marked red coloration), pro- duces acid and coagulation of the casein in litmus-nutrose-glucose solution, while no such effects are observed in litmus-nutrose-lactose solution, shows characteristic agglutination with pure culture of typhoid bacillus. Ordinarily the agglutination tests are all that are necessary after the organism has been grown on the Drigalski-Conradi or other media. However, ' Die Experimentelle Bak^naJo|U^ ^Be^^^^ii.^^ 588 DIAGNOSTIC METHODS it is to be remembered that the freshly isolated organisms show less tendency to agglutinate than do the organisms which have been grown for some time or have been transferred through several generations. The importance of blood cultures^ in cases of typhoid fever may be esti- mated by the experimental evidence that 70 to 80 per cent, of cases are posi- tive, oftentimes many days before the agglutination tests are positive. It has been further known that the organisms tend to diminish in the blood during the course of the disease until they practically disappear from the blood about the end of the third week. 2. Bacillus Paratyphosus. In certain cases, which clinically closely resemble typhoid fever, although running a somewhat milder course, organisms may be isolated from the blood, which are intermediate between the true bacillus typhosus and the bacillus coli communis. The clinical differentiation of these cases is often difficult, although the sudden, onset of vomiting, diarrhea, chill, and fever of paraty- phoid are somewhat different from the more gradual onset and step-like in- crease of temperature in typhoid; while the temperature-curve, the somewhat different early characteristics of the feces, the early appearance of herpes, the usual lack of splenic enlargement, etc., may serve as differential points in favor of paratyphoid. ^ Aside from this type, we find sporadic or epidemic cases in which the symptomatology is that of a severe gastroenteritis, the so-called "infectious meat-poisoning" usually classed as ptomaine poison- ing, which is due to the bacillus paratyphosus. These organisms are widely spread in the animal kingdom and may be found even as a contamination in water supplies. Not infrequently it is found in man, as a pure saprophyte, no clinical symptoms traceable to its presence being observed. It may be present in the pus of otitis media, orchitis, cholecystitis, osteochondritis, arthritis, lymphadenitis, also, as a secondary invader, in scarlet fever, measles, pneumonia, meningitis, etc. The examination o^ the blood is made as previously described, transfers the sub-cultures being made upon the various media. These paratyphoid bacilli are, apparently, of two types, known as "A and "B". Both types differ from the bacillus typhosus in that they produce gas in glucose media and show different agglutination reactions. Type A behaves in most other respects like the typhoid bacillus, except that the former causes fluorescence in neutral-red agar. Type B is differentiated from the typhoid bacillus and from type A of the paratyphoid bacillus by not coagulating litmus-milk but in producing acid (red coloration) and, after 5 to 10 days, in causing a conversion of this red color to a deep blue (due to alkali formation). The typhoid, colon, paratyphoid A and dysentery bacilli never cause a blue ' This work is fast becoming an absolute necessity, as the employment of the preventive inoculation for typhoid practically destroys the reliability of the Widal test of the serum of those previously Inoculated. See Moreschi, Ztschr. f. Immunitatsforch., 1914, XXI, 410; Mcintosh and McQueen, Jour. Hyg., 1914, XIII, 409; Saski, Ztschr. f. klin. Med., 19141 LXXX, 79; Carnot and Weill-Halle, Presse m6d., 1915, XXIII, 89; Hohlweg, Munch, med. Wchnschr., 1915, LXII, 538; Lipp, Ibid., 539. " See Hunt, Arch. Int. Med., 1913, XII, 64; Heigel, Wien. klin. Wchnschr., 191SJ XXVIII, 57; Dreyer, Walker and Gibson, Lancet, 1915, 1, 324 and 643; Irons and Jordan, Jour. Infect. Dis., 1915, XVgj^^ggp]^^p^g\^^d. Research, 19x5, XXXII, 399-. THE BLOOD 589 coloration of the milk, while the blue color due to the bacillus faecalis alcaligenes is produced at once. The peculiarities of the agglutination reac- tion with this organism will be discussed in the next section. 3. Bacillus Coli Commtinis. This organism is, probably, not a unitary organism, as many types of the species exist. The colon bacillus is found in all warm-blooded animals and in many cold-blooded ones as a normal habitant of the intestinal canal. These bacilli show a marked antagonism for the putrefactive bacteria, so that physiologically, they should be regarded as conservative organisms. Owing to the wide occurrence of this organism, it is frequently found as a contamination of food and water supplies unless special precautions are taken to exclude it. Although ordinarily a harmless type, yet, from the pathologic stand- point, the colon bacillus is generally regarded as a pathogenic organism under certain conditions. In this connection we must distinguish an endogenous from the exogenous colon infections. The first are found in cases of wound infection and, more especially,- in infections of the lower urinary tract into which the bacilU have gained entrance through the urethra; while the latter are observed under the following conditions. So long as the natural protective and bactericidal agents of the body are intact and so long as the intestinal epithelium is unimpaired, the colon bacilli do not pass into the organs or serous cavities of the body. However, in the course of any disease, such as typhoid, cholera and severe non-specific enteritis, in which the epithelium is destroyed, the colon bacillus may pass from the intestinal tract, either by direct contiguity of tissue or through the blood current, and assume a pathogenic r61e as a secondary invader. Moreover, should the patient be suffering with a chronic wasting disease which lowers the general resistance to infection, the continuity of the epithelium may still be preserved and the bacillus become pathogenic in many organs. Thus we find infections due to the bacillus coli communis in the kidney, urinary bladder, gall-bladder, peri- toneal cavity, ovary, uterus, and other organs. Pus poekets, which may have caused little clinical disturbance, may spread, become chronic and induce a septicemia, of which the etiologic factor may be determined only by resort to blood culture. The biologic properties distinguishing the colon bacillus from the typhoid and paratyphoid baciUi are as follows: The colon bacillus is very little, if at all, motile; it produces gas in glucose media and indol in bouillon or pep- tone media; it coagulates milk and produces acid with red coloration in litmus milk; its colonies on litmus-lactose agar are deep-red in color (the color diffus- ing into the surrounding media), while those of typhoid and paratyphoid are colorless; in neutral-red agar it causes fluorescence and marked gas pro- duction, while the typhoid bacillus produces no change in the media; it does not show characteristic agglutination reactions, while the typhoid bacillus does. 4. Pneumococcus. This organism may be recovered from the blood in practically all cases of lobar pneumonia due to infection with this t3^e, although it must be remem- bered that other organi^i9"fe§^ ^¥ Mipf&M^^c factor iij some cases. At 59° DIAGNOSTIC METHODS times the pneumococcus may be obtained as early as 12 hours after the initial chill, while in other cases it may appear as late as 48 hours after the crisis. Rosenow obtained positive results in 160 out of 175 cases, Prochaska 48 out of 50, whUe Cole and Lenhartz report only 30 per cent, of positive findings. ^ The finding of the pneumococcus in the blood does not, necessarily, indicate an un- favorable prognosis, although a large percentage, 40 to 50, of the cases in which it is found result fatally.^ The earlier the organisms are found the more grave the prognosis. The number of organisms found by culture is not as large as in other types of blood infection, so that Fraenkel is lead to be- lieve that the prognosis is bad when a large number of colonies develop from I c.c. of blood. The infection may spread through the lymphatic channels to the blood, so that we may find these organisms in the bUe, urine, milk and other body secretions. Moreover, we may have many infections in other organs as a complication of the original pneumonia. Oftentimes, we find the pneumococcus as the cause of primary patho- logical conditions in many organs, when there has been no antecedent pneu- monia. Thus we may have a pneumococcic otitis media, bronchitis, pleuritis, peritonitis, meningitis, endocarditis, pericarditis, orchitis, tonsillitis, cystitis, salpingitis, enteritis, conjunctivitis and keratitis. The causative factor may be discovered only by examinations of the various excretions related to the special condition. While the blood culture may be of little clinical value in the diag- nosis of lobar pneumonia as the chnical findings are usually so clear-cut, yet it may be the only means at our disposal in solving the mystery of an obscure infection in other parts of thebody,as for instance in a malignant endocarditis.' The blood is taken as previously outlined and is transferred to agar, blood-agar, plain, serum or ascitic bouillon, or litmus-milk. The organisms which are especially confusing from the standpoint of differentiation are the various types of streptococci. On blood-agar plates the pneumococcus shows an especially characteristic greenish hemolytic colony. The streptococcus vjridans produces the same green zone, while the streptococcus pyogenes shows no green color but a large colorless hemolytic zone. Blood bouillon shows no hemolysis with either the pneumococcus or streptococcus viridans, while a marked burgundy-red color appears in the presence of streptococcus pyogenes. The differentiation between the pneumococcus and streptococcus viridans is made by the use of bile acids. Bouillon cultures of pneumococcus are cleared while those of the viridans are unaffected. Further, the pneu- mococcus ferments inulin solutions, while most strains of streptococci do not produce this result. The detection of the capsule and the agglutination re- actions clear up the diagnosis. Rosenow's Capsule Stain. This method* is, in the writer's opinion, the best at our command for this. purpose. The technic is as follows: Make a thin smear on a perfectly 1 SeeLyall, Jour. Am. Med. Assn., 1912, LVIII, 1841. " See Hastings and Boehm, Jour. Exper. Med., 1913, XVII, 239. * See Rosenow, Illinois Med. Jour., 1912, XXI, 425; Jour. IiJect. Dis., 1912, XI, 94; also, Cole (Jour. Am. Med. Assn., 1912, LIX, 693), for a discussion of immunity in pneu- "°"V-°InfecfDi"Nfxt?i^ THE BLOOD 591 clean slide or cover-glass. If the material, such as sputum, is too thick, add enough distilled water so that it can be spread evenly by means of a piece of fine tissue or cigarette paper. In cases of cultures (blood-agar, serum, glu- cose or Loeffler's blood-serum being preferable), remove a small amount of the growth from the surface of the medium and at once mix thoroughly with a loopful of serum on the slide, or, better still, make a rather dense suspension in a few drops of distilled water and then mix an equal quantity of this suspension with serum, and spread by means of tissue paper. As the smear becomes nearly dry, cover for lo to 20 seconds with 5 to 10 per cent, aqueous solution of tannic acid; wash in water and blot; stain with carbol (saturated alcoholic solution gentian violet, i part; 5 per cent, aqueous carbolic acid, 4 parts) or anUin gentian violet half a minute to a minute, heat over flame but do not boil; wash in water again; Gram's iodin solution for J^ to i minute; decolorize in alcohol (95 per cent.) ; stain for 2 to 10 seconds, depending on the thickness of the smears, with saturated alcoholic (60 per cent.) solution of Griibler's eosin; wash in water and blot finally, clear in xylol and mount in balsam or examine directly. If the organism, like the Bacillus mucosus, is Gram-negative, the bacillus may be stained with Loefiler's or aqueous methylene blue. The pneumococci are stained deeply brownish-black, sharply differenti- ated from the capsule, which is stained pink. Beautiful results are also^ob- tained with the streptococcus mucosus. In the thickest part of the smear the space occupied by the capsule may be perfectly clear; elsewhere in the smear, if properly made, where the conditions are suitable for absorption of eosin, the capsule is stained deeply pink; not rarely a clear refraction zone (often mistaken for the capsule in former methods) may be seen periph- erally to a distinctly stained, often large capsule. In case of sputum in which the cocci are embedded in a more or less tenacious mucus, the cap- sules, at times, are not rendered stainable by the above method. In that case it is well to fix and stain simultaneously with the 2 per cent, aqueous tannic acid, 4 parts, and saturated solution of gentian violet, i part. This modification often gives beautiful results. The cocci, however, decolorize easily and the tannic acid-gentian-violet may be followed by carbol-gentian- violet and then the usual procedure. Ordinary carbol-fuchsin, diluted five to ten times, and aqueous eosin (50 per cent, saturated solution) may also be used to stain the capsule, although the saturated alcoholic (60 per cent.) eosin has given the best results. Decolorization after the modified Gram pro- cedure of tarmic acid-fixed smears is more rapid than in the case of heat- fixed smears, which fact should be borne in mind. By the use of this method it has been determined that the capsule of the pneumococcus and allied organisms is not difficult to preserve or readily soluble in water, as is generally believed. To stain the capsule is a problem of rendering it stainable rather than one of preservation. The reactions which accomplish this appear to be colloidal reactions. 5. Streptococci. In general iniections ^iMim^ Idf M^mSi^t^ of the streptococcus, blood 592 DIAGNOSTIC METHODS cultures reveal the offending organism in a large percentage of cases, de- pending, of course, upon the location and severity of the process. There are many varieties of streptococci, of which some are saprophytic and some pathogenic. Pathologically, streptococci are found in the blood iii many con- ditions. They may obtain entrance to the blood by rupture of a streptococcic abscess into a vessel. Infected thrombi, as met with in puerperal sepsis, may form the source of the invasion. Long-standing infections, such as empye- mas, may so lower the resistance of the patient that a septicemia may resxilt. An especial point of invasion is through wounds, the infection here often being extremely dangerous. A further type is the cryptogenetic septico-pye- mia in which the source of infection can not be traced. The organism may be obtained from the blood in practically all cases of mycotic endocarditis due to this streptococcus. In puerperal sepsis the results are not so frequent, only about one-fourth of the cases being positive. The cryptogenetic types associated with metastatic lesions in the joints and organs show a much larger percentage of positive findings. In scarlet fever we find, according to Hektoen, that streptococci may occasionally be found in the blood in cases which run a short, mild, and uncomplicated course; they occvu: with relatively greater frequency in the more severe and protracted cases in which there may develop local complications and signs of general infection, although they may not be demonstrable in fatal cases. The strep- tococci may also be found in diphtheria, measles and small-pox especially in the fatal cases, in which they act as secondary invaders. It is to be em- phasized that the prognosis is not, necessarily, bad when streptococci are found in the blood, although there is a large mortality. The morphology of these organisms is variable and the varieties numer- ous.^ The pathogenic tj^es, isolated directly from pathologic lesions,' appear to consist of rather long chains of more than 8 pairs of cocci when grown in bouillon, while the non-pathogenic types form shorter chains. Involution forms are common in specimens from culture. Some of them are encap- sulated, two organisms being enclosed. This may lead to confusion with the pneumococcus but the differential points given under the pneumococ- cus will serve to distinguish them. Usually the microscopic examination of the stained specimen will be all that is necessary, but occasionally careful work is needed to identify them. In doubtful cases, J^ to i c.c. of the blood withdrawn by venous puncture may be injected intraperitoneally into a mouse, when a general streptococcic septicemia will ensue. Many other organisms have been and may be isolated from the blood. Thus the staphylococci are not infrequently the cause of a bacteriemia arising from an endocarditis, osteomyelitis, or furunculosis. The gonococcus is often found in cases associated wtih gonorrheal endocarditis, arthritis, paro- titis, myositis, etc. The meningococcus, the influenza bacillus, bacillus mucosus, bacillus of anthrax and of glanders have been obtained from the 1 See Winslow, Jour. Infect. Dis., 1912, X, 285; Jungmann, Deutsch. Arclt. f. klin. Med., 1912, CVI, 283; and Bergey, Jour. Med. Research, 1912, XXVII, 67. See p. 40 for a discussion of streptococcic sore-throat. Also, Thalhimer and Rothschild, Jour. Exper. Med., 1914, XIX, 429 and 4445)amQjnSjjQiiaryUl(w M*d^ 1914, VII, 291; Rosfenow, Jour. Am. Med. Assn., 1914, LXIltf^'ii^mifGiMcWSt'd'^mtweileT, Ibid , 1915, LXV, 1159. THE BLOOD 593 blood in suitable cases. The tubercle bacillus has been repeatedly found in cases of both acute and chronic tuberculosis. However, much recent work since the publication of Rosenberger, who attempted to prove that tubercle bacilli were present in the circulating blood of incipient tuberculosis and that a primary bacteriemia was always present in these cases, has absolutely re- futed this finding and has emphasized the importance of eliminating the possibility of the presence of acid-fast bacilli in the reagents and, even, in the distilled water used.^ It is probably true that the organism at the bottom of every infectious disease of known origin may be obtained from the blood at some stage of the infection. It is important, therefore, that the clinical examination include blood cultures in any case of obscure origin, as in this way treatment and prognosis will be markedly influenced. Churchill and Clark^ have recently called attention to the importance of this work in children. VII. Serum Pathology This section of hematology is very closely associated with pathology and biochemistry and can, therefore, be taken up only in brief outline.' As a matter of fact, changes in the number of the cells as well as in the percentage of hemoglobin must be dependent, to a certain extent, upon the more obscure changes which are taking place in the plasma in various diseases. Our ability to fathom the secrets of the many physical and chemical changes of the plasma has been so slight that we have hitherto neglected to take into consideration anything but the changes in the cells, which can be so easily studied by the various methods previously outlined. Our knowledge of the various types of immunity to infection and of the many factors concerned therein has in- creased to such an extent in recent years that a complete discussion is out of place here. By the elaboration of the side-chain theory of Ehrlich and of the opsonic theory of Wright, we have come somewhat nearer to a proper realiza- tion of the importance of the serum in all infections as well as in many diseases in which great metabolic disturbance is evident. For that matter one can hardly imagine a condition in which the blood plasma may not show some characteristic change, inasmuch as the nutrition of the entire body can come through the blood. When one considers the close correlation of the various organs he may see at once that pathological changes in any of the viscera may result in an abnormal blood, which may show no variations at present capable of detection. For these reasons one hails with delight any advance in serum pathology and could but wish that his knowledge might more rapidly increase. 1 See Berry, Jour. Infect. Dis., 1914, XIV, 162; also, Rautenberg, Berl. klin. Wchnschr., 1914, LI, 348 and 492; Baetge, Deutsch. med. Wchnschr., 1914, XL, 591; Faber, Jour. Am. Med. Assn., 1914, LXIII, 1656; Jousset, BuU. I'Acad. de Med., 19x5, LXXIII, 203iLuc- ciarini, Rif. Med., 1915, XXX, 233 and 281; Austrian and Hamman, Bull. Johns Hopkins " Hosp., 1915, XXVI, 293; Kessel, Am. Jour. Med. Sc, 1915, CL, 377; Honeij, Jour. Infect. Dis., 1915, XVII, 376. ^ Am. Jour. Dis. ChUd., 1911, I, 193. ' See Kraus and Levaditi, Hiandbuch der Technik und Methodik der Immunitats- forschung, Jena, 1909-1911; Citron, Die Methoden der Immunodiagnostik und Immuno- therapie, Leipzig, 191 2; Dieudonnfi, Immunitat, Schutzimpfung und Serumtherapie, Leip- zig, 1913; Vaughan, Protein Split ^Products in .Relation io Immunity and Disease, Phila- delphia, 1913; Kolmer, IniecmSI fSMim^MMQ^SM Thempy, Philadelphia, 1915. 38 594 DIAGNOSTIC METHODS Ehrlich's Side-chain Theory.^ The early work of Ehrlich, published in 1885, advanced a theory to ac- count for various phases of immunity, especially of the action of the blood in producing antitoxins against various poisons elaborated by infectious agents. It is to be said that no such formation of antitoxin against the ordinary medic- inal poisons has been found. According to this theory, the protoplasm of the ceU consists of a central group of molecules (Leistungskern), in which the inherent vital characteristics of the cell are located and whose integrity is necessary for normal cell life. At different portions of the cell certain other molecular groups are attached exactly as side-chain groups are attached to the benzene nucleus of organic chemistry. These groups or, as Ehrlich styles them, side-chains are capable of uniting with various material which is brought into intimate relationship with the cell structure. Such materials are foods, toxins, and other injurious .Toxin united with cell. Receptors. "Toxophore. "Haptophore. Toxin molecule. Body cell Fig. 151. — Illustrating the mechanism of the toxin-cell union by the intermediation of receptors. {Da Costa.) agents. In order that foods may be taken up by the cell it must possess cer- tain groups which will enable it to combine with the groups in the side-chain of the cell. It must be, in other words, homologous or, as Ehrlich states, must bear the same relationship to the side-chain which Fischer has applied in his assumption of the "key in the lock" hypothesis regarding ferment action upon the various types of hexoses. In the nomenclature of Ehrlich the side-chains are styled receptors and the group of the food or of the toxin which combines with these receptors is known as the haptophore group. These receptors, as well as haptophores, possess specific affinity, uniting with one another only when homologous. It has been found that a toxin molecule has certain injurious effects upon the cell; it is, therefore, necessary to ascribe this action to other than the two groups above mentioned, as the union of a haptophore with a receptor ' In this connection see Sachs, Die Haemolysine, Wiesbaden, 1902; Kraus andLevaditi, Handb. d. Tech. u. Meth. d. Immunitatsforschung, Jena, 1907-1911; Adami, Principles of Pathology, New York, 1910; Dieudonnfi, Immunitat, Schutzimpfung und Serumtherapie, Leipzig, 1913; Kolle and Wassermannj Handbuch der pathog. Mikroorg., Jena, J912; Kolle and " - — ■ ■ .._.... Hetsch, Die Experimentelle Bakteriologie, Eerli DigitizecfbyMicrosbtm lin, 1911. THE BLOOD 595 would form an inert substance. Ehrlich, therefore, assumes the presence in the' toxin^ molecule of a second group which he styles the toxophore group, which exerts the untoward effect upon the cell. The toxophore group in it- self can unite with the cell only through the medium of its haptophore group. As the cell becomes irritated by the presence of the toxophore group, it endeavors to overcome this by a new formation of receptors. According to the strength of the irritation, many more receptors will be found that can combine with the toxin material present, so that many of the extra receptors pass out into the circulation in the form of free receptors. This is a graphic explanation of the fact that the cell when irritated by toxic material elabo- rates substances from its own protoplasm, which have a neutralizing effect upon the toxic substance. These free receptors (haptines) form the anti- toxins. They combine only with homologous toxin material and are, there- fore, specific Welch believes that these antitoxins have a second function Toxins united with cell. Free recep- — . tors; haE>tin; -:— — "^ Fig. Toxins united with antitoxin. Body cell. 152.^ — Illustrating the elaboration and action of antitoxin. {Da Cosla.) beside that of neutralization of toxin, namely, an irritating one upon the bac- terial invaders so that these organisms are forced to elaborate similar sub- stances for their own protection. Toxins which have been deprived of their toxophore group are known as toxoids and can combine with the receptors of the cell, exerting no untoward effect upon the cell. These toxoids may also unite with antitoxin through the medium of their haptophore group. Occa- sionally toxins are incompletely combined with the antitoxins; that is, the antitoxic material is not sufficient in amount completely to neutralize the toxin, so that such toxins may still combine with the cells and exert a mod- ified poisonous effect. Such attenuated toxins are known as toxones. It has been found that the injection into animals of bacteria, various body cells, and certain secretions of some animals, as for instance, snake venom, gives rise to the development of specific antibodies in the blood serum of the animal so treated, the substances injected being named antigens.^ , 'See Coca, Jour. Infect. Dis., 1915, XVII, 351. ^See Pick (in KoUe and Wassermann's Handb. d. pathogen. Mikroorg., 1912, I, 685) who shows that true antigens are always albumin-containing colloids. Also, Elliott, Jour. Infect. Dis., 1914, XV, 501; Salus, Biochem. Ztschr., 1914, LXVII, 357; Porter, Biochem. Jour., 191S, IX, I. Digitized by Microsoft® 596 BIAGNOSTIC METHODS Such blood or serum will be found to have a lytic (destructive) action upon cells similar to. those injected. Such sera are specific, that is, they act only upon the kind of cell used in the injection. The term hemolysis has been introduced to express the destructive action upon the erythrocytes shown by the dissolving out of the hemoglobin from the red cell. The stroma or dis- coplasm of the red cell is a membrane which shows peculiar relations to diffu- sion of various materials into the cell and to the passage of hemoglobin and other cellular material from the cell. Its chief function seems to be to pre- vent, as far as possible, any loss in hemoglobin. If this membrane becomes permeable, then we must assume the action of some toxic material. The term hemolysis has reference merely to the abnormal loss of hemoglobm and not to any disturbance beyond increased permeabihty of the stroma. The stroma of these cells remains behind.and may be seen in the centrifuged speci- men as the so-called shadows. Hemolysis must, therefore, be considered as a sign of protoplasmic death. Substances (hemolysins) bringing about Union of complement, ambo- ceptor, and cell. Receptors, /Zy.ij.Ja ^' Zymophore. t--J ! Haptophore. Complement Amboceptor. ' Complementophile. Cytophile.. Erythrocyte. Fig. 153. — Illustrating the mechanism of hemolysis. {Da Costa.) such change belong, necessarily, in the class of blood poisons. Such hemoly- sins are increased or lowered temperature, various inorganic compounds, such as distilled water, ammonium salts, and organic compounds, such as urea, bile acids, ether, alcohol, chloroform, solanin, saponin, and digitalin. The saponins are among our strongest hemolytic substances, acting in dilutions of I to 100,000. Besides these we have various secretions, such as those of the cobra, spider, and the bees which are active hemolytic agents. It has been observed by various workers that hemolysis is prevented when the serum is heated to 60°. This points to the fact that some substance is destroyed which is of great importance in this process. In addition, it has further been found that the renewing of the activity of the old heated serum' by adding a supply of fresh isologous serum wiU restore the hemolytic activity. It is evident, therefore, that there are two factors which must be taken into consideration, one is a thermostable (heat-resisting) substance, while the other is thermolabile (destroyed by heat). To the first of these Ehrlich has Digitized by Microsoft® THE BLOOD 597 given the name amboceptor and to the second the name complement. The amboceptor has been shown to have two haptophore groups, with one of which its unites to the receptor of the cell and with the other to the comple- ment. The haptophore group which unites with the cell is known as the cytophile, while the one uniting with the complement is termed the comple- mentophile. For hemolysis, therefore, we must have the cell receptor, the amboceptor, and the complement. The complement has also been shown to have two groups analogous to those of the toxin molecule. The first is the haptophore group, while the second is the zymophore group, through which the destructive action upon the cell is manifest. Receptors. 9^^ ^^J^ ^' ^' Erythrocyte. Fig. 154. — Illustrating the Mechanism of Antihemolysis. {Da Costa.) A, Interference of anticomplement with complement-amboceptor union. B, inter- ference of antiamboceptor with amboceptor-cell union. C, antiamboceptor-amboceptor union. D, anti-complement-complement union. The amboceptor is formed within the body as the result of cellular hyperactivity aroused by the irritant action of the toxic material. The com- plement is probably derived, for the most part, from the leucocytes, and acts very much as an enzyme. It can exert its toxic action only when united with the cell by means of the amboceptor, so that free complement has no injurious effect. It has been frequently observed that the red ceUs are more resistant than normally, while in many Cases they appear less resistant to hemolysis. This is explained by the side-chain theory very much as it explains the formation of antitoxin. These antihemolysins are formed within the blood plasma after inoculation with hemolytic material. The hyeractivity of the cell causes it to throw off two types of such bodies, namely, anticomplement and anti- amboceptors. The former combines with the haptophore group of the comple- ment and the latter with the cytophilic group of the amboceptor, each of these combinations making it impossible for the necessary union of cell, amboceptor, and complement to occur.. ... , , .,. „^ Digitized by Microsoft® 598 DIAGNOSTIC METHODS It has been found that frequently the serum of an animal, which has been injected with certain bacteria or with certain body cells, shows the peculiar property of agglutinating or clumping such bacteria or cells when these latter are added to it. This condition is known as agglutination and the agents bringing it about are styled agglutinins. These substances are developed in the blood of the animal during the process of adaptation toward the presence of such foreign material. Agglutinins from the standpoint of the side-chain theory are free receptors, having a haptophore group which unites with the receptor of the cell or of the bacterium, and cause agglutination through the presence of a second groUp known as the zymophore or agglutiniphore group. Agglutination, therefore, does not require the presence of a complement. In some tases, intraperitoneal injection of body fluids or of solutions of certain proteins into animals brings about a cohdition which enables the blood serum of such animals to cause a precipitation of the protein to which the animal has been adapted. This fact has been taken advantage of in formulating a medicolegal method for the detection of blood of different ani- mals and will be taken up in a later section. The precipitins consits of free receptors combining, by means of their haptophore group, with the receptor of the cell and exerting their precipitating effect through the medium of their zymophore or precipitinophore group. Phagoc3rtosis. According to the theory of Metchnikoff, the leucocytes are capable of' incorporating into their substance materials which are foreign to the blood in which they circulate. This process is known as phagocytosis and is one of the greatest protective measures which the system has for its fight against bacterial invasion. When the blood becomes laden with bacteria, as in the various infectious diseases, we find a leucocytosis in practically all cases, the exceptions having been previously noted. This is the natural sequence if the system is to rid itself of these invaders. The leucocytes are drawn by chemical attraction (chemotaxis) toward the bacteria and attempt to swallow them by throwing pseudopodia about them and drawing them into the proto- plasm. This is successful in many cases, while in others it is not, so that the question of ascendency of the leucocyte or of the bacterium wUl depend upon the degree of phagocytosis. Strangely enough much variation is shown in the susceptibility of different organisms to phagocytosis. Thus the pneumococ- cus at times is very difficultly amenable to phagocytosis, while at others it is easily acted upon. The recent work of Rosenow^ has thrown much light upon the mechanism of this action. Opsonins. Realizing that there was a more definite basis for phagocytosis than was embraced in the older conceptions, Wright^ introduced the idea of opsonins to designate the presence in the blood serum of substances which render the ^ Jour. Infect. Dis., 1906, III, 683; also, Kite and Wherry, Ibid., 1915, XVI, 109. ^ Proc. Royal Soc, i9°3,^XI^|y.^j, MicrOSOft® THE BLOOD 599 various bacteria subject ta phagocytosis. The normal blood serum contains such opsonic material for the various bacteria with which it may be infected, but this varies greatly toward the different organisms. Thus we may find individuals showing much more opsonin (a higher opsonic index) toward one organism than toward another. This explains, in a way, the well-known fact that different people are variably susceptible to the same disease, while the same individual may be strongly resistant toward infection with one organism, but easily a victim of another infection. Regarding their chemical nature very little is known. There seems to be a certain amount of evidence which points to the fact that these opsonins belong to the class of globulins, while Simon and Lamar^ have shown that they are apparently associated with the euglobulin fraction. Quite as little is known of the structure of the opsonins, so that it is at present doubtful in what position to place them in the side-chain theory. According to Hek- toen,2 they may contain a haptophoric group which unites with the bacterial or other receptors and also an opsonipherous group which brings about changes in the cell, making it capable of phagocytosis. According to Savt- chenko and Dean, the opsonins should be' regarded as amboceptors, while Greig-Smith looks upon the process of opsonification as the first stage of ag- glutination. All of these theories must wait for future confirmation. The opsonins are thermolabile and are usually destroyed by heating for lo minutes to 6o°C. They occur in all classes of vertebrates and show here a peculiar characteristic, namely, that the serum of different animals is capable of activating various organisms for phagocytosis by leucocytes of animals of different species. This would bring the opsonins into the same field as the agglutinins, precipitins, and hemolysins. From a clinical standpoint, opsonins are frequently found to be diminished in certain bacterial infections. It is, therefore, conceivable that the resist- ance of the patient or, in other words, his phagocytic power might be increased by the addition of substances which could act as opsonic material. Such substances are the bacterial vaccines of Wright, suspensions in physiological salt solution of dead cultures of the organism to which the patient shows a diminished power of phagocytosis. The relation of the phagocytic power of the patient, as evidenced by the number of organisms which a definite number of leucocytes takes up under the opsonifying influence of this serum, as com- pared with the same condition in the case of the serum of a normal individual,, is known as the opsonic index toward the organism investigated. The number of bacteria taken up by the leucocytes of the normal individual is . taken as one. For preparation of these vaccines see last chapter. According to Wright, the injection of a dose of vaccine is followed by a decrease in the opsonic index (the negative phase), which is of variable degree and duration, depending upon the dose. This negative phase is followed by an increase in the opsonic power of the leucocytes (the positive phase), which ' Bull. Johns Hopkins Hosp., 1906, XVII, 27. 'Jour. Am. Med. Assn., 1906, XL VI, 1407; Ibid., 1907, XL VIII, 1739; Illinois Med. Jour., XIII, 9; also, Zinsser g^dy^g^ Jmij^^^^Ql^., 1914, XIX, 345. 6oo DIAGNOSTIC METHODS is associated with improvement in the condition of the patient. The various doses of the vaccine should be so administered that it is never given during a negative phase. While a low opsonic index is the rule in chronic cases, high indices may be observed with active systemic manifestations of acute cases. As a rule, it is more generally beneficial to use a vaccine prepared from the discharges of the patient than it is to use an already prepared vaccine of the same organism. The reason for this is that so much difference exists in the various strains of the same organism that little or no result may follow the administration of a stock vaccine. It is not always easy to gauge the dosage of a vaccine in any particular case, as the idiosyncracy of the patient plays such an important r6le. It is better practice, therefore, to start with a small dose and obtain no reaction that it is to give an overdose which wiU prove very harmful. Failure to observe this precaution is, in the writer's opinion, the reason that tuberculin so soon fell into disrepute and is now being restored to such great favor. It has been found by various workers that the administration of a proper dose of a vaccine is followed by certain local symptoms, such as swelling, red- ness, and tenderness at the point of injection, while certain systemic symp- toms, such as rise in temperature, general malaise, and pains in the joints are, practically always evident. It has, therfefore, become the practice of many of our workers, to gauge the results of vaccine treatment more by the reaction tha,n by the determination of the opsonic indices of their patients. Diagnostic Use of Vaccines. The vaccines discussed above are used almost entirely for therapeutic pur- poses. While it is highly probable that any of the vaccines might give char- acteristic reactions in cases infected with the same organism, yet they have not been put to extensive diagnostic use. However, certain suspensions of organisms are, at present, widely employed in this way. It is evident, from the preceding discussion, that the serum of a patient contains bodies acting as neutralizing agents against the toxins of the infect- ing bacteria. This property varies with the individual and with the severity (dosage) of the infection. Patients, who have acquired immunity against certain infectious diseases, either by passing through an attack of the in- fection* or by use of protective vaccination (small-pox or typhoid fever), or who are in the process of acquiring immunity, that is, are at the time pass- ing through an active infection, show a certain changed reactivity to in- fection with the same organism. In other words such patients are not insensible to reinfection, but show a changed reactivity, as von Pirquet' has shown, especially as regards the time, quality and quantity of the reactions observed. This is the condition known as AUergy and is shown only by pa- tients immune or becoming immune (infected) to the suspected organism.^ Normal individuals do not show the reaction so quickly nor so intensely as do the infected or immune subjects, providing the dosage is properly 1 Arch. Int. Med., 191 1, VII, 259. 2 See. Gurd, Jour. Med. Research, 1914, XXXI, 205; Meigs, Jour. Infect. Dis., 1914, XV, 541; Zinsser, Arch. Int. Med., 1915, XVI, 223. Digitized by Microsoft® THE BLOOD 6oi regulated, as the former have sufficient antibodies to overcome the effects of the amount of toxin, which will produce local or general reactions in the in- fected patient.^ These are the basic principles underlying the diagnostic use of vaccines. Tuberculin Reactions. Method of Koch. . This is the earliest method for the diagnostic use of tuberculin. The normal temperature variations of the patient are first determined for 24 hours previous to the injection. The preparation used is Koch's old tuberculin, which is a water and glycerin extract of the cultures of the tubercle bacillus, of such a strength that i c.c. is equivalent to i gram of tuberculin. The injection maybe made into the deeper, tissues either in the intrascapu- lar or gluteal region by means of a properly sterilized hypodermic syringe. The amount of tuberculin to be injected depends on the age and condition of the patient. As a rule 3^ to i mg. is given to a rohust adult, while a weak patient or a child receives }4.o to p^o ™g- These strengths are easily made by diluting the original solution with the proper amount of 3^ per cent, carbohc acid solution. The temperature variations should then be followed, observa- tions being made every two hours. Arise of 3^°C. (Mo°r-) within a few hours or even as late as 36 hours, is taken as a positive result, although some ob- servers require a more marked rise before admitting a positive finding. Few other symptoms are noticed but, if the dose has been large, marked malaise, chill, high fever, and local reactions may obtain. If no rise in temperature is observed after the first injection, a second or a third one may be given, .the dose being doubled each time but exceeding, under no circumstances, 10 mg. It is possible that a single injection of 5 or 10 mg. may give more diagnostic results than repeated doses up to this amount, although it must be admitted that such large doses may react, in rare cases, positively in normal individuals. A positive result is very strong evidence of an active tubercular condition. A negative result is less reliable as some tubercular conditions, such as peri- tonitis or encapsulated foci elsewhere, may not react positively. Method of Moro. In this method use is made of a salve consisting of equal parts of old tuberculin and lanolin. A small bit of this salve is rubbed into the skin, pref- erably in the thoracic or abdominal region. After 24 to 48 hours small pale nodules, i to 2 mm. in diameter, are seen at the point of inoculation if the case be tubercular. A more severe reaction may be observed in the form of red- dish miliary nodules. These nodules are limited to the point of application of the salve and disappear in 24 to 48 hours. Occasionally, some exudation may arise from the intense reactions, but this is very rare. This method is very simple and is very reliable. ^ ^ See Hektoen, Jour. Am. Med. Assn., 191 2, LVIII, 1081; also, Vaughan, Am. Jour. Med. Sc, 1913, CXLV, 161. 2 See Krumbhaar and Musser, Am. Tour. Med. Sc, 1914, CXLVII, 540. Digitized by Microsoft® 602 DIAGNOSTIC METHODS Method of von Pirquet. This method' is very widely used, especially in cases ojE infection in children. The nner side of the forearm is cleansed with alcohol and ether and allowed to dry. Two drops of old tuberculin are placed on the skin at a distance of 6 to 8 cm. from each other. The skin covered by these drops is then punctured by a large needle or gently scarified. The tubercuhn is allowed to remain for lo to 15 minutes, when the clothing may be replaced. It is wise to make a third puncture or scarification between the two tuberculin points as a control upon the patient's reaction, no tuberculin being used here. A slight swelling is usually produced which becomes surrounded by a Kght reddish border m a few hours, and disappears in 24 hours. If the reaction be positive, the area of puncture swells within 24 to 48 hours to a papule, reddish in color and 10 to IS mm. in diameter. The center may be pale and be surrounded with small serous points. By comparison with the normal point, a reaction miay be more clearly demonstrated. The swelhng and redness begin to disappear within 48 hours, a slight pigmentation usually following this positive reaction. No general disturbance is noted as a rule. If the reaction be repeated, a marked positive result may be observed as an evidence of hypersensibility, the so-called anaphylactic reaction. This may, also, be noticed in the other methods, even though a much smaller dose be used than in the initial apphcation. Method of Calmette. This method was advanced almost simultaneously by Calmette and Wolff- Eisner. One or two drops of a i to 100 dilution of old tuberculin in physio- logicsaltsolution are place in the conjunctival sac, the lids being held apart for a few seconds to permit of absorption of the tuberculin. Only one eye is thus treated, the other being used as the control. A positive reaction appears in 6 to 24 hours and may assume one of the following types: (i) Mere reddening of the caruncle and the inner surface of the lower lid; (2) the bulbar conjunc- tiva may take part in the process; (3) suppurative conjunctivitis with marked injection of the palpebral conjunctiva; (4) hemorrhagic conjunctivitis of the palpebral and bulbar portions with profuse fibrino-purulent exudation. An associated rise in temperature may be observed in the severe types.'' Contraindications to this test are found in any inflammation of the eye or conjunctiva, in the previous treatment of the eye with tuberculin, and recent application of tuberculin elsewhere. It would not seem that this method is advisable owing to the great discomfort which may arise arid, especially, as it is not without danger of severe injury to the eye.' ' See Wachenheim, Am. Jour. Dis. Child., 1912, IV, 27; Austrian, Jour. Exper. Med., 191 2, XV, 149; Rosenberg, Ztschr. f. exper. Path. u. Therap., 1913, XII, 549; and Wachen- heim, Am. Jour. Dis. Child., 1913, V, 466; Klopstock, Ztschr. f. exper. Path. u. Therap., 1914, XV, 13; Monrad, Ugesk. i. Laeger, 1914, LXXVI, 1419; Kuchenhoff, Deutsch. med. Wchnschr., 1914, XL, 229; Salvetti, Pediatria, 1915, XXIII, 35; Fishberg, Arch. Pediat., 1915, XXXII, 20; Am. Jour. Obs., 1915, LXXI, 361; Kraemer, Miinch. med. Wchnschr., 191S, LXII, s and 46; Frazer, Med. Record, igis, LXXXVII, ST, Palmer, Jour. Am. Med. Assn., 1915, LXIV, 1312; Cattermole, Ibid., 191S, LXV, 782. ^ See Vaughan, Jour. Am. Med. Assn., 1913, LXI, 1591- 'Austrian (Bull. Johns Hopkins Hosp., 191 1 XXIII, i) advocates a similar test for typhoid fever. The antigen is a "typho-protein" prepared from a mixed culture of 80 different typhoid strains. Positive results are obtained in 95 per cent, of cases of typhoid while it is negative without excepdon in practically alLxontrols. Its great advantages are that it is a bedside test and fe'^^Qff 9M Mif^Q^M^i the disease. THE BLOOD 603 Luetin Reaction. Noguchi' has recently announced "A Cutaneous Reaction in Syphilis," which may prove of great value in the diagnosis of this condition. His mate- rial is prepared as follows: Pure cultures of the treponema palUdum (see p. 582) are allowed to grow for periods of 6, 12, 24, and 50 days at 37°C. under anaerobic conditions. One was cultivated in ascitic fluid containing a piece of sterile placenta, and the other in ascitic fluid agar also containing placenta. The lower portion of each solid culture was cut out and the tissue removed. These agar columns, containing large numbers of spirochetae, were carefully ground in a sterile mortar. This paste W9,s then gradually diluted by addii^g, little by little, the fluid culture, until the emulsion became perfectly liquid. This mixture was heated to 6o°C. for one hour in a water-bath and 0.5 per cent, carbolic acid was added. When this mixture is examined under the dark-field microscope 40 to 100 palUdae may be seen in each field. This suspension is called luetin.^ The skin of the upper arm is sterilized with alcoholic sublimate solution before the injection. The amount of luetin injected is 0.05 c.c. to 0.07 c.c. This injection is intradermic, that is, in the skin as superficially as possible. In normal cases there appears, after 24 hours, a very small erythematous area at and around the point of injection. No pain or itching is experienced. This slight reaction gradually recedes within 48 hours and leaves no induration. In certain cases the reaction may reach a stage of small papule formation after 24 or 48 hours, after which time it commences to recede. In positive cases the following types of reaction occur : (a) Papular Form. — A large, raised, reddish, indurated papule, usually 5 to 10 mm. in diameter, makes its appearance in 24 to 48 hours. The papule may be surrounded by a diffuse zone of redness and show marked telangiec- tasis. The dimensions and the degree of induration slowly increase during the following three or four days, after which the inflammatory processes begin to recede. The color of the papule gradually becomes dark bluish-red. The induration disappears within a week, except in certain instances in which a trace of the reaction may persist for a longer period. This latter effect is usu- ally met with among cases of secondary syphilis under regular mercurial treat- ment in which there are no manifest lesions at the time of making the skin test. Cases of congenital syphilis also show this reaction. (B) Pustular Form. — The beginning and course of this reaction resemble the papular form until about the fourth or fifth day, when the inflammatory processes commence to progress. The surface of the indurated round papule becomes mildly edematous, and multiple miliary vesicles occasionally form. At the same time a beginning central softening of the papule obtains. Within the next 24 hours, the papule changes into a vesicle filled at first with a semi-opaque serum that later becomes definitely purulent. Soon the pustule ruptures. The margin of the broken pustule remains indurated, while a crust quickly forms, which falls off within a few days. The induration soon disap- pears leaving almost no scar. This reaction was found almost constantly ' Jour. Exper. Med., 1911, XIV, SS7; Munch, med. Wchnschr., 1911, LVIII, 2372. 2 Luetin may now be obta©^'fi^e^ frpnWPOffesdffl^'s and Co. or Mulford Co. 6o4 DIAGNOSTIC METHODS in tertiary types as well as in secondary or hereditary forms which had been treated with salvarsan. (C) Torpid Form. — In rare instances, the injection sites fade away to almost invisible points within three or four days, so that they may be passed over as negative reactions. Sometimes these spots suddenly light up again after lo days or so and progress to small pustular formations. No marked constitutional symptoms have been observed after the use of the luetin. In most positive cases a slight rise in temperature took place lasting for one day. The conclusions of Noguchi,^ based upon a study of 400 cases, are as follows: Luetin produces a cutaneous reaction in syphilitic and parasyphilitic patients that is most constant and severe in the tertiary and hereditary affec- tions. Diuring the primary and secondary stages, the reaction is infrequent, and when present is of mild degree. An exception has been found in cases in which energetic treatment has been or is being carried out and in which clin- ical signs of syphilis are absent. ^ Such cases may show a severe reaction, • especially those treated with salvarsan. In certain cases of old infection in which no treatment has been taken and in which no symptoms have appeared for many years, and in the course of which miscarriages have occurred, this reaction has failed to appear. Despite the absence of symptoms, mothers who have young syphilitic children have usually given the reaction. It re- mains to be determined in how far this reaction can be used to supplement the Wassermann reaction. It appears probable that the Wassermann reac- tion is more constant in the primary and secondary, and the cutaneous reaction in the" tertiary and latent forms of syphilis.^ It further appears that the Wassermann reaction is more directly and immediately affected by anti- syphilitic treatment than is the cutaneous reaction.* ' See Ziegel, Arch. Int. Med., 1912, IX, 520; Miiller and Stein, Wien. klin. Wchnschr., 1913, XXVI, 408; KLlausner, Ibid., 973; Noguchi, Presse med., 1913, XXI, 757; Wolfsohn, Jour. Am. Med. Assn., 1913, LX, 1855; Kaliski, New York Med. Jour., 1913, XCVIII, 24; Schmitter, Jour. Cutan. Dis., 1913, XXXI, 549; Brown, Am. Jour. Dis. Child., 1913, VI, 171; Foster, Am. Jour. Med. Sc, 1913, CXLVI, 645; Fagiuoli and Fisichella, Berl. klin. Wchnschr., 1913, L, 1811; (iavim, Rjforma Med., 1913, XXIX, 985, 1013, 1049, and 1075; McNeil, Jour. Am. Med. Assn., 1914, LXII, 529; Joltrain, Ann. de M6d., 1914, I, 337; Fagiuoli and Fisichella, Berl. klin. Wchnschr., 1914, LI, 449; KUgore, Jour. Am. Med. Assn., 1914, LXII, 1236; Much, Med. Klin., 1914, X, 811; Blechmann and Delort, Ann. de Med. et Chir. Infant., 1914, XVIII, 406; Blechmann, Delort and Tulasne, Ibid., 465; Cannata, Pediatria, 1914, XXII, 481; Pusey and StUlians, Jour. Cutan. Dis., 1914, XXXII, 560; Noguchi, New York Med. Jour., 1914, C, 349; Muscel, DefBca and Friedmann, Munch, med. Wchnschr., 1914, LXI, 1272; Clausz, Ibid., 1933; Curti, Rif. med., 1914, XXX, 1264; Boas and Stiirup, Hospitalstid., 1914, LVII, 1417; Trossarello, Gazz. d. osp., 1914, XXXV, 457; Wolff and Zeeman, Ibid., 811; Schippers, Ibid., 817; Kafka, Berl. klin. Wchnschr., 1915, LII, is; Ross, Jour. Ment. Sc, 1915, LXI, 244; Sherrick, Jour. Am. Med. Assn., 191S, LXV, 404. ^ As this luetin reaction is an allergic one, it is not surprising to find that, in the primary and secondary stages, there are not sufficient antibodies present to produce the reaction. Although emulsion of ascitic agar itself may produce an intradermic reaction similar to that of luetin, yet this does not militate against the test as it shows a hypersensitiveness, which is in itself rather characteristic of syphilis. ' See Robinson, Jour. Cutan. Dis., 1912, XXX, 410. Sherrick (Jour. Am. Med. Assn., 1915, LXV, 404) calls attention to the fact that recent or simultaneous administration of potassium iodid will cause a positive luetin reaction in non-syphilitic individuals. ^ Irons has recently perfected a local skin reaction in cases of generalized gonorrheal infections, using as his vaccine a glycerine extract of autolyzed gonococci. After a few hours a papule is formed with a surrounding area of hyperemia, the maximum reaction being reached in 24 hours. Irons jJ^ffS^ffe^sgoB^g/^gg^jj^ions showing a total diameter of s THE BLOOD 605 Schick Reaction. * By means of this reaction individual susceptibility and immunity to diph- theria may be readily recognized in a great majority of cases. It consists in the intracutaneous injection of diphtheria toxin of definite dosage and the observance of the irritant action at the point of injection. Technic. In the first place the minimum fatal dose of the diphtheria toxin is deter- mined for a guinea-pig of 250 grams weight. ' This amount is then so diluted with sterile physiologic salt solution that each c.c. of the dilution contains 0.2 of the minimum fatal dose. Of this diluton o.i c.c. (representing one-fiftieth of the minimum fatal dose) is injected into the skin by means of an accurately graduated syringe (i c.c. record tuberculin syringe) with steel or platinum- iridium needle, which should be very fine, sharp and she rt-pointed. The site of injection is the skin of the outer side of the upper portion of the arm. Reaction. If the injection be properly done, one observes a white blister-like punctate- appearing wheal, which shows distinct markings of the openings of the hair follicles. This elevation (due to the traumatism) persists for a short time, aU signs of it usually disappearing within an hour. If there is sufi&cient antitoxin in the blood of the subject to neutralize the small amount of toxin, no further changes shoulld be observed. This is a negative reaction and indicates that the person is immune to diphtheria. A positive reaction is noted in the gradually increasing redness and infil- tration of the skin, a distinct circumscribed area of redness and infiltration measuring about i to 2.5 cm. being seen within 24 or 48 hours. This redness persists for several days and gradually fades, leaving a brownish pigmenta- tion and slight scaling. In some cases, especially in older children and adults, a pseudo-reaction is observed, which is not due to the action of the toxin but is, rather, an ana- phylactic response to the protein substance of the diphtheria bacillus or, as Kolmer and Moshage believe, to trauma of a skin which is unduly sensitive. Some local redness and infiltration may be noted, but this comes on more rapidly than does the true reaction, the redness is less sharply outlined, the local infiltration is more marked and the signs disappear in 2 to 4 days with mm. or over; negative when 3 mm. or less (Jour. Infect. Dis., 1912, XI, 77). See, also, Fronstein, Med. Obozr., 1913, LXXIX, 225. Gaj' and Force (Arch. Int. Med., 1914, XIII, 471) have introduced a skin reaction for tjrphoid fever, which consists in using a "typhoidin" solution, prepared by concentrating a glycerin bouillon culture of typhoid bacUli, and which is applied to the abraded skin. A difiference of 2.5 mm. between the areolae of the " typhoidin" point and the control point is taken as evidence of a positive reaction. See, also, Gay, Am. Jour. Med. Sc, 1915, CXLIX, 157. ' This toxin may be obtained from the larger laboratories handling biological products or may be procured from the health oflBcers in the larger cities. In this latter case a definite amount of a known potency toxin is supplied together with a diluent fluid. After the toxin is diluted it deteriorates rapidly, so that it should not be used after 12 hours' standing. The undiluted toxin, obtained from the supply houses should be kept cool to prevent deterioration. Usually the potency of the toxin is stated on the package, so that the neces- sary dilution may be directly ^gured. ,, ... „^ D^ized by Microsoft® 6o6 DIAGNOSTIC METHODS little or no pigmentation and no scaling. These falsi reactions are confusing, but should be easily differentiated by careful observation. To control the reaction, one may inject into the other arm o.i c.c. of bouillon diluted i to lo ox I to I, coo. An extreme sensibility of the skin will be indicated by slight local reaction in the control arm. Significance. In normal subjects a negative result indicates, in practically loo percent, of cases, the presence in the blood of diphtheria antitoxin and, hence, an im- munity to diphtheria. A typical positive reaction points to an absence of anti- toxin and a resulting susceptibility to infection. From the reports of the literature, it is evident that about 80 per cent, of new-born children, 50 to 60 per cent, of children, especially those between the ages of I and 5, and 90 per cent, of adxilts contain sufficient antitoxin, as shown by this test, to make them immune to diphtheria. In the presence of diphtheria, the question of exposure of other children is an important one. It is evident that immunization by injection of antitoxin is indicated only in those giving positive Schick reactions. Further, by means of this test definite separation of the diphtheria cases from non-diphtheritic cases may be made, as patients with true diphtheria have little antitoxin in the blood and hence show a positive reaction. Likewise diphtheria carriers may be separated from actual diphtheritics, as the former usually have a large amount of anti- toxin in the blood and, therefore, give negative reactions. There can be little question but that this test will prove invaluable in hospitals in selecting internes and nurses to look after the diphtheria patients. Moreover, the question of isolation of actual diphtheria patients becomes a much easier matter, with the knowledge that those showing a negative Schick reaction are immune to diphtheria and need not, therefore, be removed from chance of infection. The economic as well as the diagnostic value of this test seems to be very great.' VIII. Sero-Diagnosis In the attempt to enlarge our diagnostc procedures advantage has been taken of many of the principles previously discussed. The methods evolved are among those most frequently employed and are reliable within certain small limits which will be mentioned later. They are all based upon certain peculiar properties possessed by the serum of patients with infectious diseases and are, therefore, of special importance in differential diagnosis. 'Schick, Munch, med. Wchnschr., 1913, LX, 2608; Groer and Kassowitz, Ztschr. f. Immunitatsf. u. exper. Therap., 1914, XXIII, 108; Kassowitz and Schick, Ztschr. f. d. ges. exper. Med., 1914, II, 305; Otto, Deutsch. med. Wchnschr., 1914, XL, 542; Schick, Kasso- witz and Busacchi Ztschr. f. d. ges. exper. Med., 1914, IV, 83; Park, Zingher and Serota, Arch. Pediat.j 1914, XXXI, 481; Jour. Am. Med. Assn., 1914, LXIII, 859; Veeder, Am. Jour. Dis. Child., 1914, VIII, 154; Kolmer and Moshage, Ibid., 191S, IX, 189; Weaver and Maher, Jour. Infect. Dis., 1915, XVI, 342; Bundesen, Jour. Am. Med. Assn., 1915, LXIV, 1203; Graef and Ginsberg, Ibid., 1205; Moody, Ibid., 1206; Kolmer and Moshage, Ibid., 191S, LXV, 144; Zingher, Ibid., 329; MofEett and Conrad, Ibid., loio; Birnberg, St. Paul Med. Jour., 1915, XVII, 204; Levinson and Blatt, Arch. Diag., 1915, VIII, 201; Zuckermann, New York Med. Jour., 1915, Bl^Md by Microsoft® THE BLOOD 607 A . Agglutination Reactions. Since the work of Gruber and Durham and of Pfeiffer and KoUe in 1896, the fact has been well established that the serum of an animal, which has been rendered immune to certain bacteria, shows the property of agglutinating or clumping homologous bacteria, owing to the production of immune bodies, the so-called agglutinins. This reaction takes place in high dilutions of the Fig. 15s. — Bacillus typhosus at beginning of Widal test. (Da Costa.) serum and is practically specific for any given organism. Widal,^ in the same year, applied this principle to the diagnosis of typhoid fever, showing that clumping and loss of motility of the typhoid bacilli occur when a suspension of the actively motile types is treated with an homologous immune serum at such a dilution that normal or non-homologous serum does not react. I. Gruber-Widal Test. Fig. 156. — A pseudo-Widal reaction. (Da Costa.) Cultures. The cultures of the t3^hoid bacillus must be fresh and must show many actively motile organisms in the hanging-drop specimens, if they are to be used in the microscopic method. It is advisable always to use cultxires con- taining about the same number of bacilli, as the reaction is somewhat quan- ' Bull. m6d. Paris, 1896, X, 618 and 766. Digitized by Microsoft® 6o8 DIAGNOSTIC METHODS titative. Thus, if few bacilli be present, they may be clumped by a small amount of agglutinin both specific and non-specific, while if the number be very large, the specific agglutinin may not be sufficient to cause marked clump- ing and loss of motility of the organisms. Great differences toward aggluti- nation exist in different strains of the typhoid bacillus, so that it is essential always to employ, for diagnostic purposes, strains which passed through several generations on artificial media. A stock culture of the bacillus is kept in sealed tubes of nutrient agar in a cool dark place and, from this stock, fresh agar cultures are made every few weeks as occasion may require. From these stock agar cultures, bouillon tubes are inoculated and incubated for 15 to 24 hours. These latter are used for the agglutination tests. These bouillon cultures should be equally cloudy throughout and show no pellicle formation or gross clots of bacilli. It is wise to make up fresh bouillon cultures every other day so as to be certain of the maximum motility of the organisms. ^ Fig. 157. — A positive Widal reaction. (Da Costa.) In the use of the macroscopic method cultures killed by heat, formalin, carbolic acid, th3'mol, etc., may be employed. Many laboratories through- out the country supply these cultures for general work. Ficker's "Typhus diagnostikum" is one of the widely used types. For self-evident reasons it is wise to have some standard strength of these cultures. Hastings advocates a mixture of 5 c.c. of 5 per cent, carbolic acid solution, 10 c.c. of glycerin, and 85 c.c. of physiologic salt solution to which are added the organisms scraped from the surface of two 24-hour agar-slant cultures of bacillus typhosus (the bacilli being rubbed into the mbcture with a spatula). Bass advises a sus- pension of 10,000 million dead typhoid bacilli per c.c. in 1.7 per cent. NaCl solution to which i per cent, formalin is added. Obtaining the Blood. In this procedure much depends upon the custom of the worker as to the method followed. As this reaction is a pure serum one, it is much more logical to use the serum than the whole blood, if one has any choice in the matter. ' Riemer (Munch, med. Wchnschr., 1913, LX, 908) cafls attention to the fact that typhoid bacilli show diminished agglutination it grown in alkaline media. See, also, Gay and Claypole, Arch. Int. ^^^^j^f^^Q^'^y'^^JofQ^olf®' ^^^'^' ^^^^'' ^^^^' ^'^' "'*^' THE BLOOD 609 The simplest and best clinical method in the use of a small capillary tube (made from 3<^-inch glass tubing) with a central bulbar enlargement into which the blood (15 to 20 drops) is drawn by capillarity from a puncture of the ear. The tube is then laid flat until coagulation occurs and the serum separates. To hasten this process, the end of the tube, which contains no blood, may be sealed in the flame and the tube centrifuged. If it is desired to send the specimen to the laboratory, seal both ends and pack in such a way as to insure it against breakage. In this connection it may be well to recall that Lyons^ has shown the possibility of obtaining a culture of the bacillus typhosus from the clot settling in such tubes. This may be done by the method previously outlined. Another method of obtaining the serum is to apply a cantharides blister which will furnish enough serum in 6 to 12 hours for many tests. Venous puncture may, also, be employed, but this is rarely advisable as sufflcient serum may be more easily obtained in the above ways. Some workers use the whole blood instead of the serum. If this be done, the leucocytometer may be employed, using physiologic salt (0.9 per cent.) solution as the diluent. Draw the blood from a puncture in the ear to the mark i and the diluent to 10. This gives a dilution of the whole blood of i to 10 or I to 20 of the plasma, as the plasma forms approximately 50 per cent, of the whole blood. This dilution may then be used directly for the later work by mixing with an equal volume of the typhoid suspension. More frequent, possibly, in the hands of workers in municipal or the larger ■clinical laboratories, is the employment of dried blood sent to them on glass slides, bits of paper, mica, etc. Such specimens may be easily transported but can not yield anything but an approximate result, as an accurate dilution is out of the question unless very careful weighting of the blood is carried out. A large drop of the dried blood is dissolved in 10 drops of water, the dilution "being called i to 10. If the blood be upon paper, the dilution must be made largely by guess, experience teaching an approximate dilution from the color of the mixture. These dilutions will, of course, be doubled when the test is applied. Dilution of the Serum. The methods of diluting the whole fresh blood and the dried blood have •already been given. As diluting pipets for the serum, one uses small (J^ inch), glass tubing which has been drawn into a long capillary at one end. A rubber bulb fits over the larger end and provides the suction. It is good practice •always to have a number of these pipets on hand so that they may be dis- carded or thoroughly cleaned before a second use. If the same pipet be employed for different cases, a contamination may arise to such an extent that erroneous results obtain. A few drops of the sprum, collected as above, are drawn into the capillary tube by suction. From this tube the serum is then dropped into the mixing vessels, preferably watch crystals. Two dilutions are usually made in the "writer's laboratory, namely i to 25 and i to 50. Allow i drop of serum to fall ^ .Arch. Int. Med., 1909, IV, 64. 39 Digitized by Microsoft® 6lO DIAGNOSTIC METHODS into each of two watch crystals, the remaining serum in the capillary being blown into the original collecting tube. Now add, from the same pipet used in dropping the serum in order to insure drops of the same size, to one of these drops of serum 24 drops of physiologic salt solution and to the other 49 drops of the same diluent. Mixtures of these diluted sera with an equal volume of typhoid suspension, as employed in the test, give final dilutions of I to 50 and I to 100. Microscopic Reaction. The serum (preferably), whole blood, or dried blood is diluted as previously described. Mixtures are then made of these dilutions with an equal volume of typhoid suspension. By means of a platinum loop of stiff wire, a loopful of the diluted serum {i to 25) is placed on the center of a cover-slip. After burning the serum from the loop, a loopful of the typhoid suspension is transferred to the same cover-glass and the two fluids thoroughly mixed by means of the loop. The dilution of the serum will then, evidently, be double its original dilution, or i to 50. The ordinary hanging-drop preparation is then made by inverting the cover-slip over the hollow of the slide, which has been ringed with vaseline. Instead of using the hanging-drop method, some prefer to place the cover directly upon the slide. Usually the specimen does not dry sufficiently to introduce errors, but the results are certainly not as characteristic by this procedure in all cases. The specimen is best studied with the high-power lens, using a strong light. The organisms will be seen, in specimens properly lighted, as free actively motile bacilli moving with their rapid darting motion through the field. In the course of 45 to 60 minutes, or often in much less time, the organ- isms will be seen to be collected in cluinps and to be motionless, except for a slight vibratory movement. This is a typical positive reaction, namely marked clumping with complete loss of motility in one hour at a dilution of i to 50. Such a reaction is practically never due to any other disease than typhoid fever, if the culture of the bacillus be a pure one. In some cases marked agglutination with incomplete loss of motility obtain. In the writer's opinion this can be interpreted only as a doubtful or incomplete reaction. The probability is that typhoid fever is present, but one must admit the possibility of group agglutinins, due to the paratyphoid bacillus, as well as agglutinins of other infections. When dried blood is used, a slight tendency to agglutination may be observed, due to the presence of small particles of fibrin, so that in such cases the loss of motility should be demanded even more rigorously than when serum is used, before a positive result is reported. It has been shown that the serum of many healthy patients or that of subjects suffering with diseases other than typhoid fever frequently show a reaction similar to that described above. This statement is true only when the question of dilution of the serum is taken into account. The char- acteristic Widal reaction is shown at a dilution of i to 50 or higher, while in other diseases, with the exception of paratyphoid, the reaction practically disappears (the typhoid bacillus being, of course, used) at a dilution of i to 20. The element of time is also of great importance. In typhoid fever, the Digitized by Microsoft® THE BLOOD 6ll reaction may occur instantly at i to lo or 20, and in one-half to one hour at I to 50, yet a period ranging from one-half to one hour is necessary with other sera at a dilution of i to 10 or 20. On the other hand, there are cases of undoubted typhoid fever that show no agglutination at i to 10 or 20, while at I to 50 this is marked. What occurs here is uncertain, some crediting the paradox to bacteriolysis of the organisms at the former dilutions. Macroscopic Reaction. The method is based on the fact that the agglutination of typhoid bacilli by a potent homologous serum is visible to the naked eye, the clumps of bacilli settling to the bottom of the test-tube leaving a clear supernatant fluid. If living cultures of bacillus typhosus are employed, the best technic is the following: Make up the serum dilutions as previously described, using i to 50 and I to 100 dilutions. To these diluted sera, contained in small sterile test-tubes (11 cm. long by 88 mm. wide), is added a loopful of typhoid bacilli scraped from the surface of a 24-hour agar culture. The loop is rubbed against the inner surface of the test-tube until the bacteria have been sus- pended in the serum. The turbid suspension is then thoroughly shaken until it becomes uniformly cloudy, no clumps of bacteria being seen, and is placed in the incubator at 37°C. for one hour. The tubes are then removed and examined by strong transmitted light. The agglutinated bacteria may easily be recognized by their granular flocculent appearance. General clumping should obtain in one hour at a dilution of i to 50, while complete precipitation (settling) should follow in 24 hours, a clear supernatant fluid being left be- hind. This is the positive reaction. A negative result is shown by no clump- ing and little, if any, loss in the turbidity of the mixture. For the general practitioner, who desires to use the macroscopic method, the employment of cultures killed by heat or chemical means is recommended, as it obviates the necessity of an incubator, culture media, or microscope and permits him to have on hand material for his tests. Any of the suspensions of killed organism,s may be used, Ficker's and Hastings' being widely employed. Dilute the serum as previously described, the dilutions in this case being i to 25 and I to 50. To this diluted serum (3^ c.c), contained in the small test- tubes, add an equal volume of the suspension of killed bacteria and examine as above mentioned. The clumping should be observed in one hour and complete clearing should obtain in 24 hours, if the reaction be positive. This method gives very reliable results and is' very simple. Method of Bass and Watkins. These workers"^ have introduced a modification of this macroscopic agglu- tination test which is simple, reliable, and quick. It has the advantage that it can be carried out at the bedside, the result being known in 2 to 3 minutes. Little equipment is required. The suspension consists of 10,000 million killed typhoid organisms per c.c. in 1.7 per cent. NaCl solution to which i per cent, formalin is added. Allow a full drop of blood to fall into 4 drops of water and mix thoroughly. Instead of this, one may make a blood-smear (using approximately 3^ drop of '■ Arch. Int. Med., 1910, VI, 717. Digitized by Microsoft® 6l2 DIAGNOSTIC METHODS blood) and dissolve this on the slide with i drop of water, the mixture being stirred with a tooth-pick or similar substance. With this diluted blood (i to 4) mix an equal amount of the above suspension of typhoid bacilli on a glass slide. Tnt the slide from side to side so as to keep the mixture flowing back and forth. If the reaction is positive, a grayish mealy sediment appears within one minute, usually in less time. This sediment appears in the fluid around the edges and tends to collect there. If the agitation is continued, the clumps increase in size for 2 to 3 minutes. If the reaction does not appear in this time, it will not show up at all. When the reaction is negative, no agglu- tination occurs and the mixture remains as clear and unchanged as when placed on the slide. Recently Michaelis^ has advocated a new agglutination reaction for the bacillus typhosus as well as other bacteria. This is based on the fact that bacteria are agglutinated by acids, the rate and degree being dependent upon the concentration of hydrogen ions present. This test promises much in the future as it depends entirely upon chemical factors which are more easily and more exactly controlled than are the uncertain biologic units with which we usually work. 2 Time of Appearance and Disappearance of the Reaction. The time at which a Widal reaction may appear in the course of a typhoid is of some importance. The earliest date of appearance of a positive reaction is rather hard to determine, as workers differ in their interpretation of the beginning of the disease. Definite reports of positive results as early as the second day have been given by Fraenkel and others. Although this reaction may appear before the rose spots, splenic tumor, or diazo reaction, yet it is practically never found when the blood shows a normal or an increased num- ber of leucocytes, a leucopenia with a relative lymphocytosis being associated with a positive Widal in practically all cases. A differential blood count may, therefore, be of great value when the means of making an agglutination test are not at hand. Wood was able to obtain positive results in only 10 per cent, of his cases in the first week of the disease. The majority of observers are agreed that the first appearance of this reaction is in the second week of the disease in most cases. In a certain number, it may not appear till the third or fourth week the highest agglutinations frequently being seen toward the end of the disease and in the early stages of convalescence. It is very rare not to obtain a positive reaction at some stage of the disease, providing the tests are made at different intervals. It is possible that cases reported, in which no Widal reaction was observed throughout the course of the disease, may be due to faulty technic or to insufficient tests. A single negative test, of course, proves nothing. The persistence of the Widal reaction is likewise variable. Little seems to depend upon the severity of the attack as to the time of disappearance of the reaction. Usually this reaction persists for i to 2 months, although it may obtain for 4 to 6 months or even for years. It is undetermined 1 Folia Serologica, 1911, VII, loio; Deutsch. med. Wchnschr., 1915, XLI, 241.. 2 See Beniasch, Zeitsch. f. Immun.-Forsch., 1912, XII, 268; Gillespie, Jour. Exper. Med., 1914, XIX, 28. Digitized by Microsoft® THE BLOOD 613 whether these latter cases are due to reinfection rather than a continuation of the agglutination powers of the serum. Reports are extant of cholelithiasis in which typhoid bacilli were isolated from the gall-bladder as long as 18 years after an attack of typhoid, the Widal reaction being positive in these cases. In this connection it is to be said that "typhoid-carriers" do not show a positive Widal as a rule. Specificity of the Reaction. From the standpoint of the biologic theory, the typhoid bacillus is aggluti- nated only by an homologous immune serum. Practically, we 'find that this is the case within certain Hmits, if all conditions are properly met. A posi- tive Widal reaction is not infrequently met with in cases of jaundice, in which no specific disease is clinically evident. ^ However, it is certain that most, if not all, of these cases of jaundice are actually secondary to a typhoid cholecystitis with its accompanying sequelae. Such patients may have an "ambulatory" typhoid or may have no clinical signs other than a positive Widal. Further, in many cases of infection with bact^ia of the colon group, a "collateral agglutination" may obtain, that is agglutination of several members of the group may occur with one serum. Cases are undoubtedly met with in which the infection is due to the paratyphoid bacillus and in which the agglutination of the typhoid bacillus is stronger than the para- typhoid; while, on the other hand, cases of true typhoid may show a stronger reaction with bacillus parat)rphosus than with the bacillus typhosus. Accord- ing to Lentz, the time of such associated agglutinations is important in this connection. The paratyphoid bacillus is agglutinated more quickly by a paratyphoid serum than is typhoid bacillus by the typhoid serum. A positive reaction, all conditions outlined above obtaining, indicates infection with the bacillus typhosus in practically all cases. This test can not be regarded as of great value in the early stages, as only a very small percent- age of cases show early agglutination reactions. It is especially serviceable in the differential diagnosis from the second week of the disease on to convales- cence. In the ambulatory types or in localized typhoid infections, especially in the gall-bladder, this test is of great value. A negative reaction should not be interpreted as excluding typhoid, as many typical and severe cases show no reaction until late in the disease.^ Like all laboratory tests, it must be used only in connection with the clinical findings, if the best results are to follow. The attending physician is the one to interpret the result, not the laboratory worker. 2. Diseases Other than Typhoid Fever. Other organisms, beside the bacillus typhosus, show such agglutination re- actions. The technic is the same and the results comparable, the same ' See Mazza, Gazz. d. osp., 1914, XXXV, 921 ; Perlmann, Munch, med. Wchnschr., 1915, LXII, 435; Stuber, Ibid., 1173; Bernard and Paraf, Presse med., 1915, XXIII, 333. ^ The widespread use of typhoid vaccine as a preventive inoculation has introduced a large possible error into this reaction. The serum of injected (immune) patients shows a positive Widal reaction very quickly. This condition persists for a varying period, so that blood cultures or Austrian's ophthalmo-reaction (see p. 602) should be used in such cases. See Moon, Jour. Am. Med. ^'gitf^(3f,JSj!CMfflJ?0Sdw®ellermann, Mtinch. med. Wchn- schr., 1914, LXI, 2453. 6 14 DIAGNOSTIC METHODS specificity being shown. It is especially in the realm of infections with members of the colon group that these tests are, frequently, uncertain, yet, here also, careful variation in the dilution of the serum and observation of the time and degree of agglutination will lead to a positive result, which will be of the greatest clinical value. The organisms, frequently identified by these reactions, are the para- typhoid bacilli, cholera spirillum, Shiga bacillus, bacillus of plague, malta- fever, tuberculosis, and the diplococcus of pneumonia."^ B. Precipitin Reaction. This reaction is based upon another fact well established in serum path- ology. It has been proven that the serum of an animal, injected with blood or blood-serum of another animal, shows the property, when added to an homologous serum, of precipitating the albumin of this serum in the form of a light flocculent precipitate. This same peculiarity has been observed after injection of exudates or transudates or of pure proteins both animal and vege- table.^ These reactions are, therefore, specific for the type of protein injected, within certain definite limits. Although this test has been used in the detection of certain albuminous substances excreted in the urine (see p. 262), yet its chief interest to the laboratory worker is its application to the medico- legal detection of blood and blood-stains.' The work of Wassermann and Uhlenhuth has shown that blood-stains may reveal their origin from any particular species of animal by application of this test. It is evident that one must, of course, prove that such stains are really due to blood by other methods, as this biologic test is merely for specific pi;oteins and not for blood as such. These latter tests will be given in a later section. In this discussion the writer quotes largely from Uhlenhuth and Weidanz.^ The Anti-serum. This is the serum of the animal immunized against the proteins of the blood, exudates or secretions of a particular species of animal. These pro- teins are usually those of the human as this type is of especial importance in medico-legal investigations. However, in the larger laboratories where many different serological tests are being frequently made, animals are kept on hand, which have been immunized against the proteins of most domestic animals, so that an absolute differentiation and identification of a stain may be made. The preparation of the anti-serum is as follows: A rabbit is injected intra- peritoneally with 5 to 10 c.c. of whole blood or serum. At intervals of 3 to 5 days other similar injections are given until six or eight in all have been ^ Kolmer (Jour. Exper. Med., 1913, XVIII, 18) has shown that there is no appreciable amount of agglutinin for treponema pallidum in the sera of secondary and tertiary syphilis. See, however, Zinsser and Hopkins (Jour. Exper. Med., 1915, XXI, 576, and Kissmeyer, Deutsch. med. Wchnschr., 1915, XLI, 306. ^ See last chapter for a discussion of the phenomena of anaphylaxis brought about by such injections. ^ See Hektoen, Jour. Infect. Dis., 1914, XIV, 403; Bing and Trier, Ugesk. f. Laeger, I9i4> LXXVI, 2117. ...... * Prakt, Anleitung z. Austuhrung des biol. Eiweissdifferenzierungsverfahrens, Jena, 1909. See, also. Leers, Die f©)g(liift"|<^ Bl|)i#fi'O^S©#®ig, Berlin, 1910. THE BLOOD 615 made. About a week after the last injection, a few drops of blood are drawn from a puncture in the ear of the animal and are allowed to clot in a small test-tube. The potency of the serum is then tested by permitting a few drops of the separated serum to fall into a small test-tube containing about I c.c. of a I to 1,000 dilution of dried homologous blood in physiological salt solution. In this preliminary potency test as in the later precipitin test it is important that the dilution of the solution under investigation should be about I to 1,000. One may recognize this proper dilution by the fact that the solution forms a layer of foam on shaking and, further, on heating and adding to I c.c. of the solution a drop of 25 per cent, nitric acid, only a sUght opalescence obtains. If the addition of the anti-serum to the i to 1,000 dilution of dried blood causes a turbidity in i to 2 minutes, the serum is sufficiently active for the test' and the animal may be bled. The blood is now withdrawn, either in small amount as desired for im- mediate use or the animal may be completely bled. The blood is collected in wide-mouth test-tubes, which should be then plugged with cotton and placed in a slanting position until the blood is coagulated. The serum, which must be clear and sterile, is removed to small test-tubes, which are plugged with cotton, sealed with paraffin, and stored in a cool place. This is the anti-serum. It should be sufficiently potent to produce a turbidity almost at once, at latest in I to 2 minutes, in a i to 1,000 dilution of homologous blood in 0.9 per cent. NaCl solution. ^ Solution of the Blood or Stain. As a preliminary precaution, it is to be emphasized that all vessels and instruments used should be thoroughly clean and sterile and that all fluids be absolutely clear. . If the blood or exudate be dried upon solid material, such as glass, knife blades, stone, wood, etc., it is carefuUy scratched off with a sterile instrument. This material is powdered and dissolved, as far as possible, in chemically pure physiologic salt solution contained in a sterile test-tube. If only traces of the stain are present, one may form a well of wax about the spots and place in this some of the salt solution. It is absolutely essential that no other solvent than 0.9 per cent. NaCl solution be used, as otherwise erroneous reactions may occur or the usual precipitin reaction may not obtain. If the stain has penetrated clothing or other soft material, the spot is cut out with scissors, is finely divided, teased with a needle and placed in a small watch-glass or test-tube with a small amount of salt solution for one houi, or, if the stain be very old or has been exposed to marked changes in the weather, for 24 hoiurs in a cool place. The fluid obtained in either of the above ways is filtered, first through hardened filter-paper (Schleicher and Schiill, Nr. 575, 603, or 605) and then through a small Berkefeld filter. This filtrate, which must be perfectly clear, should be neutralized to litmus either by the addition of sodium carbonate or tartaric acid solutions, as the case may be. Further, this ' It is important to remember that such serum may contain a small amount of antigen (along with the precipitin) which may persist as long as 15 days after the last injection. See von Dungern, Centralbl. f)yB^J^^(joJ,aty|^^^^Ugg]f^ 1903, XXXIV, 355; Uhlenhuth and Weidanz, Loc. cit., 221; ZiiBser and roung, Jour. Exper., Med., 1913, XVII, 396. 6l6 DIAGNOSTIC METHODS filtrate should show distinct foam-formation on shaking. This is not always the case if the stain has been exposed to the sun and dust or has dried either upon fatty material or iron. Under these conditions, the precipitin reaction may not be seen in the usual time, but will obtain, as a rule, in five minutes. As previously stated, the dilution of this physiologic salt extract of the stain should be approximately i to i,ooo. If sufficient stain can be scratched from the surface of the material upon which it is found, this may be weighed and proper dilution made. Such procedures are usually impossible, so that we use the approximate method given on previous page. Technic of the Test. The reaction is carried out in the small test-tubes, which should be ap- proximately of the same size and thickness, should be thoroughly clean and sterile, should be numbered and arranged in a series of lo tubes in a small rack. In tubes i and 2 are placed i c.c. of the above solution of the stain to be tested. In tube 3, I c.c. of a i to 1,000 dilution of known fresh human blood in physiologic salt solution. In tube 4, I c.c. of a i to 1,000 dilution of dried human blood in 0.9 per cent, salt solution. In tube 5, I c.c. of sterile physiologic salt solution. In tubes, 6, 7, 8, and 9, i c.c. each of a i to 1,000 dilution of fresh or dried blood of such domestic animals as may be easily obtained, as chicken, dog, horse and sheep blood. In tube 10, I c.c. of a physiologic salt extract of the material upon which the stain was found. To each of the above tubes, with exception of tube 2, is now added from a carefully graduated pipet o.i c.c. of the anti-serum, which has been tested for its potency as above described. To tube 2 is added o.i c.c. of normal clear rabbit serum. The anti-serum must be carefully added in such a way that it runs down the side of the tube and does not drop directly into the fluid. After this addition the tube must not be shaken. The reaction is carried out at room temperature. If the reaction be positive, a distinct clouding or turbidity will be observed immediately, or at latest within two minutes, in tubes 1,3, and 4 (all of which contain human blood), while the other tubes should remain perfectly clear. If the anti-serum has been properly added, the turbidity may be visible as a distinct contact ring which gradually increases upward. A negative reaction is shown by the lack of turbidity in any of the tubes. Specificity of the Reaction. Although this test is extremely delicate, yet it may happen that a stain, definitely identified as a blood-stain by other tests, may not react typically to the precipitin test, owing to the small amount of protein matter in the salt extract. This is especially the case if the protein has been rendered insoluble by heat or destroyed by chemical agents or by putrefaction. Even in such cases a positive reaction may occasionally be obtained, Uhlenhuth and Beumer reporting positive results after blood had been subjected to putre- faction for two years. Digitized by Microsoft® THE "BLOOD 617 As far as the influence of time upon this reaction is concerned, it may be said that blood-stains over 50 years old have been detected. The whole point, in this connection, is whether any trace of soluble protein remains. If so, the test will react positively, providing the dilution and the time of reaction be carefully regulated to suit the lessened concentration. Heterologous precipitin reactions do not occur if the conditions of the test are properly maintained. If more concentrated blood solutions are used and the amount of anti-serum increased, such erroneous reactions may obtain. It is true that confusing reactions may arise in the differentiation of the blood of the closely related animals such, for instance, as the horse and donkey; goat, sheep and ox; dog and fox; hare and rabbit; chicken and pigeon; and especially man and the anthropoid apes (orang-outang, goriU^ and chimpanzee) . How- ever, the point of great medico-legal importance is that such close differentia- tions are rarely called for, as the question at issue i,s, usually, whether a given stain is or is not due to human blood. As the apes are relatively scarce in this country, confusion is seldom probable. This reaction may, therefore, be regarded as practically specific for the especial type of blood protein with which the immune serum reacts (all details of the test being properly regu- lated), only the proteins of the very closely related animals responding to the test in a way which might leave the slightest room for doubt as to the origin of a stain or of a protein. The substance in which the stain is found may, often, exert some retarding action on the test. Stains upon cloth or paper usually show prompt results, although those upon wall-paper may, at times, react atypically owing to the influence of the chemical of the dyes. Strong alkalies or acids prevent the reaction, but these are thrown out by neutralization before the test is applied. Tannic acid, derived from stains upon leather may prevent the reaction, al- though this is not always the case. Iron rust may retard but does not pre- vent the reaction. If the stain be upon soft clay, mortar, lime or fresh plaster so that an intimate mixture has occurred, the reaction will probably not obtain, according to Graham-Smith and Sanger, but if the stain be upon hard well- dried mortar no influence is seen, as demonstrated by Biondi. As previously stated, this active immune serum reacts promptly with the protein elements of exudates, sputum, pus, urine, feces, nasal and bronchial secretions, vaginal, lochial and seminal fluids, etc., so that a positive precipitin reaction means merely that a protein is present, which has the same origin as that against which the animal furnishing the anti-serum was immunized. ' Whether or not this is a blood protein is, in no way, a part of the test. This must be determined by other tests. C. Complement Fixation Test. As previously stated, the serum of an animal, which has been injected with bacteria or with washed blood corpuscles of an animal of a different species, acquires the property of dissolving homologous cells through the development ^ Robertson and his pupils have contributed some interesting articles on the blood rela- tionship of animals as displayed in the composition of the serum proteins. See Robertson, Jour. Biol. Chem., 191 2, XIII, 325; Woolsey, Ibid., 1913, XIV, 433; Thompson, Ibid., 191S, XX, i; Briggs, Ibid., 7-Djgjtized by Microsoft® 6l8 DIAGNOSTIC METHODS of antibodies or immune bodies, which are specific for the cells injected. This process is known as bacteriolysis or hemolysis. Such sera are known as lytic; bacteriolytic, if the action is directed toward bacteria, hemolytic, if toward blood cells. It is to be remembered, in this connection, that fresh blood serum of many species is hemolytic for the red cells of some, but not all, other species, the degree being relatively small. Moreover, we find that the blood of the same species may be markedly hemolytic for the homologous cells, as evidenced by the effects occasionally observed after transfusion. It is, therefore, evident that we may have in any serum both the immune and the natural hemolytic agents, the former being much the more effective. It has been shown that hemolysis, by means of an immune serum, may be prevented by heating the serum to 55 to s6°C. for one-half hour, the process being known as inactivation. Addition of a small amount of a fresh, isologous serum will restore (reactivate) the hemolytic activity. Hence, two separate factors must be concerned in hemolysis, the one a thermostable (heat-re- sisting) substance, the other a thermolabile (destroyed by heat) product. The first of these is called the amboceptor, the latter, the complement. Amboceptor is usually still active in serum which has been kept for a long period, while complement disappears from the serum in a short time even though it be kept on ice. The function of the amboceptor is to sensitize the cells, while that of the complement is to dissolve the cells are sensitized. Hemolysis is impossible without the combined action of both factors, either one singly having no effect. Complement can combine with the cell only through the medium of amboceptor, while this union does not take place unless amboceptor and antigen (the blood cells) are homologous. In other words, the cells can be hemolyzed only by homologous serum in the presence of complement. It is evident, on the other hand, that hemolysis must occur when the proper hemolytic trinity is present. The amboceptor and com- plement bear definite quantitative relations to one another in this process, an increase of one factor permitting the use of a less amount of the other. Complement may combine itself equally well with bacteriolytic or hemo- lytic amboceptor providing the homologous antigen be present. If, there- fore, we mix bacterial emidsions with the corresponding inactivated immune serum and complement, this latter must attach itself (become fixed) to the bacterial cell through the medium of the amboceptor, so that the later addi- tion of hemolytic amboceptor and washed cells will not result in hemolysis, owing to the fact that the complement originally present has been fixed to such an extent that none is left to combine with the hemolytic amboceptor. This phenomenon, discovered by Bordet and Gengou, is known as "fixation of complement" or as "deviation of complement." It is decidedly a quan- titative as well as a qualitative reaction. Had complement remained free or been added in excess of the amount necessary to act upon the bacterial cell, a certain amount of visible hemolysis must have occurred, and might, even, have been complete, had sufficient hemolytic amboceptor been present. These quantitative elements are the most important ones in the application of the test to be discussed later. This test has been useareTaK^nosucpiiqjoses in many infectious diseases; THE BLOOD 619 for instance, in typhoid fever by Widal and Lesourd as well as by Hirschfeld, in cerebrospinal meningitis by Cohen, in whooping cough by Bordet and Gen- gou, in scarlet fever by Besredka and Dopter and Foix and Mallein, in sys- temic gonococcic infections by Miiller and Oppenheim, Bruck, Albarran and Jungano. Its most important and frequent application is in the sero-diag- nosis of syphilis, as advanced by Wassermann, Neisser and Bruck and, inde- pendently, by Detre. It is evident that the application of this complement fixation test leads, just as in the case of the agglutination and precipitin tests, to the identifica- tion of an antibody, provided one has the homologous antigen, or vice versa, the antigen and antibody necessarily being homologous and, hence, specific, the one for the other. It had been shown by Klebs, Metchnikoff and Roux that syphilis could be transmitted to monkeys by inoculation with human syphilitic material (antigen) . Wassermann and his coworkers then found that the serum of these sj^philitic apes contained an antibody not found in normal serum. Having this antibody, it was an easy step to show its relation to syphilitic antigen and to apply the complement fixation test in the detection of antibodies in the serum of patients infected with syphilis.' As the culti- vation of the treponema pallidum has only recently been achieved by Noguchi, no direct method of detecting these antibodies is available.* As indirect methods must be used, the antigen employed in the early studies was an ex- tract of syphilitic tissues in the active stage of the disease, as these contain large numbers of the causative spirochete. Later workers have shown that this antigen need not be specific, as alcoholic extracts of certain normal or- gans, crude tissue lecithin, or, even, salts such as sodium glycocholate or oleate, may serve quite as well in the reaction. Although this fact lessens the true specificity of the test, from the biologic standpoint, there is no doubt of the clinical value of the reaction. The so-called syphilitic antigen must be either a normal substance or one possibly associated with an unknown prod- uct which actually produces a specific result. However this m9,y be, it is a well established fact that, if the serum of a patient infected with syphilis be treated with antigen and complement, no hemolysis occurs on the later addi- tion of hemolytic amboceptor and red cells because the complement is bound to the antigen through the medium of the homologous amboceptor; while if the patient is non^syphilitic marked hemolysis occurs as complement is free to act with the hemolytic system. This is the basis of the Complement Fixa- tion Test as applied to syphilis by Wassermann and his colleagues. Although the antigen need not be specific, yet the test itself is remarkably specific being given by only a few non-syphilitic conditions as will be seen later. In the actual technic of the test, many modifications have been advised, the variations consisting either in the antigen or in the cells to be hemolyzed or in both. Five essential factors enter into the reaction whatever be the modification employed. These are (i) syphilitic antigen, (2) syphilitic anti- body, (3) erythrocytes (antigen), (4) hemolytic amboceptor and (5) com- plement. These factors must be accurately adjusted to one another, as ' See Krauss, Biochem. Ztschr,, 1914, LXIV, 222. ' See foot-note on page 6Migiuzeaby Microsoft® 620 DIAGNOSTIC METHODS serious errors may arise if the quantitative relations are not observed. The writer will describe only the original Wassermann and the Noguchi methods, as these are, in his opinion, the most reliable ones to follow.^ a. The Wassermann Reaction. Preparation of Antigen. Since the discovery of the fact that the antigen used in this test was not absolutely specific, in the biologic sense, for the syphilitic antibodies, many different antigens have been advocated. The writer selects those which have promised the best results, although he believes that Noguchi's antigen and, especially, Sachs' alcoholic heart antigen with cholesterin are by far the most reliable and constant ones introduced. a. Aqueous Extracts. I. Wassermann's Method. ^ The liver or spleen of a syphilitic fetus is ground up in a mortar with sand or, preferably, minced in a meat grinder. The hash is placed in a dark-colored flask and mixed with 0.9 per cent, sodium chlorid solution containing 0.5 per cent, phenol. The proportion is i gram of substance to 4 c.c. of the salt solu- tion. This mixture is shaken for 24 hours in a shaking machine and is then allowed to settle or is centrifuged. The reddish-brown opalescent superna- tant liquid, which is the antigen, is decanted into dark bottles, tightly stop- pered and kept on ice. On standing, a precipitate forms, which must not be disturbed by shaking. This aqueous antigen appears to be rather unstable and shows, sooner or later, some inhibition (anticomplementary action) with normal serum, although Citron^ finds such antigens active even after 11 months. Before use the activity of this extract, as well as of all others, must be quantitatively determined by the method later outlined. The usual doses are 0.2 (maximum) and o.i (minimum) c.c. As a general rule, we use only such an amount of antigen, the double dose of which shows no binding of complement. h. Alcoholic Extracts. I. Method of Porges and Meier.* The liver of a syphilitic or normal fetus is hashed and extracted for 24 hours with five times its weight of absolute alcohol. Filter through filter- paper and distill the alcohol from the filtrate in vacuum at 40° C. A syrupy residue remains which is suspended in physiologic salt solution containing 0.5 per cent, phenol, using 100 parts of salt solution to i of syrup. Filter throug filter-paper. The usual dose of this antigen is 0.2 to 0.3 c.c, although less quantities may be used as determined by methods of standardization. Instead of this antigen, these workers use, also, a i per cent, solution of commercial lecithin in phenol-containing physiologic salt solution. This solution is stable if kept in the ice box. It has the disadvantage that the ' Hecht (Wien. klin. Wchnschr., 1909, XXII, 265) and later Weinberg (Ann. de I'Inst. Pasteur, 1912, XXVI, 427) utilize the natural amboceptor and complement present in unheated human serum. See, also, Gradwohl, Jour. Am. Med. Assn., \^i\, LXIII, 240. * Deutsch. med. Wchnschr., 1906, XXXII, 745. 2 Die Technik der Bordet-Gengouschen Komplementbindungsmethode. in Handbuch of Kraus and Levaditi, Jena, iQog, p. 1076; Die Methoden der Immunodiagnostik und Im- munotherapie, Leipzig, 1910. U^^JZJ&d, J6jmMig's blood. 626 DIAGNOSTIC METHODS unit of amboceptor is used hemolysis will always be incomplete, even with more than one unit of complement. Likewise, if with one amboceptor unit there is combined less than one unit of complement, hemolysis can not be complete. If with more then one unit of amboceptor there be used less than one unit of complement, hemolysis may be complete or incomplete according to the relative amounts of each factor used.^ In the presence of many units of amboceptor hemolysis may be complete when but a small fraction of the complement unit is present." Titration of Antigen. The degree of dilution in which the antigen is to be used must be deter- mined for each extract by testing it both against normal and syphilitic sera. Its concentration must be such that it does not prevent hemolysis with normal sera, while it will promptly inhibit hemolysis in presence of complement with sj^hilitic sera. Set up two parallel series of tubes. Into each tube of one series place 0.25 c.c. of a I to 5 dilution of known syphilitic serum and into each tube of the second series place 0.25 c.c. of a i to 5 dilution of normal serum. Dilutions of the antigen are now made, with physiologic salt solution as the diluent, begin- ning with I to I and running up to i to 20 or higher -if necessary. Into the first tubes of the above series place 0.25 c.c. of the first antigen dilution, into the second tubes of each series place the second dilutions and so on until all are accounted for. To all of the above tubes add 0.25 c.c. of complement (i to 10) and incubate at 37°C. for one hour. Now add 0.25 c.c. of hemolytic amboceptor in twice the maximum dilution found to cause complete hemo- lysis (two units) and 0.25 c.c. of a 5 per cent, suspension of sheep corpuscles. Place in an incubator for two hours and then in ice box over night. No hemo- lysis should be observed in the tubes containing syphilitic serum except in those containing the higher dilutions, while in the tubes with normal serum hemolysis will occur in all tubes except those with the lower dilutions of antigen. Hemolysis in the tubes with syphilitic sera is due to the normal hemolytic power of the antigen, while the lack of hemolysis in the tubes with normal sera is due to the anticomplementary action of the antigen. In carrying out the Wassermann test, antigen is used in one-half the lowest dilu- tion (largest amount of antigen) which gives complete hemolysis with normal sera, this being taken as one antigen unit. Thus if a dilution of i to 5 gave complete hemolysis, a dilution of i to 10 would be used in the actual test. If one uses the drop method, as mentioned above, i drop of antigen (undi- luted) and I drop of complement (undiluted) are mixed with 10 drops of salt solution., The mixture is incubated one-half hour and i drop of amboceptor and I drop of a 50 per cent, suspension of corpuscles are added. Hemolysis should occur within one-half hour. If it does not, anticomplementary action is marked and less antigen must be employed in the test. Having determined the strength of the antigen, a portion of the stock solu- tion is diluted with salt solution so that i c.c. equals one unit. In testing the 1 See Kromayer and Trinchese, Med. Klin., 1912, VIII, 404 and 1670, who use an abso- lute minimum of complementin thjeir ".refined." Wassermann test; also, Thompson, Arch. Int. Med., 1914, XIII, 904. Digitized By Microsom THE BLOOD 627 antigenic properties of this antigen, one may use it with a large number of posi- tive and normal controls, or, preferably, run it in parallel series with an antigen of known value. If the results agree, it may then be substituted for the old antigen. The titer being known, the antigen may be used for subsequent tests without redetermination of this value. Technic of the Test. Original Wassermann Method. All of the glassware used in the test must be kept thoroughly clean and dry, but need not be sterile. The pipets, which should be both graduated to o.oi and o.i c.c, should, preferably, be used with only one reagent. If they are employed with others, they must be thoroughly rinsed in salt solu- tion between such applications. After using, wash them well in salt solu- tion (never with soap and water), allow them to stand in a tall cylinder of distilled water for a few hours and dry them in an oven. The test-tubes used in this test are about 14 cm. in length by 1.5 cm. in diameter, while the some- what smaller ones (i by 10 cm.) may be employed in the Noguchi modification. Before actually performing the test, the reagents are prepared and stand- ardized as previously described or the amboceptor may be titrated at the begin- ning of the test, as it is not added until the preliminary incubation of the serum with the antigen and complement. The sera of the patient and of the known positive and normal controls must all be inactivated by heating to 56°C. for one-half hour.' The complement is diluted i to 10 with physio- logic salt solution and the suspension of washed sheep corpuscles prepared. When these preliminaries are attended to one may proceed with the test itself. Arrange a double row of test-tubes, the front tube of each set to receive the antigen and the back row the control tube without antigen. One set of tubes must be placed for the unknown serum, one set for the positive and one set for the normal control; while other controls are arranged as spoken of later. 1. Into both tubes of each set place 0.2 c.c. of the serum to be tested. 2. To all tubes, add 1 c.c. of complement (diluted i to 10). 3. To tubes in front row only, add 0.2 c.c. of antigen (aqueous extract of syphilitic liver). Make up volume of each tube to 3 c.c. with salt solution, mix contents by shaking and incubate at 37°C. for one hour. 4. Add to each tube i c.c. of antisheep amboceptor (two units). 5. Add to each tube i c.c. of 5 per cent, suspension of sheep corpuscles. Mix the contents of the tube thoroughly, incubate at 37°C. for two hours and place the tubes in the ice box over night. At the end of this period the results are read. Controls. 1. Positive s)^hilitic serum. To be run as above. 2. Known negative serum. To be run as above. ' Gramenitzki (Biochem. Ztschr., 1912, XXXVIII, 501) and Fenyvessy (Ibid., 1912, XL, 353) have shown that if the serum be not heated to complete inactivation, the complement is restored on allowing to stagj^j^^^afrfoJiiftf/c/'OSO/?® 628 DIAGNOSTIC METHODS 3. A tube containing complement and two units of antigen, to prove that no anticomplementary action of the antigen obtains. 4. A tube containing complement, to show that the hemolytic system is active. 5 . A tube containing antigen alone, to show absence of lytic power of antigen. AU controls are brought up to 3 c.c, incubated and, then, amboceptor and sheep corpuscles are added as above. If the test is properly conducted, hemolysis will occur in every control tube excepting in the tube containing syphilitic serum and syphilitic antigen (the positive control) and in the tube of control 5. In the series of unknown sera, no hemolysis will occur in the tubes containing syphilitic serum. This lack of hemolysis, partial or complete, is, of course, a positive reaction. Citron^ advises the use of two tubes for serum and antigen, one tube con- taining 0.2 c.c. of serum and 0.2 c.c. of antigen as given above, while the other tube contains o.i c.c. of serum and o.i c.c. of antigen. In this way the results may be represented somewhat more quantitatively, as the degree of inhibition of hemolysis varies according to the amount of S3^hilitic antibody present. If this amount be large, complete inhibition occurs in the tube containing 0.1 c.c. of serum and 0.1 c.c. of antigen as well as in tube No. i, the result being expressed as + + 4 — \-. If inhibition is incomplete in tube 2 but complete in tube I, the result is + ++• Both of these degrees are strongly positive reactions. If tube 2 is completely hemolyzed while tube i is completely inhibited, the result is H — (-. Incomplete inhibition in tube i and complete hemolysis in tube 2 is expressed as +. These two latter results are classed as weakly positive reactions. If tube i shows doubtful inhibition while tube 2 shows complete hemolysis, the result is + . Complete hemolysis in both tubes is, of course, a negative result. ^ Noguchi's Modification. This is not to be confused with Noguchi's test in which a human hemolytic system i,s used. The modification here suggested uses a different antigen and different quantities of the same reagents as employed by Wassermann. This is the method, followed in the writer's laboratory, when the sheep hemolytic system is used, a control being run with Sachs' antigen or a specific watery extract as antigen in cases showing affections of the central nervous system. 1. Set up the double row of tubes as in the preceding test using 0.2 and 0.1 c.c. of serum to be tested. 2. To all tubes add 0.1 c.c. of an undiluted complement. 3. To front row only, add 0.1 c.c. of acetone-insoluble antigen. Make up volume of each tube to 1.5 c.c. with physiologic salt solution. Mix contents thoroughly and incubate at 37°C. for one hour. 4. Add to each tube i c.c. of antisheep amboceptor (two units). '■ Handbook of Kraus and Levaditi, 1908, II, 1105. ^ Ivy (Jour. Am. Med. Assn., 191 2, LIX, 432) advocates the use of the Duboscq color- imeter as a means of estimating the exact degree of hemolysis, the control tube of each case being taken as the standard of 100 per cent. Ninety per cent, and over of hemolysis is regarded as negative. See Kaplan (New York Med. Jour., 1913, XCVII, 1172; Ibid., 1913, XCVIII, 157); and Kaplan and McClelland (Ibid., 1012) for the determination of amino nitrogen as a control. Digitized by MlCrOSOft® THE BLOOD 629 5. Add to each tube 0.5 c.c. of a 10 per cent, suspension of sheep corpuscles. The total volume is now 3 c.c. Mix contents thoroughly and incubate at 37°C. for two hours. Allow to stand four to six hours at room temperature and read results. The interpretation is as given above. Drop Method. This is the method of Landsteiner, Miiller and Poetzl and is frequently employed. The writer can not convince himself that this method is an accurate one owing to the possible variations in size of the drops, which are delivered from a capillary pip'et. 1. Set up the double row of tubes as in the preceding tests and add to each 10 drops of physiologic salt solution.- 2. Add I drop of serum to each of the two tubes used for the sera tested. 3. To front row only, add 2 drops of antigen. 4. To all tubes add i drop of complement. Mix thoroughly and incubate for one hour at 37°C. 5. Add I drop (two units) of standardized antisheep amboceptor to each, tube. 6. Add I drop of a 50 per cent, suspension of sheep corpuscles to each tube. Incubate for two hours. Allow to stand at room temperature and read results. The method of interpretation is as above. ' "' Method of Noguchi.^ It has been found that human serum contains a variable amount of natural antisheep amboceptor.' This, of course, increases the effect of the immune antisheep amboceptor used in the tests previously described. One may, therefore, obtain a negative Wassermann test under these circumstances even though a large amount of complement is fixed. Noguchi has overcome this possible error by introduction of a human hemolytic system to replace the sheep hemolytic system of the original test. He employs, also, a smaller amount of reagents and uses either fresh or in activated serum of the patient. If preferred, one may use larger quantities of patient's serum, than outlined by Noguchi; if so, the reagents are increased in the same proportion. This method of Noguchi is the one preferred by the writer and is the one followed as a routine in his laboratory, as it has been found somewhat more rehable than the original Wassermann test in obscure cases.* Sachs' antigen is used in every test. Collectioii of Serum. Noguchi obtains the patieiit's blood from a puncture on the ventral side of the last joint of the middle finger. The finger may be compressed by squeez- ing before the puncture and, afterward, massaged to press out the blood, this being collected in small tubes drawn out into capillary points. In the writer's laboratory the blood is obtained as given on page 623. 1 See Mefford, Illinois Med. Jour., 1914, XXVI, 428. ^ Jour. Exper. Med., 1909, XI, 392; Serum Diagnosis of Syphilis, Phila., 1912. 'See Dexter and Cummer, Arch. Int. Med., 1912, IX, 605; also, Bailey, Ibid., 551. * See Coca and L'Esperance (Arch. Int. Med., 1913, XI, 84) for a slight modification of the Noguchi technic. See, also, Miller, Interstate Med. Jour., 191^, XX, 145; Thompson,. Arch. Int. Med., 1913, XI, 512. See Cecil and Lamb (Arch. Int. Med., 1913, XI, 249) for the value of this test in testiig.^|g^ijj^f^g^ggg.^@ 630 DIAGNOSTIC METHODS Corpuscle Suspension. The suspension may be prepared with the blood of the patient or with that of the examiner. If with that of the former, it is especially important that every trace of serum be removed by careful washing. The standard amount of corpuscle suspension is i c.c. of a i per cent, or o.i c.c. of a 10 per cent, sus- pension for each tube. If the patient's blood is to be used, enough may be obtained for many tests as follows: Fill a graduated centrifuge tube to 9 c.c. with 2 per cent, sodium citrate in phystological salt solution. AUow the blood to drop in until it fills the tube to 10 c.c. Mix and centrifuge. Pour off the supernatant fluid and fill up to 10 c.c. with fresh salt solution. Mix thoroughly and again centrifuge. This may be repeated two or three times, in order to insure removal of every trace of serum. The deposited corpuscles may now be suspended either in 100 c.c. of salt solution (making a i per cent, suspension) or in 10 c.c. (giving a 10 per cent, suspension). This procedure, while thoroughly reliable, is not followed by the writer. It is our custom to obtain the corpuscles from a puncture of the finger of the worker, allowing 3 drops of blood to fall into 12 c.c. of salt solution in a centrifuge tube. This gives, approximately, a i per cent, suspension. The washing is done as above. This latter method enables us to obtain fresh suspensions whenever desired. Hemolytic Amboceptor. This is prepared, as previously outlined, by injecting a rabbit, intraperi- toneally, with increasing doses of washed human corpuscles, instead of with sheep corpuscles as in the Wassermann method. After the animal is immu- nized, the blood is collected and allowed to coagulate. The serum is then withdrawn and titrated as previously described. Amboceptor Paper. — This has been introduced by Noguchi to simplify the test and to enable those not in close touch with clinical laboratory facilities to perform the test. While this paper may be used, the writer advises those do- ing any number of these tests to place their reliance more in the liquid ambo- ceptor. The serum, obtained as above, is poured over sheets of filter-paper (Schleicher and SchiiU's No. 597), cut into squares of 10 x 10 cm. Allow all the sheets to become evenly wet and absorb the excess with another sheet of paper. These are then dried kt room temperature by placing each square separately upon a clean sheet of unbleached muslin. When dry, the sheets are cut into strips 5 mm. wide and are then standardized as follows: Take a series of tubes containing i c.c. of the i per cent, erythrocyte suspension and add to each tube 0.02 c.c. (as one unit) of complement. Now add the strips in increasing lengths, as i mm., 2 mm., 3 mm., etc., and incubate for two hours. The shortest strip causing coinplete hemolysis in this time represents one unit of amboceptor. These strips are then marked into sections of twice this length (two units) and cut off when the actual test is to be made. The papers should be kept dry and sealed. The papers on the market are quite variable and are not always to be relied upon. As previously stated, these papers should be used o^^^&ifM *^9^a^ceptor is not at hand. THE BLOOD ORIGINAL WASSERMANN TEST 631 e E a CO a 9 w iii • V bo a Make up volume of each tube to 3 c.c. with salt solution. Mix contents and incubate at 37° one hour ls§ a Us m ^1 Is It U-, < Hemolysis Unknown serum .... Unknown serum .... Positive control Positive control Negative control. . . . Negative control. . . . Anticomplementary contro . Hemolytic control. . . c.c. 0.2 0.2 0. 2 0.2 0.2 0.2 c.c. I C.c. 0.2 0.2 0. 2 0.4 0.2 C.C. C.C. Depends on sera. Complete. None. Complete. Complete. Complete. Complete. Complete. None Antigenic control. . . . NOGUCHI'S MODIFICATION (SHEEP HEMOLYTIC SYSTEM) B H M § 6 d 4iS3 1 "g 5 a g M-l M r- fi cd 1^1 a 0.0, III ■B°o Jl 1! ■s.s "1 Hemolysis Unknown serum .... Unknown serum .... Unknown serum . . . Unknown serum . . . Positive control. . . . Positive control Negative control. . . . Negative control. . . . Anticomplementary contro . Anticomplementary contro . Hemolytic control. . . c.c. 0.2 0.2 0. 1 CM 0. 1 0. 1 0.2 0.2 c.c. O.I O.I 0.1 0.1 0.1 0. 1 0.1 0. 1 0. 1 0.1 0.1 c.c. 0.1 O.I O.I O.I 0.2 0. I c.c. o-S °-S o-S o-S o.S o-S "-S o.S o-S o-S OS c.c. I I 1 I I I I I I I 1 Depends on sera. Complete. Depends on sera. Complete. None. Complete. Complete. Complete. Complete. Complete. Complete. NOGUCHI'S MODIFICATION (HUMAN HEMOLYTIC SYSTEM) E g a 3 4j '0 "-1 1 < as 8 J3 a u .2 I «S u 3 i II It llf 3"! go i Hemolysis Unknown serum .... Unknown serum .... Positive control. ..... Positive control. .... Negative control. . . . Negative control. . . . Anticomplementary control. Hemolytic control. . . c.c. 0.02 0.02 0.02 0.02 0.02 U.02 c.c. 0. 1 0.1 0.1 0. 1 0. 1 0.1 0.1 0.1 c.c. 0. 1 o.i 0.1 0. 1 C.c. I 2 units 2 units 2 units 2 units 2 units 2 units 2 units 2 units Depends on sera. Complete. None. Complete. Complete. Complete. Complete. Complete. Digitized by Microsoft® 632 DIAGNOSTIC METHODS Complement. Guinea-pig serum is used as complement in this system as in the others. The dilution is 40 per cent, made by mixing i o.c. of complement with i3^c.c. of physiologic salt solution. The method of obtaining it has been given above. Complement paper may be prepared but it is very unsatisfactory and should not be used if the liquid form can possibly be obtained. Antigen. The antigen, used in this system, is prepared as previously described (p. 622). The stock solution is a 3 per cent, methyl alcohol solution of the ace- tone-insoluble lipoids. From this a 0.3 per cent, emulsion is prepared for the test by mixing i c.c. of this stock solution with 9 c.c. of physiologic salt solu- tion. It seems advisable always to control this antigen by running a control test with a watery extract of specific organs, and of Sachs' antigen, especially if an affection of the central nervous system obtains. Technic. 1. Arrange the double row of tubes with same controls as in the other tests. Place in all tubes i capillary drop (0.02 c.c.) of the fresh serum to be tested. If the serum has been inactivated use 4 drops (0.08 c.c). One may use 0.2 c.c. of .fresh (not inactivated) cerebrospinal fluid. 2. Add to each tube o.i c.c. of 40 per cent, complement, or in a great emergency two units of complement paper. 3. To the tubes in front row of each set, add o.i c.c. of antigen emulsion. 4. To both tubes of each set, add i c.c. of i per cent, washed human corpuscles. Mix thoroughly and incubate for one hour at 37°C. 5 . Add to each tube 2 units of antihuman amboceptor in liquid or paper form. Mix and incubate for two hours at 37°C. Allow the tubes to stand at room temperature for a few hours and read results. The results of this test are interpreted as in the preceding ones. This human system is more adjustable and reliable than is the sheep system.. Noguchi has pointed out certain variable factors which may obtain and which may require a proper adjustment to insure proper results. These variables- are, it is to be understood, greater in the sheep system, and are far less easily controlled. I quote directly from Noguchi. (i) One sometimes meets with, instances in which hemolysis is complete within 10 to 20 minutes, and in which the positive control tubes with antigen undergo, sooner or later, gradual hemolysis. Such rapid progress of hemolysis at first mentioned is a sign of imperfect reaction. If the test is properly made, hemolysis proceeds gradu- ally, and is complete in the water-bath within half an hour or thereabout. The causes of this accelerated hemolytic process are either an abnormally weak resistance of the blood corpuscles, or an exceptionally high activity or insensitiveness to fixation of the complement employed; or it may be the re- sult of all these acting together.^ It happens occasionally that the serum of certain guinea-pigs contains an abnormally active complement. In order to- establish this point, and thus to remove this source of error, one has only to- 1 See Bailey, Jour. Exper. Med., 191 2, XV,. 470. Digilized by Microsoft® THE BLOOD 633 make the test witL a quantity of complement which corresponds exactly to two complement units. (2) There are sometimes encountered instances in which hemolysis remains incomplete even in the control tubes in which there is no antigen. Here the causes of the imperfect reaction are found either in the weakness of the complement, or the amboceptor used, or both.^ Usually the cause is the weakness of the complement, which, owing to its great lability, is likely to deteriorate. The activity of the amboceptor is far less subject to external influences which bring about its deterioration, and it is therefore ex- tremely rare to find that the imperfection in the reaction arises from this source. In testing several specimens of serum at one time it happens oc- casionally thaj; some specimens are slower in completing the hemolytic re- action than others. The cause of this slowness is not present in the comple- ment or amboceptor, but in the specimens themselves. ^ In such cases the specimens are found to contain anticomplementary substances which react with and reduce the activity of the complement. To remove this source of error, it is necessary to heat the serum to S5°C. for 20 minutes and use four drops for the test. The diflSculty may be obviated in some cases by collecting specimens of serum to be tested just before meal-time because the anticom- plementary substance is closely associated with the absorption of the chyle into the circulation soon after the meal. (3) The quality and quantity of the antigen can also be sources of error. If one uses poor antigen, either there will be no positive reaction at all, or weak positive reactions will be entirely overlooked. If, on the other hand, an excessive amount of unfractionated crude antigen is employed, certain nonspecific weak reactions may become manifest, or a false positive reaction even may be obtained, as the result of the action of anticomplementary substances sometimes contained in prepara- tions of the antigen. These sources of error can be entirely excluded by choosing an antigen that has been carefully prepared and standardized.' Diagnostic Value of the Wassermann Test. . As stated above, the Wassermann reaction, or any of its modifications, is not a specific immunity reaction. While the antibody is, presumably, syphilitic in origin, the antigen is a physiologic lipoid and not a product of the ' See Barratt, Jour. Path, and Bacteriol., 191 2, XVI, 363. ^ See Bronfenbrenner and Noguchi, Jour. Exper. Med., 1912, XV, sgS and 625. ' For discussions of many obscure points regarding the nature of the reagents and the technic of the test see Addis, Jour. Infect. Dis., 1912, X, 200; Dick, Ibid., 1913, XII, lii; Lippmann and Plesch, Ztschr. f. Immunitatsforsch., 1913, XVII, 548; Baumann, Illinois Med. Jour., 1913, XXIV, 117; Burdick, Colorado Med., 1913, X, 13s; Kolmer and Cassel- man', Jour. Med. Research, 1913, XXVIII, 369; Surface and Routt, Ibid., 441; Cummins, Ibid., 1913, XXIX, 23; Weil, Biochem. Ztschr., 1913, XLVIII, 347; Stillians, Jour. Cutan. Dis., 1913, XXXI, 316; von Gierke, Deutsch. med. Wchnschr., 1913, XXXIX, 692; Rabinowitsch, Ibid., 1210; Kolmer and Williams, Jour. Infect. Dis., 1913, XIII, 96; Spat, Biochem. Ztschr., 1913, LVI, 21. Kapsenberg, Nederl. Tijdschr. v. Geneesk., 1913, II, 1007; Wassermann and Lange, Kolle and Wassermann's Handb., I9r3, VII, 951; Lesser, Miinch. Med. Wchnschr., 1914, LXI, 7o;Langer, Deutsch. med. Wchnschr., 1914, XL, 274; Olmstead, Med. Record, 1914, LXXXV, 341; Thiele and Embleton, Lancet, 1914, I, 526; Mcintosh and Fildes, Ibid., 860; Matzenauer and Hesse, Wien, klin. Wchnschr., 1914, XXVII, 319; Sternberg, Ibid., 545; Klein and Frankel, Miinch. med. Wchnschr., 1914, LXI, 651; Wechselmann, Berl. klin. Wchnschr., 1914, LI, 304; Blumenthal, Ibid., 1316; Nathan, Ibid., 1934; Stone, New York Med. Jour., 1914, XCIX, 1242; Grulee, Am. Jour. Med. Sc, 1914, CXLVIII, 688; Lyon and Eiman, Ibid., 885; Stephens, Arch. Diag., 1913, VIII, 126; Schlesinger, Med. Record, 1915, LXXXVII, 61; Bana, Brit. Med. Jour., 1915, I, 587; Stitt and Clark, U. SDigMaQdebyMtre^U®^!!, 410; Clark, Ibid., 411. 634 DIAGNOSTIC METHODS treponema pallidum. In other words, the complement fixation test, as ap- plied to the diagnosis of s)^liilis, is not the result of an interaction between homologous (specific) antigen and antibody. Nevertheless, we find that the reaction does have a certain specificity from the diagnostic, if not from the biologic, standpoint. As a general rule it may be stated, at the outset, that a positive Wasser- mann test, all proper precautions and controls being made, indicates syphilis in practically all cases. It is true that certain nonspecific conditions, espe- cially leprosy and, occasionally, scarlet fever, give positive reactions, but the large majority of cases of other diseases, reported as giving positive results, are those in which syphilis has not been absolutely excluded. In such cases the syphilitic infection may be latent instead of the active cause of the trouble at the time of making the test, yet it must be reckoned with in deciding as to the value of this test from the diagnostic point of view.^ On the other hand, it is to be distinctly understood that a negative Wasser- mann test does not exclude syphilis, a fact that must be taken into considera- tion when the question of marriage is involved. Such cases must be judged . largely by the clinical manifestations and history rather than upon the negative character of this test. Varying percentages of positive results in known syphilitic and parasyphilitic conditions have been reported by various workers. Some of the more enthusiastic ones claim that a negative Wasser- mann reaction means that the case is not syphilitic. This view, however, must be regarded as untenable in the face of the large numbers of negative results in the literature and in the daily experience of those working in this field. The following table, compiled from Boas^ and Noguchi' shows the reported cases of syphilis with the percentages of positive results.* ^ See Kolmer, Jour. Exper. Med., 1911, XIV, 235; Field (Jour. Am. Med. Assn., 1912, LVIII, 1681) has called attention to the occurrence of positive Wassermann tests in cases of lead poisoning. Hilgermann, Deutsch. med. Wchnschr., 1912, XXXVIII, 118; Gibson, Australasian Med. Gaz., 1913, XXXIII, 300. For other diseases showing a Wassermann see Jakobovics, Jahrb. f. Kinderhkde., 1914, XXIX, 215; Fletcher, Lancet, 1914, I, 1677; Butler, U. S. Naval Med. BuU., 1915, IX, 51; Hesse, Wien. klin. Wchnschr., 1915, XXVIII, 62; Sutherland and Mitra, Indian Jour. Med. Research, igiSj 11, 984. ^ Die Wassermannsche Reaktion., Berlin, 1913. ' Serum Diagnosis of Syphilis, Philadelphia, 191 2. ^ See Lucas, Am. Jour. Dis. Child., 1912, III, 259; Churchill, Ibid., 363; Kaplan, Med. Record, 1912, LXXXI, 1132; Bates, Arch. Int. Med., 1912, X, 470; De Buys, Am. Jour. Dis. Child., 1913, V, 65; MUne, Am. Jour. Med. Sc, 1913, CXLV, 197; Wolbarst, New York Med. Jour., 1913, XCVII, 378; Grindon, Davis, Greiner and Weiss, Interstate Med. Jour., 1913, XX, 221; Sarateanu and Velican, Monatsschr. f. Geburtsh. u. Gynak., 1913, XXXVII, 89; Balcarek, Med. Klin., 1913, IX, 1552; Eliasberg, Deutsch. Ztschr. f. Chir., 1913, CXXIV, 113; Blackfan, Nicholson and White, Am. Jour. Dis. Child., 1913, VI, 162; Holt, Ibid., 166; Richards, Jour. Am. Med. Assn., 1913, LX, 1139; Kaplan, New York Med. Jour., 1913, XCVIII, 308; Mcllroy, Watson and Mcllroy, Brit. Med. Jour., 1913, II, 1002; Heidingsfeld, Jour. Am. Med. Jour., 1913, LXI, 1598; Mensi, Gazz. d. osp., 1913, XXXIV, 1032; Post, Boston, Med. and Surg. Jour., 1913, CLXIX, 777; Kaplan, Jour. Am. Med. Assn., 1913, LXI, 2214; Emery, Lancet, 1914, I, 223; Philippson, Policlinico, 1914, XXI , 20; Russo, Riv. osped., 1914, IV, 173; Descomps, GrSce, M^d., 1914, XV, 37; Lesser and Klages, Deutsch. med. Wchnschr., 1914, XL, 1309; White, Therap. Gaz., 1914, XXXVIII, 307; MacKinney, Ann. of Surg., 1914, LX, 309; Bahr, Illinois Med. Jour., 1914; XXV, 245; Corbus, Ibid., 1914, XXVI, 112; Craig, Jour. Am. Med. Assn., 1914, LXII, 1232; Thomas and Ivy, Am. Jour. Med. Sc, 1914, CXLVIII, 55; Southard, Boston Med. and Surg. Jour., 1914, CLXX, 947; Stone, Med. Record, 1914, LXXXVI, 545; Fordyce, New York Med. Jour., 1914, C, S97; Craig, Am. Jour. Med. Sc, 191S1 CXLIX, 41; Krauss, Biochem. Ztschr., 191S, LXVIII, 48; Pontano, Policlinico, 1915, XXII, 41, 77 and 113; von Gonzen- bach, Cor.-Bl. f: Schweiz. A0^f;z©c6£)^lWiiC/f6SOift®22s; Wolbarst, Interstate Med. THE BLOOD 635 Condition Number of cases Positive results Primary syphilis 974 2,762 830 1.233 861 125 64 498 360 56 . s per cent. 88.0 per cent. 80.0 per cent. 51.0 per cent. 47.0 per cent. 94.5 percent. 47 . 6 per cent. 88 . 1 per cent. 70.0 per cent. Secondary syphilis Tertiary syphilis Early latent S3^hilis Late latent syphilis Congenital syphilis Cerebrospinal syphilis General paralysis Tabes dorsalis Considerable discussion has arisen in the literature regarding the relative merits of the Wassermann and Noguchi systems. As a general rule it is found, by one running these two systems in parallel, that the positive results are somewhat higher with the Noguchi system. The following table, taken from Noguchi, gives the figures for the cases reported, in which the two tests were made upon the same serum. Condition Number of cases Wassermann positive Noguchi positive Primary- syphilis 208 669 4SS 30s 79 SS 88 per cent. 92 per cent. 74 per cent. S4 per cent. 98 per cent. 73 per cent. 94 per cent. 98 per cent. 83 per cent. 68 per cent. 98 per cent. 80 per cent. Tertiary S3T)hilis. Latent syphilis Congenital syphilis Cerebrospinal S3^hilis.. Effect of Treatment on the Reaction. Under the influence of a vigorous course of mercurials, a positive Wasser- mann test may be made to disappear. This must, however, not be inter- preted as meaning that the syphilitic infection has been cured. It is almost daily observed by those doing large numbers of these tests that a patient, having the test made while under active treatment with mercury, will fre- quently give a negative test; while the same patient may show a positive reaction if the mercury be withdrawn for two or three weeks prior to the test. On the other hand, we occasionally find cases in which the treatment has resulted in a positive Wassermann, prior tests having been negative. These facts must be borne in mind by those who may attempt to establish a diagno- sis without full knowledge of the conditions under which the test is being made. Mercurial treatment may render the infection latent, so that a nega- tive reaction may obtain until later active manifestations appear and become associated with a positive test. This variability has been shown by Craig, ^ who reports 5 2 cases treated, with 3 2 positives and 20 negatives after treatment Jour., 1915, XXII, 109; Gradwohl, Southern Med, Jour., 1915, VIII, 477; Robertson and Klauder, Jour. Am. Med. Assn., 1915, LXIV, 199; Keyes, Ibid., 804; Vedder and Hough, Ibid., 972; Weisenburg, Ibid., 975; Haberman, Ibid., 1141; Heimann, Ibib., 1463; Wile and Stokes, Ibid., 1465; Glomset, Ibid., 1915, LXV, 682; Uhle and MacKinney, Ibid., 863. 'Jour. Exper. Med., 1910, XII, 726; Arch. Int. Med., 1911, .VIII, 395. Craig and Nichols (Jour. Am. Med. Assn., 191 1, LVII, 474) and Craig (Ibid., 1913, LX, 565) call attention to the influence of alcohol in converting a positive test into a negative one within ^"^ °"'^^' Digitized by Microsoft® 636 DIAGNOSTIC METHODS ranging from two weeks to twelve years. Patients should not have had any mercurial treatment for at least two to three weeks prior to the test if any reliance at all is to be placed upon it. Patients well treated give negative reactions, while those receiving inadequate treatment, no matter for how long a period, show positive results. The reaction is, unquestionably, affected by mercurial treatment, but some cases persist in giving a positive reaction in spite of treatment. The reaction may return shortly after treatment, while it is not, by any means, settled that the disappearance of a reaction justifies one in stopping treatment. After treatment with "606" (salvarsan, dioxy-dimino-arseno benzol), the results are variable. In promptly cured cases, a positive reaction may disappear within two weeks or it may require four to five weeks to obtain a negative result. A positive reaction may persist for considerable time and then disappear, reappearing again after an uncertain period. It has been definitely established that, if a patient be syphilitic, a negative Wassermann can be converted into a positive one by an injection of salvarsan, owing to the fact that the spirochete are killed and their toxic action is temporarily in- creased, the bodies concerned in the Wassermann test being thereby increased. This is the so-called "Provocative Wassermann Reaction" and can be ob- tained in every stage of infection. The blood for the test should be taken before 'the injection as a control and then 24 and 48 hours after. Occasion- ally it is wise to take the blood one to two weeks after the injection also, in cases which are of doubtful nature. In deciding as to the proper criterion of a cure in any case a negative test following a positive one has little value. To establish a basis for the assertion of a cure, one should insist that the following points be met. One year with- out treatment, without any suspicious signs, with several negative Wasser- mann reactions and no positive ones, and with a negative provocative Wasser- mann reaction and luetin test at the end of the year.^ /3. Diseases Other Than Syphilis. ^ The complement-fixation test is, theoretically, applicable to any in- fectious disease. Acute infections, however, do not show positive results until sufficient time has elapsed to permit of absorption of enough toxin to cause a systemic response to invasion, as shown by the presence of specific antibodies in the serum tested. Sub-acute and chronic cases offer the best chances for positive results. Practically, however, the tests have not been equally satisfactory in all types of infection or invariably reliable in the same type. The principle of the test is the same as described above no matter what the condition under investigation. The hemolytic system may be either sheep or human, the reagents used being as mentioned with the exception of the antigen employed. These antigens are polyvalent in character, being pre- 1 See Nichols, Jour. Am. Med. Assn., 1912, LVIII, 603; also, Craig and Nichols, Studies of Syphilis, Bull. 3, War Dept., Office of Sur. Gen., Washington, 1913; Pusey, Am. Jour. Med. Sc, 1913, CXLVi, 497. Corbus (New York Med. Jour., 1914, C, 472) reports a case of second infection, the Wassermaun hayine been negative for one and one-half years. 2 See Ludke in Ka.ndhook&lm^S&a£mMi£dJ^m^zungshand i, igii, 518; Ztschr. f. klin. Med., 1911, LXXII, 545. THE BLOOD 637 pared from several different strains of the suspected organism. As different strains of the same organism often differ markedly one from another, the possibility exists of the serum of an infected patient fixing complement only in the presence of closely allied strains. Hence, the more strains of the or- ganism in any given antigen the more certain will be the results of the test. The antigens are prepared as follows: Cultivate as many different strains as possible of the organism on appropriate media, such as agar slants or blood- agar slants, and wash the surface of each slant with 2 or 3 c.c. of a 0.9 per cent, solution of sodium chlorid, scraping the organisms from the media with a platinum loop. Pour off the suspension into a sterile tube and shake thor- oughly. Kill the organisms by heating the tube in a water-bath at 6o°C. for one-half hour. Standardize the number of bacteria so that i c.c. of the sus- pension contains 1,000 million bacteria (see last chapter). The amount of antigen to be used in the tests must then be determined by titration just as described for the Wassermann test. Gonorrhea. In systemic gonorrheal infections the complement-fixation test is be- coming of considerable importance. The technic is carried out as given above. The antigen should consist of at least twelve strains of gonococci. In the writer's laboratory the human hemolytic system is employed exclusively and the results have been constant and satisfactory. Others prefer the sheep system. The same precautions must be followed here as in the Wassermann test, every point in the technic being carefully controlled. A positive reaction indicates a focus of active gonorrheal infection in the system; while a negative result does not exclude this condition, owing to the variability in the antigens mentioned above. The influence of treatment upon this test is not at all clear, but it must be remembered that all cases treated with vaccines wiU show strong positive reactions. Just how long after such treatment is discontinued one may expect to obtain a positive result is still unsettled, so that the absolute determination of a cure must rest largely on the clinical findings. This is an important point, especially where marriage is contemplated, as the possibility of infecting another must be kept in mind. Non-infectivity cannot be predicated on a single negative fixation test.'^ Attempts to use this method, as a diagnostic procedure, have been made in typhoid fever, tuberculosis, meningococcic infections, cholera, pertussis, small-pox, infection with pyogenic cocci and other infections but the results have not been conclusive or reliable. Likewise it has been employed in the diagnosis of infection with various animal parasites, such as in echinococcus 'See Schwartz and McNeil, Am. Jour. Med. Sc, 1911, CXLI, 693; Swinburne, Arch. Diagnosis, 1911, IV, 227; Schwartz, Am. Jour. Med. Sc, 1912, CXLIV, 369; Schwartz and McNeil, Ibid., 815; Lenartowicz, Tygodnik lekarski, 1912, VII, 368; Dermatol. Wchnschr., 1912, LV, 1179; Lespinasse and Wol£E, Illinois Med. Jour., 1913, XXIII, 26; Smith, Am. Jour. Dis. Child., 1913, V, 313; Finkelstein and Gerschun, Berl. klin. Wchnschr., 1913, L, 1817; McNeill, Arch. Pediat., 1913, XXX, 657; McDonagh and Kline, Jour. Path, and Bacteriol., 1913, XVII, SS9; Thomas and Ivy, Arch. Int. Med., 1914, XIII, 143; Smith, Am. Jour. Dis. Child., 1914, VII, 230; Williams, Interstate Med. Jour., 1914, XXI, 1198; Kolmer and Brown, Jour. Infect. Dis., 1914, XV, 6; Thomas, Ivy and BirdsaU, Surg., Gyn. and Obs., 1914, XIX, 390; Arch. Int. Med., 1915, XV, 265; Irons and NicoU, Jour. Infect. Dis., 1915, XVI, 303; Sormani, Nederl. Tijdschr. v. Geneesk., 1915, I, 1077; Uhle and MacKinney, New York Med. Jour., 1915, CII, 737. Digifized by Microsoft® 638 -DIAGNOSTIC METHODS disease; helminthiasis in general and trichinosis. Here, also, the results are still too uncertain to warrant the routine use of this test.* D. Abderhalden's Sero-diagnosis.'' I. Of Pregnancy. Abderhalden has recently introduced a biologic test for pregnancy, which, he assumes, depends upon the presence of certain ferments found in the serum of pregnant animals. This test appears to be fairly certain in its results and should prove invaluable to the gynecologist and obstetrician. It will be recalled that the injection of foreign protein into the system results in the production of certain well-established biologic properties in the serum of the animal so injected. Among these properties one finds the formation of precipitins, agglutinins, anaphylactogens, etc. Abderhalden has shown that the parenteral injection (subcutaneous, intraperitoneal or intravenous) of foreign protein or carbohydrate brings about the appearance the blood serum of proteolytic or amylolytic ferments, as an indication of the effort of the system to protect itself against the possible toxic effects of this non-hydrolyzed material. Such protective ferments are, he believes, specific and are never found in the serum of normal (non-injected) animals. The blood will be seen, therefore, to have acquired definite digestive properties apart from that resident to a more or less extent in the leucocytes. A further step, taken by Abderhalden and his pupUs, has been the demonstration that substances, which are native to the system but foreign to the blood, may arise from physiologic or pathologic changes within the system itself and, after absorption into the blood, produce quite as definite a response as if the ma- terial were introduced from without. Schmorl and Veit have shown that syncytial cells and portions of the chorionic villi may become detached from the placenta and enter the mater- nal circulation, while other placentral products (of an unknown nature) pre- sumably also are either absorbed or washed into the blood current. As these substances are, in part at least, protein in nature, we should have, if the theory holds, a production of protective proteolytic ferments in the maternal bl6od, which are capable of digesting placental protein and are more or less specific. If, therefore, the serum of a suspected animal be treated with placental pro- tein, hydrolytic cleavage must occur with the formation of products capable" of more or less easy detection, provided the serum contains such ferments. These are the principles which form the basis of the test as originated by Abderhalden. Since the introduction of this test an enormous amount of work has ap- peared dealing with its various phases. The earlier publications seemed to ^ See Kolmer, Arch. Int. Med., 1912, IX, 220; Hastings, Jour. Am. Med. Assn., 1913, LX, 1208; Besredka and Manoukhine, Ann. de I'Inst. Pasteur, 1914, XXVIII, 569; Compt. rend. Soc. de Biol., 1914, LXXVI, 197; Hastings, Jour. Infect. Dis., 1914, XX, 52 and 72; Garbat, Am. Jour. Med. Sc, 1914, C3CLVIII, 84; Friedlander and Wagner, Am. Jour. Dis. Child., 1914, VIII, 134; Olmstead and Luttinger, Arch. Int. Med., 1915, XVI, 67; Winholt, Jour. Infect. Dis., 1915, XVI, 389; HoweU, Ibid., 456; Nakajo and Asakura, Ibid., 1915, XVII, 388 and 400, Kolmer and Strickler, Jour. Am. Med. Assn., 1915, LXIV, 800. ^ Abderhalden, Abwehrfermente des tierischen Organisms, Berhn, 1914. See, also, Vaughan, Jour. Am. Med. Assn. ,1914, LXIII, 365; Taylor and Hulton, Jour. Biol. Chem., 191S, XXII, 59; Adami, Jour. Canadian Med. Assn., IQ15, V, 569. Digitized by Microsoft® THE BLOOD 639 confirm the finding of Abderhalden that specific ferments were present in certain conditions, capable of digesting only so-called homologous protein. Many of the studies were adverse, the results being attributed by Abderhal- den to faulty technic. During the last year numerous researches have been made, which seem to strike a somewhat discordant note. At the present time the status of this question may be summed iip as follows : In every fresh serum, male or female, normal or abnormal, ferments are present. in the blood which, under certain conditions, are capable of causing hydroly tic cleavage of protein. Unquestionably, these proteolytic ferments are found in increased amounts in cases of pregnancy, carcinoma and other conditions to which the Abderhalden test has been applied. These ferments are, however, not to be regarded as specific, in the sense that they digest only homologous protein added as a substrat, as it is very questionable whether they act at all on the tissue used in the Abderhalden test. Owing to the presence in the blood of normal antiproteolytic substances, these normal (and hence not defensive ferments) ferments cannot exert their activity. If, however, this so-called "antitrypsin" be removed from the blood (i) by extraction with chloroform or ether (Delezenne and Pozerski), (2) by inactivation by addition of 0.2 per cent, acetic acid to the serum (Opie), or (3) by adsorption with an organic substrat or substances such as kaolin, starch or agar (Plant, Kjaergaard and others), changes occur in the degree of dispersion of the colloidal system and the normal proteolytic ferments become free to act. Sera treated in any of the above ways will undergo self-digestion and liberate dialyzable products capable of detection by the Abderhalden or other methods. The substrat (placental or other tissue) acts exactly in this way. Further investigations have shown that this reaction is similar in many respects to that of combina- tion of antigen and antibody as previously discussed, thus bringing the Ab- derhalden reaction into the realm of other immunity tests. According to Stephan, Hauptmann and, more recently, to Bronfenbrenner, the Abderhal- den test is possible only when complement is present. If complement is miss- ing, as when the serum is heated, any fresh serum will activate the test. From the later studies of Bronfenbrenner it seems probable that the reaction occurs in two stages: (i) the substrat (antigen) combines with the antibody, thus becoming sensitized; (2) this sensitized tissue then adsorbs the anti- trypsin, leaving the normal proteolytic ferments of the blood free to digest the protein of the serum. In other words, the Abderhalden test does not depend upon the presence of specific ferments which digest only homolo- gous tissue, but upon the presence of specific (probably) antibodies, which unit with the subtract and, in so doing, absorb antitryptic bodies and render the normal proteolytic ferments active. In this sense the Abderhalden test is to be regarded as specific, as it is certain that positive reactions in suspected cases are far more numerous, under the most carefully controlled conditions, than are the negative findings. Howeyer, the specificity is far removed from that claimed by Abderhalden. ' '■ For discussions of the various points raised in this newer investigation see Gammeltof t, Ugeskr. f. Laeger, 1913, LXXV, 1247; Heilner and Petri, Miinch. med. Wchnschr., 1913, LX, 1530; Oeller and Stephan, Ibid., 1914, LXI, 12, 75 and 425; Plant, Ibid., 238; de Waele, Ibid., 364; Flatow, Ibid., ^d&C^til^ ^IVS&Fb^^ft^'^' ^^^'^■' ^°'^' Hauptmann, Ibid., 640 DIAGNOSTIC METHODS Two distinct methods of detecting the presence of these antibodies have been devised. The first, the optic method, is capable of very wide application to the diagnosis of different conditions and should prove extremely useful in solving many problems of great clinical importance. It requires, however, considerable skill and technical ability as well as rather expensive apparatus. The second, the dialyzation method, is much simpler both in technic and in necessary equipment. Both of these methods tequire the most assiduous attention to the various details given, if any dependence is to be placed upon the results of the tests. I. The Optic Method. The basis of this test is as foUows: A solution of placental peptone in physiologic salt solution has a definite power of rotating the plane of polarized light. Likewise, the serum, both suspected and normal, has a similar action. The degree of rotation, however, of either remains permanent for some time at 37°C. If a solution of peptone and normal serum be mixed and the degree of rotation of this mixture determined, no appreciable change will be observed between the initial and final polarimetric readings. If, however, a solution of placental peptone, whose polarizing action is known, be treated with a serum containing the specific ferments above mentioned, digestion of the peptone occurs with the formation of products showing rotatory powers sufficient to change the initial rotation of the mixture to quite an extent. These changes may be observed at different intervals and interpreted as described later. Preparation of Placental Peptone. The fresh placenta is made blood-free by cutting it into small pieces and placing these under running water for about 15 minutes. Dry the pieces be- tween folds of filter paper and place them in about .five times their weight of 70 per cent, sulphuric acid. Allow the mixture to stand for three days at room temperature and shake the container frequently. At the end of this time, place the container in ice-water and dilute the contents with 10 volumes of distilled water, stirring constantly. Remove the sulphuric acid by adding approximately the calculated amount of finely powdered barium hydrate and complete the precipitation with a known solution of this salt, stirring the mix- ture constantly. When the reaction of the mixture becomes neutral to lit- mus paper, filter off the barium sulphate. If the filtrate be turbid, refiltra- 1167; von Domarus and Barsieck, Ibid., 1553; Freund and Brahm, Ibid., 1664; Lindig, Ibid., 1668; Beumer, Ibid., 1999; Kjaergaard, Ztschr. f. Immunitats f. Orig., 1914, XXII, 31; de Waele, Ibid., 170; Herzteld, Biochem. Ztschr., 1914, LXIV, 103; Ibid., 1915, LXVIII, 402; Wilheim and Szandicz, Ibid., 1914, LXV, 219; Benech, Compt. rend. Soc. de biol., 1914, LXXVI, 361; Bettencourt and Menezes, Ibid., 1914, LXXVII, 102; Michaelis and Lagermarck, Deutsch. med. Wchnschr., 1914, XL, 316; Frankel, Ibid., 589; Bisgaard and Korsbjerg, Ibid., 1367; Mosbacher and Port, Ibid., 1410; Peiper, Ibid., 1467; Oeller and Stephan, Ibid., iSS7; Friedmann and Schonfeld, Berl. klin. Wchnschr., 1914, LI, 348; Lange, Ibid., 785; Csepai, Wien. klin. Wchnschr., 1914, XXVII, 804; Franz, Arch. f. Gynak., 1 914, CII, S79; Heinemann, Monatsschr. f. Geburtsh. u. Gynak., 1914, XXXIX, 768; Adachi, Ztsclir. f. Geburtsh. u. Gynak., 1914, LXXVI, 516; Jobling and Petersen, Jour. Exper. Med., 1914, XIX, 480; Ibid., 1914, XX, 37; Bronfenbrenner, Ibid., 1915, XXI, 221 and 480; Jobling, Eggstein and Petersen, Ibid., 239; Jobling, Petersen and Eggstein, Ibid., 1915, XXII, 129 and 141; Echols, Jour. Am. Med. Assn., 1914, LXIII, 370; Eggstein, Ibid., 73s; Falls, Ibid., 1172; Ibid., 1915, LXIV, 1898; Ibid., 1915, LXV, 524; Bronfen- brenner, Schlesinger and Mitchell, Ibid., 1915, LXV, 1268; Bronfenbrenner, Jour. Lab. and Clin. Med., 1915, 1, 79. Digitized by Microsoft® THE BLOOD 64I tion is necessary until a perfectly clear filtrate is obtained. The separation of the barium sulphate is much facilitated by the use of the large centrifuge, if such be at hand. Wash the precipitate with a large amount of cold water and combine the filtrate and washings. Test the mixture for both barium and sulphuric acid. If either be present, it must bere moved. Now evapo- rate this barium- and sulphuric acid-free solution to dryness on the water-bath, under reduced pressure at a temperature not exceeding 40 or so°C. It is wise to test the evaporated material at several intervals for the presence of either barium or sulphuric acid, as these sometimes appear on concentrating the mixture. It is important that these be removed, as their presence will result in further hydrolysis of the peptone and, ii> consequence, will lessen the value of the final product. A thick yellow syrup or a foamy mass remains after this evaporation. The product may be used in this form, but it is pre- ferable to purify it, if reliable results are to be invariable. This yellowish residue is dissolved in methyl alcohol with the aid of heat and the hot solution is poured into absolute ethyl alcohol. The peptone is thrown down as a yellow powder, which is soluble in water to a clear yellowish solution of weakly acid or amphoteric reaction. This powder is not hygro- scopic. A further purification is still advisable. Dissolve the above yellow powder in water up to a 5 per cent, solution and add 10 per cent, solution of phosphotungstic acid as long as a precipitate forms. Filter and wash several times with water. Rub up this precipitate in a mortar with some water and twice its weight of barium hydrate. Filter again and remove the excess of barium from the filtrate with sulphuric acid. Filter off the barium sulphate and evaporate the filtrate to dryness under reduced pressure at 40 to 50°, as outlined above. This product is snow white and is permanent. It is absolutely essential for the successful application of the optic test that the placental peptone be as pure as possible. The same product is not always obtained by the above method, as the hydrolysis may proceed further than the peptone stage. Such products are unsuitable for the test. It is wise, therefore, to work as quickly and as carefully as possible with a large amount of placental substance, so that one may obtain an appreciable amount of placental peptone. If the product be found serviceable, it may be kept for years. A further point to be considered in the use of a prepared peptone is that the solution of this placental product must give absolutely no turbidity with the serum to be tested. Such a finding is not infrequent, owing to the probable presence of precipitins in the product. Such a peptone cannot be used. A further important property of the prepared peptone must be its power of rotating the plane of polarized light. The degree must not be too small or the product will prove of little value. It will be seen, therefore, that the preparation of a serviceable and proper placental peptone is a matter of con- siderable difficulty and is paramount to the successful performance of the test. One may preserve the peptone, prepared as above, either in the solid state or in the form of a solution. The advantage of a solution is that one has on hand a large amount of material, which will give good comparative results, as the solution is P^^^^^f^^jfj^^^^^^fB^SMP^^^ ^^^^ solutions of 0.5 to 642 DIAGNOSTIC METHODS 2.5 per cent, strength. He now advises a 10 per cent, solution 01 tne placental peptone in physiologic salt solution. This solution must be absolutely clear and colorless. If not, filter through thick paper or a Berkefeld. Preserve this clear solution by overlaying its surface with toluol. When required for the test, the solution is withdrawn by apipet dipping below the toluol. If care be taken to keep a layer of toluol over the solution, the stock material will be permanent for a long period. Should this solution become turbid at any time, the material should be thrown away and a new stock solution prepared as above. The optical activity of this stock solution must be tested before each test.i Obtaining the Serum. The serum of the patient is obtained as described under the complement- fixation tests, withdrawing 10 to 15 c.c. of blood. Place the blood, as drawn, directly into a sterilized centrifuge tube, so that all cellular elements may be completely separated. For a successful test the serum must show no sign of the presence of cells. A further precaution to be taken is that no sign of hemolysis must be present in the serum. For this reason, the cells should be separated rapidly. It is wise to make the test on the same day on which the blood is taken, although, if the precautions above mentioned are observed, a delay of 24 to 48 hours does not materially aSect the activity of the serum. Technic. Having prepared the 10 per cent, solution of placental peptone and having proven that it answers all the requirements above mentioned, place i c.c. of this clear solution (withdrawn by a pipet) in a small, clean, sterile test-tube. Add 2 c.c. of the clear suspected serum and shake the tube several times. Ex- amine the mixture carefully for any turbidity or precipitation. If any be observed, the test, cannot be carried out. Add sufiicient physiologic salt solution to the mixture to fill the i decimeter polarimetric tube (see below). This mixture with salt solution is preferably made in this way, rather than to add the salt solution after the peptone solution and serum have been placed in the polarizing tube. Any turbidity may be much more easily detected. Pour the above mixture into the i decimeter tube, whose mantle has been filled with water at 37°C. Carefully determine the initial rotatory power of the mixture, checking the readings several times and controlling them by subsequent ones after 5 or 10 minutes. No change should be observed in these readings. Place the tube and its contents in the incubator at 37°C. and repeat the readings every hour for a few periods and then continue every 6 or 8 hours. Do not extend the investigation over more than 48 hours. Record all readings and interpret them as given below. Control tubes must be arranged as follows: (i) the peptone solution alone^ (2) the suspected serum alone; (3) peptone solution plus normal serum; (4) peptone solution plus known positive serum; (5) peptone solution plus inacti- vated (heated to 6o°C.) suspected serum. In all of these controls the same 1 The placental peptone may now be obtained on the market from the Hochst-Farb- werke Co., New York. Digitized by Microsoft® THE BLOOD 643 conditions must be maintained and the same length of polarizing tube must be used as in the test itself. If any turbidity occurs in any of the control mixtures or tubes, these must be disregarded in interpreting the test as turbid solutions give variable results with the polarimeter. It goes without saying that this test requires the very best equipment possible. The cheap polariscopes are absolutely useless as they are not deli- cate enough to detect the fine variations given. The three-shadow instru- ment of Landolt-Lippich, made by Schmidt and Haensch, is especially to be •recommended. The polarizing tubes used are, preferably, the i decimeter tubes which are furnished with a mantle which may be filled with water at any desired temperature. If different length tubes are used in any of the tests, a correction must be made in order that comparative figures may be obtained. In performing this test, even to a greater degree than when the instrument is used in other work, much depends upon the ability of the worker to detect slight variations in the degree of the rotatory powers of the mixtures under investigation. The method is easy to learn, but the special sensibility toward such changes cannot be taught. Abderhalden cautions any one, who shows a working error of as much as 0.04° in his observations, against attempting to interpret the test. In reporting the result of this test, Abderhalden employs the following method. Deviations within 0.04° Negative Deviations between 0.05 and 0.1° Positive (-|-) Deviations between o.ii and 0.2° Positive (-|--|-) Deviations over 0.2° Positive (-|--f-|-) 2. The Dialyzation Method. This method is much more simple than the optic method, both as regards technic and apparatus. It must not be thought, however, that any less care is necessary in carrying out the details of the test. In fact, erroneous results are, perhaps, more easily obtained by careless manipulation when this method is employed. The basis of this method is the conversion of the colloidal non-dialyzable placental protein into dialyzable products through the activity of the fer- ments above mentioned. These products are, then, detected by simple color reactions in the dialysate. Preparation of Placental Albumin. Although Abderhalden does not regard the proteolytic ferments of the serum in pregnancy as absolutely specific for a given species of animal, he, nevertheless, insists that the protein preparations used in either of his methods be prepared from, the placenta of the same species as that of the animal whose serum is to be tested. As autolysis proceeds fairly rapidly in placental tissue, the albumin should be prepared from placentas which are as fresh as possible. Remove the ex- ternal portions of the placenta, such as the membranes, and wipe away as much blood as possible. Di^ifcizehfet^alttfephdsijft® small pieces and wash for a 644 DIAGNOSTIC METHODS short time in running water. ^ While this is being done, boil about 2 liters of water to which are added 2 drops of glacial acetic acid. Throw the washed bits of placenta into this boiling water and boil for S to 15 minutes. Pour the mixture upon a loose quick-acting filter and boil the pieces again with a second portion of acidulated water for 5 to 15 minutes. Pour off this water and test with the triketohydrinden hydrate reaction given below. If a positive reaction obtains, the placental tissue must be again boiled with acidulated water until a negative reaction occurs. The essential points in this process are rapid and complete coagulation of the placental albumin and the removal of all soluble dialyzable material which may react with the reagent mentioned above. ^ As soon as a negative result is obtained with the extractive water, pour the mixture into a wide-mouth flask, add some chloroform, overlay the fluid with toluol and stopper the flask; or place the material in several smaller glass jars and overlay with toluol. This placental albumin keeps almost in- definitely and may be removed from the containers as desired. It should be tested, from time to time, to show that it contains, in itself, nothing which may react with the reagents used in the later test.^ Obtaining the Serum. The serum is obtained by venous puncture as previously described, the blood (about 10 c.c.) being drawn directly into a sterilized centrifuge tube. The cellular elements are separated as quickly as possible and the serum drawn off into a clean sterile tube. It is of especial importance in this test that the serum show no sign of hemolysis. As it has been shown that amino- acids are present in the blood during digestion and may, therefore, give a posi- tive reaction with triketohydrinden hydrate, it is wise to take the blood in the morning before breakfast in all cases; If such be not done, a less amount of serum must be used in the later test to compensate for this possible error. Selecting the Dialyzing Tubes. It is evident that this part of the preparation for the test is of extreme im- portance. The dialyzing thimbles must be permeable for peptone but not for albumin. Unless these conditions obtain, the test is valueless. Not all of the thimbles, purchasable upqii the market, are by any means available. Abderhalden advises the use of the diffusion shells No. 579 A. of Schleicher and Schiill. Not all of these will answer the purpose. It is necessary, therefore, that all the dialyzing thimbles used in the test should have been previously tested and known to answer the above require- ments. As the thimbles are usually dry and hard when obtained, soak them in cold water for a few hours, place them in boiling water for a few seconds and keep them in water covered with toluol. To test these thimbles for their permeability for albumin, proceed as follows. Remove the thimble from the water and place in it 5 c.c. of serum "^ It is essential that all visible blood be removed as its presence will introduce a large error ^ Care must be taken not to add an excess of acetic acid to the water as this may interfere with the ninhydrin test and thus give rise to an appreciable error. ' Abderhalden recommends that this placental albumin be tested with ninhydrin before being used in any test. This is a vital point. Digitized by Microsoft® THE BLOOD 645 or of a solution of egg albumin. Add a few drops of toluol to prevent bacterial action. Place 20 c.c. of distilled water in the dialyzing vessel and overlay this with toluol. This dialyzing vessel should be quite narrow, the distance between the wall and the thimble (when in place) being about J^ cm. These vessels are kept plugged with cotton and are sterilized before use. Now sus- pend the thimble with its albuminous contents in the dialyzing tube in such a way that the fluid outside is as high or, preferably, a little higher than that within the thimble. Plug the vessel with cotton to prevent contamination and put the apparatus in the incubator at'37°C. for 18 to 24 hours. At the end of this time, test the dialysate (outside fluid) for albumin by the biuret or triketohydrinden hydrate reactions given below. Those thimbles giving negative results are retained to be tested for their permeability to peptone. The shells permitting the passage of albumin cannot be used in the test. Select those thimbles showing impermeability to albumin and wash them thoroughly in water. Place in them 5 c.c. of a i to 1000 solution of Witte's or, preferably, peptone from silk (peptone La Roche) and add sufflcient toluol t6 cover the solution. Dialyze as above against 20 c.c. of distilled water, placing the apparatus in the incubator for 18 hours at 37°C. Those thimbles which permit the passage of peptone, as shown by the triketohydrinden hy- drate test, are then kept for use in these tests and the non-permeable ones are laid aside. The properly tested and selected thimbles are then preserved in water overlaid with toluol. Technic. Remove a few pieces of the coagulated placental albumin from the con- tainer, wash in distilled water and dry between filter paper.' Break this up into very small bits or grind up in a mortar. Weigh out three portions of J^ gram each. Place J^ gram in each of three tested dialyzing thimbles in such a way that none of the material touches or remains upon the top or outside of the shells. Carefully wash off the outside of the thimble by means of a stream of distilled water or hold it under running water. This is done to re- move any possible adhering placental albumin, which would vitiate the test later made. Now add to tube No. i, i to 1.5 c.c. of clear hemoglobin-free serum to be tested. This serum is withdrawn from its contaijier by means of a sterile graduated pipet. Overlay the surface of the mixture in the thimble with toluol. This thimble is then placed in a sterile dialyzing tube, as de- scribed above, containing 20 c.c. of distilled water, which should stand slightly higher than the fluid in the thimble. Overlay the external fluid with toluol and plug the dialyzing vessel with cotton to prevent contamination. Place the apparatus in the incubator at 37°C. for 18 hours and then test the dialysate for peptone as outlined below. Controls. Charge thimble 2 with J^ gram of placental albumin and i c.c. of serum of a known positive control. Overlay with toluol and arrange as above. '■ Test the material before use with the triketohydrinden hydrate reaction. Absolutely no trace of a blue coloration ^0^^ Microsoft® 646 DIAGNOSTIC METHODS Charge thimble 3 with 3^ gram of placental albumin and i c.c. of a known negative serum or with the inactivated (heated to 6o°C.) serum used in the test. A further control should be run, using i c.c. of the serum alone without the addition of albumin, to prove that it does not contain any dialyzable sub- stances which will give the later reactions. The tests with all of these controls are carried out exactly as the test it- self, every precaution being taken to prevent the introduction of errors. The tests for cleavage products of albumin are made with one of the following tests, the latter being some respects preferable. The Biuret Test. This was the test formerly employed by Abderhalden and has some advantages in that it does not react with certain dialyzable products not infre- quently present in the serum of normal subjects. It requires considerable care and skill in manipulation as well as in interpretation. Doubtful results are very frequent unless every precaution be taken. Remove about 10 c.c. of the dialysate by means of a pipet dipping below the toluol. Place this in a test-tube and add 5 c.c. of a $$ per cent, sodium hydrate solution. Mix by careful shaking and add very carefully, drop by drop, from a buret a very dilute (0.25 per cent.) solution of copper sulphate in such a way that a distinct contact ring is formed. If peptone is present, a violet-red to a pure red contact ring will be observed, sharply differentiated from the lower colorless and upper blue solutions. This is a positive reaction. A negative result is shown by the appearance of a distinctly blue ring. It is not the simplest matter to distinguish between the various shadings which occur, so that one must not make his decision without having had some ex- perience in differentiating the colorations obtained with pure albumin and peptone solutions. The Triketohydrinden Hydrate Reaction. This reagent occurs in colorless crystals readily soluble in water. It may be obtained from the Hochst-Farbwerke (Lucius & Briining) Company under the trade name of "ninhydrin." Its formula is CaH4\ /C(0H)2. It is of especial importance that every precaution be taken to prevent error when this reagent is used, as reactions may rise from the presence of substances which are not at all associated with the hydrolytic products of protein material. This reagent is not by any means specific, even for albumin, pep- tone or amino-acids, although it was formerly believed that it reacted only with substances containing an amino and a carboxyl group, the former es- pecially in the a position. It has been shown that there is a larger number of compounds which are not, in a chemical sense combinations with amino and carboxyl groups and which, nevertheless, give very characteristic re- actions. Among these we find: amines; amino-aldehydes; urea derivatives; amino-sulphonic acids; ammonium derivatives of certain organic acids, di- carbonyl compounds and halogen-aldehydes; ammonium compounds of thiosulphuric, oxy-sulphqg^gj[fg^i|i^s/5^^^S^^^; ammonium formate, am- THE BLOOD 647 monium thio-lactate, etc. Of special importance is the fact that a very small amount of basic products of putrefactive origin will give a decided reaction (hence the importance of using every means to prevent decomposi- tion of the tissue. Certain further precautions are essential. If any of the original placental albumin be left on the outside of the dialyzing thimble, a very obvious error will arise. If the serum used contains amino-acids, the amount must be determined by depth of color with the ninhydrin. One must avoid the presence of acid or ammoniacal fumes in the laboratory. Strong alkalies cause, in themselves, a coloration with the reagent, while dilute alkalies may decolorize the solution. Acids prevent the appearance of the blue color and will destroy the color already formed, even in the presence of a large amount of reacting material. It must be insisted, therefore, that the fluid to be tested be absolutely neutral. Further, all vessels and pipets must be absolutely clean and the water used must be free from bacteria.' Remove lo c.c. of the dialysate by means of a pipet dipping below the toluol and place this in a large test-tube. Add 0.2 c.c. of a i per cent, aqueous solution of triketohydrinden hydrate. Heat rapidly to the boiling point and keep the mixture boiling for one minute. If the reaction be negative, the solution remains colorless or becomes, at most, light yellow. If the reaction be positive, a deep blue color will appear either immediately or on allowing the tube to stand for a short time. After use wash the thimbles thoroughly in running water and then place them in boiling water for not over 15 seconds. The reaction is carried out in the same way with the control tubes. Tube 2 should show a distinct positive reaction, while tube 3 should give a negative result. The tube with serum alone should show a negative reaction, but oc- casionally it is positive owing to the presence of a large amount of amino- acids in the serum as drawn. If the controls are all positive, the test is, of course, valueless, as some factor has been imperfectly controlled. Bronfenbrenner's Modification. Using the known property of antibodies of uniting with antigen at low temperatures, which prevent the activity of the complement and, also, ex- clude the activity of the proteolytic ferment, Bronfenbrenner adds to 3^ gram of placental tissue, contained in a glass tube, 1.5 c.c. of the suspected serum. Place this in the ice box over night. The next morning the serum is poured off, the tissue is placed in a centrifuge tube and washed, by centrifugation and decantation, until all traces of the serum are removed. The sensitized tissue is then placed in a dialyzing thimble and 1.5 c.c. of fresh male guinea-pig serum or normal human serum are added. Follow the usual technic of Ab- derhalden from this point. A positive ninhydrin reaction, under these conditions, is almost absolute evidence of specific disease. ' ^ See Ruhemann, Jour. Chem. Soc, Trans., 1910, XCVII, 1438 and 2025; Ibid., igii, XCIX, 792; Ibid., 1912, CI, 780; Abderhalden and Schmidt, Ztschr. f. physiol. Chem., igii, LXII, 37; Ibid., 1913, LXXXV, 143; Pearce, Jour. Am. Med. Assn., 1913, LXI, 1456; Warfield, Ibid., 1914, LXII, 436; Halle, Lowenstein, and Pribram, Biochem. Ztschr., 1913, LV, 357; Neuberg, Ibid., 1913, LVI, 500; Herzfeld, Ibid., 1914, LIX, 249; Neuberg, Ibid., 1914, LXVII, 56; Deetjen and Frankel, Miinch. med. Wchnschr., 1914, LXI, 466; Howe Biochem. Bull., 1914, III, 26Pi9ifized by Microsoft® 648 DIAGNOSTIC METHODS If the suspected serum, which has been in contact with the placental tissue in the ice box, be placed in a dialyzing thimble and kept at 37°C. for 18 hours, the dialysate will show a positive reaction due to the removal of the antitryp- sin and the later self- digestion of the serum protein. The untreated serum, used as a control, should show no ninhydrin reaction. Value of the Test. This test, applied by either one of the above methods, is. extremely valu- able. Abderhalden's results, which have been definitely confirmed (see bibli- ography at the end of this discussion), show that in the majority of cases in which the reaction was positive the patient has been proven to be pregnant; while negative reactions have been given only in non-pregnant cases. In other words, a negative result is definite, a point of distinction from a negative result with the complement-fixation test; while a positive result is evidence of the presence of specific elements within the blood (pregnancy), either at the time of the test or at least within a period which does not exceed two weeks (Schwarz) . In the absence of a true pregnancy, hydatidif orm mole and other pathologic conditions of the.chorionic villi may be accountable for a positive reaction. A negative test should be considered as eliminating pregnancy. As this reaction is evidence of pregnancy, in most cases giving positive results (an error of lopercent. being probable) ,it is especially interesting to find that it is positive from the middle of the second month of pregnancy on and disappears within 10 to 15 days after the termination of the pregnancy. It is, therefore, of great value in early diagnosis as well as in the diagnosis of a second pregnancy following so closely upon a previous one that other signs are not pres- ent. Differential diagnosis should be greatly improved by the use of this test and fewer laparotomies performed under the mistaken diagnosis of tumor. A further value of the testis found in the fact that medico-legal cases may be established on a firm basis of definite knowledge of the conditions present.^ ' As this test is of so much importance I give a fairly full bibliography of the subject for reference. Abderhalden, Freund and Pincussohn, Prak. Ergeb. d. Geburtsh. u. Gynak., 1910, II, 367; Abderhalden, Handb. d. biochem. Arbeitsmeth., 1911, V, 575; Ibid., 1912, VI, 223; Abderhalden and Kiutsi, Ztschr. f. physiol. Cham., 1912, LXXVII, 249; Abderhalden, Ibid., 1912, LXXXI, 90; Ibid., 191 2, LXXXII, 109; Munch, med. Wchnschr., 1912, LIX, 1190, 1305, 1939, and 2172; Deutsch. med. Wchnschr., 1912, XXXVIII, 2160; Abderhalden and Weil, berl. Tierarztl. Wchnschr., 1912, XXVIII, 665; Frank and Heimann, Berl. klin. Wchnschr., 1912, XLIX, 1706; Franz and Jarisch, Wien. Idin. Wchnschr., 1912, XXV, 1441; Veit, Ztschr. f.'Geburtsh. u. Gynak., 1912, LXXII, 463; Petri, Zentralbl. f. Gynak., 1913, XXXVII, 23s; Schwarz, Interstate Med. Jour., 1913, XX, 19s; Henkel, Arch. f. Gynak., 1913, XCIX, 56; Lindig, Munch, med. Wchnschr., 1913, LX, 288; Abderhalden, Ibid., 411 and 462; JeUinghaus andLosee, Bull. Lying-in Hosp., New York, 1912, IX, 68; Gutman and Druskin, Med. Record, 1913, LXXXIV, 99; Engelhorn, Munch, med. Wchnschr., 1913, LX, 587; SchUmpert and Hendry, Ibid., 681; Freund and Brahm, Ibid., 685; Freund, Ibid., 700 and 763; Abderhalden, Ibid., 701 and 763; Lindig, Ibid., 702; Heimann, Ibid., 91s; Stange, Ibid.' 1084; Rubsamen, Ibid., 1139; SchifF, Ibid., 1197; King. Ibid., 1198; Maccabruni, Ibid., 1259; Ann. di ostet., Milano, 1913, 1, 486; Abderhalden, Miinch. med. Wchnschr.,' 1913, LX, 1386; Lamp6 and Papazolu, Ibid., 1423 and 1533; Frank and Rosen- thal, Ibid., i425;Lichtenstein, Ibid., 1427; Heilner and Petri, Ibid., 1530 and 1775; Steising, Ibid., 1535; Frank, Rosenthal and Biberstein, Ibid., 1594; Abderhalden and Weil, Ibid., 1703; SchUmpert and Issel, Ibid., 1758; Bruck, Ibid., 177s; Goudsmit, Ibid., 177s; Abder- ' halden and Schiff, Ibid., 1923; Plotkin, Ibid., 1942; Lamp€ and Fuchs, Ibid., 2112; Tschud- nowsky. Ibid., 2282; Behne, Zentralbl. f. Gynak., 1913, XXXVII, 613; Parsamow, Ibid., 934; Mayer, Ibid., 1181; Porchownick, Ibid., 1226; SchUmpert, Berl. klin. Wchnschr., 1913, L 1136; Rosenthal, Ibid., 1149; Gottschalk, Ibid., 1151; Veit, Ibid., 1241; Aschner, Ibid., 1243; Schafer, Ibid., J^^^/^^^^Ifl^c/J^^^y^eler and Lohnberg, Ibid., 1898; THE BLOOD 649 2. Of Other .Conditions. The principles upon which Abderhalden's sero-diagnosis is supposedly based have been considerably altered by the newer researches. However, the method has found, and will continue to find, wide application in the diagnosis of varied conditions. ^ Although the basic idea of the presence of Williams and Pearce, Surg., Gynec. and Obst., 1913, XVI, 411; McCord, Ibid., 418; Judd, Jour. Am. Med. Assn., 1913, LX, 1947; Am. Jour. Med. Sc, 1913, CXLVI, 391; Pari, Gazz. d. osp., 1913, XXXIV, 727; Ekler, Wien. klin. Wchnschr., 1913, XXVI, 696; Jaworski and Szymanowski, Ibid., 922; Daunay and Ecalle, Compt. Rendu Soc. de biol., 1913, LXXIV, 1190; Sunde, Norsk Mag. f. Laegevid., 1913, LXXIV, i234;'Schlimpert, Brit. Med. Jour., 1913, II, 1003; Deutsch. med. Wchnschr., 1913, XXXIX, 1225; Jonas, Ibid., logg; Naumann, Ibid., 2086; Evler, Med. KMn., 1913, IX, 1042 and 1086; Bauer, Ibid., 1797; Abderhalden, Monatschr., f. Geburtsh. u. Gynak., 1913, XXXVIII, 24; Wolff, Ibid., 394; Schwarz, Interstate Med. Jour., 1913, XX, 393; Jour. Am. Med. Assn., 1913, LXI, 484; Abderhalden, Gynak. Rundschau, 1913, VII, 467; Decio, Ibid., 436; Ann. di ostet., 1913, I, 198; Abderhalden and Fodor, Ztschr. f. physiol. Chem., 1913, LXXXVII, 220; Abderhalden and SchifiE, Ibid., 225 and 231; Gabastou and Widakowich, Semana Med., 1913, XX, 813; Scherer, Berl. klin. Wchnschr., 1913, L, 2183; Williamson, Jour. Obst. and Gynec. Brit. Emp., 1913, XXIV, 211; Werner and von Winiwarter, Wien. klin. Wchnschr., 1913, XXVI, 1841; Labusquiere, Ann. de Gynec. et d'Obst., 1913, XL, 664; Fraenkel, Berl. klin. Wchnschr., 1913, L, 2287; Kabanoff, Med. Obozr., 1913, LXXX, 312; Sabin, Presse mJd., 1913, XXI, 1616; Williams and Ingraham, Colorado Med., 1913, X, 364; Abderhalden, Miinch. med. Wchnschr., 1913, LX, 2774;Lamp5, Ibid., 2831; Meyer, Ibid., 2906; Wegener, Ibid., 1914, LXI, 15; Bronstein, Ibid., 74; OeUer, Stephan and Mayer, Ibid., 12 and 75; Behne, Zentralbl. f . Gynak., 1914, XXXVIII, 74; Akimoto, Ibid., 81 ; Diner, New York Med. Jour., 1914, XCIX, 478; Holmes, Chicago Med. Recorder, 1914, XXXVI, 213, 475 and 593; Schwarz, Jour. Am. Med. Assn., 1914, LXIII, 371 ; Lindemann, Ztschr. f . d. ges. exper. Med., 1914, IV, 177; Guggenheimer, Ztschr. f. Chemotherap., 1914, III (2TI) 210; McLester, Am. Jour. Med. Sc, 1914, CXLVIII, 75; Pearce and Williams, Jour. Infect. Dis., 1914, XIV, 351; Pregl, Fermentforsch., 1914, 1, i; de Crinis, Ibid., 13; Abderhalden, Ibid., 20; Hirsch, Ibid., 33; Strauss, Ibid., 55; Paquin, Ibid., 58; Ross and Singer, Arch. Int. Med., 1914, XIV, 552; Ibid., 1915, XV, 724; Lange, Biochem. Ztschr., 1914, LXI, 193; Thar and Kotschnieff, Ibid., 1914, LXIII, 483; Salus, Ibid., 1914, LXV, 381; Parsamow, Ibid., 1914, LXVI, 269; Neuberg, Ibid., 1914, LXVII, 56; Halsen, Ibid., 277; Frankel, Ibid., 298; Thar and Kotschnieff, Ibid., 1915, LXIX, 389; Abderhalden, Deutsch. med. Wchnschr., 1914, XL, 268 and 401; Hirsch, Ibid., 270; AJlmann, Ibid., 271; Pincussohn, Ibid., 425; Abderhalden, Ibid., 428; Lamp6 and Stroomann, Ibid., 635; Lamp^, Ibid., 1213; Melikjanz, Ibid., 1369; Nieszytka, Ibid., 1519; Hirsch. Ibid., 1560; Otto and Blumen- thal. Ibid., 1836; Eder, Ibid., 1838; Abderhalden, Miinch, med. Wchnschr., 1914, LXI, 233 and 401; Voelkel, Ibid., 349; Singer, Ibid., 350; Lamp6, Ibid., 463; Kammerer, Clausz and Dieterich, Ibid.; 469; Swart and Terwen, Ibid., 603; Abderhalden and Grigorescu, Ibid., 767; Abderhalden and Fodor, Ibid., 765; Schiff, Ibid., 768; Abderhalden, Holle and Strauss, Ibid., 804; Abderhalden and Paquin, Ibid., 806; Deetjen and Frankel, Ibid., 826; Abder- halden and Wildermuth, Ibid., 862; Rosenthal and Biberstein, Ibid., 864; Abderhalden and Ewald, Ibid., 913; Lichtenstein and Hage, Ibid., 915; Freymuth, Ibid., 916; Griesbach, Ibid., 979; Abderhalden and Grigorescu, Ibid., 1209; Herzfeld, Ibid., 1503; Abderhalden, Ibid., 1879; Jaff6 and Pribram, Ibid., 2125; Nieden, Ibid., 2200; JafE6 and Pribram, Ibid., 1915, LXII, 614; Puppel, Monatsschr. f. Geburtsh. u. Gynak., 1914, XXXIX, 764; Fetzer, Ibid., 1914, XL, 598; Petri, Ibid., XLI, 309 and 388; Baumann, Ibid., 1915, XLII, 199; WaUis, Jour. Obs. and Gynec. of Brit. Emp., 1914, XXV, 53; Paine, Boston Med. and Surg. Jour., 1914, CLXX, 303; Ecalle, Arch. mens. d'Obs. et de Gynfic, 1914, III, 257; Partos and d'Ernst., Ibid., 333; Jellinghaus andLosee, Am. Jour. Obs. and Dis. Women and Child., 1914, LXIX, 593; Kolmer and Williams, Ibid., 1915; LXXI, 899; Ibid., 1915, LXXII, loi; Grey, Bull. Johns Hopkins Hosp., 1914, XXV, 117; Echqls, Wis. Med. Jour., 1914, XIII, i; Stoner and Skeel, Cleveland Med. Jour., 1914, XIII, 392; Bullock, Lancet, 11, 225; Gavronsky, Ibid., 1915, I, 119; Leitch, Brit. Med. Jour., 1914, II, 161 and 330; Hinselmann, Zentralbl. f. Gynak., 1914, XXXVIII, 258; Kjaergaard, Ibid., 264; Schott- laender. Ibid., 425; Primsar, Ibid., 438; Hussy, Ibid., 897; Gentili, Ibid., 1159; Saxl, Berl. klin. Wchnschr., 1914, LI, 824; Brieger and Schwalm, Ibid., 839; Ebeler and Lohnberg, Ibid., 191S, LII, 319; Abderhalden, Med. Klin., 1914, X, 665; Lamp6 and Paregger, Ibid., 725; Abderhalden and Grigorescu, Ibid., 728; Forster, Ibid., 772; von Graff and Saxl, Ibid., 1387; Lindstedt, Hygiea, 1915, LXXVII, 833; Goldstone, Med. Rec, i9i4,LXXXVI, 67; Keitler and Lindner, Wien. klin. Wchnschr., 1915, XXVIII, 549; Goodman, Ann. Surg., 1915, LXI, 149. 1 See Wolff and Frank, Berl. klin. Wchnschr., 1914, LI, 875; Gumpertz, Beitr. z. Klin, der Tuberk., 1914, XXX, 2q|^'^(fjjg5^t)3l"VWM^iWa^ Munch, med. Wchnschr.; 1914, 650 DIAGNOSTIC METHODS specific ferments in the blood in pathologic conditions must, probably, be changed, yet the test appears to be somewhat specific in the sense that certain antibodies are probably present in the serum, which antibodies so sensitize the substrat that the antiproteolytic substance of the blood is withdrawn, thus leaving the protein of the serum free to undergo self-diges- tion by the normal proteolytic ferments of the blood. The possibihty un- questionably exists that the use of a definite substrat (homologous in nature) may enable us to detect and differentiate pathologic processes much more sharply than has hitherto been the case. Although the specific nature of this test, as originally advocated by Abderhalden, has been attacked and shown to be dependent on other factors than those originally assumed, yet it is certain that there is much diagnostic value to the test, whatever may be its explanation. Considerable study must, however, be made before we are in a position properly to interpret our findings in all cases. (a) Cancer. In no other condition is a certain and constant diagnostic test so much to be desired as in cancer. Many methods have been advocated but Abderhalden's test is by far the most promising. The technic of the test is as described above. As the protein material to be sensitized, cancerous tissue is employed, the material being cut into small bits and prepared exactly as is placental tissue. It would seem neces- sary to use homologous tissue in this work, that is carcinomatous or sarcoma- tous as the case may be. This point has not been definitely established but the results in any given case would, probably, be more specific. The dialyza- tion fluid is distilled water. Erpicum advises the use of a 2 per cent, solution of sodium fluorid as an antiseptic, but this is unnecessary if toluol is used freely as should be done. Sterility of all material and glass-ware is essential in this test. The results reported have been more than encouraging in that benign growths seem to give, almost constantly, negative results while the malignant forms show an extremely large percentage of positive findings. The writer has used this test frequently and has corroborated his findings by pathological examination of the tissues removed at operation in many of the cases. In some of the cases the results have been far from satisfactory.'^ More detailed study is necessary before the value of this test in cancer is determined. LXI, 980; Ammenhauser, Ibid., 2000; Schultz and Grote, Ibid., 2510; Baeslack, Jour. Am. Med. Assn., 1914, LXII, 1002; Ibid., 1914, LXIII, 559; Luxenburg, Med. Klin., 1914, X, 1104; Varney and Morse, Michigan State Med. Jour., 1914, XIII, 515; Hegner, Cor.-Bl. f. schweiz. Aerzte, 1914, XLIV, 1292; Vladesco and Popesco, Compt. rend. Soc. de. biol., 1914, LXXVII, 586; Smith, Jour. Infect. Dis., 1915, XVI, 319; FaUs, Ibid., 466; Voelkel, Miinch. med. Wchnschr., 1914, LXI, 349; Wohl, Am. Jour. Med., Sc, 1915, CXLIX, 427. 1 See Abderhalden, Munch, med. Wchnschr., 1913, LX, 2385; Labb6, Gaz. m^d. de Nantes, 1913, 2 me. S., XXXI, 461; Frank and Heimann, Berl. klin. Wchnschr., 1913, L, 631; Markus, Ibid., 776; Munzer, Ibid., 777; Epstein, Wien. klin. Wchnschr., 1913, XXVI, 649; Deutsch and Kohler, Ibid., 1361; Erpicum, Bull, de I'Acad. roy. de m6d. de Belg., 1913, XXVII, 624; Ludke, Gaz. d. h6p., Paris, 1913, LXXXVI, 1064; von Gambarofi, Miinch. med. Wchnschr., I9I3,LX, 1644; Brockman, Lancet, 1913, II, 1385; Abderhalden, Deutsch. med. Wchnschr., 1913, XXXIX, 2391; Hara, Ibid., 2359; Fried, Miinch. med. Wchnschr., 1913, LX, 2782; Schwarz, Am. Jour. Obst., 1914, LXIX, S4; Ball, New York Digitized by Microsoft® THE BLOOD 65 1 (b) Dementia Prsecox. In the realm of nervous disorders differential diagnosis is not always as exact as could be desired. Abderhalden's test has been applied by many in such cases with excellent results. In this work the selection of the proper material for the substrat seems to be the matter of greatest importance. It is probable that testicular tissue should be used for male patients and ovarian tissue for female sufferers, although cortical tissue and thyroid have been used by some but positive results with the latter are few. The adoption of cortical tissue is highly improbable as differential diagnosis is not subserved by its use. General paralysis, epilepsy with dementia, and manic-depressive insanity all respond promptly to brain tissue but very rarely to testicular or ovarian material. A further point is that all syphilitic and parasyphilitic disorders seem to cause hydrolysis of cortical brain tissue but not of other organs. The antibodies found in the serum of these patients are, therefore, more or less specific, as a reaction with testicular or ovarian tissue is almost positive evidence of dementia praecox.^ E. Herman-Perutz Reaction. As the Wassermann test for syphilis is so complex and requires so much experience for its proper performance and interpretation, attempts have been made to find a serum reaction for syphilis which could be used by the general worker and which, at the same time, would give results comparable to those of the Wassermann test. Such a test is that of Herman and Perutz,^ which is a Med Jour., 1913, XCVIII, 1249; Jour. Am. Med. Assn., 1914, LXII, 599; Halpern, Mitt a. d. Grenzgeb. d. Med. u. Chir., 1914, XXVII, 340; Piorkowski, Berl. klin. Wchnschr., 1914, LI 254; Frankel, Ibid., 356; Fasiani, Wien. klin. Wchnschr., 1914, XXVII, 267; Manoiloff, Ibid., 269; Oeller and Stephan, Miinch. med. Wchnschr., 1914, LXI, 579 and 583; Schawlow, Ibid., 1386; Weinberg, Ibid., 1617, 1685 and 1732; Frankel, Deutsch. med. Wchnschr., 1914, XL, 589; Hara, Ibid., 1258; Cytronberg, Mitt. a. d. Grenzgeb. der Med. u. Chir., 1914, XXVIII, 243; Heimann and Fntsch, Arch. f. Chir., 1914, CIII, 659; Lan- zarini, Gazz. d. osp., 1914, XXXV, 1057; Trubina, Deutsch. Zeitsch. f. Chir., 1914, CXXXI, 520; Fulchiero, Rif. med., 1914, XXX, 1261; Van Slyke and Vinograd, Proc. Soc. Exper. Biol, and Med., 1914, XI, 154; Dick, Jour. Infect. Dis., 1914, XIV, 242; Kahn, Arch. Diag., 1914, VII, 356; Biehn, 111. Med. Jour., 1915, XXVII, 206; Goodman, Surg., Gyn. and Obs., 1914, XIX, 797; Levin, New York Med. Jour., 1914, C, 621; Bullock, Lancet, 1915, 1, 223; Goodman and Berkowitz, Surg., Gyn. and Obs., 1915, XXI, 463; Ball, Jour. Am. Med. Assn., 1914, LXII, 599; Ibid., i9i4,LXIII, 1169; Lowy, Ibid., 1915, LXIV, 1559; Levin and Van Slyke, Ibid., 1915; LXV, 94s; Berghausen, Interstate Med. Jour., 1915, XXII, 228. ' See Fauser, Deutsch. med. Wchnschr., 1913, XXXIX, 304; Wegener, Munch, med. Wchnschr., i9i3,LX, 1197; Schulz, Deutsch. med. Wchnschr., 1913, 39, i399;Lampe, Ibid., 1774; Bundschuh and Roemer, Ibid., 2029; Fischer, Ibid., 2138; Fuchs, Munch, med. Wchnschr., 1913, LX, 2230; Binswanger, Ibid., 2321; Theobald, Med. Klin., 1913; IX, 1850; Berl. klin. Wchnschr., 1913, L, 2180; Beyer, Miinch. med. Wchnschr., 1913, LX, 2450; Kafka, Med. Klin., 1914, X, 153; Fauser, Munch, med. Wchnschr., 1914,; LXI, 126; Kastan, Deutsch. med. Wchnschr., 1914, XL, 319; Arch. f. Psychiat., 1914, LIV, 928; Grigorescu, Med. Khn., 1914, X, 418; Neue, Ibid., 1217 and 1259; Simon, Jour. Am. Med. Assn., 1914, LXII, 1701; Sterne, 111. Med. Jour., 1914, XXVI, 327; Gehrmann, Ibid., 335; Holmes, Ibid., 332; New York Med. Jour., 1914, XCIX, 567; Chicago Med. Recorder, 1914, XXXVI, 644; Ibid., 1915, XXXVII, 88; Bouman and Hasselt, Nederl.Tijdschr. v. Geneesk., 1915, I, 423; Mayer, Miinch. med. Wchnschr., 1915, LXII, 580; Sioli, Arch. f. Psychiat. u. Nervenkr., 1914, LV, 241. " Med. Khn., 1911, VII, 60. Landau (Wien klin. Wchnschr., 1913, XXVI, 1702; see, also, Presse m6d., 1914, XXII, 335, and Riv. osped., 1914, IV, 641) has introduced a test for syphilis, using as his reagent a i per cent, solution of iodin in carbon tetrachlorid. To 0.2 c.c. of fresh serum he adds o.i c.c. of the above reagent, shakes the tube and allows the mixture to stand for four hours. Positive results are shown by a clear yellow serum above the decolorized reagent, indicatinft-dgiigdaa-ajLttoJ^fei probably by the lipoids. If the I 652 DIAGNOSTIC METHODS modification of an older test of Porges. Its simplicity should recommend it, especially as its results are usually definite and easily interpreted. For this test two solutions are used. Solution A. Sodium glycocholate, 2.00 grams Cholesterin, 0.40 gram 95 per cent, alcohol, 100.00 c.c. At the time of making the test, this solution is diluted with distilled water in proportion of i to 20. Solution B. A 2 per cent, aqueous solution of sodium glycocholate. This solution must be made up fresh at the time of making the test, as it is not permanent. Further, the solution is not a perfect one so that the bottle should be shaken before using. Technic. Withdraw the blood as for the Wassermann test. As only a small amount is needed, puncture of the ear or finger is usually sufficient. Col- lect the blood in a sterile tube, allow it to clot and centrifuge. The serum must show no hemolysis. Pipet off the clear serum. Although Herman and Perutz believed the test was better if the serum was inactivated by heating to s6°C. for one-half hour, later work has shown that this is not essential. To 0.4 c.c. of the clear serum add 0.2 c.c. of the i to 20 dilution of solution A and 0.2 c.c. of solution B. Shake thoroughly, plug the tubes with cotton and allow them to stand not longer than 18 to 24 hours at room temperature. On examining the tubes, a positive result is indicated by a flocculent precipi- tate, while a negative result is shown by no precipitation. The results may be expressed as +, -f -+-, + + + depending on the degree of precipitation. This gives the results so that comparison with the Wassermann may be made easily. This test practically parallels the Wassermann test, except that in secondary syphilis the results are not quite so uniform with the former. The percentage of variation is greater than with the Wassermann test, so that our interpretation of the result would be guarded. In other words, a positive result indicates syphilis, while a negative result does not exclude syphilis. This reaction should find a place in the laboratory of every general worker serum is an opaque grayish white, the reaction is negative. While this test gives results in many cases which parallel those of the Wassermann test, yet it shows a large percentage of positive results in non-syphilitic cases. Further, a negative Landau test is of Uttle value as many known syphilitics give negative tests. This test has very little diagnostic value. See Golay, Rev. mid. de la Suisse roman., 1914, XXXIV, 571; Villaret and Pierret, Presse m6d., 1914, XXII, 582; Correa, Brazil-Medico, 1914, XXVIII, 39s; Kolmer, Jour. Am. XXXVr4^3,^Rec^S iS|'^^?^^/Hife/8sB)fe '964; CapeUo, Gazz. d. osp., 1915, THE BLOOD . 653 who does not have facilities for performing the Wassermann test, but should not be relied upon to the exclusion of the Wassermann test.^ F. Tests before Transfusion. As transfusion of blood is becoming a matter of more or less frequent occurrence, it is advisable to test the blood for isoagglutinins and isohemolysins in every possible case. " That donor should be chosen whose blood shows no inter-agglutination or hemolysis with the patient's serum and corpuscles. If such a donor cannot be obtained, it is safer to use a person whose serum is agglutinative toward the patient's cells than one whose cells are agglutinated by the patient's serum" (Kolmer). Technic. Two or three c.c. of blood are obtained from each donor by the usual sero- logical method. Place 0.5 c.c. at once in a centrifuge tube containing 5 c.c. of a I per cent, solution of sodium citrate in normal salt. Place the re- mainder of the blood in a small dry test-tube and allow the serum to separate. Obtain 3 or 4 c.c. of blood from the recipient in the same manner and follow the same procedure with this blood as with that of the donors. Centrifuge the sodium citrate tubes, pipet off the supernatant fluid and wash the cells again with normal salt solution. Repeat if necessary in order to free the cells from every trace of serum. After the final washing, add enough normal salt solution to the cells to make a total volume of 5 c.c. Centrifuge the serum tubes and separate the clear serum. This serum should show no trace of hemolysis. The following series of tests should then be set up. In this work the blood should not be allowed to stand for a period of time before the elements are separated, and the actual tests should be begun within 24 hours of the withdrawal of the blood. Use small sterile test-tubes about 8 by i cm. 1. Four drops of donor's serum -|- i drop of recipient's red-cell emulsion. 2. Four drops of recipient's serum + i drop of donor's red-cell emulsion. 3. Control: Foiu: drops of donor's serum + i drop of donor's red-cell emulsion. 4. Control: Four drops of recipient's serum + i drop of recipient's red- cell emulsion. 5. Control: One drop of donor's red-cell emulsion -j- 4 drops of normal salt solution. 6. Control: One drop of recipient's red-ceU emulsion + 4 drops of normal salt solution. One c.c of normal salt solution is added to each tube and the tubes are shaken and. placed in the incubator for two hours, being inspected about every one-half hour. Agglutination in tubes i and 2 may be recognized ' See Gammeltoft/Hospitalstid., 1912, V, 471; Deutsch. med. Wchnschr., 1912, XXX- VIII, 1934; Jensen and Feilberg, Hospitalstid., 1912, V, 493; FeUberg, Ibid., 973; Ipsen and Helweg, Ibid., 1341; Thomsen and Boas, Ibid., 1152; Jensen, Ibid., 1516; Pontoppidan, Ugesk. f. Laeger, 1912, LXXIV, 1377; Jensen and Feilberg, Berl. klin. Wchnschr., 1912, XLIX, 1086; Lade, Deutsch. med. Wchnschr., 1913, XXXIX, 693; Kallos, Ibid., 1885; Leschly and Boas, Hospitalstid, 1913, VI, 640; Thomsen and Boas, Ztschr. f., Immunitats- forsch., 1913, XVI, 430: Olitsky and Olmstead, Jour. Am. Med. Assn., 1914, LXII, 293. DigifizedDy Microsoft® 6S4 DIAGNOSTIC METHODS macroscopically by the clumping of the red cells. Hemolysis is noted as in previous tests. If there is any question about hemolysis, let the tubes stand in the ice box over night and read them as in the complement-fixation tests. Tubes 3, 4, 5, and 6 should show no hemolysis or agglutination.^ Besides the above tests, it is advisable to test the donors with the Wasser- mann test. Further, immediate members of the patient's family offer the least risk in transfusion and should be obtained whenever possible. IX. Medicolegal Aspects In medicolegal examinations one is frequently called upon to determine the identity of various suspected stains. Such examinations may tax the entire resources of the worker and have, in many cases, led to absolutely negative results. It is not sufficient at the present day that the examiner state that a certain stain is blood, but he must be prepared to say what kind of blood it is. This latter point has been made possible by recent work so that one is fairly sure of limiting this statement at least to the blood of very closely associated animals. (i) Red Cells. It is not always a simple matter to determine that a stain on cloth, wood, iron, etc., is really due to blood. The color of the stain may be of any hue from dull red to a dirty gray, depending upon its exposure to various elemental conditions. The action of various substances, such as mortar, brick, or lime in any form, strong acids and alkalies, leather, chemicals in wall-paper, starched clothing, etc., may so change the blood or its reactivity to certain tests that no definite conclusions can be obtained. It is self-evident that the demonstration under the microscope of blood- cells is the surest proof of the presence of blood, the size and shape of the corpuscles frequently giving a clew as to the source of the blood. It becomes, therefore, necessary to make a suspension of the stained material preferably in isotonic (0.9 per cent.) sodium chlorid solution. The degree of solubility of the stain will depend upon the age of the stain, the heat to which it has been subjected, the amount of sunlight or of moisture to which it has been exposed, and the material upon which the stain is formed. Various fluids have been advanced, from time to time, as dissolving agents for the stains, many of these are active laking or hemolytic agents, especially for dried red cells, so that one sees in such preparations only the shadow of the red cell. Such laking agents are distilled water, 0.85 per cent, ammonium chlorid solution, 50 per cent, glycerin solution, and many others. Frequently a strong 30 per cent, potassium hydrate solution will enable the worker to obtain a very good idea of the presence or absence of red cells. Marx's fluid (made as follows, hydrochlorate of quinin 10 c.c. of a i to 1,000 solution added to 10 c.c. of 33 per cent, potassium hydrate solution, and tinted with eosin which will stain the erythrocytes a characteristic reddish hue) is a very excellent examining fluid. "• See Rous and Turner (Jour. Am. Med. Assn., 1915, LXIV, 1980), for an alternative method of testing the blood J3,-^^|^^s^ MicrOSOft® THE BLOOD 655 As the presence of the red cells is so hard to demonstrate, search for them is frequently omitted and reliance placed more upon the demonstration of the hemoglobin in such stains. If the stain is upon a hard surface it may be scraped off with a clean piece of glass. If it be upon cloth, a portion of the stained part and also of the unstained part is removed and cut into small pieces with scissors which are absolutely clean. Such small pieces not broader than I mm. are very easily handled and can be readily teased out if desired. (2) Guaiac Test. This test originally devised by van Deen is one of our oldest for the detec- tion of blood coloring matter, but seems to be much more reliable in its nega- tive phase than when positive. The principle of the test is as follows: To a watery solution of the suspected stain is added an equal portion of fresh tinc- ture of guaiac. This tincture is best made by dissolving the guaiac resin in al- cohol when needed. The addition of the guaiac tincture to the suspected solution causes a milky turbidity. On now adding ozonized oil of turpentine, peroxid of hydrogen, or oil of eucalyptus, so that these latter substances float upon the guaiac and blood solution, a distinct blue color will be manifested at the point of contact, and, on shaking the tube, the coloration will spread throughout the mixture. This test seems to be very deUcate, demonstrating the presence of blood in a dilution of several thousand. The blue coloration observed in this test is due to the oxidation of the guaiac to guaiaconic acid, which is then further oxidized into guaiac blue by the catalytic action of the oil of turpentine. It has been shown by Taylor that this reaction is also given by many substances among which we find manganate and permanganate of potassium, peroxid of manganese, peroxid of lead, chlorin, bromin, icdin, nitric and chromic acids, ferric chlorid, salts of copper, ferro- and ferricyanid of potassium, gum acacia, gluten, unboiled milk, raw potato pulp, pus, and any living cell' or its intracellular enzymes. Various enzymes such as the oxidases will give this reaction. Buckmaster advises the use of the pure guaiaconic acid along with peroxid of hydrogen in the place of the guaiac resin and believes that the test increases in delicacy and accuracy under these conditions. He states that fluids containing hemoglobin or most of its de- rivatives give this test when it is impossible to detect pigments by any other methods and rightly adds that boiling the fluid suspected of containing blood does not interfere with the reaction but, on the contrary, throws out of consideration the action of milk, pus, fibrin, or of any enzyme. For the proper performance of this test the fluid to be tested, should not be alkaline and only very slightly, preferaibly not at all, acid. (3) Schaer's Test. This test is similar in many ways to the van Deen test, but employs, instead of guaiac, a i to 4 per cent, solution of aloin in alcohol. On adding this tincture to the suspected solution ared color soon becoming distinctly cherry-red appears upon the addition of ozonized oil of turpentine. Many other substances give a pink color, but only after the lapse of one or two hours, while the color with blood appears in a very short time. The tincture of aloin should always be freshly prepared, as o|)j^^ gm^pgl^j^color change after standing. 656 DIAGNOSTIC METHODS (4) Phenolphthalin Test. This test/ originally employed by Meyer ^ and, later, by Utz,' has been so • improved by Kastle and Amoss* and Delearde and Benoit^ that it is, probably, our most delicate one for the detection of blood, especially in stains, its limit of delicacy being about i part of blood in 8 million of water. As in the pre- vious tests, the hemoglobin acts as an oxygen carrier, the active oxidizing agents being ozonized turpentine or hydrogen peroxid. The oxidases, peroxi- dases and catalases play small r61es in this process in the case of blood-stains but, in the various secretions and excretions of the body, their influence is very great. Phenolphthalin is a product of the reduction of phenolphthalein by zinc in alkaline solution. When oxidized in alkaline solution, it is converted into phenolphthalein with production of an intense red color. The reagent may be obtained in the market or, preferably, prepared as follows. Phenolphtha- lein is dissolved in a considerable excess of 30 per cent, sodium hydrate solu- tion and boiled with an excess of zinc dust until a few drops of the strongly alkaline liquid no longer give a red color after neutralization with HCl and sufficient alkali to alkalinize the solution. The solution is then decanted from the excess of zinc dust and the phenolphthalin is precipitated by acidifying with HCl. Collect the precipitate on a filter and purify by repeated crystalli- zation from water and alcohol. This purification is continued until a white crystalline compound is obtained free from every trace of phenolphthalein (as shown by absence of red coloration on addition of alkali). Dry at room temperature or in the oven at 50° to 8o°C., care being taken to avoid contact with metallic surfaces. This compound should be kept in tightly stoppered bottles in a dark place, as oxidation gradually occurs. Rarely do we find that the coloration is sufficient to interfere with the tests, but in the delicate forensic tests it may be necessary to repurify a product which has stood for some time.* The solution, as used in the test, is as follows: Mix a slight excess of phenolphthalin, prepared as above, with i c.c. of N/io sodium hydrate solu- tion and a few c.c. of redistilled (from glass) water, shake thoroughly and filter. To the filtrate add 20 c.c. of N/io NaOH solution, o.i c.c. of 3 per cent, hydrogen peroxid solution and make the mixture up to 100 c.c. This solution shows no trace of pink coloration when fresh, but gradually acquires a color, which may become so intense that the reagent cannot be employed. In forensic work, use only freshly prepared solutions. To I part of the aqueous solution of the stain or of the secretion or excre- 1 See Kastle, BuU. 51, Hyg. Lab. U. S. P. H. & M. H. S., 1909. * Munch, med. Wchnschr., 1903, L, 1489. ' Chem. Ztg., 1903, XXVII, 1151. * Bull. 31, Hyg. Lab. U. S. P. H. & M. H. S., 1906, * Comp. Rend. Soc. de biol., 1908, LXIV, 990. ^ For routine laboratory work this reagent may be prepared as follows: Two grams of phenolphthalein, 20 grams of sodium hydrate, 10 grams of zinc dust and 100 c.c. of distilled water are mixed and heated until the solution becomes colorless. Filter while hot and pre- serve in an amber bottle. This solution is prone to show a slight reddish tinge from time to time owing to reoxidation, so that it may be necessary to heat it with zinc dust again and "^^''"- Digitized by Microsoft® THE BLOOD 657 tion to be tested add 2 parts of the reagent and allow to stand for a few min- utes. In the presence of blood a pink to red color appears, the intensity de- pending on the amount of blood present. This reaction is retarded by the extracts of various animal tissues or various secretions of the body. For this reason we are never able to detect as small amounts of blood in the secretions as in watery solutions of pure blood, the limit of delicacy of all reactions being far less in the former than in the latter case. Boiling the solutions before applying the test removes most of the interfering factors. If the secretion be treated with a thick cream of albuminium hydrate suspension, the precipitate will carry down the blood pigment and thus concentrate it. A small amount of this precipitate, whether derived from saliva, urine, feces suspension, milk, or exudates, will show a decided red color when added to 2 c.c. of the reagent. Of course, in applying the test to the aqueous solution of the blood stain, no such treatment need be employed. Fig. 158. — Hsmin Crystals from Human Blood. [Hai^'k.) Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University of Pennsylvania. This test is especially recommended for the detection of blood in all secre- tions and excretions (boiling them before applying the test.) and, particularly, in the examination of suspected stains for forensic purposes. All possible fallacies must be guarded against and confirmatory tests used if necessary. '^ (5) Teichmann's Test. This is one of the most important tests for the presence of blood and when positive is conclusive proof of the kind of stain with which one is work- ing. A drop of blood or a portion of the suspected stain is spread upon a glass slide and covered with a drop of a very dilute solution (o.oi per cent.) of com- ' Vas (Deatsch. med. Wchnschr., 1912, XXXVIII, 1412) calls attention to the possi- bility of the presence of phenolphthalein as a purgative (if feces be tested). The addition of alkaU alone will give a red color under this condition. See Cade and Mulsaut, Lyon Med., 1912, CXIX, 885; also de Jager, Berl, klin. Wchnschr., 1914, LI, 795; Schirokauer, Deutsch. med. Wchnschr., 1914, XL, 1472 and 1617; Schneider and von Teubern, Ibid., 1673; Boas, Ibid., 1915, XLi, 549. Digitized by Microsoft® 6s8 DIAGNOSTIC METHODS mon salt.^ The salt solution is then evaporated at a low temperature. A f ew drops of glacial acetic acid are then placed upon the salted stain and the preparation is covered with a cover-glass. The acid is now slowly evaporated by holding the slide over a flame in such a way that the fluid steams but does not boil. As the acid evaporates, more is allowed to run under the cover- glass, this addition and evaporation being done thrice. The specimen is cooled and mounted in glycerin or distilled water, after which it maybe exam- ined with the dry lens. A successful specimen, which is not always obtained, will show the presence of numerous brownish rhomboid crystals, which are separate or grouped in sheafs or rosettes. These are the hemin crystals or hydrochlorate of hematin, called, after their discoverer, Teichmann's crystals. It very frequently happens that the specimens are not successful, so that several slides should be made before a negative report is given. Reasons for failure may be found in the excessive heat applied to evaporate the acid, in the fact that the salt solution may have been too concentrated, or that the stain may have been very old. The addition of salt seems to be unnecessary except in the case of old blood stains or when the blood is poor in salts. It should even then be added in only very small amounts. As pointed out by Rose, when blood is mixed with iron rust the hemin test is usually negative.^ (6) Spectroscopic Examination of Blood. Certain characteristic appearances of the blood are noted on spectroscopic examination. The spectrum of the various blood pigments has been given in previous sections so that only a few additional remarks are necessary in this place. In spectroscopic examinations, the use of the ordinary hand spectro- scope is all that is necessary, the more complicated ones adding little to the differentiation of the various spectra. If the blood be fresh and the hemo- globin unaltered the spectrum is, of course, that of oxyhemoglobin; but as the usual stains submitted for examination are frequently very old ones, such a spectrum is practically never seen. The end-product of the alteration of hemoglobin as found in old blood stains is hematoporphyrin, which is iron- free and can be identified easiest by the spectroscope. The suspected stain is dissolved in concentrated sulphuric acid yielding a reddish- violet fluid, which is then examined speetroscopically. The spectrum of hematoporphyrin has been given previously. Many blood stains can be recognized only by this test, so that it is always advisable to make a spectroscopic examination, espe- cially if other tests for hemoglobin have failed.' (7) Precipitin Test. The precipitin test has been discussed in great detail previously (page 614). This is the absolute identification test for a stain, providing the stain has been shown to be a blood stain by the tests given above. The precipitin ' Nippe (Deutsch. med. Wchnschr., 1912, XXXVIII, 2222) advises one to mix the speci- men with a few drops of a solution of o.i gram each of potassium bromid, iodid and chlorid in 100 grams of glacial acetic acid. This modification shows up the crystals even when blood is mixed with rust. See, also, Symons, Bipchem. Jour., 1913, VII, 596. 2 See Symons, Biochem. Jour., 1913, VII, 596; Beam and Freak, Ibid., 191S, IX, 161. ' See Sutherland and Mit^,-^^^j; Semaine M6d., 1903, X^?'^i?F ^Y MlCrOSOft%> 2 grams. 3 grams, 10 c.c. 10 c.c. 00 c.c. 668 DIAGNOSTIC METHODS catarrhalis, resemble the gonococcus both in morphological and staining char- acteristics, so that a differentiation by this method is not always possible. Fortunately, however, the cultural peculiarities of these organisms absolutely differentiate them.' Gram's Stain. The principle of the staining of various organisms by Gram's method is that certain organisms retain the primary color after treatment with de- colorizing agents, while others lose this primary stain and must be treated with a contrast stain for their recognition. If the organism retains the primary blue color it is called Gram-positive, while if it lose the primary color and take on the contrast stain it is called Gram-negative. Among the organ- isms which are Gram-negative we find the gonococcus, meningococcus, micro- coccus catarrhalis, influenza bacillus, typhoid bacillus, colon bacillus, Koch- Weeks bacillus, and the Morax-Axenfeld bacillus; while the tubercle bacillus, smegma bacillus, diphtheria bacillus, pneumococcus, streptococcus, staphylo- coccus, and various saprophytic cocci found in the smears both of the male and female urethra are Gram-positive. The solutions required in Gram's method are: (i) an anilin oU^ — -gentian violet mixture, consisting of 84 c.c. of anilin water (water saturated with anihn and filtered), and 16 c.c. of a saturated alcoholic solution of gentian violet; (2) a solution of iodin consisting of one gram of iodih, 2 grams of potas- sium iodid, dissolved in 300 c.c. of water; (3) a dilute solution of carbol fuchsin, a dilute aqueous solution of safranin, or a i per cent, aqueous solution of Bisrnarck brown as a contrast stain. Technic. Smears of the exudate are made, in the manner previously described for making blood smears, by receiving a drop of the purulent material upon one end of a glass slide and spreading it in a thin even layer by means of a second slide. These smears are then fixed by passing several times through the flame. When the slides have cooled, they are covered with the gentian- violet solution which is allowed to act for one to three minutes. The solution is then poured off and the excess removed by washing in water. Without; dr)dng, the iodin solution is placed on the slide and allowed to act for one- half to one minute. It is then washed in water and the preparation treated with 95 per cent, alcohol until no more color is removed by it. The alcohol is then removed by washing with water and the smear is covered with one of the contrast stains above mentioned, in the writer's laboratory the safranin solution, which is allowed to act for only a few seconds. The stain is then washed off with water and the slide dried between folds of filter-paper. The specimen under the microscope shows the Gram-positive organisms stained deep blue, while the Gram-negative bacteria and the bodies of the 1 See Warden (Jour. Infect. Dis., 1913, XII, 93), who emphasizes the importance of a slightly acid reaction in the media containing preferably human body fluids, such as ascitic or hydrocele fluid. Warden (Jour. Infect. Dis., 1913, XIII, 124) calls attention to the fact that, oftentimes, what appears to be the gonococcus in smear preparations is shown to be a staphylococcus on culture. This awaits confirmation. See, also, Whitney, Boston Med. and Surg. Jour., 1914, D^fi^ejOt^MieepSBiiff^ork Med. Jour., 1914, C, 1255; Wolbarst, Ibid., 1915, CI, 146; Warden, Jour. Infect. Dis., 1915, XVI, 426; Pearce, Jour. Exper. Med., 1915, XXI, 289. PLATE XXXIII. > %:^ 9^ Kathar.n? )-li 11 GONOCOCCI IN URETirRAL DISCHARGE. (GrAM's StAIN.) Digitized by Microsoft® Digitized by Microsoft® TRANSUDATES AND EXUDATES 669 pus cells take the red safranin stain. In such specimens the gonococcus, if present, will be observed both intracellularly and extracellularly, the former being the more characteristic. The ordinary pus cocci may likewise be intracellular, but these are distinctly Gram-positive, while the gonococcus is Gram-negative. In the purulent exudate from the urethra, large masses of mucoid material may be present, which are known as gonorrheal threads. These may be found in the urinary sediment and are usually easily recognized. They con- tain masses of pus cells, within which may be found numerous gonococci. These shreds may persist for years in anyone with a history of a previous gonorrhea, but they may then contain no organisms. The cytology of gonor- rheal pus presents nothing characteristic beyond the presence of numerous eosinophiles with large numbers of polynuclear neutrophiles and an occasional lymphocyte. Smegma Bacilli. In the exudate of- the preputial follicles, known as smegma preputii, are found fat globules, ammonium soaps, cholesterin crystals, and a few epithelial cells. In this smegma are found many bacilli, smegma bacilli, which show the same morphological and practically the same staining characteristics as the tubercle bacillus. This is differentiated, as described under Sputum. If the urine is to be examined for tubercle bacilli, it is much better technic to use a catheterized specimen than it is to resort to methods of questionable differentiation. If this is done any acid and alcohol-fast bacilli may be regarded as the tubercle bacilli, but in all cases the smegma bacilli must be excluded.* Ducrey's Bacillus. This bacillus is the organism causing soft chancre and is known as the bacillus ulceris cancrosi. It is found in the purulent discharge from the chancre, but rarely, if ever, in pure culture. The sections of tissue may, however, show none but this organism. In making preparations for study of this organism, the ulcerated surface is scraped with a platinum loop, the pus spread upon a slide, dried in the air, and fixed with alcohol-ether or over the flame. It stains readily with all bacterial stains, but decolorizes with Gram's stain. In stained specimens the organism appears as a short, thick, oval bacillus with rounded ends and two lateral indentations, which occasionally give it the appearance of a figure 8. Ordinarily the extremities are more deeply stained than the central portion, this appearing almost clear. The organism has a tendency to form chains or groups, which are rarely found in the pus cells but are frequently seen within the epithelial cells. Spirochaeta Pallida. The Treponema Pallidum, discovered by Schaudinn and Hoffmann,^ is now definitely established as the causative factor of s3^hiUs. It may be obtained from the primary chancre, the incised papules, condylomata, mucous patches, inguinal glands, as well as from the internal organs in ' See Brereton and Smith, Am. Jour. Med. Sc, 1914, CXLVIII, 267. ' Arbeiten aus der kais. °i but may be, in a few cases, 1,005 to 1,010 or 1,050 to 1,055. Though the contents of the proliferating cyst may have a variable composition, still it may be character- ized, in typical cases, by its slimy or ropy consistency; by its grayish-yellow, chocolate-brown, sometimes whitish-gray color; and by its relatively high specific gravity. Such a liquid does not ordinarily show a spontaneous coagulation ' ' (Hammarsten) . Microscopical examination of the sediment shows red and white blood- cells, large epithehal cells, which may be filled with vacuoles, cylindrical or goblet cells, granular cells showing more or less fatty degeneration, fatty 1 See Szecsi, Folia Haemat., 1912, XIII, i. See RoUeston (Brit. Med. Jour., igi4, I, 238) and Weishaupt (Arch. f. Gynak., 1914, C, 496) for a discussion of eosinophilia in peritoneal exudates; also, CaBH^jSSii.OyMi&IK^pii®, III, x. PLATE XXXVII. ExuDATF IN Malignant Tleueisy. (Eusix-iiejiatoxylin Stain. After Deguy axd (JUILLAUMIN. ) Digitized by Microsoft® Digitized by Microsoft® TRANSUDATES AND EXUDATES 677 granules, cholesterin crystals, and colloid corpuscles in the form of large, circular, highly ref tactile bodies.' Chemically, these cysts are characterized by the presence of colloid, which is not a distinct chemical entity. It is a gelatinous substance, insoluble in water and acetic acid, soluble in alkalies, and yields a reducing body on boiling with acids. Not infrequently pseudomucin (metalbumin) is found, especially in the extremely viscid fluids. For its detection the serum albumin must be previously removed by the addition of acetic acid, boiling, and filtering. The filtrate is treated with alcohol when a thready precipitate forms. If this precipitate be boiled with HCl, a substance is formed which reduces copper solutions quite markedly. Pseudomucin is distinguished from true mucin by the fact that it is not precipitated by acetic acid. Mitjukoff has isolated a further colloid body from certain ovarian cysts, to which he gives the name of paramucin. It is precipitated by acetic acid and is soluble in an excess. If treated with alkali it first swells up and then dissolves in an excess of the re- agent. It differs from mucin and pseudomucin in the fact that it reduces copper solution without previous boiling with acids. McConnelP has recently reported the finding of a multi-locular cyst of the ovary, which contained true mucin, (c) Papillary Cysts. These are intraligamentary types and contain a yellow, yellowish-green, or brownish-green fluid which contains only traces of pseudomucin. The specific'gravity ranges between 1,032 and 1,036. {d) Dennoid Cysts. These may be seen in the shape of small cysts not larger than a pea, but usually they are much larger, in some cases reaching the size of a man's head., The cyst usually contains a fatty unctuous material, which is derived from the epidermal lining of the cyst, and associated with it fat, desquamated epitheUal scales, hair, teeth, bone, cartilage, etc. (2) Parovarian Cysts. These cysts of the organ of Rosenmuller contain a clear, pale yellow, or colorless, hmpid fluid or occasionally one showing shght opalescence. The specific gravity ranges from 1,002 to 1,010 and differs from that of the ovarian cyst by its usual hmitation to these lower figures. Albumin may be present in small amounts or be entirely absent, while pseudomucin is rarely, if ever, present. (3) Hydrocele. The contents of such a cyst are usually clear, show a color which may range from yellow to green, have a specific gravity of 1,015 to 1,030, and usually coagulate spontaneously. They contain a relatively large percentage of al- bumin, of which about 50 per cent, is globulin. In ordinary hydrocele many large oval cells may be seen, which have an eccentric nucleus and may be grouped in masses, although more frequently they appear as isolated cells; ' Dienst (Miinch. med. Wchnschr., 1912, LIX, 2731) lias called attention to the fact that fibrin is absent in the fluid from ovariajLcysts while it is constantly present in ascitic fluids. Digitized byMicrosofm ^ Jour. Med. Research, 1909, XX, 105. 678 DIAGNOSTIC METHODS in some cases many eosinophiles, but this is rare in uncomplicated hydroceles. If the hydrocele be of tubercular origin a marked lymphocytosis usually exists. (4) Spermatocele. Fluids from such cysts are usually thin, colorless, and cloudy like thin milk. They may have an acid reaction, but are ordinarily alkaline. The specific gravity ranges between i ,006 and i ,0 1 o. Such fluids do not coagulate either spontaneously or after the addition of blood. Microscopically, one observes cell detritus, fat granules, leucocytes, and spermatozoa. (s) Hydronephrosis. This is a true retention cyst of the kidney, due primarily to obstruction of the ureter, which may be either congenital or acquired. Material aspirated from a renal cyst is usually clear, but may be yellowish or reddish and dis- tinctly turbid. Its specific gravity varies from 1,010 to 1,015, while the chem- ical composition is usually suggestive of urine. For some time the presence of urea and uric acid in hydronephrotic cysts was supposed to be pathognomonic, but it has been shown that these substances may be present both in ovarian and pancreatic cysts and may even be lacking in old renal cysts. Occasion- ally epithelial cells, derived from the uriniferous tubules may be found, but such cells are not always present nor are they sufficiently characteristic to be of great importance from the diagnostic standpoint. (6) Hydatid Cysts. The fluid obtained by puncture of an echinococcus cyst is usually clear and of an alkaline reaction, has a specific gravity varying between 1,005 ^.nd 1,015, is practically free from albumin, and contains a large amount of inorganic salts, especially of sodium chlorid. The characteristic findings of such a cyst are the booklets, scolices, and shreds of faintly laminated membrane. In some cases no trace of any morphological elements can be found, but usually the diagnosis is rendered certain, especially if careful search be made, by the presence of some portion of the parasite or the cyst membrane. (7) Pancreatic Cysts. The puncture fluid from a pancreatic cyst varies in its physical properties, depending upon the nature of the cyst as well as the length of time the fluid has remained in the cyst. It is usually bloody in character, has a specific gravity ranging from 1,010 to 1,030 and may contain methemoglobin, hema- tin, and cholesterin. As characteristic constituents of such a cyst one finds ferments, which will digest all types of food material.^ Such tests may be performed as outlined in the sections on Gastric Contents and Feces. VI. Cerebrospinal Fluid Since the introduction of lumbar puncture by Quincke, the cerebrospinal fluid has gained more or less importance from the diagnostic point of view. ' See Christian (Arch. Int. Med., 1912, IX, 143) who reports peculiar findings in the fluid of an epigastric cyst; also Besley, Jour. Am. Med. Assn., 1914, LXII, ion; Ipsen, Hospitalstid., 1914, LVII, 889 and 921; Speese, Ann. Surg., 1914, LX, 673; Rosenthal, Arch. f. Verdauungskr., 1914L..XX, 6L9a Euatachio EiL Med., 1915, XXXI, 424; Willis and Budd, Surg., Gyn. and §^S?%gmW§^PSO"^ TRANSUDATES AND EXUDATES 679 So much may be learned, either from the standpoint of direct or differential diagnosis, that every practitioner should be able to perform a lumbar puncture and to examine the fluid obtained. There is little danger in the procedure as the spinal cord does not reach to the point of puncture and the fibers of the Cauda equina are sufficiently movable to escape the needle. While few bad effects are observed in the ordinary run of cases, a few have been reported in which symptoms of collapse were evident. It should be a rule, therefore, to stop proceedings if such symptoms arise and also to keep the patient quiet in bed for at least 24 hours following the puncture, so that the pressure in the cerebrospinal cavity may become equalized. ^ Ltunbar Puncture. The patient is placed upon his left side near the edge of the bed, the knees should be flexed upon the abdomen, and the site of puncture prepared as for any surgical procedure. The needle used for puncture should be from 5 to 10 cm. long and have a lumen from i to 2 mm. in diameter. It is always wise to provide the larger needles with a stylet, so that tissue fragments or blood may not gain entrance accidentally to the tube and thus lead to possible ■diagnostic errors. This stylet may be removed the moment the needle penetrates the dura mater. The site of puncture should be on a level with the junction of the third and fourth lumbar vertebrae at a point about i cm. to the side (preferably the upper) of the median line. The needle is directed slightly upward and inward, the depth to which the puncture should be made varying with the age of the patient, the younger the child the less the depth. This puncture ■should be made carefully and yet with suflScient force to penetrate easily the musculature. If any marked resistance arises, it is probable that the needle has struck the vertebra, in which case the pressure must be reduced or the needle may break. This is not an infrequent occurrence with those not used to the technic, so that it may be advisable for the student to practice the procedure upon the cadaver. As soon- as the dural sac is reached, the cerebrospinal fluid will flow from the canula, the rate of flow indicating in a general way the pressure of the fluid. No aspiration should be used at any time, as this procedure is extremely dangerous. It is frequently advisable to know exactly what this pressure is, so that one may resort to the following method as used by Sahli. As soon as the needle penetrates the dura, a connection is made with a mercury manometer by means of a rubber tube filled with a i per cent, solution of carbolic acid. The portion of the manometer above the level of the mercury, forming the con- nection between it and the carbolic acid tube, must also be filled with the fluid. The manometer is filled with mercury to the zero point and held in ■such a manner that this point is on a level with the point of the aspirating ' .For discussions regarding the source of the spinal fluid and variations in it see Dixon and Halliburton, Jour. Physiol., 1913, XLVII, 215; Ibid., 1914, XLVIII, 129 and 317; Dandy and Blackfan, Jour. Am. Med. Assn., 1913, LXl, 2216; Frazier, Ibid., 1914, LXIII, 287; Gushing, Jour. Med. Research, 1914, XXXI, i; Weed, Ibid., 21, 51 and 93; Wegefarth, Ibid., iig and 149; Wegefarth and Weed, Ibid., 167; Frazier and Peet, Am. Jour. Physiol., 1914, XXXV, 268; Weed and Gushing, Ibid., 1915, XXXVI, 77; Frazier, Jour. Am. Med. Assn., 191S, Lxiv, II 19. Digitized by Microsoft® 68o DIAGNOSTIC METHODS needle, which is possible with ordinary manometers only when the connecting tube is of considerable length.^ Under normal conditions the dural pressure, in the dorsal position, ranges between 5 and 7.5 mm. of mercury, or 60 to 100 mm. of water if a water manometer be used. In pathologic conditions, such as meningitis or brain tumor, it ranges between 15 and 60 mm. of mercury or 200 to 800 mm. of water. Normal cerebrospinal fluid is colorless, limpid, and free from morphologi- cal elements. Its specific gravity ranges between i ,002 and i ,010. ^ It is alka- line in reaction, the degree of alkalinity varying between 15 and 20. It con- tains a trace of protein and about o.i per cent, of glucose. The salt content of this fluid is, according to Zdarek, 0.836 gram, of which 0.429 gram is referable to sodium oxid and 0.017 to potas- sium oxid. This would rather militate against the older statements that a large amount of potassium salts as compared with sodium salts was pres- ent. The relation of KCl to NaCl is, according to Nawratzki, i to 18, while Zdarek gives this ratio as i to 40. The former figure agrees closely with that obtained by various workers with pathological cerebrospinal fluids.' The amount of fluid obtained by lumbar puncture is extremely variable. In normal individuals this amount is unknown as puncture is rarely per- A, formed upon normal cases. Patholog- ically, the amount obtained varies be- tween ID and 100 c.c. Naturally, if the communication between the subarachnoid spaces of the brain and of the spinal cord is blocked by a tumor or inflammatory adhesions, or if the aque- duct of Sylvius or the foramen of Magendie be obliterated, little fluid may be obtained by puncture, although large amounts may be present above the obstruction. The largest amounts are seen in cases of serous or tubercular meningitis, so that such conditions may usually be ruled out if a small amount of fluid is obtainable. Pathologically, we may observe a very cloudy fluid, due to the presence of leucocytes, erythrocytes, and endothelial cells. This cellular admixture may be so extensive that the fluid resembles pure pus. In cases of cerebral hemorrhage frOm the ventricles, hemorrhagic pachymeningitis, or traumatic lesions of the spinal cord, so much blood may be present as to give the appear- 1 See Strauss (Jour. Am. Med. Assn., 1914, LXII, 1327) for a special spinal-puncture needle with manometer attachment. ^ See Stanford (Ztschr. f. physiol. Chem., 1913, LXXXVI, 43 and 219), who shows that this density is higher in progressive paralysis and epilepsy than in other conditions. 3 See Rosenbloom and An^j^^^Jfy^Tf^j^^f^fi, XIV, 536. Fig. 159. — ^Lumbar puncture: Quincke's site; b, Maran's site; c, Chipault's site. {Tyson.) TRANStTDATES AND EXUDATES 68 1 ance of practically pure blood, the color varying from a bright red to a brown- ish or greenish-red, depending upon the length of time it has remained in con- tact with the remaining portion of the fluid. This admixture with blood may lead to the spontaneous coagulation of the fluid. ^ This may serve as a differentiating point between inflammatory and noninflammatory lesions. Thus, in tubercular meningitis very slight coagulation may be observed while in the epidemic cerebrospinal meningitis the coagulum may be very firm. The chemical examination of the cerebrospinal fluid offers some points of clinical value. While the albumin content normally is much less than o.i per cent., it may vary under pathologic conditions to as high as 0.8 per cent. The total protein, especially the euglobulin portion, is increased in all cases of acute exudative inflammations of the meninges, in hydrocephalus as Polanyi^ has shown and, also, in syphilitic and parasyphilitic diseases of the cere- brospinal tract. Glucose is usually present but may entirely disappear under pathologic influences due to the autolysis controlled by the leucocytic fer- ments, the glucose being converted into lactic acid.^ Cholin is present nor- mally in traces, while pathologically it may vary, according to Donath, between 0.021 per cent, and 0.046 per cent. Koch* has introduced the following method for examination of this fluid, which should extend our knowledge. The fluid is centrifuged and the sedi- ment used for the bacteriological and cytological examinations. Ten c.c. of this clear fluid are placed in a beaker, 3 c.c. of a saturated solution of ammo- nium nitrate and 5 drops of nitric acid are added, and the mixture heated on a water-bath until complete coagulation has occurred. This precipitate of protein^ is filtered through a perforated Gooch crucible, washed with o.i per cent, ammonium nitrate solution and then with alcohol, dried at ioo°C. and weighed. The filtrate is taken before the washing with alcohol is begun, concentrated to 10 to 15 c.c. and 2 c.c. of i per cent, barium nitrate solution added while boiling. The barium sulphate precipitate is allowed to settle over night, is filtered off, dried and weighed. With the filtrate a phosphorus estimation is made by the molybdic method, with subsequent precipitation as magnesium ammonium phosphate in the usual way. Increase in the phosphorus and sulphur content is associated with degenerative changes in the nerve tissue. A test for reducing substances is made with i c.c. of the original fluid. 1 Schwarz (Deutsch. Ztschr. f. Chir., 1913, CXXIV, 346) shows that a yellowish tint (xanthochromasia) is frequently due to conversion of the blood pigment into urobilin and, hence, that this is a valuable indication of intracranial hemorrhage. See, also. Babes, Compt. rend. Soc. de bioL, 1914, LXXVII, 67. For total nitrogen and urea of spinal fluid see Adler and Ragle, Boston Med. and Surg. Jour., 1914, CLXXI, 769; Woods, Arch. Int. Med., 1915, XVI, 577; and CuUen and Ellis, Jour. Biol. Chem., 19x5, XX, 511. Weston (Jour. Med. Research, 1915, XXXIII, 119), discusses the cholesterol variations in this fluid. ^Biochem. Ztschr., 1911, XXXIV, 205. See, also, Ferrier, Lancet, 1913, II, 1107. 'See Kopetzky (Trans. Am. Acad, of Ophth. and Oto-Laryngol., 191 2, I, 261) who believes the absence of the copper-reducing body to be the earliest sign of bacterial invasion of the central nervous system. See Szabo, Ztschr. f. d. ges. Neurol, u. Psychiat., 1913, XVII, 14s; Soper and Granat, Arch. Int. Med., 1914, XIII, 131. •Arch. Neurol., 1907, III, 331; Arch. Neurol, and Psychiat., 1909, IV, i. ' Mott advises the following method for protein: 10 c.c. of the centrifuged fluid are faintly acidified with acetic acid and 20 c.c. of absolute alcohol are added. The mixture is boiled and filtered, the pred^giiize^tl^iMier^e)^^ and weighed. 682 DIAGNOSTIC METHODS Microscopic Examination. By far the most important part of the clinical examination of cerebro- spinal fluid is a study of the bacteriology and cytology of the fluid. Normally the fluid contains practically no morphologic elements, while under patho- logic conditions large numbers of various types of cells may be present. The material for examination is obtained as previously described in the section on cytology. In true tubercular meningitis a lymphocytosis is almost inva- riably observed, while in the epidemic type, due to the meningococcus of Weichselbaum, the cells are of the polynuclear type. In the chronic cases of epidemic meningitis as well as during convalescence from this disease, the lymphocytes may be present to such an extent that a slight degree of lympho- cytosis is present, but never to the same extent as observed in the tubercular type.' A lymphocytosis is also observed in syphilitic lesions of the central nervous system. This is important from the standpoint of differential diag- nosis. In certain cases of brain tumor, of non-specific origin, a lymphocyto- sis may obtain along with an increase in the protein-content of the fluid. In the meningitis due to the pneumococcus, a polynucleosis is the rule, al- though occasional cases are seen in which a lymphocytosis obtains.^ In the prodromal stages and early days of acute anterior poliomyelitis a marked increase of fluid is accompanied by an increase in number of cells, which are largely of the mononuclear type. These are replaced by the polymorphonu- clear forms as the number diminishes. In the microscopic examination of the cerebrospinal fluid it is sometimes of interest to know the number of cells per c. mm., of the fluid. The technic is as follows: The following mixture is employed for staining the white and dissolving the red cells: Methyl violet 0.2 gram, glacial acetic acid 5 c.c, water to 100. Fill the pipet with stain to the mark i and then fill to point 1 1 with uncentrifuged cerebrospinal fluid. Shake thoroughly, let stand for five minutes and count, using a Fuchs-Rosenthal counting chamber. The ruled surface of the cell contains 3.2 c. mm. of fluid. Count all of the white cells in the entire ruled area, multiply by 11 and divide by 32. The result is the number of cells per c. mm., which varies from i to 10 normally.^ Epidemic Cerebrospinal Meningitis. In these cases the fluid may be transparent, but is usually somewhat opalescent and may be thick and purulent. The cellular elements are usually polynuclear in type and red cells may be more or less numerous. Smears made from the sediment show the presence of numerous diplococci, which closely resemble the gonococcus in morphological and staining char- acteristics. This organism, the diplococcus meningitidis intracellularis of 1 Wassermann andLange (Berl. klin. Wchnschr., 1914, LI, 527) believe that these lym- phocytes are, at least, one source of the substances responsible for the positive Wassermann reactions obtained with spinal fluid. 2 See, however, Brady, Jour. Am. Med. Assn., 1913, LX, 972. ' See Blatteis and Lederer, Jour. Am. Med. Assn., 1913, LX, 811; Karpas, Ibid., 1913, LXI, 262; Engman, Buhman, Gorham and Davis, Ibid., 735; Brem, Ibid., 742; Maruyama, Wien. klin. Wchnschr., 1913, XXVI, 1233; Ball, Interstate, Med. Jour., 1913, XX, 1109; Rubenstone, New York Med. Jour., 1913, XCVIII, 1210; Gordon, Ibid., 1914, XCIX, no; Mitchell, Darling and Newcomb,. Jourj Nervous and Mcnt. Dis.; 1914, XLI, 686; Cottin, Rev. m6d. de la Suisse RomfP^l^^fS^vi'ff^^S^ow, Cleveland Med. Jour., 1915, XIV, 196. TRANSUDATES AND EXUDATES 683 Weichselbaum, appears as a diplococcus, each element forming a hemisphere with its parallel side contiguous to that of its mate.^ It is sometimes seen in the form of tetrads or as isolated cocci, which appear as true spheres of vari- able size and showing a clear space in their interior. It is stained with the ordinary dyes and is negative to Gram's stain. For its cultural peculiarities see the last chapter of this book. Not infrequently one observes specimens of the meningococcus which show a Gram-positive reaction, so that it is dif- ficult to distinguish them, especially when they are in the form of isolated cocci or in groups of two or four, from the pneumococcus. This type has been described as the meningococcus of Bonome, while Jaeger and Heubner describe a diplococcus which is Gram-positive and may be confused with the unusual types of Weichselbaum' s meningococcus. Recently Flexner has succeeded in preparing an antimeningococcic serum which appears to have remarkable results in controlling this hitherto un- manageable disease. In using this serum, the injections must be made directly into the spinal cavity. ^ More or less frequent injections of the serum and examinations of the lumbar fluid are made and the influence of the serum estimated by bacteriological and cytological examinations. In the cases of mixed cerebral infection, in which the meningococcus is associated with the pneumococcus, streptococcus, typhoid bacillus, staphylococcus', and other organ- isms, this serum does not seem to have as much influence as in the pure meningo- coccus infections.' It is to be remembered that a purulent meningitis may be secondary to infection with practically all of the pus-forming organisms found within the system. It is, therefore, essential that any infection showing meningeal symptoms should be investigated by an examination of the cerebrospinal fluid. In sleeping sickness, a study of the cerebrospinal fluid frequently reveals the presence of the trypanosoma Gambiense. These parasites are not found in all cases, but when present usually furnish a grave prognosis.' Recent investigation of the' cerebrospinal fluid, applying 'Wassermann's serum reaction for syphilis, has shown that in the large majority of nervous cases of syphilitic origin a positive reaction is obtainable. ' See Frost, Pub. Health Rep., igi2, XX"VII, 97; Broers and Smit, Nederl. Tijdschr. v. Geneesk.j-igis, H, 1175; also, Hort, Lakin and Benians (Brit. Med. Jour., 1915, i, S4i), Foster (Ibid., 543) and Donaldson (Lancet, 1915, i, 1333), who suggest that the meningococ- cus is only a phase in the life history of the true infective agent. 2 See Dubois, Jour. Am. Med. Assn., 1913, LX, 820; also, 'Wollstein, Jour. Exper. Med., 1914, XX, 201; Dubois and Neal, Am. Jour. Dis. Child., 1915, IX, i. ' CoUignon and Mod (Presse m6d., 1911, XIX, 732) advocate the following test to differentiate meningococcic from other types of meningitis. To a few c.c. (50 drops) of centrifuged spinal fluid add one to four drops of antimeningococcus serum and incubate the mixture (along with a control- tube containing only spinal fluid) at s6°C. for 12 hours. If the serum and spinal fluid be homologous, a definite precipitation will be observed. This test appears to be ^peoif.c^jgjtJzed by MicrOSOft® Fig. 160. — Diplococcus intracellularis meningitidis. (Councilman.) 684 DIAGNOSTIC METHODS Tubercular Meningitis. The fluid in such cases is usually clear, but may be slightly opalescent. The cellular elements are largely mononuclear while a few red cells may be present. If tubercle bacilli cannot be found after careful search, the presence, of a lymphocytosis along with an increased protein-content will be at least suggestive, while animal inoculation or the tuberculin test will clear up the diagnosis in most cases. (See Lucas.') Acute Anterior Poliomyelitis. This disease has been definitely established as infectious through the work of Flexner and his associates. The causative agent was known to belong to the ultra-microscopic t3^e and to pass the Berkefeld filter. In other words it is a filterable virus, the point of ingress and egress of which is the nasal and pharyngeal mucosa. This virus is transmitted by means of the biting stable- fly (stomoxys calcitrans) and probably, also, by the common fly and bedbug. ^ Cultivation of this organism has been accomplished by Flexner and Noguchi.' The culture medium consists essentially of sterile human ascitic fluid to which has been added a fragment of sterile fresh kidney tissue of the normal rabbit. Solid media is made by the addition of 2 per cent, agar to above. The exclusion of oxygen is necessary for the initial culture, but it suffices to cover the media with a deep layer of sterile paraffin oil. Films prepared from the lower layers of the fluid cultures are made, are air-dried and fixed in methyl alcohol for one hour. Wash in distilled water and im- merse in Giemsa's solution (i drop of stain to i c.c. of distilled water) for two to twelve hours. Examination reveals minute globoid bodies arranged in pairs or short chains, or in small aggregated masses. These bodies are stained violet and average about 0.2 micron in diameter. Their pathogen- icity has been proven by inoculation of monkeys. The changes in the spinal fluid, while not absolutely diagnostic are sugges- tive. The amount of fluid usually increases early in the disease, this increase being associated with a relative increase in the mononuclear types . These cells may first be of the small lymphocytic variety but they soon changed to the large mononuclear form. As the disease progresses, the cells diminish in number, the polymorphonuclear forms assuming the ascendency. The total protein is in- creased in amount (as detected by the Noguchi or Ross- Jones method), while fibrin formation appears to be an early sign, although this may soon disappear.' ^ Am. Jour. Dis. Child., 1911, 1, 230; also, Mandelbaum, Deutsch. Arch. f. klin. Med., 1913, CXIII, 92; Roby, Jour. Am. Med. Assn., 1915, LXV, 1027. ^ Rosenau, Discussion in 15th Cong, on Hyg. and Demog., Abs. in Jour. Am. Med. Assn., 1912, LIX, 1314; Anderson and Frost, Pub. Health Rep., 1912, XXVII, 1733; Howard and Clark, Jour. Exper. Med., 1912, XVI, 850; Mitzmain, Pub. Health Rep., 1913, XXVIII, 34S; Lucas and Osgood, Jour. Am. Med. Assn., 1913, LX, 16,11; Rosenau, Ibid., 1612; Sawyer and Herms, Ibid., 1913, LXI, 461. ^ Jour. Exper. Med., 1913, XVIII, 461. See, also, Amoss, Ibid., 1914, XIX, 212. ■"See "A Clinical Study of Acute Poliomyelitis" by Peabody, Draper and Dochez, published by the Rockefeller Institute in 191 2; Flexner, Science, 19 12, XXXVI, 685; Fraser, Jour. Exper. Med., 1913, XVIII, 242; Kling and Levaditi, Ann. de I'lnst. Pasteur, 1913, XXVII, 718; Cassel, Deutsch. med. Wchnschr., 1913, XXXIX, 2507; Lust and Rosen- berg, Munch, med. Wchnschr., 1914, LXI, 121; Flexner and Amoss, Jour. Exper. Med., 1914, XIX, 411; Ibid., 1914, XX, 249; Ibid., 1915, XXI,so9; Flexner, Noguchi and Amoss, Ibid., 91; Flexner, Bull. Johns Hopkins Hosp^, 1915, XXVI, 180; Am. Jour. Dis. Child., 1915, IX, 353. See, also, MoBtga^Bidib^MiBrdDSej^^onr., 1914, CLXX, 373; Corbus, Jour. Am. Med. Assn., 1914, LXIII, 550; Neisser, Berl. klin. Wchnschr., 1915, LII, 486. TRANSUDATES AND EXUDATES 685 Cerebrospinal Sjrphilis. Although such cases do not show pathognomonic findings in the cerebro- spinal fluid, yet certain factors are of importance in diagnosis. A lymphocy- tosis is usually observed together with a marked increase in the globulin con- tent, which is estimated as follows :' Noguchi's Butyric Acid Test. One part (o.i or 0.2 c.c.) of spinal fluid is mixed with 5 parts (0.5 c.c.) of a 10 per cent, butyric acid solution in physiologic salt solution. This mixture is heated to boiling and immediately i part (o.i c.c.) of normal (4 per cent.) sodium hydrate solution is added and the mixture again boiled for a few seconds. The presence of an increased content of protein in the fluid is in- dicated by the appearance of a granular or flocculent precipitate which gradu- ally settles under a clear supernatant liquid. This precipitate appears with- in a few minutes in a specimen containing a considerable increase in protein, while two hours may be required to obtain a distinct reaction in specimens weaker in protein. May'' has called attention to the advisability of using larger portions of the fluid, preserving the same proportions of reagents as used by Noguchi. This reaction appears regularly in the cerebrospinal fluid of patients with syphilitic and parasjrphilitic affections and also in all cases of inflammation of the meninges caused by such organisms as the meningococcus, pneumococcus, influenza bacillus, tubercle bacillus, etc. These latter cases are, however, easily differentiated from the sj^hilitic affections. Normal fluid gives a tur- bidity but the granular precipitate does not occur at all or only after many hours. Although this reaction is given in many non-sjrphilitic conditions, it has a certain value. It is not specific and, when present, does not necessarily indi- cate a syphilitic infection. On the other hand, it can be employed to estab- lish or confirm a deduction based upon the clinical history and the results of the Wassermann reaction and cytodiagnosis, thus becoming of great indirect diagnostic value. A S3^hilitic infection is, however, practically excluded by a negative reaction. In this respect this test has advantages over the Wasser- mann reaction, in which a negative result is not always reliable as indicating absence of syphilitic infection.' Nonne's Test. This test,^ like the above, is for the detection of an increase of the protein material in the spinal fluid. It is, however, not limited to syphilitic condi- tions, although it is widely used for such cases. Nonne has advanced two steps which he styles "phases." 1 See Kaplan and Casamajor, Arch. Int. Med., 1912, IX, 262; also, Ball, Jour. Am. Med. Assn., 1912, LIX, 1272. Ellis (Jour. Exper. Med., 1913, XVIII, 162) asserts that no syphi- litic patient should.be regarded as cured without a complete negative examination of the cerebrospinal fluid. 2 Arch. Int. Med., 1911, VIII, 183. ' Nichols and Hough (Jour. Am. Med. Assn., 1913, LX, 108) report the demonstration of spirochaeta pallida in the cerebrospinal fluid and the successful inoculation of a rabbit therefrom. ' Arch. f. Psychiat. uo Nervenkrankh., 1907, XLIII, 433; Miinch. med. Wchnschr., 1907, LI V, 2 1 1 7 . Digitized by Microsoft® 686 DIAGNOSTIC METHODS Phase I. To a small portion of the clear cerebrospinal fluid add an equal volume of a hot saturated solution of ammonium sulphate. A trace of opalescence or a distinct turbidity, usually appearing within three minutes, indicates a posi- tive reaction. Normal fluids never show any appreciable change with this test, while pathologic types always react promptly. The test is positive in all infections of the nervous system and is of special interest in general paralysis, tabes and obscure cerebrospinal syphilis. Although not absolutely differ- ential, it is, yet, of great importance, as a negative test excludes syphilis and parasyphilis. Phase n. In case no turbidity is observed in Phase I, add a drop or two of acetic acid to the above mixture. A distinct turbidity appears at once. This phase is given, not infrequently, with normal fluids so that little value is attached to it. Ross- Jones Test. This^ is a modification of Phase I of Nonne and is, in my opinion, some- what more delicate. Two c.c. of a saturated solution of ammonium sulphate are placed in a test-tube and i c.c. of the cerebrospinal fluid is gently run on to the surface so as to form a contact layer. A clear-cut, thin, grayish- white ring is observed at the junction of the liquids if the reaction be positive. This ring should appear within three minutes. The value of the test is the same as that above. Lange's Colloidal-gold Test. As neither the Nonne nor Ross- Jones tests exclude inflammatory processes in the brain or cord, a more differential test is to be desired. This seems to have been found in Lange's test. It is based on the fact that protein material precipitates colloidal gold at a dilution which is constant for definite proteins. If the protein be in less concentration a protective power over the colloidal particles is exerted and no precipitation occurs. In the case of spinal fluid, the protein present in different pathologic conditions shows a different precipi- tating point in syphilitic and non-syphilitic disorders. This testis, therefore, much more differential than are the preceding ones given. Preparation of the Colloid Solution. Place 500 c.c. of double-distilled water in a Jena flask of 1,000 c.c. ca- pacity and heat slowly. When the temperature is about 6o°C., add, while still heating, 5 c.c. of i per cent, solution of gold chlorid (Merck's C. P.) and follow immediately with 5 c.c. of 2 per cent, solution of potassium carbonate. Heat rapidly to boiling and discontinue heating. Add quickly 5 c.c. of i per cent, dilution of formalin (not of formaldehyd). All these solutions must be added with a pipet as the amounts must be accurate in order "to insure success in the preparation of the fluid. Shake the flask vigorously until a change of color occurs, beginning with a darkening of the fluid and passing through faint blue, dark blue, andpurple to a final red. This is the finished colloidal 1 Brit. Med. Jour., 1909, ]PM4?ecf by MicrOSOft® TRANSUDATES AND EXUDATES 687 solution and should be a distinct magenta and absolutely clear. Preserve in a dark-colored bottle. Technic. Place in a rack a series of twelve test-tubes. In the first, place with a pipet 1.8 c.c. of 0.4 per cent, freshly made sodium chlorid solution. In the other eleven tubes place i c.c. of the chlorid solution. To the first tube add 0.2 c.c. of spinal fluid, which must be free from blood. The dilution in tube i is, therefore, i to 10. Mix this thoroughly and place i c.c. of this first dilution in the second tube. This dilution is i to 20. Proceed in the same way with the remaining tubes, getting dilutions up to i to 20,440. After the dilutions are prepared, add to each tube 5 c.c. of the colloidal solution and let the tubes stand for 24 hours at room temperature. The read- ing of the test is as follows: The original tube showed a medium magenta shade. As the precipitation of the gold occurs, the color of the tubes con- stantly manifests a bluer tinge and finally becomes clear and colorless. The dilution at which the greatest precipitation occurs is noted and the results at various dilutions expressed by the + or — sign. Thus — indicates no precipitation; -|-, a red with beginning blue tinge; + +, red-blue and blue- red shades; + + +, violet and dark blue colorations; + -|- + +, light blue color ; + + + + +. complete precipitation with a clear and colorless solution. ' In syphilitic and parasyphilitic cases the tendency of the precipitation is constantly toward the left of the series, that is toward the i to 10 dilution. The maximum effect in such conditions is i to 40 or i to 80. It never con- tinues past I to 640 and infrequently past i to 160. In non-syphilitic cases the tendency is toward the right of the series, that is toward the i to 20,000 dilution, the maximum being observed at some point above i to 80. Tubercu- lous meningitis shows the most intense reaction at i to 160 or i to 320, while suppurative meningitis gives most marked reactions at i to 1,200 and up. This reaction promises to be of great value, especially in congenital syphilis. BIBLIOGRAPHY 1. Adami. Principles of Pathology. Philadelphia, 1908. 2. Kraus und Levaditi. Handbuch der Immunitatsforschung. Jena, 1908. 3. Jordan. General Bacteriology. Philadelphia, 1908. 4. Mestrezat. Le Liquide C^phalo-Rachidien. Paris, 1912. 5. Oppenheimer. Handbuch der Biochemie. Jena, 1908. 6. Plant, Rehm, and Schottmuller, Leitfaden zur Untersuchung der Zerebro- spinalfiussigkeit. Jena, 1913. ' Berl. klin. Wchnschr., 1912, XLIX, 897; Ztschr. f. Chemotherap., 1912, I, 44. See, also Grulee and Moody, Jour. Am. Med. Assn., 1913, LXI, 13; Eicke, Munch, med. Wchn- schr., 1913, LX, 2713; Kaplan and McClelland, Jour. Am. Med. Assn., 1914, LXII, 511; Miller and Levy, Bull. Johns Hopkins Hosp., 1914, XXV, 133; Kafka and Rautenberg, Ztschr. f. d. ges. Neurol, u. Psychiat., 1914, XXII, 353; Lee and Hinton, Am. Jour. Med. Sc, 1914, CXLVIII, 33; Debenedetti, Rif. Med., 1914, XXX, 906; Brock, 111. Med. Jour., 1914, XXVI, 422; de Crinis and Frank, Munch, med. Wchnschr., 1914, LXI, 1216; Kafka, Ibid., 191S, LXII, 105; Solomon and Koefod, Boston Med. and Surg. Jour., 1914, CLXXI, 886; Solomon and Welles, Ibid., 1915, CLXXII, 398 and 625; Weston, DarUng and New- comb, Am. Jour. Insan., 1915, LXXI, 835; Grulee and Moody, Am. Jour. Dis. Child., 1915, IX, 17; Barnes and Ives, Interstate Med. Jour., 1915, XXII, 792; Swalm and Mann, New York Med. Jour., 191S1 CI, 710; Rubenstone and Schwartz, Ibid., 1273; Busquet, Bull. I'Acad. Med., 1915, Lxxiv, wtgitized by Microsoft® CHAPTER X SECRETION OF THE MAMMARY GLANDS I. General Considerations The normal secretion of milk takes place in the mammary glands of the female after delivery. It is true that a small quantity of milk may be secreted by the new-born of both sexes for a few days after birth. Moreover, cases have been reported in which the adult male secreted sufficient milk to act as a wet-nurse, but these must be regarded as cases of extreme rarity. During the course of a normal pregnancy a small amount of a thin, yellow- ish fluid may be expressed from the mammary glands, but as a rule the first real secretion is observed following delivery of the child. This secretion is thin and watery, more or less translucent, and shows a distinct yellowish color. . r\ a Q -;^U Q^*^,"?o --'ivifi ■■■■) .. / -/ Fig. i6i. — Normal Milk and Colostrum. (Hawk.) a, Normal milk; b, colostrum. Microscopic examination shows the presence of rather large cells in which are many fat granules and occasionally a distinct nucleus. This secretion, which is called colostrum,^ continues for three to four days and is distinguish- able from the later secretion by the fact that it contains relatively more salts than normal milk and, according to the usual statements, more sugar. In a series of determinations of breast milk collected during the fourth day after delivery, the writer could not show any marked increase in the sugar in all cases, but in three out of eight cases examined a percentage higher than eight of lactose was obtained. ' See Engel and Bauer, Die Biochemie und Biologie des Kolostrums, Wiesbaden, 1912, Bergmann; Benestad, ^^''^^'[^f^^j^e^tyMcrOsjM' ''^'^' ^' "'^^ SECRETION OF THE MAMMARY GLANDS 689 It is probable that the function of the colostrum is gradually to accustom the child to the taking of food by diminishing to a slight extent the elements, protein and fat, which are more apt to cause digestive disturbances than is the lactose. The secretion of true milk begins about the fourth day and continues for a variable length of time. Marked variations are observed both in the quan- tity and the quality of this secretion in various women so that no hard and fast rule can be given as to the composition of normal mother's milk. While it is probably true that the normal woman should nurse her child during a large part of the first year, it is rare to find, especially in private practice, many such cases. Either the milk becomes scanty and loses in nutritive power or becomes excessive and consequently diluted. In either case the child is not receiving the' most suitable nourishment, so that breast-feeding is abandoned under these circumstances. It seems to be a general rule, which is difficultly explicable, that the more highly socially developed the woman the less apt she is to nurse her child with any success. It is possible, and this seems to the writer the most probable explanation, that the child usually receives more or less constant attention from the physician, and variations in the breast milk are more frequently noticed than in the case of the poorer child who rarely has the advantage of medical attention unless more or less serious illness occurs. II. Physical and Chemical Properties ' Various figures have been given for the composition of human milk so that it is difficult to strike an average. So much depends upon the nourishment of the mother, upon the amount of exercise taken by her, and upon the general condition of the system that marked variations exist in the proportion of the chemical constituents. The following table, taking averages of examinations reported by various writers, may serve as one representing more nearly the normal condition. '^ Water, 87.24 Salts, 0.26 Protein, i-So Fat, 4.0Q Lactose, 7.00 These figures are not as high as regards protein as those usually given, but are higher as far as the lactose is concerned. Although many writers have given the percentage of protein much higher than 1.5 per cent., the writer has never been able, in several hundred examinations of milk in his laboratory, to find many showing a protein percentage of two or more. As it is frequently necessary to modify cow's milk so that it may more ' See Schloss, Monatsschr. f. Kinderhkde., 1912, X, 499; also, Meigs and Marsh, Jour. Biol. Chem., 1913, XVI, 147; Talbot, Am. Jour. Dis. Child., 1914, VII, 44s; Grulee and Caldwell Ibid., 191S, IX, 37^^^^^^^^if^ff^§^;f^^- Biol. Chem., 1915, XXI, 239. 690 DIAGNOSTIC METHODS nearly approach mother's milk in composition, the writer inserts the following table for comparison. Water, 87.25 Salts, 0-7S Protein, 3 -SO Fat, 4.00 Lactose, 4-5° It will be seen that cow's milk shows a higher per cent, of protein and a lower per cent, of carbohydrate. It is necessary, therefore, that this be modi- fied by diluting the milk so as to diminish the protein and by adding lactose to make up for the deficiency of carbohydrate. In the dilution the fat content will necessarily be lowered so that this may be remedied, as suggested by Backhaus, by the addition of cream. The writer must refer to works on pediatrics for the various methods of modifying cow's milk. It is to be re- membered that no modification is equal to mother's milk The casein of human milk forms a much finer clot with the gastric juice than does that of cow's milk so that the latter may not be well tolerated by the child. More- over, some unknown principle present in human milk is accountable for a distinct biologic difference in these two types of nutritive material. (i) Appearance and Color. Normal human milk is a white fluid which usually has a slight bluish tinge 'except immediately after birth when the color may be distinctly yellowish from the presence of colostrum. If the percentage of fat be relatively high the color will be more nearly a pure white with little of the blue tone. (2) Specific Gravity. The specific gravity of human milk varies between 1,028 and 1,034. An increase in the percentage of fat will usually lower the specific gravity while the protein and carbohydrates will increase it. Cow's milk shows approximately the same specific gravity. If a low spe- cific gravity is obtained it is evidence either of an increased percentage of fat or of dilution with water. If the percentage of fat be low the milk is un- questionably a watered one. If the milk tested shows a high specific gravity the chances are that most of the fat has been removed either by skimming or by centrifugation. The determination of the specific gravity may be made by a special in- strument known as Quevenne's lactodensimeter or by the use of the ordinary hydrometer used in urine work. As it is never of any clinical importance or even of any marked practical importance that the specific greCvity should be absolutely accurately determined, the writer is accustomed to use the urino- meter for such determinations. Corrections for variations in temperature are necessary only when great differences exist between the temperature of the room and the tempemturaat/ whiek- fha.inafacument is calibrated. SECRETION OF THE MAMMARY GLANDS 691 (3) Reaction. Normal human milk as well as cow's milk shows an amphoteric reaction to litmus-paper and an acid reaction to phenolphthalein, cow's milk being somewhat more acid than mother's milk toward the latter indicator. ^ (4) Coagulation. If milk be allowed to stand, the reaction gradually becomes more and more acid owing to the development of bacteria, especially of the bacillus acidi lactici. When the degree of acidity reaches a certain point, casein sepa- rates first in the form of flocculi and later the entire fluid may coagulate to a jelly-like mass. This mass soon contracts and settles out leaving a slightly turbid fluid known as milk plasma or acid whey.^ In order to inhibit the development of bacteria and prevent this coagulation, known as souring, certain preservatives are frequently added to market milk and should be capable of detection by the practitioner. These will be discussed in a later section. Besides this type of coagulation of cow's milk a second form is observed which takes place under the influence of chymosin without any change in the reaction of the fluid. In this case the whey is sweet and contains practically all of the lactose originally present in the milk. (5) Total Solids. Five to ten c.c. of the well-mixed milk are placed in a weighed platinum dish, evaporated to dryness on a water-bath, and dried to constant weight in the oven at 105°. The difference in weight between the platinum dish and its contents, on the one hand, and the platinum dish, on the other, gives the amount of total solids in the milk taken. A simple calculation will yield the percentage of total sohds. Under normal conditions the total solids of both human and cow's milk should average between 12 and 13 per cent. Variations in this figure are, of course, due to fluctuations in the various constituent elements. (6) Ash. The platinum dish containing the dried residue of the milk is heated over a direct flame until the residue is completely incinerated. This is then placed in the desiccator and dried to constant weight. The difference in weight between the dish and contents and the dish along represents the amount of salts present in the milk originally taken. ^ (7) Protein. The methods for the determination of protein are divided into those for estimation of the total protein material present and into those in which sepa- rate determinations are made of the casein, on the one hand, and albumin and globulin, on the other. ' See Clark, Jour. Med. Research, 1914, XXXI, 431; Milroy, Biochem. Jour., 1915, IX, 2IS- ^ See Orla-Jensen, Ztschr. f. physiol. Chem., 1914, XCIII, 283. ' Bosworth, Jour. Biol. Chem., 1915, XX, 707; Holt, Courtney and Fales, Am. Jour. Dis. Child., 1915, X, 229. Digitized by Microsoft® 692 DIAGNOSTIC METHODS (a) Total Protein. Method of Sebelien. Ten c.c. of milk are diluted with 90 c.c. of water, 5 c.c. of a saturated so- dium chlorid solution, and 15 c.c. of Almen's tannic acid solution are added. The mixture is thoroughly stirred and the dense precipitate which forms is allowed to settle. The composition of Almen's tannic acid solution is as follows : Tannic acid, 4 grams. Acetic acid (25 per cent.), 8 c.c. Alcohol (50 per cent.), qs., ad., 200 c.c. The precipitate, which consists of the total protein of the milk and a large portion of the fat cairried down by the precipitate, is then filtered off through a fine filter and is wElshed with cold water. The filter-paper and its contents are then placed iii a Kjeldahl flask and a nitrogen determination made as described under Urine. It is advisable to use 20 c.c. of sulphuric acid instead of the ten employed in the case of urine, as the mixture oxidizes much more rapidly under these Conditions. If the nitrogen obtained in this determina- tion be multiplied by 6.37, the result will be the protein in 10 c.c. of milk. 1 I Method of Bog^is. For routine woirk this method is perhaps more advisable for the general practitioner than is the preceding, but it does not always give confirmatory re- sults. It is based Upon the fact that the total protein of milk is precipitated by phosphotungstic acid in hydrochloric acid solution, the amount of precipitate being measured in ^n Esbach tube. The reagent used has the following composition: Phosphdtungstic acid, 25 grams. Concentrkted hydrochloric acid, 25 c.c. Distilled water, q.s., ad., 250 c.c. , It has been found that the milk should be diluted with water before adding the reagent if the rfesults are to be accurate. As a rule, a dUution of i to 10 for human milk and i to 20 for cow's milk suffices. The diluted milk is poured into the Esbach tube^ to the mark U and the reagent added to the mark R. The tube is then closed with a stopper and inverted several times thoroughly to mix the contents. It is then set aside for 24 hours when the percentage of protein in the milk is read off di- rectly from the calibrations on the tube in case the dilution was i to 10, while with a dilution of i to 20 the figures are multiplied by 2. This method, while convenient, is open to the objection that many factors may influence the depth to which a precipitate settles. Moreover, the Esbach tubes reading as high as 12 parts do not give as satisfactory results as those with readings from one to seven. Such being the case the method must be used more for clinical purposes than for scientific estimations. ^ If a Purdy centrifuge tube be filled with the diluted milk to the 10 c.c. mark and- the reagent added to 15 c.c, the amount of protein may be determined quickly by centrifuging for 3 minutes and reading the jjercmtage directly. The figures obtained agree quite closely with those of the gravimetri(fl^>«K5e0 by MlCrOSOlt® SECRETION OF THE MAMMARY GLANDS 693 (b) Casein. Twenty c.c. of well-mixed milk are measured into a beaker and approxi- mately 380 c.c. of water are added. The mixture is thoroughly stirred and very dilute acetic acid added drop by drop with constant stirring until a floc- culent precipitate is observed. When this point is reached a stream of car- bon dioxid is passed through the mixture for one-half hour, after which the vessel is allowed to stand until the next day. The above part of the technic is directly applicable to cow's milk. If human milk is being examined it is necessary to heat the vessel to 4o°C. during the passage of the carbon dioxid. After the mixture has stood overnight, it is filtered through a nitrogen- free filter and washed with water. The residue on the filter contains casein which is mixed with a portion of the fat present. The filter-paper and con- tents are then placed in a Kjeldahl flask and a nitrogen determination is made as previously described. Multiplication of the nitrogen value by 6.37 yields the amount of casein in the 20 c.c. of milk originally taken.^ (f ) Albumin and Globulin. The filtrate from the above precipitation of casein contains the remainder of the protein material and the carbohydrate of the milk. This filtrate is placed in a porcelain dish and heated for a few minutes to the boihng-point. The protein material is coagulated and may be filtered through a nitrogen- free filter and washed several times with cold water. A nitrogen determina- tion is then made and the value multiplied by 6.37 to obtain the amount of albumin and globuUn present in the 20 c.c. of milk. The filtrate from this latter precipitation contains the lactose, which may then be determined by titration with Fehling's solution, as described under Urine. It is to be remembered that 10 c.c. of Fehling's solution are reduced by 0.0678 gram of lactose, and not by 0.05 as in the case of glucose. (8) Fat. It is important in the determination of the fat content of milk that a thoroughly mixed specimen be examined.^ As the fat tends to rise to the surface of the milk, the fluid should be poured from one vessel into another several times to insure thorough mixing and an immediate measurement made of the portion to be tested. For clinical purposes as well as for examination of market milk the method of Babcock is to be recommended. For accurate results, however, this method is not to be advised. Babcock's Method. This method consists in the destruction of the organic matter, except the fat, by means of sulphuric acid. The &it is then separated by centrif ug- ing and determined by reading off the percentage from the calibrations in the neck of the bottle used. In the case of cow's milk or where sufficient human milk may be ob- ' See Van Slyke and Bosworth, Jour. Biol. Chem., 1915, XX, 135. ^ See Brodrick-Pittard, Biochem. Zt-chr., 1914, LXVII, 382; Osborne and Wakeman, Jour. Biol. Chem., igis, XXI, 539. Digitized by Microsoft® 694 DIAGNOSTIC METHODS tained, 17.6 c.c. of milk are measured into the bottle and 17.5 c.c. of sul- phuric acid added. These fluids are then mixed by shaking and rotating the bottle in such a way that no curds pass into the neck of the bottle. As soon as the mixture becomes homogeneous and dark brown or even black in color, the bottles are placed in a special centrifugal machine and whirled for five minutes. If the room be very cold it is advisable to fill the holders of the centrifuge with boiling water in order to keep the fat melted while it is being centrif uged. At the end of five minutes, centrif ugation is discontinued and the neck of the bottle filled with boiling water. The melted fat will rise in the neck of the flask and may be read off from the calibration. In order to facilitate this the bottles are again centrifuged for one minute. If a small amount of milk only is available the smaller tubes shown in cut may be used. Milk is added to the mark five and sulphuric acid poured in so as to fill the body of the tube. It is usually necessary to add the milk and acid by means of thin narrow pipets, as the neck of the tube is too small to permit of easy entrance of the fluids otherwise. The milk and sul- phuric acid are mixed by rotation of the tube until a homogeneous fluid results. The mixture is |||||H{|{ \\ centrifuged for a few minutes, the neck of the tube being filled with a mixture consisting of equal parts of concentrated hydrochloric acid and amyl alcohol. The percentage of fat is then read off from the calibra- tions on the tube. 5C,C Fig. 163.— Bottle for human milk. Extraction Method. A few grams of dried washed Fig. 162. — Babcock Bottles, sand are placed in the extraction a, Milk bottle; b, cream bottle, ^j^^jj ^f ^ Soxhlet apparatus and 10 c.c. of well-mixed milk are allowed to fall upon it drop by drop. This is dried at a temperature of ioo°C. for one to two hours and is placed in the tube of the extraction apparatus. The fat is extracted in the usual way by the use of gasoline or anhydrous ether, complete extraction usually requiring from two to three hours. The apparatus is disconnected, the ether evapo- rated from the distilling flask, the residue in the flask dried at ioo°C., and the flask and contents dried to constant weight in the desiccator. The differ- ence between the original weight of the flask and its weight including the ex- tracted residue, yields the ambtiht of fat in the 10 c.c. of milk. This method is the most accurate oije, but is not as convenient as the preceding for the general practitioner. ^.^.^^^^ ^^ Microsoft® SECRETION OF THE MAMMARY GLAKDS 69s (9) Lactose. In general routine analyses of milk the lactose may be determined by difference. By this is meant that subtraction of the values for the sum of water, ash, protein, and fat from 100 will yield the percentage of lactose. For clinical pur- poses this is usually sufficient, but for the more accurate work it does not give exact figures, as slight amounts of other undeter- mined substances are present. For any direct determination of lactose it is necessary that the larger portion of the protein material be removed previously. This may be done by the method outlined under Determination of Casein, Albumin, and Globulin; or for clinical purposes sufiSciently accurate results may be reached by 'acidifying with acetic acid, boiling, and filtering. It is advisable always to take the time to saturate the mixture with carbon dioxid after the casein "Aas been precipitated with acetic acid, as the results are more satisfactory.^ The fluid is then titrated by the use of Fehling's or Purdy's solution, using all the precautions mentioned under these tests in the section on Urine. Ten c.c. of Fehling's solu- tion are reduced by 0.0678 gram of lactose, while the 35 c.c. of Purdy's solution are re- duced by 0.02712 gram of lactose. If the polarimeter is to be used for the estimation of lactose in milk the following procedure may be used. Fifty c.c. of the well- mixed milk are placed in a flask, 25 c.c. of a solution of neutral lead acetate are added, the flask is closed with a stopper through which passeS a glass tube approximately 30 cm. in length. The mixture is then heated over a small flame to boiling. After the mixture has ■cooled it is filtered through a dry filter into a dry vessel and polarized. Fig. 164. — Soxhlet (Hawk.) apparatus. (10) Preservatives in Cow's Milk. {a) Sodium Carbonate. To hide the acid reaction qf a spoiled sample of milk sodium carbonate is often added, and may be detected as follows: Ten c.c. of milk are mixed "with ten c.c. of 96 per cent, alcohol and a drop of rosolic acid solution. Pure unadulterated milk produces a brownish-yellow color, but, in the presence of sodium carbonate or bicarbonate, a rose color is obtained. For greater ' See Jackson and Rothera, Biochem. Jour., 1914, VIII, i; Palmegiani, Pediatria, 1914, xxn, 739; Hill, Jour. Biol. (^f^tif§&>bflMit;T^oft® 696 DIAGNOSTIC METHODS precision in doubtful cases the questionable sample should be compared with known unadulterated milk, Phenol-phthalein solution may be used as an in- dicator in place of rosolic acid. By tHis method 0.05 per cent, of carbonates may easily be detected. {b) Salicylic Acid. Twenty c.c. of milk are treated with two or three drops of sulphuric acid and shaken with an equal amount of ether. The greatest possible part of the ethereal solution is drawn off and evaporated, the residue extracted with 40 per cent, alcohol, filtered, and 5 c.c. of the filtrate treated with a few drops of ferric chlorid solution. A violet color shows the presence of sahcylic acid or some other hydroxy derivative of benzol. (c) Formaldehyd. Two c.c. of concentrated sulphuric acid are placed in a test-tube and a drop of ferric chlorid solution added. A few c.c. of milk are allowed to run from a pipet upon the surface of the mixture in such a way that a distinct line of contact forms. A violet color at the point of contact of the two liquids is characteristic of formaldehyd in the presence of casein. (d) Boric Acid and Borax. Fifty c.c. of milk are alkalinized with milk of lime, evaporated to dryness and incinerated. The resulting, white crystalline residue is treated with a few drops of tincture of turmeric and very dilute hydrochloric acid and is then dried on the water bath. The presence of the slightest trace of boric acid gives to the djy residue a beautiful vermilion or cherry red color. It is possible by this method to detect o.ooi per cent, of boric acid in mUk. Only very dilute hydrochloric acid must be used in testing for boric acid, since the concentrated acid itself gives with tincture of tvirmeric a red color. The coloration produced by boric acid is distinguished from that produced by hydrochloric acid by the fact that it does not disappear by treatment with water in the cold, but only after long boiling, while the color caused by hydro- chloric acid disappears as soon as it is diluted with water. If the crystalline residue obtained as above described be treated with alcohol and the alcohol ignited, a flame tinged with a beautiful emerald-green color is obtained in the presence of boric acid. III. Bacteriological Examination or Milk The bacteriological examination of human milk is frequently desirable from a clinical standpoint as milk may become contaminated as it passes along the lacteal ducts. In pathologic conditions many types of organisms, such as typhoid bacilli, pneimiococci, and tubercle baciUi may be obtained, although the latter are extremely rare. With cow's milk the chief question at issue is whether the milk contains sufficient bacteria to be harmful to the child. Aside from the presence of the tubercle bacillus in the milk of infected animals, large numbers of saprophytic Digitized by Microsoft® SECIiETION OF THE MAMMARY GLANDS 697 organisms must find their way into this fluid and will, if in large numbers, influence the intestinal activity of the child. Moreover, it must be remem- bered that many epidemics of milk-borne disease, such as typhoid fever and streptococcic sore-throat, may arise owing to improper handhng of the milk or to inefiicient pasteurization.' The methods of examining milk for bacteriologic differentiation are the same as for any other fluid and will be discussed in the next chapter. It is, not infrequently, a matter of importance to the general practitioner and of untold value to health officers to determine the number of bacteria in the milk suppHed to the community. The outline of the method to be followed in this work is as follows: Collect the specimen as freshly as possible. Keep it on ice from the time of collection until you are ready for the examination. Sterilize a quantity of distilled water in the autoclave. Plug a number of pipets (i and 10 c.c.) with cotton and sterilize in the hot oven. A number of Erlenmeyer flasks, plugged with cotton, a 100 c.c. graduate, and a number of Petri dishes are sterilized at the same time. Fig. 165. — Wolfhiigel's colony counter. By means of the graduate place 09 c.c. of sterfle distilled water in an Erlen- meyer flask, 90 c.c. in a second and 99 c.c. in a third and replace the cotton. By means of a sterile i c.c. pipet transfer i c.c. of the well-shaken specimen to flask number i. The dilution of the milk in this first flask is, therefore, i to 100. Shake this dilution thoroughly and transfer 10 c.c. of this dilution, by means of a pipet, to the second flask containing 90 c.c. of water. The second dilution is i to 1,000. By means of a second i c.c. pipet transfer i c.c. of the i to 100 dilution to the last flask, containing 99 c.c. of water, thus obtaining a dilution of i to 10,000. These dilutions are usually all that are necessary. Others may be made as desired. In the examination of the best market milks, the I to 100 dilution is sufficient. Liquefy a number of tubes of litmus-lactose agar, litmus-lactose-bile agar and plain agar and cool them down to 40° to 45°C. Transfer i c.c. of each dilution of the milk to each of two sterile Petri dishes and add the liquefied culture medium. Mix by careful manipulation and allow the media to harden. Place in incubator for 48 hours and count the number of colonies, using a colony counter such as shown in the illustration. Multiply the number of ' See discussion of "Streptococcic Sore Throat" on page 40; also Rogers and Dahlberg, Jour. Agricul. Research, 1914, 1, 49i. Shippen (Jour. Am. Med. Assn., 1915, LXIV, 1289) shows, in conformation of the work of De Jong and De Graef, that colon bacilli may sur- vive the pasteurization P^'^'^^Ajfj^Afff^ti^^^^'^Ax^Wfh^o be taken as evidence of later contamination. " " 698 DIAGNOSTIC METHODS colonies by the dilution to obtain the actual number of colonies per c.c. of original milk.^ The number of bacteria in milk may vary from a few thousand to many millions. It is an impossibility to obtain a specimen which is sterile so that the presence of as high as 100,000 organisms should not be regarded as danger- ous unless pathogenic types are present. If the freshly examined specimen contains between 10,000 and 50,000 bacteria per c.c, it is probable that all possible precautions have been taken to prevent contamination, although the .writer has found some specimens showing a count of only 100 per c.c. In the examination of milk it is customary also to make a cytological examination. It was formerly supposed that normal fresh milk contained only a few scattered leucocytes and epithelial cells, any marked increase in their number being taken as evidence of infection. However, by use of more careful methods it has been shown that the number of these cells may run as high as 500,000 or more and yet no abnormality be present. It is necessary in this work to distinguish between the ordinary leucocytes, which are con- stantly discharged in large numbers in the milk, and the true pus cells, which are evidence of disease. No detailed work along this line has been reported. The association of large numbers of bacteria, especially of staphylococci and streptococci, is probably evidence of infection of the animal and consequent unfitness of the milk for use, although the significance of the increase in cellular elements is not, by any means, settled. The methods used in this cytological examination are the same as outlined previously. ^ BIBLIOGRAPHY 1. CzERNY UND Keller. Die Nahrungs-Pathologie. Berlin, 1907. 2. Holt. Diseases of Infancy and Childhood. New York, 1906. 3. Langstein and Meyer. Sauglingsernahrung und SauglingsstofEwechsel. Wies- baden, 1914. 1 See Brew, New York Agricultural Exper. Station, Geneva, N. Y., Bull. 373, IQ14; also, Goodrich, Jour. Infect. Dis., 1914, XIV, 512. 2 See Lewis, Am. Jour. Dis. Child., 1913, VI, 225; Hachtel, Jour. Am. Med. Assn., 1913, XI, 565; Stokes and Stoner, Ibid., 1024; Breed, Bull. 380, New York Agricultural Exp. Station, Geneva, N. Y., 1914; Hewlett and Revis, Lancet, 1915, I, 855; Frost, Jour. Am. Med. Assn., 1915, LXIV, 821. Digitized by Microsoft® CHAPTER XI CLINICAL BACTERIOLOGY I. General Considerations In no field of medical or other scientific research have there been recent advances of such far-reaching importance to humanity as in the realm of Clinical Bacteriology. The study of the relationship of bacteria and their metabolic products to disease in general and the identification of the specific organism of many of our most dreaded diseases has led to such an enormous output of work upon the various ways in which the system may react to bac- terial invasion, that our knowledge of the subject of immunity, both natural and acquired as well as active and passive, has advanced with leaps and Fig. i66. — ^Lautenschlager Hot-air Sterilizer. bounds. Indeed, so greatly has our knowledge increased that preventive medicine is fast taking the place of curative medicine. It is evident, there- fore, that the scientific aspect of bacteriology, great as it is, is, for the general worker and for the public at large, overshadowed by the success attached to the practical application of the knowledge acquired from the hard and earnest work of scores of scientific investigators. Scientific and applied bacteriology must go hand in hand, each playing its important part in the struggle against disease. The object of the writer in presenting this section is not to include a dis- cussion of the advances in bacteriology, as this would, indeed, be absurd. He does wish, however, to present certain general features of the subject, so that Digitized by ^^crosoft® 7-00 DIAGNO&TIC METHODS the student and practitioner may be given such a working knowledge as will enable them properly to perform and interpret many of the tests, which they may have occasion to undertake. Many such points have been discussed throughout this book but many have been omitted, so that the writer feels that this discussion will not be untimely. II. Sterilization As is well known, bacteria are practically ubiquitous, being foimd in air, water, food and soil. It is evident, therefore, that all material and glass-ware, which may be used in bacterio- logical work, must be clean and sterile before any reliable results may be obtained. Glass-war.e should be thoroughly cleaned by boiling in soapsuds or by soaking for a few hours in the following cleaning mixture. Potassium bichromate, 60 grams. Water, 300 c.c. Concentrated sulphuric acid, 460 c.c. After washing or soaking, rinse the glass thoroughly in running water and dry. Plug the test-tubes and flasks with non-absorbent cotton and place in a hot-air sterilizer or oven for one hour at a temperature of i70°C. The internal temperature of the sterilizer must be carefully regulated or the variation may be so great in different portions of the sterilizer that the more resistant organisms and spores will not be killed. Culture media are best sterilized by the use -of the autoclave, an apparatus for holding steam under pressure. The usual pressure used is 1 5 pounds, the media being kept in con- tact with steam at this pressure (temperature about i20°C.) for 15 to 20 minutes. The gas is then turned off, and the apparatus allowed to cool. While most media may be sterilized by this method, gelatin media would better not be heated longer than five minutes and. blood-serum and carbohydrate-containing media may undergo chemieaL change if the heating is prolonged. For thia reason, such media as last mentioned or all media, in the absence of an autoclave, may be sterilized in the Arnold sterihzer, an apparatus so con- structed as to produce steam from a small amount of water, the tank being fed by the water of condensation. As many bacterial spores are not killed by liVf> =;tpam it is necessary to use the so-called discontinuous sterihzation when nvesieam, bigitized by Microsoft® Fig. 167. — Autoclave. CLINICAL BACTERIOLOGY 701 the Arnold is employed. Heat the media for 30 minutes after the steam has filled the sterilizer and allow the media to stand at room temperature for 24 hours. Repeat the process on each of two other successive days. In this way spores develop into vegetative organisms,which are killed by the later heating. Certain fluids, such as serum or ascitic fluid, cannot be sterilized by heat- ing owing to coagulation. These are sterilized by passing through a Pasteur, Chamberland or Berkefeld filter. Fig. 168. — .\rnold Sterilizer. III. Preparation or Culture Media For the development of bacteria certain factors are essential. The most important of these are (i) oxygen; (2) food; (3) moisture; (4) a proper reaction of the culture media; and (5) an optimum temperature for growth. Regarding the question of proper food it is to be said that the number of culture media advocated is legion. I cannot, therefore, attempt to give any but those in general use in laboratory routine. Many special media have been discussed in other sections of this book and will be referred to later in the discussion of special organisms. Nutrient Broth (Bouillon; Beef Broth). Infuse 500 grams of chopped lean meat with 1,000 c.c. of distilled water for 24 hours in the ice box. Instead of this meat, one may use 3 grams of Liebig's meat extract, but the medium is not as nutritious. Strain the infu- sion through cotton flannel, the juice being well pressed out. Add i per cent, peptone (10 grams). Warm on water bath, stirring until the peptone is dissolved. Heat over hclS^^zedt^ iMi6Cesisft®r 30 minutes. Restore the 702 DIAGNOSTIC METHODS original volume of fluid. Titrate the acidity of the broth, using N/20 sodium hydrate solution and phenolphthalein as an indicator.' Adjust the reaction to +1 by adding the calculated amount of normal sodium hydrate. Boil two minutes over free flame, stirring constantly. Restore loss by evaporation and filter through paper or absorbent cotton. Titrate and accurately adjust the final reaction to +1. Place the broth in test-tubes, using 10 c.c. in each tube. Plug tubes with cotton and sterilize in the autoclave. ^ Sugar-free Broth. • Make the infusion of meat (do not use meat extract) as above. Add to this 10 to 20 c.c. of a 24-hour broth culture of bacillus coli communis and incu- bate at 37°C. for 18 hours. Boil the mixture to kill the organisms and pro- ceed as above, using this material as if it were ordinary meat infusion. Sugar Broths. Add to the sugar-free broth above mentioned i per cent, of dextrose, lac- tose or other sugar. Adjust the reaction of these broths so that it is neutral to phenolphthalein. Sterilize by the discontinuous method.^ Nutrient Gelatin. To the nutrient broth add 10 per cent, of "gold label" sheet gelatin. Warm on water bath, stirring till gelatin is dissolved but not allowing the temperature to rise above 5o°C. Heat over steam bath for 30 minutes and restore loss by evaporation. Titrate, after boiling one minute tb expel carbon dioxid, and adjust the reaction to -|-i as above described. Boil two minutes over free flame, stirring constantly. Make up loss by evaporation and filter through paper or absorbent cotton. If a clear filtrate be not obtained, it may be assured by cooling the mixture to 6o°C. and stirring in the white of an egg beaten up in 30 c.c. of water. Titrate and adjust final reaction. Tube as above and sterilize in the autoclave at i2o°C for five minutes. On being re- moved from the autoclave the gelatin is placed at once in the ice-box. Nutrient Agar.' Prepare the meat infusion as given under nutrient broth. Weigh the fil- tered infusion and add 2 per cent, of Witte's peptone. Warm on water bath stirring until the peptone is dissolved and not allowing the temperature to go above 6o°C. While this mixture is being prepared, boil 15 grams of thread '■ The optimum reaction for such culture medium is one having a neutral reaction to l|itmus and a degree of acidity requiring i c.c. of normal (4 per cent.) sodium hydrate to neutralize 100 c.c. of the medium. This is known as +1. See Clark, Jour. Infect. Dis., 191S, XVII, 109. Place s c.c. of the medium to be tested in a porcelain dish and add 45 c.c. of distilled water. Boil to expel carbon-dioxid. Add i c.c. of J^ per cent, solutionjof phenolphthalein in 50 per cent, alcohol. Titrate, while hot, with N/20 sodium hydrate up to a faint but distinct pink color. Read from the buret the amount of N/20 NaOH used to neutralize the 5 c.c. of medium and calculate the amount of normal NaOH necessary to neutralize the entire quantity of bouillon. Thus, if i.s c.c. of N/20 NaOH were used for 5 c.c, this would represent 30 c.c. for 100 or 300 for 1,000 c.c. But 300 c.c of N/ 20 NaOH is equivalent to 15 c.c. of normal NaOH. As we desire the medium to have an end-reaction of -fi per hundred or 10 per liter, we must evidently add 5 c.c. of normal NaOH to bring this medium up to the proper standard. ' See Holman, Jour. Infect. Dis., 1914, XV, 209; also Vedder, Ibid., 1915, XVI, 385. ' See Report of Committee on Standard Methods of Water Analysis, Jour. Infect. Dis., 1907, Supplement III, 108; ^^Q^^f^^i^Ry MfSr^sSM^'^' ^^^"^' '°^- CLINICAL BACTERIOLOGY 703 agar in 500 c.c. of water for one-half hour and make up the weight to 500 grams. Let it cool to 6o°C. To 509 grams of the meat-peptone infusioa add 500 grams of the 3 per cent, agar, keeping the temperature below 6o°C. Heat over steam bath for 30 minutes and restore loss by evaporation. Titrate and proceed as given above for nutrient gelatin. Great difficulty is sometimes experienced in filtering this preparation, but if the filter paper and funnel be wet with boiling water, the medium usually passes through- easily. Sterilize in the autoclave. Glycerin Agar. Prepare nutrient agar in the usual way. After filtering add 6 to 8 per cent, of glycerin. Mix thoroughly, readjust the titer if necessary and place in test-tubes. Sterilize in autoclave. This medium is preferable to plain agar in cultivating such organisms as the tubercle bacillus, which does not grow at all well on plain agar. Lactose and Dextrose Litmus Agar. To nutrient agar, prepared from sugar-free broth instead of nutrient broth, I per cent, of lactose or dextrose may be added just before sterilization, which must be in the Arnold sterilizer. Adjust the reaction to the neutral point to phenolphthalein. If the medium is to be used in tubes, the sterilized litmus solution or preferably azolitmin solution shall not be added until just before the final sterilization. If it is to be used in Petri dishes, the sterilized solu- tions shall not be added until the medium is ready to pour into the dishes. The litmus solution used is a i per cent, solution of Merck's purified litmus extract. Of this 5 or 6 per cent, is added. It is, probably, preferable to use instead of this, a i per cent, solution of Kahlbaum's azolitmin. "Ameba" Agar. This is the medium used by Musgrave and Clegg in cultivating the ameba and differs from nutrient agar in its strength. It is prepared in the same way but has the following formula: Extract of beef. Sodium chlorid, Agar, Water, The reaction is made — i, that is, it is i per cent, alkaline. Variations may be made in this medium, where the very delicate organisms are concerned, by adding a very small amount of peptone. Blood Agar. Prepare about 40 tubes of nutrient agar and, while still unsoHdified, place them in a slanting position so that a large surface is given. Such tubes are called slants. Place 50 c.c. of nutrient agar in a flask and melt it. Allow it to cool to about 4S°C. and add 15 c.c. of human blood, which has been drawn by venous puncture. Mix the contents of the flask by shaking and pour 2 c.c. of this mixture over the surface of each agar slant. Place these slants in position again sb that the added material may harden. These tubes cannot be resterilized. ^. ... , . ... „^ Digitized by Microsoft® 0.3 to 0.5 gram. 0.3 to 0.5 gram 20.0 grams. 00.0 c.c. 704 DIAGNOSTIC METHODS Instead of this method, which is preferable, one may add i to 2 c.c. of the human blood directly to xo c.c. of agar melted and cooled to 40 or 4S°C. Mix by shaking and slant the tubes. This medium is especially valuable for routine work as it will grow practically all organisms. Ascitic or Hydrocele Agar. Dissolve 15 grams of agar in 1,000 c.c. of water. Filter and place 5 c.c. in each test-tube. Sterilize in the autoclave. To each agar tube add 5 c.c. of sterile ascitic or hydrocele fluid and mix by rotating the tube. Cool so as to form slants. , This medium cannot be resterilized. It is not always the simplest procedure to prepare this medium as the body fluid may be contaminated. Litmus Milk. Fresh milk, as pure as possible, is obtained and steamed for fifteen min- utes in the Arnold. It is then placed in the ice box over night to allow the cream to separate. The cream is drawn off and the skimmed milk used for the medium. The reaction should be adjusted to +1. Add i per cent, azolitmin solution and tube 10 c.c. in test-tubes and sterilize for five minutes in the autoclave or in the Arnold for three successive days. Blood-serum. This is the medium devised by Loffler for the cultivation of the diphtheria organism and is very frequently used for others as well. It is prepared by adding i part of neutral nutrient broth containing i per cent, dextrose to 3 parts of sterile beef-aerum. The mixture is then mixed thoroughly and placed in culture boxes or, preferably, in short tubes. In theJatter it should be slanted. Coagulate at 75°C. After being held at this temperature until coagulated, the serum may be very slowly heated to about 95°C. The above are the more important culture media used in routine work. Special media will be mentioned in discussing specific organisms. IV. Incubation Although bacteria may develop at temperatures above that of 37°C. (body temperature) or at much lower temperatures, yet the optimum tem- perature for most bacteria is 37°C. It is true that many organisms develop more luxuriantly and more rapidly at 2o°C., but these bacteria are, for the most part,' non-pathogenic. It is essential, therefore, that some sort of apparatus be at hand, which will permit of the maintenance of a regular and uniform temperature of 37°C. Such an arrangement is called an incubator, many types of which may be ob- tained. Some of these are heated by gas and others by electricity, the latter beingby far the "more preferable. A heat-regulator is attached so that varia- tions in the temperature inside the incubator may be reduced to a minimum. In these incubators must be placed a dish of water to supply the necessary moisture, the development of bacteria proceeding the better the more nearly saturated the atmospherg)^^Jijj^){^-|^gQ^@ CLINICAL BACTERIOLOGY 705 Cultures when made upon the various media are placed in the incubator for periods ranging from 24 hours to 10 days or more depending on the organ- ism under investigation. As a rule 24 or 48 hours is the usual period of in- cubation. Owing to the rapidity of liquefaction of the medium, gelatin-con- taining media cannot be placed in a temperature of 37°C. Such a medium must be used at room temperature (about 20°C.). Fig. 169. — Incubator. V. Preparation of Cultures As previously stated, oxygen is absolutely essential for the development of all bacteria. Some organisms are capable of growth in free oxygen (atmos- pheric air), while others cannot live in such an atmosphere but obtain their oxygen from the culture media upon or in which they develop. The former bacteria are known as t3f0£e'if^MbhSM^ ^^^ ^^"'^'^ ^^'P^* ^^^ called 7o6 DIAGNOSTIC METHODS anaerobic bacteria. The majority of the pathogenic organisms belong in the aerobic division, although many important pathogens are anaerobic. A . Aerobic Organisms. In this group we find the diphtheria bacillus, tubercle bacillus, influenza bacillus, typhoid bacillus, cholera spirillum, gonococcus, meningococcus, pneumococcus, and many others. By means of the platinum needle, which is absolutely indispensable in bacteriological work, the material to be investigated is spread over the surface of the proper culture medium (in routine work agar or blood agar) and- the cotton plug replaced. These plugs do not prevent access of air but keep out dust and contaminating bacteria. Place in the incubator and allow the culture^to develop for 24 or 48 hours. Instead of the medium in the tubes, one may use liquefied agar or blood agar. Place the liquid material under investigation (such as milk or water) in a sterilized Petri dish and pour in the liquefied medium, which has cooled down to 40 or 4S°C. Fig. 170. — ^Platinum Needles. Study the macroscopic characteristics of the culture and prepare smears from the culture for microscopic examination. If the cultures are pure, that is if only one type of organism is present, sub-cultures piay be made upon special media in order absolutely to identify the organism. If several types are present, each kind of colony is transferred to other media and isolated in pure culture and then identified by subvcultures. Where simple diagnosis is desired, these sub-cultures are rarely necessary, but where a pure culture is at issue, as in the preparation of a vaccine, very careful differentiation is essential. B. Anaerobic Organisms. In this class one finds the tetanus bacillus, treponema pallidum, Vincent's spirillum, organism of anterior poliomyelitis and many others. Cultures are made as in the case of aerobic organisms. However, these anaerobic types must develop in complete absence of free oxygen. To insure the absence of oxygen four methods are available. (i) Boiling the culture media. Allow the medium to cool down to 40 to 45°C. and inoculate. This method is not at all reliable, owing to the possible access of air after boiling. (2) Displacement of the oxygen, of the air by an inert gas, usually by hy- drogen. Inoculate the culture medium as above. Place in a larger vessel provided with entrance and exit tubes and tightly sealed. Pass a stream of purified hydrogen through the vessel continuously for several hours, close the stoppers and place the apparatus in the incubator. Digitized by Microsoft® CLINICAL BACTERIOLOGY 707 (3) Absorption of the oxygen. This is the more commonly employed method. Place the culture tubes on a glass or metal support inside a larger tube or bottle. Place in the bottom of the bottle 10 grams of pyrogallol and pour on it 100 c.c. of i per cent, sodium hydrate solution. Tightly stopper the bottle and place in the incubator. ^ (4) The use of a vacuum. This method consists in exhausting, with an air pump, the air in a large vessel containing the inoculated culture media. Close the tubes to prevent reentrance of air and place in the incubator. After the air is exhausted, hydrogen may be passed into the vessel, thus giving double assurance of freedom from oxygen. —a, Fig. 171. — Anaerobic Cultivation in Hydrogen Gas (Heinemann). a, Novy jar; b, glass cock; c, gas generator; d, sulphuric acid; e, sodium hydrate solution; /, opening of Novy jar; g, stopper. After the cultures have developed they are studied and examined as men- tioned above. Sub-cultures and isolation of the different types are not so frequently essential with these anaerobic organisms, as the contamination is usually due to aerobic bacteria, which do not, of course, develop in these cultures. VI. Staining The general methods of staining bacteria depend upon the special organ- ism under investigation. As a rule Loffier's methylene blue (see p. 21) and Gram's stain (see p. 668) are the ones which give all the general informa- tion desired. Certain organisms, however, require special staining, which methods will be discussed under the specific organism. Organisms are di- vided into two groups according to their reaction to Gram's stain, so that the writer would advise the constant use of this stain in order properly to classify an organism as positive or negative to this stain. Staining of Spores Certain organisms show within their protoplasm a small bright refractile spot, known as a spore. These are very resistant and develop into vegetative organisms very rapidly. The spore is not stained readily, but when once ' The following method is very successful for routine work. Push the cotton plug into the tube a short (fistance, place dry pyrogallic acid on the upper portion of the cotton and pour on it 5 drops of 10 per cent, sodium hydrate solution. Stopper tightly with cork and seal it in with paraffin. Digitized by Microsoft® 7o8 DIAGNOSTIC METHODS Stained is very resistant to decolorization, the body of the bacillus losing its color before the spore does. In ordinary stained preparations the spore shows as an unstained spot in the body of the stained bacillus. Of course, the spore may be stained with the simple stain, if the action be prolonged or if the stain be heated, but this does not give clear differentiation.^ Holler's Method. Make a smear of the culture. Dry in air and fix in absolute alcohol for two minutes followed by chloroform for two minutes. Dry. Drop a few drops of 5 per cent, aqueous solution of chromic acid on the smear and leave for two minutes. Wash in water. Cover the smear with Ziehl's carbol fuchsin (p. 20) and warm for one minute. Wash in water and decolorize in 5 per cent, sulphuric acid for a few seconds, completing the decolorization, if necessary, with absolute alcohol. Wash in water. Stain with methylene blue for one- half minute. Wash in water, dry and examine. The spores are stained red and the body of the organism is blue. Staining of Capsules. Many methods have been advocated for this purpose, but the writer has found the method of Rosenow (see p. 590) the most satisfactory, giving beautiful pictures and uniform results. Staining of Flagella. Van Ermengem's Method. Place the film for five minutes at so°C. (or 30 minutes at room tempera- ture) in the following freshly prepared solution. 20 per cent, aqueous solution of tannic acid, 60 c.c. 2 per cent, solution of osmic acid, ' 30 c.c. Glacial acetic acid, 4 or 5 drops. Wash in water and then in absolute alcohol. Cover the smear with ^2 per cent, aqueous solution of silver nitrate for a few seconds and, without washing, transfer the slide to the reducing bath for a few seconds. This reducing bath is as follows: Gallic acid, 5 grams. Tannic acid, 3 grams. Fused sodium acetate, 10 grams. ■Distilled water, 350 c.c. Without washing, place the slide in the 3^^ per cent, silver nitrate solution and move the liquid back and forth over the smear until this becomes black. Wash in water, dry and examine. Successful preparations by this method, or any other, depend upon the obtaining of thin speciniens. Take a small amount of a young agar culture of the organism and make a homogeneous emulsion in a watch glass with dis- tilled water. Place a drop or two of this emulsion on a perfectly clean glass slide and allow the drop to spread out. Dry in air and stain as above. ' See Huntoon, Jour. Am. Med. Assn., 1914, LXII, 1397. Digitized by Microsoft® CLINICAL BACTERIOLOGY 709 VII. Identipication of Organisms After cultures have been prepared as above described and have been incu- bated for 24 to 48 hours, the diagnosis of a given infection may be possible by direct examination of the cultures and stained slides made therefrom.' This is true in cases in which search is being made for a specific organism without any reference to the associated bacteria. However, in the closer study of cul- tures, which is required in many cases, especially in the preparation of autoge- nous vaccines, sub-cultures on special media must be made in order properly to purify the culture and to permit of differentiation and recognition of the organism causing the infection. For this reason the writer deems it advisable to discuss briefly the cultural characteristics on special media and the means 'of absolute identification of some of the more important pathogenic bacteria.^ I. Diphtheria Bacillus ^ebs-Loffler Bacillus). The medium best adapted for the development of the diphtheria bacillus is Loffler's blood serum.' By means of a stout platinum needle or a sterile swab remove a portion of the suspicious material or membrane from the nose, throat or tonsils and spread this over the surface of the above culture medium. If the material is to be sent to a laboratory and no culture medium be at hand place the swab in a sterile test-tube or homeopathic vial and plug this with cotton. It is very essential that the proper material be obtained for these cultures or otherwise negative results will obtain. If necessary tear away the membrane in order to be certain of your material. Place the culture in the incubator at 37°C. for six to eight hours and ex- amine. The cultures should rarely be examined after more than 20 hours to insure the best results. The growth appears as a mass of grayish- white points or colonies varying irj size from a small to large pin-head. They are somewhat more opaque in their center than at their margin, which may be tinged a light yellow. The longer the period of growth, the larger the colonies, the more irregular their borders (early cultures usually show a regular margin) and the greater the tendency for the colonies to show radial striations. These colonies on blood serum always remain discrete and do not flow together. Smears made from such cultures are stained with methylene blue and with Neisser's or Gram's stain as given on page 39. Such smears show the organ- isms with the characteristic morphology and staining peculiarities given there. It is to be remembered that such cultures and smears may show no diphtheria bacilli, although the case is clinically one of diphtheria. This may be due to 1 It is important to remember that transmutations may appear within the groups pf bac- teria so that pneumococci, for instance, may change into true streptococci by variations in the culture medium, by changing the oxygen tension within the culture tube, etc. See Thiele and Embleton, Ztschr. f. Immunitatsforsch. und exper. Therap., 1913, XIX, 643; Rosenow, Jour. Infect. Dis., 1914, XIV, i; Jordan, Proc. Nat. Acad. Sc, 1915, I, 160. * For a diflferential method of study of various bacteria by the addition of various aniline dyes to the culture medium see Churchman, Jour. Exper. Med., 1912, XVI, 221; Ibid., 1913, XVII, 373; Churchman and Michael, Ibid., 1912, XVI, 822; Krumwiede and Pratt, Ibid., 1914, XIX, 20 and 501; Simon and Wood, Am. Jour. Med. Sc, 1914, CXLVII, 524- ' See Drigalski and BierastJDeutsch. med.-WchnscJK-i 1913. XXXIX, 1237) who advise the addition of ox bile to thi^^M? 0/ M/CrOSOff® 710 DIAGNOSTIC METHODS the taking of improper material for the culture, to the fact that antiseptic solu- tions have been used prior to the time of taking the culture or to the fact that too long a period of incubation has been used. Under the latter condition other organisms will so far outgrow the diphtheria bacilli that the latter are unrecognizable. For these reasons it must be insisted upon that a negative bacteriologic examination should never be regarded as excluding diphtheria. In some very urgent cases recourse is made to the examination of direct smears from the swab without preliminary cultivation. This method rarely, if ever, gives results which may be relied upon but it occasionally does. It would seem to the writer that cultures should be insisted upon in all cases, as the time necessary for obtaining a growth is so slight that one is warranted in waiting for a more definite diagnosis. All suspicious throats, especially in children, should be regarded as diphtheritic until proven to be otherwise by several cultures. This precaution is best both for the patient and for his associates. See Schick's immunity test (p. 605). It not infrequently happens that certain organisms, such as Hofmann's bacillus (pseudo-diphtheria bacillus) and the xerosis bacillus, complicate the diagnosis, as these organisms resemble the diphtheria bacillus in many ways. Cultures of Hofmann's bacillus, grown on LofHer's medium, show smaller and whiter colonies than those of the diphtheria bacillus. The growth of the former is much more rapid and there is not the same tendency for the colonies to take up the pigment of the medium and become yellowish in color. If Hof- mann's bacillus be grown for 48 hours in glucose broth to which litmus has been added, no acid is produced; while the diphtheria bacillus readily pro- duces acid (red coloration of medium). This fact has been mentioned on page 40, whereon are given the different sugars upon which these conflicting organisms act with production of acid but no gas formation. This fermen- tative test is of great value in differentiation. Further, the morphology of Hofmann's bacillus is not the same as that of the Klebs-LofHer bacillus, although at times the two may become confusing. The former is shorter than the latter (although the latter occasionally shows a short form) ; the former is oval, stains deeply throughout except for one un- stained portion and shows no granules. Occasionally on^ may observe a few beaded forms but these are rare. The more or less regular morphology of the pseudo-diphtheria bacillus is quite in contrast to the pleomorphic type of the diphtheria bacillus. Negri and Mieremet' and Bunting and Yates^ report the successful cultiva- tion of a pleomorphic diphtheroid organism in cases of Hodgkin's disease, 1 Centralbl. f. Bakteriol., 1913, LXVIII, 292. '' Arch. Int. Med., 1913, XII, 236. See, also, Steele, Boston Med. and Surg. Jour., 1914, CLXX, 123; Billings and Rosenow, Jour. Am. Med. Assn., 1913, LXI, 2122; Kusunoki, Virchow's Arch., 1914, CCXV, 184; Yates, Bunting and Kristjanson, Jour. Am. Med. Assn., 1914, LXIII, 2225; Verploegh, Kehrer and van Hoogenhuyze, Munch, med. Wchnschr., 1914 LXI, 1158; Mellon, Am. Jour. Med. Sc, 1915, CL, 245; Harris and Wade, Jour. Exper. Med., 1915, XXI, 493; Fox, Jour. Med. Research, 1915, XXXII, 309 and 325; Arch. Int. Med., 191S, XVI, 46s; Rhea and Falconer, Ibid., 1915, XV, 438; Bloomfield, Ibid., 1915, XVI, 197; Bunting, Bull. Johns Hopkins Hosp., 1915, XXVI, 179; Olitsky, Jour. Am. Med. Assn., 1915, LXIV, 1134; Yates and Bunting, Ibid., 1953; Hatcher and Lemmon, Ibid., 1915, Lxv, I3S9- Digitized by Microsoft® CLINICAL BACTERIOLOGY 711 which organism had been previously demonstrated in the tissues by Frankel and Much. The former workers used Bordet's potato medium and call the organism isolated the Corynebacterium granulomatis maligni, while the latter investigators employed Dorset's egg medium and glycerin-phosphate-agar and suggest the name of Corynebacterium Hodgkini {Bacillus hodgkini). Rosenow has reported the growth of these organisms on Loffler's blood serum and blood agar. The morphology of this organism is very pleomorphic: thus plump short rods, some so short as to resemble cocco-bacilli; small thin bacilli with polar staining; comma-shaped bacilli; granular rods of variable size; branching forms; club-shaped involution forms and large spherical types. The organism is Gram-positive, is non-acid fast and does not form spores. 2. Influenza Bacillus (Pfeifier's Bacillus). This organism is found most frequently in the sputum and nasal dis- charges of those affected with influenza. It is often recognizable in the routine sputum examinations, but it is often overlooked owing to its small size. As hemoglobin is essential for the development of this organism, blood agar is the best medium upon which to cultivate it. In obtaining the speci- men for culture work, select a solid portion of the sputum and wash it several times with sterile distilled water to remove the excess of mucus. Or, if de- sired, make an emulsion of the sputum in sterile water. Take up the solid part of the washed sputum and rub it over the surface of a blood-agar plate in a Petri dish. Incubate at 37°C. for 24 or 48 hours. Macroscopic ex- amination shows a number of small delicate dew-drop Uke colonies, which never become confluent. This organism is rarely found in pure cultures, the ancillary types being pneumococci, streptococci and staphylococci. Other types of colonies will, therefore, be seen on the plates. Select a colony having the characteristics above mentioned and prepare smears as usual. The morphology and staining properties are as given on page 26. 3. Pertussis Bacillus (Bordet and Gengou's Bacillus). The best medium for the cultivation of this organism is the potato medium of Bordet and Gengou, which is prepared as follows. To 200 c.c. of a 4 per cent, aqueous solution of glycerin add 100 grams of finely chopped potato. Heat in the autoclave for five to ten minutes. The supernatant fluid is now a concentrated glycerin extract oLpotato. To 50 c.c. of this extract add 150 c.c. of a 0.6 per cent, sodium chlorid solution and 5 grams of agar. Heat this mixture in the autoclave and, while hot, filter it into test-tubes, adding 2 to 3 c.c. to each tube. Sterilize in the autoclave. Allow the medium to cool down to 40 to 4S°C. and add an equal amount of sterile defibrinated human or rabbit blood. Mix thoroughly and prepare slants of the mixture. This medium is especially valuable, also, in cultivating the influenza' bacillus, gonococcus and meningococcus. This bacillus should be looked for in the early stages of the disease when coughing begins. In the later stages is is not so easily found owing to the large number of other bacteria. Select a solid piece of the sputum and wash in sterile distilled water. D/gj^raad tt>o*Se/tlliSC^ftSace of a Petri plate prepared 712 DIAGNOSTIC METHODS from the above medium. These plates are more satisfactory for the bacilli of pertussis and influenza than are the slants, as these organisms require much oxygen. The primary cultures are difficult to obtain, as the growth is so scanty during the first 24 hours, and often in 48 hours, that little is visible. Sub-cultures made from this initial growth give a more copious development. On the third day the colonies appear white, slightly elevated and sharply outlined. From generation to generation the growth becomes quicker and more luxuriant. The morphology and staining characteristics of this organism have been given on page 26. So close is the similarity of the influenza and the pertussis bacilli that it seems advisable to differentiate these organisms. The growth of the former is quicker and less luxuriant than that of the latter. The colonies of influenza bacilli are slightly bluish and transparent, while those of the pertussis organism are whiter and thicker. If stroke cultures are made upon slanted potato-blood-agar, the growth of the pertussis bacfllus is thicker than that of the influenza bacillus; its border is abrupt while that of the influenza bacillus is more spread out. The growth of the influenza bacillus is always trans- parent, shows an irregular border and has a moist appearance. Further, the pertussis bacillus hemolyzes the blood medium, so that the line of culture ap- pears clearer than the surrounding. The influenza bacillus does not cause hemolysis. Mallory, Horner and Henderson (Jour. Med. Research, 1913, XXVII, 391) have shown the etiologic relationship of this' organism to pertussis, finding it in the characteristic lesions between the cilia of the epithelial cells lining the trachea and bronchi. 4. Typhoid Bacillus (Eberth-Gaffky Bacillus). The characteristic cultural properties of this organism have been discussed on pages 130 and 587, while its agglutination reactions are given on page 607. The isolation and detection of the typhoid bacillus in the various excretions and secretions of the body is a matter of considerable difficulty, often calling upon all the resources at the command of the worker. Study of the cultural characteristics, of the agglutination reactions and the staining properties of this organism will readily differentiate it from the colon and associated bacteria, but careful work is necessary. It is questionable whether the ty- phoid bacillus has been isolated from water and milk supplies in a pure state, but this is not at all necessary as the detection of the colon bacillus, which is more easily identified, is all that is necessary to establish pollution. The typhoid bacillus may be found in the dejecta of convalescent and recovered patients for some time, so that careful measures should be adopted to prevent spread of disease through these carriers. '^ 5. Colon Bacillus (Bacillus Coli Communis). This organism has been discussed on page 589 and its cultural character- istics and points of differentiation given. The colon bacillus is coming into great prominence, owing to its discovery in so many of the secretions and ex- ^ See Jordan, Jour. Am. Med. Assn., 1914, LXII, 1772; Hirschbruch, Berl. klin. Wchn- schr., 1914, LI, 1176. Digitized by Microsoft® CLINICAL BACTERIOLOGY 7 13 cretions of the body under pathological conditions. ^ The use of vaccines made from cultures of colon bacilli is varied and the results usually excellent. In the bacteriological examination of milk and water, the detection of the colon bacillus is of especial importance as an indicator of sewage contamina- tion. In times past the custom has been to use, for this work, litmus-lactose agar for the plate cultures and glucose broth for the study of the fermentative properties of the organisms in the water. It is well known that both water and milk, as well as other fluids, contain acid- and gas-producing organisms which are not colon bacilli, so that one is not justified in calling all such bacteria colon bacilli. In order to ehminate these extraneous gas- and acid- producing bacteria, the use of bile has been advocated. The addition of bile to the usual media inhibits the other organisms but was not supposed to affect the colon bacilli. The media used in this later study and now adopted for all sanitary examinations o"f water and milk supplies are the lactose bile medium, consisting of i per cent, lactose dissolved in ox bile and sterilized, and lactose-bile-agar. • The bile agar is prepared as follows. In 1,000 c.c. of fresh neutral ox bile dissolve 15 grams of agar, 10 grams of Witte's peptone and 10 grams of lactose, boiling as little as possible. When dissolved, filter the medium without titration, place in tubes and sterilize in the autoclave for three minutes. Rector^ has advocated a slight modification of this medium, which he claims is much better. His formula. is as follows: Dried ox bile, 100 grams Witte's peptone, 10 grams Agar, 15 grams Lactose, 10 grams Neutral red (i per cent, solution), 10 c.c. Distilled water, 1000 c.c. While it is unquestionably true that this bile medium has an advantage over non-bile media in that interfering organisms are eliminated and, there- fore, the acid- and gas-producing organisms may more certainly be classified as colon bacilli, yet Jordan' has shown that this medium exert an inhibiting effect on colon bacilli themselves to the extent of 33 }'s to 50 per cent, of the viable colon bacilli present in the material under investigation. Such being the case much more care must be exercised in reporting upon a specimen of water or milk, especially as to numbers of colon bacilli and as to the safety of such specimens for general use. 6. Dysentery Bacillus (Shiga-Kruse BaciUus). In bacillary dysentery certain organisms are found which closely resemble the typhoid and colon bacilli in -morphology but which differ in certain cul- tural characteristics from the latter bacteria. In the course of an epidemic of 'See Conradi and Bierast, Kolle and Wassermann's Handbuch, 1913, VI, 483; also Bornand, Centralbl. f. BakterioL, Abt. 2, 1913, XXXVIII, 516; Kligler, Jour. Infect. Dis., 1914, XV, 205; Browne, Ibid., 580; Rogers, Clark and Davis, Ibid., 1914, XVI, 411; Clark andLubs, Ibid., 1915, XVII, 160. ' Am. Jour. Pub. Health, 1913, III, IS4. ' Jour. Infect. Dis., 1913, 1913, III, IS4- Wlgitrnd by Microsoft® 714 DIAGNOSTIC METHODS dysentery in Japan, Shiga discovered this bacillus, but it remained for Kruse to elaborate and fix its characteristics. Since that time, similar organisms have been found in various epidemics throughout the world, but in many cases certain differences were observed in the behavior of the organisms to- ward different sugar-containing media. On the basis of fermentative tests with acid production in sugar-broths, and, also, upon variability in the agglu- tination reactions of the organisms isolated, at least four distinct types of the dysentery bacillus must be considered as established. I. Shiga-Kruse type. II. Flexner-Strong type. III. Hiss-Russell, El Tor I strain, "Y" type. IV. Harris- WoUstein type. The following table, taken from Jordan,^ will show at a glance the varia- tions in the action of these similar organisms! Bacillus dysenteriae Acid production from: Indol Mannits Maltoss Saccharose Dextrose duction + + + + -f- -1- -1- -1- -1- -t- Type II (Flexner-Strong). Type III (Hiss-Russell) Type IV (Harris-Woilstein) -1- + + The detection of these organisms in the stool is not a matter of great difficulty, by cultural methods, if the case is examined relatively early in its progress. However, after some time the examination becomes more and more uncertain until the organisms are rarely found after the ithird week. The material for the preparation of cultures is obtained from a freshly passed stool. Flecks of mucoid material are selected and placed in sterile water or physiologic salt solution, in order to remove the colon bacilli with which the material is contaminated. This washing is repeated three or four times, fresh water being, of course, added each time. If there be no visible mucus in the stool, emulsify a portion of the stool in nutrient broth. The washed mucoid particles, or a portion of the fecal emulsion, are now rubbed over the surface of three or four litmus-lactose agar plates or of plates made of Drigal- ski and Conradi's medium (see p. 130) without the addition of the crystal violet. Allow these plates to dry in the air by keeping the cover slightly open for one-half hour, place them in the incubator at 37°C. with the bottom side up and examine at the end of 24 hours. Any colon bacilli left after the washing, and there are always some, will appear as red colonies with a gradual shading of color into the blue medium. The Shiga bacillus does not change the color of the medium, its colonies appearing as translucent, iridescent, ' General Bacteriology, Philadelphia, 1913. See, also, Lentz, KoUe and Wassermann's Handb., 1913, III, 927; Barber, Philippine Jour. Sc, 1913, VIII (B), 539; Bluhdorn, Deutsch. med. Wchnschr., 1914, XL, 782; Monatsschr. f. Kinderhkde., 1914, XIII, Orig., 37; Schild, Ibid., 51; Musgrave and Sison, Philippine Jour. Sc, 1914, IX (B), 241; Flexner and Amoss, Jour. Exper. Med., 1915, xx^j^^^ed by Microsoft® CLINICAL BACTERIOLOGY 715 dew-drop like growths with irregular margins and centers more opaque than the edges. This growth is very similar to that of the typhoid bacillus on this medium. Test one of the latter colonies for motility of the organisms by the hanging drop method and, also, test its agglutination with a potent dysentery serum. If these tests are satisfactory, namely, if no motility is observed and agglutination is definite, sub-cultures of these colonies are made upon slant agar, neutral red-glucose-agar (glucose agar to which three or four drops of a saturated aqueous solution of neutral red are added), litmus- mUk and plain milk. The cultures on agar and neutral red-glucose-agar show the same characteristics as do those of the typhoid bacillus, but the lack of motility excludes this latter organism. These cultures have a peculiar sperm-like odor. On litmus-mannite-agar the Shiga bacillus produces no acid, while the typhoid bacillus develops a distinct red color. Litmus-milk is not changed in color, nor is plain milk coagulated. It will be seen, therefore, that the Shiga bacillus differs from the typhoid bacillus in its action toward litmus-mannite-agar and, especially, in its lack of motility and its different agglutination reactions. From the colon bacillus, the cultural characteristics differentiate it sharply. The morphology and staining properties of this organism have been given on page 132. It is Gram-negative^ does not form spores, nor does it possess flagella. In smear preparations, the dysentery bacillus resembles the typhoid bacillus, but the former is thick and often polymorphous. Culturally the other types of dysentery bacilli are differentiated from the Shiga bacillus by the fact that their cultures on litmus-lactose-agar are more irregular and have a reddish- violet tinge, while the latter organism shows colo- nies of a translucent dew-drop like appearance. Further, the Shiga bacillus does not produce indol, while all the other types, as well as the colon bacillus, do. In confirming this differential diagnosis, the agglutination tests are valuable. The Shiga bacillus should agglutinate in a dilution of i to 20 of the patient's serum, while the Flexner type agglutinates at a dilution of i to 80. For differentiation of the other types, the fermentative tests must be utilized, as agglutination is' not typical. 7. Cholera Vibrio (Koch's Comma Bacillus). "this organism" has been briefly discussed on page 129. It appears in stained preparations as a small, short slightly curved rod, which has led to the name of "comma" bacillus. It is about 2 m long and 0.5 ^ broad. It is not infrequent to observe long, straight involution forms, especially from liquefied gelatin cultures. This bacillus has a single flagellum at one end and shows very active motility. It stains easily with the ordinary stains and is Gram- negative. The spirillum of cholera grows well on practically all media at room tem- perature, with the exception of potato. The medium must be alkaline in re- action to insure the growth. This alkalinity is obtained by adding 3 c.c. of 10 per cent, sodium hydrate to 100 c.c. of neutral medium. The growth on gelatin plates at room temperature is more or less char- acteristic. These colonifi''a^^,^#'^ipM'§, as round and regular white 7l6 DIAGNOSTIC METHODS points, which later become irregular and show a granular appearance as if the colony were covered with ground glass. Within 48 hours liquefaction begins, the colony sinking into the depression. On gelatin stab-cultures, the lique- faction begins as a cup-shaped depression at the surface, a bubble of air being retained in the depression. The later liquefaction takes the form of a funnel, being much broader at the surface. Later the medium is entirely liquefied. Some of the vibrios resembling the cholera bacillus, as especially the Finkler- Prior vibrio, produce a more rapid liquefaction of the gelatin and do not show the bubble of air which is practically always present in the cholera cultures. This point is not, however, distinctive enough for strict diagnostic purposes. It is to be remembered, in this connection, that the other types of organisms found in the intestinal canal do not liquefy gelatin media. On agar plates the colonies appear as pale, flat, thin growths which are transparent and show, by transmitted light, a peculiar opalescence. As the cholera spirillum is a strongly aerobic organism, we find a copious surface growth in i per cent, peptone solution containing J^ per cent, sodium chlorid. Other organisms do not develop readily in this medium, so that we not in- frequently may obtain an almost pure culture of this simple medium. The addition of a few drops of concentrated sulphuric acid to such a culture pro- duces a burgundy-red color, due to the formation of nitroso-indol. Thus the cholera spirillum produces indol and, at the same time, reduces the nitrates of the culture medium to nitrites. While the colon bacillus produces indol, it is necessary to add a trace of nitrite before the nitroso-indol reaction is obtained. This cholera-red reaction, as it is called, was supposed to be distinctive for the cholera vibrio but it has been shown that other cholera-like organisms also react. A negative result with this test is, therefore, of greater value than a positive one. In the bacteriological diagnosis of cholera, several points must be borne in mind. Select some of the " rice particles " and inoculate a peptone water cul- ture as above, or plant i c.c. of fecal material in 50 c.c. of this medium. From this surface growth, at the end of six to eight hours at 37°C., sub-cultures are made upon other media and smears are examined and studied for the char- acteristic organisms. Glycerin and agar plates are inoculated and incubated for 24 to 48 hours, the former at room temperature and the latter in the incu- bator. It should be a rule to inoculate Dieudonne's alkali-hlood-agar at the same time. This medium is prepared by mixing equal parts of defibrinated beef blood and normal (4 per cent.) sodium hydrate; heat in autoclave for one-half to three-fourths hour. Add 30 parts of this mixture, while still hot, to 70 parts of neutral 3 per cent. agar. Mix and sterilize in the autoclave. When cooled, pour into Petri plates. Allow the plates to dry by standing open in incubator for several hours and do not use these plates earlier than 24 hours after preparing. Colon bacilli and other extraneous organisms do not grow on this medium, while the cholera organism develops rapidly. The colonies are large, circular, crystal-clear by transmitted light and by reflected light gray. Microscopic examination of the organisms from these colonies show many degenerative /BjgfftepsfdiBy MiGBl9SffliS5jg>typical comma forms. CLINICAL BACTERIOLOGY 717 After these cultures are prepared test the motility of the organism in hang- ing drop and follow this by the agglutinating reactions and, especially, by the application oi Pfeiffer's bacteriolytic test. This latter test is as follows: An emulsion of the organisms in question is injected into the peritoneal cavity of a guinea-pig, which has been immunized against a definite species of the vibrio. In 20 to 30 minutes remove, with a trocar, some of the peritoneal fluid and examine under the microscope. The organisms present will be non- motile and appear as more or less granular spherical masses instead of the typical forms, if the reaction is positive; if negative, the organisms will appear as in the usualy preparations.^ 8. Gram-positive Cocci. A. Pneumococcus (Diplococcus of Fraenkel; Streptococcus Pneumoniae). This organism is practically always found in the normal throat, so that its presence therein does not indicate a pathologic process. It is found, also, as the primary etiologic factor, in a wide range of infectious processes, so that it must not be regarded as limited to pneumonic types of infection. The mor- phology and staining characteristics of this organism have been given on page 25, while its cultural peculiarities, especially, have been discussed on page 589. It is to be remembered that, from the morphological standpoint, it is often- times impossible to distinguish the pneumococcus from the streptococcus, as the former not infrequently appears in chains, and the typical lanceolate diplococci give place to forms which are more nearly true coccoid types. Cultural methods must, therefore, be employed and the special peculi- arities of the pneumococcus and streptococcus differentiated by the use of the blood-agar plates, the blood bouillon tubes, the use of bile acids, and the fermentative test with inulin as outlined on page 590. The agglutination test should, also, be employed with specific immune serum in all cases of doubtful diagnosis. 2 It is to be remembered that there are distinct varieties of pneumococci, which are distinguishable from one another only by careful study of the cul- tural peculiarities as well as by observation of their effects on animals. Tran- sition forms from the pneumococcus to the streptococcus and vice versa are observed under certain cultural conditions, so that it is often a more than difii- cult problem properly to classify these organisms. One of these organisms, the streptococcus mucosus capsulatus, is probably to be considered with the true pneumococci. It occurs as round, or very slightly lance-shaped cocci, which are encapsulated, the capsule being wider than that of the pneumo- coccus. The organisms usually appear in pairs, but there are always seen a few in chains of four to eight organisms. The colonies on blood agar have a mucoid slimy consistency and have a greater tendency to become confluent than do those of the true pneumococcus. ■■ See "The Bacteriological Diagnosis of Cholera," Pub. Health Rep., 1912, XXVII; 371; also, KoUe and Schiirmann, Kolle and Wassermann's Handb., 1912, IV, i; Kabeshima, Centralbl. f. BakterioL, Abt. i, 1913, LXX, 202; Craster, Jour. Exper. Med., 1914, XIX, 581. " See Dochez and Gillespie, Jour. Am. Med. Assn., 1913, LXI, 727; also, Floyd and Wolbach, Jour. Med. Research, 1913, XXIX, 493; Dochez and Avery, Jour. Exper. Med., 191S, XXI, 114; Lyaii, ibid.,£^Y/zecf by Microsoft® 7l8 DIAGNOSTIC METHODS B. streptococci. There are several varieties of streptococci, which are not readily distin- guished by their morphology but which cultural peculiarities usually suflSce to identify. These cultural points have been discussed on pages 590 and 591. It is to be said, however, that this differentiation is not a simple proposition, as these varieties readily change, the one into the other and, also, into forms which are hardly distinguishable from the typical pneumococcus. The chief types of the streptococcus are a. Streptococcus pyogenes (hemolyticus) , a long-chained form which pro- duces no green coloration on blood agar but shows a large colorless hemolytic area about its colonies. b. Streptococcus viridans, a short-chain type producing green colonies on blood agar but only slight hemolysis. c. Streptococcus mucosus, an encapsulated organism discussed above, pro- ducing no green colonies nor any hemolysis. Many other types have been isolated in pathological conditions and their exact classification is rather difl&cult. Usually the organisms have shown characteristics which place them in classes intermediate between those given above and even have indicated their close relationship to the pneumococcus. One of these virulent organisms has been discussed on page 40, being called the streptococcus epidemicus and causing streptococcic sore-throat. In these later days much work has been done on this group of organisms and niuch of value has been forthcoming. Some of this work is so important that the writer must discuss it briefly, in order properly to correlate the sub- ject of streptococci to various pathological processes. Davis' has called attention to the close association of streptococcic infec- tions to chronic arthritic troubles and finds that the atrium of infection in these arthritides is usually the tonsil or peritonsillar structures. As is dem- onstrated the streptococcus hemolyticus, the diplococcus (streptococcus) rheumaticus and the streptococcus epidemicus (see p. 40) are very prone to attack the joints; while the streptococcus viridans rarely does so. Such cases can be separated by the clinical findings and history of tonsillitis in the pa- tients from the other types of deforming arthritides. The bacteriologic ex- amination of cultures from the tonsil clears up the etiology. In taking the cultures from the tonsil, the material must be obtained from the depths of the crypts after the tonsil has been incised. In such cultures the streptococ- cus hemolyticus is usually found. The streptococcus viridans is found in surface cultures, but this is disregarded as it is less virulent and is rarely associated with arthritic involvements. It is to be recalled that the strepto- coccus hemolyticus may be found on the surface of the tonsils in ordinary streptococcic tonsillitis, and even on normal tonsils, but these do not neces- sarily have any special general value in the absence of clinical history of joint troubles. In the treatment of these cases of arthritis due to streptococcic infection, proper vaccines often work like magic. Two hundred million of ' Jour. Infect. Dis., 191 2, X, 148; Jour. Am. Med. Assn., 1913, LXI, 724. See, also, Richards, Ibid., 1914, LXII.^j^^^Jyign, ^^;.(^)^nd BacterioL, 1915, XIX, 478- CLINICAL BACTERIOLOGY 719 the killed organisms are given at a dose with five-day intervals between the administrations. Rosenow^ in his work on the pneumococcus and streptococcus has isolated certain organisms from infected joints, blood cultures and tonsillar infections in cases of acute articular rheumatism and finds organisms corresponding to the micrococcus rheumaticus of Poynton and Payne. He finds, however, that this organism resembles very closely the streptococci, its virulence being less than that of the streptococcus hemolyticus and greater than that of the streptococcus viridans. By animal passage and other means three of the strains isolated from acute rheumatism have been converted into typical hemolytic streptococci on the one hand and into pneumococci on the other. The cultures made from the involved joints and regional lymph nodes have revealed three types of streptococci. One produces green colonies on blood agar and forms long chains; a second produces a narrow zone of hemolysis and forms short chains; while the third forms a grayish-brown colony with- out affecting perceptibly the blood in the medium. This latter organism appears as a diplococcus, in short chains and as single cocci. The most distinctive cultural feature of these types is their production of a very high acid reaction in dextrose-broth and abundant growth at low temperatures. Progressive transmutation from a streptococcic type to a pneumococcus is easily shown. Billings,^ on the basis of this work of Davis and Rosenow, has studied the clinical aspects of arthritic cases and finds that a chronic focal infection is as- sociated with many of these arthritides. Clinically, he finds the focal dis- ease in the faucial tonsil, dental alveoli or jaw, antra of head, etc., in a large number of cases. In other cases chronic colon, gonorrheal or streptococcic infection of the seminal vesicles and prostate were found to be the focal point of the arthritic involvement. Cultures were prepared from the material obtained from the focal infection, sub-cultures made and the dominant organ- ism isolated in pure culture. From these cultures vaccines were prepared and used with excellent results. Billings is especially emphatic upon the point of using the auto-vaccine from the dominant isolated organism and believes that only by this method may good result from vaccine treatment. C. Staphylococcus Pyogenes. These organisms grow well on all types of media. Their cultures on agar are best adapted to the development of the pigment, which is the character- istic differentiating point of members of- this species. Cultivation at room temperature frequently brings out the pigment better than growth in the incubator. These organisms are readily recognized, in stained preparations, 'Jour. Am. Med. Assn., 1913, LX, 1233; Jour. Infect. Dis., 1914, XIV, i and 61; Rosenow, New York Med. Jour., 1914, XCIX, 270; Jour. Am. Med. Assn., 1914, LXII, 1146; Ibid., i9i4,LXIII, 903; Davis, 111. Med. Jour., 1914, XXVI, 158; Billings, Ibid., 164; Jour. Am. Med. Assn., i9i4,'LXIII, 899; Smith and Brown, Jour. Med. Research, 1914, XXXI, 4ss; Hopkins and Lang, Jour. Infect. Dis., 1914, XV, 63; Thro, Ibid., 234; Holman, Ibid., 293; Crabtree, Ibid., 309; Davis, Ibid., 378; Rosenow, Ibid., 1915, XVI, 367; Thro., Ibid., 1915, XVII, 227; Broadhurst, Ibid., 277; Brawley, 111. Med. Jour., 1915, XXVIII, '' lArch. Int. Med., 191., %g^fi^^yj^c}S%^6M''' '^''' ^^^' ^''- 720 DIAGNOSTIC METHODS by their morphology. They appear as distinct cocci, five to' fifty in a cluster often compared to a bunch of grapes. Rarely do they occur singly or in pairs. Occasionally short chains may be observed, but most of them are clumped. • In purulent material these organisms may be both intra- and extracellular, but their staining characteristics readily distinguish them from the Gram- negative types of organism. 9. Gram-negative Cocci. A. Gonococcus (Diplococcus of Neisser). The morphological and staining characteristics of the gonococcus have been discussed on page 668. It is to be emphasized that this organism can- not, in every case, be distinguished by its morphology, staining characteristics and intracellular position from other organisms, especially from the meningo- coccus, micrococcus catarrhalis and a few saprophytic diplococci, such as diplococci flavus, crassus and cinereus.^ In judging of the possibility of a certain organism being the gonococcus, the origin of the culture must be known and something of the clinical history must be given. Further, it is to be recalled that the intracellular grouping of the organisms is liot especially limited to the gonococcus nor is it always present in every gonorrheal case. Such arrangement is given both by the meningococcus and micrococcus catarrhalis. In the early cases of gonorrhea, when pus cells are very few in number, the gonococci are for the most part extracellular, some of them lying upon the epithelial cells, but practically none being found within the pus cells. As the pus cells increase in number, the intracellular types become predomi- nant owing to the active phagocytosis. In the later stages and in the chronic forms of infection, the extracellular types become more frequent, very few intracellular forms being observed. As the gonococcus does not grow readily, if at all, on the ordinary culture media, special media is necessary. Albumin, preferably of human origin, is ■essential in any medium to be used. The best medium is Wertheim's serum agar prepared by adding i part of human serum to 2 to 3 parts of nutrient agar. The serum is warmed to 40°C. and added to the liquefied agar of the same temperature. Instead of the serum, ascitic or hydrocele fluid may be substituted as suggested by Kiefer and Steinschneider. In fact, ascitic agar is 'probably more frequently used than is the serum agar. Blood agar also affords an excellent medium for the development of the gonococcus. On the above media, colonies of the gonococcus appear in 24 hours as gray- ish-white points, which merge later into growths of a grayish-blue tinge with slight iridescence. These colonies have a slimy consistency, are round but soon become confluent. The gonococcus is very sensitive to drying, so that the culture medium must have plenty of water of condensation. It survives exposure to air for only a short time, although in masses of dried pus it may live, exceptionally, for six or seven weeks. In favorable culture media it rarely maintains its vitality more than 48 to 72 hours at room temperature but will live longer if kept in a refrigerator (Jordan). The gonococcus is bacteriologically identified by its characteristic morphol- 1 See Kligler, Jour. ExpeiE);^fl^e»??yMik^/?® CLINICAL BACTERIOLOGY 72 1 ogy, its Gram-negative staining, its frequent intracellular position and by its ready growth on blood or ascitic agar and its lack of growth on ordinary media. These characteristics are, however, not sufficient to differentiate it from the closely related organisms, which will be discussed below. It is to be said that a diagnosis of a gonorrheal infection outside of the genital tract is never safe if based entirely on microscopic evidence, and the cultural evidence is not always clear. The micrococcus catarrhalis is not infrequently the etiologic factor in a genito-urinary infection as well as in other parts of the system, while the meningococcus very often causes certain genito-urinary complications, which may be associated with the presence of this latter organism in this tract (see Koch^). Further, in chronic cases of true gonorrhea, it is very frequently a difficult matter to detect the gonococcus either microscopically or culturally. The organisms found may be Gram- variable and few will be intracellular, so that great care must be exercised in diagnosis. In such cases one should use the provocative test of administering some irritant or allowing the patient to partake freely of alcohol. Such a procedure followed by milking the pros- tate will usually result in the appearance of typical gonococci in the purulent material expressed. Of course in these cases one may use the complement- fixation test or the vaccination test as suggested by Irons, and later, by Fron- stein (see p. 604). From the clinical point it may tax the resources of the physician to estab- lish a diagnosis of gonorrhea in any given case, outside of those cases of acute gonorrhea in which the history and symptomatology are clear. However, in cases of meningitis with genito-urinary complications or of general systemic gonorrheal infection with meningeal symptoms, the diagnosis is not so simple, as a differentiation must be established between the meningococcus and gonococcus. In this differentiation, cultivate the material obtained from the urethral discharge or from lumbar puncture upon faintly alkaline ascitic agar for 24 to 48 hours. The meningococcus grows more luxuriantly than the gonococcus. The colonies of the former are larger (2 to 3 mm. in diameter) than those of the latter (i to 1.5 mm.). On microscopic examination of these colonies with the low power, the colonies of the meningococcus are found to be flat, homo- geneous, usually round, very slightly irregular border but never notched; while those of the gonococcus are irregular, border usually notched and center always elevated. On cultivating these organisms on litmus-ascitic-agar media containing various sugars, the gonococcus will show acid production in the dextrose media only; while the meningococcus produces acid in both dex- trose and maltose, but in no others. The micrococcus catarrhalis, with which the gonococcus may be often con- fused, grows well on ordinary media. It does not produce acid in ^.ny sugar- containing medium. Its morphology, staining characteristics and intra- cellular position are, however, exactly like those of the gonococcus. B. Meningococcus (Diplococcus Intracellularis Meningitidis). The morphological and staining characteristics of this organism of 1 KoUe and Wassermann'£)^'a6ifltlt3)4MpaiS!$(SS^ 46 722 DIAGNOSTIC METHODS Weichselbaum have been given on page 682. For the bacteriological diagno- sis of a meningococcic infection, one may use spinal fluid, blood, or purulent products from metastatic points, secretions of nose and bronchi. This ma- terial should be examined as soon as possible after obtaining, as the meningo- cocci are not especially resistant to various changes. For the development of this organism, especially of the first generation, media are necessary which contain animal, preferably human, albumin. The blood agar or ascitic agar given above are the best to use. The cultural peculiarities of the meningococcus on this medium have been given. From the cultures the organisms not infrequently appear variable in size and show variable depth of staining, although they are always Gram-negative. In examining spinal fluid for these organisms, centrifuge the fluid in a sterile tube and plant two or three ascitic agar plates with the sediment. Iso- late the colonies by sub-culture if necessary and identify the organism mi- croscopically. Inoculate special sugar-containing media and differentiate by the results mentioned above. The meningococcus produces acid only in dextrose and maltose media. In examining material from the nose or throat simple microscopical exami- nation is of little avail, as we have the possibility of finding the micrococcus catarrhalis as well as the saprophytic diplococci previously mentioned. Cul- tural work and differentiation on sugar media are absolutely essential. These saprophytic cocci, especially the diplococcus crassus, are found in both the lumbar fluid and nasal secretions. This latter diplococcus produces acid in dextrose, maltose, galactose, levulose, cane-sugar and lactose media. This organism is probably identical with Jager's meningococcus. Some of the varieties of the diplococcus flavus ferment levulose in addition to dextrose and maltose, while other types (Diplococcus flavus III) acts exactly like the men- ingococcus.^ It will be seen, therefore, that the fermentation test should be applied in all differential work, where the meningococcus is concerned. C. Micrococcus Catarrhalis. This organism is discussed on page 17. It is found in catarrhal condi- tions of nose and throat and, also, in infections of the genito-urinary tract. It grows readily on all media. On ascitic agar plates the colonies are white, firm, irregular, dry and somewhat smaller than the growths of meningococcus. Under low microscopic power they appear brown and granular, while those of the meningococcus are yellow and homogeneous and of the gonococcus gray- ish and slimy. The firm and dry quality of the colony is characteristic of the micrococcus catarrhalis. From the ordinary staphylococci, it is differen- tiated by its non-liquefaction of gelatin. Preparations made from culture media show involution forms, some of the organisms being very large. In the direct smears of purulent material the organisms are Gram-negative, intracellular or extracellular biscuit-shaped diplococci. For its identification cultural work is necessary and a study of its fermentative powers is essential. The micrococcus catarrhalis does not pro- ' See Kutscher, KoUe and 15efi2&yfe'|'^t?S^'S5/?@'3, IV, 589- CLINICAL BACTERIOLOGY 723 duce acid in any sugar-containing medium. An organism described as the Micrococcus cinereus is probably identical with this organism of catarrh. VIII. Vaccines In previous sections of this book the problems of immunity in its relation to opsonins, agglutinins and precipitins have been discussed briefly. It was shown that the injection of a vaccine (a suspension, in physiologic salt solu- tion, of a certain number of killed organisms) into the body of a patient suffer- ing with a chronic infection due to the organism injected brought about a systemic reaction by which the resistance of the patient was increased. In this way it was hoped that the natural resources of the body would be brought into activity by the stimulation of antibody formation and that this would result in great benefit to the patient. Further, by the use of vaccines, the resistance of the human organism against bacterial invasion could be so in- creased that immunity was conferred, for a variable period of time, toward the organism so injected. The most recent use of this latter principle is the antityphoid vaccination, which will be discussed later. It will be remembered that patients, who have passed through an attack of an infectious disease, have acquired, in most infectious diseases, an immunity against reinfection. An attempt is often made, especially in the treatment of infections with diphtheria organisms, tetanus bacilli and meningococci, to aid the patient in his efforts to overcome the invasion, that is to help him in acquiring immunity, by injection of serum from an animal previously immun- ized against such infection. This serum or antitoxin supplies antibodies to the infected patient, with which he may overcome the toxic products of bacterial development in the system. In other words, serum therapy confers immunity, shown in recovery from the acute infection, by aiding the system through introduction of antibodies produced outside the system of the pa- tient. Vaccine therapy, on the other hand, produces immunity by causing the production of antibodies within the system itself. It must be recalled that patients who have been inoculated with serum (protein) often become sensi- tized to its action to such an extent that a later injection of the same serum, even in much smaller dosage, may provoke serious and even fatal results. This is the phenomenon of anaphylaxis and must be remembered whenever one is dealing with the parenteral introduction of protein material into the system.^ In this section the writer wishes to discuss the method of preparation of vaccines, both of the stock and autogenous varieties. The elaboration of the 1 See Besredka, in Kraus and Levaditi's Handbuch, 1911, Erganzungsbd. I, 209; Biedl and Kraus, Ibid., 255; Abderhalden, Ztschr. f. physiol. Chem., 1912, LXXXII, 109; Wells and Osborne, Jour. Infect. Dis., 1913, XII, 341 ; Weil, Jour. Med. Research, 1913, XXVIII, 243; Auer and Van Slyke, Jour. Exper. Med., 1913, XVIII, 210; Zinsser, Ibid., 219; Vaug- han. Jour. Am. Med. Assn., 1913, LXI, 1761; Friedberger, Nederl. Tijdschr. v. Geneesk., 1913, II, 1065 and 1278; Nixon, Brit. Med. Jour., 1913, II, 1351; Weil, Jour. Med. Re- search, 1913, XXIX, 233; Leschke, Ztschr. f. exper. Path. u. Therap., 1914, XV, 23, Seligmann, " Anaphylaxie," in Handb. d. Biochem. des Menschen u. d. Tiere, Erganzbd., 1913, 248; Vaughap, "Anaphylaxis and Infection," Harvey Lectures, 1913-14, p. 132; Bradley and Sansum, Jour. Biol. Chem., 1914, XVIII, 497; Wallace, Jour. Am. Med. Assn., 1914, Lxii, 1 166. Digitized by Microsoft® 724 DIAGNOSTIC METHODS principles upon which their use is based as well as a discussion of the thera- peutic value finds no place here. I. Preparation of Vaccines. A. Stock Vaccines. The material for this work is obtained, under the strictest precautions to prevent contamination, from a focus of infection with the organism whose vaccine is desired It is at once plated upon general or special medium, de- pending upon the organism in question, and the organism isolated in pure culture. This may be a simple matter or it may be a very complicated one. In any case, all the differential points previously given for the varying or- ganisms must be kept in mind, so that proper medium and methods of isola- tion may be employed. It is absolutely essential that the final culture be pure, if the vaccine is to be of any value. In order to obtain a large amount of the pure culture, transfers from the original pure culture are made to three or four tubes of the medium best suited for its development and these tubes incubated for 24 to 48 hours. The cul- tures are then washed- from the surface of the medium, using sterile physio- logic salt solution as the menstruum and scraping the surface lightly with the platinum needle to insure the removal of the organisms. Pour this sus- pension into a sterile flask or large tube and shake thoroughly to break up the bacterial masses. This is an important point in the technic, as otherwise the suspension will not be uniform. If necessary, one may filter out the large clumps by means of a sterile filtering arrangement. Having prepared the suspension, this is then sterilized by heating in a water-bath to 6o°C. for one hour. From this tentatively sterilized suspen- sion, cultures are made upon proper media by transferring several loopsful of the suspension to the surface of the slanted media and incubating for 48 hours. If no growth occurs, the suspension may be regarded as sterile.^ Standardization. — The sterilized vaccine is now examined for the number of bacteria per c.c. of the suspension. The method usually adopted is to com- pare the number of bacteria in a definite volume of suspension with the num- ber of red blood cells in the same volume of freshly drawn blood. The technic is as follows. By means of a graduated capillary pipet place equal volumes of blood and of bacterial suspension on a glass slide, mix thoroughly and spread as for an ordinary blood slide. Stain with Wright's or other blood stain. Count the number of red cells and of bacteria in a number of microscopic fields and obtain an average of each. As the number of red cells per cu. mm. is arbitrarily fixed at 5 million, the relation of bacteria to the red cells, as shown by the counts, will readily give the number of bacteria in the cu. mm. The number in i c.c. is obviously 1,000 times greater.^ 1 King and Davis (Am. Jour. Pub. Health, 1914, IV, 917) have shown that potassium tellurite is an excellent indicator of sterility. Characteristic black compounds are pro- duced if a little of the salt be added to fluids containing viable bacteria. 2 See Hopkins (Jour. Am. Med. Assn., 1913, LX, 1615) for a method of standardizing vaccines based on centrifugation and determination of percentage of bacterial sediment; Glynn, Powell, Rees and Cox, Jour. Path, and Bacterid., 1914, XVIII, 379; Blaker, Indian Jour. Med. Research, 1914, I, 726; Harvey and Acton, Ibid., 1914, II, 648; Fitch, Jour. Am. Med. Assn., Z91S, LXIV^S^^^^ ^^ MicrOSOft® CLINICAL BACTERIOLOGY 725 Having obtained the concentration of the original suspension, the vaccine must be standardized so that each c.c. shall contain a standard number of bacteria. This is done by diluting with sterile physiologic salt solution (which contains sufficient cresol to bring the concentration of the final product up to 0.2 to 0.4 per cent, cresol) until the proper strength is reached. The addition of the cresol prevents future contamination of the vaccine and is an additional safeguard as to sterility of the product. The standard maximum dosage per c.c. of some of the more usual stock vaccines is as follows: Staphylococcus, 500 million Streptococcus, 100 million Colon bacillus, 100 million Gonococcus, 100 million Typhoid, 1,000 million Pneumococcus, 100 million Of course this maximum is not suited to all cases. The dosage must be varied for the individual and for the type of infection. B. Mixed Stock Vaccines. A prerequisite to the use of any vaccine should be the bacteriological identification of the infection in question. The use of a simple or mixed stock vaccine is never justified without such a precaution. It is questionable whether one should ever use a mixed stock vaccine, but if the bacteriological examination has revealed a mixed infection, a proper mixture may be used in the proportions suitable for each separate constituent of the vaccine.- In recent years the number and varieties of mixed stock vaccines have increased to such an extent that they are to be regarded as questionable therapeutic agents. To quote from Billings, "There is a value in vaccine therapy but to obtain benefit from vaccines, they should be rationally used. To use vaccines, simple or mixed, in the treatment of a patient, without first ascer- taining the nature of the disease, and, if infectious, the kind of invading or- ganism, is unscientific, reprehensible and wrong. When the mixed vaccines or the filtrate of culture-broth of countless organisms are used, it is like the shotgun prescription of our ancestors of the profession. They were and are intended to hit something." Such usage must be regarded, therefore, as mere guesswork. C. Autogenous Vaccines. These are the proper agents to use whenever a vaccine is to be employed. The specific toxins of the organism call into activity the specific antibodies, which are necessary to overcome the effects of the organism causing the infection, for which the autogenous vaccine is being prepared. "Instead of adding a heavier burden to the individual's immunizing mechanism, already taxed in its struggle against the invading bacteria, these bacteria, reintro- duced as autogenous vaccines, if successful stimulate further therapeutic immunizing responses." It can be considered as established that these autogenous vaccines are the ones giving the best results. These autovaccines ^^^S^-k^ MififfP^Bf discharges, excretions, blood,. 726 DIAGNOSTIC METHODS etc., of the patient. The effort to obtain pure cultures and definitely to identify the organism or organisms which are dominant must be painstaking, both aerobic and anaerobic cultures being made in many cases. If there be a mixed infection, each organism is isolated in pure culture and the suspen- sions of the pure cultures are then mixed in such a way that the resulting combiriation will contain the organisms in their usual strength per c.c. In this standardization of the autogenous vaccines a little more latitude is al- lowed than in the case of stock vaccines, as the dosage of the aytovaccine . must be adjusted more to the individual than to the standard. In other words, one must use these autovaccines in graduated dosage, beginning with a small dosage and working up. It is true that occasionally mixed autovaccines are prepared which have not been sufficiently differentiated. This method is uncertain and crude. The method employed is to obtain a culture of all the organisms present in the discharge and prepare a suspension which shall have a standardized content within the range of usefulness of the organisms present. Here we are con- fronted with the same proposition as in the case of mixed stock vaccines. All the organisms should be identified, if not they should not be used. In this. way a more reliable and more scientific product will be furnished. 2. Antityphoid Vaccination. The basis of the use of vaccines, as a preventive immunizing inoculation against typhoid fever, is the observation that one attack usually confers im- munity from further attacks. This use of vaccines has become widespread and is promising great results, especially in those subjected to variations in the sanitary surroundings. The doses are given at ten-day intervals; the first dose contains 500 million organisms and the second and third doses i billion bacteria each. A local reaction is usually present but this subsides within 48 hours, while a general systemic reaction is not always observed. As this measure is of such importance to health and sanitary officers, as well as to physicians in general, I give, herewith, some references for consultation.' '■ See Wright, Lancet, 1896, II, 807; Brit. Med. Jour., 1897, 1, 16; Leishman, Jour. Roy. Inst. Pub. Health, 1910, XVIII, 394; Firth, Jour. Roy. Army Med. Corps, 1911, XVII, 49S; Russell, Boston, Med. and Surg. Jour., 1911, CLXIV, i; Nelson and Hall, Jour. Am. Med. Assn., 1911, LVII, 1759; Schereschewsky, Pub. Health Rep., 1911, XXVI, 1508; Davis, Jour. Am. Med. Assn., 1912, LVIII, 537; Russell, Ibid., 1331; Maverick, Ibid., 1672; Engelbach, Interstate Med. Jour., 1912, XIX, 537; Albert and Mendenhall, Am. Jour. Med. Sc, 1912, CXLIII, 232; Williams, Ibid., 352; CaUison, Ibid., 1912, CXLIV, 350; Russell, Bull. 2, War Dept. Office Surg. Gen., Washington, 1913, 7; Craig, Ibid., 15; Nichols, Ibid., 23; Hunt, Am. Jour. Med. Sc, 1913, CXLV, 826; Hiss, Jour. Med. Research, 1913, XXVIII, 385; Russell, Jour. Am. Med. Assn., 1913, LXI, 666; Metchnikoff and Besredka, Ann. de I'Inst. Pasteur, 1913, XXVII, 597; Russell, Am. Jour. Med. Sc, igi3, CXLVI, 803; Foxworthy, Military Surg., 1914, XXXIV, 332; Sachs, Med. Klin., 1914, X, 1538; Russell, Jour. Am. Med. Assn., 1914; LXII, 1371; Krumbhaar and Richard- son, Am. Jour. Med. Sc, 1915, CXLIX, 406; Thomsen, Hospitalstid., 1915, LVIII, 211; Stursberg and KJose, Miinch. med. Wchnschr., 1915, LXII, 380; Cecil, Jour. Infect. Dis., 191S, XVI, 26; Biedl, Wien. klin. Wchnschr., 1915, XXVII, 125; Paltauf, Ibid., 125; Kirschbaum, Ibid., 208; Eggerth, Ibid., 209; Csernel, Marton and Feistmantel, Ibid., 229; Sladek and St. Kotlowski, Ibid., 389; Goldscheider and Aust, Deutsch. med. Wchnschr., 1915, XLI, 361; Kisskalt, Ibid., 393; Schneider, Ibid., 393; Reibmayr, Miinch. med. Wchnschr., 1915, LXII; 610; Rhein, Ibid., 427; Bourke, Evans and Rowland, Brit. Med. Jour., 1915, I, 584", Widal, Bull, de I'Acad. Med., 19x5, LXXIV, 205; Vincent and Chantemesse, Ibid., 225; Harris and Ogan, Jour. Am. Med. Assn., 1915, LXIV, 3; Garbat, Ibid., 489; Trowbridge, Fin^fras^^TQori^jQ^g^f^Elmei, Ibid., 1147; Lyster, Ibid., 1915, LXV, 510; Jobling and^Pefersen,T:6id., 515; Sawyer, Ibid., 1413. CLINICAL BACTERIOLOGY 727 BIBLIOGRAPHY 1. Besson. Practical Bacteriology, Microbiology and Serum Therapy. London, 1913- 2. Hewlett. A manual of Bacteriology. St. Louis, 1914. 3. Irons. Billings-Porchheimer Therapeusis, vol. 5. New York, 1914. 4. Jordan. General Bacteriology. Philadelphia, 1913. 5. KoLLE AND Hetsch. Die Experimentelle Bakteriologie und die Infektions- krankheiten. Berlin, 191 1. 6. KoLLE AND Wassermann. Handbuch der Pathogenen Mikroorganismen. Jena, 1912 and 1913. 7. Kraus and Levaditi. Handbuch der Technik und Methodik der Immuni- tatsforschung. Jena, 1909-1913. 8. McParland. Text-book on Pathogenic Bacteria. Philadelphia, 1913. 9. Park and Williams. Pathogenic Microorganisms. Philadelphia, 19 14. General Clinical Diagnosis . I. Boston. A Text-book of Clinical Diagnosis. Philadelphia, 1905. 2. Brown and Ritchie. Medical Diagnosis. Edinburgh, 1906. 3. Brugsch and Schittenhelm. Lehrbuch klinischer Untersuchungsmethoden fiir Studierende und Aerzte. Berlin, 1911. Technik der Speziellen Klinischen Untersuchungsmethoden. Berlin, 1914. 4. Butler. Diagnostics of Internal Medicine. New York, 1905. 5. Da Costa. Medical Diagnosis. Philadelphia, 1890. 6. Debove et Achard. Manuel de diagnostic medical. Paris, 1900. 7. Deguy et Guillaumin. Microscopie clinique. Paris, 1912. 8. Emerson. Clinical Diagnosis. Philadelphia, 1913. 9. French. Medical Laboratory Methods and Tests. Chicago, 1912. 10. Guiart et Grimbert. Precis de diagnostic. Paris, 1912. 11. Greene. Medical Diagnosis. Philadelphia, 1907. 12. Hare. Practical Diagnosis. Philadelphia, 1908. 13. Hutchinson and Rainy. Clinical Methods. Chicago, 1908. ,14. JagiC. Klinische Mikroskopie. Wien, 1913. 15. KoRANYi und Richter. Physikalischc Chemie und Medizin. Leipzig, 1908. 16. Von Jaksch. Klinische Diagnostik. Berlin, 1910. 17. Kxemperer. Grundriss der klinischen Diagnostik. Berlin, 1906. 18. Kraft. Analyt. Diagnostikum. Leipzig, 1913. 19. Lenhartz. Mikroskopie und Chemie am Kjrankenbett. Berlin, 1913. 20. Leube. Specielle Diagnose der inneren Krankheiten. Leipzig, 1908. 21. Morris. CKnical Laboratory Methods. London, 1913. 22. MussER. Medical Diagnosis. Philadelphia, 1904. 23. Von Noorden. Handbuch der Pathologic des Stoffwechsels. Berlin, 1906. ] 24. Panton. Clinical Pathology. London, 1913. 25. SAiiLi. Lehrbuch der klinischen Untersuchungsmethoden. Wien, 1913. 26. Schilling. Aerztliche Technik. Wurzburg, 1906. 27. Simon. A Manual of Clinical Diagnosis. Philadelphia, 1914. 28. Todd. Manual of Clinical Diagnosis. Philadelphia, 1912. 29. Wesener. Medizinisch-kKnisch Diagnostik. Berhn, 1907. 30. Wood. Chemical and Microscopical Diagnosis. New York, 191 1. 31. Zuelzer. Chemische und Mikroskopische Diagnostik. Leipzig, 1906. Digitized by IVIicrosoft^ Digitized by Microsoft® INDEX Abderhalden's sero-diagnosis of cancer, 650 of dementia praecox, 651 • of pregnancy, 638 Abortion, 384 Abscess, blood in, 546 indican in, 257 of liver, sputum in, 33 of lung, sputum in, 33 Absorptive power of stomach, 86 Acanthia lectularia, 164 Acarus scabiei, 162 Accidental albuminuria, 261 Acetic acid in gastric contents, 64, 73, 77 Aceto-acetic acid, 191, 324 Acetone bodies, 191, 317 in blood, 451 in urine, 191, 317 determination of, 322 significance of, 317 tests for, 320 Acetonemia, 451 Acetonuria, 317 Acholic stools, 107, 116 Achorion Schonleinii, 165 Achroiocythemia, 426 Achromatophilia, 496 Achroodextrin, 36, 82 Achylia gastrica, 78, 89 Acid, acetic, 64, 73, 77, 249 aceto-acetic, 191, 324 alloxyproteic, 208, 248 amino, 82, 191, 246 bile, 61, 109, 191, 331 butyric, 64, 73, 77, 249 cholalic, 117, 191 chondroitin-sulphuric, ' 191, 208, 253 diacetic, 191, 324 diamine, 83, 92, 246 fatty, 13, 115, 123, 191, 249, 449 formic, 249 glycocholic, 117, 191 glycosuria, 333 glycuronic, 191, 314 hippuric, 191, 248, 345 homogentisic, 191, 333 hydrochloric, 51, 65 hydroquinone-acetic, 333 lactic, 64, 73, 74, 249 nucleinic, 232, 253 oxalic, 191, 250 oxaluric, 191, 250 i8-oxybutyric, 191, 325 oxymandelic, 191 oxyproteic, 191, 208, 248 phosphoric, 191, 198 Acid, picric, 272 propionic, 249 rosacic, 254 rosolic, 21 sulphuric, 206 taurocholic, 117, 191, 331 uric, 191, 232, 439 uroferric, 208 uroleucic, 191, 333 Acid-fast organisms, 20 Acidity of gastric juice, 64 of urine, 184, 186 Acidophilic cells, 496 granules, 499, 506 Acidosis, 227, 317 Acid stains, 477 unit, 186 Actinomyces in sputum, 27 Actinomycosis, 27 Acute anterior poliomyelitis, 684 bronchitis, sputum in, 31 gastritis, gastric juice in, 90 hemorrhage, anemia due to, 535 infectious conjunctivitis, 47 diseases, blood in, 549 leukemia, 543 nephritis, 178, 181, 190, 194, 201, 217, 265 rheumatism, blood in, 557 yellow atrophy of the liver, urine ^ in, 220, 246, 343 Addison's disease, blood in, 548 Adenin, 240 Adler's test for blood, 110 Adolescent albuminuria, 264 Aerobic cultures, 706 organisms, 706 ^stivo-autumnal malaria, 566 Agar, ameba, 703 ascitic, 704 blood, 703 dextrose, 703 glycerin, 703 hydrocele, 704 litmus lactose, 703 nutrient, 702 Age, effect of, on red blood ceUs,'^493 on white blood cells, 52 1 Agglutination, 598, 607 Agglutinins, 598 Agglutinophore, 598 Agonal leucocytosis, 516 Air in sputum, 4 Albumin, determination of, 272 in exudates, 664 in feces, 122 in milk, 693 in sputum, 5 Digitized byMcrosoft® 73° INDEX Albumin, in transudates, 663 in urine, 261 of blood, 428 quotient, 275, 429 removal of, 274 serum, 261 significance of, in urine, .261 tests for, 266 Albuminuria, accidental, 261 adolescent, 264 after baths, 262 alimentary, 262 colliquative, 265 constitutional, 263 cyclic, S63 false, 261 febrile, 264 functional, 261 hematogenous, 265 hypostatic, 264 intermittent, 263 lordotic, 263 mixed, 280 . ■ neurotic, 265 of the new-born, 262 of pregnancy, 262 orthostatic, 263 orthotic, 263 physiologic, 261 post-infectious, 264 postural, 263 renal, 265 structural, 265 thermolytic, 278 toxic, 265 traumatic, 265 true, 261 vesicular, 261 with definite renal lesions, 265 Albumon, ,427 Albumose, Bence-Jones, 276 in blood, 429 in feces, 122 in gastric contents, 82 in urine, 276 Albumosuria, 276 alimentary, 280 digestive, 280 enterogenous, 280 febrile, 280 hematogenous, 280 hepatogenous, 280 myelopathic, 277 pyogenic, 280 renal, 280 significance of, 279 tests for, 279 Alcohol-fast organisms, 20 Aleukemic myelosis, 544 Alimentary albuminuria, 262 albumosuria, 280 chloruria, 193, 372 galactosuria, 305, 312 glycosuria, 285 levulosuria, 305 lipuria, 346 pentosuria, 308 Alkaline phosphates, 199 tide of urine, 186 Digitized by Alkalinity of blood, 395 Alkalinuria, 200 Alkaptonuria, 333 AUantoin, 191, 248 Allergy, 600 AUoxur bases, 122, 191, 240, 341 AUoxyproteic acid, 191, 208, 248 Almfe-Nylander's test for glucose, 292 Alm^n's tannic acid solution, 692 Aloin test for blood, 109, 655 Altitude, effect of, on red cells, 493 Alveolar epithelial cells in sputum, 1 1 Ambard's coefficient, 438 Amblyochromatic erythroblasts, 491 Amboceptors, 618, 624, 625, 630 Amboceptor paper, 630 Ameba agar, 703 Ameba coli in feces, 134 in mouth, 42 in sputum, 28 in urine, 366 pulmonalis, 28 Amebic dysentery, 135 pyorrhea, 42 American hook-worm, 152 Amino-acetic acid, 246 Amino acids in urine," 191, 246 a-aminoisobutyl-acetic acid, 343 Ammonia in blood, 443 in urine, 227 Ammoniemia, 443 Ammonium magnesium phosphate in feces, 120 in sputum, 13 in urine, 348 urate calculi, 368 sediment, 347 Amniotic fluid, 383 Amount of blood, 387 of cerebrospinal fluid, 680 of feces, 104 of gastric juice, 57 of sputum, 2 of urine, 177 Amyloid kidney, globulin in urine of, 275 urine in, 178, 182, 253, 266, 275 Amylopsin, 102 Amylosis, 34 Anachlorhydria, 70 Anaerobic cultures, 706 organisms, 706 Anaphylaxis, 723 Ancylostoma duodenale, 151 Ancylostomiasis, 151 Anemia, 528 aplastic, 534 Biermer's, 531 chlorotic, 529 definition of, 528 Ehriich's, 534 due to acute hemorrhage, 535 to acute infections, 538 to bad air, 537 to blood poisons, 538 to chronic diseases, 557 to chronic hemorrhage, 536 to inanition, 536 M;crOSO/lf®^"*sst^"^l parasites, 152, 537 INDEX 731 Anemia, febrile, 538 hemolytic, 538 infantum pseudoleukemica, 533 yon Jaksch's, 533 leukanemia, 534 lymphatic, 543 of the South, 152 of the tropics, 493 primary pernicious, 531 progressive pernicious, 531 secondary, 534 simple primary, 528 splenic, 532 Anemic degeneration, 497 Anesthesia, changes in urine after, 319 effects of, on blood, 547 Angioneurotic hematuria, 355 Anguillula aceti in urine, 366 intestinalis et stercoralis, 147 Anhydremia, 390 Animal gum in urine, 314 Animal parasites in blood, 561 in ear, 47 in feces, 133 in gastric contents, 63 in sputum, 28 in urine, 366 Ankylostomum duodenale, 151 Anopheles maculipennis, 561 Anterior urethritis, 353 Anthracosis, 4, 34 Antiamboceptors, 597 Antibodies, 595, 619 Anticomplement, 597 Antiformin method for tubercle bacilli, 18 Antigens, 595, 619, 620, 622, 626 Antihemolysins, 597 Antihuman amboceptor, 630 Antimeningococcic serum, 683 Antisheep amboceptor, 624 Antitoxins, 594 Antityphoid vaccination, 600, 726 Anuria, 180 Aplastic anemia, 534 Appearance of blood, 393 of exudates, 664 of feces, 105 of gastric contents, 57 of leucocytes; 502 of milk, 690 of red cells, 486 of semen, 376 of spinal fluid, 680 of sputum, 4 of transudates, 663 of urine, 180 Appetite juice, 51 Arabinose, 309 Arginin, 247 Arneth's classification of neutrophils, 505 Arnold's test for diacetic acid, 325 Arnold- Volhard method for chlorids, 195 Arterial blood, 394 Arthropoda, 161 Ascaridas in feces 145 , Digitized ^scaris alata, 146 ^ Ascaris caniculae, 146 canis, 146 canis et martis, 160 cati, 146 felis, 146 graecorum, 147 lumbricoides, 145 lumbricus canis, 146 marginata, 146 mystax, 146 teres, 146 trichiura, 149 tricuspidata, 146 vermicularis, 147 visceralis et renalis, 160 werneri, 146 Ascitic agar, 704 fluid, cytology of, 676 Asexual cycle of malarial parasite, 564 Ash-free diet of Taylor, 195 Asiatic cholera, feces in, 106, 129 organism of, 129 Aspergillus flavus, 16 fumigatus, 16 in aural secretion, 47 in sputum, 16 niger, 16 . subfuscus, 16 Assimilation limit, 284 Asthma, bronchial, 33 eosinophilia in, 10, 33, 519 sputum in, 33 Atrophic gastritis, 92 Aural secretion, 46 bacteria in, 46 larvae in, 47 molds in, 47 Autotoxic enterogenous cyanosis, 496 Autovaccines, 600, 725 Azoospermatism, 378 Azotorrhea, 114 Babcock's method for fat in milk, 693 Bacillary dysentery, 132 index, 599 Bacilluria, 366 Bacillus aerogenes capsulatus, 133 anthracis, 26 coli communis, 48, 129, 366, 589, 712 comma, 61, 129, 715 Ducrey's, 669 hodgkini, 544, 711 icteroides, 583 leprae, 24 mallei, 27 mucosus capsulatus, 25, 46 of Boas-Oppler, 62 of Bordet-Gengou, 26, 711 of bubonic plague, 27 of diphtheria, 38, 709 of dysentery, 132, 713 of Finkler-Prior, 61, 130 of Friedlander, 25, 46 of glanders, 27 of Hansen, 24 of Hofiman, 710 of influenza, 26, 711 by /W^feSf^ ?P^ Yersin, 27 ^ofHer, 38, 709 732 INDEX Bacillus of Koch, i8, 132, 364, 592, 667 of Koch- Weeks, 48 of Morax-Axenfeld, 48 of ozena, 46 of Perez, 46 of Pfeiffer, 26, 33, 711 of Sanarelli, 583 of Shiga, 132, 713 of soft chancre, 669 of tuberculosis, 17, 132, 364, 592, 667 of Tunnicliff, 45 of Vincent, 40 of Welch, 133 of whooping-cough, 26, 711 paratyphoid, 131, 588 pertussis, 26, 711 pestis, 27 pseudo-diphtheria, 40, 710 pyocyaneus, 4 rhinitis, 45 smegma, 24, 364, 669 timothy, 24 typhosus, 26, 130, 366, 587, 712 ulceris cancrosi, 669 xerosis, 40 X of Sternberg, 583 Bacteria in blood, 585 in conjunctiva, 47 in ear, 46 in exudates, 667 in feces, 128 in gastric contents, 62 in miUi, 696 in mouth, 36 in nasal secretions, 44 in sputum, 14 in urine, 363 Bacterial flora of feces, 129 of vagina, 380 Bacterial vaccines, 598, 723 Bacteriemia, 585 Bacteriology of blood, 585 of cerebrospinal fluid, 682 of exudates, 667 of feces, 128 of milk, 696 of sputum, 14 of urine, 363 Bacteriolysins, 595 Bacteriuria, 363 Balantidium coli, 138 Baldwin's method for oxalic acid, 251 Bang's test for albumose, 279 for sugar, 296 Banti's disease, blood in, 532 Barberio's test for semen, 379 Barber's itch, organism of, 166 Bases, aUoxur, 122, 191, 240, 341 hexone, 83, 92, 247 nuclein, 122, 191, 240, 341 purin, 122, 191, 240, 341 xanthin, 122, 191, 240, 341 Basic stains, 477 Basket cells, 509 Basophile leucocytes, 506 Basophiles, 506 Basophilia, 498, 506 Basophilic degeneration of « Basophilic stippling of reds, 498 Bass and Watkin's macroscopic agglutina- tion test, 611 Baths, albuminuria following, 262 effect of, on red cells, 494 Beckmann apparatus, 405 Bed bug, 164 Beef tape-worm, 141 Bence- Jones, body, 276 protein, 276 amount of, 276 significance of, 276 tests for, 277 Benedict and Murlin's testjfor amino acids in urine, 247 Benedict's test for sugar, 290 Benzidin test for blood, no Benzoic acid, 248 Bial's test for pentose, 310 Biermer's anemia, 531 Bile acids in blood, 451 in feces, 117 in gastric contents, 61 in urine, 191, 331 media, 713 pigments in blood, 451 in exudates, 664 in feces, 108, 117 in gastric contents, 59, 61 in sputum, 3 in urine, 191, 329 significance of, 329 tests for, '329 Bilharzia hematolsia, 29, 585 Bilharziasis, 585 Bilicyanin, 117 Bilifuscin, 117, 329 Bilihumin, 117 Biliprasin, 108, 329 Bilirubin, 59, 108, 117, 329, 345 Biliverdin, 59, 108, 1 17, 329 Biologic test for blood, 614 Bismuth oxid in stools, 107 test for glucose, 292 Biuret test for protein, 279 Black's method for ;3-oxybutyric acid, 326 Black sputum, 4 urine, 182 -water fever, 281 Bladder, inflammation- of, 187, 352 tuberculosis of, 365 Blastomycetes in skin, 169 in sputum, 17 Blastomycosis, 17, 169 Blennorrhea, 381 Blood, 385 acetone in, 451 after anesthesia, 546 after splenectomy, 560 after surgical intervention, 546 agar, 703 albumin in, 428 alkalinity of, 395 amino acids in, 445 ammonia in, 443 bacteriology of, 585 biliary constituents in, 45 L .;,. Micros&f^^^^^^^^ ^"' 445 :iB.. INDEX 733 Blood, casts, 359 cells in, 486 chemical properties of, 407 chemical tests for, 109, 655 cholesterin in, 450 coagulation of, 401 color-index of, 426 color of, 394 constituents of, 394 counting of cells of, 455, 468, 510 creatinin in, 444 crises, 491 cryoscopy of, 405 cultures, 586 dust, 525 electric conductivity of, 406 enumeration of cells of, 455, 460, 468, 505, 510 fat in, 449 ferments of, 454 fixation of smears of, 473 formation of, 386 fresh, 470 gases in, 454 glucose in, 446 hemoglobin of, 409 in abscess formation, 546 in acute infections, 549 in acute rheumatism, 557 in Addison's disease, 548 in aplastic anemia, 534 in bilharziasis, 585 in carcinoma, 559 in chlorosis, 529 in chronic diseases, 557 in chronic tuberculosis, 557 in diabetes mellitus, 547 in diphtheria, 556 in distomiasis, 585 in filariasis, 577 in gout, 548 in kala-azar, 576 in leprosy, 559 in leukanemia, 534 in leukemia, 539 in malaria, 561 in measles, 554 in myxedema, 549 inorganic constituents of, 452 in pernicious anemia, 531 in pertussis, 556 in pneumonia, 550 in primary anemia, 528 in pseudoleukemia, 544 in relapsing fever, 574 in rickets, 549 in Rocky Mountain spotted fever, 584 in scarlet fever, 553 in secondary anemia, 534 in sleeping sickness, 575 in splenic anemia, 532 in syphilis, 558, 578 in typhoid fever, 552, 587 in varicella, 555 in variola, 555 in whooping-cough, 556 in yellow fever, 583 Ojgjtized by limitations of examinations of, 659 Blood, medico-legal aspects of, 654 tests for, 654 morphology of, 469 needle, 392 nitrogen of, 430 non-protein nitrogen of, .430 obtaining of, 392 odor of, 394- osmotic pressure of, 405 parasitology of, 561 pathology of, 528 general, 546 special, 528 physiology of, 386 pigments of, 409 • plates, 468, 523, 526 properties of, 393 proteins of, 427 reaction of, 395 red cells of, 486 serum reactions of, 606 smears of, 470 solids of, 409 specific gravity of, 399 spectroscopic tests for, 410, 658 staining of smears of, 476 tests for, 109, 654 total solids of, 409 urea in, 435 uric acid in, 439 value of examinations of, 659 viscosity of, 401 vital staining of, 485 volume of, 387 volume relations of elements of, 390 white cells of, 502 Blood casts, 359 cells in exudates, 665 in feces, 120 in gastric contents, 61 in sputum, 3, 5, 11 in urine, 328, 354 plates, 523 appearance of, 523 counting of, 468 function of, 525 number of, 524 size of, 524 staining properties of, 524 poisons, 538 siriears, fixation of, 473 preparation of, 470 staining of, 476 staining, 476 Bloody sputum, 3,5, 11 Boas' method for lactic acid, 76 for estimating gastric motility, 85 test for free hydrochloric acid, 67 test-meal, 55 Bof s-Oppler bacillus, 62 Body louse, 163 Boggs' coagulometer, 403 method for protein in milk, 692 Bone-marrow, 386, 525 function of, 386 morphology of, 525 M/&flB®iOfi^reservative in milk, 696 Bordet's potato medium, 711 734 INDEX Boric acid as preservative, 696 Bothriocephaloidea in feces, 144 bothriocephaluslatissimus, 144 bothriocephalus latus, 144 bothriocephalus sp. Ijima et Kuri- moto, 145 dibothriocephalus cordatus, 144 Bottcher's crystals, 14, 376 Bouillon, 701 Bradshaw's myelopathic albumosuria, ■ 277 Breakfast, test, 54 Bremer's blood-test in diabetes, 547 Bricklayers' anemia, 153 Brodie-Russell coagulometer, 403 Bronchial asthma, 33 stones, 8 Bronchioliths, 8 Bronchiolitis exudativa, 7 Bronchitis, acute, 31 chronic, 32 eosinophilic, 10 fetid, 32 fibrinous, 32 putrid, 32 Broncho-pneumonia, sputum of, 31 Bronfenbrenner's modification of Abder- halden test, 647 Bunge-Trantenroth's method for tuber- cle bacilli, 24 Burri's method for spirochaete, 670 Busk's intestinal fluke, 158 Butyric acid in gastric contents, 64, 73, 77 Cabot's ring bodies, 499 Cachexial fever, 576 Cadaverin, 123 Caffein, 240 Calcium carbonate calculi, 370 sediment, 349 of urine, 211 oxalate calculi, 368 crystals in sputum, 14 in urine, 250, 341 phosphate calculi, 368 sediment, 346, '347 soaps in feces, 120 sulphate sediment, 345 Calculi, ammonium urate, 368 biliary, 117 bronchial, 8 calcium carbonate, 370 oxalate, 368 classification of, 367 cystin, 370 examination of, 369 formation of, 367 hepatic, 117 intestinal, 118 nasal, 46 phosphatic, 368 pulmonary, 8 renal, 367 table for examination of, 369 ureteral, 367 urethral, 367 uric acid, 368 urosteaiith, 370 Digitized by MicrosMwrne, 366 Calculi, vesical, 367 xanthin, 370 Calmette's tuberculin reaction, 602 Cammidge's reaction, 311 Cancer (see Carcinoma) Cane sugar, absorption of, 123 digestion of, 64 in urine, 314 Capsule, staining of, 590 Carbohydrates, digestion of, 36, 64, 82 in blood, 445 in exudates, 664 in feces, 116, 123 in milk, 695 in urine, 283 Carbol-fuchsin solution, 20 Carbonates in urine, 211 Carbon dioxid hemoglobin, 412 in blood, 454 monoxid hemoglobin, 412 poisoning, 412 Carcinoma, Abderhalden's test in, 650 blood in, 559 cells in exudates, 675 fragments in feces, 118 in gastric contents, 63, 93 in urine, 363 of cervix, 384 of kidney, 354, 363 of pleura, 675 of rectum, 118 of stomach, 91 of uterus, 384 Cardiac albuminuria, 266 edema, 179 pleurisy, 675 Carniri, 240 Casein in feces, 115 appearance of, 115 Leiner's test for, 115 in milk, 693 Casts, 356 blood, 359 chemistry of, 356 colloid, 360 epithelial, 359 fatty, 359 fibrinous, 359 in sputum, 8 granular, 357 hyaline, 356 mixed, 356 origin of, 356 prostatic, 378 pseudo, 361 pus, 359 significance of, 357, 361 size of, 356 staining of, 357 testicular, 378 true, 356 waxy, 358 Catalase, 522 Catarrhal stomatitis, 38 Cellulose in feces, 117 Centrifugation, 338 Cercomonads in feces, 138 in sputum, 28 INDEX 735 137 Cercomonads in vaginal secretions, 381 Cercomonas coli hominis, 137 hominis, 138 intenstinalis, 138 seu Bodo urinarius, Cerebrospinal fluid, 678 bacteriology of, 682 chemistry of, 681 cytology of, 682 in nasal secretion, 45 microscopy of, 682 obtaining of, 679 pressure of, 679 properties of, 680 syphilis, 685 Cerumen, 46 Cestodes in feces, 139 in sputum, 29 Chalicosis, 4, 34 Chancre, organism of, 669 Character of blood, 393 of exudates, 664 of feces, 105 of sputum, 4 of urine, 176 Charcoal in feces, loi Charcot-Leyden crystals in feces, 12c in sputum, 14, 30,. 31 Cheesy masses in sputum, 6 Chemical fixation of smears, 475 Chemotaxis, 598 Childhood, red cells in, 500 white cells in, 521 Chinese liver-fluke, 159 Chinovose, 308 Chloremia, 426 Chlorid excretion in urine, 192, 372 retention, 192 Chlorids of the blood, 452 of the urine, 192 amount of, 192 estimation of, 195 variations of, 192 Chloromata, 4 Chlorosis, 529 Chlorotic anemia, 529 Chloruria, 192, 372 Cholecyanin in feces, 107 in urine, 329 Cholelithiasis, 117 Cholemia, 451 Cholera red reaction, 716 spirillum, characteristics of, 715 in feces, 129 Cholesterin blood, 450 crystals in feces, 120 in sputum, 13 in urine, 346 Choletelin in urine, 329 Choluria, 329 Chondroitin-sulphuric acid, 191, 253 Chromogenic bacteria m sputum, 4 Chromogens in urine, 254 Chronic bronchitis, sputum in, 32 diseases, blood in, 557 gastritis, nephritis, urine in Chyloid exudates, 665 Chylous exudates, 665 Chyluria, 182, 346 Chymosin, 64, 81 CimEeomonas hominis, 138 Cimex lectularius, 164 Cladocoelium hepaticum, 157 Clark's method for elastic tissue, 12 Clay-colored stools, 107, 117 Cleaning glass-ware, 469 Clearing of urine, 298, 300 Coagulation of blood, 401 of exudates, 664 of milk, 691 of urine, 283 time of blood, 403 Coagulometer of Boggs, 403 of Dorrance, 402 of Rudolf, 403 of Russell-Brodie, 403 Coal pigment in sputum, 4, 34 Coarsely granular cells of Schultze, 506 Coating of the tongue, 38 Coccidioidal granuloma, 170 Coccidioides immitis, 170 Coccidium hominis, 137 perforans, 137 Coccobacillus foetidus ozenae, 46 Coefficient, creatinin, 242 of Ambard, 438 of Haeser, 189 of Haines, 189 of Long, 189 refraction, 389, 429 Colitis, catarrhal, 116 malignant, 118 mucous, 112 Collection of feces, 99 of gastric contents, 52 of puncture fluids, 663 of sputum, 2 of urine, 176 CoUignon and Pilod's test, 683 Colloid casts, 360 cysts, 676 Colloidal nitrogen, 248 Colon bacillus, 48, 129, 366, 589, 712 Color-index of blood cells, 426 of blood, 394 of exudates, 664 of feces, 106 of gastric contents, 58 of sputum, 3 31S 201, 266 of urine, 181 Colorimeter, 244 Colostrum, 688 Coma diabeticum, „ Combined hydrochloric acid, 71 Comma bacillus of Koch, 61, 129, Common flea, 165 liver-fluke, 157 Complement, 597, 618, 624, 625 fixation test, 617, 636 Complementophile, ,'i97 Composition of blood, 407 of milk, 689 gastric juice in, 90 of, urine, 190 715 208, ns, biliary, 117 in sputum, 8 736 INDEX Concretions in bronchioliths, 8 in pneumoliths, 8 intestinal, Il8 coproliths, ii8 enteroliths, ii8 nasal, 46 renal, 367 vesical, 367 Conductivity, electric, of blood, 406 of urine, 371 Congo-red test, 66 Conjugated glycuronic acids, 124, 314 Conjunctival secretions, 47 Conjunctivitis, diphtheritic, 47 gonorrheal, 48 infectious, 47 vernal, 49 Consistency of blood, 401 of feces, 105 of gastric contents, 59 of milk, 689 of spinal fluid, 680 of sputum, 2 of urine, 181 Constipation, 105 Coproliths, 118 Corpora amylacea, 377 Corynebacterium granulomatis maligni, 711 Cough, whooping, blood in, 556 organism of, 26, 711 Counting of blood plates, 468 of pus cells, 354 of red ceUs, 455 of white cells, 465 Crab louse, 163 Creatin in urine, 241, 245 Creatinin coefficient, 242 estimation of, 243 metabolism of, 241 tests for, 243 variations of, 241 Creatorrhoea, 114 Crenation, 487 Crescents in blood, 567 Crises, blood, 491 Cryoscopy of blood, 405 of urine, 371 Crystals in feces, 120 in gastric contents, 63 in semen, 376 in sputum, 13 in urine, 338 Culex mosquito, 583 Cultures, aerobic, 706 anaerobic, 706 blood, 586 media, 701 throat, 39 urine, 363 Curds in feces, 115 Curschmann's sjjirals, 7, 30, 31 Cutaneous reactions, 600, 603 Cyclic albuminuria, 263 Cylindroids, 360 Cylindruria, 361 Cyst, colloid, 676 i^^:V Digitized by Cyst, hydatid, 678 hydrocele, 677 hydronephrotic, 678 myxoid, 676 ovarian, 676 pancreatic, 678 papillary, 677 parovarian, 677 serous, 676 spermatocele, 678 Cystein, 208 Cysticercus cellulosse, 141 Cystin, 208 calculi, 370 sediment, 342 Cystinuria, 342 Cystitis, 187, 352, 365 Cystorpermium hominis, 137 Cystotaenia solium, 140 Cytology in cardiac pleurisy, 676 in malignant pleurisy, 676 in nephritic pleurisy, 676 in pneumococcus pleurisy, 674 in primary tubercular pleurisy, 674 in secondary .tubercular pleurisy, 674 in streptococcus pleurisy, 675 in typhoid pleurisy, 675 of ascitic fluid, 676 of cerebrospinal fluid, 682 of exudates, 672 of peritoneum, 675 of pleura, 674 of normal-fluids, 673 of sputum, 10 technic of, 672 Cytophile, 597 Dahlia stain, 507 Daland's hematocrit, 390 Dare's hemoalkalimeter, 396 hemoglobinometer, 420 method for alkalinity of blood, 396 Darling's method of staining ameba, 136 Day urine, 178 Dechloridization, 194 Deficit of hydrochloric acid, 73 Definitive host, 140 Degenerated forms of red cells, 498 of white cells, 509 Degeneration, anemic, 497 hemoglobinemic, 499 Degree of tolerance, 284 Delayed chloroform poisoning, 319 Dementia praecox, Abderhalden's test in, 651 Demodex folliculorum, 162 Dermacentor Andersoni, 584 venustus, 584 Dermoid cysts, 677 Desmoid bag, 87 Deutero-albumose, 82, 276 Dextrin in urine, 314 Dextrose agar, 703 Diabetes alternans, 234 M/croMiu^%7,%82 INDEX 737 Diabetes mellitus, blood in, 547 Bremer's test in, 547 lipemia in, 547 urine in, 179, 182 Williamson's test in, 548 phosphatic, 201 renal, 284 Diabetic coma, 318 Diacanthos polycephalus, 1 53 Diacetic acid in urine, 324 Diagnosis, functional, 370 Diagnostic value of agglutination test, 613 of complement fixation test, 633 of precipitin test, 616 Diamines in urine, 123, 342 Diamino acids, 83, 92, 246 Diaminuria, 342 Diarrhea, 105 Diastase in urine, 251 Diazo reaction, 334 Dibothriocephalus cordatus, 144 latus, 144 Dibothrium latum, 144 Diet of Folin, loi of Schmidt and Strasburger, 100 of Taylor, 195 Dieudonne's medium, 716 Differential counting, 510 Diffusible alkalinity of blood, 395 Digestion, gastric, 82 intestinal, 103, 121 leucocytosis of, 513 products of, 82, 103 Digestive insufficiency, 113 Dilatation of stomach, 83, 84 Diluting fluids for blood, 459, 466 Dimethylaminoazobenzol test, 66 Dimethylaminobenzaldehyd reaction, 336 Dimorphous muris, 138 Diphtheria, baciUus of, 38, 709 blood in, 556 taking smear in, 39 Diphtheritic conjunctivitis, 47 laryngitis, 39 Diplacanthus nana, 142 Diplococcus cinereus, 720 crassus, 720, 722 flavus, 720, 722 intracellularis meningitidis, 682, 722 lanceolatus, 25, 31 of Bonome, 663 of Praenkel, 25, 31, 589, 717 of Jaeger and Heubner, 683 of Neisser, 667, 720 of Weichselbaum, 682, 722 pneumoniae, 25, 31, 589, 717 Diplogonoporus grandis, 145 Diplomellituria, 287 Dipylidium caninum, 141 cucumeHnum, 141 Distoma capense, 585 pulmonale, 29 Ringeri, 29 Westermanii, 29 Distomiasis, 585 Distomum buski, 158 cavise, 147 47 Digitized by Distomum conus, 159 crassum, 158 hematobium, 30, 585 hepaticum, 157 hepatis endemicum seu perniciosum 159 hepatig innocuum, 159 japonicum, 159 lanceolatum, 159 sibiricum, 159 sinense, 159 spathulatum, 159 tenuicoUe, 159 Ditrachyceros rudis, 153 Dittrich's plugs, 6 Dochmius ancylostomum, 151 duodenalis, 151 Ddhle's inclusion bodies, 554 Donne's test for pus, 354 Donogany's test for hemoglobin, 282 Doremus ureometer, 222 Dorrance's method for coagulation-time , 402 Drigalski and Conradi's media, 130 Drop method for Wassermann test, 629 Dropsical cells, 489, 529 Dropsy of chorionic villi, 384 Drugs, effects of, on blood, 429, 516 reactions of, in urine, 183 Dry test of feces for blood, 1 1 1 Ducrey's bacillus, 669 Durham's hemocytometer, 467 Dum-dum fever, 576 Dwarf tape- worm, 142 Dysentery, amebic, 135 bacillary, 132 Dysmenorrhea, 383 Dyspepsia, 91 Earthy phosphates, 199, 201 Eberth's bacillus, 26, 130, 366, 587, 712 Echinococcus in feces, 145 in sputum, 9 in urine, 366 Ectasis gastric, 85 Eel, vinegar, 366 Effusions, pleuritic, 674 Egg-yellow reaction, 335 Egyptian chlorosis, 153 Ehrlich's anemia, 534 anemic degeneration, 497 classification of leucocytes, 510 dahlia stain, 507 diazo reaction, 334 dimethylaminobenzaldehyd reac- tion, 336 egg-yellow reaction, 335 hemoglobinemic degeneration, 499 side-chain theory, 594 tri-acid stain, 480 triple stain, 480 Einhorn's method for total acidity, 72 saccharometer, 303 Elastic tissue in feces, 1 15 in sputum, 12 Electric conductivity of blood, 406 of urine, 371 M/|-Mffik WaUs,"42 ^'^ 738 INDEX Endamoeba coli, 137 gingivalis, 42 histolytica, 134 tetragena, 135 Endotheliosis, 674 Enteritis, catarrhal, 116 malignant, 118 membranous, 112 mucous, 112 Enterokinase, 1 02 Enteroliths, 118 Enthelmintha, 139 Entozoa in feces, 139 Enumeration of blood cells, 455, 465, 468 of pus cells, 354 Eosin-hematoxylin stain, 479 methylene-blue stain, 47 Eosinophiles, 506 Eosinophilia, 519 Eosinophilic bronchitis, 10 Epicritic elimination ol nitrogen, 216 polyuria, 179 Epidemic cerebrospinaj meningitis, 682 Epiguanin, 240 Episarkin, 240 Epistaxis, Gull's renal, 355 Epithelial casts, 359 cells in feces, 119 in gastric contents, 62 in serrien, 377 in sputum, 1 1 in urine, 349 Erepsin, 102 Error in cell counting, 466 Erythrasma, 169 Erythroblasts, 490 Erythrocytes, 486 appearance of, 486 color-index of, 426 counting of, 455 crenation of, 487 degenerations of, 498 formation of, 386 functions of, 501 isotonicity of, 499 nucleation of, 490 number of, 492 pathological types of, 488 recognition of, in stains, 654 resistance of, 499 rouleaux formation of, 486 shape of, 488 size of, 488 staining properties of, 496 structure of, 486 variations of, 492 Erythrocytometer, 456 Erythrocytosis, 495 Erythrodextrin, 36, 82 Esbach's method for albumin, 272 Essential albuminuria, 261 pentosuria, 308 renal hematuria, 355 Esterification method of Fischer, 247 Estivo-autumnal malaria, 566, 569 Ethereal sulphates, 207, ^10 Euchlorhydria, 70 Euglobulin, 275 European cat-fluke, 159 Digitized by European hook-worm, 151 Eustrongylus gigas, 160 visceralis, 160 Ewald test-meal, 54 Ewald and Siever's method for gastric motility, 85 Exercise, effect of, on red cells, 494 leucocytosis due to, 517 Extraction method for fat, 694 Extraneous material in sputum, 9 Extruded intracellulars, 564 Exudates, 662 bacteriology of, 667 chyloid, 665 chylous, 665 conjunctival, 47 cytology of, 672 formation of, 662 hemorrhagic, 665 obtaining of, 663 peritoneal, 675 pleural, 674 properties of, 663 purulent, 666 putrid, 666 serofibrinous, 664 serous, 664 urethral, 668 False albuminuria, 261 •Famine fever, 574 Fa^ciola hepatica, 157 humana, 157 Pasciolopsis buski, 158 Pasting stomach, contents of, 60 Fat in blood, 449 in exudates, 665 in feces, 115, 123 in milk, 693 in urine, 346 Fatty acids in blood, 449 in exudates, 665 in feces, 115, 123 in sputum, 13 in urine, 249 casts, 359 granules in leucocytes, 509 stools, 107 Favus, 165 Febrile albuminuria, 264 albumosuria, 280 anemia, 538 diseases, blood in, 549 urine, 179 Fecal vomitus, 61 Feces, 99 amount of, 104 bacteriology of, 128 bile acids in, 118 biliary pigments in, 108, 117 blood in, 108 carbohydrates in, 116, 123 • chemical examination of, 121 color of, 116 concretions in, 11 81 consistency of, 105 crystals in, 120 fat in, 114, 123 INDEX 739 Feces, formed, 105 macroscopic examination of, 103 marking of, loi microscopic examination of, 119 morphological elements in, 119 mucus in, 11 1 normal, 99 odor of, 106 parasitology of, 133 protein in, 114 pus in, 113 reaction of, 121 tissue fragments in, 118 total nitrogen of, 122 total solids of, 121 unformed, 105 Pehling's test for glucose, qualitative, 291 quantitative, 295 Female secretions, 380 Fermentation method of Schmidt, 124 test for diphtheria bacillus, 40 for glucose, 293, 303 Fermentative dyspepsia, 117' Ferments in blood, 454 in feces, 102 in gastric juice, 64, 77 in leucocytes, 454 in sputum, 6 intestinal, 102 in urine, 251 pancreatic, 102 Ferrocyanide test for albumin, 271 Ferrometer of JoUes, 453 Fibers, elastic, 12, 115 muscle, in feces, 115 in gastric contents, 59 Fibrin ferment, 402 in blood, 404 in urine, 283 network, 404 significance of, 404 tests for, 283 Fibrinogen, 402 Fibrinous casts in sputum, 8 in urine, 359 Fibrinuria, 283 Filaria Bancrofti, 577 in blood, 577 in urine, 346, 366 nocturna, 577 sanguinis hominis, 577 Filariasis, 577 Finely granular cells of Schultze, 504 Fischer s esterification method, 247 test-meal, 55 Fish tape- worm, 144 Fittipaldi's method for albumose, 279 Fixation of complement, 617 of smears, by chemicals, 475 by heat, 473 Fixed alkalinity, 187 Flagella, staining of, 708 Flagellata in feces, 137 in sputum, 28 in urine, 366 Plat worms, 139 Pleischl-Miescher hemometer, JJ7 . . Flexner's serum, 683 IJigttlZea by Florence's test for seminal fluid, 379 Fluids, diluent for blood, 459, 466 Fluke- worms, 145, 156 Folin's method for acetone, 323 for acidity of urine, 185 for ammonia, 230 for creatin, 245 for creatinin, 243 for free mineral acidity, 185 for indican, 260 for sulphates, 210 for urea, 223 for uric acid, 235 standard diet, loi Folin and Denis' method for ammonia, , 444 for non-protein nitrogen, 430 urea, 435 uric acid, 439 Folin and Parmer's method for total nitrogen, 431 Folin and Macallum's method for am- monia, 230 Foreign bodies in sputum, 9 Form of stools, 105 Formaldehyd as preservative, 177, 696 Formalin method for ammonia, 231 Formation of blood, 386 of casts, 356 of exudates, 662 Fraenkel's diplococcus; 25, 30, 589, 717 Fragments of tissue in feces, 118 in gastric contents, 63 in sputum, 12 in urine, 363 Free hydrochloric acid, 65 amount of, 69 detection of, 66 determination of, 67 formation of, 51 significance of, 70 variations of, 70 Freezing point of blood, 405 or urine, 371 Fresh blood, 470 Friedlander's bacillus, 25, 46 Frommer's test for acetone, 321 Fucose, 308 Functional albuminuria, 261 diagnosis, 370 hematuria, 355 Functions of gastric ferments, 84 of intestinal ferments, 102 of leucocytes, 522 of red cells, 501 Fusaria mystax, 146 vermicularis, 147 Fusiform bacillus of Vincent, 40 ^ Futcher and Laz ear's fixation method, 476 malarial stain, 484 Gabbet's staining method, 2 1 Gabritschewsky's polychromatophilia, 497 i GaflEky's table, 23 Galactosuria, 305, 312 Gall stones in feces, 117 ter, 417 .. ...Gall stones in leces, 117 Vigftited by Microsg^krance of, 117 740 INDEX Gall stones, composition of, 117 Gamete, 564 Gametocyte, 564 Gametoschizonts, 572 Gas bacillus, 133 Gases in blood, 454 in feces, 125 in gastric contents, 83 Gastric carcinoma, 91 contents, 50 acetone in, 84 after test meals, 62 amino acids in, 83 bacteria in, 62 blood in, 59, 61, 83 crystals in, 63 digestion products in, 82 epithelial cells in, 62 food remnants in, 62 from fasting stomach, 60 from vomitus, 60 gases in, 83 indirect examination of, 87 macroscopic examination of, 57 microscopic examination of, 62 mucus in, 60, 62, 91 obtaining of, 52 protozoa in, 63 pus in, 62 tissue fragrnents in, 63 crises, 89 juice, 57 acetic acid in, 64, 73, 77 acidity of, 64 amount of, 57 butyric acid in, 64, 73, 77 combined hydrochloric acid in, 71 composition of, 64 deficit of hydrchloric acid in, 73 ferments of, 64, 78 free hydrochloric acid in, 65, 92 hyperacidity of, 65, 71, 88 hypersecretion of, 89 hypoacidity of, 65, 70 in disease, 88 lactic acid in, 74, 92 organic acids in, 73 Pawlow's work on, 51 properties of, 57 secretion of, 51 motility, 84 ulcer, 91 Gastrin, 51 Gastritis acute, 90 atrophic, 90 chronic, 90 Gastrosuccorrhea, 89 Gelatin, nutrient, 702 Genital organs, secretions of, 376 Genito-urinary tuberculosis, 352, 365 Gerhardt's test for diacetic acid, 324 , Ghoreyeb''s stain for spirochsete, 670 Gieinsa's stain, 483, 670 Gigantoblasts, 491 Gigantocytes, 489 Glanders, bacillus of, 27 Globulin-albumin in blood, 275, 429 in exudates, 663 in milk, 693 in urine, 274 significance of, 275 tests for, 275 Glomerular insufficiency, 371 Glossina-palpalis, 575 Glucose agar, 703 in the blood, 446 in the urine, 284 determination of, 29's significance of, 284 tests for, 288 Glutoid capsules, 103 Glycemia, 446 Glycerin agar, 703 Glycocholic acid, 117, 191 GlycocoU, 246 Glycogen in the blood, 449 Glycosuria, 284 alimentary, 285 after poisoning, 287 after use of drugs, 287 diabetic, 286 e saccharo, 284 ex amylo, 284 masked, 293 neuro-hepatogenous, 286 occult, 293 physiologic, 285 salt, 284 transitory, 285 Glycosuric acid, 333 Glycuronic acid, 314 Glycyl-tryptophan test, 93 Gmelin's reaction for biliary pigments, 330 Goldhorn's stain, 580 Goldschmiedt's test for glycuronic acid, 316 Gonococcus, 667, 720 Gonorrhea, complement-fixation test in, 637 Gonorrheal conjunctivitis, 48 stomatitis, 41 threads, 353, 669 urethritis, 669 Goodman and Stern'^ method for albu- min, 273 Gout, blood in, 548 perinuclear granules in, 505 urine in, 234 Gowers' hemoglobinometer, 421 Gram-negative organisms, 668 Gram-positive organisms, 668 Gram's stain, 668 Granular casts, 357 cells in blood, 504, 506, 507 in prostatic fluid, 378 in sputum, 10, 11 degeneration, 497 Granules in blood, acidophjle, 506 basophile, 498, 506 of Grawitz, 498 Ehrlich's a, 506 ^, .506 Microsoft<$; I07 INDEX 741 Granules in blood, Ehrlich's e, 504 eosinophile, 506 fatty, 509 glycogen, 509 Grawitz, 498 hemoconien, 525 in malaria, 563, 565, 567 mast cell, 506 melanin, 563, 565, 567 Neusser's, 505 neutrophile, 504 oxyphilic, 506 perinuclear, 505 sudanophile, 509 in sputum, 1 1 Grape-sugar in urine, 284 Gravel in urine, 367 Grawitz' basophilia, 498 Green sputum, 3 vomitus, 61 Griess-Ilosvay reagent, 36 Grinders' rot, 4 Gross' method for trypsin, 102 Ground itch, 152 Gruber-Widal reaction, 607 Guaiac test for blood, 109, no, 655 Guanin, 240 Gull's renal-epistaxis, 355 Gum, animal in urine, 314 Gummatous lymphoma, 545 Gunning's mixture, 217 test for acetone, 321 Gunzburg's package, 87 reagent, 66 test for free hydrochloric acid, 66 Gynecophorus haematobius, 367, 585 Haeser's coefficient, 189 Haines' coefficient, 189 test for glucose, qualitative, 291 quantitative, 299 Haldane and Smith's method for volume of blood, 388 Halitus sanguinis, 394 Hammarsten's test for biliary pigments, 331 Hammerschlag's method for specific gravity, 400 for pepsin, 79 Haptines, 595 Haptophore, 594 Hard chancre, organisms of, 669 Hart's method for |8-oxybutyric acid, 326 Harvest bug, 162 Hayem's solution, 459 Hay fever, 45 Hay's test for bile acids, 331 Head louse, 163 Heart disease cells, i, 32 pleurisy of, 675 Heat fixation of smears, 473 Heat test for albumin, 267 Hehner-Maly method for organic acids, 73 Heller's table for examination of calculi, 369 , test for albumin, 268 for hemoglobin, 283Digitized by Microsaft&ritis, 355 Hemameba malarias, 565 vivax, 563 Hemamebiasis, 561 Hematemesis, 3, 61 Hematin, 413 hydrochlorate, 414, 658 Hematoblasts, 523 Hematochyluria, 578 Hematocrit, 390 Hematogenous albuminuria, 265 albumosuria, 280 urobilinuria, 255 Hematoglobulin, 41 1 Hematoidin in the blood, 414 in sputum, 11, 13 in urine, 328 Hematopoietic organs, 386, 525 Hematoporphyrin in blood, 414 in feces, 108 in stains, 658 in urine, 328 Hematoporphyrinuria, 328 Hematuria, 354 angioneurotic, 355 constitutional, 355 essential, 355 extra-renal, 355 functional, 355 idiopathic, 355 renal, 355 Hemin, 414, 658 Hemoalkalimeter of Dare, 396 Hemochromogen, 412 Hemoconien, 525 Hemocytometer of Durham, 467 of Oliver, 468 of Thoma-Zeiss, 456 Hemoglobin in blood, 409 amount of, 425 derivatives of, 413 estimation of, 415 properties of, 409 variations of, 425 in sputum, 3, 5, 11, 13 in urine, 281, 328 tests for, 282 quotient, 426 value, 426 Hemoglobinemia, 281 Hemoglobinemic degeneration, 499 Hemoglobinometer of Dare, 420 of Oliver, 422 of Tallqvist, 424 Hemoglobinuria, 281, 328 paroxysmal, 281 significance of, 281 tests for, 282 Hemolysins, 596 Hemolysis, 596 Hemolytic anemia, 537 Hemometer of Fleischl-Miescher, 417 of Sahli, 421 Hemophilia, renal, 355 Hemoptysis, 3 Hemo-renal index, 452 Hemorrhage, anemia due to, 535 occult, 108 Hemorrhagic exudates, 665 742 INDEX Hemosiderin, ii, 415 Hepatic insufficiency, 125, 222, 286, 305 Hepatogenous albumosuria, 280 urobilinuria, 255 Herman-Perutz reaction, 651 Herpes tonsurans, 166 Heteroalbumosuria, 277 Heterochylia, 89 Heteroxanthin, 240 Hexamitus duodenalis, 138 Hexone bases, 83, 91, 247 Hippuric acid in' urine, 248, 345 Histidin, 247 Histoplasma capsulatum, 576 Histoplasmosis, 576 Hodgkin's disease, 544 Hoffman's bacillus, 710 Homogentisic acid, 333 Hopkin's method for uric acid, 235 Hoppe-Seyler's colorimetric pipet, 416 Howell's immature nucleated reds, 490 mature nucleated reds, 490 Huppert-Messinger method for acetone, 322 Hyaline casts, 356 ' Hydatid cysts, 143, 678 Hydatidiform degeneration, 384 Hydremia, 389 Hydrobilirubin in stools, 107 Hydrocele agar, 704 fluid, 677 Hydrochloric acid in gastric juice, 51 amount of, 69 combined, 71 deficit, 73 estimation of, 67 free, 65 physiologically active, 72 tests for, 66 Hydrogen sulphide in gastric contents, 83 in urine, 208 Hydronephrosis, 678 Hydrops folliculorum Graafii, 676 Hydroquinone-acetic acid, 333 Hydruria, 178 Hymenolepis diminuta, 142 flavopunctata, 142 murina, 142 nana, 142 Hypalbuminosis, 428 Hyperacidity of gastric juice, 65, 71 Hyperalbuminosis, 428 Hyperchlorhydria, 7.1, 88 Hyperglycemia, 446 Hyperinosis, 404 Hypermotility of stomach, 85 Hypersecretion of gastric juice, 89 Hypertonic solutions, 500 Hyphogenous sycosis, 166 Hypinosis, 404 Hypochlorhydria, 70 Hypostatic albuminuria, 264 Hypotonic solutions, 500 Hypoxanthin, 240 Idiopathic enterogenous cyanosis, 412 pentosuria, 308 Ilosray's reagent, 36 „ Digitized by Immature nucleated reds of Howell, 490 Immunity, 593 Inactivation of serum, 596 Inanition, anemia due to, 536 Incubation, 704 Index, bacillary, 599 color, 425 hemoglobin, 426 opsonic, 599 phagocytic, 599 volume, 391 India ink method for spirochsete, 670 Indican, 256 Indicanuria, 256 Indigo blue in urine, 256 red in urine, 258 Indirect examination of gastric contents, Indol-acetic acid, 260 Indoxyl-potassium sulphate in urine, 256 Infectious diseases, blood in, 549 Inflttenza, bacillus of, 25, 711 sputum in, 33 Infusoria, in feces, 138 in sputum, 28 in urine, 366 Inorganic constituents of blood, 452 or urine, 192 Inoscopy, 667 Inosite in urine, 314 Insecta, 163 Insufficiency, digestive, 113 glomerular, 371 hepatic, 125, 222, 286, 305 motor, 84 renal, 371 tubular, 371 Intermittent albuminuria, 263 Intestinal concretions, 118 digestion, 102 juices, 102 obstruction, 114 parasites, 133 sand, 118 Iodide of potassium test, 86 Iodoform test for acetone, 321 for lactic acid, 76 lodophilia, 509 Iron in the blood, 453 in urine, 213 Irritation forms of leucocytes, 509 Isoagglutinins, 653 Isohemolysins, 653 Isomaltose in urine, 314 Isotonicity of red cells, 499 Isotonic solutions, 499 Itch parasite, 162 Jaeger and Heubner's diplococcus, 683 Jaffa's test for creatinin, 243 for indican, 257 von Jaksch's anemia, 533 Japanese liver-fluke, 159 Jaundice, blood in, 452 sputum in, 4 urine in, 182, 329 INDEX 743 Jenner's stain, 481 Jigger, 165 Jolles' ferrometer, 453 Jousset's fluid, 667 Juice, gastric, 57 intestinal, loi Justus' test for syphilitic blood, 559 Kahler's disease, 277 Kala-azar, blood in, 576 parasite of, 576 Karyomorphism of neutrophiles, 505 Kastle and Loevenhart's method for lipase, 252 Kathrein's test for bile pigments, 330 Kelling's test for lactic acid, 75 Kendall and Day's method for typhoid bacilli, 131 Kidney, abscess of, 352 acute inflammation of, urine in, 178, 180, 190, 194, 201, 217, 266 amyloid disease of, urine in, 178, 181,253,266,275 cancer of, 354, 363 chronic inflammation of, urine in, 178, 179, 181, 190, 194, 201, 266 echinococcus cysts of, 366 hemorrhagic lesions of, 354 hydronephrotic cysts of, 678 malignant disease of, 354, 363 stones, 367 suppurative lesions of, 352 syphilitic disease of, 266 tubercular, 352 Kjeldahl's method for nitrogen, 217 Klebs-Lofiler bacillus, 38, 709 Knop-Hufner method for urea, 222 Koch's bacillus, 18, 132, 364, 592, 667 comma bacillus, 61, 129, 715 tuberculin, 18, 601 Koch-Weeks bacillus, 48 Kohlrausch's method for electric con- ductivity, 371, 406 Kowarsky's method for sugar in blood, 447 KJrabbea grandis, 145 Kreatin (see Creatin), 241 Kreatinin (see Creatinin), 241 Lab, 64, 81 Labor, albuminuria following, 262 Lactic acid in blood, 450 in gastric contents, 74 in carcinoma, 92 significance of, 74 tests for, 75 in urine, 249 Lactose in milk, 695 in urine, 312 Lactosuria, 312 Lactose-Utmus agar, 703 Laiose, 306 Laking of blood, 394 LambUa intestinalis, 138 Landau's test, 652 Lange's colloidal-gold test, ESgi/f/zecf by Large lymphocytes, 502 mononuclear leucocytes, 503 Larvae in aural secretions, 47 in feces, 148, 149, 153 Laveran's malarial organism, 561 Layers of sputum, 5 Lead, anemia due to, 538 basophilia in poisoning, 498 Lecithin globules in semen, 377 Legal's test for acetone, 320 Leiner's test for casein, 115 Leishman-Donovan bodies, 576 Le Nobel's test for acetone, 320 Leo's method for chymosin, 81 Leprosy, baciUus of, in sputum, 24 blood in, 559 Leptodera intestinalis et stercoralis, 147 Leptothrix buccalis, 38 in sputum, 15 Leptus autumnalis, 162 Leube's test of gastric motility, 85 Leucin in sputum, 13 in urine, 246, 343 Leucocytes, 502 appearance of, 502 basophilic, 506 counting of, 465 degenerated forms of, 509 differential counting of, 510 eosinophilic, 506 ferments of, 454 formation of, 387 functions of, 522 granules in, 498, 506, 507 in blood, 502 in exudates, 673 in feces, 120 in gastric contents, 62 in milk, 698 in sputum, 10 in urine, 351 irritation forms, 509 karymorphism of, 505 large mononuclear, 503 lymphocytes, 502 mkst-cell, 506 myelocytes, 507 neutrophilic, 504 number of, 511 oxyphilic, 506 pigmented, 568 polymorphonuclear, 504 small mononuclear, 503 splenocytes, 503 transition forms of, 504 types of, 502 variations in number of, 511, 521 Leucocytic crystals in sputum, 14 Leucocytometer, 457 Leucocytosis, 512 agonal, 516 antemortem, 516 cachectic, 515 eosinophilic, 519 infectious, 515 inflammatory, 515 mast-cell, 521 Micros&fti^, 518 744 INDEX Leucocytosis of digestion, 513 of pregnancy, 514 of the new-born, 514 polymorphonuclear, 512 post-hemorrhagic, 516 therapeutic, 516 Leucohydrobilirubin in feces, 107 Leucopenia, 521 Leucorrhea, 381 Leucourobilin, 107 Leukanemia, 534 Leukemia, 539 acute, 543 lymphatic, 543 mixed, 539 splenomyelogenous, 539 Levaditi and Manouflian's method for staining treponema, 671 Levulose in urine, 305 determination of, 306 recognition of, 306 significance of, 305 tests for, 306 Levulosuria, 305 Lieben's test for acetone, 321 Lientery, 113 Limitations of blood examinations, 659 Limnasa truncatula, 158 ., Lipacidemia, 450 Lipaciduria, 249 Lipase in gastric juice, 64, 82 in pancreatic juice, 102 , in urine, 252 Lipemia, 450, 547 Lipliawsky's test for diacetic acid, 325 Lipuria, 346 Liquor sanguinis, 393 Lithemic diathesis, 234 Litmus-glucose agar, 703 lactose agar, 703 milk, 704 Liver, abscess of, sputum''in, 33 insufficiency of, 125, 222, 286, 305 Lobar pneumonia, blood in, 550 chlorids in urine of, 194 organism of, 25, 31 sputum in, 31 Lochia alba, 383 cruenta, 383 rubra, 383 serosa, 383 LofSer's antiformin method, 18 methylene blue, 2 1 Lohnstein's saccharometer, 303 Long's coefficient, 189 Lordotic albuminuria, 263 Luetin reaction, 603 Lumbar puncture, 679 Lung, abscess of, sputum in, 33 fluke in sputum, 29 inflammation of, 31, 194, 550 stones, 8 Lymphatic leukemia, 543 pseudoleukemia, 544 Lymphemia, 543 Lymphocytes, 502 Lymphocytosis, 518 Lymphopenia, 518 _. .^. Digitized by Lymphosarcoma, 545 Lysins, 596 Lytic action, 596 Macrocytes, 489 Macrocythemia, 489 Macrocytosis, 489, 531 Macrogamete, 565 Magnesium ammonium phosphate in feces, 120 in sputum, 13 in urine, 348 phosphate in urine, 198, 348 salts in urine, 211 ' soaps in feces, 116 Malaria, blood in, 572 fresh blood in, 563 mosquito theory of, 561 parasites of, 563 estivo-autumnal, 566, 569 quartan, 565, 569 tertian, 563, 568 stained smears in, 568 Malarial pigment, 415 Male secretions, 376 Malfatti's method for ammonia, 231 Malignant disease, blood in, 559 gastric juice in, 91 urine in, 354, 366 lymphoma, 545 pleurisy, 675 Mallory's stain for Negri bodies, 173 Malone's test for pregnancy, 247 Maltose in urine, 313 Mammary secretions, 688 Maragliano's endoglobular degeneration, 497 Marechalt's test for bile pigments, 330 Marshall's method for urea, 225 Martius and Liittke's method for HCl, 71 Marx s fluid, 654 Mast-cell granules, 506 leucocytosis, 521 Masturbators, albuminuria of, 263 Mature nucleated reds of Howell, 490 May-Grunwald stain, 482 McCrudden's method for calcium, 212 Meals, test, 54 Measles, blood in, 554 Medicinal leucocytosis, 516 Medico-legal aspects of blood, 654 of semen, 378 Megaloblasts, 491 Megalocytes, 489 Megalogastria, 85 Megastoma entericum, 138 intestinale, 138" Melanin, 182, 332, 415 Melanogen, 182 Melanuria, 332 Membranous dysmenorrhea, 383 enteritis, 112 ureteritis, 252 Meningeal fluid, examination of, 678 Meningitis, epidemic cerebrospinal, 682 tubercular, 684 Meningococcus of Bonome, 683 of,Weirhselbaum, 683, 721 Microsoft® INDEX 745 Menstruation, 382 Messinger method for acetone, 322 Metalbumin in ovarian cysts, 677 Metamyelocytes, 505 Methemoglobin, 41 1 Methylene azure stains, 481 blue in urine, 183, 335 stains, 21, 478 test for functional activity, 372 Methylphenylosazon, 307 Methylxanthin, 240 Mett's method for pepsin, 79 Meyer's test for blood, 656 Microblasts, 490 Micrococcus, catarrhalis in sputum, 17, 722 tetragenus in sputum, 17 Microcytes, 488 Microgametes, 565 Microgametocytes, 565 Microscopy of blood, 469 of exudates, 672 of feces, 1 19 of gastric contents, 62 of milk, 696 of semen, 376 of sputum, 9 of urine, 336 Microsporon Audouini, 167 furfur, 167 minutissimum, 169 scorteum, 166 Miescher's hemoglobinometer, 417 Milk, 688 appearance of, 690 ash of, 691 bacteriology of, 696 coagulation of, 691 composition of, 689 cow's, 689 curds in stools, 115 fat of, 693 human, 689 lactose of, 695 microscopy of, 696 preservatives in, 695 properties of, 689 protein of, 691 reaction of, 691 specific gravity of, 690 sugar of, 695 total solids, 691 Milk-curdling ferment, 64, 81 Milky zone, 563 Mineral acidity of urine, 185 Mintz's method for free HCl, 68 Mixed infection in tuberculosis, 23 leucocytosis, 518 Molds in aural secretions, 47 in buccal secretions, 36 in sputum, 16 Moller's method for spores, 708 Monocalcium phosphate in urine, 346 Monocercomonas hominis, 138 Monochromatophilia, 496 Mononuclears, basophile, 508 eosinophile, 508 large, 502 neutrophile, 508 Digitized by Mononuclears, small, 502 Moore and Wilson's test for alkalinity^ 395 Morax-Axenfeld diplobacillus, 48 Morner-Sjoqvist method for urea, 225 Morner's mucin-like bodies in urine, 253, test for tyrosin, 345 Morning sputum, i Moro's tuberculin reaction, 601 Morphology of blood, 469 of blood-forming organs, 525 Mosquito, anopheles, 561 culex, 583 cycle of malarial paras'tes, 571 stegomyia, 583 theory of malaria, 561 of yellow fever, 583 Motility of intestine, 104 of stomach, 84 detection of, 85 types of, 84 Motor insufficiency, 84 Moults, 161 Mouth, inflammation of, 40 catarrhal, 40 gonorrheal, 41 mycotic, 41 ulcerative, 40 ulceromembranous, 40 secretions of, 35 Much's method for tubercle bacilli, 22 Mucin in ovarian cysts, 677 in sputum, 5 'in urine, 252 Mucinophiles, 507 Miicoid material in urine, 349 sputum, 5 Mucopurulent sputum, 5 Mucor in sputum, 16 Mucous corpuscles, 349 threads in urine, 353, 669 Mucus in feces, 1 1 1 appearance of, 11 1 detection of, in significance of, 112 in gastric contents, 60, 62, 91 in sputum, 5 in urine, 252 MuUern's blood stain, 478 Murexid test, 339 Muscle fibers in feces, 114 in gastric contents, 60 in sputum, 12 Mycelial casts in sputum, 8 Myelemia, 539 Myelin granules in sputum, 1 1 Myeloblasts, 503 Myelocytes, 507 basophile, 508 eosinophile, 508 neutrophile, soS Cornil's, 508 Ehrlich's, 508 Myelocytosis, 518 Myelogenous leukemia, 539 Myeloid leukemia, 539 Myelopathic albumosuria, 277 Myxedema, blood in, 549 ^dium stegomyiae, 583 746 INDEX Myxoid cyst of ovary, 676 Myxoma of the placenta, 384 Nakayama's test for bile pigments, 330 Naphthoresorcin test, 316 Nasal secretion, 44 bacteria in, 44 composition of, 44 concretions in, 46 pathology of, 44 spinal fluid in, 45 Negri bodies, 172 Neisser's diplococcus, 667, 720 stain for diphtheria bacillus, 39 Nematodes in feces, 145 in urine, 366 Nephritic albuminuria, 265 hematuria, 355 oliguria, 179 pleurisy, 675 Nephritis, acute, 178, 180, 190, 194, 201, 217,265 albuminuria of, 265 chronic diffuse, 190, 194, 201, 265 interstitial, 178, 182, 265 parenchymatous, 178, 180, 265 hemorrhagic, 355 suppurative, 352 syphilitic, 266 unilateral, 371 Nervous dyspepsia, 90 type of albuminuria, 265 of pol3niria, 179 Neubauer and Fischer's test for gastric carcinoma, 93 Neuberg and Wohlgemuth's method for pentose, 310 Neuberg's test for glycuronic acid, 316 for levulose, 306 Neusser's granules, 505 Neutral calcium phosphate in urine, 346 dyes, 477 stains, 477 sulphur, in urine, 208 Neutrophile cells, 504, 508 granules, 504 Neutrophilic karyolobism, 505 New-born, albuminuria of, 262 leucocytosis of, 514 Night urine, 178 Nikiforoff's method of fixation of smears, 475 Nitric acid test for albumin, 268 Nitrites in saliva, 36 Nitrogen of sputum, 2 or urine, 213 • partition of blood, 430 of urine, ^4 Nitrogenous balance, 213 bodies in blood, 430 in exudates, 664 in feces, 122 in gastric contents, 93 in milk, 692 in sputum, 2, 5 in transudates, 664 in urine, 213 allantoin, 248 Digitized by Nitrogenous bodies in urine, alloxypro- teic acid, 208, 248 amino-acids, 246 ammonia, 227 creatinin, 241 hippuric acid, 248, 345 oxyproteic acid, 248 purin bases, 240 total, 213 undetermined, 246 urea, 219 uric acid, 232 equilibrium, 213 Nitroprussid'test for acetone, 320 Nocht's malarial stain, 484 Noguchi's antigen, 622 butyric acid test, 685 luetin reaction, 603 method for spirochete, 581, 671 modification of Wassermann test, 629 Nondiffusible alkalinity of blood, 395 Nonne's test of cerebrospinal fluid, 685 Nonprotein nitrogen of blood, 430 Normal feces, 99 salt solution, 500 Normoblasts, 490 Normocytes, 486 Nose, secretions of, 44 Nubecula, 180 Nubecular threads, 180, 349 Nucleated red cells, 490 Howell's immature, 490 mature, 490 Nuclein bases, 122, 191, 240, 341 Nucleinic acid, 232, 253 Nucleo-albumin in urine, 252 Number of blood plates, 524 of leucocytes, 511 of red cells, 492 of stools, 104 of tubercle bacilli in sputum, 22 Nummular sputum, 5, 6 Nutrient agar, 702 broth, 701 gelatin, 702 Nutrition, effect of, on blood, 493, 536 Nycturia, 178 Nylander's test for glucose, 292 Obermayer's test for indican, 257 Obermeier's spirillum, 574 Obtaining blood, 392, 585 exudates, 663 gastric contents, 52 Occult blood in feces, 108 Ochronosis, 333 Odor of blood, 394 of exudates, 664 of feces, 106 of gastric contents, 59 of sputum, 4 of urine, 183 Oidium albicans, 16, 41 Oil test-breakfast, 102 Oligemia, 388 M;9fe§^^^"^^'42^ INDEX Oligocythemia, 495 Oliguria, 179 Oliver's hemocytometer, 468 hemoglobinometer, 422 test for bile acids, 332 Oocyst, 572 Ookinet, 570 Operation, blood after, 546 Opisthorchis felineus, 159 sinensis, 159 Oppler-Boas bacillus, 62 Opsonic index, 599 Opsonins, 598 Optical activity of carbohydrates, 300 of conjugated glycuronates, 301 of glycuronic acid, 301 of urine, 190 Oral secretiojis, 35 • Orcein stain, 12 Orcin test for pentose, 310 Organic acidity of urine, 185 acids in gastric contents, 73 in urine, 249 Organized sediments in urine, 349 bacteria, 363 blood cells, 354 casts, 356 epithelial cells, 349 mucoid material, 349 parasites, 366 pus cells, 351 spermatozoa, 363 tissue fragments, 363 Origin of casts, 356 of leucocytes, 387 of red cells, 386 Orthostatic albuminuria, 263 Orthotic albuminuria, 263 Osier's disease, 496 Osmotic pressure of blood, 405 of urine, 371 Otornycosis, 47 Ova in feces, 133, 154 in sputum, 29 in urine, 367 of anopheles, 561 * of intestinal parasites, 154 Ovarian cysts, 676 colloid, 676 dermoid, 677 myxoid, 676 papillary, 677 serous, 676 Ovoids in malarial blood, 567 Oxalate of calcium in sputum, 14 in urine, 250, 341 calcvdi, 368 Oxalic acid in urine, 250 amount of, 250 determination of, 251 origin of, 250 variations of, 250 Oxaluria, 250 Oxaluric acid, 250 Oxid of bismuth in feces, 107 j8-oxybutyric acid in urine, 325 determination of, 326 significance of, 326 tests for, 326 Digitized by 747 acid. Oxyhemoglobin, 411 p-oxyphenyl- a-amino-propionic 344 Oxyphilic cells, 496 granules, 506 Oxyproteic acid, 191, 248 Oxyuris vermicularis, 147 Ozena, 46 Palpation, albuminuria due to, 264 Paludism, 561 Pancreatic cysts, 678 disease, feces in, 114 fluid, 678 juice, 102 composition of, 102 ferments of, 102 insufiiciency of, 114 Panoptic staining, 478, 481 Papillary cysts of the ovary, 677 Pappenheim's amblyochromatic erythro- blasts, 491 heteroplastic promyelocytes, 508 method for tubercle bacillus, 2 1 stain for blood smears, 481, 484 trachyochromatic erythroblasts, ,490 Paracresol, 333 Paragonimus westermanii, 29 Paramecium coU, 138 Paramucin, 677 Parasites, anemia due to, 537 eosinophilia due to, 520 in blood, 561 in feces, 133 in sputum, 28 intestinal, 133 in urine, 366 malarial, 561 , of the skin, 161 Parasitology of the blood, 561 of the feces, 133 of the skin, 161 Paratyphoid bacillus, 131, 588 Paraxanthin, 240 Parenchymatous nephritis, acute, 178, 180, 190, 194, 201, 217, 266 chronic, 178, 179, 266 Parhemoglobin, 409 Parovarian cysts, 677 Paroxysmal hemoglobinuria, 281 polyuria, 179 Pathogenic bacteria in blood, 585 in exudates, 667 in feces, 128 in gastric contents, 62 • in milk, 696 in sputum, 17 in urine, 363 Pea-soup stools, 130 Pediculus capitis, 163 pubis, 163 vestimenti, 163 Penicillium glaucum, 16 Pentose in urine, 308 determination of, 310 significance of, 308 tests for, 309 M/BpDfeSft^. alimentary, 308 748 INDEX Pentosuria, essential, 308 idiopathic, 308 intrinsic, 308 Penzoldt and Faber's test, 86 Pepsin in gastric juice, 64, 77 activity of, 77 detection of, 78 determination of, 79 significance of, 78 in urine, 251 Pepsinogen, 77 Peptic glands, 50 Peptone in the blood, 429 in gastric contents, 82 in urine, 281 Peptonuria, 281 Perez' bacillus, 46 Perforating empyema, sputum in, 34 Pericardial fluid, 662 Perinuclear granules of Neusser, 505 Periodic albuminuria, 263 polyuria, 1 79 Peritoneal exudates, 675 composition of, 675 cytology of, 675 Permeability of red cells, 405, 499 renal, 370 Pernicious anemia, 531 Pertussis, blood in, 556 organism of, 26, 711 Pessary forms of red cells, 486 PetrefE's method for tubercle bacilli, 19 Pettenkofer's test, 332 Pfeifler's bacillus, 26, 33, 711 Phagocytic cells, 4, 598 index, 600 Phagocytosis, 598 Pharyngomycosis leptothrica, 38 Phenol in feces, 99 in urine, 332 Phenolphthalin test, 656 Phenolsulphuric acid, 332 Phenolsulphonephthalein test, 373 Phenoltetrachlorphthalein test, 125 Phenylglucosazon, 294 Phenylhydrazine test for glucose, 294 Phloridzin test, 373 Phloroglucin test for pentose, 309 vanillin test for HCl, 66 Phosphates, calcium, 199, 212 in blood, 453 in sputum, 13 in urine, 198 magnesium, 198, 213, 348 magnesium-ammonium, 13, 120, 348 triple, ig, 120, 348 Phosphatic calculi, 368 diabetes, 201 sediments in urine, 346, 347 Phosphaturia, 200, 347 Phosphorus containing proteins, 253 poisoning, blood in, 538 urine in, 221 Phthirius inguinalis, 163 Phthisis, blood in, 557 hemoptysis in, 3 melanotica, 1 1 sputum in, 30 stone-cutters', 4 Digitized by Physiological albuminuria, 261 glycosuria, 284 salt solution, 500 variations in blood cells, 492, 513 Physiologically active HCl, 72 Physis inteStinalis, 153 Pigment, bile, in blood, 451 in feces, 108, 117 in gastric contents, 59, 61 in sputum, 3 in urine, 329 blood, in feces, 108 in gastric contents, 59, 61, 83 in sputum, 3, 5, 11, 12, 14 in urine, 328 coal, in sputum, 4, 34 in leucocytes, 4, 573 in red cells, 497, 563 of blood, 409 of urine, 254, 328 Pin worm, 147 Pineapple test, 77 Piroplasma hominis, 584 Piroplasmosis, 584 von Pirquet's tuberculin reaction, 602 Placenta cells, 384 Plague bacillus, 27 Plasma, 393 Plasmodium falciparum, 566 quotidianum, 565 malariae, 565 precox, 566 tenue, 563 vivax, 563 variety minuta, 563 Platelets, blood, 523 Platodes in feces, 139 Plehn's karyochromatophilic granules, 569 Plethora, cellular, 389 serous, 389 true, 389 vera, 389 Pleuritic effusions, 674 cytology of, 674 withdrawal of, 663 Plugs, Dittrich's, 6 prostatic, 378 Pneumococcus of Praenkel, 25, 31,, 589, 717 pleurisy, 674 Pneumoliths, 8 Pneumonia, blood in, 550 chloridsinurinein, 194 organism of, 25, 31 sputum in, 31 urine in, 194 Pneumonoconioses, 34 Pneumonomycosis aspergillina, 16 Poikilocytes, 489 Poikilocytosis, 489 Poisons, blood, 538 Polariscope, 300 Polariscopic method for glucose, 302 PoKomyelitis, acute anterior, 684 Polychromasia, 496 PolychromatophiUa of Gabritschewsky, 497 INDEX 749 Polychrome dyes, 481 Polychromemia, 495 Polyglobulia, 495 Polymorphonuclear basophiles, 506 eosinophiles, 506 neutrophiles, 504 Polymorphonuclear neutrophiliosis, 512 Polynucleosis, 512, 674 Polyplasmia, 389 Polyuria, 178 epicritic, 179 paroxysmal, 179 periodic, 179 Poor, anemia of the, 536 Pork tape-worm, 140 Posterior urethritis, 353 Post hemorrhagic anemia, 535 infectious albuminuria, .264 Postural albuminuria, 263 Potassium acid urate sediment, 340 ferrocyanide test for albumin, 271 iodide test, 86 of blood, 409 of urine, 211 - sulphocyanate in saliva, 35 Precipitinophore, 598 Precipitins, 598 Precipitin test for blood, 614 Pregnancy, albuminuria of, 262 ammonia in urine of, 228 anemia of, 536 , blood in, 514 tj leucocytosis of, 514 sero-diagnosis of, 638 urine test in, 247 Preparation of blood smears, 47 1 culture media, 701 vaccines, 724 Preservation of urine, 176 Primary anemia, 528 pernicious anemia, 531 proteoses, 276 tubercular pleurisy, 674 Products of gastric digestion, 82 of intestinal digestion, 102 Progressive pernicious anemia, 531 Promyelocytes, 508 Propepsin, 51, 78 Prostatic casts, 378 fluid, 376, plugs, 378 secretion, 376 Prostatitis, 353 Prostatorrhea, 378 Protalbumose, 276 Protein in blood, 427 in exudates, 664 in feces, 114 in gastric contents, 82, 93 in milk, 692 in sputum', 5 in urine, 260 quotient of serum, 429 Proteoses in urine, 276 Prothrombase, 402 Protoryxomyces coprinarius, 137 Protozoa in blood, 561 ■ in feces, 134 r-.- -x- _i u in gastric contents, efilQltlzed by Protozoa in sputum, 28 in urine, 366 Provocative Wassermann test, 636 Prowazek-Greeff trachoma bodies, 48 Prune-juice sputum, 3, 31 Pseudo casts, 361 diphtheria bacillus, 40, 710 elastic tissue, 12 gall stones, n8 globuHn, 275 hemoglobin, 411 leukemia, 544 infantum, 533 mucin, 677 nucleation, 497 parasites, 153 rhabditis stercoralis, 147 Ptomaines in feces, 123 in urine, 342 Ptyalin, 35, 36, 82 Ptyalism, 37 Puberty, albuminuria of, 264 Puerperal infection, 382 Pulex irritahs, 165 penetrans, 165 Pulmonary actinomycosis, 27 gangrene, 33 hemorrhage, 3 tuberculosis, 30 Punctate basophilia of Grawitz, 498 Purdy's method for albumin, 274 for chlorids, 198 for glucose, 298 for phosphates, 205 for sulphates, 210 Purin bases in urine, 191, 240, 34 1 Purpura hemorrhagica, blood in, 53 1 Purpurin, 254 Purulent exudates, 666 sputum,, 5 urine, 351 Pus casts, 359 ceUs in feces, 113 in gastric contents, 62 in sputum, 10 in urine, 351 enumeration of, 354 significance of, 353 tests for, 353 Putrescin, 123 Putrid bronchitis, 32 exudates, 666 Pycnometer, 188, 400 Pycnotic nucleus, 490 Pyelitis, urine in, 351 productiva, 252 Pyelonephritis, 351 Pyloric glands, 50 stenosis, 86, 92 Pyogenic albumosuria, 280 Pyonephrosis, 351 Pyorrhea alveolaris, 42 Pyrocatechin, 333 Pyuria, 351 Quantity of bloodj 387 of gastric juice, 57 M;C^SM^malarial parasite, 565 7 so INDEX Quartan, asexual cycle of, 565 sexual cycle of, 566 Quotient, albumin, 275, 428 protein, 275, 429 volume, 391 Ratio of N to CI output, 193 to PsOb output, 200 to SO3 output, 206 Ray fungus, 27 Reaction of blood, 395 of feces, 121 of gastric contents, 57 of milk, 691 of spinal fluid, 680 of sputum, 3 of urine, 184 Reactivity of blood, 395 Receptors, 595 Rectum, blood in cancer of,'io8 Red cells (see Erythrocytes), 486 in exudates, 665 in feces, 109, 120 in gastric contents, 59, 61, 83 in sputum, 3,5, 11 in suspected stains, 654 in urine, 354 Red, indigo in urine, 258 jigger, 162 sputum, 3 Refraction coefficient of serum, 389, 429 Reichmann's disease, 89 Relapsing fever, 574 Relative value of phosphoric acid, 201 Removal of albumin, 274 of glucose, 294, 303 of tttrbidity, 267 Renal abscess, 351 albumosuria, 280 aneurism, 355 calculus, 367 concretions, 367 diabetes, mellitus, 284, 373 diagnosis, 370 epistaxis, 355 epithelial cells in urine, 349 hematuria, 355 hemophilia, 355 insufficiency, 370 Rennin, 64, 81 Resistance of red cells, 499 Resorcin test for free HCl, 67 Rhabdonema intestinalisj 147 strongyloides, 147 Rhamnose in urine, 308 Rheumatism, blood in, 557 Rhinitis, 45 Rhizopoda in feces, 134 Rice-water stools, 106, 129 vomitus, 61 Rickets; blood in, 549 Rickett's organism of spotted fevgr, 584 Riegel's method for chymosin, 81 test meal, 55 Rigg's disease, 42 Ring bodies in red cells, 498 of Cabot, 498 worm of the beard, 166 of the body, 166 r-.- -xr ^ u ^ Digitized by Ring worm of the scalp, 167 Ringer's solution, 500 Rivalta's test for exudates, 664 Roberts' method for glucose, 304 Roch's test for albumin, 271 Rocky Mountain spotted fever, blood in, 584 organism of, 584 Romanowsky's stain, 481 Ronchfese's method for ammonia, 231 Rosacic acid, 254 Rosenbach's method for bile pigments, 330 for skatoxyl, 258 Rosenow's capsule stain, 590 Rosin's test for bile pigments, 330 Ross- Jones test of cerebrospinal fluid, 686 Rot, grinders', 4 Rouleaux formation, 486 Round worms in feces, 145 Rowntree and Geraghty's test, 373 Rubner's test for lactose, 313 Rudisch and Kleeberg's method for uric acid, 237 Rudolf's method for coagulation time, Ruhemann s uricometer, 239 Russell and Brodie's coagudometer, 403 Russo's test for typhoid, 335 Rusty sputum, 3, 31 Saccharometer of Einhorn, 303 of Lohnstein, 303 Saccharomyces cerevisiae, 15, 288 Saccharose in urine, 314 Sagitula hominis, 153 Sago-like granules in sputum, i, 11 Sahli's desmoid reaction, 87 hemometer, 421 test-meal, 56 Salicylic acid as preservative, 696 test for gastric motility, 85 Saliva, 35 amount of, 35 bacteria in, 36 cells in, 36 chemistry of, 35 ferments in, 35 microscopic examination of, 36 nitrites in, 36 obtaining of, 37 pathologic changes in, 37 ■ potassium sulphocyanate in, 35 _ ptyalin in, 35, 36 Salivary corpuscles, 36 Salivation, 37 Salkowski method for alkalinity o blood, 397 Salkowski-Ludwig method for uric acid, 236 Salol test of Ewald and Sievers, 85 Salomon's test for gastric carcinoma, 93 Salt glycosuria, 284 Salzer's test-meal, 56 Sand flea, 165 intestinal, -118 in urine, 367 M/cros^o^# 367 INDEX 751 Sanguineus exudates, 665 sputum, 5 Saprophytes in feces, 128 in sputum, 14 in urine, 363 Sarcinae in feces, 129 in gastric contents, 63 in sputum, 17 in urine, 363 ventriciili, 63 Sarcoma, blood in, 545 Sarcoptes scabiei, 162 Saturation deficit, 73 Scarlet fever, blood in, 553 Schaer's test for blood, 109, 655 Scherer's method for albumin, 272 test for leucin, 344 Schick reaction, 605 Schistocjrtes, 488 Schistosomum hematobium in blood, 585 m urine, 367 Schizogone, 564 Schizont, 572 Schlosing's method for ammonia, 229 Schmaltz' specific gravity tubes, 399 Schmidt and Strasburger's standard diet, 100 Schmidt's fermentation method for feces, 124 Schugner's granules, 569 Schultze's granular cells, 504, 506 Sclerostoma duodenale, 151 Scybala, 105 Seat worms, 147 Sebelien's method for protein in milk, 692 Secondary anemia, 534 proteoses, 278 tubercular pleurisy, 674 Secretin, 102 Secretion of gastric juice, 51 of genital organs, 376 of mammary glands, 688 of urine, 176 Sedimentation, 336 Sediments in urine, 3156 bacteria, 363 bilirubin, 345 blood cells, 354 calcium carbonate, 349 oxalate, 341 phosphate, 346 sulphate, 345 casts, 356 cholesterin, 346 cystin, 342 epithelial cells, 349 fat, 346 hematoidin, 345 hippuric acid, 345 indigo, 257 leucin, 343 . , , magnesium ammonium phosphate, phosphate, 348 mucoid material, 349 mucous threads, 349 ^. ... nubecular threads, iSoQl^lzed by Sediments, organized, 349 parasites, 366 phosphates, 346, 347 preservation of, 176 pus cells, 351 spermatozoa, 363 tissue fragments, 363 tyrosin, 344 unorganized, 338 urates, 339 uric acid, 338 xanthin, 341 Sedimentum lateritium, 339 Seliwanoff's test for levulose, 306 Semen, 376 chemistry of, 376 medico-legal aspects of, 378 microscopic examination of, 376 pathology of, 378 recognition of stains of, 379 spermatic crystals in, 376 spermatozoa in, 377 Seminal stains, 379 medico-legal aspects of, 378 Septic pleurisy, 674 Sero diagnosis, 606 of pregnancy, 638 of syphilis, 617 of typhoid, 607 Serous cysts of the ovary, 676 exudates, 664 plethora, 389 pleurisy, 664 sputum, 5 Serum albumin in blood, 428 in urine, 261 diagnosis of syphilis,^6i7 of typhoid, 607 globulin, 274 determination of, 276 significance of, 274 test for, 275 variations of, 274 pathology, 593 reactions, 606 refraction coefficient of, 389, 429 special properties of, 593 Sex, variations of blood cells due to, 492 Sexual cycle of malarial parasite, 570 secretions, 376 Shadows of leucocytes, 509 red cells, 486 Shaffer's method for acetone bodies, 327 Shiga's bacillus, 132, 713 Showers of casts, 357 Side-chain theory of Ehrlich, 594 Siderosis, 34 Signet rings in malarial blood, 563, 567 Significance of acetonuria, 318 of albuminuria, 261 of albumosuria, 279 of Bence Jones proteinuria, 276 of cylindruria, 357, 362 of free HCl in gastric contents, 70 of globulinuria, 275 of glycosuria, 284 of hematuria, 355 M;crOSdflf@tic acid in stomach, 74 752 INDEX Significance of leucocytosis, 512 of levulosuria, 305 of mucous threads in urine, 349 of mucus in feces, 1 1 1 of nitrogen-partition of urine, 214 of /3-oxybutyric acid in urine, 325 of pentosuria, 308 of pepsin in gastric juice, 78 of pyuria, 351 Sjoqvist's method for urea, 225 Skatoxyl-potassium sulphate' in urine, 258 ■Skin, blood in diseases of, 520 parasites of, 161 Sleeping sickness, blood in, 575 organism of, 575 Small-pox, blood in, 555 ■Smears, preparation of, 471 of blood, 471 of exudates, 668 of feces, 119 of pus, 668 of sputum, 9 of syphilitic material, 670 Smegma bacillus in buccal secretions, 37 in exudates, 669 in sputum, 24 in urine, 364 preputii, 669 Smith's test for bile pigments, 330 Soaps in feces, 116 Sodium acid urate, 339 carbonate as preservative of milk, 695 chlorid retention, 193 in nephritis, 194, 372 in pneumonia, 194 in blood, 409 in urine, 211 Soft chancre, organism of, 669 Soluble starch, 36, 82 Solvents for blood stains, 654 Specific gravity of blood, 399 of cerebrospinal fluid, 680 of exudates, 664 of milk, 690 of serum, 399 of transudates, 663 of urine, 187 Specificity of agglutination test, 613 of complement-fixation test, 633 of precipitin test, 616 -Spectrophotometer of Hiifner, 416 Spectroscopic examination, 658 tests for blood, 410, 658 Spermatic crystals, 376 Spermatocele, 678 Spermatorrhea, 363, 378 Spermatozoa, 363, 377 Spermin crystals, 14, 376 Spiegler's test for albumin, 271 Spirals of Curschmann, 7, 30, 31 Spirillum of Asiatic cholera, 61, 129, 715 of Obermeier, 574 of relapsing fever, 574 of Vincent, 40 •Spirocheta, buccaiis, 36 Digitized by Spirocheta, pallida, 579 characteristics of, 579, 582 cultivation of, 581 in blood, 579 in exudates, 669 • in tissues, 669 staining of, 580, 669 refringens, 580 Spit cups, 2 Spleen, diseases of, blood in, 532 removal of, 560 Splenectomy, blood after, 560 Splenic anemia, 532 Splenocytes, 503 Splenomegaly, blood in, 532 tropical, 576 Splenomyelogenous leukemia, 539 Spore cyst, 572 Spores, staining of, 708 Sporoblast, 572 Sporogone, 561 SpOrogony of malarial parasite, 570 Sporothrix Schenckii, 171 Sporotrichosis, 171 Sporozoa in feces, 137 Sporozoits, 572 Spotted fever, organism of, 584 Sputum, I air in, 4 albumin in, 5 amount of, 2 bacteria in, 14 biliary pigments in, 3 blood in, 3, 5, 11 character of, 4 cheesy particles in, 6 chemistry of, 5 chromogenic bacteria in, 4 coal pigment in, 4, 34 coctum, 32 collection of, 2 color of, 3 concretions in, 8 consistency of, 2 cotton fibers in, 4 crudum, 31 crystals in, 13 Curschmann's spirals in, 7, 30, 31 cytology of, 10 deportment on standing of, 5 Dittrich's plugs in, 7 echinococcus membranes in, 9 elastic tissue in, 12 epithelial cells in, 1 1 extraneous matter in, 9 fatty acids in, 13 ferments in, 6 ferric oxid in, 4 fibrinous casts in, 8 flour in, 4 foreign bodies in, 9 fundum petens, 4 heart disease, cells in, 11, 32 hemoglobin derivatives in, 11, 13 in abscess of the lung, 33 in actinomycosis, 27 in acute bronchitis, 31 in bronchial asthma, 33 M;C/*Oiaf/¥®ho-pneumonia, 31 INDEX 753 Sputum, in chronic bronchitis, 32 in croupous pneumonia, 31 in fibrinous bronchitis, 32 in gangrene of the lung, 33 in influenza, 33 in jaundice, 4 in perforating empyema, 34 in pneumonoconioses, 34 in pulmonary tuberculosis, 30 in putrid bronchitis, 32 leucocytes in, 10 macroscopic examination of, 6 microscopic examination of, 9 morning, i mucin in, 5 mucoid, 5 mucopurulent, 5 myelin granules in, 1 1 nitrogen of, 2 nummular, 5, 6 odor of, 4 origin of, i parasites in, 28 prune-juice, 3, 31 purulent, 5 pus cells in, 10 reaction of, 3 red blood cells in, 1 1 sanguinous, 5 serous, 5 spit-cups for, 2 stone dust in, 4 tenacity of, 3 types of, 5 Staining characteristics of tubercle ba- cillus, 20 methods, principles of, 476 of bacteria, 668 of blood smears, 478 of casts, 357 of elastic tissue, 12 properties of cells, 496 vital, 485 Stains, blood, 478 Bunge and Trantenroth's, 24 Burri^s, 670 dahlia, 507 Ehtlich's tri-acid, 480 triple, 480 eosin hematoxylin, 479 methylene blue, 478 Gabbet's, 21 Ghoreyeb's, 670 Giemsa's, 483, 670 Goldhorn's, 580 Gram's, 668 india-ink, 670 iodine, 507 Jenner's, 481 •Leiner's, 115 Loffler's methylene blue, 21 Mallory's, 173 May-Grunwald's, 482 Mtillern's, 478 . Neisser's, 39 Nocht's, 484 orcein, 12 osmic acid, 116 Digitized by Pappenheim's blood, 4^1, 484 Stains, for tubercle bacillus, 21 polychrome, 481 Romano wsky's, 481 safranin, 668 scharlach R, 116 seminal, 379 Sudan III, 116 thionin, 112, 484 Tunnicliff's, 670 Turk's iodine, 507 Unna-Tanzer's, 12 Van Gieson's, 173 Weigert's fibrin, 8 Williams and Lowden's, 172 Wright's, 482 Zenoni's, 5 Ziehl-Neelsen, 20 Staphylococcus pyogenes in sputum, 26 • Starch in feces, 124 ' detection of, 124 digestion of, 36 estimation of, 124 Steatorrhea, 115, 123 Stegomyia, fasciata, 583 Stercobilin, 107, 255 Sterility, 378 Sterilization, 700 Stippled cells, 498, 567 Stock vaccines, 724 Stomach, absorptive power of, 86 carcinoma of, 91 contents, 50 dilatation of, 84, 85 diseases of, 88 ectasia of, 85 fasting, 60 function of, 84 histology of, 50 inflammation of, 90 motility of, 84 tube, 52 ulcer of, 91 washing, 93 Stomatitis, catarrhal, 40 gonorrheal, 41 mycotic, 41 ulcerative, 40 ulceromenbranous, 40 Stone cutters' phthisis, 4 Stones, bronchial, 8 gall, 117 in bladder, 367 in kidney, 367 in lung, 8 intestinal, 118 in ureter, 367 in urine, 367 nasal, 46 renal, 367 ureteral, 367 vesical, 367 Stools (see Feces), 99 acholic, 107, 116 clay-colored, 107 curds in, 115 frequency of, 104 M;crosEi#®oup, 130 rice-water, 106, 129 754 INDEX Strasburger's method for bacteria in feces, 128 Strauss' test for lactic acid, 75 Streptococcic sore throat, 40 Streptococcus hemolyticus, 718 mucosus capsulatus, 717 pleurisy, 675 pyogenes in sputupi, 26 in blood, 591 viridans in blood, 592 Streptothricosis, 27 Streptothrix eppingeri, 14 Striatula, 153 Strongyloides intestinalis, 147 Strongylus duodenalis, 151 gigas, 160 quadridentatus, 151 renalis, 160 Structural albuminuria, 265 Sudanophiles, 509 Sugar broth, 70B Sugar-free broth, 702 Sulphates of urine, 207 easily split, 207 ethereal, 207 preformed, 207 total, 207 unoxidized, 207 Sulph-hemoglobin, 412 Sulphhemoglobinemia, 412 Sulphocyanates in saliva, 35 Sulpho-salicylic acid test for albumin, 271 Sulphur compounds in urine, 206 amount of, 206 determination of, 209 neutral, 208 types of, 206 variations of, 207 Sulphur test for bile in urine, 331 Surgical interference, blood after, 546 Syphilis, albuminuria of, 266 blood in, 558 hemoglobin test of Justus in, 559 organism of, 578 serum test of Wassermann in, 620 Tabanus striatus fabricus, 576 Table for examination of calculi, 369 Gaflfky's, 23 Tasniidae in feces, 140 tasnia aegyptica, 142 canina, 141 cucumerina, 141 cucurbitina, 140 dentata, 140 diminuta, 142 echinococcus, 143 elliptica, 141 flavopunctata, 142 inermis, 141 lata, 144 leptocephala, 142 t»nia mediocanellata, 141 minima, 142 moniliformis, 141 nana, 142 saginata, 141 Digitized by Taeniidas solium, 140 varerina, 142 Tallqvist's hemoglobinometer, 424 Tape- worms in feces, 139 Tartar of the teeth, 38 Taurocholic acid, 117, 191, 331 Taylor's ash-free diet, 195 Teichmann's crystals, 657 test for blood, 657 Tenacity of sputum, 3 Tertian malarial organism, 563 asexual cycle of, 564 sexual cycle of, 563, 570 Test meal of Boas, 55 of Ewald, 54 of Fischer, 55 of Riegel, 55 of Sahli, 56 of Salzer, 56 oil, 102 Testicular casts, 378 Thecosoma hematobium, 585 Theobromin, 240 TheophyUin, 240 Therapeutic measures, effect of, on blood, 494> 516 Thermolabile substances, 596 Thermostable substances, 596 Thionin stain, 112, 484 Thiosulphuric acid in urine, 191 Third corpuscles of blood, 523 Thomas and Weber's method for pepsin, 80 Thoma-Zeiss hemocytometer, 456 Thorn-apple crystals, 347 Threads, mucus in urine, 180, 349, 353 Thread worm, 147 Throat cultures, 39 Thrombase, 402 Thrush, 41 Tick fever, 584 Tide, alkaline, of urine, 1 86 Timothy bacillus in sputum, 24 Tinea barbae, 166 circinata, 166 favosa, 165 sycosis, 166 tonsurans, 167 versicolor, 169 Tissue, elastic in sputum, 12 fragments in feces, 118 in gastric contents, 63 in sputum, 9 in urine, 363 Toisson's fluid, 460 Tollen's orcin test for pentose, 310 naphthoresorcin test for glycuronic acid, 316 phloroglucin test, 309 test for pentose, 309 Tongue, coating of, 38 Tonsillitis, leucocytosis in, 515 Topfer's method for combined HCl, 72 for free HCl, 68 test for free HCl, 66 Total acidity of gastric juice, 64 components of, 64 determination of, 64 IVIicrosofm^^^ of. 65 INDEX 755- Total acidity of urine, 185 nitrogen of blood, 430 of feces, 122 of gastric juice, 82 in carcinoma, 93 of urine, 213 amount of, 213 determination of, 217 variations of, 214 solids of blood, 409 of feces, 121 of mUk, 691 of urine, 189, 192 non-protein nitrogen of blood, 430 volume of blood, 387 Towel test for hemoglobin, 416 Toxemia, hepatic, 222, 286, 305 intestinal, 123 renal, 370 Toxogenic protein decomposition, 216 Toxoids, 595 Toxones, 595 Toxophore, 595 Trachoma bodies, 48 Trachyochromatic erythroblasts, 490 Transfusion, tests before, 653 Transitional leucocytes, 504 Transudates, 662 coagulation of, 664 obtaining of, 663 properties of, 663 Traumatic albuminuria, 265 Treatment, effect of on Wassermann's test, 635 Trematodes, 156 in feces, 145 in sputum, 29 Treponema pallidum (see Spirocheta pallida), 579, 581, 669 Triacid stain of Ehrlich, 480 of Pappenheim, 481 Trichina spiralis, 149 Trichinella spiralis, 149 Trichinosis, 149, 520 Trichiuris trichiura, 149 Trichocephalus, dispar, 149 hominis, 149 mastigodes, 149 trichiuris, 149 Trichomonas hominis, 137 intestinalis, 137 vaginalis, 381 in urine, 366 Trichophyton megalosporon endothrix, 166 microsporon, 167 Trichotrachelidae, 149 Triple phosphates as calculi, 369 in sputum, 13 in urine, 348 Tripperfaden in urine, 353, 669 Trombidiosis, 163 Trombidium irritans, 162 Trommer's test for glucose, 288 Tropeolin test for HCl, 67 Tropical splenomegaly, 576 Tropics, anemia of, 493 Trousseau's test for bile pigments, 330 True albuminuria, 261 oygy^y^ed by Trypanosoma Brucei, 576 equiperdum, 576 Evansi, 576 Gambiense, 575 in the blood, 575 in spinal fluid, 576, 683 Trypanosomiasis, 575 Trypsin in feces, 102 in pancreatic cysts, 678 in urine, 251 Tryptophan test, 93 Tsetse flies, 575 Tsuchiya's method for albumin, 273 Tube casts in urine, 356 Tubercle bacilli in the blood, 592 in exudates, 667 inoscopy, 667 in feces, 132 in sputum, 17 culture medium for, 19 morphology of, 21 number of, 23 staining of, 20 value of examination for, 22 in urine, 364 . . . • ■ 1 Tubercular meningitis, 684 pleurisy, 674 Tuberculin, 18 reactions, 601 Tuberculosis, blood in, 557 of bladder, 364 of intestine, 132 of kidneys, 352 of lymph glands, 545 of meninges, 684 of peritoneum, 676 of pleura, 674 pulmonary, blood in, 557 sputum in, 30 Tuberculous cystitis, 364 Tubular insufficiency, 370 Tumor shreds in feces, 118 in gastric contents, 63 in urine, 363 Tunnel workers' anemia, 153 TunnicliflE's bacillus, 45 stain for spirochete, 670 Turk's iodin stain, 507 counting chamber, 458 Two-glass test, 353 Typhoid bacillus in the blood, 587 in feces, 130 Drigalski and Conradi's media, 130 Kendall and Day's media, 131 in urine, 366 fever, blood in, 552, 587 feces in, 130 Widal reaction in, 607 pleurisy, 675 Typhoidin reaction, 605 Tyrosin in sputum, 13 in urine, 246, 344 Uffelm^nn's test for lactic acid, 75 Uhlenhuth's antiformin method for tu- bercle bacilli, 18 , ..Ulcer c^the stomach, 91 Microsoft® ^ 7S6 INDEX Ulceromembranous angina of Vincent, 40 Unaltered bile in feces, 108 Uncinaria Americana, 1 52 duodenalis, 151 Uncinariasis, 151 Undetermined nitrogen of urine, 246 Unilateral nephritis, 371 Unit of counting chamber, 463 Unna-Tanzer's stain, 12 Unorganized sediments in urine, 338 Unoxidized sulphur of the urine, 207 Uranium method for phosphates, 202 Urates in urine, 339 Urea in blood, 435 in urine, 219 amount of, 220 determination of, 221 variations of, 220 Urease method for urea, 225, 436 Uremia, blood in, 437 urine in, 370 Uremic coefficient, 438 constant, 438 Ureometer of Doremus, 222 of Hinds, 223 Ureteral calculi, 367 Ureteritis membranacea, 252 Urethritis, anterior, 353 posterior, 353 Uric acid, 232 calculi, 368 diathesis, 234 in blood, 439 in the urine, 232 determination of, 235 metabolism of, 232 variations of, 233 sediment, 338 Uricacidemia, 442 Urine, 175 acetone in, 191, 320 acidity of, 184 albumin in, 261 albumoses in, 276 alkaline tide of, 186 alkapton bodies in, 182, 333 alloxur bodies in, 191, 240, 341 amino-acids in, 246 ammonia in, 227 amount of, 177 animal gum in, 314 animal parasites in, 366 appearance of, 180 ash of, 192 bacteria in, 363 Bence- Jones protein in, 276 bile acids in, 331 biliary pigments in, 329 black, 182 ■ blood cells in, 354 pigment in, 328 blue, 183, 257 calcium in, 211 calculi in, 367 carbohydrates in, 283 carbonates in, 211, 349 casts in, 356 changes on standing of, igjjg/f/zed by Urine, chemistry of, 190 chlorids in, 192 cholesterin in, 346 chromogens in, 254 chyle in, 182, 346 clearing of, 298, 300 collection of, 176 color of, 181 composition of, 190 consistence of, 181 creatin in, 241, 245 creatinin in, 241 cryoscopy of, 371 cultures of, 363 cystin in, 208, 342, 370 dextrin in, 314 dextrose in, 284 diacetic acid in, 324 diastase in, 251 drug reactions in, 183 Ehrlich's benzaldehyde reaction in, 336 diazo reaction in, 334 egg-yellow reaction in, 335 electric conductivity of, 371 epithelial cells in, 349 fat in, 346 fatty acids in, 249 ferments in, 251 fibrin in, 283 foreign bodies in, 366 free mineral acidity of, 185 organic acidity of, 185 functional diagnosis from, 370 glucose in, 284 glycosuric acid in, 333 glycuronic acid in, 314 green, 182 hematoporphyrin in, 328 hemoglobin in, 288, 328 hippuric acid in, 248, 345 homogentisic acid in, 182, 333 indican in, 256 indigo in, 256 • inosite in, 314 iron in, 213 lactic acid in, 249 lactose in, 312 laiose in, 306 leucin in, 246, 343 leucocytes in, 351 levulose in, 305 magnesium in, 211 maltose in, 313 melanin in, 182, 332 microscopy of, 336 mucin-like substances in, 252 mucoid material in, 349 neutral sulphur in, 208 nitrogen in, 213 nitrogenous bodies in, 213 nubecula in, 180, 349 nuclein bodies in, 240 nucleo-albumin in, 252 odor of, 183 optical activity of, 190 organized sediments of, 349 oxalic acid in, 250 INDEX 757 Urine, ^-oxybutyric acid in, 325 parasites in, 366 pentoses in, 308 peptone in, 281 phosphates in, 198, 346, 347 physical properties of, 177 pigments in, 254, 328 potassium in, 211 preservation of, 176 protein of, 260 proteoses in, 276 ptomaines in, 342 purin bases in, 240 pus in, 351 quantity of, 177 reaction of, 184 Russo's reaction in, 335 sediments of, 336 serum-albumin in, 261 globulin in, 274 skatoxyl in, 25,8 sodium in, 211 solids of, 189, 192 specific gravity of, 187 spermatozoa in, 363 sugar in, 284 sulphates in, 207 sulphur compounds in, 206 tissue fragments in, 363 total solids of, 189, 192 tyrosin in, 246, 344 urates in, 338 urea in, 219 uric acid in, 232, 338 urobilin in, 255 urochrome in, 254 uroerythrin in, 254 urohematin in, 256 tirorosein in, 260 xanthin bases in, 240, 341 Urinod, 183 Urinometer, 188 Urinous odor, 183 Urobilin, 255 Urobilinuria, 255 Urochrome, 254 Urochromogen, Weisz' test for, 254 Uroerythrin, 254 Uroferric acid, 208 Uroleucic acid, 191, 333 Urophain, 260 Urorhodin, 258 Uroroseinogen, 260 Urorubin, 258 Urostealith calculi, 370 Uterine secretions, 382 Vaccination, antityphoid, 600, 726 Vaccines, 600, 723 autogenous, 725 diagnostic use of, 600 preparation of, 724 stock, 724 Vaccine therapy, 600 Vacuolization, 497 Vaginal secretions, 380 Vaginitis, catarrhal, 380 gonorrheal, 381 Diaitized Value of blood examination^"^'^'-^'^" Value of functional renal diagnosis, 370 of search for tubercle bacilli, 22 Van Deen's test for blood, 109, 655 Van Ermengem's stain for fiagella, 708 Van Gieson's stain for Negri bodies, 173 Van Slyke and Cullen's method for urea, 226 Vaquez' disease, 496 Variations in number of leucocytes, 511, . 521 of red cells, 492, 500 Variola, blood in, 555 Venous blood, 394 puncture, 393, 585 Vermiculus, 570 Vernal conjunctivitis, 49 Vesicular albuminuria, 261 mole, 384. Vincent's angina, 40 bacillus, 40 spirillum, 40 Vinegar eel in urine, 366 Viscosity of blood, 401 Vitali's test for pus, 353 Vital staining of blood cells, 485 Volatile alkalinity of urine, 187 Volhard's method for chlorids, 195 Volume index of blood, 391 of blood, 387 quotient, 391 value, 391 Vomitus, 60 bile in, 61 blood in, 61 fecal, 61 green, 61 mucus in,' 61 odor of, 61 pancreatic fluid in; 61 parasites in, 61 pus in, 6i rice water, 61 Wagner's test for blood, 1 1 1 Wang's method for indican, 259 Wariield's test for pregnancy, 247 Wassermann's serum reaction for syphilis, 620 Waxy casts, 358 Weber's test for blood, no Weidel's test for xanthin, 341 Weinstein's test for gastric carcinoma, 93 Weisz' test for urochromogen, 254 Welch's gas bacillus, 133 Weyl's test for creatinin, 243 Whetstone crystals of uric acid, 339 of xanthin, 341 Whip worm, 149 White blood cells (see Leucocytes), 502 Whooping cough, bacillus of, 26, 711 blood in, 556 Widal reaction, 607 Williams and Lowden's stain for Negri bodies, 172 Williamson's blood test in diabetes, 548 by Mi^M^^I^.^ method for gastric motility. 758 INDEX Winternitz', Henry and McPhedrans' test for catalase, 522 Wolff and Junghans' test, 95 Wright's opsonic method, 599 stain for blood smears, -482 vaccine therapy, 600 Wright and Kinnicutt's method for blood plates, 469 Xanthin bases in feces, 123 in urine, 191, 240, 341 calctili, 370 Xerosis bacillus, 40 Xylose in urine, 308 Yaoita's method for ova, 133 Yeast cells in feces, 126 in gastric contents, 62, 63 in sputum, 15 in urine, 293 Yellow fever, blood in, 583 mosquito theory of, 583 Ziehl-Neelsen method for tubercle ba- cilli, 20 Zygotes, 572 Zymogens in gastric juice, 77, ,81 Zymophore, 598 Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft®