LABORATORY METHODS m mmmiLmsf wwmm McCampbell. CORNEL L UNIV ERSITY THE Jiflntupr Bftprtnary ICtbrary FOUNDED BY ROSWELL p. FLOWER- for the use of the N. Y. State Veterinary College 1897 This Volume is the Gift of .?.??.¥. A. Moore 356 Cornell University Library QR 183.M12 Laboratory methods for the experimental 3 1924 000 262 042 Cornell University Library The original of tliis book is in tlie Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000262042 Laboratory Methods FOR The Experimental Study of Immunity BY EUGENE F. MCCAMPBELL Associate Professor of Bacteriology, Ohio State University, Columbus, Ohio. Compliments of the Jiuthor. COLUMBUS, OHIO The F. J. Heer Printing Co. f909 Laboratory Methods FOR The Experimental Study of Immunity BY EUGENE F. MCCAMPBELL Associate Professor of Bacteriology, Ohio State University, Columbus, Ohio. COLUMBUS, OHIO The F. J. Heer Prinflngr Co. (909 Copyright, 1909, By E. F. McCAMPBELL PREFACE. The study of the immunity of the body to the various infectious diseases has been receiving the attention of a large number of investigators throughout the entire world. During the last decade great progress has been made in the elucidation of the mechanisms of defense and the fac- tors of safety possessed by the animal body. In the following laboratory outline the aim will be to present for consideration only those general topics in con- nection with the phenomena of immunity with which the student of experimental medicine must be familiar before attempting to investigate any of the more intricate prob- lems of immuno-pathology. It is presumed that the student undertaking work in an advanced subject of this nature has had an elementary training in chemistry, general biology, bacteriology and pathology. It is imperative that the student be familiar with the ordinary technique in the handling of pathogenic micro-organisms and the care of inoculated animals. The experiments outlined are those which have proverl by experience to be the most serviceable in the illustration of the points under discussion in the lecture course which accompanies the experimental work. The experiments out- lined comprise one year's work. For suggestions in regard to the presentation of this outline the writer is indebted to Professor H. T. Ricketts of the University of Chicago. E. F. Mc. Ohio State University, October, 1909. INDEX. Page General Notes 7 List of Apparatus 8 Notes on the Experimental Inoculation of Animals' 9 Notes on the Collecting of the Body Fluids 11 Notes on the Collecting and Washing of Erythrocytes and Leucocytic Exudates ■. 12 The Hemolytic Scale 13 Exercise 1. The Standardization of Bacterial Cultures 14 Agar Slant Cultures. Broth Cultures. The Platinum Loop Method. Exercise 2. Natural and Acquired Immunity 20 Natural Immunity. Acquired Immunity. Active and Passive. Exercises. The Immunization of Animals 24 Different Methods. Exercise 4. The Mechanism of Action of Toxic Substances. . 32 The Action of Soluble Toxins. The Action of Animal Toxins (Cobra Venom). The Action of Endotoxins. The Action of the Bacterial Proteins. The Mechanical Action of Bacteria. The Action of Ptomains. Exercises. The Selective Action of Toxins 40 Exercise 6. The Opsonins 44 Suspension of Bacteria. Suspension of Leucocytes. The Preparation of Serum. The Technique of the Opsonic Test. The Technique of the Opsonic Test, Modified. The Phagocytic Index. The Opsonic Index. The Percentage Index. The Technique of Making Smears and Staining. Exercise 7. The Mechanism of Action of Opsonins 58 Exercise 8. Practical Work in Opsonic Index Determina- tion 62 Exercise 9. The Specificity of Opsonins 64 Exercise 10. The Hemopsonins 68 Exercise 11. The Preparation of Soluble Toxins 72 Diphtheria Toxin. Tetanus Toxin. 5 6 Page Exercise 12. The Precipitation of the Tetanus Toxin 78 Exercise 13. Tetanolysin 80 The Thermal Death Point of Tetanolysin. Exercise 14. Tetanospasmin 84 Exercise 15. The Preparation of Tetanus Antitoxin 86 Exercise 16. The Standardization of Tetanus Antitoxin 90 Exercise IT. The Preparation of Diphtheria Antitoxin 94 Exercise 18. The Standardization of Diphtheria Antitoxin. . 98 Exercise 19. The Concentration of Diphtheria Antitoxin.... 106 Exercise 20. The Preparation of Tuberculin 110 Koch's Old Tuberculin and others. The Standardization of Tuberculin. Exercise 21. The Tuberculin Reaction 114 The Cutaneous Tuberculin Reactions. The Ophthalmo-Tuberculin Reaction. Exercise 22. The Preparation of Mallein. . . . , 120 Exercise 2.S. The Mallein Reaction 122 Exercise 24. The Preparation of Antibacterial Substances.. 124 Bactericidal Action in vitro. Bactericidal Action in vivo. Exercise 25. The Bactericidal Action of Normal Sera 128 Exercise 26. The Bactericidal Action of Whole Blood and Serum 132 Exercise 27. The Hemolysins 136 The Sensitization of Erythrocytes by Amboceptors. The Combination of Complement with Erythrocytes. Exercise 28. The Hemolytic Action of Cobra venom 144 Exercise 29. The Preparation of Endocoraplement 148 Exercise 30. The Complementing Action of Lecithin ISO Exercise 31. The Agglutinins 154 The Microscopic I\Iethod. The Macroscopic Method. Group Agglutination. E.xercise 32. Practical Work in the Diagnostic Use of Ag- glutinins , 162 Exercise 33. The Precipitins 166 Exercise 34. The Cytotoxins 170 Exercise 35. The Anaphylaxis Reaction 174 Exercise 36. The Deflection of the Complement as a Test for Antibodies .'. . . 178 The Wasserman Reaction. Exercise 37. The Preparation of Bacterial Vaccines 186 Exercise 38. The Preparation of Vaccine for Anthrax 190 Exercise 39. The Preparation of Vaccine for Symptomatic Anthrax 192 Exercise 40. The Preparation of Vaccine for Hog Cholera. . 194 GENERAL NOTES. All glassware is to be thoroughly cleaned before using by a cleaning fluid composed of, Bichromate of potash 60 parts Sulphuric acid 460 parts Water 300 parts and subsequently washed thoroughly in tap water, then in distilled water, after which it is to be thoroughly dried in the hot air oven. Cover-glasses and slides after cleaning in the above manner and washing in distilled water may be conveniently kept in bottles containing 95% alcohol and dried and burned for a moment in the Bunsen flame immediately be- fore using. Bichloride of mercury (i-iooo) and phenol (carbolic acid) 5%-io% will be found in the laboratory for pur- poses of disinfection. All animals should be burned after necropsies have .been made. In order to prevent infection it is necessary that the student see that the hands are free of abrasions which are not properly protected and that the hands are frequently disinfected during the progress of the work. It is expected that the student keep accurate notes in ink of all experiments performed on the supplementary blank sheets and also that these sheets be used for lecture notes on the particular subject under experimentation. It is important that the experiments contemplated be carefully read over in advance by the student in order that it may be possible to prepare the materials and reagents in adequate time. In certain experiments in order to facilitate work, the class is divided into groups. Each student in the group will, however, report and make notes on the entire series of experiments. 7 LIST OF APPARATUS. 2 Sec glass syringes graduated in i/io cc. I clinical thermometer. 1 thermometer loo C. graduated in i/io°. 2 500 cc. Erlenmeyer flasks. 4 lOD cc. Erlenmeyer flasks. 2 500 cc, flat flasks (whiskey flasks). 2 toxin flasks. 6 100 cc. volumetric flasks. 12 I cc. pipettes graduated in i/ioo cc. 6 I cc. pipettes graduated in i/io cc. 6 5 cc. pipettes graduated in i/io cc. 12 conical test glasses. 12 Petri plates. 36 small test tubes. 36 large test tubes. 3 funnels, 2 in., 4 in., 6 in. 3 test tube racks. I nest of beakers. r Bunsen burner. I tripod. I ring stand. I wire gauze. I gross of slides. I box No. I cover glasses. 24 hanging drop slides. 3 packages of filter paper. I wax pencil. I platinum needle, long. I platinum needle, short. I rack of staining reagents containing: i) Alcohol 95%. 2) Grain's Solution. 3) Gentian violet, analin. 4) Loeffler's methylene blue. 5) Gabbet's methylene blue. 6) Carbol-thionin blue. 7) Carbol-fuchsin. 8) Distilled Water. Animals, porcelain filters, scales, special stains, chem- icals, and special pieces of apparatus will be provided by the laboratory. NOTES ON THE EXPERIMENTAL INOCULA- TION OF ANIMALS. The technique of inoculation into the various labora- tory animals is usually modified to suit the particular or- ganism injected and the end desired. The technique must also be varied with the different species af animals used. All inoculations should be made with a sterile syringe graduated in i/io cc. or platiiiuni or steel needle, and the instruments should be carefully sterilized after using. All animals should be placed in properly labeled cages and daily temperatures and weights accurately recorded. I . — Subcutaneous inoculation : This method of in- oculation can be conveniently used in most experimental animals. In using a syringe care should be exercised that the needle is introduced just beneath the dermis and not into the muscles. Often subcutaneous inoculations are made by the use of the so-called skin pocket. This pocket is made by making an incision in the skin with a sharp scalpel and dissecting away the subcutaneous fascia for a short distance under the skin. Inoculation is then made into this pocket with a sterile platinum needle carrying the micro-organisms. Sterilize the skin with I -1000 HgCL and 60% alcohol before injection. lO 2. — Intramuscular inoculation : In this method of inoculation the needle of the syringe is introduced directly through the skin deep into the muscular tissue. This method can be us^d with practically all animals. Sterilize the skin as above. 3. — Intravenous inoculation : In making intrav- enous injections it is imperative that the injection be made slowly and that there be no air or oil in the syringe. The method can be used with most any animal in which the veins can be easily exposed. It is very frequently used in the case of rabbits in which case the metliod is as follows: I. Carefully shave and sterilize the ear with i-iooo HgClj, and 60% alcohol. 2, Place a bull-dog forcep on the ear near the base so as to cause dilatation of the posterior auricular vein. 3. The vein having been distended the needle of the syringe is introduced directly into it for a distance of i cm. and a bull-dog forcep placed over it so as to hold it solid in the vein. The forcep at the base of the ear is then removed and the contents of the syringe injected slowly. A forcep should always be placed on all veins before the needle is introduced. 4. — Intracardiac inoculation : This method is fre- quently used in small animals such as guinea pigs where it is difficult to make intravenous injections. The method is as follows: i. Shave the chest of the guinea pig over the heart and sterilize it with i-iooo HgCl, and 60% al- cohol. 2. Introduce the needle in the 2 interspace, in about the median line, on the left side of the sternum. Point the needle down at an angle of about 45 degrees and pltinge into the left ventricle and draw up a little blood into the syringe in order to make sure that the needle is in the heart, then inject the contents of the syringe slowty. This method may also be used in drawing blood from the heart of a live animal. 5. — Intraspinal inoculation : It is often desired to inject toxins and micro-organisms into the cerebro-spinal II canal. The technique is varied with the animal used, it being easier to perform lumbar puncture on the larger ani- mals such as dogs. The method is as. follows: i. The animal is securely fastened to the operating table with the back up and the hair is shaved off in the region extending from the first. lumbar vertebra to the sacrum and over a space about three inches in width. The skin is sterilized with i-iooo HgCL and 60% alcohol. 2. The animal is slightly bent to the right by an assistant pulling the head and pelvis toward each other. 3. The needle, which must be somewhat longer that that commonly used in other work, is then introduced between the 3 and 4 lumbar vertebrae on the left side of the animal and directed up at an angle of 45 degrees. After passing in between the vertebrae the needle comes in contact with the dura and this must be punctured carefully and an amount of cerebro-spinal fluid allowed to escape which corresponds to the amount of ma- terial which it is desired to inject. 4. The syringe is then connected and the contents injected slowly. The above method' may be used ' in securing cerebro-spinal fluid for diagnosis or for cultures. 6. — Intraocular inoculation : Inoculations into the cornea or anterior chamber of the eye can be most con- veniently made with a short, fine steel needle carrying the infecting material. The wound should be made obliquely. Inoculations into the posterior chamber should be made with a syringe bearing a fine needle after cocainizing the eye. NOTES ON THE COLLECTING OF THE BODY FLUIDS. Blood: Blood may be secured from live animals such as rabbits and guinea pigs by bleeding directly from the heart by means of an aspirating syringe. Rabbits can also be bled from the ear veins. Fowls are usually bled 12 directly from the median veins of the wing. Goat, sheep, ox and horse blood and the blood of the other larger animals can be easily secured from the jugular vein by the intro- duction of a canula. Dogs are most conveniently bled from the femoral vein after making a small incision through the skin. If it is desired to draw off all of the blood from an animal it should be done by severing the carotid artery. Cerebro-spinal fluid: Cerebro-spinal fluid can be se- cured by lumbar puncture as described under the section on intraspinal inoculation. Pleural, pericardial and peritoneal fluids or exudates : These fluids can be most easily secured by the introduction 6i sharp pointed sterile glass pipettes into the cavities after making incisions of the tissues into the serous membranes. Aqueous and Vitreous Humors: These fluids should be secured with an aspirating syringe with a fine needle. Urine: Urine is obtained by the aspiration of the bladder by means of a long needle connected with a sterile syringe or by a sterile catheter. Note : -^ Human blood can be best secured by placing a tourni- quet around the upper arm and passing a large size hypodermic needle into the median basilic vein. NOTES ON THE COLLECTING AND WASHING OF ERYTHROCYTES AND LEUCO- CYTIC EXUDATES. Washed erythrocytes and leucocytes are frequently used in immunological work. They should- be prepared ac- cording to the following technique : 1. — Procure the blood or exudate according to the methods given in the previous notes. Defibrinate by "whipping" or collect by the addition of sodium citrate solution.* 2. — Place the defibrinated or citrated blood or exu- date in centrifuge tubes and mark on the side of the tube 13 the height of the fluid. When using citrated blood it is necessary to keep accurate account of the amount of sodium citrate solution added. 3. — Centrifugate and remove the serum or serum and citrate solution with a sterile pipette. 4. — Add 20 volumes of 0.85% NaCl sol. to each tube, shake and centrifugate. 5. — Repeat 3 times, carefully removing the super- natant fluid each time with a sterile pipette. 6. — Restore original volume of serum with 0.85% NaCl sol. 7. — A suspension of the desired percentage may be prepared, 0.85% NaQ sol. being used as the diluting fluid. *NoTE : — 1% sodium citrate in 0.85% sodium chloride solution composes the citrate solution used to prevent the coagulation of blood. THE HEMOLYTIC SCALE. This scale is used to record the different degrees of hemolysis of erythrocytes which may be produced. The hemolysis may also be recorded by comparison with a per- centage scale: 1 — Complete. 2 — Almost complete. 3 — Marked. 4 — Medium. 5 — Slight. 6 — Trace. 7 — Faint trace. 8 — Negative. '4 EXERCISE I. The Standardization of ^scterial Cultures. A. — Cultures grown on agar slant tubes : The hard glass test tubes used for media work should contain lo cc of agar and should be slanted so that the base of the slant coincides with the bottom of the tube. The slant tubes must always be inoculated in a uniform manner in all work in order that accurate comparative results may be obtained. After growing the culture in the incubator at 37°C for 24 hours a definite amount of sterile sodium chloride solution (0.85%) or broth is added to the agar slant culture tube. (Usually 5 cc to 10 cc are used for this purpose.) The growth is then carefully scraped off the surface of the agar with a sterile glass rod with a rounded end so as not to cut into the media. The resulting suspension in the sodium chloride solution or broth is then carefully shaken. By the use of varying fractions of this suspension any desired portion of the 24 hour growth of the organism may be obtained. B. — Cultures grown in broth: The hard glass test tubes used for media work should contain 5 cc. to 10 cc. of broth of the desired reaction. The broth must be inoculated in a uniform manner and the culture allowed to grow 24 hours. At the end of this time the broth should be carefully shaken and by the use of varying fractions any desired portion of the 24 hour growth of the organ- ism may be obtained. Cultures grown on other liquid or solid media may be standardized in the same way. C. — The platinum loop method of standardization : The standard platinum loop is made of No. 27 platinum 15 i6 wire which has been looped around a No. lo steel wire nail. This loop is supposed to contain two milligrams when level full of culture. It is important to standardize this loop after it is made and to preserve it unaltered. Standardization may be accomplished by repeated attempts to inoculate a lo cc. broth tube with a definite arnount of culture and the subsequent plating of i/io cc. of this broth. The resulting colonies should be counted and the plates compared. The results should correspond within narrow limits. If it is so desired fractions of a loop may be -obtained in the the following way : One loop of culture is mixed with lo cc. of broth in a test glass. The loop of culture should be gradually rubbed up with the broth. With a sterile pipette graduated in i/ioo cc. the following frac- tions of the loop of culture may be obtained : 1 cc 1/10 loop 0.5 cc 1/20 loop 0.1 cc 1/100 loop 0.1 cc of orignal suspension plus 0.9 cc of broth... 0.5 cc 1/200 loop 0.1 cc 1/1000 loop 0.01 cc 1/10000 loop In this way anyjJesired fraction may be obtained. Normal sodium chloride solution is sometimes used as a diluent but it is often bactericidal. All dilutions should be poured into lo cc. of agar at 45° C mixed and plated in Petri dishes. Results should be tabulated in 24 hours after incubation at 37°C. Special Directions, i. — Prepare 15 agar tubes of 10 cc. each. 2. — Prepare 15 broth tubes of 10 cc. each. 3. — Procure from the instructor a culture of Micro- coccus pyogenes and a culture of Bacillus typhosis. 4. — Inoculate 2 different agar slants with as nearly the same amount of each the original culture as possible. 17 i8 and plate i/ioo of 24 hour growth according to the usual bacteriological technique. Tabulate results on both cul- tures. Use a counting plate. 5. — By the loop method of standardization plate 1/50, 1/500 and i/iooo of the 24 hour culture of B. typhosis and A[. pyogenes aureus. Count .and tabulate results in 24 liours. 6. — Repeat Nos. 4 and S twice or three times in order to acquire a uniform and reliable technique. 19 20 EXERCISE 2. Natural and Acquired Immunity. The term immunity or resistance to disease can only be used in a relative sense. Practically all species of ani- mals are susceptible to the invasion of pathogenic micro- organisms under certain conditions. The power of re- sistance usually varies with the species of animal and the micro-organism which attempts to invade it. In certain animals the mechanisms of defense are almost perfect under natural conditions but when these conditions are varied in the least the animals become susceptible. However, in a large number of instances an animal which is naturally sus- ceptible may increase its powers of resistance and become immune. Immunity, therefore, may be divided into two classes : natural immunity and acquired immunity. In each of these types various factors may come into play which will be considered in the lecture course. In the following experi- ments only certain of these factors will be illustrated : Natural Immunity: 1 — Procure two chickens of approximately the same weight. 2 — ■ Place chicken No. i in a cage in the incubator room at 37" C and keep it there with food and water for 4 days. At the close of this time inoculate with i/io cc. of a 48 hour culture of B. tetani. 3 — Inoculate chicken No. 2 with i/io cc. of a 48 hour culture of B. tetani at the same time. 4 — Place both chickens in a cage and note results. 21 22 a — Discuss the phases of immunity and susceptibility involved in this experiment. b — Discuss natural antitoxins and natural antibac- terial substances. Note : — The 1/10 cc of culture should be mixed with 2 cc of 0.85% NaCl sol. and the injection made into the pectoral muscles with a sterile syringe. Acquired Immunity: Active Type: This type of immunity maj- be brought about in several ways. (Tabulate in notes.) 1 — Procure two rabbits from the instructor. One of these is a normal rabbit, the other has received 2 inocula- tions 7 days apart of i/io of a 24 hour culture of B. ty- phosus killed by heat; 50° C, 20 minutes. 2 — Inoculate both rabbits with 1/5 of a 24 hour agar culture of B. typosis in 2 cc of 0.85% NaCl solution. 3 — Place rabbits in a cage and carefully note weight and temperature on successive days and general results. Note : — Make the subcutaneous inoculations in 2 cc 0.85% NaCl solution. Passive Type: i — Procure from the instructor 1000 units of diphtheria antitoxin and three guinea pigs. 2 — Inject 2 guinea pigs with 500 units each. 3 — Inject all 3 guinea pigs (after 3 days) with o.i cc. of diphtheria toxin which has been supplied by the ''n- structor. 4 — Place animals in cage and note weight and tem- perature on successive days and general results. Note : — Make subcutaneous inoculations in 2 cc 0.85% NaCl solution, a — ■ Discuss in detail both types of immunity as shown in the above experiments. 24 EXERCISE 3. The Immunization of Animals. In order to produce immunity in the animal bod\- of an active type it is necessary to cause the body cells to react chemically to the injected agent whatever that may be. The intensity of the reaction depends upon the organ- ism or substance used and the manner in which it is in- jected. The substance injected always possesses a certain degree of toxicity for the animal receiving it. Animals may be immunized to almost any protein, be it animal or plant cells themselves or some constituent thereof such as serum albumins and globulins, milk, egg white, etc. The serum of the immunized animal possesses varied effects upon the substance used to bring about the immunity and the bodies of other animals into which it may be injected. On account of the toxic effects of the proteins it is necessary that the student proceed cautiously in order not to kill the animal. The weight of the inoculated animal, providing the animals are fed in a regular manner, is the best indication as to the reaction which is taking place. The weight, together with the temperature should be accurately recorded. Injections should only be made when these two conditions are normal. The resulting reaction after each injection should determine the amount and time of the subsecjuent injection. Usually this time extends over a period of from 2 to 10 days. Injections may be made in a variety of ways and in a variety of locations in the animal body. In the subsequent experiments these points will be indicated in the directions for each case. Extreme care must be observed as thp 2.5 26 animals are to be used in a series of experiments which follow. Follow directions explicitly, otherzvise death of the animal may result from the injection of too large an amount of material or from too frequent injections. All in- jections should be made with a sterile syringe and the material injected slowly. This is especially true in the case of intravenous injections. Animals may be actively immunized in the following ways : 1. K}- the injection of living bacteria. ■_'. P.y the injection of bacteria of reduced virulence. 3. By the injection of dead bacteria. 4 By the injection of the secretory or excretory products of the bacteria. 5. By the injection of the disentegration products of bacteria liberated after the death of the cells. G. By the injection of bacteria or bacterial products which are in no way related to the bacterium against which immunity is conferred. Special Directions. Animals to be immunized : 1. Immunize 5 guinea pigs by injecting them with a killed culture of M. pyogenes aureus. Heat a 24 hr agar culture in 0.85% NaCl solution 50° C for 30 minutes. Be- gin injections with 1/50 24 hour culture, give 4 injections ending with i/io 24 hour culture. Total volume of fluid injected should be 2 cc. and the injection should be given intraperitoneally. The blood serum of these animals will be used in experiments on opsonins. 2. Immunize 2 rabbits by injecting them with a live culture of B. typhosis. Inject 1/500 of a 24 hour culture and give increasing amounts as often as the weight of the animal permits. (No injections should be given within four days.) Total amount of fluid injected should be 2 cc. and injection should be given subcutaneouslv first and later intraperitoneally. The blood serum will be used in experiinents on agglutinins. 27 28 3- Immunize 2 rabbits to fresh cow's milk by giv- ing them increasing amounts by intraperitoneal injection. Begin injections with 2 cc and increase to 5 cc and 10 cc as soon as the weight of the animal permits. The blood serum of these animals will be used in work on precipitins. 4. Immunize 4.300 gram guinea pigs to a solution of egg white in 0.85% NaCl solution. The solution of egg white should be i to 10. Give injections of 2 cc, 3 cc, 5 cc and 10 cc every 3 to 4 days by intraperitoneal method. The blood serum of these animals will be used in experi- ments on precipitins. 5. Inject 4,300 gram guinea pigs with a solution of egg white as described above giving i cc by the intra- peritoneal method. These animals will be used in experi- ments dealing with hypersusceptibility or anaphylaxis. A pregnant female guinea pig should be included among those which are injected. 6. Immunize 2 full grown rabbits with the defibrinated blood of the goat. (Consult instructor and Notes as to method of securing goat blood). It is better to wash the erythrocytes several times with sterile 0.85% NaCl solu- tion in order to get rid of the serum. Make a 20% solution of them in 0.85% NaCl solution. (Consult instructor.) In- ject 5 cc intraperitoneally every three days if the weight of the animal permits until four injections have been given. Tlie blood serum of these animals will be used for hemolytic experiments, and also for hemopsonic work. 7. Procure i guinea pig from the instructor, kill it by chloroform and extirpate the kidneys. Cut the kidneys in small pieces and grind them together with some sterile sand in a sterile mortar. Wash in 0.85% NaCl solution by centrifugation. Add 20 cc of sterile 0.85% NaCl and filter through sterile paper filter. Immunize 2 rabbits by giving them injections intra- peritoneally of 3 cc of the above filtrate as often as their weight will permit. Give at least six injections. (Filtrate 2g 30 should be kept on ice between injections and thoroughly shaken before using). The blood serum of these animals will be used in experiments on cytotoxins. 8. Immunize 2 guinea pigs by injecting them intra- peritoneally, first, with a killed 24 hour culture of Microspira comma in gradually increasing doses (1/10-24 hr.), then follow by small and later by gradually increasing doses of the live micro-organisms. Heat culture to 65°C for 15 minutes to destroy it. Injections of the live bacteria should not be given within 7 days of each other. Note weight and temperature. The blood serum of these animals will be used in bactericidal experiments. 9. Immunize 2 guinea pigs, i) to 24 hour culture of B. prodigeosus, 2) to Ps. pyocyanea. Give injections often. Note weight of animals. These animals will be used in experiments on immunity in anthrax. These animals need not be immunized until later in the course. 31 32 EXERCISE 4. The Mechanism of Action of Toxic Substances Pro- duced by Bacteria and Other Plant and Animal Cells. It is a well known fact that bacteria and other cells produce their effects on the various animals and plants in different ways. In some infectious diseases the etiological organism acts in several different ways on the host while in others there is but one way in which the disease is produced. A. — The Action of Soluble Toxins : In certain diseases such as diphtheria and tetanus the bacteria are localized at a certain point in the body and from this point disseminate their toxins. These toxins are carried by the blood, lymphatics and sheaths of nerves depending on the particular toxin and its affinities. I. Procure from the instructor a solution of tetanus toxin which has been prepared after the manner given in Exercise 11. Inject 'i guinea pig subcutaneouslv with 0.3 cc of this toxin and carefully note results. a. What is the effect of this toxin on the various parenchymata of the body? b. How is the toxin carried? c. What bacteria produce toxins ? B. — The Action of the Toxins Produced by the Animal Cells. (Cobra venom) : Various snakes, am- phibians, insects, etc., produce by cellular activity soluble toxins. These toxins act in different ways upon the various species of animals inoculated. I. Procure from the instructor a 2% solution in 0.85% NaCl solution of the venom of the cobra and inject 33 34 I rabbit and i guinea pig with 2 cc each, the former intra- venously, the latter intracardiacly. The venom of the cobra after being taken from the snake is dried and when kept in this condition on ice and away from the light may be preserved for some time. Detailed experiments on cobra venom will be per- formed in a later exercise. At this time its action on the animal is the only point which it is desired to demonstrate. Carefully note the results of the experiment. a. Is there, any difference in the susceptibility of the rabbit and guinea pig? C. — The Action of Endotoxins : By far the ma- jority of bacteria produce their effects by means of the so- called endotoxins. These endotoxins are held in combina- tion with the protoplasm of the cell and are liberated when the cell dies and is disintegrated. 1. Autolize the bacteria in a 24 hour culture of B. typhosis by suspending them in 10 cc of a 0.85% NaCl solution and incubating them for 48 hours at 37° C. At the end of this time filter through a Berkefeldt filter. 2. Inject 2 cc intravenously into a rabbit and note results. a. What bacteria produce endotoxins? D. — The Action of the Proteins of the Bacterial Cell: In certain diseases such as tuberculosis, un- doubtedly the proteins of the bacterial cell are the real toxic agents. Other factors may, however, be present as contributory. 1. Procure from the instructor an agar slope culture of B. tuberculosis which has been growing for some time. 2. Scrape the growth off into a sterile mortar and triturate, keeping the mortar covered with a filter-paper saturated with i/iooo HgClj. Add 10 cc of sterile 0.85% NaCl solution and triturate further. 3. Place material in 2 centrifuge tubes and centrifu- gate 20 minutes after which time remove the supernatant 35 36 fluid (save), add as much 0.85% NaCl sol. as the tube will conveniently hold and centrifugate for 20 minutes. Pro- ceed as above a third time and after removing all but 5 cc of the supernatant fluid from each tube : 4. Inject 2 guinea pigs by the intraperitoneal method with 5 cc of the emulsion. These animals should be placed in a cage and kept under observation for some time. 5. Inject 2 guinea pigs with 5 cc of the supernatant fluid (ist washing). a. In what diseases are the proteins of the cell con- cerned ? b. What has been removed by washing the bacteria in salt solution? Observe great care in the above experiment in order to prevent infection. E. — The Mechanical Action of Foreign Cells : Without doubt the presence of foreign cells in the body in certain localities exerts an injurious effect upon that body. The presence of large numbers of bacteria in the circulation mechanically prevents the body cells from functionating properly and this is a rather important factor in some of the infectious diseases. 1. Procure from the instructor a culture of B. an- Ihracis. 2. Inject 2 guinea pigs with 2 cc of a thick emulsion of the bacteria in 0.85% NaCl solution directly into the heart. (Consult instructor.) 3. Kill the animal by chloroform in 48 hours if not already dead and take sections of the lung, liver, spleen and kidney. These tissues will be imbedded in celloidin and sec- tioned, after which they will be returned to the student for staining and examination. 4. Stain sections with methylene blue and eosin (Con- sult instructor). 37' 38 5- Carefully examine sections for mycotic emboli and infarcts. F. — The Action of the Products Produced by Bacteria Acting on Proteins: Under certain conditions some bacteria in splitting up protein compounds produce very virulent toxic substances from these compounds. Such substances when ingested often give rise to serious intoxications. A like result is obtained by the absorption of certain of the split-products which are produced in the inter- stines by the action of certain bacteria. These toxic pro- ducts are commonly known as ptoinains. 1. Procure i pound of meat and grind fine. 2. Add 500 cc of tap water and mix thoroughly. 3. Inoculate with a culture of B. vulgatus or some common saphrophyte and place in a large bottle with a glass tube to carry off gases. 4. Incubate for 2 weeks at 37°C and filter off ma- terial through muslin. 5. Pass liquid filtrate through a coarse Berkefeldt filter and concentrate ^ at a Icfw temperature on a water bath. 6. Inject varying quantities into guinea pigs and note results. 7. Inject the filtrate of a broth culture of B. vulgatus or other bacteria used (incubated for 2 weeks at 37° C) as a control. 8. Read the assignments as to the methods of puri- fication of ptomains. a. Can antibodies be produced for ptomains? 39 4D EXERCISE 5. The Selective Action of Toxins for Certain Tissues of the Animal Body. Bacterial and other plant and also animal toxins pos- sess a selective action often for certain tissues of the body. For example, tetanus toxin has an affinity for nerve tissue, diphtheria toxin has an affinity for the tissue of certain parenchymatous organs such as the kidney as well as nerv- ous tissue and certain toxins in cobra venom have an affin- ity for the endothelium of the blood vessels. 1. Procure from the instructor a guinea pig of 300 grams weight. Kill the animal by chloroform and care- fully extirpate the brain. 2. Triturate the brain in a sterile mortar and grad- ually add 10 cc of sterile 0.85% NaCl solution. 3. Procure some tetanus toxin (1-200) from the in- structor and proceed as follows : 4. Arrange three small test tubes in a rack and num- ber them I, 2 and 3. 5. To Xo. I add 3 cc of the emulsion of the brain of the guinea pig. To No. 2 add 3 cc of the emulsion of the brain of the guinea pig plus o.oi cc of tetanus toxin. To No. 3 add o.oi cc of tetanus toxin. ^fake volumes in all three tubes to 4 cc, incubate 2 hours at 37°C, and proceed as follows : 6. Procure 3 guinea pigs of approximately 300 grams weight from the instructor and inject the contents of each tube into a separate guinea pig by the subcutaneous method. (Shake tubes thoroughly). 7. Carefully record the descriptions of the animals and note results. 41 42 8. Duplicate 5, adding i/io unit of tetanus anti- toxin to No. 2 after 2 hours at 37°C, incubate 30 min- utes and inject. 9. Inject I guinea pig as an additional control with .01 cc tetanus toxin plus i/io unit of tetanus antitoxin. a. What conclusions do you deduce from the ex- periments ? b. Does tetanus antitoxin aid the brain in neutraliz- ing toxin? c. Tabulate a list of the various toxins and the tissues for which they seem to have an especial affinity. 4J 44 EXERCISE 6. The Opsonins. In the immunity of any given body one, two or three factors may be concerned. These factors are antitoxic substances, bactericidal substances and phagocytic cells. The principle phagocytic cells of the body are the poly- morphonuclear leucocytes. Practically all varieties of cells in the body, with the possible exception of some forms of connective tissue cells, may act as phagocytes. The ac- tivity of the phagocytes of the body is dependent upon the presence in the blood serum and other body fluids of the animal of certain substances which act upon the bacteria or other cells such as erythrocytes in some chemical or phy- sical manner and make them susceptible to phagocytosis. It is with these substances that we are concerned in the pres- ent exercise. The tropic substances above described are called opsonins. They are specific and are present in nor- mal sera for a large number of bacteria but may be mark- edly increased by the immunization of the animal with the specific bacteria or other cells. All bacterial cells are susceptible to phagocytosis under certain conditions. The more virulent the bacteria the less the phagocytosis by the leucocytes and other cells. This is nicely shown in a com- parison of virulent and non-virulent pneumococci. With- out the serum containing the opsonins there is a small amount of so-called spontaneous phagocytosis by the leu- cocytes. In the following experiments on opsonins three con- stituents which are normally present in the body are used but are separated from each other in order that we may have accurate information as regards their mode of action. These substances are leucocytes, bacteria and serum. 45 46 A. — Suspension of Bacteria: i. Inoculate an agar slope tube with M. pyogenes aureus and incubate for 24 hours. 2. Prepare a suspension by scraping off the growth with a sterile platinum needle and triturating it in a small amount of 0.85% NaCl solution in a test glass, gradually adding more salt solution until the solution shows a slight opalescence. This procedure is necessary in order to break up the clumps of bacteria. This may be further facilitated by drawing the suspension up in a sterile pipette of a small calibre and forcibly blowing it out against the side of the test glass several times and finally filtering it through scraped filter paper. In order to find out the approximate number of bacteria in the suspension equal parts of defribi- nated human blood may be mixed with it and a smear made and stained. Assuming that there are 5,000,000 erythro- cytes to the cubic millimeter, the average number of bac- teric per cubic centimeter may be obtained by comparative estimation. B. — Suspension of Leucocytes: Leucocytes may be secured in different ways. If a pure suspension of leucocytes is desired they may be obtained by inoculating a solution of beef broth or a suspension of aleuronot into the peritoneal or pleural cavity of an animal and the subsequent collec- tion of the resulting exudate in 20 to 24 hours after. In collecting this exudate proceed as described in the prelimi- nary notes. Usually in opsonic work the leucocytes are obtained by drawing the blood of the desired animal and defibrinating it or drawing it in a 1% solution of sodium citrate in 0.85% NaCl solution (1-5). The latter method is to be preferred. The leucocytes are then collected from the so-called "blood cream." In any event the leucocytes must be thoroughly washed with 0.85% NaCl solution so as to remove all serum. Proceed as follows: I. Collect the blood of a guinea pig by bleeding from the carotid in citrate solution as described above. 47 48 2. Centrifugate and remove serum and citrate solu- tion. 3. Add 0.85% NaCl to compensate for fluid removed in 2, mix and centrifugate again. (JNIix by the use of a pipette). 4. Repeat again 3. 5. With a fine sterile glass pipette carefully draw off the upper layer of the blood ("cream") which contains the leucocytes and place in a small test tube. Keep the tube on ice until ready for use. In some work the leucocytes may be mixed with the whole blood after the last centrifugation and used in this way. C. — Serum: The serum is most conveniently col- lected by bleeding the animal directly into a centrifuge tube, allowing the blood lo clot and subsequently centrifu- gating it and pipetting off the serum. Serum may also be prepared from defibrinated blood. 1. Bleed a guinea pig from the carotid artery and' prepare the serum as directed above. Having prepared the leucocytic and bacterial suspen- sions and the serum proceed as follows : Technique of Opsonic Test: i. Dilute serum 10 times with 0.85% NaCl solution. 2. Prepare a fine capillary pipette of uniform calibre. (Consult the instructor.) 3. Make a mark with a wax pencil on this pipette 2 cm from the end. 4. Carefully draw the leucocytic suspension up to the mark and then further up in the pipette. 5. Draw the serum up to the same mark and then on up in the pipette. 6. Draw the bacterial suspension up to the same mark and then on up into the dilated portion of the pipette where the three ingredients are thoroughly mixed and again blown down into the capillary portion of the pipette and the pipette sealed in the flame. Some operators prefer to mix the in- 49 50 gredients in a small test tube and then draw the mixture up into the pipette and seal it. 7. Incubate the pipette 15 minutes at 37" C frequently rotating it in the incubator. 8. After incubation break the tip of the pipette and make slide or cover glass smears. 9. Stain smears with carbol-thionin or a modified Ro- inanowsky stain. Note: — A control pipette, with leucocytes, 0.85% NaCl solu- tion instead of serum and bacterial suspension should be prepared, incubated and stained. Special Directions: Perform the same experiment with the blood serum of the guinea pig immunized in Ex- ercise 3. Draw a small amount of blood from the heart. Compare the results of the two experiinents and draw conclusions. Use a mechanical stage on the inicroscope to facilitate the counting of the bacteria on the leucocytes. Phagocytic Index : The phagocytic index of .a serum is based on the average number of bacteria, or other cells, which are taken up by 50 to 100 polymorphonuclear leuco- cytes. This number is, of course, dependent upon the con- centration of the opsonins in the serum. Percentage Index: The percentage index is ob- Uiin'^d by determining the average number of polymor- ])honuclear leucocytes which are phagocytic for a definite liacterium after counting 500 leucocytes. Opsonic Index: The opsonic index is obtained by dividing the phagocytic index of the sertim of a diseased animal with that of the serum of a normal animal, using the same leucocytic suspension. In diseased conditions in, which the leucocytes and opsonins are factors the phagoc3'tic index ma)' be increased by vaccination so that the opsonic index will be normal, i. e., i.o or even more than i.o. The same increase in opsonic power is noted in the serum of animals immunized with a specific bacterium. SI 52 Special Directions: Determine the phagocytic and opsonic indices of the two guinea pigs which were used above for M. pyogenes aureus. Technique of the Opsonic Test (Modified-McCamp- bell) : The ordinary technique of opsonic index de- termination is somewhat laborious and where clinical work is being carried on accurate information may be gained as to the opsonic content of a serum as follows : 1. The bacterial suspension is prepared as above after having grown a 24 hour culture of the organism in ques- tion and to this suspension 0.8% sodium citrate is added. 2. Use the leucocyte pipette of a hemocytometer and draw the bacterial suspension up to the mark 0.5 on the pipette then on up into the bulb. 3. Draw the blood of the diseased animal or person up to the mark 0.5 then on up into the bulb where it must be thoroughly mixed with the bacterial suspension and again blown down into the capillary portion of the pipette and a strong rubber band placed around the ends. 4. Incubate 15 minutes, rotating often, and at the end of this time make smears, stain and count the average bac- teria in 50 polymorphonuclear leucocytes. 5. Proceed as above with the blood of a normal in- dividual. The above method differs from the ordinary technique in that two different suspensions of leucocytes are used and that whole blood containing opsonins in the serum as well as the leucocytes is used and the ingredients not sepa- rated. The citrate of sodium prevents the blood from clotting and while it is slightly antiopsonic in its action this effect is constant in both normal sera and that of the dis- eased individual, so accurate comparative results may be obtained. Special Directions: Determine the opsonic index of the immunized guinea pigs used above by this method to 53 54 M. pyrogenes aureus. Use a normal guinea pig which has been obtained from the instructor. Technique of Making Smears on Sides and of Staining in Opsonic Work. A smear of the material taken from the opsonic pipette is best made by blowing the drop on the end of a cover glass or slide and then, by the use of another cover glass or slide, held at an angle of 45 degrees, pushing the drop across the surface of the original cover glass or slide. Staining by Modified Romanowsky Stains — Jen- ner's or Wright's: i. Cover the preparation after be- ing thoroughh' dried in the air with the stain for one minute. As the stain contains methyl alcohol this process serves to iix the film to the slide. 2. Add distilled wateij to the stain until it is rather dilute and allow the active process of staining to go on 5 minutes. Time and dilutions of stain should be varied in order to obtain the best results. 3. Wash stain off with distilled water and immediately blot with filter paper and allow to thoroughly dry in the air. The specimen may then be mounted in balsam and examined. Use oil immersion objective in examination. Staining by Carbol-thionin : i. Prepare film as above, allow to dry in the air and fix by absolute alcohol or heat. 2. Cover the preparation with carbol-thionin and stain I to 2 minutes. 3. Wash with water, blot, dry in the air, mount in balsam and examine. Use oil immersion objective in examination. Carbol-thionin : Thionin saturated in 60% alcohol 20 cc Phenol 2% 80 cc 55 56 Staining by Giemsa Stain: i. Prepare film as above and allow to dry in the air. 2. Fix film for 5 minutes with absolute alcohol. 3. Mix stain in a test tube by adding 0.2 cc of stain to 3 cc of distilled water. (i-iS). 4. Stain preparations 10 minutes. 5. Wash in tap water, blot rapidly, dry in the air, mount in balsam, and examine. Use oil immersion objective in examination. 57 58 EXERCISE 7. The Mechanism of Action of Opsonins. It is said that the opsonins act chemicall)' or fjhysically on the bacteria. It is further stated that the leucocytes can- not norniall}- phagocytize a bacterium which has not come in contact with the opsonins in the serum. The student will 'perform the following experiments in the hope of demonstrating the exact mode of action of the opsonins : 1. Prepare an suspension of washed leucocytes from the blood of the guinea pig. 2. Prepare some guinea pig serum from a normal ani- mal. Dilute 10 times with 0.85% NaCl solution. 3. Prepare a 24 hour suspension of M. pyogenes aureus. 4. Make the following combinations in capillary pipettes. 1. Equal parts of leucocytes, serum and bacterial sus- pension. 2. Equal parts of leucocytes and bacterial suspension. 5. Make the following combinations in small test tubes using 2 cc of each ingredient. Shake often. often. 3. Equal parts of leucocytes and serum. 4. Equal parts of serum and bacterial suspension. 6. Incubate pipettes i and 2 and tubes 3 and 4 for 15 minutes at 37" C. 7. Make smears of contents of i and 2 and stain with carbolthionin. 8. Wash leucocytes in 3 with 20 volumes of 0.85% NaCl solution. Restore volume. 9. Wash bacteria in 4 with 20 volumes of 0.85% XaCl solution. Restore volume. 59 6o 10. Prepare capillary pipettes by adding, a — To 3, equal parts of bacterial suspension, b — To 4, equal parts of leucocytes. 11. Incubate a and b 15 minutes at 37°C rotating frequently. 12. Make smears and stain with carbol-thionin. 13. Carefully compare the slides of i and 2 and a and b and draw conclusions as to the mechanism of ac- tion of opsonins. a. What is the spontaneous phagocytosis and has it occurred in an}' of the above experiments? b. In an infected individual with a low opsonic index for the specific infecting organism, would a leucocytosis be of any value in the development of an immunity? 6i 62 EXERCISE 8. Practical Work in the Determining of the Opsonic Index. At this time the student will make determinations of the opsonic index of various laboratory animals and patients in the hospital. It is expected that a series of different diseases will be examined and accurate records kept. Later in the course students will be assigned to different cases which are being treated by bacterial vacqines and will be expected to be perfectly familiar with the technique of determining the opsonic index. 63 64 EXERCISE Q. The Specificity of Opsonins. The serum of an animal may contain opsonins for a variety of bacteria. These opsonins are specific and can be removed by absorption with the bacteria in question. 1. Prepare a 24 hour suspension of M. pyogenes aureus. (Concentrated and as thick as possible.) (Con- • suit instructor.) 2. Prepare some guinea pig serum from a normal animal. (Undiluted.) 3. Prepare an suspension of the washed leucocytes of the blood of the guinea pig. Proceed as follows : 4. To I cc of the concentrated bacterial suspension add 0.5 cc of undiluted serum in a small test tube and incubate at 37" C for 30 min. 5. Centrifuge 4. and pipette oS supernatant fluid. (Serum in dilution). 6. Dilute supernatant fluid with 5 volumes of 0.85% NaCl solution. 7. Dilute a portion of i until the suspension corre- sponds to that of the previous suspensions used in opsonic work. 8. Dilute a portion of 2 with 10 volumes of 0.85% NaCl solution. 9. Make the following combinations in capillary pipettes: (Technique of opsonic test). A — Equal parts of leucocytes (3), serum (6), and bacterial suspension (7). B — Equal parts of leucocytes (3), serum (8), and bacterial suspension (7). 6s 66 10. Incubate pipettes at 37" C for 15 minutes, fre- quently rotating. 11. Make smears and stain with carbol-thionin. Ex- amine and compare. a. What conclusions do you draw as to whether any absorption of opsonins has taken place? 12. Prepare a 24 hour suspension of B. typhosis. 13. Prepare a 24 hour suspension of Streptococcus pyogenes. 14. Make the following combinations in capillary pipettes. (Technique of opsonic test). C. — Equal parts of leucocytes (3), serum (6), and bacterial suspension (12). D. — Equal parts of leucocytes (3), serum (8), and bacterial suspension (12). E. — Equal parts of leucoc3'tes (3), serum (6), and bacterial suspension (13). F. — Equal parts of leucocytes (3), serum (8), and bacterial suspension (13). 15. Incubate pipettes at 37° C for 15 minutes, fre- quently rotating. - 16. Make smears and stain with carbol-thionin. Ex- amine and compare. b. What conclusions do you draw as to the .specificity of the opsonins? 17. Carefully compare the phagocytic indices of all serums with the different bacteria and tabulate results, 67 68 EXERCISE 10. Hemopsonins. Phagocytosis of erythrocytes by polymorphonuclear leucocytes and various other cells has been observed. It has been shown that there are certain substances in the serum which act on the erythrocytes and render them sus- ceptible to phagocytosis. These substances are called hemopsonins and are present in larger amounts in the serum of animals which have been immunized to the corpuscles in question. Very often in diseased conditions phagocytic cells are noted containing large numbers of erythrocytes. The hemopsonins are separate and distinct from the bacterial opsonins, hemagglutinins and hemolysins. 1. Carefully draw 2 cc of the blood of the rabbit im- munized to goat blood in Exercise 3, allow it to clot and collect the serum. 2. Prepare a 5% suspension of goat erythrocytes in 0.85% NaCl solution. (See Notes on pages 11, 12 as to technique of drawing blood and washing erythrocytes.) (Whole blood may also be used.) 3. Prepare an suspension of the washer leucocytes of the rabbit either from defibrinated or citrated blood or aleuronot exudates. 4. Heat the serum obtained in i for 30 minutes at 60° C to destroy the hemolytic amboceptor. (Hemopsonins are not affected.) 5. Proceed as follows, mixing in small test tubes, 0.1 cc heated rabbit serum (4). 0.2 cc 5% goat erythrocytes (2). 0.5 cc rabbit leucocytes (3). 6g 70 6. Control, I 0.2 cc 5% goat erythrocytes (2). 0.5 cc rabbit leucocytes (3). 0.1 cc 85% NaCl solution. 7. Incubate 5 and 6 for 60 minutes at 37" C. 8. Make smears, stain faintly with eosin, dry and examine. It is not necessary to always use the leucocytes from the species furnishing the serum (hemopsonins) in the experiments. To demonstrate this proceed as follows: 9. Prepare an suspension of the washed leucocytes of the guinea pig from defibrinated or citrated blood. 10. Duplicate 5-6-7-8. 1 1 . Prepare an suspension of the washed leucocytes of the dog from defibrinated or citrated blood. 12. Duplicate 5-6-7-8. Carefully record each experiment, read the literature on hemopsonins and draw conclusions. 71 72 EXERCISE II. The Preparation of Soluble Toxins. The soluble toxins are produced by a relatively small number of bacteria. B. tetani and B. diphtheria are the principal producers of soluble toxins together with Ps. pyocyanea and B. botulinus. The toxins can be very easily prepared by growing the bacteria in fluid medias of suitable composition. The maximum toxin production is obtained by growing the cultures in a media of a particular compo- sition at 36° C to 37° C for periods varying from 7 to 14 days depending on the specific bacterium. Soluble toxins are also produced by various plant and animal cells and can be obtained in various ways. Some of these will be .'■tudied later. At this time we are concerned with only the bacterial toxins. Often the toxins produced by various cells may be separated into several specific toxins. The student will prepare diphtheria and tetanus toxin as fol- lows : A. — Diphtheria Toxin: i. Procure i lb. of lean meat, grind and add 1000 cc of water. 2. Inoculate the infusion (i) with a culture of B. coli. and incubate at 37" C for 24 hours. 3. Strain infusion through cloth. 4. Boil and filter through filter paper. 5. Make up volume to 1000 cc and add 0.5% NaCl and 2^{ peptone. 6. Titrate with X/20 XaOH and make" 0.5% acid with X/i NaOH. (If necessary add N/i HCl.) 7. Filter into toxin flasks. Sterilize in autoclav at 15 lbs. for 10 minutes. 8. Add 0.2% dextrose. (Instructor will supply sterile soiution of 20% dextrose in water.) 73 74 g. Inoculate with a virulent culture of B. diphtheriae. (Float culture on surface of medium.) 10. Incubate 8 days at 56° C to 37° C. 11. Examine culture to determine purity. 12. Add 0.5% phenol and allow the culture to stand 24 hours. 13. Filter culture through a Berkefeldt filter into small flasks and keep on ice until ready for use. The strength of this toxin will be determined as de- scribed below using guinea pigs as test animals. B. — Tetanus Toxin: i. Procure i lb. of lean meat, grand and add 1000 cc of water. 2. After infusion has stood 24 hours strain through a cloth. 3. Add 0.5% NaCl to the filtrate, boil and again filter through paper filter. 4. Titrate and make 0.2% alkaline with N/i NaOH and filter into toxin flasks. 5. In order to secure anaerobiosis cover the culture medium with i cm. of neutral Russian paraffin oil and sterilize flasks in the autoclav at 15 lbs. for 10 minutes. 6. Inoculate when cool, through the oil, with a virulent culture of B tetani. (Use a large number of organisms.) 7. Incubate at 37° C for 10 days. 8. Add 0.5% phenol and allow culture to stand for 48 hours. 9. Filter through a Berkefeldt filter into small flasks and keep on ice until ready for use. Note : — Instead of 5, anaerobiosis may be secured by incu- bating flasks in an atmosphere of H gas as in other anaerobic work. Toxins prepared in this way contain both tetanolysin and tetanospasmin. Tetanojysin is unstable and degenerates rapidly when in solution. The strength and constitution of this toxin will be determined as described below using white mice and guinea pigs as test animals. 75 76 a. What are toxoids and what do they produce in immunization? b. What are toxons? Are they of importance? c. Discuss in detail the "toxin spectrum" of Ehrlich. 77 78 EXERCISE 12. The Precipitation of Tetanus Toxin. In order to preserve the tetanolysin and the tetano- spasmin of the tetanus toxin unaltered it is necessary to precipitate and dry the toxin. The student will proceed as follows using a modifica- tion of Rickett's method. 1. Prepare tetanus toxin as described previously. 2. Add ammonium sulphate until the toxin broth is saturated and add i this amount of ammonium sulphate in addition. 3. Place in large Petri dishes and keep at 37" C for 24 hours. 4. Filter off the scum on the surface of the fluid using hard filter paper and a vacuum pump to draw off all the water. 5. Collect precipitate and dry over sulphuric acid. 6. Preserve in the ice chest over sulphuric acid. M'aterial prepared in this way is highly toxic and may be conveniently used in a 0.2% solution in 0.85% NaCl sol. Special Directions: Use 500 cc. of tetanus toxin and precipitate as directed above, 8o EXERCISE 13. Tetanolysin. Tetaiiolysin is one of the special and comparatively unessential toxins produced by B. tetani. It has an especial affinity for erythrocytes. Tetanolysin readily degenerates and in the following experiments it is necessary to use either fresh tetanus toxin or that having been precipitated and dried as described above. Proceed as follows : 1. Prepare a 5% suspension of the washed erythro- cytes of the rabbit. Erythrocytes should be washed three times in 20 volumes of 0.85% NaCl solution. 2. Prepare a 0.2% solution of tetanus toxin which has been dried. 3. Arrange a series of test tubes small in the rack and add i cc of the 5% suspension of rabbit erythrocytes to each tube. 4. Determine the amount of toxin which will produce complete hemolysis by adding varying amounts. 5. The total volume should be made to 2 cc with 0.85% NaCl sol., and the tubes should be shaken and incu- bated at 37° C for at least 3 hours. A reading should be taken at the end of this time as well as after the tubes have settled in the ice chest. a. Does normal rabbit serum show any antitoxic action for tetanolysin? The Thermal Death Point of Tetanolysin. As before stated tetanolysin is a very labile toxin. In order to demonstrate this properly proceed as follows: 8i 1. Determine the minimum hemolytic dose of the te- tanolysin for the erythrocytes of the rabbit. (Use results of previous experiments). 2. Arrange two series of 5 small test tubes each and add to each 10 times the minimum" hemolytic dose of 0.2% sol. of tetanus toxin. 3. Arrange a water bath and expose the tubes for varying lengths of time at different degrees of temperature. 4. The class will be divided into two sections and will proceed as follows : a — Run one series at 40°C, 45°C, 50°C, 55°C and 60°C for 30 minutes. b — Run one series at 40°C, 45°C, SCC, 55°C and 60°C for 15 minutes. 5. Determine the minimum hemolytic dose for each tube in the series. a. Is the action of tetanolysin important in in- fections with B. tetani? 83 84 EXERCISE 14. Tetanospasmin. Tetanospasmin is the most stable of the toxins pro- duced by B. tetani. This toxin has an especial affinity for the nervous system as can be demonstrated by the follow- ing experiment: 1. Procure some tetatius toxin broth which has been prepared in the manner prescribed before. 2. Heat 5 cc of this toxin broth 50 °C for 30 minutes. 3. Inject a serjes of guinea pigs with varying amounts of the heated toxin beginning with i/ioo cc and ending at i/io cc. 4. Determine the concentration of tetanospasmin which acts the most rapidly. 5. Carefully record results the observations extending over several days. a. How is tetanospasmin carried and where is it concentrated in the nervous system of the infected animal? b. What has been destroyed by heating the toxin in 2? 8s 86 EXERCISE IS. The , Preparation of Tetanus Antitoxin. Tetanus toxin having been prepared after the method described in Exercise ii, the students will proceed to pre- pare the corresponding antitoxin as follows : 1. Test the potency of the toxin by injecting a series of guinea pigs to find the minimum lethal dose. (MLD). This will probably lie in dilutions in the 3rd decimal place. 2. Use the toxin which shows the highest MLD. 3. Use a healthy goat as the source of the antitoxin. 4. To 10 MLD's for guinea pig add 0.05% iodine trichloride and inject the goat subcutaneously. Make up to 3 cc with 0.85% NaCl solution before injection. 5. Weigh and take temperature of goat daily. The same amount of food should be provided daily. 6. As soon as the temperature and weight of the ani- mal return to normal give another injection of a larger amount of toxin plus trichloride of iodine. 7. After the 5th injection when the animal's tempera- ture and weight have returned to normal inject 10 MLD's for guinea pig without the iodine. 8. Give increasing doses of toxin as often as pos- sible for 2 or 3 months. 9. Test antitoxic content. (This may be omitted). 10. Bleed goat from jugular vein with a trochar into sterile glass graduates of 200 cc capacity to the extent of 600 cc. (Consult the instructor.) 11. The blood thus collected should be placed in the ice chest and when the serum has separated it should be removed by means of sterile bulb pipettes and placed il sterile flasks. 87 8g 12. Filter serum through a Berk'efeldt fiUer into small flasks or bottles and add 0.5% phenol as preservative. 13. Test sterility of serum in both aerobic and anae- robic cultures. 14. The serum should be kept in the ice chest and in the dark until it can be standardized. 15. Standardize according- to the method subsequently given. a. In what ways may the toxin be attenuated? b. Will tetanus antitoxin added to tetanus toxin and injected increase the antitoxin produced? Note: — Often after injections of toxin the goat, may show tetanic symptoms. Do not give injections imtil these symptoms have subsided. 89 90 EXERCISE i6. The Standardization of Tetanus Antitoxin. Tetanus antitoxin having been prepared by immuniz- ing a goat it must be tested as to its potency. In a stand- ardization a solution of Standard Test Toxin is used. This toxin is supplied in the dry powder by the Hygienic Lab- oratory of the Public Health and Marine Hospital Service. The minimum lethal dose (MLD) to this toxin is 0.000,006 gm for a 350 gm. guinea pig. The L-|- dose of this toxin contains 100 MLD's for a 350 gm. guinea pig. The toxin is standardized against a standard test antitoxin. Tetanus Immunity Unit: "The immunity unit for measuring the strength of tetanus antitoxin shall be 10 times the least quantity of antitetanic serum necessary to save the life of a 350 gram guinea pig for 96 hours against the official test dose of a standard toxin furnished by the Hygienic Laboratory of the Public Health and Marine- Hospital Service." The L+ dose is the smallest quantity of tetanus toxin which will neutralize i/io of an immunity unit, plus a quantity of toxin sufficient to kill the animal in just 4 days. The students will proceed as follows, in each division: 1. Carefully weigh out 0.05 gm. of dried Standard Test Tetanus Toxin. (Use accurate quantitative balances). 2. Dissolve in 83.33 cc of 0.85% NaCl sol. (Use burette), i cc of this solution equals 0.000,6 gm. of the original dried toxin (100 MLD's) (L+ dose). 3. Dissolve the antitoxin to be tested, i cc of serum to 99 cc 0.85% NaCl sol. (I cc = .01 cc antitoxin), 91 92 4- Rtm the following series and others if necessary: No. of guinea pig Weight of guinea pig Mixture of Toxin and Antitoxin Time of death cc. gms. cc. 1 1 .0006 + 0.001 1 .0006 + .0015 1 .0006 + .002 1 .0006 + .0025 1 .0006 + .003 1 .0006 + .0033 •) 3 4 5 6 The guinea pig dying in 4 days has received i/io an immunity unit and from the fraction of a cc. injected the number of units of antitoxin per cc. of serum can be cal- culated. Special Directions: a — Make all injections of toxin-antitoxin mixtures subcutaneously in the same place. AVhy? Inject just below umbilicus. b — All mixtures of toxin-antitoxin should be made up to 4 cc with 0.85% NaCl sol. and kept for i hour at room temperature in the dark before injection. c — Guinea pigs should weigh as near 300 grams as possible. d — All dilutions should be made with calibrated pipettes graduated in i/ioo cc. Graduated test tubes should be used to hold mixtures before injection. e — A series of sterile syringes should be used in mak- ing the injections. Read Bulletin 43. Hygienic Laboratory, U. S. Marine Hospital Service for details as to various methods of standardization, 94 EXERCISE 17, The Preparation of Diphtheria Antitoxin. Diphtheria toxin having been prepared according to the method described previously, the students will proceed to prepare the corresponding antitoxin as follows : 1. Test the potency of the toxin prepared on a series of guinea pigs in order to find out the minimum lethal dose (MLD). 2. The toxin of the highest potency in the class hav- ing been determined this will be used in the preparation of the antitoxin. 3. A healthy goat will be used for this work instead of the customary horse. 4. Weigh the goat and take temperatures daily. The same amount of food should be provided daily. 5. Inject the goat subcutaneously with 10 times the MLD of the toxin for guinea pig. Make up to 3 cc with 0.85% NaCl solution before injection. 6. As soon as the temperature and weight of the ani- mal returns to normal give another injection of a larger amount. 7. Give injections every 5 to 7 days, if possible for 2 months. 8. Test the antitoxic content of a sample of blood drawn from the jugular vein by the method described in Notes on page 11.* 9. An antitoxic serum of some potency having been obtained the goat should be bled from the jugular vein by * If antitoxin is low, less than 200 units per cc, immunize goat for a longer time. 95 96 means of a trochar into sterile glass graduates of 200 cc capacity to the extent of 600 cc. (Consult instructor). 10. The blood thus collected should be placed in the ice chest until the serum has separated and subsequently this should be removed into sterile flasks by means of sterile bulb pipettes. 11. Filter the serum through a Berkefeldt filter into flasks or bottles and add 0.5% phenol as a preservative. 12. Test the sterility of the serum by making aerobic and anaerobic cultures. 13. The serum should be kept on ice in the dark until ready for standardization. 14. , Inject a guinea pig with o.i cc of the antitoxin in order to exclude contaminating toxins. a. What is the character of the immunity conferred upon the goat by the increasing injections of toxin? b. Describe the v.arious ways in which diphtheria antitoxin can be prepared by immunization. 97 98 EXERCISE i8. The Standardization of Diphtheria Antitoxin. Diphtheria antitoxin having been prepared by immu- nizing a goat it must be standardized. For the purpose Standard Test Antitoxin is used. The Standard Antitoxin is dried powdered horse serum of such strength that i gram contains 1,700 immunizing units. (Royal Prussian Institute for Experimental Therapy). The dried antitoxin is dissolved in equal parts of glycerine and 10% NaCl solu- tion and diluted so that i cc is equal to i immunizing unit. Diphtheria Immunity Unit : The immunity unit for meas- uring the strength of diphtheria antitoxin is that quantity of serum which is sufficient to protect a 250 gram guinea pig for 4 days against 100 fatal doses (IMLD's) of diph- theria toxin. In order to standardize the freshly prepared antitoxin it is necessary to first standardize some diphtheria toxin in which toxoid formation has come to a standstill. The toxin used in the immunization of the goat can be conveni- ently used. The standardization is accomplished by adding varying amounts of toxin to i unit (i cc) of the Standard Test Antitoxin so that one fatal dose of the toxin remains unbound. This is called determining the L-)- * dose. The L° dose is also determined. The L° dose is that quantity of toxin which just neu- tralizes I immunity unit (i cc) as is shown by a necropsy done 48 hours after the subcutaneous injection of the mix- ture into a guinea pig. The site of inoculation should be scarcely visible. A slight congestion of the site of inocula- *L= (Limit) from limes, boundary or zone. -|- = Fatal result. 99 lOO tion determines the L° dose. Edema at the point of inocu- lation may be noted in animals receiving larger amounts of toxin. The L+ dose is the smallest quantity of toxin which will neutralize i immunity unit (i cc) plus a quantity wof toxin sufficient to kill a guinea pig at least by the 4th day after injection. The students in each division will determine the L-|- dose of the toxin and also the L° dose of the toxin by run- ning the following series : THE L-f DOSE. Immunity unit Toxin cc. iRestilt in 4 days. 1 1 cc 0.19 2 .21 ''2 '.h .24 '' 1 cc 3 — 1 cc 4 — 1 cc 6 — 1 cc Run a longer series if necessary, dying after 4 days. Exclude all animals THE L DOSE. Immunity unit Toxin cc. Results at necrosy after 48 hr. 1 — 1 cc 0.14 .15 .16 .17 .18 .19 2 — 1 cc .3 — 1 cc 4—1 cc 5 — 1 cc 6 — 1 cc It is possible that it may be necessary to run a longer series than the one indicated in order to obtain the L° dose. loi I02 The toxin contains an excess of toxons and secondary toxins if the difference betiveen the L-\- and the L° dose is more than 15 MLD's. It is important to know the L° dose for this reason. The L° dose equals 100 MLD's of toxin. The Antitoxic Unit: That quantity of the new antitoxin which when mixed with the L+ dose of the toxin causes the animal to die in from 4 to 6 days contains i unit of antitoxin. Having determined the L+ dose of the toxin the stud- ents in each division will run the following series to deter- mine the number of antitoxic units per cc. of the serum. Toxin LH- cc. New Antitoxin cc. 1 0.01 .008 .006 .006 .004 .002 2 3 4 5 6 Results within 4 to 6 days. Special Directions: i. Obtain the limits for the new antitoxin and run a series between to 4th decimal place. The antitoxic units should be very accurately ascer- tained, 2. Calculate the number of units per cc. and place antitoxic serum in sterile bottles, 3. Before bottling test for sterility by making cul- tures. 4. Test phenol content by injection into a mouse. (With 0.5% phenol the mouse should show only slight tremor). 103 I04 Special Directions: a — In all injections make up the volume of the solutions to 4 cc with sterile 0.85% NaCl solution. b — Arrange a series of sterile glass syringes in a rack and add serums to them. (Sterile albolene may be used to temporarily plug the needle during the time the syringes are being filled). c — Make dilutions with volumetric flasks and cali- brated pipettes. d — Make all injections subcutaneously in the same place on the abdominal wall of the guinea pig. e — Necropsy all guinea pigs and record results. Read Bulletin 21. Hygienic Laboratory, U. S. Hos- pital Service for detailed study of standardization of diphtheria antitoxin. roS io6 EXERCISE 19. The Concentration of Diphtheria Antitoxin. Diphtheria antitoxin may be concentrated by precipi- tating, redissolving and dialyzing. Gibson was the first to devise this method of concentration. Slight modifications may be made in the method in various laboratories. The student will proceed as follows : 1. To 500 cc of antitoxic serum add an equal volume of a saturated solution of ammonium sulphate and allow to stand 24 hrs. 2. Filter and collect precipitate on heavy filter paper. 3. Re-dissolve in HjO, distilled, and re-precipitate as above. 4. Dissolve in saturated NaCl solution, 2 volumes to I volume of the original serum. 5. Precipitate by the gradual addition of 0.25% acetic acid (80%). 6. Allow to stand 24 hours and filter through hard filter paper, 7. When nearly dry collect precipitate and place in parchment sacs, 8. Dialyze in running water for 48 hours. 9. Neutralize with 20% NajCog. I p. Dialyze in running water for 48 hours, 11. Test with 5% BaClj to prove that all the am- monium sulphate has been removed ; if not, dialyze longer in running water, 12. Filter through hard filter paper under suction. 13. Pass filtrate through a Berkefeldt filter into sterile flasks or bottles and add 0.3% chloroform. to7 io8 The antitoxin should show a concentration of 2 to J times that of the original serum. Immunization with Antitoxins. In the preceding experiments the protective action of antitoxins has been demonstrated. No special work in addition along this line will be done unless clinical material is available. 109 no EXERCISE 20. The Preparation of Tuberculin. In 1891 Koch described a toxic preparation made by growing the B. tuberculosis in glycerine broth. This preparation which is known as Koch's "old tuberculin" has been used in diagnosis of tuberculosis and to some extent in the treatment of the disease. Various modifications of the original product have since been made and are used in diagnosis and in treatment. The student will prepare the following: Koch's Old Tuberculin: i. Prepare i liter of broth from i lb. of lean meat according to the usual method. (Use 500 cc and sterilize the rest.) 2. Add 2% peptone and 0.5% NaCl. 3. Add 5% glycerine and make neutral to phenol- phthalein with N/i NaOH. 4. Place 250 cc in each of 2 toxin flasks aud sterilize. 5. Inoculate one with a virulent culture of bovine B. tuberculosis and the other with a virulent culture of human B. tuberculosis. (Carefully float > culture). 6. Incubate at 37° C for 4 to 5 weeks. (Growth should cover the surface of broth). 7. At the end of this time heat culture at 100° C for I hour. 8. Concentrate to i/io volume on a water bath in a vacuum at a low temperature. 9. Add 0.5% phenol as a preservative. 10. Filter through Berkefeldt filter into sterile flasks or small bottles. Keep in a cool place. a. How are the following tuberculins prepared? 1. Tuberculin "TA". 2. Tuberculin "TO" Ill 112 3. Tuberculin "TR" or Koch's "new tuberculin". 4. Tuberculin "BF" or Denys tuberculin filtrate. 5. Dixon's Tubercle Bacilli Extract. 6. Dixon's Suspension of Dead Tubercle Bacilli. 7. Tuberculin precipitatum, Calmette. 8. Tuberculin unguentum, Moro. 9. Margaliano's tuberculin toxins. 10. Tuberculocidin,_ Klebs. 11. Tuberculoplasmin, Btichner. 12. Oxytuberciilin, Herschfelder. 13. Tuberculin, Behring or "TD" and "TDR". The Standardization of Tuberculin; Tuberculin cannot be standardized with accuracy. Koch estimates the amount of tuberculin necessary to kill tuberculous animals such as infected guinea pigs and compares normal guinea pigs injected with the same amount. Often 0.5 cc to i. cc will produce no reaction in a healthy guinea pig while a tuberculous one will be killed in 24 to 60 hours, von Lin- gelsheim standardizes by intracerebral injections, i/i8o as much being necessary to produce death as when injected subcutaneously or intraperitoneally. von Behring uses healthy guinea pigs as a basis for standardization. Ac- cording to this method "i cc ^ loooM" which means that I cc of tuberculin should be diluted so as to be fatal for 1000 grams of guinea pig. The toxicity of i cc varies with the various preparations of tuberculin, 11,-^ 114 EXERCISE 21. The Tuberculin Reaction. The fact that tuberculpsis could be diagnosed by the use of tuberculin was first pointed out by Koch. Im- mediately following the introduction by Koch it was also used a