McGRAW-HILL PUBLICATIONS IN THE AGRICULTURAL AND BOTANICAL SCIENCES EDMUND W. SINNOTT, Consulting Editor TEXTBOOK OF AGRICULTURAL BACTERIOLOGY McGRAW-HILL PUBLICATIONS IN THE AGRICULTURAL AND BOTANICAL SCIENCES Edmund W. Sinnott, Consulting Editor Adams — Farm Management Babcock and Clausen — Genetics in Relation to Agriculture Babcock and Collins — Genetics Laboratory Manual Belling — The Use of the Microscope Boyle — Marketing of Agricultural Products Braun- Blanquet and Fuller and Conard — Plant Sociology Brown — Cotton Carrier — Beginnings of Agricul- ture in America Cruess — Commercial Fruit and Vegetable Products Cruess and Christie — Laboratory Manual of Fruit and Vegetable Products Fames and MacDaniels — Intro- duction to Plant Anatomy Eckles, Combs and Macy — Milk and Milk Products Emerson — Soil Characteristics Fawcett and Lee — Citrus Diseases and Their Control Fitzpatrick — The Lower Fungi — Phycomycetes Gardner , Bradford and Hooker — Fundamentals of Fruit Pro- duction Gardner , Bradford and Hooker — Orcharding Gdumann and Dodge — Compara- tive Morphology of Fungi Hayes and Garber — Breeding Crop Plants Heald — Manual of Plant Diseases Horlacher — Sheep Production Hutcheson and Wolfe — Production of Field Crops Jones and Rosa — Truck Crop Plants Loeb — Regeneration LOhnis and Fred — Textbook of Agricultural Bacteriology Lutman — Microbiology Maximov — A Textbook of Plant Physiology Miller — Plant Physiology Piper and Morse — The Soybean Pool — Flowers and Flowering Plants Rice — The Breeding and Improve- ment of Farm Animals Sharp — Introduction to Cytology Sinnott — Botany: Principles and Problems Sinnott — Laboratory Manual for Elementary Botany Sinnott and Dunn — Principles of Genetics Smith — Fresh-water Algae of the United States Swingle — A Textbook of System- atic Botany Thatcher — The Chemistry of Plant Life Thompson — Vegetable Crops Waite — Poultry Science and Practice Weaver — Root Development of Field Crops Weaver and Bruner — Root Devel- opment of Vegetable Crops Weaver and Clements — Plant Ecol- ogy There is also a series of McGraw-Hill Publications in the Zoological Sciences, of which A. Franklin Shull is Consulting Editor. TEXTBOOK OF AGRICULTURAL BACTERIOLOGY BY F. LOHNIS, Ph.D. BACTERIOLOGIST, UNITED STATES DEPARTMENT OF AGRICULTURE AND E. B. FRED, Ph.D. PROFESSOR OF BACTERIOLOGY, UNIVERSITY OF WISCONSIN First Edition Fourth Impression McGRAW-HILL BOOK COMPANY, Inc. NEW YORK AND LONDON 1923 Copyright, 1923, by the McGraw-Hill Book Company, Inc. PRINTED IN THE UNITED STATES OF AMERICA PRESS OF BRAUNWORTH A CO. BOOK MANUFACTURERS BROOKLYN, Xs KNNRMINr DEPT.. LIBRARY . c\ ’b U9> ^ 51 PREFACE This “Textbook of Agricultural Bacteriology” was written to give the reader an accurate and fairly complete view of this new and wide field of knowledge. Most of the material presented in the book was collected and used by the senior author while teaching at the University of Leipzig (1903-1914), and under the title “Vorlesungen fiber land- wirtschaftliche Bakteriologie” was published in 1913. Several requests for an English translation have been received since then. It was deemed preferable, however, to await an opportunity when the whole matter could be thoroughly revised and rearranged in such a manner as to make the book most useful for the American and British student. The junior author has used the “Vorlesungen” for his course at the University of Wisconsin, while the senior author’s time since 1914 has been devoted exclusively to research work. It is hoped that all the varied experiences incorporated in the book will have added to its usefulness. Inevitably in a joint work of this character there were differences of opinion between the authors on certain points. Inasmuch, however, as the book was designed as a text, it was felt that it would be inadvisable to introduce any evidences of differences of opinion, either by footnote or individual statements. The first half of the book is devoted to a discussion of fundamental facts, while in the second half the practical application of bacteriology to agriculture has been fully considered. Many problems of great importance to the farmer are treated in the chapters on Dairy and Soil Bacteriology, but their accurate understanding is not assured unless the preceding chapters have also been studied. The book is neither a laboratory manual nor a reference book, but the quotations given in the text and in footnotes will direct the reader to such literature if this is desired. We are indebted to Professor E. G. Hastings for valuable criticisms. With a few exceptions the illustrations are originals, mostly made in the senior author’s laboratory at Leipzig. About twelve new ones have been prepared by F. L. Goll of the U. S. Department of Agri- culture. Washington, D. C. F. LoHNIS. Madison, Wis. g g Fred. December, 1922. v 281124 Digitized by the Internet Archive in 2016 with funding from Duke University Libraries https://archive.org/details/textbookofagricu01lohn CONTENTS PAGE Introduction 1 Relation of bacteriology to agriculture — History of bacteriology — ■ Literature. PART I GENERAL MORPHOLOGY AND PHYSIOLOGY OF BACTERIA AND RELATED MICROORGANISMS CHAPTER I. Morphology of Bacteria and Related Microorganisms .... 15 Form and size of cells — Variability of the cell form; involution forms — Monomorphism and pleomorphism — Cell compounds; branched growth — Structure of cells — Flagellation. II. Development of Bacteria and Related Microorganisms ... 25 Multiplication of cells — Formation of colonies — Conjunction, conjugation, copulation — Reproductive organs and resting cells — Autolysis and sym- plastic stage. III. Classification of Bacteria, Fungi, and Protozoa 34 Artificial and natural classification — Nomenclature — Various systems of bacteria, lower fungi, and protozoa. IV. Relations of Microorganisms to Their Environment .... 39 1 . Bacterial Nutrition 39 Chemical composition of cells — Nutrients — Stimulants — Alkaline and acid reactions. 2 . Physical Factors 44 Moisture — Air — Temperature — Light — Various other factors. 3 . Symbiosis and Antagonism 54 4- Resistance of Resting Forms 58 5 . Distribution of Microorganisms in Nature 60 Occurrence in soil, air, water, milk, and manures — Adaptation to the environment. V. Counting, Isolating, Cultivating, and Testing Bacteria and Related Microorganisms 67 Counting bacteria, fungi, and protozoa — Plate cultures — Single-cell cultures — Testing pure cultures. VI. Sterilization, Pasteurization, Antisepsis, and Asepsis ... 78 Effect of various methods — Physical treatment — Chemical treatment — Combined treatments. vii 281124 CONTENTS viii CHAPTER PAGE VII. Activities of Bacteria and Related Microorganisms .... 86 Efficiency and virulence — Physical and chemical actions. 1. Production of Color, of Light, and of Heat 88 2. Transformation of Organic Substances 93 Putrefaction, decay, fermentation — Nitrogen-carbon ratio — Enzymes. 8. The Cycle of Nitrogen 95 Destruction of organic nitrogenous compounds — Ammonification — Nitri- fication — Nitrate reduction — Assimilation of amino, ammonia, and nitrate nitrogen — Liberation of nitrogen — Fixation of nitrogen. 4- The Cycle of Carbon, Oxygen, and Hydrogen 117 Metabolism of carbohydrates, alcohols, and organic acids — Formation and destruction of humus — Formation and assimilation of carbon dioxide — Formation and metabolism of carbon monoxide, methane, and hydrogen. 5. Transformation of Mineral Substances 130 Metabolism of phosphorus compounds — Bacterial action upon carbonates and silicates — Sulfur bacteria — Iron bacteria. 6. Pathogenic Action of Microorganisms 138 Virulence and infection — Immunity and immunization — Vaccination, serum treatment, and chemotherapy. PART II DAIRY AND SOIL BACTERIOLOGY VIII. Bacteria and Related Microorganisms in Foodstuffs . . . 149 Germ content — Participation of bacteria and fungi in the making of hay, silage, and sauerkraut — Spoilage of fodder — Activities of bacteria in the digestive tract. IX. Bacteria and Related Microorganisms in Milk 161 1. Germ Content of Milk 161 Modes of contamination— Reduction and increase in numbers — Different qualities of milk — Biological milk tests. 2. Activities of Bacteria in Milk 176 Normal and abnormal alterations of milk — Formation of acids, alcohol, and gas — Decomposition of casein and fat — Changes in taste, flavor, color, and viscosity. 8. Milk Pasteurization — Fermented Milks 184 X. Bacteria and Related Microorganisms in Butter .... 188 1 . Germ Content of Butter 188 Origin of butter organisms — Changes in germ content — Types of butter organisms. 2. Bacterial Action and Quality of Butter 191 Influence upon taste and flavor — Changes in storage butter — Rancidity Other abnormal alterations. 8. Cream Pasteurization — Use of Starters 195 CONTENTS IX CHAPTER PAGE XI. Bacteria and Related Microorganisms in Cheese .... 197 1. Germ Content of Cheese 197 Origin of cheese organisms — Frequency and species of microorganisms in cheese. 2. Bacterial Activities and the Ripening of Cheese 201 Enzymatic and bacterial activities — Normal and abnormal alterations in ripening cheese. 3. Means of Regulating the Activity of Microorganisms in Cheese . . . 210 Influence of technique— Use of pasteurized milk and of starters XII. Sewage Disposal 215 Various methods — Septic tanks — Trickling filters — Activated sludge — Chemical treatment. XIII. Bacteria and Related Microorganisms in Barnyard Manures . .221 1. Germ Content of Barnyard Manures 222 Frequency and groups of microorganisms. 2. Bacterial Activities in Barnyard Manures 224 The rotting of manure — Decomposition of carbonaceous and nitrogenous compounds — Liberation and fixation of nitrogen. 3. Prevention of Losses of Plant Food from Barnyard Manure .... 234 Mechanical, chemical, and biological methods. XIV. Bacteria and Related Microorganisms in Soils 237 1 . Germ Content of Soils 237 Quantity and quality of soil organisms — Their relation to the produc- tivity of soils — Boil sickness — Biological soil tests. 2. Bacterial Activities in Soils 244 Carbon metabolism — Humus, tilth, and productivity of the soil — Nitrogen metabolism — Losses and gains in nitrogen. 3. Means of Regulating the Activities of Microorganisms in the Soil . . 262 Influence of soil management (tillage, irrigation, manuring, liming, crop- ping, fallowing) — Soil disinfection — Inoculation of soils and seeds. Index 273 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY INTRODUCTION During the last decades bacteriology has become of great importance to agriculture, because bacteria may act in many ways, both useful and harmful to the farmer. Molds, yeasts, lower algae, and protozoa frequently participate in such processes. A general term applicable to all these minute living beings is “microorganisms” or “microbes.” 1 Accordingly, “microbiology” is another name for this branch of science, and this term is really more nearly accurate than the commonly used denomination “bacteriology.” However, the latter expression is fairly well established not only because it has been in vogue for several decades, but also on account of the fact that in most of the processes concerned, bacteria usually exert the greatest influence. Aims and Scope of Bacteriology. — Bacteria and related microor- ganisms may participate in the following processes: 1. Human diseases 2. Animal diseases 3. Plant diseases 4. Normal and abnormal alterations of foodstuffs, of milk, butter and cheese. 5. Numerous biochemical processes taking place in sewage, in manure, and in soil. Bacteria have gained their widest, though unfavorable, reputation as causative agents of human and animal diseases. Many people con- sider the term bacteria as practically synonymous with infection, disease, and death. This belief, however, is no less incorrect than it would be to assume that all green plants are dangerous, because a few of them are poisonous. Nevertheless, general interest was first attracted by medical bacteriology, and the discovery of bacteria as causative agents of human and animal contagious diseases made the term bacteriology 1 Derived from the Greek words nwpbs (mikros) small, and /Sios (bios) life. 2 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY familiar to civilized mankind. Bacteriology soon became an important branch of both human and veterinary medicine and has attained an enormous development within less than half a century. Plant diseases also have been thoroughly studied for a considerable length of time. In most cases fungi were discovered as causative agents ; but more recently some important plant diseases were found to be caused by bacteria. Their study, therefore, has become a part of plant pathology. Agricultural Bacteriology. — The two main objects of agricultural bacteriology are: (a) the study of bacteria and other microorganisms in their relation to foodstuffs, milk, and dairy products, usually called dairy bacteriology ; and (b) the study of the bacterial processes in manure and in soil, usually termed soil bacteriology. The enormous amount of knowledge accumulated with regard to the causative agents of human, animal, and plant diseases, as well as the multitude of problems awaiting further investigation, have made it unavoidable that these subjects were again subdivided among special- ists. The same tendency of specialization is also noticeable in agricul- tural bacteriology. Some laboratories are reserved for dairy bac- teriology, others for soil bacteriology, a few for still more specialized work. The thoroughness required for successful research work neces- sitates far-reaching specialization, but in order to obtain a broad and well balanced knowledge of the whole field of agricultural bacteriology attention should not be centered too much upon certain special prob- lems, although they may at once attract high interest on account of their great practical importance. Sound knowledge of the fundamental facts is the basis upon which all specialization must rest, and the agriculturist who possesses such knowledge will not find it very difficult to gain a correct understanding of new findings in agricultural bac- teriology, and to make proper practical application of them, if this is feasible. Cycle of Matter. — To what extent agriculture and even the con- tinuity of Life itself depend on the incessant and energetic activity of bacteria and related microorganisms can easily be demonstrated. Figure 1 shows in a schematic manner how the eternal cycle of matter is used and regulated by agriculture and industry for the benefit of mankind. The mineral constituents of the soil help to build up plant products. These may be either directly used as human food, or they may be converted into animal products, such as meat and milk, or into industrial products, such as linen and cotton garments, vegetable oils, etc. Animal and industrial products again may be either directly used, or they may undergo another transformation before they serve our INTRODUCTION 3 purposes; for instance, wool is transformed into cloth, industrial residues like oil cakes are used as fodder, etc. But all these con- structive processes would soon come to an end if there were not a complete cycle of matter, that is, if the material used by plants, animals, and men, would not ultimately and regularly return to its origin. All organic residues must again be mineralized, otherwise the earth would long since have been littered with corpses, and all life would have become extinct. It is true that by the respiration of living plants, animals, and men considerable quantities of organic substances are being constantly broken up into carbon dioxide and water, but this fact does not materially change the general necessity of a per- manent equilibrium between constructive and destructive processes, between Life and Death. Work of Higher and of Lower Organisms. — That the constructive part of the cycle of matter is closely connected with the life of organ- isms was always self-evident to thinking mankind. On the other hand, only during the last decades was it discovered that nearly every step Food L ^and Clothing Organic Constituents of Soil Fig. 1. — Cycle of matter. 4 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY in the retrograde transformation of organic substances represents the work of minute organisms, which can not be seen except by the use of most powerful optical instruments. Occasionally purely physical and chemical processes participate in these destructive, as well as in the constructive, changes of matter. Nevertheless, the dominating and directing influence of living organisms is now equally beyond doubt in both cases. The microorganisms in milk, in butter, in cheese, in manure, and in soil are just as important to the farmer, although he may never see them, as are the milk cows hi his stable and the growing crops in his fields. The task of the medical bacteriologist usually centers upon the problem of becoming acquainted with the disease-producing germ, and to find out how it can be successfully fought and eliminated. The farmer, however, should know under what conditions he will be able to secure the most favorable results from the cooperation of the useful bacteria, and how to avoid the detrimental effects of the activities of harmful microorganisms. It was an old belief among practical agriculturists that barnyard manure adds “life” to the soil, that the surface soil is more active than the “inert” subsoil, that the “ripen- ing” of cream and cheese depends to a great extent on the use of well “working” starters. These and similar expressions indicate clearly how by practical experience a fairly correct insight was gained long before exact bacteriological investigations had become possible. Environmental Conditions. — The excellent results obtained by care- ful selection and breeding of the cultivated plants and domesticated animals led many to the belief that it should be the foremost task of the agricultural bacteriologist to select and to cultivate the most efficient strains of useful bacteria in order to make them available for the practical agriculturist. However, only in a few cases can such direct results be expected, as for instance in the use of selected bacterial cultures for the preparation of starters in the dairy, or for the inocula- tion of leguminous seeds. In all other cases the conditions under which these useful microorganisms live and work must first be in- vestigated very thoroughly. Even the most active bacteria can not dis- play their ability under unfavorable conditions, just as the best milk cow cannot show a high productivity when improperly kept and fed, nor will the best seed ever produce heavy crops on a badly tilled, weedy soil. To secure a complete and detailed knowledge of these environmental conditions is by no means an easy, though a very important task of agricultural bacteriology. At the present much remains to be done in this direction, and frequently one must be satisfied if at least the INTRODUCTION 5 general principles have been worked out which are governing bacterial life in the different phases of the transformation of matter. Year by year more details will be discovered; but a clear impartial conception of their accuracy and importance will always be dependent on a sound knowledge of the underlying general principles. Therefore these will have to be considered before the various problems of dairy and soil bacteriology can be approached intelligently. A short historical survey of the development of bacteriology will be given first. Earliest Bacteriological Hypotheses. — A more or less indistinct feel- ing that many of the processes now known to be caused by bacteria were an expression of some invisible life may be traced back through many centuries. About two thousand years ago an agricultural text- book was written by Marcus Terentius Varro, wherein it is emphasized that farm buildings never should be erected on swampy ground. As one of the reasons for this advice the author states that in such land “certain minute invisible animals develop which, transferred by the air, may enter the body through mouth or nose, and may cause serious diseases.” In its original form this interesting piece of antique bacteriology reads as follows : 1 Advertendum etiam, si qua erunt loca palus'tria, . . . quod in iis crescunt animalia quaedam minuta, quae non possunt oculi c-onsequi, et per aera intus in corpus per os ac nares perveniunt atqne effieiunt difficiles morbos. It must be left in doubt whether Varro himself was the first to con- ceive this remarkably accurate idea, or whether he merely copied it from an older unnamed source. Palladius, author of another book “On Agri- culture,” wrote again about 400 years later: 2 Palus omui modo vitanda est, . . . propter pestilentia vel animalia inimica, quae generat. (Swamps must be avoided because of the plagne or the dangerous animals which develop therein.) The beneficial effect of the nitrogen fixing bacteria now known to be active in the root nodules of leguminous plants, was also fairly well known among the agricultural writers of ancient Rome. Planting of lupine, vetch, bean, etc., was declared to enrich the soil and to act like an applica- tion of barnyard manure. In Columella’s book “De re rustica,” written in the first century of the Christian era, we find lupine, alfalfa, vetch, bean, lentil, chick-pea, and pea enumerated as plants which either enrich 1 Varronis de re rustica, Lib. I, cap. XII, printed in 1536 by Joannes Gymnicus in Cologne, together with contributions “De re rustica” by Cato, Palladius, and Columella. 2 Palladii de re rustica, — Lib. I, tit. VII. 6 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY the soil or at least preserve its fertility, while all others are said to exhaust the fields. 1 Earliest Bacteriological Observations. — In numerous mediaeval pub- lications the doctrine of the “contagium animatum” (the living con- tagion) was treated again and again; certain “animalcula” were sup- posed to be responsible for various infectious diseases. The first investigators who actually succeeded in seeing bacteria were probably the two Dutchmen Anthony van Leeuwenhoek and Christian Huygens. Leeuwenhoek himself made the lenses for his manifold studies, upon which he reported in numerous letters to the Royal Society of London. A complete collection of these communications was printed in 1695 at Delft, where he resided, under the title “Arcana Naturae Detecta” Fig. 2. — Drawings of bacteria made by A. van Leeuwenhoek in 1683 (Figs. A-G on left side) and in 1693 (Figs. A-D on right side), reproduced in “Arcana Naturae Detecta,” 1695, pp. 42 and 335. (Nature’s Secrets Unveiled). The first reference to bacterial life is to be found in a letter dated October 9, 1676, and two very interesting sets of drawings of bacteria were presented in two other letters, written in 1683 and in 1692; both are reproduced in Fig. 2. A few years older than these are some drawings made by Chr. Huygens in a manuscript dating from 1678. 2 Leeuwenhoek used for his sketches material taken from his teeth, which, as he emphasizes, were perfectly clean and healthy. Some of the bacteria were found to be actively motile, or as the Dutch author says “very gayly moving” under his lenses (indicated by the curved dotted line C-D in Fig. 2). In rainwater and in watery infusions of various 1 Columella, Lib. II, cap. X, XI, and XIV. 2 Beijerinck, Jaarboek der K. Akad. Amsterdam, 1913. INTRODUCTION 7 organic substances he saw similar organisms ; but he did not enter into any hypotheses or investigations concerning the role these minute “animals” were possibly playing in nature. Earliest Bacteriological Experiments. — An abstract of Leeuwen- hoek’s letter of 1683 was published ten years later in the Philosophical Transactions of the Royal Society of London (Vol. XVII, 1693). A few weeks afterwards Sir Edmond King, a member of this society, con- firmed the correctness of the Dutch author’s findings, 1 and also pointed out some important physiological facts which, however, were soon for- gotten. To ascertain exactly whether these minute corpuscles were really living beings, he added with a needle small amounts of sulfuric acid, ink, salt, sugar, or fresh blood to the droplets containing the bacteria under his microscope. Sulfuric acid and fresh blood proved to be most injurious; they quickly killed the bacteria, while the other substances Fig. 3. — Drawings made by Sir Edm. King, Philos. Transact. Roy. Soc. (London) Vol. XVII, 1693, No. 203. merely caused a temporary shrinking or swelling of the cells. By adding fresh water the original cell form could be reestablished, provided that the alteration had not gone too far and had not yet caused the death of the organism. This report shows that three facts, generally considered to be quite recent discoveries, i.e., the bactericidal action of blood, the plasmolysis and plasmoptysis of bacterial cells (to be discussed in Chapters I and VII, 6) are clearly described in this early, but long for- gotten paper. Some drawings made by King are reproduced in Fig. 3 ; they are unquestionably inferior to those of Leeuwenhoek. Earliest Bacteriological Classification. — During the eighteenth cen- tury many more or less ingenious speculations were contributed by various authors, but only one real advance in bacteriology was to be recorded. It is represented by the appearance of a beautifully illustrated book on “Infusoria,” written by the Danish investigator O. F. Muller , 2 1 King, Phil. Trans. Roy. Soc., vol. XVII, 1693, pp. 861-865. 2 “Animalcula infusoria fluviatilia et marina.” Hauniae, 1786. 8 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY Several of the generic names introduced by him (Monas, Vibrio, Proteus) are retained up to the present time. Earliest Practical Application of Bacteriology. — Early in the nine- teenth century the first practical results in bacteriology were secured: The Frenchman Appert discovered and taught the principles of success- fully preserving animal and vegetable foods. 1 A better knowledge of the various possibilities of thorough disinfection was also gained. That in some respects our forefathers had indeed fairly accurate ideas is to be seen, for instance, from what was known at that time about the cause and remedy of the blue discoloration of milk kept in cellars. Some kind of fungus was believed to settle on the surface of the milk; fumigation by burning sulfur and treatment of the vessels with hydrochloric acid were strongly recommended. 2 In 1837 Th. Schwann 3 stated definitely that all fermentative and abed. Fig. 4.— Drawings of bacteria published by Pasteur in 1864 (Compt. rend, tome 58, p. 142). (a) Urea bacteria. (5) Lactic acid bacteria and yeasts, (c) and ( d ) Butyric acid bacteria. putrefactive processes are caused by living organisms (“infusoria” and fungi). Two years later it was emphasized by Donne 4 that the altera- tions in milk should be investigated not only by chemical methods, but under the microscope too. Soon after C. J. Fuchs 5 succeeded in clearing up the bacterial causes of the souring as well as of several abnormal changes of milk. Louis Pasteur and His Contemporaries. — The real foundation of modern microbiology, however, was laid by the famous French chemist, Louis Pasteur. Since 1857 he published in the “Comptes rendus de l’Academie des sciences a Paris” numerous papers on fermentation, formation of lactic acid and butyric acid, transformation of urea to ammonia, etc. It is true that his first object was to disprove the old 1 “L’art de conserver toutes les substances animales et vdge tales,” 1810. 2 A. Thaer, “Grundsatze der rationellen Landwirtschaft,” Bd. 4, 6. Hauptstuck, § 54. 3 Annalen der Physik und Chemie, 2. Folge, Bd. 41, p. 184. 4 Compt. rend. Acad. Paris, tome 9, pp. 367, 800. 6 Magazin fur die gesamte Thierheilkunde, Bd. 7, 1841, pp. 150, 174, 180-194. INTRODUCTION 9 hypothesis of spontaneous generation, which was revived once more at that time, but soon his studies turned to more important and more practical problems, and they stimulated effectively similar research work, especially in France and in England. An immediate practical application of Pasteur’s discoveries to agri- cultural problems was advocated as early as 1862 in a booklet written by a German farmer named W. Iiette. 1 It was emphasized therein that in addition to the chemical points of view, as taught at that time by J. Liebig and his disciples, the biological aspects also should be con- sidered, especially with regard to the effect of stable manure and green manures upon the tilth of the soil. Decades passed, however, before this advice was heeded. Development of Dairy Bacteriology. — More rapid progress was made in the microbiology of milk and dairy products. For ex- ample, von Hessling 2 wrote in 1866 quite positively that as the various fermentations in milk so also the ripening of cheese is caused by lower fungi ; and in a book entitled ‘ ‘ Etudes sur la fabrication de f romage, ’ ’ published in 1867 by L. II. de Martin, the differences among the various kinds of cheese were explained as the results of the activity of different species or of various varieties of microorganisms. The intelligent use of starters for cream and cheese ripening was explained and recom- mended in several books of that time. More detailed information con- cerning the bacteria connected with the ripening of cheese was sought and secured by E. Duclaux in France, 3 and by Manetti and Musso in Italy, 4 while the famous British surgeon John Lister 5 worked on the problem of excluding all bacteria from the milk by observing the greatest cleanliness in every respect. Soon after, in 1884, another Englishman, 0. Ernest Pohl, made use of these investigations and was indeed able to produce on his farm milk of very low germ content 6 by anticipating those methods which are now recommended by the American Medical Milk Commissions for the production of certified milk. Development of General and of Soil Bacteriology. — Very thorough botanical investigations upon the morphology and physiology of the bacteria were started in 1872 by Ferdinand Cohn at the University of Breslau, 7 and it was in this laboratory that Robert Koch developed 1 “Die Ferments tionstheorie gegeniiber der Humus-, Mineral- und Stickstoff- theorie.” Berlin, 1862. 2 Virchow’s Archiv f. pathol. Anatomie, Bd. 35, p. 561. 3 Ann. agronomiques, 1879, Ann. de I'lnstitut national agromique, 1879-80. k Landw. Versuchsstationen, Bd. 21, 1878, p. 224. 6 Quarterly Jour, of Microscopical Science, New Series, vol. 18, 1878, p. 179. 6 Helbig, Pharmazeutische Zentralhalle, Bd. 51, 1910, p. 1051. 7 Beitrdge zur Biologie der Pflanzen, 1872-1876. 10 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY his ingenious methods which became the basis of modern bacteriology. In 1876 his classical work on the anthrax bacillus was published 1 ; a few years later he also completed the first extensive work on the bacterial content of the soil. 2 Prior to these studies, however, were the very thorough investigations on nitrification in soil, made by the French chemists TTi. Schlosing and A. Muntz, 3 which were later confirmed and extended at the Bothamsted laboratory in England by R. Warington , 4 who also worked on denitrification and other important biological proc- esses taking place in the soil. Discoveries on Nitrogen Fixation. — At Bothamsted, as at other places, interesting data had been collected with regard to the fixation of nitrogen by leguminous plants, but it remained for the German chemists Hellriegel and Wilfarth 5 to secure complete and final proof that again bacteria, living in the nodules peculiar to the roots of these plants, are directly connected with this process. In 1888 the Dutch bacteriologist M. W. Beijerinck succeeded in obtaining pure cultures of these organ- isms, and soon after he was able to show that they indeed are the causes of root nodules and nitrogen fixation. 0 Until 1921, Beijerinck continued to work at Delft (the same ancient town where more than 200 years earlier Leeuwenhoek had made his first contributions to bacteriology)? and many discoveries of great importance to agricultural bacteriology have originated in his laboratory. Best known among them is perhaps his work on Azotobacter, the most vigorous of the numerous bacteria capable of enriching the soil by the fixation of atmospheric nitrogen. 7 Other nitrogen assimilating bacteria had been cultivated before by Mar- cellin Berthelot 8 in France, and by S. Winogradsky 9 in Bussia. The latter also succeeded for the first time in the difficult task of growing pure cultures of the nitrifying organisms. 10 Present Status of Medical and of Agricultural Bacteriology. — After Bobei't Koch and his disciples had solved, in the early eighties of the 1 In Cohn’s Beitragen zur Biologie der Pflanzen, Bd. 2, Heft 2, p. 277. 2 Mitteilungen aus dem Kaiserl. Gesundheits-Amte, Bd. 1, 1881, p. 34. 3 Compt rend. Acad. Paris, tome 77, 1873, tome 84 and 85, 1877, tome 86, 1878, and tome 89, 1879. 4 U. S. Dept. Agr., Exp. Sla. Bull. 8, 1892. 6 Landw. Versuchsstationen, Bd. 33, 1886, Bd. 34, 1887, and “Untersuchungen uber die Stickstoffnahrung der Gramineen und Leguminosen,” Beilageheft z. Zeitschr. d. Vereinsf. Rubenzuckerindustrie, Nov., 1888. 6 Botanische Zeitung, Bd. 46, 1888, Bd. 48, 1890. 7 Centralbl. f. Bakt., II. Abt., Bd. 7, 1901, p. 567. 8 Compt. rend. Acad. Paris, tome 116, 1893, p. 843. 9 Compt. rend. Acad. Paris, tome 116, 1893, p. 1385. 10 Annales de Vlnstitut Pasteur, tome 5, 1891; Archives des sciences biologiques, St. Petersbourg, tome 1, 1892. INTRODUCTION 11 last century, the old questions concerning the causative agents of such dreaded diseases as cholera, tuberculosis, typhoid, etc., the medical branch of bacteriology spread rapidly to all civilized nations, and laboratories for medical bacteriology were established everywhere. With agricultural bacteriology, progress was slower. In Germany, the one-sided chemical point of view, as established by J. Liebig, remained predominant. In France, in England, and in other European countries the investigators were not numerous enough to exert a marked influence. So it became the opportunity of America to offer a promising field to bacteriological investigators. In addition to numerous research laboratories for medical bacteriology, the agricultural branch of this science is equally well represented at the American agricultural experiment stations. During the last decades much progress has been made in this country, and at present about 1000 workers are united in the ‘ ‘ Society of American Bacteriologists.” If the opportunities offered are adequately used, many valuable results may be expected, because many problems are awaiting thorough investigation. Literature. — A classified list of books and periodicals devoted wholly or in part to agricultural bacteriology is given below. The large reference books, mentioned under C, will furnish information on special subjects. A. Textbooks on General Bacteriology W. Benecke, Bau und Leben der Bakterien, 1912. E. O. Jordan, Textbook of General Bacteriology, 1922. W. Kruse, Allgemeine Mikrobiologie, 1910. E. Mace, Traite de Microbiologie, 1912-1913. Ch. Marshall, Microbiology, 1921. B. Textbooks on Agricultural Bacteriology H. W. Conn, Agricultural Bacteriology, 1918. J. E. Greaves, Agricultural Bacteriology, 1922. E. Kayser, Microbiologie agricole, 1921. S. Orla-Jensen, Dairy Bacteriology, 1921. E. Pantanelli, Prinzipali Fermentazioni dei Prodotti Agrari, 1912. J. Percival, Agricultural Bacteriology, 1920. H. L. Russell and E. G. Hastings, Agricultural Bacteriology, 1921. C. Reference Books on General and Agricultural Bacteriology E. Duclaux, Traite de Microbiologie, 1898-1901. F. Lafar, Handbuch der Technischen Mykologie, 1903-1915. F. Lohnis, Handbuch der Landwirtschaftlichen Bakteriologie, 1910. Gino de Rossi, Microbiologia Agraria e Tecnica, 1921-1922. D. Books on Bacteriological Technique and Diagnostics. F. D. Chester, Manual of Determinative Bacteriology, 1901. E. B. Fred, Laboratory Manual of Soil Bacteriology, 1916. C. Gunther, Einfuhrung in das Studium der Bakteriologie, 1906. 12 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY P. G. Heinemann, Laboratory Guide in Bacteriology, 1911. K. B. Lehmann und R. 0. Neumann, Bakteriologische Diagnostik, 1920. F. Lohnis, Laboratory Methods in Agricultural Bacteriology, 1913. E. Periodicals Abstracts of Bacteriology . Centralblatt fur Bakteriologie, I. und II. Abteilung. Journal of Bacteriology . Journal of Dairy Science. Soil Science. Part I GENERAL MORPHOLOGY AND PHYSIOLOGY OF BACTERIA AND RELATED MICROORGANISMS CHAPTER I MORPHOLOGY OF BACTERIA AND RELATED MICROORGANISMS Morphological and physiological characters of cultivated plants and domesticated animals determine the degree of usefulness of these organisms. Therefore such knowledge is of fundamental importance to the agriculturist. The same holds true with regard to bacteria and other microorganisms useful or harmful to agriculture. Because most of these organisms can be seen clearly only with a very powerful microscope, a discussion of their morphological features will help in gaining an accurate understanding of their peculiar nature, which is at the root of their surprisingly great activity. Form and Size of Cells. — While all higher plants and animals repre- sent very complicated and finely adjusted structures of cells and cell compounds, it is the single cell that acts as the living unit as far as bac- teria, yeasts, molds, and protozoa are concerned. When these single cells grow and multiply, it often happens, of course, that temporarily a num- ber of cells will be more or less closely connected. Especially the lower fungi (molds) frequently form threads or chains of cells, which some- times may be seen with the naked eye. But here again the single cell remains the living unit ; long threads break up into short joints, so-called oidia, which process can be clearly observed with the common white mold ( Oidium lactis) frequently visible as a white fur-like cover on sour cream. The most characteristic forms of bacteria, lower fungi, and protozoa are pictured on Plate I. The shape of the single cell is fundamentally the same as in higher organisms : globular, oval, cylindrical, or spiral. There are smaller or larger differences and variations in every case ; and intermediate shapes between ovals and short rod-forms, as well as between cylindrical and spiral cells, so-called comma-shaped organisms, are not infrequent. Generally the cells of yeasts, molds, and protozoa are con- siderably larger than the bacteria, but also this rule has its exceptions. Azotobacter, shown in Fig. 5, Plate I, one of the most important soil organisms, reaches, for instance, a rather conspicuous size, while some- times yeasts and protozoa may remain much smaller. Usually special treatments — staining with aniline dyes, or mixing the unstained cells 15 16 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY with India ink — are applied to get a clearer picture than is obtainable with the living cells suspended in water. How incredibly small bacteria really are, will become clear from the following consideration. At 1000-fold magnification many of the rod- shaped bacteria measure about 0.5 by 1.75 mm. A man magnified on the same scale would appear as a giant 1700 meters tall and 500 meters broad. Between such an immense being and men of normal size exactly the same relation in size would exist as between men and the bacteria 1000-fold magnified. Therefore, to reach an accurate concep- tion of the real size of the bacteria, another step of the same relation would have to be made, but this is almost beyond imagination. Measuring - Bacteria. — On account of their minute size bacteria and other microorganisms are measured by “micro-millimeters. ” One micro- millimeter, usually abbreviated “micron” (plur. micra) and written 1/x, is equivalent to 1/1000 mm. Most of the globular bacteria, usually called cocci, 1 have a diameter of about 1/4. The short rod forms, as they are found, for instance, in the root nodules of leguminous plants, measure usually 1 / 2 - 3 AyO--^ 1 / 2 l x , while the long rods reach 4 to 6/4 or more in length. Among all the bacteria thriving in milk, butter, cheese, manure, and soil, only a few will be found smaller than 1 4/4 or larger than 10/4. Instances of exceptionally large bacteria are found in the case cf organ- isms connected with the transformation of sulfur compounds (Chapter VII, 5) ; their length may reach 40 to 60/x or more. The cells of the causative agent of pleuropneumonia of cattle, on the other hand, measure only 0.1 to 0.2/4. After the ultra-microscope w T as discovered, some authors were of the opinion that they had found a special group of “ultra- microorganisms,” much smaller than the smallest bacteria known. Un- doubtedly such very minute forms exist, but it seems as if they are merely peculiar growth types of larger bacteria or of protozoa. Size and Efficiency. — Single cells of yeasts, molds, and protozoa are usually 5- to 10- to 20-fold larger than bacteria. For two reasons these differences are of great physiological importance. First, the smaller a body, the greater the area of its surface in relation to its volume. Sec- ond, as the exchange of substances in most of the metabolic processes, caused by bacteria or fungi, takes place through the cell wall, the relative size of its surface naturally determines to a large extent the efficiency of the active cell. Figure 5 shows why the smaller size of the bacteria i Derived from 6 k6kkos (kokkos) = fruit kernel. Strictly taken, the term globular bacteria should not be used, because the name bacterium comes from fSc Urpov (baktron) or paKTrjpia (bakteria), Greek words for rod. But at present the term bacteria is so generally used for all of these organisms, quite irrespective of their shape, that it has practically lost its original meaning. Lohnis-Fred, Text book Plate I 1-4. Globular bacteria, stained with methylene blue, 1000 1. Micrococci 2. =3. Streptococci from milk 4. Sarcina 5-8. Rod-shaped bacteria, stained with fuchsin, X 1000 5. Azotobacter 6. Nodule bacteria 7. Bact. casei 8. Hay bacillus 9-12. Curved and spiral bacteria, India ink preparations, X 1000 9. Proteus 10. Vibrio sp. 11. Spirillum sp. 12. Spirochaeta sp. 13-16. Yeasts, molds, and protozoa, living, X 1000 13-14. yeasts of different shape 15. Oidium lactis 16. Protozoa MORPHOLOGY OF BACTERIA AND RELATED MICROORGANISMS 17 renders them more efficient than are the larger fungus cells. Rectangle A represents the surface of a cube whose length of edge is 1 mm. If the calculation is simplified by ascribing to bacteria and fungi cubical shape and an average size of 1/a 3 and of 10/a 3 respectively, it follows that the 1 mm. cube will be filled by 1 million fungus cells or by 1000 million bacteria cells. The total surface of 1 million 10/a cubes is 600 mm. 2 (rec- tangle B), that of 1000 million 1/a cubes, however, is 6000 mm. 2 (rec- tangle C) . A 100- or 1000-fold reduction in size results in a 100- or 1000- fold enlargement of the active surface and also — at least to a certain extent — of the efficiency of these organisms. Fig. 5. — Rectangle A: Surface of a cube whose length of edge is 1 mm. B: Total surface of 1 million cubes whose length of edge is 10/a. C : Total surface of 1000 million cubes whose length of edge is l/i. One thousand million cells within 1 cubic millimeter, about the size ox a pin head, is again something well beyond imagination. If five men would each count two cells per second throughout every one of 300 days in a year, they would finish within this period not more than 100 millions, which is only one-tenth of the total sum. Size and Number. — Considering the minute size of the bacteria it is easily understood why such large numbers of them may be present in soil, water, air, food, etc. Figure 6 shows three glass containers (1/10 original size) which were filled with 20 kg. milk, butter, and Swiss cheese, respectively. In the centers of the front panes small cubes of black glass were fastened, indicating how much space would be filled by the bacteria present in that amount of milk, butter, or cheese, if they all could be collected in these places. 18 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY Wherever bacteria display great activity, rod-shaped cells are most prevalent. This again is easily understood, when it is taken into account that the active surface is comparatively much larger with a rod than with a globule. But globular cells are most frequent among the bacteria in the air ; there is little chemical activity, and small globules are naturally bet- ter able than rods to float in the air for a long time. Variability of the Cell Form. — It is a well known fact that the form of higher organisms always varies to some extent, as is especially notice- able with cultivated plants and domesticated animals. Therefore, it is Fig. 6— Glass containers with milk, butter, and cheese (xV orig. size). Germ content in milk 2^ milli ons per c.c.; in butter 20 millions per g.; in cheese 500 millions per g. not surprising that the small single cells of bacteria ana related organ- isms exhibit similar tendencies. And if one considers how much more they are exposed to the modifying influences of their environment, it will at once become obvious that even much greater variations of the cell forms are to be expected in these cases. Figure 9 on Plate I presents a bacterium, especially inclined to assume various shapes, which on account of this behavior has been named Proteus, in memory of the old Homeric sea-god, of whom it was told that he could change into every form imaginable. But on closer examination the nodule bacteria (Fig. 6, Plate I), as well as the streptococci 1 taken 1 A chain of beads, used as necklace, was called by the Greeks ffrperrbs (streptos). MORPHOLOGY OF BACTERIA AND RELATED MICROORGANISMS 19 from milk (Figs. 2 and 3, Plate I), also display certain variations in their cell forms and the cocci some deviation from the typically globular shape. But the alterations of bacterial cell forms are not restricted to such comparatively small variations. Figure 1 on Plate II indicates what changes may occur with the slender straight rods of Bacterium casei, shown in Fig. 7 on Plate I. Figure 2 on Plate II should be compared with Fig. 6 on Plate 1, and Figs. 3 and 4 on Plate II with Fig. 8 on Plate I (the normal form of Bacillus Malabarensis is very similar to that of the hay bacillus). Involution Forms. — Cells of atypical shape are often termed “involution forms,” and this name is indeed quite appropriate as far as such changes are really due to cell degeneration. Involution is contrary to evolution, and synonymous with degeneration, that is retrograde devel- opment leading to death. Unfortunately, it has become a very wide- spread habit to speak of involution forms wherever a type of growth be- comes visible which the observer considers to be atypical. The opinion that bacteria must be always globules, rods, or spirals, and that only these are typical or “legitimate” forms, has so firmly taken hold of so many bacteriologists that it is usually considered entirely superfluous to make a thorough investigation of the viability and the further behavior of such assumed involution forms. But whenever such investigations were made, it was frequently discovered that these changed cells were by no means pathological or in course of degeneration. The irregular cell forms of the nodule bacteria appear, for instance, when development is at its height, and they are very active in fixing nitrogen from the air. Other bacteria, participating in the process of nitrogen fixation, display similar changes. And increased knowledge has shown that pathogenic organisms, too, may appear, while fully active, in shapes widely differing from those often called typical. Monomorphism and Pleomorphism. — After Robert Koch had de- veloped his methods of isolating the bacteria and of growing them in pure cultures, it was soon discovered that under constant conditions a con- spicuous uniformity in growth was to be observed. This fact was con- trary to earlier opinion. It had been thought before that bacteria, like fungi and protozoa, were able to assume many different forms; they all were considered to be polymorphous or pleomorphous. 1 But as the bac- teria thus far studied were practically all grown in mixed cultures, and the new results, recorded by R. Koch and his pupils, apparently proved without exception that pure cultures of bacteria did not display such 1 Derived from iro\6s (polys) = many, tt\Icov (pleon) = more, and txopfp-q (morphe) = form. 20 TEXTBOOK OF AGRICULTURAL BACTERIOLOGY pleomorphism, the opposite point of view gained great strength among bacteriologists. The bacteria were now declared to be strictly mono- morphous, and the theory of monomorphism was taught nearly every- where ; only comparatively few bacteriologists did not accept it. However, as more and more data accumulated, it became increasingly difficult to reconcile the facts observed with this theory. "When the ex- periments were conducted under strictly uniform and constant conditions, as a rule, uniform and constant results were secured. But exceptions were not entirely absent, and changes in the environmental conditions led to still greater deviations. By declaring one type of growth in each case to be typical, and by discarding other forms as ‘ ‘ atypical ’ ’ or as signs of ‘ ‘ in- volution, ” the monomorphistic dogma could and can be preserved for a while. But if the facts are weighed impartially, no doubt remains that like lower fungi, algae, and protozoa, which have long been known to be pleomorphous, the bacteria too are able to assume different cell forms in the course of their full development, although under constant conditions uniformity and constancy are frequently observed. Cell Compounds ; Branched Growth. — With all lower organisms the single cell is the living unit, but cell compounds may be temporarily formed by bacteria, and with the fungi this type of growth is more fre- quent and more permanent. The globular cells of the cocci may occur in tetrads or in irregular clusters (Fig. 1, Plate I), in which case they are sometimes called staphylococci, 1 or in short or long chains as streptococci (Figs. 2 and 3, Plate I), or in regular cubical bundles, made up of 8, 16, 32, or more cells (Fig. 4, Plate I), to which the name Sarcina is usually applied. 2 Compounds of rod-shaped cells are either chains or threads ; in the chain the single units are still easily discernible, while in the threads the dividing cell walls are less clearly visible or have entirely vanished. Fig. 7. — Chain of budding yeast cells (X500). Fig. 8. — Branching thread of a mold ( X500). 1 Derived from os (lophos) = tuft. CHAPTER II DEVELOPMENT OF BACTERIA AND RELATED MICRO- ORGANISMS For propagating higher plants, vegetative parts of the mother plant (cuttings, tubers) may be used, or new growth is secured from seeds, that is from special reproductive organs of sexual origin. The same two ways are open for the lower organisms, although the purely vegetative multi- plication of cells is much more frequent. The simple, but efficient struc- ture of the vegetative cells of the bacteria favors this kind of develop- ment. Multiplication of Vegetative Cells. — After a cell has reached its full size, it divides into two cells, these when grown sufficiently, separate into four, four into eight, and so on. Because of this simple fission the bac- teria have been called schizomycetes, which means fission fungi, 1 although fundamentally the same mode of cell multiplication takes place in all lower as well as in the higher organisms. Quite unique, however, is the rapidity of multiplication of bacterial cells. Under suitable conditions a new fission, or a doubling of the cells takes place after 20 to 30 minutes, and if this would go on in the same manner for a day or two, the follow- ing stupendous multiplication would result : One bacterium would produce after 1 hour 4 bacteria after 2 hours 16 bacteria after 3 hours 64 bacteria after 8 hours 65,536 bacteria (in round figures 60,000) after 15 hours 1000 million = approximately 1 mm. 3 after 23 hours 65,000 mm. 3 = 65 cm. 3 after 35 hours 1000 million cm. 3 = 1000 m. 3 Therefore, the possibility exists that the progeny of one single bac- terial cell represents after iy 2 days of steady multiplication a bacterial mass that would fill 200 trucks of 5 tons capacity each. It is self-evident that natural conditions will never allow such excessive multiplication. Lack of food, the detrimental effects of metabolic products, the antagonis- tic action of other organisms, and various other influences will always 1 Derived from