BACTERIOLOGY BACTERIOLOGY SECOND EDITION A Study of Microorganisms and their Relation to Human Welfare ./ Discussing the History of Bacteriology, the Nature of Micro- organisms, and their Significance in connection with Pathology, Hygiene, Agriculture and the Industries BY H. W. CONN, Ph.D. Formerly Professor of Biology atXWesleyan University and Bacteriologist of the State Board of Health of Connecticut AND HAROLD J. CONN, Ph.D. Soil Bacteriologist at the New York Agricultural Experiment Station WILLIAMS & WILKINS COMPANY 1924 BALTIMORE Copyright 1924 WILLIAMS & WILKINS COMPANY Made in United States of America First Edition Published, September, 1923 Second Edition Published, March, 1924 ALL RIGHTS RESERVED COMPOSED AND PRINTED AT THE WAVERLY PRESS By the Williams & Wilkins Company Baltimore, Mb., U. S. A. TABLE OF CONTENTS Preface 9 PARTI. GENERAL BACTERIOLOGY 1. History of Bacteriology Chapter I. The Period of Speculation 13 The beginnings of bacteriology 13 A century of wonder-1760-1860 16 Chapter II. The Period of Fundamental Discoveries-1860-1880 .. 24 Pasteur and fermentation 24 Relation of microorganisms to disease 26 Classification of bacteria 34 A remarkable quarter-century 38 Chapter III. The Development of Methods 39 The microscopic study of bacteria 39 Pure cultures 41 Further developments 48 Modern methods 49 Chapter IV. The Era of Development-1881 till Today 55 Tracing diseases to their causes 55 Protective inoculation 56 The mastery of anthrax : 57 Protection against rabies 59 French and German rivalry 60 Diphtheria antitoxin 61 Theories of immunity 63 Plant pathology 64 Agricultural and industrial bacteriology 65 2. Microorganisms and Their Activities Chapter V. Two Great Opposing Processes in Nature 67 Building-up processes in nature 68 Breaking-down processes in nature 69 Putrefaction and decay 71 Fermentation and enzymes 73 Chapter VI. The Organisms Concerned 80 Protozoa 80 Fungi 81 Yeasts 84 2 CONTENTS Bacteria 85 Intermediate forms 91 Ultramicroscopic organisms 91 Chapter VII. Classification and Relationships of Bacteria 92 Are they animals or plants 92 Relationships of bacteria 93 Classification of bacteria 94 Chapter VIII. Physiology of Bacteria 102 Relation to external conditions 102 Bacterial associations 104 Metabolism of bacteria 105 Three important physiological groups 106 PART II. NON-PATHOGENIC ORGANISMS 1. Microorganisms in the Dairy Industry Chapter I. Milk Bacteria and Their Activities 113 Development of dairy bacteriology 113 Sources of milk bacteria 115 Growth of bacteria in milk 119 Kinds of bacteria in milk 121 Phases of growth of normal bacteria in milk 129 Chapter II. Market Problems 132 Factors preventing high bacterial counts 133 Controlling the quality of market milk 138 Bacterial analysis of milk 142 Chapter III. The Relation of Milk Bacteria to Public Health... 145 Sanitary significance of bacteria in milk 145 Protection of milk against bacteria 149 Chapter IV. Manufactured Milk Products 153 Ice cream 153 Butter 153 Cheese 158 2. Microorganisms in Relation to the Fertility of the Soil Chapter V. Soil Microorganisms in General 165 Importance of microorganisms in the soil 165 Soil as a habitat for bacteria 167 Kinds of microorganisms adapted to these conditions 170 Chapter VI. Effect of Bacteria on Plant Nutrients 174 Bacteria in relation to organic matter 174 The carbon cycle 177 Chapter VII. Effect of Bacteria on Plant Nutrients (Continued). 180 The nitrogen cycle 180 CONTENTS 3 Organic matter in the soil 193 Bacteria in relation to mineral matter 194 Chapter VIII. Practical Lessons from Soil Bacteriology 198 Control of bacteria in the soil 198 Maintaining permanent fertility 205 Handling manure 207 3. Bacteria in Relation to Miscellaneous Industries Chapter IX. The Preservation of Food 209 The spoiling of food 209 Preservation of food liable to bacterial decomposition 211 Preservation of food not liable to bacterial decomposition 217 Food poisoning 219 Chapter X. The Practical Use of Fermentations 222 Alcoholic beverages 222 Bread-raising 225 Vinegar making 228 Treatment of fodder 234 Miscellaneous fermentations 236 Application to new processes 239 Chapter XI. The Manufacture of Pure Cultures and Sera 240 Smallpox vaccine 241 Other vaccines 242 Diphtheria antitoxin 243 Other antitoxins 244 Tuberculin and mallein 245 PART III. PATHOGENIC ORGANISMS 1. Human and Animal Diseases Chapter I. Microorganisms as the Cause of Disease 249 The germ theory of disease 250 Infectious diseases 252 The transmission of disease 254 The science of public health 256 How microorganisms produce disease 257 Chapter II. Resistance and Immunity 259 Types of immunity 260 To what is immunity due 263 The biological theory of immunity, or phagocytosis 264 The chemical theory of immunity 267 Compromise theories 270 Bacteriophage 271 Chapter III. The Practical Value of the Phenomena of Immunity. .. 275 The artificial production of immunity 275 4 CONTENTS Practical results from the chemical theory of immunity 275 Practical results from the theory of phagocytosis 279 Methods of producing immunity• 283 The diagnosis of disease with the aid of the phenomena of immunity.. 285 Chapter IV. Sanitation 290 Control of disease in its relation to the method of transmission. 290 The results of sanitation 300 Chapter V. Strictly Human Diseases 302 Diseases caused by parasites with no important natural habitat out- side the human body 302 Diseases caused by parasites that can also live a non-parasitic life.... 322 Chapter VI. Diseases Caused by Parasites Attacking Both Human Beings and Lower Animals 334 Spread through lower animals as intermediate hosts 334 Spread primarily from man to man without intermediate hosts 346 Chapter VII. Animal Diseases That Do Not Ordinarily Attack Human Beings 359 Diseases of mammals 360 Diseases of poultry 366 Diseases of insects 367 2. Bacteriology of Water and Sewage Chapter VIII. Number and Kinds of Bacteria in Water 369 Source of bacteria in water 370 Variations in numbers 373 Action of bacteria upon water 374 Kinds of bacteria in water 375 Chapter IX. Water in Its Relation to Disease 378 Water-borne diseases 379 Means of detecting pollution of water 382 Chapter X. Purification of Drinking Water 386 Sedimentation 387 Filtration 388 Chemical treatment 389 'Charter XI. The Bacteriology of Sewage 392 Kinds of bacteria 392 Sewage purification 393 3. Bacteria in Relation to Plant Diseases Chapter XII. The Science of Plant Pathology 400 History 400 Terminology 402 Control 403 5 CONTENTS Chapter XIII. Bacterial Diseases 404 Certain well known diseases 404 Chapter XIV. Fungus Diseases 409 Group of fungi concerned 409 Phycomycetes- 410 Fungi imperfecti 411 Ascomycetes 412 Basidiomycetes 415 Myxomycetes 417 PART IV. APPENDIX I. Media-Making 421 II. Characterization of Bacteria 424 III. Reaction of Media and Hydrogen-Ion Concentration 433 PREFACE Early in 1916, a conversation between the authors of this book brought out the fact that there was need of a textbook in bacteri- ology which should deal with general bacteriology and its applications both to medicine and to agriculture. This book, growing out of that conversation, is intended primarily for college classes in first year bacteriology, composed partly of students who intend further specialization and partly of those who plan to take no further work along this line and wish a general knowledge of the important applications of bacteriology, as well as of the principles that underlie it. It is so arranged that the instructor, by passing quickly over Part II or Part III, can adapt it to introductory lessons in pathological or in agricultural bacteriology, respectively. Another thought kept in mind in planning the book was the historical treatment of the subject. There is no science that has had a more intensely interesting history than bacteriology; and yet few textbooks treat it in more than an incidental fashion. No dis- cussion of the history of bacteriology was known by the authors which was brief enough for use as an introduction to a course in bacteriology and at the same time sufficiently interesting to appeal to the student's imagination; yet, as a matter of fact, there is no quicker way of arousing a student's interest in bacteriology than by giving him an account of the discoveries that led to its development. For this reason, the conventional method of presenting the subject has been departed from in this book to the extent that no detailed description of bacteria is given until the very end of the first part of the book. This has been done as the result of class room ex- perience showing that students are much more eager to learn things about bacteria after hearing something about the interesting dis- coveries which first brought them to men's attention. In order to give an adequate treatment to the history of the subject, it has been necessary to include many more dates and other details than it is possible, or even desirable, for the elementary student to keep in mind; and it is realized that if the student has to learn them all the very end for which this historical introduction 8 PREFACE was written will be defeated. The selection of the details important enough for students to learn must, of course, be left largely to the judgment of the instructor; but in general it may be said that the dates given in parenthesis, instead of bodily in the text, are of minor importance and are given for reference only. One other matter that has not been handled in the conventional way is the inclusion of plant pathology in the part of the book dealing with animal and human diseases. The logic of this is evident; and the only justification for the usual arrangement of plant diseases with agricultural bacteriology is that the knowledge of them is of practical value to agricultural rather than to medical students. It is hoped that the plan adopted in this book will give the student the idea that animal and plant diseases are not, after all, so very different. The death of the senior author in the spring of 1917 made it neces- sary for the junior author to complete the wrork alone. At that time, however, the first draft of those sections which the senior author was writing was practically finished; so there is good reason to believe that the general plan of the book is essentially the same as it would have been had he lived to help in completing it. His death, together with the disturbances of the last few years, have, however, resulted in delaying its publication. Harold J. Conn. Geneva, Neiv York, 1922 PREFACE TO SECOND EDITION Although the present edition follows the first within a com- paratively short period of time, certain material is now added which it is hoped will make the book a more serviceable text. A section has been added to the chapter on immunology dealing with the recently observed phenomena ordinarily ascribed to "bacteriophage." References to literature have been added at the ends of chapters. These references are not claimed to constitute a complete bibliog- raphy-which to cover such a broad subject as that treated in this book would have to be a very long one. They are added primarily to give the teacher an idea where to look for more detailed infor- mation on the subjects treated in the different chapters. New illustrations have also been added in this edition, including certain portraits of early bacteriologists which it had been planned to insert in the first edition but which were omitted rather than delay publication until the desired photographs could be obtained. It is hoped that these additions will make the book both more useful and more attractive. Geneva, New York, 1994 Harold J. Conn. PART I GENERAL BACTERIOLOGY 1. HISTORY OF BACTERIOLOGY CHAPTER I The Period of Speculation The history of bacteriology divides itself naturally into three main periods. The first, a period of speculation, brought out but few scientific facts, and ended with the first important discoveries of Pasteur between 1855 and 1860. The second was the period of fundamental discoveries, which lasted from 1860 until 1880. The third period, that of rapid development, began with the introduc- tion of modern methods in 1881. The points which separate these periods are the most important landmarks in the history of bacteriology. THE BEGINNINGS OF BACTERIOLOGY First observations of bacteria. Apparently, Kircher, a Roman Catholic monk in 1671, was the first to see the organisms which we now call bacteria. Although he gave no special description of them, it seems probable that among the "invisible worms" which he claimed to have seen with the microscope, some were bacteria. For their study he had only simple lenses, and did not attempt their descrip- tion; but his studies extended to a variety of substances, such as Pus, decaying matter, blood, water and so forth, which surely contained bacteria. It is true that some of his "worms" were Undoubtedly chains of blood corpuscles; but the probability is that others were real bacteria. He even speculated concerning the Possible causal relation of these tiny "worms" to disease. His speculations were more nearly correct than many that were made by those who followed him. Whether or not he was the first to see bacteria, we may never know, but it is certain that only shortly after, in 1683, the Dutch- uian, Leeuwenhoek, known as the father of microscopy, did see and describe them. Leeuwenhoek was a common citizen of Delft who bad learned the art of grinding small lenses as a young man, and 14 BACTERIOLOGY later gave his entire energy to the perfection of this craft. By making his lenses very small and very carefully polished, he was able to obtain greater magnifications (from 40 to 160 diameters) than had previously been obtained. No modern microscopist thinks he can do anything with his subject unless he has a compound microscope with a magnifying power of at least 1000 diameters; but for the seventeenth century Leeuwenhoek's lenses were re- markably fine. He mounted his lenses between two copper, silver Fig.1. Leeuwenhoek's Microscope (After Loeffler) Fig. 2. Leeuwenhoek's Sketches of Bacteria or gold plates, fastened to which was a small adjustable table upon which the object to be examined could be mounted and brought into focus (fig. 1). With these simple microscopes, he began to examine everything he could think of that might be interesting upon mag- nification. The field was an entirely new one, and no matter what he examined, he made many surprising observations. Among other things, he examined material scraped oft the teeth; and to use his own words: "with greatest surprise I saw that every- where in this material, many very tiny animalcules were to be found, which moved around in the most surprising fashion." He even made PERIOD OF SPECULATION 15 figures of what he saw (fig. 2), which were crude, but nevertheless sufficiently accurate to make us confident that he had seen bacteria. We cannot expect his descriptions to have been accurate; but con- sidering the limitations of his lenses, they were wonderfully good. He found these "animalcules" in many other objects that he ex- amined. He noticed, for example, how abundant they were in a green scum on the water of a vivarium in his garden, and this sug- gested to him that we must swallow countless numbers of them in the water that we drink. Speculations as to their nature. Other microscopists soon found similar bodies in many substances that they examined, and by the beginning of the eighteenth century their existence was quite gener- ally known. Many who were fortunate enough to have the use of a microscope of any kind began to add observations to those of Leeu- wenhoek and to speculate as to the nature of these tiny bodies, until became a sort of mania from which no microscopist could keep entirely free. Even the famous botanist Linneus, who considered microscopic life of so little importance that he placed it all together m a single genus, mentioned the possibility that some of these in- visible forms of life might cause disease. The idea that diseases might be spread by particles too small to be seen by the eye had been proposed much earlier (1546) by Fracastoria of Verona. His ideas of contagion were not unlike those held today, but he had no evidence to support his theories and had Uo clear conception as to what the disease germs were like. A century and a half later, however, when Leeuwenhoek had observed bis "animalcules," the suggestion that diseases, which had always Puzzled the human mind, might be caused by them quickly seized the imagination of scientists, and the idea was developed by one after another. Apparently the first to formulate the germ theory °f disease in anything like its modern form was an Austrian physician, Plenciz (in 1762). The most important idea brought out by Plenciz was the specificity of the disease germs: that is, the conception that each disease had its own specific agent which could cause that disease and no other. He also explained that the microscopic agents might be distributed through the air, enter the body and multiply there, Producing the disease, and might later spread to other human beings by means of discharges, excreta, and so forth. His writings sound strangely modern, considering that they were pure speculation and 16 BACTERIOLOGY were written a full century before the germ theory of disease was definitely established. Plenciz also suggested that the rising of bread was due to an animalcule present in the leaven, and observed in the leaven a microscopic body which he considered to be the actual cause of the fermentation. At this date, in short, the imagination was vivid and guesses were rife; but there was almost no real knowledge, and no evidence was brought forward to show' that the ideas were anything more than unfounded speculation. The only bit of actual knowledge in all this mass of theory was that minute organisms do exist, so small as to require the microscope to see them. The realization that practically all that was written about them was without any real evidence led to the discrediting of all the theories concerning their significance. The suggestions as to their relation to disease were soon forgotten, or if remembered were only mentioned for the purpose of ridicule, until (in 1820) Ozanan wrote: "I will not waste time in refuting such an exploded theory." A CENTURY OF WONDER-1760-1860 Following the work of Plenciz, nearly a century passed before any real light began to be thrown upon the nature of these organisms. During the first three or four decades of the nineteenth century the knowledge of them stood almost as it had at the beginning of the eighteenth; and even at the middle of the century the knowledge was still very fragmentary, for the simple reason that no one knew' how to study them. Even into the third decade of the century the sum and substance of actual knowledge consisted in the simple fact that there are many microscopic living creatures in our waters and in many other places, showing more or less variety in size and shape. Not even the most vivid imagination has the slightest conception of the wonderful revelations which the years to follow' were to disclose regarding them. Classification of the microorganisms. There was especial ignorance in these days as to the different kinds of microorganisms. It oc- curred to no one at first that, such tiny objects could be classified and systematized. Gradually, however, the idea of classifying them began to appeal to scientists. Even the poor microscopes of those days showed that they differed greatly in size and shape; and as early as the eighteenth century O. F. Muller proposed a classification PERIOD OF SPECULATION 17 and gave various names to the forms which he saw. His classifica- tion was very crude; but considering his poor microscopes and the ignorance in those days of methods for staining microscopic prep- arations, he made very good headway in arranging the micro- organisms. His work need not detain us; but in passing it is interest- ing to notice that some of his names of microscopic animals are still m use today. Thus the matter stood until nearly the middle of the nineteenth century. Naturally when microscopes were so poor that nearly all these microscopic creatures looked alike, no one was much interested in classifying them, or if interested, could accomplish much. No °ne dreamed of the wealth of species and even of families and classes which were to be found among them as methods of study improved. The first classification worth recording was that of Ehrenberg (1838). He distinguished between rods and spherical forms, straight and spiral forms; and, although his descriptions are vague if judged by niodern standards, some bacteriologists today claim that certain of his species can be recognized. Of special interest is the fact that he was the first to use the terms Bacterium, Spirillum and Spiro- chaeta, all three of which are in use today as generic names for bacteria or bacteria-like organisms. Ehrenberg and his contemporaries all considered these creatures to be animals. With their low-powered microscopes, they discovered only the motile forms; and the only creatures previously known that could move were animals. Soon, however, it was discovered that some plants produce motile spores, and then it began to be wondered h motile microorganisms were necessarily animals. About the middle of the century certain scientists were bold enough to classify them definitely among the plants. The first to do so was a biologist Earned Perty; but he was quickly followed in this by the well known student of bacteria, Ferdinand Cohn. At about the same time that these organisms were first regarded as plants, another important step was taken in their classification, when Naegeli separated the green microorganisms from the colorless °nes. Naegeli did this realizing that the forms with green coloring Platter were more like the Algae and higher green plants in their Physiology, while the colorless forms were more like the Fungi. Green plants and colorless plants play very different roles in Nature's Processes, and the sharp separation between them, which Naegeli 18 BACTERIOLOGY recognized, was a valuable contribution to the classification of plants. The colorless microscopic plants were placed by him in the group of Fungi, the name Schizomycetes (i.e., fission fungi) being given to those forms that multiply by the process called fission (see p. 87). This is the technical name which scientists still give to the group of bacteria. The origin of microscopic life. While a few microscopists were thus trying to classify microorganisms, many more were speculating as to where they came from-a problem which pushed itself forward almost as soon as they were first observed. The development of bacteriology was greatly influenced by the belief in spontaneous generation. The spontaneous origin of living things had been taken for granted by the ancients. Aristotle, for instance, taught that eels could come from mud. Indeed, no one ventured to ques- tion that living can come from the non-living till in the seventeenth century (1688) w7hen Francesco Redi tried an experiment to see if fly maggots would appear in meat if the adult flies were kept away from it. He easily showed that if the flies did not get access to the meat to lay their eggs in it, no maggots would appear-a startlingly new idea in his day. From this he drew the far-reaching conclusion, quite unwarranted by the facts then known, that no living thing arises except from some living parent (or, in the much quoted words of Harvey, "omne vivum ex vivo"). This conclusion was so contrary to the beliefs of the day that it was vigorously combated and a dispute began whose echoes we still hear and which we cannot say is wholly settled yet, since even in these days we occasionally run across the claim that the production of living things has been brought about from the non-living. Certain phases of the dispute have been settled, however; and a science of bacteriology was impossible until they were settled. It did not take long to prove that, so far as concerns the larger animals and plants, Redi's claim that they arise only from living ancestors was true. But in regard to the smaller creatures this was not so easy to prove. The early investigators found that if they made a clear infusion of meat and allowed it to stand for a few days, it be- came cloudy and the microscope showed it to be filled with living organisms. Did they come from the air, or did they develop spon- taneously out of the infusion? About the middle of the eighteenth century Needham tried to settle the question by boiling such an PERIOD OF SPECULATION 19 infusion to kill all living things in it and sealing it hermetically so that nothing more could get in from the air. These boiled, sealed flasks, when set aside, sometimes remained clear, but often became cloudy after a few days, and were then found to be filled with living organisms, He believed that these must have developed spon- taneously in the liquids, and he had plenty of followers in this con- clusion for the idea fell quite in accord with the beliefs of his day. Meanwhile another school of experimenters, foremost among whom stood Spalanzani, opposed this belief, insisting that the experiments had not been carefully enough performed to warrant the conclusion. They performed experiments of their own in opposition to the Needham school in which they showed that when sufficient care was taken to destroy thoroughly all living matter in the infusions and to seal the flasks completely, no signs of life would appear in the in- fusions. If, however, there was the slightest crack in the glass or in the seal, living things quickly made their appearance, a fact which, it was insisted, showed that the living organisms actually came from the air and were not generated spontaneously in the liquid. This was again denied and again affirmed, and the dispute went on merrily for over a century. Oddly enough the question was settled in a practical way even while the scientists were vehemently disputing about if. It had always been known that moist food will not keep long, and in the years following Leeuwenhoek it had betm found that the spoil- ing of food was accompanied by the growth of myriads of the "animalcules." Now early in the century (1809) Appert discovered that if such perishable food be placed in proper containers, be then subjected to heat and afterwards hermetically sealed, it would keep without spoiling and without the development of living organ- isms within it. The process of canning food was really a demon- stration that living things come only from living ancestors. It must not be supposed that the scientists did not know of the preservation of food by Appert's methods. They claimed that boiling the solutions in the vessels containing food would drive out all the air, and since oxygen is a necessity for life, living organisms could not be expected to arise under such conditions. This criti- cism was met (in 1836) by Schulze, who reintroduced air into his experimental flasks, first passing it through concentrated sulphuric acid, and in the following year by Schwann, who passed air through 20 BACTERIOLOGY a red hot tube and then allowed it to enter his flask of fermentable material. Either of these devices would furnish the material with the air necessary for life, but would kill all the living things contained in the air before it entered the flasks. Even these experiments were criticized by members of the Needham school who claimed that the air thus heated or chemically treated lost some of its life-giving properties; so the dispute still continued. Later (1854), however, Schroder and von Dusch discovered a better method, allowing air to reenter the flasks, but filtering it through a plug of cotton which removed all dust particles and thus all living things that might be in the air. It was finally shown independently by Hoffman in 1860 and Pasteur in 1861 that no filter was necessary provided the neck Fig. 3. Pasteur's Culture Flask. (After Loeffler) of the flask was drawn out into a fine tube and bent once or twice so that nothing could fall in through it (fig. 3). Finally Tyndall (1876) showed that flasks of sterilized material could remain open indefinitely in a small chamber without comtamination provided the air of that chamber was so free of dust particles that none could be Been in a bright ray of light passing through it. As experiments were refined more and more, the reasons for be- lieving that even microscopic life could come into existence spon- taneously grew less and less. It was found possible to capture the dust particles from the air, sow them in solutions and actually see living things in them grow. It was proved that keeping dust parti- cles out of infusions would prevent the appearance of life, provided all living things had been previously killed. It was also shown that some forms of living things-the spores of bacteria-are so resistant PERIOD OF SPECULATION 21 to heat that simple boiling will not kill them, a fact which explained some of the contradictory results of early experiments. In short, it was finally thoroughly demonstrated that under experimental conditions the minute organisms, just as truly as larger animals and plants, always arise from others like themselves by the common process of multiplication. Until this conclusion was reached, manifestly, the development of bacteriology could not progress; for if it were possible to suppose that microorganisms could arise without any ancestry, their logical study would hardly be possible. Fermentation. Even before the matter of spontaneous generation was settled, and considerably before a satisfactory classification of the microorganisms had been proposed, the difference between bacteria and yeasts was recognized. Yeasts, because of their com- paratively large cells and their method of multiplication by budding (see p. 84), were quite conspicuous objects to the early micro- scopists; nevertheless, knowledge of them would not have progressed as rapidly except for their agency in fermentation. Fermentations have been utilized by man for centuries. Perhaps there is no race of man that has not known something in regard to these phenomena. All savage tribes make some alcoholic beverage by fermentation of sweet juices. Leavened and unleavened bread are mentioned in oldest Hebrew records, leavened bread being that which had been allowed to ferment and thus become porous. The Romans possessed some method by which they prepared a yeast which they put artificially in the dough for the purpose of raising it. How they prepared this yeast has not been recorded except that they produced it in some way from grapes. From the Romans down through the centuries the use of yeast for this purpose has been more or less common. Among the ancients, of course, this process Was not understood; but it had been found that if a little of a satis- factory batch of the dough after rising were put aside till the next day and then used to start the process for the next batch of bread more constant results could be obtained. The risen dough thus saved for use another day was called leaven. The early microscopists examined leaven and rising dough and found there minute bodies which they correctly assumed to be the cause of the fermentation. Their lenses, however, did nothing more than barely reveal the existence of these bodies and in these early days no one thought of obtaining pure cultures of them to use in 22 BACTERIOLOGY bread rising. The commercial preparations of yeast now so common in the market were not introduced until very recently. It was known even in the early days that there were other forms of fermentation besides that causing the rising of dough. The fer- mentation of fruit juices giving rise to alcoholic drinks was known but was not realized to be caused by the same ferment as that which produced bread rising. Other gaseous fermentations of sweet solutions were also known. These were all classed together, and with them were placed other purely chemical processes like the evolu- tion of gas when acid acts on metals. The distinction of these various types of fermentation from each other came about as the microorganisms that caused them were studied. The discovery of yeasts was made (in 1837) independently by two investigators, Latour and Schwann. They showed that the bodies observed in fermenting sugar solutions have all the characteristics of living things and that they grow and multiply by budding. By a series of ingenious experi- ments Schwann showed that the alcoholic fermentation of sugars does not take place without these organisms and that the organisms grow during the fermentation. This conclusion should have stimulated the study of microscopic life in general and its connection with other phenomena besides fermentation; but the development of the subject was delayed by the position of the chemist Liebig, who at this time had achieved such a commanding reputation that any view he advocated was sure to command acceptance. Liebig repudiated the idea that fermenta- tions and similiar phenomena are produced by living agents and claimed that they are purely chemical processes. His idea was that albuminous matter has a tendency to undergo spontaneous chemical decomposition, the large molecules of these substances breaking down into simpler and more stable ones. This process of breaking down he assumed to require no outside agency in the case of albumins, whose molecules, he considered to be so unstable that they start to break down spontaneously, the process progressing more actively hour by hour (putrefaction). He believed sugars were a little more stable than albumins and would not of themselves enter into such a series of decomposition changes; but if the process were once started, they would decompose, much the way a fire will con- tinue if once kindled. Liebig's explanation of the action of yeast was that the albuminous matter in it was sufficient to start the PERIOD OF SPECULATION 23 decomposition, kindling the fire, as it were, not from their living activities or as the result of their growth, but simply because the breaking down of their albuminous substance started the same proc- ess in the sugar. This conception of fermentation seemed so convincing as a logical theory that it was not easy to doubt it; and, supported by the name of Liebig, it acquired a standing that could not be overcome by anything except extraordinary evidence. Thus Liebig actually delayed the development of bacteriology many years. It was not until another master mind attacked the subject that Liebig's theories began to be discredited and this branch of science started again on the right road. The new master mind was Pasteur, who finally took up the subject and did not leave it until the modern science of bacteriology had been well launched upon its startling career. REFERENCES Friedrich Loffler. Vorlesungen uber die gesichtliche Entwickelung der Lehre von den Bacterien. 252 pp. Leipsig, 1887. Ferdinand Cohn. Untersuchungen uber Bacterien. Beitrage zur Biologie der Pflanzen. Bd. 1, Hft. 2, p. 127. 1872. CHAPTER II The Period of Fundamental Discoveries-1860-1880 PASTEUR AND FERMENTATION Shortly after the middle of the nineteenth century began the most important period in the development of bacteriology-the period in which the fundamental discoveries were made that opened up the field for the later investigators. This period was ushered in by the work of Louis Pasteur, and indeed, Pasteur was the genius around whom the bacteriological work of this period centered. His career was all the more striking because of his humble start in life. His father was a tanner, and he began life without any special advantages; but in spite of this origin, he became the most prominent man of his day, one of the leading men of the century. His work was appreciated not only by fellow-scientists, but by the rank and file as well. When, early in the present century, a Paris newspaper took a vote among its readers as to the most notable man France had produced, by far the largest vote was given to Pasteur, the second, Victor Hugo, falling much below him. Pasteur's original profession was chemistry. At the age of twenty- five he had already achieved recognition because of some work on the molecular structure of crystals. During his early thirties, he became interested in the phenomena of fermentation, which he believed to be due to biological agencies, thus opposing the position of Liebig. It was a simple matter to confirm the work of Latour and of Schwann, showing that the alcoholic fermentation could take place only in the presence of yeast; but he was not content with this simple demonstration. He soon proved that there are many types of fermentation, each caused by its own specific agent-a task which must have taxed his ingenuity and patience to the utmost. Up to his day there had been no clear idea that there was more than one kind of fermentation. Putrefaction had been recognized, and Liebig had claimed that it was a process related to fermentation due in the same way to a chemical destruction of unstable molecules; but beyond that, there was little conception of the various different Louis Pasteur PERIOD OF FUNDAMENTAL DISCOVERIES 25 fermentative processes. Even those who believed fermentation to be of biological origin did not think of associating different micro- organisms with the decomposition of different materials. The trouble was that, when dealing with such extremely small organisms, no one knew how to separate any one kind from the others and find out what it could do alone. Pasteur solved this problem in a very simple but very ingenious way. He noticed that when any natural substance such as milk or fruit-juice underwent fermentation, microorganisms developed in it, but in different kinds of fermentation different kinds of microorgan- isms appeared. His conclusion was that the different kinds of fermentation were caused by different ferments. To test this theory he made up artificial mixtures containing the sugars whose fermentation he was studying. He sterilized these solutions and inoculated them with a little of the sediment from the fermented fruit juice, sour milk or whatever he was studying. If his artificial culture solution underwent the typical fermentation and showed the same kind of microorganism as was present in the original material, he would conclude that this organism was the cause of the fermenta- tion he was studying. He tested this out by inoculating other material with this culture and observing the type of fermentation that took place. He found, for instance, that a certain type of microorganism always grew in large numbers in ordinary sour milk and also appeared in his solutions of milk sugar when inoculated with sour milk. If, however, the milk were first boiled, he found that it fermented in a different way, with the production of butyric instead of lactic acid; and if sterilized solutions of milk sugar were inoculated from this milk they underwent butyric fermentation, showing under the microscope a different type of microorganism from that growing in normal sour milk. Milk inoculated from these artificial cultures developed lactic or butyric acid according to whether his cultures had been obtained from normal sour milk or from milk soured with butyric acid. This showed quite plainly that the fermentations were each caused by their respective ferments. He studied in the same way a tartaric fermentation, an acetic fer- mentation in cider and a slimy fermentation in wine, concluding that these are all distinct from each other and from the alcoholic fermentation, and that each is caused by its specific microorganism. He spoke of these ferments as "new yeasts"-the "lactic yeast," 26 BACTERIOLOGY "butyric yeast" and so forth-not realizing that he was studying organisms belonging to an entirely different group of living things from the alcoholic ferment, a group which is even more important in nature. These conclusions of Pasteur proved to be surprisingly nearly correct; for while in some details the statements of his early writings have been corrected by subsequent experiments and ob- servations, in general they have been borne out by later work. All of this effectually disposed of Liebig's idea that fermentation is a spontaneous decomposition of the complex unstable molecules and it opened the road for the new discoveries that were soon to come. Pasteur demonstrated the highly important' fact that fer- mentation could no longer be looked upon as a single process, but rather as consisting of a considerable variety of distinct phenomena, each brought about by distinct and definite microorganisms. The most practical of all these early investigations of Pasteur was his work on the undesirable fermentations in wine. This study was begun as an investigation of the "diseases" of wine and their control; in this it was successful, but it also resulted in a far more important discovery-the process known as pasteurization. Pasteur dis- covered that these undesirable fermentations can be prevented by heating the wine to kill the ferments; but as boiling spoils the wine, he devised the scheme of applying less heat, enough to kill the microorganisms but not sufficient to injure the quality of the wine. In later years this same process has been applied to other food products, and in the milk industry has become of great practical importance. RELATION OF MICROORGANISMS TO DISEASE During the years in which the ideas concerning fermentation had been developing, the knowledge of microorganisms had been pro- gressing in other equally important lines. The thing which brought these microscopic organisms into general prominence and set so many hundreds of students at work with them was the demonstra- tion of their relation to diseases. Interesting though they were to the microscopist, and important though their agency in decomposi- tion might be, it was the recognition of their powers to cause disease in animals and in man that produced the insistant demand for their study. But for this, bacteria might have received no more study than other small organisms, and might still be regarded as interesting only to the scientist. PERIOD OF FUNDAMENTAL DISCOVERIES 27 The suggestion that these organisms might be the cause of disease was first made, as already mentioned, in the seventeenth century, but was soon discredited. Discredit attached to the idea until the fourth or fifth decade in the nineteenth century when, at first vaguely and then more definitely, the conception was revived. There was no real evidence that diseases were produced by such organisms, but it began to be felt that the idea was not so wild and ridiculous as some had thought. No sooner had the idea begun to gain ground again than numerous contradictory observations, based upon in- sufficient data and some of them far from the truth, began to be published, and the idea might easily have fallen again into disrepute. Even before Pasteur's day however, Henle, had seen the weakness in all such work and had pointed out (in 1840) the steps needed to prove that any disease was produced by a definite microorganism. Henle's argument was that to prove the causal relation of a micro- organism to disease, it must be found constantly present in cases of the disease, it must be isolated and tested by inoculating into healthy animals. This argument is generally known today in the form which it was stated some time later (1882) by Robert Koch: (1) the or- ganism must be shown to be present in abundance in the tissues, blood, or discharges of animals suffering from the disease; (2) it must be isolated and studied in pure culture; (3) it must be shown capable of producing the same disease in healthy animals; (4) it must subsequently be found again in abundance in the experi- mentally inoculated animals. Because of the concise form in which Koch stated these requirements and because of the emphasis which he laid upon them in his work, they are generally known today as Koch's postulates; but it must not be forgotten that the idea was originally Henle's. Henle was born ahead of time. With his clear logic, if he had followed Pasteur instead of preceding him, he would undoubtedly have been one of the leading lights in bacteriology. In Henle's day, in fact, these postulates were impossible to carry out because there was no way of isolating the microorganisms, and therefore the complete proof could not be obtained. For this very reason, Henle deserves all the more credit for keenness of mind in emphasizing the necessity for a procedure impossible with the technic of his day. Although he suggested it eighty years ago, it has not been improved upon, and even today these steps are followed in the attempt to 28 BACTERIOLOGY demonstrate the cause of a germ disease, although the last two steps must sometimes be omitted, as certain human diseases cannot be given to animals. Pebrine. About the middle of the century, the silk-worms in southern France were affected with a serious disease known as pebrine, that bade fare to ruin the silk industry. The raising of silk-worms had long been an important industry among the French peasants, and the appearance of this disease caused widespread alarm. Pebrine spread rapidly through the country, attacking farm after farm, and wherever it made its appearance, it at once curtailed the production of silk. In a period of thirteen years silk production had fallen from an average of 65,000,000 pounds down to 8,000,000 pounds and it required no far prophecy to see that the industry was doomed unless something could be done to check this rapidly spreading disease. Of course this period of years had not passed without the most strenuous endeavors to find the cause of the disease and some method of combatting it. Commissions were formed, and most exhaustive study was made into the subject from every standpoint that was then within the reach of science. But all of these had been in vain, and pebrine continued to spread. In this crisis Pasteur was approached (1865) by some of the silk- worm raisers, because he was known to be a brilliant scientist who had done some interesting work in connection with fermentation. He hesitated a while before accepting, as his chosen profession was chemistry and he felt that if he once undertook a biological study of this sort, he might never return to his original subject. In this he guessed correctly. The world, to be sure, will never regret that he took the step; but many years later he is reported to have said: If I have a regret, it is that I did not follow that route, less rude it seems to me, and which would have led, 1 am convinced to wonderful discoveries. A sudden turn threw me into the study of fermentation, fermentations set me at diseases, but 1 am still inconsolable to think that 1 have never had the time to go back to my old subject. It seems a little strange that Pasteur succeeded in a few years in accomplishing what the best minds of France had been unable to accomplish in all their study of this disease. He not only found the cause of the disease but devised the lines of defense that could be established in protecting the industry against this threatening dis- PERIOD OF FUNDAMENTAL DISCOVERIES 29 aster. The reason for this conquest was simple and lay in the fact that Pasteur attacked the problem with a new weapon and by new methods. His previous studies upon fermentation and putrefaction had convinced him of the significance of microscopic life in producing many changes in Nature. The first idea that occurred to him, therefore, was to study pebrine with the microscope and to see whether he might not have here a phenomenon similar to those he had been studying before. While others had used the microscope occasionally for the purpose of studying silkworms suffering from pebrine, no one had seriously considered this as a practical aid in the solution of the problems at hand. Pasteur's microscope showed him promptly that the afflicted animals were filled with microscopic objects that were evidently living organisms. His tests showed him that it was possible to carry the disease from animal to animal simply by transportation of material containing these microscopic bodies and that the presence or absence of the disease in the silk-worm depended upon the presence or absence of these living organisms. He did not know how to isolate them or to obtain complete proof by inoculating them into healthy animals; but with his patience and clear mindedness he was able to collect such a lot of facts and to correlate them so convincingly that his conclusions were fully justified. We cannot here enter into the details of his experiments, epoch-making though they were. Briefly the results were to show that pebrine is produced by a microscopic germ, carried from animal to animal, through the egg, through the moth and through the adult-thus for the first time demonstrating the actual agency of a microorganism in causing disease. Subse- quent study has shown that the organisms concerned in this case were not bacteria, belonging rather with unicellular animals than with plants; but in Pasteur's day these two groups of micro- organisms were not distinguished. Pasteur was not content with any purely academic demonstration, for all his experimental work had its practical side. He therefore set to work at once to find some practical method of controlling the disease. The method which he devised was by the use of pure strains of silk-worms, from any source available, provided they were uncontaminated with these germs, and then by carefully watch- ing the eggs, the larvae and the adult, promptly destroying any animal in which the disease made its appearance, and retaining for 30 BACTERIOLOGY breeding purposes only those which were shown by the microscope to be free from these microscopic organisms. The details of this method were fairly simple, but were at first ridiculed by the practical raisers of silk, and no one had sufficient confidence in the method to put it into practice. This fact only stimulated Pasteur to a deter- mination to prove that he was right. He arranged to have placed at his disposal a silk-worm farm that had been abandoned because of the disease, and established himself at this farm applying upon it his own methods of raising healthy silk-worms. In a comparatively short time he freed his farm of pebrine, obtained a quantity of stock free from the disease and made the farm one of the best silk-worm farms in France. This practical demonstration of the truth of his conceptions rapidly removed the prejudice against his methods and led to their adoption elsewhere. The silk-worm industry was rapidly revived and has not seriously suffered from this menace since then. It must not be imagined that all this was accomplished in a few weeks or that Pasteur had no setbacks in the course of the work. The five years that he was working on silk-worm troubles were perhaps the most discouraging of his whole life. They were made hard for him not only by slow progress with the work, but by the death of a daughter and by an attack of paralysis. He had no more than conquered pebrine when he discovered that a second silk-worm disease was common, which was frequently confused with pebrine and was responsible for a good share of the ravages. This second disease was finally mastered, but not before much harsh criticism had been heaped upon him; and the wide recognition that finally came to him was no more than a just reward for the worries of the years that had gone before. Demonstration of the germ theory of disease. This discovery saved no one knows how much money to the silk industry; but it was worth even more to mankind in general because of the stimulus it gave to investigations of germ diseases. This was the first time that a specific microorganism had been shown to be associated with a definite disease, and it was regarded by many scientists as furnishing final proof of the germ theory of disease. This conclusion was not universally accepted, however, and for several years quite a heated dispute raged as to whether living microorganisms could cause disease. The theory was first put to practical use by Lister, who introduced antiseptic surgery (1867). Lister reasoned that the in- PERIOD OF FUNDAMENTAL DISCOVERIES 31 flammation and pus-formation that so often accompanied wounds must be due to microorganisms and therefore could be prevented in surgery if the germs were rigidly excluded from the wounds. As these germs are everywhere, in air, water and on surgical instru- ments and dressings, Lister concluded that everything coming in contact with the exposed flesh should be thoroughly sterilized with some disinfecting agent. Lister used carbolic acid mostly, even carrying on some of his operations in a spray of this disinfectant. Lister's technic proved so successful that it not only saved the lives of countless people, but furnished additional proof that certain diseases could not occur in the absence of microscopic life. The cause of anthrax. One of the most dreaded animal diseases m Pasteur's day was anthrax. This disease was so serious to sheep and cattle raisers that it had been already the subject of careful mvestigations. Up to Pasteur's time no one had learned how to control it or had even discovered its cause. Various investigators had observed certain immotile bodies in the blood of anthrax victims, hut their causal relation, though suspected by some, was as vigorously denied by others. In the early sixties, however, Davaine, struck by the similarity between these organisms and those found by Pasteur to cause fermentation, carried on some experiments to see if they Haight cause anthrax. He determined that a drop of blood contained eight or ten million of them; so he diluted the blood a million times aod found it still able to cause the disease in healthy animals. From this he concluded that the microorganisms must be the cause of the disease. Davaine's experiments were not conclusive, however, and the mat- ter remained in dispute for about fifteen years longer. With the crude knowledge of bacteria and the methods of handling them in these early days, the experiments resulted in confusion, and no def- !uite results were reached. There was a total failure at the time to the large numbers of different kinds of microorganisms Xvhich resemble each other under the microscope. Investigators f°und objects in ordinary decaying liquids that looked much like those found in the blood of the anthrax animals, and then observing that the inoculation of healthy animals with decaying liquids would yot produce anthrax, they drew the conclusion that the organisms 111 the blood of the animal had nothing to do with the disease. The c°nfusing and contradictory results of experiments in different 32 BACTERIOLOGY laboratories could not be explained until after more careful methods of study were devised, and some of the sources of error eliminated. The demonstration that the organisms that had been found were the actual cause of the disease was finally given independently (in 1876-1877) by Pasteur and by Robert Koch. The work of these two investigators-the acknowledged peers among bacteriolo- gists in these early days-was carried on by different methods. In both cases, however, the demonstration finally came from the success of the experimenters in obtaining pure cultures of the organisms from the blood of the animals suffering from the disease and sub- sequently proving that the inoculation of healthy animals with these pure cultures would invariably reproduce the disease. Pasteur cultivated the organisms from the blood of an anthrax animal in yeast water or in urine, while Koch used the aqueous humor from the eye as a culture medium. Both of them succeeded in obtaining a pure culture which they could carry on from generation to generation by simply reinoculating new masses of sterilized media in which the germs would grow. Koch's most important contribution was the microscopic study of his cultures. By inoculating small amounts of anthrax blood into aqueous humor which he placed in a warmed chamber under his microscope, he was able to watch the actual growth of the organisms. He showed that it was a tiny rod which grew in long chains (see fig. 4) and that under some conditions each tiny rod could produce within it a little glistening body which Koch correctly decided to be a spore. These spores germinated, forming rods again. Pasteur's important contribution to the study of anthrax was the conclusive demonstration-one year after the above work of that this microorganism was the actual cause of the disease. He obtained cultures of the organism by inoculating yeast-water of urine with blood from anthrax victims, and then made fresh cultures by inoculating more material from his first cultures. After keeping up these transfers for many generations, he found that it was still possible to produce a case of anthrax in any susceptible animal by simple inoculation with a small quantity of these cultures. By carrying on these cultures in the laboratory for many weeks and months, and transferring every few days a single drop of one mediuPJ in which the germs had grown to another vessel of the medium, it was possible to get rid of all of the original drop of blood inoculated Robert Koch PERIOD OF FUNDAMENTAL DISCOVERIES 33 lato the first tube, except such bodies as had been multiplying in the culture fluid. The purpose of these repeated transfers was to meet the objection that the drop of blood from the anthrax victim might have had some direct poisons in it that were the cause of the disease, these poisons, of course, being eliminated by the successive transfers. The only objects present in the final culture which could be traced hack to the original drop of blood were the rods or bacteria, which had been multiplying and were shown by the microscope to be as ahundant in the last culture as in the first or as in the original drop °t blood. The fact that the most minute quantity of the final culture would reproduce the disease and that the bacteria present in Fig. 4. Koch's Sketches of the Anthrax Organism that drop of culture fluid multiplied rapidly in the inoculated animals and became very abundant in their blood, was sufficient demon- stration that the organisms in question were the actual cause of authrax. . This demonstration of the cause of anthrax, although not the first tirUe that a microorganism was shown to cause disease, was in a way more important than Pasteur's discovery of the cause of the sflk-worm diseases. The silk-worm parasites had not been obtained ln pure culture, nor had Pasteur made any microscopic study of their development; but the anthrax organism was watched by Koch throughout its entire life history, was described and sketched so carefully that Koch's drawings look accurate today, and was studied 34 BACTERIOLOGY by both Koch and Pasteur in unquestionably pure culture. The importance of this and of the production of the disease in healthy animals by means of these pure cultures can hardly be overestimated. There no longer existed any possible grounds for denying the germ theory of disease. CLASSIFICATION OF BACTERIA Pasteur's work showed clearly that microscopic life plays an important part in nature's processes and also proved that there are plainly several kinds of microorganisms. He did not define the different kinds, however, and used various indefinite names to de- scribe them. He was more interested in the results of their action than in naming and classifying them. But the growing interest in the microscopic organisms led to wider study and to the discovery of new types, and it was soon realized that they must be named and classified in order that one investigator might know what kind of organism another was mentioning. To classify the microorganisms it was necessary to know some- thing of their structure and constancy of form. It was observed that some of them were round, some oval, some rod-shaped and some in the form of spirals, while some were much smaller than others; but it was a question whether these different forms were distinct organisms or merely different stages of some few types. This question was vigorously debated, some experimenters claiming that bacteria are pleomorphic, i.e., that the same microorganism could be traced through a long series of changing forms. The early work which apparently showed such confusing and varying stages was soon explained as due to the examination of cultures in which two or more kinds of microorganisms were mixed, and the conclu- sion was generally accepted that the microscopic organisms are as constant to type as other forms of life. This conclusion was neces- sary before any headway could be made in description or classifica- tion: and the attempts at classification made while these ideas of pleomorphism held sway appear quite fantastic to the modern stu- dent of bacteria. Every little while, however, even in later years, the belief that one kind of bacteria can change into another has kept manifesting itself and with various modifications has persisted up to the present time. One cannot safely say today that pleomorphism has been disproved since many bacteriologists claim that bacteria PERIOD OF FUNDAMENTAL DISCOVERIES 35 may go through many successive stages, appearing in totally dif- ferent form under different conditions. Such modern ideas, however, differ from the earlier ones in that few reputable bacteriologists today think they can convert one well known type into another well known type, but merely assert that some species we think we know thoroughly occur in other totally different forms that have escaped recognition. The modern theories do not prevent the development of classfication; but the mere possibility that one and the same organism may assume widely different forms at different stages in its history makes it necessary to be cautious in attempting a detailed classification of bacteria. For this reason only a very general classification need concern us (see Chapter VI). When ideas are so uncertain today, it can readily be understood what confusion must have existed in the days before it was possible to obtain bacteria in pure culture. At that time there was little conception as to the enormous numbers of different kinds of micro- organisms. It was known that the pebrine organism, the anthrax organism, and bread yeasts were different species, not only because they did different things but because they looked different; but it was not dreamed how many kinds of organisms there were which could not be distinguished in appearance from the anthrax organism. The great diversity that actually existed was first suggested by the Work on the pigment-producing bacteria. The production of colors by bacteria had been a matter of interest for some time. It had been noticed that milk or potato exposed to the air sometimes de- velops colors that were apparently due to microorganisms. Finally (at about 1879) a systematic study of these chromogenic bacteria Was undertaken by Schroeter. Schroeter found that on pieces of cooked potato exposed to the air, there often appeared little slimy drops of bright red color. By transferring a little of this slimy growth to other substances he could get the red color to develop there. He also found similar little drops of other colors, blue, yellow, orange and so forth. These drops all contained organisms that would produce the same color when placed m sterile media. From this he drew the logical conclusion that, although many of these bacteria looked alike, he was actually dealing with a number of different kinds, each capable of producing its own specific color. Evidently Schroeter, making use of Nature's assist- ance in sprinkling the bacteria over his sterile potato, had obtained 36 BACTERIOLOGY pure cultures of bacteria by an entirely new method, quite different from those employed by Pasteur and by Koch. Neither Schroeter, however, nor Cohn, in whose laboratory he was working realized that these experiments were actually pointing out the way to obtain pure cultures of bacteria in general, nor that this method when developed a little further by Koch, would revolutionize bacteriology. Schroeter's work emphasized more than ever before, the need of names by which to denote the different kinds of microorganisms. In attempting to apply names, much confusion had naturally arisen. Many names had been in use during the preceding century, but in such an indefinite way that it was almost impossible to know what the different names meant. Yeast, Vibrio, Bacillus, Bacterium, Monas, Spirillum and several other names were in use without any definite or accepted significance. Cohn realized the lack of system in this nomenclature and with his contributions to the subject some- thing like order first appeared in the naming of bacteria. Cohn recognized clearly two distinct groups, one multiplying by budding, the other by fission. The former included the sugar-fermenting yeasts, together with some other types of similar character. The fission types he grouped together under one head which he called Bacteria, Cohn first giving us this name. The term Bacterium had been in use for some time, being applied previously to certain organisms found in decaying matter, but Cohn adopted this ierm for his whole group of fission fungi and from that date we begin to read in the literature about bacteria. This particular group, as years passed, came to be recognized as far more abundant and varied than yeasts, and its importance is being more and more clearly realized all the time. Cohn's first classification (1872) was published at about the -fame time as Schroeter's work on the pigment bacteria and was probably somewhat influenced by the latter's findings. At this time he classi- fied bacteria into four tribes: the first tribe (spherical bacteria) contained the genus Micrococcus; the second tribe (short, isolated rods) the genus Bacterium; the third tribe (long rods, forming fila- ments) the genera Bacillus and Spirochaeta. These generic names are still in use today in much the sense originally used by Cohn; but the names which he gave to the four tribes are no longer used. Three years later-that is, just before Koch's work on the anthrax organism, Cohn devised a further, more detailed classification. The 37 PERIOD OF FUNDAMENTAL DISCOVERIES ■chief importance of this second classification is that it emphasized hie close relationship between bacteria and certain of the lower algae. In fact, he combined these two groups together to such an extent that no one since has ever been willing to follow him. This classification, therefore, need not be given here; but some of Cohn's ether studies are of more interest. Among the more striking of Cohn's writings is a calculation which appeals to the imagination and gives some little idea of the great Powers of the microorganisms. So minute are the bacteria ef an inch in diameter, more or less) that at first thought they might s®em insignificant. But Cohn found by watching them under the microscope that under favorable conditions some species are able multiply once an hour or sometimes once every half hour. Cohn called attention to the fact that, multiplying once an hour a single bacterium would produce in twenty-four hours about 17,000,000 descendants, and in the next twenty-four hours each of these might Produce 17,000,000. In three days the same descendants would weigh about 18,000,000 pounds. In five days, at this rate, the descendants would be sufficient to fill all the oceans of the world, la short, their prodigious powers of multiplication would soon leave r°°m in the world for nothing else. Of course they do not continue multiply at this rate; but recognizing this as a possibility, we get a little conception of the power they possess, a power that is con- stantly held in check by lack of food, or some other adverse condition, possibility of rapid growth under favorable conditions gives them almost unlimited powers and explains how they can be-all things considered-among the most powerful agents on the earth. Another important study of Cohn's resulted in the discovery of bacterial spores. We have already seen how Koch found spores in the anthrax organism; but just previous to Koch's discovery, Cohn had found them in a non-pathogenic form which he named Bacillus subtilis. Cohn not only watched these spores germinate into rods hke the parent rods, but also showed their significance. The spores, he showed, possessed greater powers of resistance to heat and dis- mfectants than the rod forms, and therefore probably helped the Organisms to live through adverse conditions. This is so true that n°w, whenever we wish to sterilize anything, the first question we must answer is whether spore-producing bacteria are likely to be Present: for if so sterilization is a much more difficult problem. 38 BACTERIOLOGY A REMARKABLE QUARTER-CENTURY We have now seen how in a quarter of a century much more prog- ress was made in the study of bacteria than had been made in the century and a half following the observation of bacteria by Leeu- wenhoek. The genius of one man, Pasteur, gave such an impetus to the study that a new science was created. Pasteur's demon- stration that bacteria could cause both fermentations and diseases was enough to awaken the interest of everyone in these minute organisms. One discovery followed another until some were willing to attribute almost any mysterious natural phenomenon to the agency of microorganisms. Interest was so great in these tiny creatures, that if skill in handling them had grown equally fast progress would have been even more rapid. Every would-be student of bacteria, however, ran immediately across the fact that such small objects were not easy to study. No one could study them unless he were endowed with unusual qualities of mind like Pasteur, Koch or Cohn. It required a genius to study bacteria. Before they could be worked with by the average student, new methods were necessary to simplify the task of handling them. REFERENCES Vallery-Radot. The Life of Pasteur. 484 pp. Translated by Mrs. R. L. Devonshire. Doubleday, Page & Co., New York. 1920. Emile Duclaux. the History of a Mind. Translated by E. F. Smith and Florence Hedges. 263 pp. W. B. Saunders Co., Phila- delphia, 1920. Ferdinand Cohn. Untersuchungen fiber Bacterien. II. Beitrage zur. Biol, der Pflanzen. Bd. 1, Hft. 3, p. 141. 1875. L. Descour. Pasteur and His Work. Translated by A. F. and B. H. Webb. 250 pp. Unwin, London, 1922. CHAPTER HI The Development of Methods In spite of thirty years work, bacteria remained only slightly under- stood even as late as 1880. The difficulties surrounding their study were many, but chiefly due to the fact that bacteria are not only excessively minute, but are colorless, almost transparent, with- out sharp distinguishing marks and frequently actively motile. To make a satisfactory study of such minute, colorless, transparent, motile organisms baffled all students for a long time. They could not be seen easily, and it was impossible to handle them separately. The difficulty of seeing them, however, was gradually overcome as the microscopic technic was developed. THE MICROSCOPIC STUDY OF BACTERIA Microscopes in Pasteur's day were very different from those used by Kircher and by Leeuwenhoek, although primitive compared with those in use today. The art of grinding lenses had been more and more perfected, more and more powerful combinations of lenses were put into use, until at this time microscopes were sufficiently powerful to reveal objects as tiny as bacteria, provided they could be made to stand out sharply enough. The two things needed to make the bacteria show plainly were methods of coloring and methods of obtaining proper illumination. It is hard to say who first used stains in the study of bacteria; because at this time the use of various dyes was becoming quite common in microscopic work and it natu- rally occurred to different men to try to make bacteria in infected tissues visible by such stains. Some fair degree of success was obtained in this; but it was Koch who first showed the practical Use of stains in the general study of bacteria. He realized that the chief difficulty in studying bacteria under the microscope was that they were either actively motile or else, because of their minute size, were affected by the molecular movements of the surrounding liquid and were constantly dancing. This prevented accurate descriptions. It was also difficult to stain cultures of these small 40 BACTERIOLOGY creatures because the surplus stain could not easily be washed off them. Koch saw that both these difficulties could be overcome if some method were devised for fastening the bacteria down, so that stains could be poured over them and washed off, leaving a permanent preparation that could be examined whenever desired. He therefore (1877) tried the experiment of drying a liquid contain- ing bacteria upon a microscopic cover-glass, and then staining it. He expected only fair results, knowing that microscopic animals and plants generally lose their shape upon drying; but much to his delight he found that drying bacteria upon a cover-glass fixed them upon it in their natural shape and size. These dried bacteria could then be stained readily with certain aniline dyes, especially those known as methyl violet and fuchsin. These preparations, as Koch demonstrated, can be photographed, and the photographs preserved permanently. This method of killing, fixing, and staining the bacteria enabled Koch almost immediately to learn more about these organisms than long years of most laborious study of the living objects had dis- closed. Their shape could be definitely determined, their size could be measured, and their method of grouping could be seen. Various unsuspected characteristics could now be detected among these organisms which had hitherto seemed to have almost no distinguish- able features. This method of studying bacteria is still in use prac- tically without modification. It is so simple and practical that no bacteriologist could do without it today. Another difficulty in studying bacteria was the matter of proper illumination. It was a fairly simple matter to throw the necessary light upon the microscopic field as long as comparatively low magni- fications were used; but with high power lenses the problem was much more difficult. The field observed with a high power micro- scope is so small that the amount of light reaching it, if merely reflected up from a mirror beneath the stage, is not enough to illu- minate the objects it contains. In Koch's first work, he used direct sunlight, which gives sufficient illumination but makes the objects appear out of shape and off-color. He therefore experimented with other illuminating devices, and finally showed the superiority of the system of lenses known as the Abbe condenser, which had just been invented but is now in use in all bacteriological laboratories. Koch was also the first to use ''homogenous immersion" objectives DEVELOPMENT OF METHODS 41 in studying bacteria-that is, objectives which are used immersed in a drop of oil placed on the slide, the oil chosen having the same refractive power as glass. Koch, it must be understood, did not invent these devices, but he was the one to show their great value in studying bacteria. PURE CULTURES A far greater contribution to the advance of bacteriology was Koch's method of obtaining pure cultures. To determine the charac- teristics of any particular species of bacteria it is manifest that one of the first requisites must be to obtain a culture of this particular species unmixed with others-that is, to obtain what is now called a pure culture. But to do this was a task of surprising difficulty. Bacteria cannot ordinarily be handled separately, although under certain special conditions, modern technic has mastered even this difficult point. For all practical purposes they must be dealt with as masses, and in Koch's day no other possibility could be conceived of. Now, bacteria are so abundant and widely distributed that any attempt to cultivate a mass of them is almost sure to yield a mixture of several different kinds together. The early bacteriol- ogists, indeed, had no method of knowing whether they had one or several different species mixed together in any mass of bacteria with which they were experimenting. Impure or mixed cultures could never produce uniform results, and no definite advance in the study of bacteria could take place until methods of obtaining pure cultures were perfected. This was not so thoroughly appreciated in the early days of bacteriology as it has been in recent years, because of the failure to recognize the great variety of microscopic organisms that look alike under the microscope. Nevertheless, in the early experimental days when Pasteur was doing his epoch-making work, the necessity of obtaining pure cultures began to be realized and various methods were devised for obtaining such cultures. It was first noticed that pathogenic bacteria generally occur in pure cultures in the tissues of the animals they attack and can be obtained pure in artificial culture by inocu- lating sterilized culture media with bits of the infected tissue. This, it will be recalled, is the principle upon which Koch and Pasteur each obtained pure cultures of the anthrax organism. But this method was not always successful, and was of no use in the case of 42 BACTERIOLOGY non-pathogenic organisms. It was therefore realized that some more satisfactory method was necessary. "Darvxinizing." One of the first attempts to obtain pure cultures of non-pathogenic organisms was by a method that has sometimes been called "Darwinizing" because it depends upon the principle of natural selection. The method was first used by Klebs (1874), who suggested it as a means of getting rid of the contaminating bacteria in an impure culture of some organism. After a culture had developed considerable growth, he removed a minute amount of sediment with a capillary tube and transferred it to fresh sterile media. Then in the same manner he transferred a small amount of the sediment from this culture to fresh media and so on until only one kind of bacteria was present. He called this "fractional culture." Klebs did not appreciate, however, that the organisms which was obtained pure at the end was not necessarily the one most abundant at the start but the one best adapted to growth in the particular medium chosen for cultivation. The method, there- fore, has its uses, but not exactly for the purpose to which Klebs thought it could be applied. A later application of this same method is to select some medium which is known to be especially adapted to the organism under investigation and to cultivate the organism in this medium until it predominates over all the other organisms mixed with it. Milk, for instance, is especially adapted to the growth of the organism which sours it. If sterile milk, therefore, is inoculated with a drop of sour milk perhaps all the bacteria present may grow, but the ones particularly favored grow the most rapidly. If the bacteria are allowed to grow but a few hours in such milk and then more sterile milk is inoculated from the first lot, and this process repeated for a few times, the lactic acid organism can soon be obtained in pure culture. The principle underlying the method is that the organism in question, growing more rapidly than any other, soon out-distances the more slowly growing bacteria, and if the successive inoculations into fresh media are made at just the right times, the organism under investigation is soon left alone. It has sometimes proved possible to obtain pure cultures by this method alone; but it is seldom that a medium well enough adapted to the growth of any particular organism can be found so that none but the desired organism will grow. The method is never entirely reliable. Its chief use has been DEVELOPMENT OF METHODS 43 found to be in the preparation of cultures for some of the other methods of purification mentioned below. The dilution method. When Klebs made use of his fractional culture method, it was a mere matter of chance what particular kind of organism he obtained in pure culture at the end of his experiment. The first successful attempt to purify a non-pathogenic organism, selected for study in advance of its purification, was made by Lister (1877). Lister had been studying the bacteria which sour milk, and by transferring minute quantities of sour milk to sterile media of various kinds found that these bacteria seemed to be of various different shapes and sizes in the different media. His first conclu- S1on was that they actually changed their form, but his mind was too keen to accept that conclusion for very long without question. He began to wonder whether there might not be more than one kind of bacteria in sour milk, and whether even though he used for his inoculations only the minute quantity that could cling to the tip °f a needle, some of these impurities might not be carried over into the sterile media, and develop there instead of the organism he was trying to study. He reasoned further that as the organism that soured milk was present in larger numbers than all the others, it ought to be possible to dilute the milk sufficiently so that he could measure out an amount containing but a single bacterium, which m all probability would be the species he desired to study. This reasoning proved correct. It was a fairly simple matter to compute the number of bacteria in a given quantity of milk. Lister ac- complished this by spreading the milk out on a specially devised slide in a layer of which he knew the exact thickness and by counting the bacteria he could see under the microscope in a small area of known size, then by calculation determining the number in the drop °f milk. Today it is possible to accomplish the same thing by a more simple technic (see p. 143), which merely requires the smearing °f a measured quantity of milk over a known area on the slide, drying, fixing, dissolving out the fat, and staining; but in Lister's day no such technic was available. When Lister had made this determination he figured out how much to dilute the milk in order that the smallest quantity he could measure out would contain no more than a single organism. Using this dilution, he inoculated five tubes of sterile milk; four of them remained sterile, but the fifth curdled in three days into a solid very sour mass. He rightly con- 44 BACTERIOLOGY eluded that he had a pure culture in this fifth tube, and used it for carrying on his experiments. He found that when various media were inoculated with this pure culture, there was no change in the shape or size of the organism. In other words, he showed plainly that the explanation of his earlier results was impure cultures. This method of obtaining pure cultures is practical and is occasionally resorted to at the present time for organisms that can be purified in no other way, but it is rather slow and cumbersome and succeeds only in the hands of an unusually careful experimenter. Solid media. The dilution method and the "Darwinizing" technic were both rather too complicated to put into the hands of ordinary students with any expectation of getting reliable results. In con- sequence the work that was done was irregular, variable, and the conclusions were never definite. For this reason the development of bacteriology proceeded rather slowly, even after the subject had been so brilliantly inaugurated by the researches of Pasteur. It was not until after 1880 that new methods placed research in bac- teriology within the reach of ordinary experimenters. The most important of these methods was the use of solid media by Robert Koch. Solid media such as potato and a few other cooked vegetables, bread, and so forth, had been in use since the beginning of bacteri- ology. Schroeter, indeed, as already mentioned, used potato as a means of separating one kind of organism from another. The drops of pigment which appeared on the slices of potato which he exposed to the air originated in most cases undoubtedly from single organisms that had fallen onto the potato; and the cultures that he made froifl them were therefore in many cases pure cultures. Schroeter, how- ever, did not realize the great abundance and variety of bacteria in nature sufficiently to appreciate the importance of what he had done. He did not call much attention to the method, and for ten years no one developed the technic any further. In 1881, however, Koch devised and described a new method of cultivating bacteria in solid media, by means of which they could be more easily handled. This method was really epoch-making, but it was extremely simple. Recognizing that in handling bacteria greater difficulties arose from their rapid motion through liquids than from their extreme minuteness, he devised a method of fixing them fast. He prepared some culture medium in which many DEVELOPMENT OF METHODS 45 kinds of bacteria had been found to grow readily and stiffened it by adding a certain quantity of gelatin. This jelly could be steri- lized by heat and would harden upon cooling. Koch's technic was to melt tubes of sterile jelly at a low temperature, then to inoculate them with small quantities of the material containing the bacteria under investigation, to shake the tube gently so as to distribute the organisms evenly throughout the whole tube of medium, and then to pour it upon a sterilized sheet of smooth glass to harden. This was known as a gelatin plate. By this means the bacteria in the original drop of inoculating material were distributed over the whole of a thin layer of gelatin, and as soon as it had hardened they became fixed in their positions and could no longer swim or float around. These plates were carefully covered to keep off the dust, and placed at a proper temperature for growth. The bacteria then began to feed upon the culture medium, grew and multiplied, but being fixed in their positions, could not separate themselves from each other. The result was that the descendants of each individual bacterium remained grouped around their ancestor and in the course of a short time the plates were covered with little specks, each speck consisting of the descendants of a single organism. These specks, or colonies as they are generally called, can be picked out of the gelatin with a sterile needle or rod and placed in a tube of sterile culture medium. Koch found that this ordinarily gave rise to a pure culture. Occasionally such a culture proved to be impure. It will be seen that this method of obtaining pure cultures depends upon the assumption that in general the bacteria are separated when shaken up in the liquified gelatin so that upon hardening each individual gives rise to a separate colony. Under such circumstances the cul- tures obtained would be pure; but it naturally happens sometimes that the bacteria are not entirely separated from each other but that two or more adhere together and give rise to a colony consisting of two or more kinds of bacteria mixed together. This makes it necessary to examine every supposedly pure culture carefully to see if it is actually pure; if not, the culture has to be purified. The puri- fication of such a mixed culture is the plating process repeated again, mixing a small amount of the impure culture with the melted gelatin. By repeating this purification process once or twice, it is ordinarily possible to obtain cultures eventually that contain one kind of bacteria only. 46 BACTERIOLOGY This method of producing pure cultures has been followed from the day of Koch up to the present time, although modifications of it have been necessary in some respects. By this method of liquefi- able solid media, bacteriologists can handle visible colonies, and no longer have to attempt to handle single bacteria. The colony is Fig. 5. Colonies of Bacteria ordinarily the starting point in modern methods of experimentation with bacteria (fig. 5). One of the chief drawbacks to Koch's technic was the difficulty in covering a flat glass plate with an even layer of gelatin and in keeping it protected from contamination until the colonies had time to develop. This suggested to another bacteriologist, Petri, the idea of devising a special glass dish for the gelatin cultures. These Petri dishes, as they are called, are essentially very shallow round DEVELOPMENT OF METHODS 47 glass boxes, consisting of two shallow cylindrical dishes one of which ls slightly smaller than the other and fits inside of it (see fig. 6). In these plates the gelatin cultures are well protected from dust. A further advantage of Petri dishes is that one can be piled on top of another so that a large number of them can be placed in a small amount of space. They are so well suited to gelatin plate cultures that they are in general use today in all bacteriological laboratories. Another modification of Koch's technic is through the use of a substitute for gelatin. Gelatin has proved to be very useful, but *t has two disadvantages that prevent its use for certain purposes. It melts at a temperature of about 23°C. (75°F.), while some bacteria Fig. 6. A Petri Dish "~notably the pathogenic forms-cannot grow at such a low tem- perature. Furthermore, quite a number of bacteria attack the gelatin and liquefy it, so that plates inoculated with them are con- verted in a day or two into a liquid which will not harden. Since the use of gelatin depends upon its solidifying power, it manifestly cannot be used for bacteria that require high temperatures, nor very easily in the presence of many bacteria that liquefy the gelatin. To meet these difficulties, a substitute for gelatin is found in agar agar (popularly called agar), a substance prepared in Japan from a sea weed. Agar has ten times or more the jellying power of gelatin, does not melt until about 60°C., and is not liquified by any of the common bacteria. Agar has not entirely taken the place of gelatin and probably never will, for it has certain disadvantages of its own 48 BACTERIOLOGY and much better results can be obtained with gelatin in some kinds of work; but the manifest advantage of resisting moderately high temperatures has caused it to be used more widely than gelatin in bacteriological work. FURTHER DEVELOPMENTS The perfection of these methods had an almost unbelievable effect upon the study of bacteriology. Previously to 1880 the subject had been so difficult that only a genius like Pasteur or Koch could obtain reliable results. Pure cultures of bacteria were almost unknown and much of the work before that date had been done with mixtures of different species of bacteria. Little reliable work was possible with impure cultures. But no sooner did this simple method of obtain- ing pure cultures appear than the subject began to advance with extreme rapidity. It no longer required a genius to obtain reliable results; so at first scores and then hundreds of young scientists turned their attention to the newly opened field, where such striking results seemed to be promised. In the next twenty years discovery followed discovery with a rapidity too great to allow proper assimi- lation of old ideas before new ones appeared. Aside from the pioneer work of Pasteur, who really first called attention to the activities of microorganisms, nothing contributed so much to the development of bacteriology as Koch's methods of obtaining pure cultures. All modern bacteriology is based upon them or upon modifications of them. Improvements in methods did not stop with Koch. During the new era of bacteriology introduced by his invention of liquefiable solid media, many new bacteriological problems began to appear, the solution of which brought about many new applications of bacter- iology to practical life; and nearly every new problem required new methods. It is probably no exaggeration to say that each year since the work of Koch has seen the appearance of more new methods in bacteriology than were devised during all the years of its previous history. Many new media have been devised for special purposes. Some of these special media are capable of supporting the life of certain types of microorganisms, although they will not allow others to grow. Other special media have been devised for enriching cul- tures, that is for stimulating the growth of certain types of bacteria at the expense of others, so that an impure culture containing origi- DEVELOPMENT OF METHODS 49 Hally but few of the desired organism and many of other kinds might Ui the end have this relation reversed-in other words media for applying the "darwinizing" procedure to certain specified types of organism. New and important methods have been devised for dis- tinguishing one kind of bacteria from another, as well as new methods tor detecting the presence of certain definite species of bacteria when Present in a mixture of numerous different kinds. The modern methods are legion, and each, as it has been devised, has added some- thing to the growing science of bacteriology. None of them however has ever accomplished what Koch's methods did, for while each has aided the advancement of bacteriology along certain lines, none has produced the complete revolution of experimental work as did Koch's simple device of using solid instead of liquid culture media. MODERN METHODS The beginning student in bacteriology can hope to master only the simpler methods and those of most general application. His ac- quaintance with these must come largely through practical work in the laboratory; and all that will be needed here is an outline of the general principles which underly modern bacteriological technic. Sterilization. All modern bacteriological technic is dependent upon the thorough sterilization of all the dishes and culture media m which the bacteria are grown. The ehrly workers supposed that mere boiling or steaming was sufficient to kill the bacteria; but when the great resisting powers of bacterial spores was discovered, it was realized that greater heat was necessary. There are three methods of sterilization commonly used in modern bacteriological laboratories: dry sterilization, steam pressure sterilization, and "fractional sterili- zation." Dry sterilization is applicable to glassware and other things that are not hurt by dry heat. An ordinary gas oven with a thermom- eter inserted is a satisfactory dry sterilizer although special steri- lizers of this type have been devised for bacteriological work. Dry heat of 150°C. (300°F.) for one hour is ordinarily sufficient to kill even the most resistant spores present on glassware. Steam pres- sure sterilization is applied to all culture media that are not injured by temperatures higher than the boiling point. This type of steri- lization is carried on in a form of sterilizer known as an autoclave (see fig. 7), in which steam can be retained under pressure, thus giving temperatures higher than the boiling point. In ordinary bacteriolog- 50 BACTERIOLOGY ical work a pressure of 15 pounds is used, which gives a temperature of 120°C. Steam at this temperature will sterilize culture media in from fifteen minutes to half an hour unless they are in such large containers that it takes considerable time for the heat to penetrate to the center of the mass. The objection to steam pressure sterili- Fig. 7. Two Different Styles of Autoclave zation is that some kinds of media are changed chemically by these high temperatures. "Fractional sterilization" has been devised to overcome this objection. This third method of sterilization is ordi- narily accomplished in flowing steam, which never rises to a tempera- ture over 100°C. (212°). Heating to this temperature kills all the bacteria present except the spores, some of which generally escape; DEVELOPMENT OF METHODS 51 but these spores will germinate if the media are removed from the sterilizer and kept twenty-four hours, and in this short time new spores will not ordinarily be formed. Hence a second heating in flowing steam on the second day ordinarily sterilizes the media com- pletely; but in practice, another heating on the third successive day is generally employed to make it more certain that all bacteria are destroyed. For some special media especially susceptible to heat, methods have been devised for making three successive daily heat- ings at temperatures considerably below the boiling point. Culture media. Much change has been made in the forms of cul- ture media since the days' of Pasteur. Originally almost anything the experimenter happened to lay his hands on was used provided the bacteria could grow on it; but now there are certain fairly definite kinds of media in use in all laboratories. Each investigator gener- ally devises his own media for special purposes; but for general work he uses the same kinds of media that others use. Under the name of broth or bouillon, bacteriologists generally refer to a solution of beef extract to which has been added a material known as peptone. Solid media are formed by adding enough agar or gelatin to this broth to form a jelly. The preparation of these media is described on p. 421. For certain purposes some sugar, generally dextrose (grape sugar), lactose (milk sugar), or saccharose (cane sugar), is added. Certain natural media are also used by the modern bacteriologist. The most commonly used is milk, which is valuable because some bacteria curdle it, others digest its casein, causing it to change into a clear disagreeable smelling liquid, while still others leave it un- changed; and these differences often help to distinguish different bacteria from each other (fig. 8). Another natural medium in com- mon use is potato. Potato has been in use as a bacteriological medium ever since the day of Schroeter, and for some time stood in considerable favor because of the striking colors produced upon it by certain bacteria and the characteristic wrinkled growth produced by others. At the present day, however, potato is not used so often because it is realized to be of varying chemical and physical nature, while present day bacteriological methods emphasize the importance of media that are of unvarying composition and consistency. One of the very important matters in preparing an artificial medium for the growth of microorganisms is its acidity. Most kinds of bacteria prefer a neutral or slightly alkaline medium, while yeasts 52 BACTERIOLOGY and molds prefer media that are somewhat acid. To satisfy the requirements of the organisms in this respect, bacteriologists are accustomed to adjust the reaction of their media before use. This is accomplished by the methods described at the end of the book (see pp. 421, 433 to 439), which depend upon the use of certain indicators, chemicals that change from one color to another as they pass through a definite range in acidity or alkalinity. Litmus is a Fig. 8. Effect of Different Kinds of Bacteria on Milk From left to right: normal milk; smooth curdled milk; curdled milk with gas bubbles; digested or peptonized milk. well known example. Frequently the bacteriologist finds it de- sirable to observe changes in acidity produced by the bacteria he is studying; and to do this he often adds one of these indicators to some medium (see, for example, litmus milk, p. 428) and is able to observe changes in acidity by watching the change in color. Isolation and study of cultures. The methods used for obtaining pure cultures are essentially the same as those devised by Koch. Gelatin or agar plates of media poured into Petri dishes are used, as already described. A little of the material from any desired colony 53 DEVELOPMENT OF METHODS on these plates is removed with a platinum needle or loop (fig. 9) previously sterilized by passing through a flame and transferred into sterile media-generally into an agar medium in a test tube, hardened in a slanting position (as shown in fig. 10) and known as an "agar slant." Fig. 9. Platinum Needles and Loops, Mounted in Glass Rods as Used by the Bacteriologist Fig. 10. An "Agar Slant" Study of pure cultures. These pure cultures are studied micro- scopically and by inoculation into anything desired. If they are suspected to be pathogenic they are inoculated into some animal that 18 likely to be susceptible-of which rabbits and guinea pigs are most commonly used. They are also inoculated into various laboratory 54 BACTERIOLOGY media and their behavior in the media serves to give the bacteriolo- gist some idea as to what kinds of bacteria he is studying. He notices, for instance, whether they ferment sugars, and if so which ones, re- cording whether the fermentation is shown by the production of acid, of acid with gas or of alcohol with gas. He also notices whether they curdle the milk, or digest it and whether they change nitrate into nitrite or free nitrogen, or cause certain other chemical changes. All these facts are recorded, and in case he is dealing with a well known organism, they help tell him what its identity is. Counting bacteria. One other common application of Koch's technic is in the counting of bacteria. If in the material that is mixed with gelatin or agar, every microorganism is separate from every other one and grows into a colony, it is obvious that the number of colonies represents the number of microorganisms in the material plated. Also if the material mixed with the medium has been measured, it is plain that a determination can be made of the number of bacteria per gram or per cubic centimeter of the material. This method is commonly used in estimating the number of bacteria in water, milk, soil or other materials; although to get a small enough number of colonies on the plates to count it is often necessary to dilute the material hundreds, thousands, or even millions of times, according to the richness of its bacterial population. This method is so convenient that it is widely used; but unfortunately many bacteria do not grow on the culture media ordinarily used, while those that do grow often occur in large clumps that do not break up when plated-both of which facts cause the plate count to be con- siderably below the actual number of bacteria present in the material investigated. The microscope is sometimes used in counting bacteria, in order to avoid these errors. The method is not new; for, as we have seen, Lister used it before Koch had devised the plate technic. New ap- plications of it, however, have been made and modern methods of counting bacteria by means of the microscope are quite different from those of Lister. Briefly modern methods consist of drying a measured amount of the material on a piece of glass, staining with some dye that brings out the bacteria and counting the number of organisms that show in a microscopic field of measured size. This method has proved especially serviceable in the study of milk and some other foods. CHAPTER IV The Era of Development-1881 Till Today It is now necessary to go back again to the time of Koch and follow Up the discoveries that have been made. The digression in the last chapter in regard to methods should serve to show some of the diffi- culties which the early bacteriologists met; for these methods were slowly perfected during the period considered in this chapter. At the beginning of this period, most of these methods were lacking, and bacteriologists were that much handicapped; but the most important of all the methods, the poured plate of gelatin, and the dried and stained microscopic preparations, were already at their disposal, and these methods inaugurated the modern period in the history of bacteriology. With these new methods accurate work was possible, more and more experimenters turned their attention to bacteria, and many new lines were investigated. Each year saw more advance made than decades had seen before, and each decade more than any previ- ous century. These modern discoveries cannot be taken up in de- tail in their chronological order, but must be considered topically m the remaining sections of the book. This chapter merely con- tains a survey of those which stand out most prominently in the history of the times. TRACING DISEASES TO THEIR CAUSES The field which proved to be particularly interesting was the re- lation of bacteria to disease, and pathological bacteriology attracted an especially large number of students. It had taken a genius like Pasteur some thirty years to establish the fact that these micro- organisms are the cause of certain diseases and to place two animal diseases, pebrine and anthrax, definitely in the list of germ diseases; but now it required only a very short time for a number of other im- portant diseases to be classed in the same list, not on the basis of speculation, but as demonstrated truths. At the very outset of this period (1882-1884), Koch himself, using his own method, dis- 56 BACTERIOLOGY covered the cause of the Great White Plague to be the tubercle bacil- lus. This discovery is especially important, because tuberculosis was the first disease to be worked out consciously according to the requirements previously laid down by Henle. In fact it was in Koch's first paper on the tubercle organism that "Koch's postulates" (which, as already mentioned, were a restatement of Henle's re- quirements) were formulated. Learning the cause of tuberculosis was no simple problem, even though the development of the new methods made the task simpler than had been the discovery of the anthrax organism. Koch's first step was to show that a micro- organism was always present in tubercular tissue; his second to de- vise methods of staining it in the tissues; and the third and most convincing step was to isolate it by means of his plating technic and to produce the same disease in healthy animals by inoculation. This work was so complete and thorough that Koch's publication on the subject is still regarded as a masterpiece. The discovery of the bacillus of typhoid fever followed speedily, as did the demonstration of the fact that diphtheria, glanders, cholera and tetanus are each caused by specific bacteria, easily recog- nized and isolated. These organisms were all studied by experi- mental methods, and some of them were proved definitely to cause their respective diseases by means of animal inoculation; although for others, like typhoid, which attacks human beings alone, such definite proof could not be established. The discovery of other bacterial diseases followed close upon these; so that a period of ten years sufficed to bring out the cause of the most important diseases caused by bacteria. Fifteen years after Koch's discoveries, all except one or two of the diseases which are now known to be caused by bacteria had been successfully investigated. Those diseases of which the causes were not discovered until recently are due not to bacteria but to other kinds of microorganisms that are not capable of being studied by Koch's technic. PROTECTIVE INOCULATION More important than the discovery of the causes of diseases were the new methods of defense against bacteriological diseases that developed in the hands of bacteriologists. The beginning of this important line of work was due, like many other important sub- jects, to an entirely accidental investigation. In 1880 Pasteur was ERA OF DEVELOPMENT 57 Working on a disease known as chicken cholera. He had obtained cultures of the organism causing the disease and had found them capable of infecting healthy fowls. Returning from a vacation, however, he found that his cultures had lost their virulence and no longer caused the disease upon inoculation. He obtained fresh virulent cultures and inoculated them into these same fowls as well as into others that had received no previous inoculation. What was his surprise to find that the ones previously inoculated with the old culture had become immune and did not succumb to the disease, although the other fowls contracted it as usual. By pure accident Pasteur had discovered the principle of protective inoculation, one of the most important facts in disease control that has yet been brought to light. The possibility of combating diseases upon this principle had been dimly perceived for centuries. The Chinese and subsequently the European nations had developed a method of protection against the great scourge of smallpox by inoculating people with infective mate- rial obtained from a light case of the disease. This, as a rule, pro- duced a mild type of smallpox, protecting the individual from the more pronounced type; but the method was uncertain, because some- times the protective inoculation brought on a severe case. Later Jenner made his important discovery that equal protection was afforded by inoculation with cowpox, a similar disease in cattle, which never caused any severe symptoms in man. Out of this work of Jenner's came the method of vaccination, which to this day enables us to control the dreaded scourge of previous centuries. This, however was purely an empirical discovery, bringing out no principle that helped control other diseases. Pasteur, on the other hand, appreciated immediately the important principle he had stumbled onto in connection with chicken cholera, and proceeded to apply it to other diseases; and thus began one of the most interesting chapters in the whole subject of medical science. THE MASTERY OF ANTHRAX Pasteur first thought of applying this method to the control of anthrax; for at this date anthrax was the most severe disease of which the causal organism had been carefully studied. The first difficulty he encountered was to learn whether anthrax actually confers im- munity upon the animals it attacks. It is such a fatal disease that 58 BACTERIOLOGY an animal rarely recovers from it, so it was ordinarily impossible to see whether an anthrax victim could contract a second case of the disease. But at last, after wide enquiry, Pasteur succeeded in find- ing one animal that had recovered from the disease. Inoculating this animal with anthrax bacilli, he found, much to his delight, that the animal was immune. The problem therefore presented itself to him of finding some method of producing a mild type of the disease which would not make the animals sick but would protect them from the more severe type. In the case of chicken cholera Pasteur had produced a mild type of the disease by using old cultures. This method was not success- ful in the case of anthrax, however, because the anthrax spores could be kept for a long period without losing their virulence. He therefore had to try an entirely different method. Finding that if the organism is cultivated at a temperature slightly above that best suited to its growth, it grows without producing spores, he cultivated it at high temperatures for a considerable time, and eventually found that these cultures had lost their power of producing anthrax in healthy animals. These weakened or attenuated cultures, as they have been called, did not establish complete immunity against virulent anthrax germs, but they did bring about such a condition in the animal that slightly less attentuated cultures could subse- quently be used without producing the disease. These second cul- tures, in turn, protected the animal from a more virulent strain of the organism; so that by successive inoculation with two or more strains of varying degree of attentuation, the animals were eventually made immune against an attack of the most virulent anthrax or- ganism. The mastery of anthrax by this method was publicly demonstrated to the world in 1881 in an ever-famous experiment. Pasteur ob- tained the opportunity to test out his theories upon 50 susceptible animals, and invited to a public demonstrated a large body of ex- perts, including veterinarians, surgeons, members of the French Academy, and other persons of note. He called their attention to the animals, half of which he stated he had protected against anthrax by his methods of inoculation, the other half being unprotected. . In the presence of his visitors he then inoculated all 50 animals with a virulent culture of the anthrax germ obtained from a dead sheep, and requested the visitors to return in two days to see the result. ERA OF DEVELOPMENT 59 When they returned at the specified time, it is said that the sight that met their eyes called forth a shout of admiration from every man. All the animals which had been previously treated with his attenuated cultures were contentedly feeding, showing no trace whatsoever of the disease; while the unprotected ones were dead or dying! The account of this test spread rapidly over the world, and produced almost at once a tremendous demand for the "anthrax vaccine" as it was called; and in twenty-five years more than 10,000,000 animals had been treated. The principle revealed by these two discoveries of Pasteur's seemed to have unlimited possibilities. It seemed to indicate that if the virulence of any pathogenic organism could only be weakened, could be used to protect an individual against severe attacks of the disease. Great prophecies were made for the method at once. It was predicted that in time the human race would be protected against all infectious diseases by this inoculation method. The Problem, however, has not proved quite as simple as this, partly because human nature refuses to be inoculated against diseases of which the danger of attack is remote, and partly because the methods used for chicken cholera and anthrax are not applicable to all germ diseases. Pasteur happened in fact to have the good fortune to select in anthrax one of the few diseases which could be mastered by methods similar to those which he had discovered in the case of chicken cholera. Nevertheless, the recent history of bacteriology has fully justified this belief as to the value of Pasteur's discovery, and at the present time similar methods are known for protecting mdividuals against nearly all of the infective diseases. They have not all, however, proved practical to apply to man, and they have not by any means all been along the same line as Pasteur's protec- tion against anthrax; but the principle then discovered has expanded m more recent years into the most striking phase of modern medical science. PROTECTION AGAINST RABIES The next disease to which Pasteur turned his attention was that known as rabies or hydrophobia. It is said that Pasteur always had a horror of this disease because of having lost one of his friends as a boy through the bite of a rabid wolf. The memory of this, working On his unusually sensitive nature, is supposed to have had much to 60 BACTERIOLOGY do with his vigorous attack against rabies. This disease he mastered by exactly the same principle as that which he had discovered in the case of chicken cholera, except that he realized that no one would submit to inoculation on the bare chance that some day he might be bitten by a mad dog or wolf. This made it necessary to apply the protective inoculation after infection had occurred-a procedure which generally proved successful because of the length of time be- tween the bite and the first appearance of symptoms. Pasteur an- nounced this cure in 1885; but having had no possible way of experi- menting previously on human beings, he was not so confident of success as he professed to be. His first patient appeared very promptly, a boy seriously bitten in many different places on his body. This boy was inoculated with Pasteur's weakened cultures and escaped the disease. To the public this was convincing proof and they hailed it as another of Pasteur's triumphs. It was later shown that the method sometimes failed, but only when applied too long after the patient had been bitten or when the bite had been near the brain. So important was the method considered that an institute was founded by public subscription-known as the Pasteur Institute -for the preparation of the necessary attenuated cultures and for treatment of victims of rabid animals. More recently other Pasteur Institutes have been founded in other parts of the world. FRENCH AND GERMAN RIVALRY The subsequent history of bacteriology is closely bound up with a rivalry between France and Germany that had begun with the Liebig-Pasteur controversy, but was intensified by the Franco- Prussian war. This war occurred in 1870, at the time when Pasteur was doing some of his earlier work in bacteriology. He had just received an honorary M.D. degree from Bonn University in recogni- tion of his work on the silk-worm diseases. Intensely patriotic as he was, the war embittered him against all things German, and he returned the Bonn diploma. Nothing could make him believe that he was not first of all a Frenchman. And his countrymen, sharing his attitude, took full credit to their own nation for his triumphs; so the subsequent rivalry between French and German bacteriologists was only natural. France had produced Pasteur; but Germany was the home of Koch. Between them the honors were quite evenly divided. Pas- ERA OF DEVELOPMENT 61 feur, of course, was the pioneer, and was the first to prove a micro- organism to be the cause of a disease; but Koch was ahead of Pas- teur in demonstrating the cause of anthrax. It was Koch who Revised the plate method, thus opening up the field of bacteriology to students in general; but it was Pasteur, in the very same year, who discovered the principle of protective inoculation. Each man, m spite of intense nationalism, admitted the value of the other's xv°rk; and but for the greater volubility of the French in defending their hero, perhaps we of today would not grant quite so much the hrger share of the honors to Pasteur. fhe rivalry reached its greatest intensity in connection with the discovery of antitoxin. This really was a more practical discovery than that of anthrax vaccine, for it led immediately to the control °f a dreaded human disease. In brief, it was the discovery that after an animal has recovered from one of a certain class of infectious diseases, its blood contains substances capable of destroying the toxic power of the poisons produced by the organisms causing this Particular disease. The practical value of this discovery was enor- mous, since it resulted presently in the production of diphtheria anti- toxin and in its use to control that dreaded scourge of childhood. dhe first step in the production of antitoxin was made by two of Pasteur's students, Roux and Yersin. They discovered that the symptoms of diphtheria were due to a poison or toxin produced by the diphtheria organism. This toxin if injected into an animal in sufficient quantity is fatal; but if used in smaller amounts causes sickness with subsequent recovery. The symptoms produced by the toxin slightly resemble those of diphtheria, but recovery is more mpid than when the bacteria themselves are present in the body. Phe next step was the discovery by Kitasato and Behring, two of Koch's pupils, of the toxin produced by the tetanus (lockjaw) organ- ism. They further discovered that if this tetanus toxin is injected mto an animal in small enough quantities so that the animal recovers an antitoxin is produced in its blood. They succeeded in obtaining a small amount of this antitoxin from the blood of an animal thus treated and used it (in 1891) in treating a child suffering from lockjaw. DIPHTHERIA ANTITOXIN 62 BACTERIOLOGY Behring very quickly followed up this work with similar work on diphtheria. He produced diphtheria antitoxin in guinea pigs by giving them small doses of diphtheria toxin, and with this antitoxin he cured the disease in other guinea pigs inoculated with diphtheria germs. This discovery was hailed with delight as everyone realized its probable importance. Behring admitted that the work of Roux and Yersin on diphtheria toxin was the foundation of his later work; but his very magnanimity only added to his own reputation and thus increased the honors granted to Germany. The only drawback to Behring's work was that it had no practical application, for the amount of antitoxin produced in the blood of a guinea pig was too small to have any curative effect on a human being. The experi- ment was then tried elsewhere of producing it in goats, but without great success. Finally (in 1894) Roux solved the problem by using horses, as they were about the largest animal that could easily be obtained. Roux himself claimed little of the credit, as he had merely applied the discovery already made by Behring; but France received his work with characteristic enthusiasm. It is said that the Frenchmen at the meeting where Roux made his work public went wild with delight; and everywhere in France it was hailed as a triumph over Germany. The method by which diphtheria antitoxin is produced today is essentially the same as the method devised by Emile Roux, and is briefly as follows: Diphtheria bacteria are cultivated in a broth in which they grow readily, and after a few days, the broth is found to be filled with a considerable quantity of diphtheria toxin, the sub- stance which poisons a child and produces the symptoms of the disease when the bacteria are growing in the child's throat. This toxin is next injected in small quantities into the body of a horse, certain definite intervals being allowed to elapse between the injections- The first injection has no noticeable effect on the horse but it renders him capable of standing a second injection of a larger amount, and this protects the animal from a still larger amount at a third injection- These injections of toxin are kept up for a number of weeks until experience has shown the maximum result to be obtained. During this period a large quantity of antitoxin had been produced in the horse's blood. At intervals thereafter some of the horse's blood is removed, allowed to clot, and the serum which separates from the clot is used to fight diphtheria in man. Paul Ehrlich ERA OF DEVELOPMENT 63 The discovery of diphtheria antitoxin was one of the epoch-making discoveries of medicine. Although in the first few years various questions were raised concerning it, and criticisms were made as to its use, these have gradually disappeared, until it is now a universally acknowledged means for controlling this formerly dreaded disease. Improvements in methods of manufacture have been made which have removed some of the objections to its use and more information has been accumulated as to the methods by which it should be ap- plied. Since its use has become generally applied in medicine, the death rate from diphtheria has markedly fallen. The deaths from diphtheria are now very few, less than 10 per cent, and the medical profession in general is convinced that if the disease be treated by antitoxin in the first two or three days of its progress there should be no failures of complete recovery. The deaths that still remain are those of persons in whom the disease has been allowed to progress so far that cure is no longer possible. Occasional evil results have come from the use of antitoxin, and a few deaths have been attributed to it, but these are largely prevented by modern methods, and cannot be regarded as weighing at all against the hundreds of thousands of lives that have been saved by its use. THEORIES OF IMMUNITY Naturally when such practical results had been obtained by utiliz- ing the immunity which is developed in an animal by an attack of a disease, there were many and various speculations as to the cause of the immunity. During the decade between the production of an- thrax vaccine and the discovery of antitoxin two theories had been proposed to explain immunity of this sort, one by Ehrlich, a German, and the other by Metchnikoff, a Russian who became one of Pas- teur's pupils and settled in France. Each of these theories came to be held by the pupils of the man who first proposed it: and thus in this matter also there arose a rivalry between France and Germany. The two schools that held these thoeries had considerable influence upon the development of bacteriology. Briefly it may be said that Ehrlich's theory was purely chemical, consisting of very intricate and technical speculations as to the com- position and formation of antitoxins and other substances concerned in immunity. Metchnikoff's theory on the other hand was one which appealed much more to the imagination, as it assumed almost intelligent action on the part of the white corpuscles in the blood. 64 BACTERIOLOGY Metchnikoff and his followers believed that the white corpuscles are scavengers capable of destroying bacteria that enter the body and that in the case of an attack by disease organisms a battle takes place between the white corpuscles and the invaders; and they further assumed that after one such battle the veteran white corpuscles were more skilful in driving out later invaders than they had been before the first attack. It must be plain to anyone who has the slightest understanding of French and German natures that one of these theories would appeal to the French type of mind, the other to the German; so the French and German schools came to differ not only in the men they championed but in the theories they supported. The first triumph for either theory was Behring's discovery of anti- toxin. Behring was a strong supporter of Ehrlich's theory and natu- rally explained his results in terms of this theory. There seemed, indeed, to be no way of applying Metchnikoff's theory to Behring's antitoxin. So although a Frenchman did get ahead of Behring in making a practical application of antitoxin, Behring's discovery was primarily a triumph for the German school. From this date on the rivalry between the two schools centered on matters of immunity; and this subject was intensely studied, not only to bring out new facts but because each side of the controversy hoped to triumph over the other. Metchnikoff's followers soon found plenty of arguments to explain antitoxin according to their theory; and certain later dis- coveries seemed to indicate the importance of this theory. Today in fact, the commonly accepted views of immunity embrace the im- portant parts of both theories, so it can be said that the final out- come of the controversy is a compromise, not a triumph for either school. PLANT PATHOLOGY While the knowledge of infectious diseases of animals was increas- ing at this rapid rate, a few investigators were studying the relation of microorganisms to plant diseases. The first plant disease to be definitely ascribed to a specific microorganism was the disease of apple and pear trees known as fire blight, which was studied in 1878 by an American, Burrill. A few years later a Dutchman, Wakker, studied a bacterial disease of hyacinths; and it was soon found that many plant diseases were caused by plants more complex than bacteria, belonging to the group known as fungi. Somewhat later the question was raised in Germany, principally by Alfred Fischer, Elie Metchnikoff ERA OF DEVELOPMENT 65 whether bacteria could cause plant diseases. Regarding all evidence as incomplete, Fischer named various reasons, all purely theoretical, why bacteria were not adapted to growth in plant tissues. This suggested to another American, Erwin Smith, that the cause for plant diseases rested much where that for animal diseases had before the days of Koch; so Smith undertook to establish definitely the causal agency of certain plant pathogens by working out Koch's postulates. He did this successfully; and although the dispute between him and Fischer continued for some time longer, there was no question to anyone who studied the evidence presented that Smith had the truth on his side. We may therefore consider that the agency of bacteria in plant diseases was not definitely established until about the beginning of the present century. AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY Not only did the study of pathogenic bacteria develop during this period, but bacteriology rapidly branched out into other fields. Although it had long been known that bacteria are widely distributed in nature, their great significance in nature's processes was not sus- pected ; but during this period of development, the relation of micro- organisms to agricultural problems came rapidly to the front. Gradually it became evident that bacteriology is as intimately as- sociated with agriculture as with medicine. Only a few years after Koch's method was promulgated, it began to be apparent that bac- teria have an important relationship to soil fertility, and the dis- covery that microorganisms are closely associated with nitrification and nitrogen fixation (see p. 182) changed the entire aspect of the problems connected with the maintainance of fertility. At about the same time, too, it began to be realized that bacteriology was intimately concerned with dairy problems. The conclusions reached as to the relations of these microorganisms to milk products were so fundamental and important that in a few years a complete revolution ensued in practically every phase of the dairy industry and in the methods of handling dairy products. Meanwhile questions connected with the preservation of foods rapidly resolved themselves into problems of protecting the food from the action of microorganisms. The preparation of silage for a winter fodder, and in some countries, even the methods of preserving hay were found to be phenomena bound up with microscopic or- ganisms. Various industrial problems, also, showed themselves to be really of bacteriological nature. The industries of alcoholic 66 BACTERIOLOGY fermentation were so dependent upon microorganisms that they soon realized the necessity of having microscopists in their establish- ments. Many large milk distributing companies eventualy recog- nized the value of similar scientific control. Railroads began to employ bacertiologists for various purposes connected with the hand- ling of their employees and passengers as well as the disinfecting of their cars. The maceration of flax and hemp became the subject of special investigation, for here, too, are processes of bacterial action which might possibly be better controlled and more profitably handled by strict scientific methods. Many new industries have arisen in connection with modern methods of sanitation and the control of public health. It has be- come more and more thoroughly understood each year that the basis upon which sanitation stands is the control of microorganisms, and out of this application of bacteriology has arisen the modern science of public health, which is year by year employing a larger number of men and is proving of greater and greater usefulness in our communi- ties. Modern conditions of war with the necessary crowding of many men into hastily prepared quarters have given an additional stimulus to sanitation. As a result of all this, several new lines of engineering, connected especially with such questions as water supplies and sewage disposal, have arisen out of the study of bacteria. In short, the forty years following the appearance of Koch's method have shown an extraordinarily rapid development, and the study of microorganisms has spread so rapidly in various directions and has assumed so many new phases that we often say a new branch of science has developed. The new science is not, however, confined to the study of bacteria, for other organisms are sometime concerned as intimately as bacteria with the processes studied by it. But the important discoveries that were made during this period soon showed that there is a class of natural phenomena associated with micro- scopic organisms which are so intimately concerned in life's processes that they have to be considered in a branch of science by themselves. For want of a better term this branch of science is generally called bacteriology, since it developed around the study of bacteria, although admittedly they are not the only organisms concerned. Robert Koch. Die Aetiologie der Milchbrand-Krankheit, begriindet auf die Entwickelungsgechichte des Bacillus anthracis. Beitr. zur Biol. Der Pflanzen. Bd. 2, Hft. 2, p. 277. 1876. REFERENCE 2. MICROORGANISMS AND THEIR ACTIVITIES CHAPTER V Two Great Opposing Processes in Nature So much has been learned about bacteria during the last thirty years and the additions to our knowledge have be°n in so many differ- ent directions that an historical treatment of the subject is Impossible. So we will turn now to general considerations. We shall first study the general chemical activities taking place on the earth and see what a large part microorganisms play in these activities. These micro- organisms, themselves-and bacteria in particular-will then re- quire more detailed study. Chemical atoms and molecules in nature are in ceaseless activity, constantly combining with each other and as constantly being dis- sociated; and life phenomena are possible only so long as this atomic activity continues. In this activity there are an endless variety of chemical changes; but they are all easily arranged into two types: the constructive or synthetic, and the destructive or analytic. Compounds are being constantly built up, on the one hand, by the combination of many atoms to form larger and larger molecules, and on the other hand as constantly being broken down into simpler and simpler molecules. These two processes are diametrically opposed to each other; yet one is as necessary to life as the other. Life is possible in the world only as long as these two processes are in equilibrium, that is as long as this series of back-and-forth changes continues. If chemical compounds were built up faster than they could be broken down, eventually the atoms would be all so combined together that activities would have to cease, while if the breaking-down processes predominated, the elements necessary for life would come to an end. It is only because the two processes are both going on at the same time, one counterbalancing the other, that all life did not cease on this earth long ago. These two processes are well known to the chemist. Both proc- esses can take place in the laboratory under his control, but with this very important difference: synthetical processes in general, require energy, analytical processes ordinarily give off energy. 68 BACTERIOLOGY BUILDING-UP PROCESSES IN NATURE What is true in the laboratory is also true in nature. Building-up processes require the application of external energy. Some of the simplest synthetical processes can take place under the direct action of light or of the earth's or the sun's heat, but all the more compli- cated processes-which are the most important-such as those that result in the formation of organic compounds, can be performed only through some biological activity. Nearly all forms of life-plants and animals, large and small-can build up certain complex sub- stances from simpler ones; but all of them except green plants have to obtain the necessary energy from the breaking-down of some other compound. When a living cell, like the Amoeba, grows and thus constructs more complex molecules, it does so only by breaking down other molecules-its food-and utilizing the energy thus liberated. In this process the total quantity of destructive change exceeds the quantity of constructive change; and such activities in the end would result in the complete disintegration of all complex molecules. In order that the constructive processes in nature may equal the de- structive, it is necessary that there be some form of life able to build up complex material without having to break down other material in order to obtain the energy required. Utilizing the sun's energy. Green plants are the organisms that are able to do this. Their green color is due to a material within the cells known as chlorophyl, which in some way not yet under- stood, enables the plants to utilize the light of the sun as energy. With this supply of energy the green plants can live on such simple ■compounds as carbon dioxide, water and nitrates, building them up into the highly complex organic matter of which plant tissues are composed. A certain amount of the material thus synthetized is used by the plant for its own life, breaking them to pieces and liberat- ing the energy that they hold, finally reducing them into simple compounds more like those from which they were originally produced. The larger part of the compounds thus built up, however, are not uti- lized by the plants but remain as an accumulation of organic matter. Green plants, therefore, are to be regarded as the most important agents in the constructive processes of nature. Vast quantities of complex organic compounds are produced by their activities. Constructive processes among bacteria. The larger number of bacteria are destructive in their action; but some extraordinarily TWO GREAT OPPOSING PROCESSES IN NATURE 69 interesting forms are distinctly constructive. Some of the soil organisms (see p. 183) have been found able to grow and hence to build up organic matter without either sunlight or organic food. These autotrophic bacteria (as they are called) can grow in darkness and obtain sufficient energy and food from simple compounds like ammonium salts, hydrogen sulphide, methane, carbon monoxide, and hydrogen. As yet we know too little about these organisms, but their existence is of great interest. They seem to be widely distributed in soil the world over. BREAKING-DOWN PROCESSES IN NATURE Green plants manufacture far greater amounts of organic materials than they need for themselves. Were there no means of reducing these again into simple forms, they would accumulate until the face of the globe would be covered by dead vegetation to the exclusion of everything else. Furthermore, the materials out of which the plants make these organic substances are limited in quantity on the earth, some of them very limited, and each year's vegetation uses up a considerable amount of this limited supply. To prevent the exhaus- tion of this supply and the consequent cessation of nature's activities all this vast mass of complex material must be broken down again into its original condition, i.e., carbon dioxide, water, and other simple molecules, so that its elements can be used over and over again. For the continuation of life, the analytical processes are just as necessary as the synthetic, since either one of them if continued alone would soon bring all life activity to a stop. The importance of the green plants in building up foods for animal life is generally understood and appreciated; but the equal necessity for the opposite process-destruction-is less commonly realized. Some of the agencies at work in breaking down organic matter, such as the combustion of wood, are purely chemical. It was formerly supposed that direct chemical action was responsible for nearly all, if not all, of these activities. As already mentioned, Liebig considered complex organic compounds to be inherently unstable and to break down of themselves by purely chemical means; but the evidence accumulating during the last sixty years has shown that nearly all of these disintegrating changes, formerly attributed to purely chemical forces, are really brought about through the agency of life. Whenever an animal uses a bit of food for its own purposes, 70 BACTERIOLOGY it starts a series of analytical changes which in the end result in the reduction of the food molecules into forms that can be used again by green plants. The breath which we exhale, for instance, takes from our bodies, in the form of carbon dioxide and water, part of the substance we took in as bread and butter; and our bodies are thus great centers of chemical disintegration. All other animals and non-chlorophyl-bearing plants take part in this same process of destruction. These breaking-down processes, whether biological or purely chemical, all result in the liberation of energy. When a piece of wood burns, the liberated energy-heat and light-is evident to anyone. When an animal consumes food, the energy manifests itself in body heat and muscular activity. When organic matter decays, however, the energy produced is liberated more slowly and is not so readily appreciated; yet the energy slowly liberated by decomposing organic matter is of more significance in nature than the violent form of energy given off by burning wood. The energy liberated in breaking down organic matter is utilized by the biologi- cal agent that brings about the process. This agent may be a large animal or it may be a microscopic organism; but in either case the energy liberated from the decomposition of the organic matter gives it power to carry on its life activities, just as sunlight gives the neces- sary energy to green plants. Thus it will be seen that the living agents in decomposition utilize organic matter for two purposes: as a food, and as a source of energy. Microscopic agents of destruction. Much of the organic matter built up by green plants is not of a nature to serve as food for animals; and much of the food which animals consume is only partly broken down in the animal body. All this material must be disposed of in other ways if it is again to be made available for green plants; and for this reason green plants and animals alone could not continue to exist permanently on the face of the earth. Complete decomposi- tion of those materials not fully broken down by animals is brought about by very small organisms, generally microscopic in size. So important are these minute agents of destruction that a branch of science has developed around them; and it is their influence, some- times for good and sometimes for ill, upon nature's economy in general and upon human welfare in particular with which this book is concerned. TWO GREAT OPPOSING PROCESSES IN NATURE 71 The organisms carrying on these activities belong to several different groups: bacteria, yeasts, fungi and protozoa. For this reason it is hard to find a general term which adequately names this branch of science. It is generally called Bacteriology, because bacteria are the most important of the organisms concerned; but the importance of other microorganisms in this line of activity is now realized to be so great that today no text-book in bacteriology is complete unless it gives considerable space to yeasts, fungi and pro- tozoa. For this reason the term Microbiology has been suggested. It is equally inadequate, however, since this science is concerned With some fungi that are not microscopic, but does not consider Unicellular green plants nor the majority of microscopic animals. As a matter of fact there is no logical reason for studying these particular organisms together, from the standpoint of classification. The reason for studying them together becomes evident when we recognize the similarity in the results of their life processes and the tendency for members of all these groups to assume parasitic life, thus producing disease. The organisms embraced in this study constitute a logical physiological group, even though they are so heterogeneous as to defy us to select a consistent name for this branch of science. If we call it Bacteriology, we must recognize that it is concerned not only with bacteria but also with the other minute organisms that influence human welfare. PUTREFACTION AND DECAY Long before the real significance of the phenomena of organic chemical destruction was understood, the more common processes of this sort were known and had received various common names, decomposition, putrefaction, decay, and fermentation are words of old usage. Their exact meaning, however, has never been clearly defined even among scientists. Nevertheless, as the processes have been studied more carefully, they have gradually been classified into more or less distinct groups, and it is possible to use one of the above names for each of the groups of phenomena, although the different groups are not always wholly distinct from each other. Decomposition is, of course, the most general term and applies to the whole series of breaking-down processes. Fermentation, on the other hand, has the narrowest meaning, yet is Used in the greatest variety of senses of any of these terms. In the 72 BACTERIOLOGY strictest sense it refers to the destruction of sugars and alcohols. So important are fermentations generally that they will be discussed more fully in the next section. Putrefaction is the term applied today to the decomposition of organic substances, chiefly of highly complex nature like proteins, which takes place under the influence of bacteria in the complete or almost complete absence of oxygen. Under these conditions the decomposition of the protein is only partial and the decomposition products, although simpler than the protein, are still rather complex molecules. They are commonly characterized by strong, unpleas- ant odors, and they are not infrequently of a poisonous nature. Putrefaction is consequently an unpleasant and generally an un- desirable process, although it is one of the necessary steps in the destruction of complex organic compounds. Decay may be defined as a destruction of organic matter taking place under the action of microorganisms in the presence of oxygen. It is generally brought about by different bacteria from those that produce putrefaction and it is much more complete. It results in the formation of very simple compounds. Organic material is made up chiefly of the chemical elements carbon, oxygen, hydrogen, and nitrogen, with considerable sulfur and minute quantities of other elements. After the process of decay is finished these elements assume the form of CO2, H2O, H2S, NH4, CH4, N and H. These substances are all gases and with the exception of CH4, no energy can be obtained by their further decomposition. Under ordinary decay in nature they pass off into the air as fast as they are formed. The decaying body thus gradually disappears, all of it except a small amount of mineral matter passing into the atmosphere. This is the condition which it is desirable for decomposing matter to assume finally; for in general the elements are then in a form in which they may be used again by green plants, converted into organic compounds and thus continue in the endless cycle. While it is easy to distinguish by definition between these different processes, they are by no means sharply defined in nature. Putre- faction and decay grade into each other with such a series of inter- mediate steps that it may be questioned whether any line of de- markation should be drawn. The whole phenomenon of protein destruction is a continuous one. At first certain bacteria that can grow in the absence of oxygen attack the material and begin its TWO GREAT OPPOSING PROCESSES IN NATURE 73 decomposition. As the process goes on, more oxygen finds its way into the decomposing mass and other bacteria, that require oxygen, are able to continue the action, reducing still further the partly decomposed products. Gradually the aerobic processes predominate until decay is complete. Thus, while it may be convenient to recog- nize two stages in organic decomposition, we must understand that both processes occur together in nature. Man has often found it to his advantage to control these processes artificially, sometimes to retard them, sometimes to hasten them. In the preservation of foods decay is retarded or even prevented entirely. Decomposition is hastened when waste materials are burned; for burning produces practically the same end result as de- cay. Sometimes moreover, we hasten the bacterial activities that bring about decay, as in the modern methods of disposing of sewage. Sewage is only partly decomposed as it flows from the sewers, and it contains too much water to be burned. If discharged into a stream its decomposition continues, using up the oxygen dissolved in the water which fishes need for respiration, and making the stream un- pleasant generally to sight and to smell. To prevent these unde- sirable results, the decomposition of sewage is now caused to take place rapidly, in filter beds and so forth, so that when finally discharged, the decomposition is complete and the stream is not materially injured. Another method of controlling decomposition is em- ployed in certain cases when intermediate products in the decay of food material have desirable flavors, so that we wish to allow them to form and then to stop the process. This is the case in the ripen- ing of cream for churning, in the manufacture of cheeses and salt pickles. These products are partly decomposed materials that owe their distinctive flavors to the action of microorganisms, whose activity must be controlled in order to prevent the process from going too far or developing undesirable by-products. The control of de- composition in these various manners is one of the chief subjects to be considered in the later sections of this book. Among the simplest and most important of the decomposition Processes is the breaking-down of carbohydrates, i.e., sugars and starches, through the agency of bacteria and yeasts. The action of yeast on sugar is especially well known and serves as a good illustra- FERMENTATION AND ENZYMES 74 BACTERIOLOGY tion of this type of decomposition. Yeasts have the power of split- ting up the sugar molecule into two molecules of alcohol and two of carbon dioxide according to the equation: CsH6(OH)5 • CHO = 2 C2H6OH + 2 CO2. (dextrose) (alcohol) The CO2 passes off into the air, causing bubbles in the fermenting mass if a liquid, and in the air it is available again for use by green plants. Alcohol is easily oxidized by direct chemical action into CO2 and H2O, and thus all the elements of the sugar become available again as plant food. The action of bacteria upon sugars is slightly different, but the end result is similar. They break up the sugar molecule, although not quite so completely as do the yeasts. Instead of producing alcohol they produce some organic acid. Sometimes they produce gas as well as acid, in which case the gas is generally a mixture of H and CO2; but often no gas is formed. The organic acids are not as readily oxidized as alcohol and generally require the action of other bacteria before their decomposition is complete, but eventually they too are converted into CO2 and H2O. Starches have a slightly differ- ent history. As long as they remain starch they are not quite so readily decomposed by bacteria; but some convert it into sugar, as do certain chemical agents (e.g., diastase, p. 76) produced by many animals and plants, including many bacteria and fungi. After being converted into sugar, it is easily attacked by yeasts and bacteria, as just described. These processes are occurring all around us everywhere. The sugar of decaying fruits, the starch of rotting vegetables and grain, disappear into the air under the fermenting action of yeasts and bacteria. These processes of carbohydrate-decomposition are generally known as fermentation; but the term is not sharply defined and there is more or less confusion in its use. Sometimes it is used in a broader, sometimes in a more limited sense. Originally it referred to various actions in liquids accompanied by the evolution of gas, not only biological activities, but also purely chemical reactions such as the action of acid on carbonate of lime. This confusion was soon cleared up and the term was confined to the action of yeast on sugar. Later it was discovered that other processes, such as the souring of milk, quite distinct from the alcoholic fermentation, were very similar in TWO GREAT OPPOSING PROCESSES IN NATURE 75 their nature, and they also were called fermentations. A gradual broadening of the term has resulted. Sometimes it is limited to the decomposition of carbohydrates with evolution of gas, but more often it is applied to any form of decomposition of sugar or starch. Further confusion arose upon the discovery that whereas the fer- mentation of sugar to form alcohol is caused by a living organism, another kind of progressive change in organic materials is produced by substances that are non-living. In this class fall the changes in foods taking place while they are being digested, such as the conversion of starches into sugars; but many of these digestive processes, such as the conversion of proteid into peptone, act upon complex nitrogenous matter, not upon carbohydrate. There is such a similarity between all these processes that the term fermen- tation was extended to include them all, thus covering indefinitely all the processes we have been considering under the designation of organic decomposition. The term is still used occasionally to apply to various decomposition processes other than the destruction of carbohydrates. It is plain, therefore, that the term fermentation has no very definite meaning and anyone using it should take care to explain in just what sense he employs it. On the whole it seems best to apply it to any decomposition of carbohydrates, but to remember that in a limited sense it often refers only to their decom- position with evolution of gas. The alcoholic fermentation was known for centuries before it was dreamed to be an activity of living organisms, even the ancients having known how to manufacture alcoholic drinks. At first, when microscopes were invented and the presence of yeasts was discovered, it was thought that they were the result rather than the cause of fermentation. But, as already mentioned, Pasteur's work estab- lished beyond question the fact that some fermentations are due to microorganisms. Soon, however, the difference was observed between this kind of fermentation and that which was produced by non-living bodies. It was found that certain fermentations, such as the conversion of starch into sugar in the presence of malt, were not stopped by mild antiseptics like alcohol or glycerin; and it was also discovered that fermentations of this kind can go on even when no microbrganosms can be seen under the microscope. The agents in this latter kind of fermentation were found to be unable to repro- duce themselves and thus to increase in quantity. Yet the similarity 76 BACTERIOLOGY between the two types of processes was such that the agents in both cases were called ferments. Those ferments that were capable of reproducing themselves were called organized ferments, the others unorganized ferments. The points of similarity between these two kinds of ferments are so important as to deserve further mention: (1) Both are brought about either directly or indirectly by living agents (even the unor- ganized ferments always being produced by animals or plants). (2) Both occur most vigorously within certain narrow temperature limits, usually not far from the body temperature. (3) The pres- ence of a special ferment is necessary in order to insure the activity, but extremely minute quantities of the ferment are sufficient. (4) These ferments become inactive if the temperature is too low or too high; after being submitted to low temperatures their activity may be renewed by warming, but very high temperatures destroy them entirely. (5) The fermentation can go on indefinitely without the addition of more ferment, provided there is plenty of raw material to act upon the products of the activity are prevented from accu- mulating; but the process is stopped entirely by too great accumula- tion of the products of the fermentation. Nevertheless the differences between the two kinds of fermentation are so great that in the early days of bacteriology they were thought to be absolutely distinct. Brew'ers had long known that from sprout- ing grain they could obtain a material-malt-which, added to starchy material, produces a progressive change, causing the starch to dissolve and become fermentable. Brewers did not at first know that the starch was converted into sugar in this process; but chemists showed this to be the case, and finally it was found that malt con- tains a substance (that has been named diastase) to which this fermentation is due. Similarly the digestive juices of the stomach were found to contain a substance (which has been called pepsin) that causes the digestion of insoluble protein into soluble peptones. Diastase and pepsin are not alive, and yet they are the cause of their respective fermentations just as truly as yeast is the cause of the alcoholic fermentation. One kind of ferment is a living organ- ism, the other a chemical substance. The non-living ferments are today known as enzymes. The nature of these enzymes has been a mystery from the begin- ning, and it remains almost as much of a mystery today as ever. TWO GREAT OPPOSING PROCESSES IN NATURE 77 They are produced as secretions from living organisms, and ap- parently in no other way. Thus, while not alive, they come wholly from living organisms. They seem to be chemical substances of a complex nature; but they are entirely different from any other chemicals known to man. Ordinarily when one chemical brings about a chemical change in another substance, the first substance in part enters into the composition of the other; thus, the addition of zinc to hydrochloric acid liberates hydrogen from the acid, the zinc meanwhile forming a new combination with the acid radical, zinc chloride being the result. When starch is converted to sugar, however, by the action of diastase, the diastase does not combine with the starch in the least. A very small quantity of diastase is capable of producing a very large quantity of sugar, and so far as can be detected by any means yet at our disposal, the diastase is not used up in the slightest during the process and can continue to act indefinitely as long as the end-products are removed from the fer- menting mass. The nearest approach to the action of enzymes by other chemical activities is that which is known as catalysis. Finely divided platinum, for example, has a catalytic action of this sort in stimulating ammonia to combine with oxygen and form nitric acid. These simple catalizing agents, however, are extremely limited in their powers compared to enzymes and are not as sensitive to effects of heat or poisons; nevertheless the similarity is great enough to be suggestive. This similarity has naturally led to the theory held by some that enzymes are merely complex proteid compounds having catalytic powers not unlike those of inorganic catalysts although much more complex. But other scientists have held the theory that they are not chemical substances, but forces-energy instead of matter, still others consider them in a class by themselves, "semi- living" in nature. These widely diverse theories show how little is actually known about the action and nature of enzymes. Theories to explain the action of organized ferments have also been varied. Pasteur held a very attractive theory concerning the activities of microorganisms which he characterized as life without oxygen. He supposed that yeasts carried on the fermentation of sugar best in the absence of oxygen. The yeast, he said, like all other living organisms, requires oxygen, and extracts it from the sugar if there is no free oxygen in the fermenting mass. When the oxygen is removed, the whole sugar molecule falls to pieces like an 78 BACTERIOLOGY arch with its keystone removed, the resulting fragments being alcohol and carbon dioxide. Attractive as this theory was, it did not stand the test of advancing knowledge and had to be abandoned. A more recent theory is that the microorganisms produce enzymes and that the enzymes cause the fermentation. This theory is widely held today, and in regard to some fermentations, indeed, has been con- clusively established. It has been shown beyond question that some of the bacteria which curdle milk without the production of acid do so by means of an enzyme closely resembling the rennin produced in the stomach of animals. Not only that, but these same bacteria secrete another enzyme that digests the curdled milk very much as do the enzymes of the pancreatic juice of mammals. Various other fermentations originally thought to be caused directly by microorganisms have been found to be due to enzymes produced by them. Yeast, for example, seemed to act directly on the sugar, for no enzyme could be obtained from it. But finally it was shown that an enzyme is produced by the yeast, only in this case it is not eliminated from the yeast cell. It can be extracted by crushing the yeast cell. In this way an enzyme, named zymase, can be separated from the yeast which is capable of causing the alcoholic fermentation without the agency of living yeast cells. It is true that there are still many fermentations in which the intermediate action of an enzyme has not been demonstrated; but the feeling is growing that enzymes are concerned in all or nearly all of the chemical activities of miero- organisms. This idea has led to a search for enzymes in all direc- tions, and they have been found very numerous and very abundant. They are found in the animal body, not only digesting the food, but also in the blood, regulating the various factors of nutrition, the growth of the body and other more obscure functions. In the plant world they are equally abundant. So common, indeed, have the enzymes shown themselves and so important are their functions that practically all life activities seem to be under their direct in- fluence, and even life itself appears to some to be simply a series of enzyme activities. The yeasts and bacteria apparently digest their food according to the same principle as the higher organisms: that is, by the secre- tion of digestive enzymes. But as they are too small to engulf their food, they secrete the digestive juices onto the food outside TWO GREAT OPPOSING PROCESSES IN NATURE 79 their bodies and then after it is digested they absorb the dissolved food material into their bodies. In other words, the distinction between organized and unorganized ferments has largely broken down. If organized ferments act only through the agency of unorganized ferments, no absolute distinction between the two kinds is possible. All fermentation is in one sense unorganized and in one sense organized, since it is produced by enzymes secreted by living organisms. It is an interesting example of the way in which tendencies of thought swing back and forth to find fermentations once more considered to be chemical processes. Liebig and his school considered them to be of purely chemical nature, taking place without the angency of a living organism and occurring spontaneously when the physical conditions were right. Later investigations showed the necessity of a biological agent, either directly or indirectly; while recent work indicates that in most cases, if not all, the biological agent acts only indirectly, and that the actual ferment is chemical. The difference between this view and Liebig's of course, is that according to the more modern theory fermentations cannot take place spontaneously but that an external agent of biological origin is needed to cause the activity. This modern conception does not in the slightest degree diminish the significance of microorganisms themselves since it is the latter that produce many of the enzymes. Neither does the enzyme theory explain fermentation more than partially, for the action of the en- zymes themselves is as mysterious as ever. It is important to remember that enzymes produced by higher animals and plants are widely distributed in nature and may often be the cause of fermentations that seem at first glance to be the result of microorganisms. The changes that take place in corn stored in a silo, for instance, were long considered to be due to bacteria; but oow it is believed that a large part is played by enzymes secreted by the corn itself. Similarly, it is not impossible that enzymes secreted by plant roots are responsible for some of the less understood activities that take place within the soil. The subject of enzymes and their relation to fermentation is a very broad one, and deserves a good deal more thorough study than it has ever been given in the Past. CHAPTER VI The Organisms Concerned The organisms that take part in the processes we are considering belong to four groups: protozoa, fungi, yeasts, and bacteria. Of these, protozoa alone are animals, and we are concerned with only a few protozoa, most of which are of interest because they cause disease. Fungi we are to take up here only in a very general way. Yeasts and particularly bacteria are the organisms about which this study must center. PROTOZOA Protozoa are the smallest of all known animals; yet they vary considerably in size. Some are so tiny that it requires the highest powered microscopes to see them, while others are all but visible to the naked eye. In shape they vary still more. The simplest forms of all consist of a jelly-like mass which has no definite shape and is able to flow over the surface of objects under water and to engulf food material that it comes across. Some others, including the smallest of protozoa, are also simple in structure, although they do have a definite shape; they are merely short elliptical bodies w'ith one or two rather long hairs (known as flagella) at one end, which serve as organs of locomotion. The larger ones have a more com- plicated structure, sometimes even having a mouth through which they take in food and are often entirely covered with hairs (cilia) to use in swimming. These three different forms are shown in figure 11. Protozoa have the power of resisting unfavorable condi- tions by surrounding themselves with a thick wall known as a cyst. Encysted protozoa are generally round and bear no resemblance to the active forms. In this form they can resist drying; and then when conditions become favorable again for active life, the cyst breaks and the organism emerges. Multiplication among protozoa generally takes place by a process known as fission-a very common process among the simpler forms of life. The first step in this process is for one individual to grow 81 ORGANISMS CONCERNED until it is much longer than usual. Then gradually each half of it takes on the characteristics of an entire individual, and finally a constriction appears between the two halves, which deepens until they are entirely separated. Sometimes the two individuals thus formed remain attached for a while, but generally they swim apart immediately. This, it will readily be seen, is the simplest form of reproduction possible to living organisms. A more complicated process of multiplication than this is sometimes observed among protozoa. This process starts with the formation of a cyst. Some- times one individual encysts as already described, or sometimes two individuals fuse together and form a single cyst-a very rudimentary Fig. 11. Different Kinds of Protozoa sort of sex union. Within a cyst of this sort a number of new individuals are formed, and when it breaks they are set free. Protozoa are to be considered here chiefly because some of them can cause disease. They do not take part in the general process of decay in nature. Their chief food seems to be bacteria, and for this reason they may be of great significance in limiting the development of bacteria. FUNGI The fungi form a large and diverse group having but two charac- teristics in common: lack of green coloring matter (chlorophyV), and the tendency of the cells to be arranged in microscopic filaments. Some scientists include yeasts and bacteria within the fungi, because both of these groups lack chlorophyl, and some members of each group show a tendency to grow out into filaments; but today botanists generally prefer to place them outside the fungi, and this classification will be followed in this book. The higher fungi are often plants of considerable size. Good examples of such fungi are the mushrooms. But the portion of 82 BACTERIOLOGY these higher fungi that we see with the naked eye is not the main part of the plant. In the ground or decaying wood beneath every mushroom is an immense network of delicate microscopic threads. This network is known as the mycelium and is the essential part of the fungus. The visible part above is merely a fruiting body, whose sole function is the production and spread of spores, the reproductive bodies. The way in which these spores are borne differs greatly in different fungi. Some fungi produce big mushrooms to bear their spores, but others, that live beneath the bark of trees, merely crack the bark and push out fruiting bodies, which are too small to be seen without magnification. There are also other differences in the way the spores are borne, and although these differences cannot ordinarily be distinguished without a microscope, they are considered impor- tant enough by botanists to form the basis of classification of the group. There is no need of going into this classification here, as the greater number of the fungi are of no practical significance. Some of the larger and more highly developed fungi to be sure, are of importance in causing the decomposition of wood; but it is princi- pally the lower simpler fungi that assist in the processes we are studying. These simpler fungi are often called molds. The molds. The organisms we call molds stand between the higher fungi on the one hand and the yeasts and bacteria on the other. Molds are quite easily distinguished from bacteria and yeasts; but there is no sharp line separating them from the higher fungi. In fact no such group as molds is recognized by botanists. Botanists classify the fungi into three or four large groups according to the method of producing spores (see section on Plant Diseases pp. 400 to 417), and the lowest members of any of these groups may be loosely spoken of as molds. But although they do not constitute a true botanical group, it is convenient to speak of molds as distinct from the higher fungi, thus grouping together the forms with which bacteriology is most concerned. Molds consist of a mass of microscopic threads-the mycelium- with spores borne on special fruiting bodies that differ from those of higher fungi principally in their simplicity and smaller size (see fig. 12). Figure 13 shows a few of the different kinds of fruiting bodies of molds. In the simplest forms the spores {conidia) are borne at the ends of filaments, being formed as mere segments separated from the parent thread. In other species the conidia are 83 ORGANISMS CONCERNED similarly formed, but the threads that bear them are frequently branched so as to form little brushes (as in the green cheese mold). In others, heads are produced at the ends of filaments from which radiate chains of conidia; while in still others the threads bear at the ends little hollow spheres that contain the conidia (the common black bread mold, for example). In one sense the molds are not Fig. 12. Diagrammatic Sketch of a Fungus Showing Mycelium and Fruiting Bodies Fig. 13. Fruiting Bodies of Three Different Kinds of Molds microorganisms, as the fruiting bodies are almost always visible to the naked eye (e.g., the green masses on moldy cheese), and if growing °n the right substances even the masses of mycelium can be seen. They are included among the microorganisms here, however, be- cause the individual threads of the mycelium are always microscopic, they can be studied only by the use of the microscope, and in many °f the places where they are most important they grow in such a way as to be absolutely invisible to the naked eye. 84 BACTERIOLOGY Molds are of considerable significance from a practical standpoint. More than half of all the known plant diseases are caused by them. The decomposition of wood takes place largely through the action of molds. Certain kinds of cheese owe their flavors to the growth of mold. Lastly, these minute organisms are often responsible for the spoiling of food. YEASTS The yeasts constitute a botanical group known as Saccharomy- cetes. They are distinguished from molds and the higher fungi by the fact that they produce no mycelium. Each individual con- sists of a single round or oval cell. These cells are on the average about 5000 °f an inch in diameter, larger than the smallest protozoa, although considerably smaller than the largest ones. They vary Fig. 14. Yeasts Fig. 15. Yeast Cells with Spores somewhat in size although not nearly so much as do the protozoa. They multiply by a characteristic method known as budding: a minute bud appears on one side of the parent cell, and grows until it becomes as large as the parent itself. Sometimes these two cells break apart as soon as the younger one is full grown; but often they remain attached, sometimes until an irregular mass of several cells has been formed (see fig. 14). Some yeasts have also been observed to reproduce by spores (fig. 15). In such cases a cell slightly en- larges and within it are formed a definite number (usually four) of spores, each of which is capable of becoming a new individual. It is not known whether all yeasts are capable of producing spores wider proper conditions; those species in which it has been observed produce spores only under specially favorable conditions. Yeasts are of interest to us in this study because of certain charac- teristic fermentations produced by them. Most important of these is their action on sugar, which they break up into alcohol and carbon dioxide. This is known as the alcoholic fermentation and is very important in nature. ORGANISMS CONCERNED 85 BACTERIA . Bacteria constitute another fairly distinct botanical group, called Schizomycetes (fission fungi). They are more abundant than yeasts or molds and from our point of view are more important. Nearly all forms of putrefaction and decay are due to them. The fertility of the soil is maintained by their activity in rendering or- ganic matter available for plant nutrition. All but a very few dis- eases of animals and human beings are caused by bacteria, and some plant diseases are due to them. Their abundance is best realized by recalling the fact that organic matter always decays unless sterilized and protected from exposure to the air. Milk, for in- stance, normally sours as a result of bacterial action. It can be sterilized, however, by heating considerably above the boiling point; and then if placed in a container that will not allow the en- trance of bacteria-such as a test-tube plugged with cotton or a bottle hermetically sealed-the milk will not sour or decay in any other way. But it is only by the most careful protection from con- tamination that decay can be prevented. Sometimes even the exposure to air for a few seconds will allow bacteria to enter, and decay begins. The air, however, is not the only place to look for bacteria. In fact the air contains very few of these organisms compared to soil, market milk, or even water. Soil contains millions of bacteria per gram, and milk that has become sour may contain billions per cubic centimeter. No spot on earth has ever been found-except possibly the peaks of snow-covered mountains-that is free from bacteria. Such being the case, it is easy to understand why they always manage to find their way into any substance that furnishes good conditions for their growth, unless it is hermetically sealed. Size and shape. Like yeasts, each individual bacterium is a single cell. They vary less in size than do the yeasts, but are generally smaller, averaging about 25,000 of an meh in diameter. Figure 16 gives an idea of the relative size of protozoa, yeasts and bacteria. The extreme minuteness of bacteria can be realized from the fact that eight billion of them could be crowded into a mass about the size of a pin-head. In shape they vary more than yeasts, although they are all very simple in shape. There are three general forms of bacteria. Some of them are spherical as shown in figure 17. Others are more than their diameter in length, varying from oval to rod- 86 BACTERIOLOGY shaped (see fig. 18). A third shape, sometimes observed, although not such a common form as either of the others, is the comma or spiral form, as shown in figure 19. Bacteria are sometimes motile, sometimes immotile. The power of motility, when present, is due to minute hair-like processes, called flagella, which sometimes sur- round the organism, and sometimes occur only at one end (see Fig. 16. Sketch Showing Comparative Size of Protozoa, Yeasts and Bacteria a, A large protozoon; b, a small protozoon; c, a yeast; d, a bacterium The shape of the organism and the arrangement of the flagella are both important points in the classification of bacteria. Besides these usual shapes of bacteria, certain irregular forms are known to occur. Club-shaped, Y-shaped, T-shaped and variously swollen or elongated forms are sometimes observed in cultures and the beginner in bacteriology is often puzzled by them. Some- times these irregular forms occur only in old cultures and are prob- able degeneration forms (involution forms, to use the technical term); ORGANISMS CONCERNED 87 but in other cases they seem to occur regularly in certain cultures and undoubtedly represent definite stages in the life cycles of these organisms. Bacteriologists are still unable to account for all of them. Fig. 17. Cocci °> Streptococci: b, micrococci; c, sarcinae Fig. 18. Rod-shaped Bacteria Fig. 19. Spirilli Reproduction. As already mentioned, multiplication of bacteria °rdinarily takes place by the process of fission (see fig. 20). This is tile simplest possible method of reproduction, as it merely requires °ne cell to lengthen and then to divide transversely into two indi- viduals. In this respect bacteria are much like protozoa and the Amplest algae. By means of this process one individual becomes two new individuals. This would seem to be a slow rate of repro- duction compared to that of higher animals and plants, among which Olle individual may bear countless spores or eggs or seeds; but as each one of the two new bacteria resulting from a single act of fission ls able almost immediately to develop into two more, the rate of Multiplication is actually prodigious. Other methods of multipli- cation seem to occur in connection with the irregular forms above Mentioned; but these methods are not yet entirely understood. 88 BACTERIOLOGY A certain group of bacteria produce spores, but these spores do not serve the same purpose as the ordinary spores of fungi, ferns and so forth. In the latter plants, spores serve to spread and multiply the species; but when bacteria produce spores, each individual bears but one spore and gives up its own existence to produce the spore. Such a spore cannot serve the purpose of multiplication. Its actual purpose is to enable the species to resist unfavorable conditions. These spores are, therefore, to be compared with the cysts of pro- tozoa already mentioned rather than with the spores of fungi. The spores of bacteria can remain alive for long periods of time, can resist drying and can even be submitted to temperatures as high as boiling water without being killed for some time. For this reason spores are of considerable practical importance, as they make it impossible to sterilize anything completely by mere boiling. Fig. 21. Types of Bacterial Spores Fig. 20. Successive Stages in Mul TIPLICATION BY FlSSION A spore is formed in the following manner: Within the rod which is to bear the spore a small round body appears which is more highly refractive than the rest of the rod and does not take stains as readily. It grows until it becomes as broad or even broader than the parent organism and perhaps half as long. Then the remains of the original organism die away. Figure 21 shows various shapes of spores and four different ways in which the spore may be borne in a rod, and in the top of figure 18 is a chain of spores, some of them with the re- mains of the rod still attached to them. In this state the individual can live almost indefinitely. When finally conditions are favorable for further development, its contents burst through the spore wall and develop into a new rod. Figure 22 shows five different ways by which a spore may develop into a new rod form. Such a rod is capable of growing and multiplying by fission like the ones from which the spore was derived. Structure. The structure of bacteria is a matter over which there has been considerable discussion. Higher plants and animals are composed of "cells." These cells are typically round masses of ORGANISMS CONCERNED 89 living material surrounded by a thin wall, and having a central body of different chemical composition, called the nucleus. The exact function of the cell nucleus is unknown, but it is plainly im- portant, because when a cell divides to form two new cells, the nu- cleus also divides and each new cell gets its own share of the original nucleus. Inside the nuclei of all cells large enough to be studied have been found still smaller bodies that stain so deeply with his- tological stains that they are called chromatin bodies. This chro- matin is believed to be a very important material, and when a cell divides it, too, is divided between the two new cells by a complex Fig. 22. Germination of Bacterial Spores a, Equatorial; 6, oblique; c, polar; d, equatorial, rod bowed; c, by stretching process which assures equal division. Large organisms are com- posed almost entirely of cells. The smallest animals, protozoa, are single isolated cells, different from those which go to make up the bodies of larger organisms, and yet showing the same typical struc- ture. Yeasts and certain of the algae are also single cells of typical cellular structure. Bacteria are as simple in structure, if not simpler than protozoa, yeasts, or algae; yet there is some doubt whether they are actually cells, for, they do not show the typical cellular structure. No one has ever demonstrated the presence of a true nucleus within a bacterium. The whole body of a bacterium shows the same stain- mg reactions as the nucleus of typical cells and within them have sometimes been observed bodies that closely resemble chromatin. For this reason it has sometimes been stated that bacteria are really isolated nuclei with the rest of the cell lacking. Recent work, 90 BACTERIOLOGY however, seems to indicate that bacteria are not mere nuclei but are complete cells with the nuclear material distributed throughout the entire cell instead of being gathered together in one body. If this is the case, the bacteria are of simpler structure than the yeasts and one-celled algae; for in the latter true nuclei are present. The difficulty in solving this problem is due to the small size of the bacteria. The same fact makes it difficult to learn anything else in regard to their structure; but we do have a few isolated bits of information on the subject. It is known, for instance, that some bacteria often develop-particularly when growing in media rich in carbohydrate-bodies that seem beyond question to be drops of fat. Undoubtedly these fat drops serve the bacteria as reservoirs of stored-up food material, just exactly as fatty tissue serves the Fig. 23. Bacteria with Fat Drops Compared with Normal Rods Fig. 24. Bacteria with Metachro- matic Granules higher animals. Figure 23 shows some bacteria growing on dex- trose agar that have become so loaded with fat-drops as to bear no resemblance to their normal form. Other bodies within bacterial cells have been observed over which there is more doubt. Unlike fat-drops, they stain deeply, and have sometimes been called meta- chromatic granules (see fig. 24). By some it is claimed that these granules are also reserve food, but that fact cannot be regarded as proven. The most important fact about these metachromatic granules is that they occur regularly in the diphtheria organism, giving it such a striking appearance that it can generally be dis- tinguished from all other bacteria. This greatly facilitates the diag- nosis of diphtheria. INTERMEDIATE FORMS It must be remembered that there are no sharp lines in nature. Although yeasts and bacteria are both distinct botanical groups, neither of them is separated from fungi by a very sharp line. There are mold-like yeasts and mold-like bacteria, neither of which has been placed definitely by botanists in one group or the other. The ORGANISMS CONCERNED 91 mold-like yeasts need not concern us especially; but apparently intermediate between bacteria and molds is the group of Actinomy- cetes, which is important because it contains a few pathogens and many soil organisms. The Actinomycetes consist of delicate branch- ing threads much like the mycelium of molds. The ends of the filaments often break up into bodies that serve as spores but look so much like bacteria that they cannot be distinguished from them under the microscope. Although the present tendency is to place this group with the fungi it has often been included among the bacteria. ULTRAMICROSCOPIC ORGANISMS Recently the discovery has been made that there are some forms of life too small to be seen even with the highest possible power of the microscope. The organisms causing certain diseases have been found to pass through filters so fine as to hold back the very smallest bacteria. What these ultramicroscopic organisms are, whether they are plants or animals, no one knows. Their importance in nature is also unknown. It is impossible to say whether they are widely distributed and of great importance, or whether they are of no signifi- cance except in the few diseases in which their activity has been discovered. It has been suggested that they are merely a stage in the life history of certain larger organisms; but although some pro- tozoa have been shown to have an ultramicroscopic stage, this theory can hardly be regarded as proven for the ultramicroscopic organisms in general. C. E. Marshall, et al. Microbiology. 2nd edition. 900 pp. Blakiston & Co., Philadelphia, 1917. REFERENCE CHAPTER VII Classification and Relationships of Bacteria Although we are more or less interested in all the organisms mentioned in the last chapter, it is with bacteria that we are pri- marily concerned, as their activities are of so much more significance. We are concerned with so very few of the protozoa, molds and yeasts that no further descriptions or classification of them is necessary; but of the bacteria we must make a more detailed study. ARE THEY ANIMALS OR PLANTS? It was shown in the early chapters of this book that the first knowledge of bacteria was very fragmentary and was intertwined with speculation. Only a few bacteria had been seen, the facts learned about them were unreliable, and it was practically impossible to distinguish one kind from another. Under such conditions it is not surprising that false ideas of the nature of bacteria were held. When bacteria were first discovered, for instance, they were supposed to be animals. Naturally the motile bacteria were the first to be seen, and it was thought that motile organisms must necessarily be animals. In the early literature they are referred to as animalcules. In 1852, however, Perty raised the question whether they were not more nearly related to plants; and indeed showed that they bear more resemblance to algae than to protozoa. Gradually this view became generally accepted, and today bacteria are almost always classed with plants. Occasionally, however, even now someone claims that bacteria are really animals, largely because they are more like animals in physiology. This argument is based on the fact that the activities of plants are primarily synthetical and those of animals primarily analytical; in which respect bacteria are more like animals. But the activities of the fungi are analytical, like those of bacteria, and no one considers them to be animals. Some biolo- gists try to solve the difficulty by making a third kingdom of life, the Protista, to include all one-celled organisms; but as a matter of fact, this classification increases rather than decreases the difficulties. CLASSIFICATION AND RELATIONSHIPS OF BACTERIA 93 To recognize Protista makes it necessary to draw two lines instead of one; and one-celled organisms are not separated from higher ani- mals or from higher plants by any sharp line. The easiest way to answer the question whether bacteria are animals or plants is to admit that in the lower forms of life there is no sharp distinction between the two kingdoms, but that for convenience's sake bacteria should be classed with plants. The reasons why it is more con- venient-and indeed more natural-to class bacteria with plants can be seen from studying their relationships to other organisms. RELATIONSHIPS OF BACTERIA The two groups of organisms most closely related to bacteria are the algae and the fungi. Algae differ from bacteria in having chlo- rophyl, but in morphology some of them are much like bacteria. Certain members of the group known as blue-green algae are eX- Fig. 25. Sketch of an Alga Similar to a Streptococcus tremely simple in shape and structure, showing much resemblance to the round bacteria, as shown by figure 25. There is, moreover, one group of filamentous organisms, the Chlamydobacteriaceae, which is generally classed with bacteria but shows such resemblances to algae that it is regarded as standing midway between the two groups. The relationship between bacteria and fungi is not at first glance so evident. Fungi resemble bacteria in the absence of chlorophyl, to be sure, but in form they are quite different. The reason why bacteria are thought to be closely related to fungi is that there have been found various intermediate types between the two groups. Some bacteria, such as the one which causes tubercu- losis and the one which causes legume nodules, often produce brandi- ng forms, resembling simple mold filaments. These bacteria with branched forms may easily be related to the intermediate group A-ctinomycetes, which as we have just seen are often considered true fungi. 94 BACTERIOLOGY On the other hand, it is possible to show relationships between bacteria and animals. There are certain rather uncommon bacteria that are spiral in form (Spirillaceae). Some of these are motile with flagella at one end. Now there are certain very small protozoa (Spirochaetae) which have much the same form and may be closely related to the Spirilli. This possible relationship is about the only justification for classing them as animals; and as the Spirillaceae are unusual bacteria, and not very common, while those that show relationship to the mold-like forms are quite abundant, it seems wisest to conclude that bacteria are more closely related to plants than to animals. One theory accepted by some biologists affords an explanation of these varied relationships of bacteria without making it necessary to commit oneself by calling them either animals or plants. This theory assumes bacteria to be the most primitive of living organisms. From them arose the plants on one hand and the animals on the other. On this theory it is easy to understand why there are inter- mediate groups seeming to connect bacteria with both kingdoms. This theory is by no means generally accepted, but to the bacteriol- ogist it is peculiarly attractive. CLASSIFICATION OF BACTERIA When bacteria were first studied it was not suspected what a vast number of different kinds there are. At first no attempt was made to classify them; and then when some sort of grouping proved necessary, the early classifications were very crude. But finally, during the years after Koch's epoch-making methods were devised, one kind of bacteria after another came to be definitely recognized, some causing well-known diseases, others associated with important phenomena like the souring of milk or nitrification in the soil. The discovery of these numerous kinds has made a more detailed classi- fication necessary. In drawing up such a classification there has been a good deal of disagreement and much confusion. Part of this confusion has arisen because of the difficulty in recognizing species among bacteria. The term species has been used for a long time in connection with the higher orders of animals and plants, although biologists are not always in perfect agreement as to what constitutes a species. If it is not known just what is meant by the term among higher animals CLASSIFICATION AND RELATIONSHIPS OF BACTERIA 95 and plants its meaning is still less definite among bacteria. Some bacterial types, to be sure, are very definite and seem to remain so. The diphtheria bacillus, for example, has been known for a long time and hundreds of thousands of different cultures have been studied, all of them alike in all essential particulars. The same may be said of quite a number of the other best known bacteria. But others seem to be more or less subject to change under different conditions, the changes described sometimes being so great as to lead to the question whether there is any constancy of types among bacteria. Some have thought, therefore, that bacteria are not as fixed as higher animals and plants, and that they are constantly undergoing changes. The sudden appearance of new diseases has led to the suggestion that common harmless types of bacteria may at times acquire patho- genic powers, thus bringing about a new plague to trouble mankind. All these ideas, however, are without any sufficient evidence. When bacteria were first observed no one anticipated this great difficulty in recognizing species among them. It was supposed that there were comparatively few kinds, all fairly easily distinguished from each other. They were therefore separated from each other by these early writers purely on the basis of the simplest features of morphology, such as size and shape. Later, spores were discovered, and still later it was learned that the different motile bacteria have their flagella arranged in different ways. These facts were soon made use of in classifying the organisms, but even so, the classifica- tion was not complete enough to include the great variety of different kinds. As a result it proved necessary to use what are called cul- tural characteristics-such as the form of growth on different media, because the different kinds of bacteria grow in different ways, there ls a great variety in the appearance of the growth on media. Some Produce glistening growth, others dull, some smooth and others Crinkled, some hard and others soft. These differences, indeed, are so striking that they were used for the purpose of classification about as soon as modern methods were used in bacteriology. Re- cently, however, it has been found that these cultural characteristics are too inconstant to be used except in special cases, and some other basis of classification has proved necessary. That most generally adopted depends on what are known as the biochemical or physio- logical reactions of the bacteria, such as: ability to liquefy gelatin, ferment sugars, reduce nitrates, and so forth. Sometimes these 96 BACTERIOLOGY reactions are quite valuable in distinguishing between different bacteria. It is known, for instance, that the common lactic acid organism always produces acid in broth containing glucose or lactose but not in broth containing no sugar but sucrose. These simple tests, therefore distinguish it from numerous other bacteria that produce acid in glucose broth alone, and also from those that acidify sucrose. Now if enough biochemical tests of this sort were devised, it might be possible to distinguish each individual kind of bacteria from all others by means of its physiological reactions. The impor- tance of these physiological tests is so great that some bacteriologists even hold the extreme view that they should be made the principal basis of classification, and insist that differences jn morphology are secondary to differences in physiology. If a classification could be worked out on physiological lines, it would have considerable practi- cal value; for it is the physiology of bacteria rather than their size and shape that is of importance from the practical standpoint. The general opinion, however, is that the broad divisions between groups of bacteria must be made on the basis of morphology, but that the divisions between the individual species may be based on physiology. In other words, there is an extreme difference in opinion among bacteriologists as to how to classify bacteria. Various classifica- tions have been proposed which differ entirely from each other, and there is as much authority behind one as behind the other. As a result it is only confusing for the beginner in bacteriology to try to learn any one of these classifications. It is better to learn the large groups and only those of the smaller groups upon which there is something like general agreement.1 1 It is inevitable that as the knowledge of bacteria advances the names of the individual organisms and their classification into groups must constantly change. The Society of American Bacteriologists has recently taken steps toward securing uniformity by appointing two committees, the first on classi- fication and characterization, and the second on the preparation of a manual in determinative bacteriology. The reports of the first committee are given among the references at the end of this chapter, as is also Bergey's Manual which is a result of the work of the second committee. The generic names and classifications in the publications of the two committees are similar but differ in important respects. Neither classification has any special authority behind it until sanctioned by general usage; and the committee on the Manual is still working on the classification, planning to change it from time to time. For this reason it does not seem necessary for a beginning student to learn the details of either classification. In a general way the classification in this CLASSIFICATION AND RELATIONSHIPS OF BACTERIA 97 Bacteria can well be divided into two large groups: 1. The true bacteria, sometimes called Eubacteria or Haplo- bacteria, in which the individuals are composed of single cells. 2. The filamentous or higher bacteria, sometimes called Tricho- bacteria, comprising certain less common forms, with relationships toward the algae and fungi. The true bacteria The true bacteria contain by far the greater number of forms with which we are concerned. There are three families of this group which are generally recognized: Coccaceae, the spherical bacteria Bacteriaceae, the rod-shaped or oval forms Spinllaceae, the spiral or comma-shapecf forms These three families need to be described in a little more detail. Coccaceae. This family is characterized by the fact that the individual cells are never more than one diameter in length. They are generally spherical, but sometimes when dividing rapidly in one direction the diameter in the direction in which growth is occurring is shorter than the transverse diameter. Spore production is rare, if it occurs at all. The great majority of them'are immotile, but a few motile cocci have been described. Four genera are recognized in nearly all methods of classification: Streptococcus. In this genus the successive planes of division are parallel, so that the cells form chains (fig. 17a). Micrococcus. Division occurs in this genus in two planes at right angles to each other. Typically a cell first divides in one direction, forming two, then each of these divides in a direction at right angles to the first, forming a square of four. This process continuing, causes the formation of sheets instead of chains (fig. 17b). Species are never parasitic on man or animals. Staphylococcus. Essentially like Micrococcus in morphology, but differing in certain particulars of physiology. All or practically all the forms are parasitic on man or animals. chapter follows the same line as these committee classifications; but it is far less detailed, and it mentions usages of some of the generic names that are pot recognized in the detailed classifications by the two committees. If it *8 desired to study these classifications in detail, they had best be consulted 111 their original form. 98 BACTERIOLOGY Sarcina. Division occurs in three planes at right angles to each other, resulting in the formation of cubical packets (fig. 17c). This genus is not nearly so common as the three preceding. Besides these four generally recognized genera there are a few other generic names of less definite application. The terms Piano- coccus and Planosarcina are sometimes given to the rare micrococcus and sarcina forms that are motile. Bacteriaceae. This family is characterized by the fact that the cells are more than one diameter in length. Generally they are rod- shaped but sometimes short oval. The oval forms, when multi- plying rapidly, closely resemble streptococci. Some produce spores. Some are motile; and when motile their flagella may occur in one of three arrangements: peritrichic (completely surrounded by flagella, as in figure 18, a and b), lophotrichic (a clump of flagella at one pole, as in figure 18c), or monotri chic (a single polar flagellum, as in figure 18d). There is great confusion in the generic names used in the Bacteri- aceae. Some use a single generic name, either Bacillus or Bacterium, for every organism in the family. Others try to distinguish between Bacillus and Bacterium, adding perhaps one or two other genera; but unfortunately they disagree as to whether these genera should be separated on the basis of flagellation or spore-formation. Still others try to recognize several different genera. The best a be- ginner can hope to do is to learn how the most commonly used names are employed. Bacillus. The typical members of this genus produce spores and have peritrichic flagella. There are, howTever, peritrichic non- spore-formers and non-motile spore-formers. Authorities differ as to which of these atypical forms to include in Bacillus. Some include all peritrichic forms whether they produce spores or not; while others include all spore-formers whether motile or not. At the present time the greatest weight of authority seems to favor the latter view. Sometimes especially by medical men, this name is used to apply to any rod-shaped organism even though immotile and producing no spores. Thus we hear of Bacillus tuberculosis and Bacillus bulgaricus. This form of expression is common, but is not considered the best usage. Clostridium. The members of this genus are typically organisms that produce a spore so much larger than the normal diameter of the rod that they are swollen at sporulation into spindles. Typically, also, they are unable to grow in the presence of free oxygen. Some 99 CLASSIFICATION AND RELATIONSHIPS OF BACTERIA bacteriologists use one of these characteristics and some the other to define the genus; so it is sometimes doubtful as to whether certain forms belong in this genus or in Bacillus. By some the term Plec- tridium is used for those organisms that have a spore formed at the end instead of the middle of the rod, like the organism that causes tetanus. Pseudomonas. This term is applied to rod-shaped organisms with a single flagellum or a tuft of flagella at one end (fig. 18, c and d). These organisms seldom, if ever, produce spores. Spore-forming bacteria with polar flagella have been described occasionally, but they have been found so seldom and there are so many chances for mistakes in their study that the existence of such forms is somewhat questionable. Bacterium. This name is especially loosely used. Some who recognize Bacillus by its spore-production, distinguish Bacterium by the absence of spores. By those who use the term Bacillus for the peritrichic rods, Bacterium is used for the non-motile ones. Some try to confine its use to some single small group of bacteria; while others go to the opposite extreme and apply it to any rod indiscriminately. On account of this very indefinite use, together with the fact that it is the singular of the general term Bacteria and thus somewhat confusing when used in a generic sense, it is not impossible that the generic use of Bacterium will become less com- mon in the future. Besides these general names, there are quite a number of generic names used by various authors to refer to special groups of bacteria. Thus: Azotobacter, Nitrosomonas, Nitrobacter are used for certain organ- isms that take part in the transformation of nitrogen in soil; see p. 183. Aerobacter is applied to a group of bacteria primarily characterized by the production of gas from sugar (the colon group; see p. 124). Lactobacillus is a name given to a certain group of organisms that produce a peculiar type of sour milk, sometimes known as Bulgarian buttermilk; see p. 125. Rhizobium is a name given to the organism or organisms causing nodules on legume roots; see p. 190. Similarly there are various other generic terms in use for special groups of bacteria; but they need not concern us here. Spirillaceae. This family consists of spiral or comma-shaped organisms (fig. 19). The comma-shaped forms are usually a part of 100 BACTERIOLOGY a spiral, and if several of them remain attached in a chain, the chain becomes a spiral. Sometimes they are motile, sometimes immotile. These bacteria are not nearly so common as the other two families and have not been so thoroughly studied. As a result there is even less agreement as to their nomenclature. Three genera are usually agreed upon: Spirosoma. Large, immotile spirals, generally in gelatinous masses. Microspira. Short, comma-shaped cells, motile with one or rarely two or three flagella. Genus is sometimes called Vibrio. Sometimes combined with the following genus. Spirillum. Cells are long, corkscrew-like, threads or shorter spirals, with a tuft of flagella at one or both poles. Some writers place the genus Spirochaete with the Spirillaceae. Spiriochaete, however, differs from the other genera in that the spirals are flexible and its motility is produced by some other means than by flagella. Most writers place it with the Protozoa. The filamentous bacteria The filamentous (or higher) bacteria are characterized by the fact that at some stage during their life the organisms exist as fila- ments. Although many filamentous bacteria have been described and variously classified, only three genera are common enough and so generally recognized that they need be mentioned here. Cladothrix (or Sphaerotilus). In this genus the filaments are ■composed of small separate cells surrounded by a sheath. The threads branch, but show only what is called false branching; that is, the cells do not branch, but the terminal cell is pushed to one side, a new cell takes its place and both go on multiplying. The differ- •ence between true and false branching can be seen plainly by com- paring the sketches of Actinomyces and Cladothrix shown in figures 26 and 27. Cladothrix multiplies by means of motile cells, known .as gonidia, that swarm out of the threads, attach themselves some- where else and grow out into new threads. Beggiatoa. The cells form fairly long unbranched filaments, and are characterized by the sulphur granules which they contain. The filaments are slightly motile, but do not possess flagella. No spores of any sort have been observed, reproduction taking place by division of the threads. Actinomyces (sometimes incorrectly called Streptothrix or Nocar- dia). There is much doubt as to whether this genus belongs with CLASSIFICATION AND RELATIONSHIPS OF BACTERIA 101 the bacteria, the present tendency, as we have already seen, being to place the group with the fungi. As it is often included among bacteria, however, a brief description of it will be given here. On ordinary bacteriological media these organisms grow in the form of delicate branching filaments that show no division into cells. Sometimes the filaments bear (or possibly break up into) small bodies that look exactly like small cocci or bacilli. They are called Fig. 26. An Actinomyces a, A colony slightly magnified b, a fragment of mycelium highly magnified Fig. 27. Cladothrix conidia and serve as spores, being resistant to drying but not to high temperatures. This genus is striking because of its remarkable Power of producing brilliant pigments upon certain media. REFERENCES Flugge. Die Microorganismen. F. C. W. Vogel, Leipsig. 1st edition, 1883; 2nd edition, 1886; 3rd edition, 1896. Lehmann and Neumann. Atlas und Grundriss der Bacteriologie. J. F. Lehmann, Munich. 1st edition, 1896; 5th edition, 1912. Migula. System der Bacterien. Gustav Fischer, Jena, 1900. D. Chester. Determinative Bacteriology. Macmillan, New York, 1901. Committee on Characterization and Classification, Soc. of Amer. Bacteri- ologists. Jour. Bact., ii, p. 505, 1917; Jour. Bact., v, p. 191, 1920. ergey's Manual of Determinative Bacteriology. Williams & Wilkins Co., Baltimore, 1923. CHAPTER VIII Physiology of Bacteria The physiology of bacteria is a very practical subject. Physiology is the study of function, and the great importance of bacteria is due to their varied functions in nature. In a sense, all the rest of this book is concerned with the practical aspects of their physiology. It is worth while, therefore, to study the important general principles of their physiology. RELATION TO EXTERNAL CONDITIONS Effect of light upon bacteria. Green plants cannot live without sunlight. Bacteria, on the other hand, not only flourish in total darkness as well as in the light, but are even harmed by direct sun- light. It has been shown repeatedly that if cultures of bacteria are placed in direct sunlight for an hour or two, the bacteria are almost if not entirely killed. This makes sunlight the cheapest disinfectant obtainable, and shows the unwisdom of shutting it out of places where dangerous bacteria might collect. Quite opposed to most bacteria in this respect, are a very few that have been found to utilize sunlight for energy much as green plants do. These bacteria are extremely interesting from theoretical con- siderations; but they are rare and their activities are not important enough to be given much time in a study of this sort. Effect of temperature. For each microorganism there is some one temperature at which the most rapid growth is possible. This is called the optimum temperature for growth. For the majority of bacteria it lies between 25° and 30°C. (75° to 85°F.). Some, however, have an optimum temperature considerably out of this range. Ani- mal pathogens, naturally,- grow best at the body temperature of the animal in which they ordinarily live (i.e., 37°C. or 98°F. in the case of warm blooded animals); while a few bacteria have been found that actually grow best at extremely high temperatures, such as 50° to 60°C. (120° to 140°F.). Still others have been found that grow best only a little above the freezing point. PHYSIOLOGY OF BACTERIA 103 Above or below the optimum temperature, the rate of growth falls off more or less rapidly until at last too high or too low tem- peratures are reached. The highest temperature at which growth can occur is called the maximum temperature for growth, the lowest is called the minimum. These extremes vary, if anything, more than does the optimum. Pathogens are usually unable to grow unless the temperature is very close to the optimum, while many of the common decay bacteria and soil organisms can grow at temperatures ranging from near the freezing point up to perhaps 40°C. (105°F.). If heated much above their maximum temperature for growth, the thermal death point is finally reached. The bacteria that do not produce spores are killed by temperatures of 65° to 70°C. as a rule. The vegetative forms of the spore-producers are also killed by these temperatures, but their spores are considerably more resistant to heat. The temperature of boiling water kills some spores, but a few kinds of spores are so resistant to heat that much higher temperatures than this have to be employed to sterilize anything completely if spore-formers are present. Spores can even resist dry heat as great as 140°C., for a considerable length of time, although moist heat of about 120°C. for twenty or thirty minutes is enough to kill any bacterial spore. Low temperatures do not kill bacteria so readily. Cultures have even been submitted to the temperatures of liquid air without being all killed. Low temperatures, if continued long enough, kill bacteria, but in any lot of organisms a few always resist the cold for such a long time that it is not a satisfactory means of sterilization. Effect of moisture. Water is necessary to the growth of bacteria. If any substance is not moist enough to give the bacteria sufficient moisture, they will not grow. For this reason, drying is one of the best means of preventing decay. Relation to oxygen. Oxygen is one of the greatest necessities of all bacteria. Most bacteria obtain their oxygen from the air just as animals do. These are the aerobic bacteria. Other bacteria are able to obtain their oxygen from some compound which is rich in it, such as a sugar. Those that always use combined oxygen and are unable to live in the presence of air are called strictly anaerobic, while those that can live in the presence of air but can also make use of combined oxygen when growing in the absence of air are called facultative anaerobic. 104 BACTERIOLOGY The aerobic bacteria are limited in their growth by the amount of oxygen they can obtain. Growing in liquids such as milk or broth, they depend upon the air dissolved, and naturally grow better the larger the surface that is exposed to the air. In soil, on the other hand, each tiny particle of mineral matter is surrounded by a very thin film of water and this film is surrounded by soil air. The bac- teria grow in this thin film and therefore are in very intimate contact with the gases in the soil air. Their supply of oxygen is limited only by the rapidity with which the soil oxygen consumed by the micro- organisms can be replaced from the atmosphere. Effect of germicides. Bacteria, like all other forms of life, are poisoned by certain chemicals. In general these chemicals are poi- sons to all forms of life, but certain of them are peculiarly bacterial poisons-if used in the right concentrations. This fact is of impor- tance, because of the times when it is desired to kill bacteria without danger to human beings. To this property is due the popularity of such germicides as chlorinated lime, hydrogen peroxide, boric acid, salicylic acid and sodium benzoate, all of which are used either in preserving food, or in disinfecting water or parts of the body where an ordinary poison would not be safe. Effect of density of solution. Some bacteria grow in distilled water, while others thrive in thick brines or syrups, so it is evident that there is no one specific gravity required by bacteria in general. Nevertheless, the organisms used to dense solutions do not thrive well in pure water, or vice versa. Even more important than this is the fact that bacteria living in a dense solution will often be killed by being placed directly into pure water; while by gradually accustoming them to less dense solutions, it is eventually possible to cultivate them in solutions of low specific gravity. BACTERIAL ASSOCIATIONS Another equally important factor influencing the activities of bacteria is their effect upon each other. It is well known that some bacteria are harmful to each other, while others are mutually bene- ficial. Very often when two kinds of bacteria are growing together the resulting activities are very different from those of either kind alone. This is an important fact, as bacteria do not occur in nature as pure cultures but as mixtures of many different kinds. In soil, for instance, there are many kinds of microorganisms which all PHYSIOLOGY OF BACTERIA 105 influence each other and also interact with the plants growing in the soil, the result being such a complexity of interactions that no one has begun to understand them as yet. In milk, however, the kinds of microorganisms are fewer, their interactions more simple, and some study has been made of their, action upon each other. It is known, for example, that a pure culture of the lactic acid organism produces a different kind of curd from that which is ordinarily found in naturally soured milk. This undoubtedly means that the other bacteria ordinarily in milk have some influence upon the production of curd, even though they themselves do not curdle the milk. Some of them have been isolated and grown in milk together with the lactic acid organism; and it has been found that associative action of this sort actually occurs. Undoubtedly such associative action is very common in nature; but the problem is such a complex one that little has yet been found out about it. METABOLISM OF BACTERIA Metabolism is the name given to the process of converting food material into body tissue. The process differs greatly in different groups of organisms. The conversion of the food taken into the stomachs of higher animals into bone, muscle, and so forth, is very different from the formation of plant tissue from carbon dioxide, Water, nitrates, and a few other equally simple compounds. The metabolism of bacteria is still different; and the different kinds of bacteria, moreover, differ greatly from each other in their physiology. The activities of some are extremely simple and those of others highly complex. In fact, one of the marvels of bacteriology is that organisms so similar in morphology should differ so greatly in physiology. Like higher organisms, bacteria require certain definite chemical dements. The most important of these elements are carbon, mtrogen, and oxygen. Of lesser importance are the elements sodium, Potassium, phosphorous and to some extent iron and sulfur. Carbon, mtrogen and oxygen are needed by all bacteria, although there is much difference among bacteria as to the exact form in which these dements are most available. No bacterial growth is possible unless these elements are present in available form; but sometimes minute Quantities of them will suffice, it having been shown, for instance, that some bacteria are able to grow upon the inconceivably small amounts of impurities found in distilled water. Besides the presence 106 BACTERIOLOGY of these food elements in available form, bacteria need some source of energy. The most common source of energy is the oxidation of carbonaceous matter, such as starches or sugars; but bacteria vary greatly in this respect, some even being able to obtain their energy from sunlight, like plants. In this process, naturally, many chemical changes are produced. The end-products of these changes are of three different kinds: the final products of metabolism, such as the protein that makes up the bacterial cells; the products of the oxidation from which the bacteria have obtained their energy; and lastly the by-products of these two processes, which represent chemical compounds that have not served any purpose, but are merely broken off the other com- pounds which the bacteria have used. Bacteria differ greatly in the production of these compounds, and for that reason the chemical changes produced in media of known composition furnish some of the best guides we have in establishing the identity of different organisms. THREE IMPORTANT PHYSIOLOGICAL GROUPS As already stated, bacteria are primarily concerned in the destruc- tion of organic compounds. In general they cannot feed directly upon the mineral world, but have to make use of material that has already been built up into organic matter by some other organism. Green plants, however, feed directly upon mineral matter, and a very few bacteria are like them in this respect. These few bacteria, known as autotrophic bacteria, are a very interesting, if not a numerous group. A few of them will be taken up in more detail in connection with soil bacteriology; but for the present it is enough to remember that they constitute one of the three great physiological groups of microorganisms. All the microorganisms except the autotrophic bacteria are busily engaged in the destruction of organic matter. Under certain conditions, however, organic matter has the power of resisting the attack of some of these microorganisms. While in living condition, for example, protein material is capable of resisting nearly all kinds of microscopic invaders. When the bacteria causing common putre- faction of protein material are inoculated into living tissues, they are unable to secure a foothold, soon cease to grow, and die. If, however, these same organisms are inoculated into the same tissue PHYSIOLOGY OF BACTERIA 107 after the animal has died, they begin to feed upon it, multiply, and soon produce a decomposition of the protein. This is a phenomenon of greatest importance, that living tissue has the power of resisting the attack of most of these microorganisms. This is not a simple phenomenon, and bacteriologists are still searching for its cause, but this much is known: that living tissues contain certain peculiar, little understood substances that have a destructive action upon most types of bacteria. These substances, whatever they may be, disappear upon the death of the tissue, and from the moment that death ensues, organic matter is subject to attack by an almost end- less variety of microscopic organisms. The class of organisms which can thus attack organic matter after death but not in the living condition are called saprophytes. This is the second of the three great physiological groups. It includes by far the larger part of the bacteria, yeasts, and fungi. There are, however, some microorganisms capable of attacking tissues while still living or, stating it in other terms, the resisting Powers which living tissues have against the invasion of micro- organisms are not sufficient to protect them from all kinds of microscopic life. The organisms with this power of overcoming the resistance of living tissues and growing as parasites in the living body of an animal or plant constitute the third great physiological group of microorganisms. It is clearly the parasitic class of micro- organisms alone that is capable of producing disease, for infectious diseases, as we now know, are always produced by the growth and Multiplication of microorganisms within the living body of plant or animal. The phenomena associated with the life of parasites on the one hand and non-parastites (i.e., saprophytes and autotrophic bacteria) °n the other, are widely distinct, so very distinct, indeed, as to form two almost separate sciences. The study of the parasitic organisms developed first, because People in general are very much interested in the causes of diseases and their cure. So much stress wras laid on this phase of the subject that the whole group of bacteria have acquired a very bad reputation and the public in general has been inclined to look upon bacteria as thoroughly mischievous organisms. The study of these parasitic Organisms and their relations to their hosts has developed many special problems that have been intensively and extensively studied 108 BACTERIOLOGY -problems concerning the distribution of the organisms, their method of invasion of the living body, the personal protection of the individual against them, the methods of destroying them, of fighting disease and preventing epidemics. These problems are all so closely related to medicine that this phase of bacteriology has been studied wholly by the medical profession and those specially interested in medical problems. This subject has developed and extended year by year until medical bacteriology and kindred topics have become very complicated subjects, and those who are interested in them have neither time nor inclination to study the other phases of bac- teriology that have been developing in the meantime. The non-parasitic bacteria, therefore, have been studied by a totally different class of students, that are not specially interested in the medical sciences. The topics they have studied have practi- cally nothing to do with disease and therefore do not attract the men who have been giving their attention to the parasitic micro- organisms. The problems are totally different. Sometimes it may be to check the growth of microorganisms, but in other cases it is to stimulate their growth. The problems are wider than those that interest the students of the parasitic microorganisms, for they apply to many phases of Nature's life processes, not to the somewhat narrow topic of human or animal disease. These problems lie at the foundation of agriculture, and their partial solution has already produced revolutions in the dairy industry and in some others. It is coming to be realized that they apply to many social problems and to many industrial pursuits. So very distinct are these two phases of the study of micro- organisms that there has been a tendency to separate them com- pletely. Even in the bacteriological societies, the students of para- sitology have pretty generally kept by themselves, while those who are interested in bacteriology from any other standpoint have formed societies of their own. Textbooks on bacteriology have shown this same tendency. There are exhaustive books on medical bacteri- ology, and others upon agricultural bacteriology, dairy bacteriology and industrial bacteriology. In some books on "general bacteri- ology" so-called, the tendency has been to give special emphasis to the particular topics the author is interested in, whether it be medical or general bacteriology, and then, for the sake of completeness, to add one or two brief, unsatisfactory chapters upon the other phases PHYSIOLOGY OF BACTERIA 109 of bacteriology, in which the author has no special interest. The result has been an unbalanced discussion of the subject, by the study of which the general student and the medical student each get an improper perspective. Some separation of these phases of bacteriology is perhaps neces- sary, as the result of educational conditions, but their entire separa- tion is certainly undesirable even for special students. The two phases of the subject really form a logical whole. The fact that some of the microorganisms concerned can develop within the body of a living host is only a small phase of the general study of these organisms; but because of its great interest to our own lives it has been given the largest amount of study and in the past has entirely overshadowed the more general phenomena. Hence the two main physiological groups of microorganisms are both considered in the following pages; but as different methods of study have proved necessary for these two groups, and as two separate branches of bacteriology have developed around them, they have to be treated Separately. Part II discusses the non-parasitic microorganisms, their relation to agriculture and the industries; Part III, the para- sites and their relation to animal and plant diseases. PART II NON-PATHOGENIC ORGANISMS 1. MICROORGANISMS IN THE DAIRY INDUSTRY CHAPTER I Milk Bacteria and Their Activities DEVELOPMENT OF DAIRY BACTERIOLOGY Milk, when in the. proper condition, is one of the best foods for anyone, and for children the best food known. It is, however, our most perishable food and the most difficult to handle in the market. Long before the development of bacteriology, the public as well as the dairymen had learned that milk must be furnished fresh each day and cannot be accumulated during periods of surplus production and stored until a change in the market conditions, as in the case of foods like molasses or vinegar. Even the more perishable of other foods, like meats and fruits, will remain in fairly good condition for several days at least, whereas the process of spoiling begins in milk a very few hours after it leaves the cow and, unless specially guarded, the product becomes unpalatable in a day or at most two days, and unfit for use a few days later. The study of bacteriology has dis- closed the reason for this long recognized fact. Milk is an excellent food for bacteria as well as for the human race. Indeed, it is one of the best natural culture media used in the bacteriological laboratory, during the journey from the udder to our tables, moreover, it is handled in such a way as to expose it to contamination with bacteria from various sources. Furthermore, the body heat of the cow Warms it to a temperature at which most bacteria grow with greatest rapidity. It is consequently subject from the very beginning to the destructive action of bacteria. One class of bacteria attacks the milk sugar; another class has its action particularly upon the albumin °r casein in the milk; while still other bacteria act, rather more slowly, on the fat. As a result of this activity, the milk is soon c°mpletely changed. Although not necessarily spoiled for human consumption, it is no longer comparable with fresh milk, but is Quite a new product. This rapid growth and activity of the bacteria the milk has given rise to nearly all, if not all, of the special dairy Problems. 114 BACTERIOLOGY Long before bacteria had been discovered, methods of dairying had been unknowingly adapted to bacteriological phenomena. The experience of centuries had shown dairymen that they must keep their milk cool if it was to remain in good condition. The more wide-awake dairymen had also decided that thorough cleaning of the milk pails assisted in keeping milk sweet. Butter-makers had discovered that they could produce a different kind of product according to whether or not the cream was allowed to stand for a considerable time before churning to undergo a process called ripen- ing. Cheese-makers also had found practical methods for extract- ing a considerable quantity of water from milk and converting the more solid part into a highly flavored product that would not undergo decomposition and could be marketed at leisure. All of these ob- servations and discoveries, together with many other facts con- cerning dairy processes, were phenomena of bacterial growth and bacteriological control, although the dairymen in former times did not dream it. The discoveries were made and the practical lessons from them adopted long before bacteria were known. Although the dairy industry had thus become already adapted to various phases of bacterial action, nevertheless the industry has been almost completely revolutionized by the discoveries of modern bacteriologists. There is hardly a phase of the industry, from the cow standing in her stall, to the milk, butter and cheese on our table, that has not been profoundly affected or even totally changed during the last few decades by the application of bacteriological science. At the present time this industry, wThich is one of the most gigantic in the world, involving the investment of larger sums of money and the employment of more people than almost any other, is based fundamentally upon bacteriology-in spite of the fact that the aver- age dairyman does not realize this and indeed knows little about bacteria. It is only because bacteriologists have been able to apply their laboratory knowledge to the practical handling of dairy prod- ucts that the dairy industry has been able to keep pace with the rapidly growing demand of our large cities for increasing quantities of milk and milk products. This is so well realized today that the larger dairy companies at the present time employ bacteriologists to control their methods. MILK BACTERIA AND THEIR ACTIVITIES 115 SOURCES OF MILK BACTERIA Milk is probably sterile when secreted into the tiny sacs that collect it deep in the milk gland of a healthy cow; but its contamina- tion with bacteria may begin immediately. Bacteria have been demonstrated within the tissues of the gland, and if the udder is diseased these bacteria may be very numerous and may involve the very gland cells themselves. Milk collected from a cow's teats under absolutely sterile conditions contains a fair number of bacteria even if the cow be in perfect health; while in the case of an udder attacked by tuberculosis or some inflammatory trouble like mastitis, the milk as secreted may contain large numbers, even millions of bacteria per cubic centimeter. The study of recent years has shown that such udder troubles are more common than formerly supposed and a considerable portion of milk samples taken from miscellaneous cows are generally found to have been infected with bacteria before leaving the udder, sometimes in sufficient number to increase appre- ciably the bacterial content of mixed milk from the herd. Neverthe- less the number of bacteria naturally occurring in the milk of a healthy cow is so small as to be insignificant in comparison to the number that enter from other sources. Exterior contamination may begin before the milk leaves the udder. Bacteria from the outside get into the teats and are able to live in- side the teats and milk ducts. Some of them are washed out at milking. Hence it may readily be seen that milk is by no means free from bacteria when it is drawn into the pail. The amount of this contamination varies with different cows and there are no means by which the dairyman can prevent it. The number of bacteria in milk when drawn, however, is comparatively small if the cow be free from udder disease. Experiments indicate that as a rule the number may be kept down below 1000 per cubic centimeter of milk, if all further contamination is avoided, and in some cases it is still lower, possibly no more than 200 or 300 per cubic centimeter. Considering this comparatively small number of bacteria present in the milk when drawn, it is a little surprising to find that immediately afterwards very large numbers may be present, such as 50,000 to 200,000 per cubic centimeter. Inasmuch as the bacteria that con- taminate the milk in the pail cause nearly all of the troublesome phenomena met in handling the product, it has proved very important to learn whence these organisms come. The chief sources of milk 116 BACTERIOLOGY bacteria are given in the following paragraphs in the order of their relative importance. Unclean milking utensils. It is practically impossible to wash milking utensils, such as milk pails or cans, or especially milking machines, sufficiently to remove all bacteria from them. This can be done only by thorough careful sterilization. The ordinary wash- ing, or even the ordinary steaming, is not enough to produce steriliza- tion. The bacteria which remain in these milk utentils, even after such washing, are alive and begin to grow at the first opportunity. This opportunity comes very quickly in the case of the can that is not thoroughly dried, especially if it be stood upside down on a flat surface or be kept tightly covered so that the moisture within cannot evaporate. Especially favorable to the growth of bacteria is the practice adopted unfortunately in some of the most scrupulously clean dairies, due to an unreasonable fear of dust, of leaving the cans for twelve hours in the warm moist air of the "sterilizer" where they have been steamed but not really sterilized. Under such conditions enormous numbers of bacteria may be present at the next milking, ready to contaminate the next milk collected in the vessel. These imperfectly sterilized or improperly washed and dried utensils constitute the chief source of the bacteria found in milk immediately after the milking. This source of contamination may be practically eliminated by the proper handling of the utensils. In the case of pails or cans this means a scrupulous washing with hot water, preferably followed- by scalding or steaming, then standing right side up for a short time to allow the steam to pass off, subsequently being inverted to drain, but so supported that the mouth is not closed to the entrance of air. The dairyman should notice carefully the con- dition of the cans returned to him by the milk company to whom he delivers his milk; for in many cases the milk companies are worse offenders in this respect than the dairymen, a mere whiff of odors from the returned cans being enough to demonstrate the presence of bacteria as effectively as a laboratory analysis. Milking machines are now becoming so common and present so many special problems that they need more consideration than other milk utensils. In many city supplies, milking machines have proved to be the greatest source of bacterial contamination. This is so generally recognized that some boards of health are forbidding the use of machines. Such legislation is very unwise, because in- MILK BACTERIA AND THEIR ACTIVITIES 117 telligent care is enough to eliminate this source of contamination, and a properly used milking machine is one of the best labor-savers that can be put on a farm. One of our experiment stations has been using a milking machine on its herd for about fifteen years, obtaining uniformly good milk of low bacterial content. The man in charge of this outfit, however, is considerably above the average farm hand intelligence. The dairyman who will economize on labor by the Fig. 28. A Milking Machine in Operation Use of milking machines must use more brain power to make up, if he would continue to produce good milk. The long rubber tubes of uulking machines are very difficult to clean and furnish a fine place the multiplication of bacteria between milkings. This can be °vercome by boiling the tubes and teat-cups after milking provided they are of a heat resistant rubber or by rinsing first in a solution of gashing soda and then in hot water, allowing them after that to stay luimersed until next milking in some disinfecting solution like brine uiixed with chloride of lime. This treatment helps keep the teat- 118 BACTERIOLOGY cups free from bacteria; but it proves well at least every week to take the teat-cups apart and clean them thoroughly. Some machines are so constructed that leaks occurring in the cups allow the milk to pass into the vacuum pipe, where the bacteria grow rapidly and at times may drip down into the milk pails. Although well constructed machines do not allow this, it shows one of the things against which the milker must be on the guard, and emphasizes the need of intelli- gence upon the part of the man who manages the machines. Filth from the cow. Anyone familiar with the methods in the ordi- nary dairy will easily understand how filth from the cow gets into the milk during milking. The habits of the cow and the physical nature of her droppings make it inevitable for her flanks and udder to become more or less soiled with manure, unless the barn is so constructed as to prevent it, and it is quite impossible to milk a cow thus soiled into a wide-mouthed pail without letting some of this filth drop into the milk. While this is the most unpleasant source of bacteria, it is of far less importance from the standpoint of numbers than the first one mentioned. Every precaution, of course, should be taken to prevent the entrance of manure or any other form of filth into milk; but manure is one of the smaller, rather than one of the larger, factors in explaining the bacteria that are found in milk. The milker. From the dirty clothing or soiled hands of the milker a considerable number of bacteria may enter the milk. This is especially true in the case of the so-called "wet-milking," the practice which some milkers have of dipping their hands in the milk before grasping the cow's teats. The actual number of bacteria thus in- troduced, when compared with those that enter from dirty utensils, is almost insignificant; but from one standpoint these few bacteria may be of great importance. Milk is a common means of distribut- ing disease germs, and the bacteria present on human beings are more likely to be the cause of disease than those that come from cows or any form of dirt, even manure. Epidemics of typhoid fever, diph- theria and scarlet fever have been traced to the contamination of milk by employees in some dairy who have been affected by these diseases, sometimes in very mild form. For this reason, the bacteria that get into the milk from the milker play a very important part in dairy problems although in actual numbers they are compara- tively few. MILK BACTERIA AND THEIR ACTIVITIES 119 The air. It was once believed that a large source of bacteria in milk was the air. Under some circumstances quite a number of bacteria may find entrance in this way. Bacteria are always floating about in the air, especially in a dusty barn where there is considerable hay, and more particularly when the hay is thrown down for the cows to munch upon during the milking. Under such circumstances it is inevitable for some of them to fall into the milk during the milking Process. The actual number that find entrance in this way, however, is really very small, and moreover, there is no special reason for believing the organisms from this source of any very great significance. While it is, of course, desirable that milk should be drawn in clean, fresh air, the bacteria that are derived from the air may be almost Neglected in the study of the bacteriological problem. The number of bacteria in milk when freshly drawn varies from a few hundred to many thousand or even a million, according to whether the milker uses scrupulously clean methods, poorly washed nailk pails, or uncared-for milking machines. Whatever the original dumber, there is always sure to be a larger number present when the milk is a day old; and the difference in numbers between different milk samples is even greater in such milk than in fresh milk. This because milk is a good food for bacteria, and if conditions are right they multiply in it very rapidly. The one condition more than any other which favors their growth is warmth. Between body tempera- ture and a temperature of 70°F., milk bacteria multiply so rapidly that uncooled milk may have hundreds of millions or even a billion bacteria per cubic centimeter before it is two days old; while if cooled immediately after the milking and kept near the freezing point, it Hiay not contain appreciably more bacteria in twenty-four hours than when drawn. At temperatures between 40° and 50°F., such as found in a good refrigerator, bacteria do grow, but grow fairly slowly, so that the numbers do not increase to such a prodigious extent if the milk is stored a day or two. Different species of bacteria multiply at different rates and have different optimum temperatures. As a result there is some variation m the character of the flora present according to the temperature at which the milk is kept-a matter to be considered further after studying the normal types of milk bacteria. Although there are GROWTH OF BACTERIA IN MILK 120 BACTERIOLOGY known to be some bacteria adapted to growth at lower temperatures, they do not develop so rapidly in the cool milk as the other kinds of bacteria do in the uncooled milk; so there need be no modifications on this account of the statement that warm milk contains more bac- teria than cool milk. These facts of course explain at once the significance of low tem- peratures in the keeping of milk. They have also been found to be the reason for the long cherished belief that thunderstorms cause the souring of milk. Experimental tests show that a thunderstorm has no influence on the souring of milk, provided the milk be kept sufficiently cold. Thunderstorms, however, occur upon days when the temperature is unusually high, and milk which is not kept artifi- cially cooled on such days is sure to grow sour whether a thunderstorm occurs or not. On such days, moreover, the great humidity of the air renders the insulation of the ordinary refrigerator a better con- ductor of heat than on dry days, thus causing the temperature inside to rise. These facts are the only foundation for the belief that a thunderstorm has the power of souring and curdling milk. If a sample of milk is found which contains millions of bacteria per cubic centimeter, it may usually be assumed that they are due to multiplication and not to primary contamination. The contamina- tion of milk from stable filth, even under the most unsatisfactory conditions rarely approaches 100,000 per cubic centimeter, and is generally much below this. If such milk is received in pails or cans that are imperfectly cleaned, containing the remains of the last lot of milk, in which bacteria have been multiplying rapidly, hundreds of thousands per cubic centimeter may be added; while if milked through improperly kept machines, a count in the millions is not uncommon while the milk is still in the stable; but the very high counts found in market milk are generally due to subsequent mul- tiplication. Extremely high numbers of bacteria in market milk, therefore, mean either unusually filthy milking utensils-especially milking machines-or excessive multiplication of the bacteria due to poor refrigeration, or commonly both. The only exception to this statement is in the case when the milk comes from cows with inflamed udders; but this condition can generally be detected by microscopic examination of the milk, for it is accompanied by the presence of long chains of streptococci characteristic of infections of this nature. 121 MILK BACTERIA AND THEIR ACTIVITIES KINDS OF BACTERIA IN MILK Predominating types There are certain kinds of bacteria that are so commonly found in milk that they constitute what may be called a milk flora. Some are so common that they may be regarded as normal in milk, although they actually come from external sources. These may be grouped under the following heads. Micrococci and streptococci. Milk drawn under the most rigid precautions from the udders of ordinary healthy cows is practically always bound to contain larger or smaller numbers of cocci, usually Micrococci, though sometimes Streptococci (see fig. 29, b and c). These organisms clearly exist in the milk ducts, often without any effect upon the cow although sometimes producing inflammatory troubles. These organisms bear much resemblance to Staphylococ- cus pyogenes and the Streptococcus pyogenes which are universally distributed and are the cause of various types of pus-forming diseases in mankind (see p. 347). It is doubtful if they are identical with them, but the question is not yet positively answered. There is no Very good evidence that the micrococci are capable of pus formation; but there is more probability of the streptococcus forms being patho- genic. There is a streptococcus forming long chains in milk, which *n many cases indicates an inflammatory condition of the udder. This organism cannot always be distinguished from the short chain streptococcus discussed in the following paragraph, but there seems to be no good reason for considering the two forms identical. This streptococcus is so commonly associated with inflammation in the udder that there is good reason to suspect it of causing trouble to the person who drinks the milk. There is, indeed, some evidence that epidemics of so-called infectious sore throat are due to strepto- cocci obtained from milk, possibly the same kind of organism that lnfects the udders of cows. The lactic acid organism. The most common organism in milk is the one which produces ordinary souring. It was one of the first bacteria recognized, having apparently been seen by Pasteur, who called it lactic yeast. It was later isolated by Lister and named Bacterium lactis, and then studied by others in the early days of bacteriology. Later studies have shown it to be very widely dis- tributed over the world and to be almost beyond question the most 122 BACTERIOLOGY common dairy organism. It is an extremely short rod, or else a coccus which ordinarily occurs in pairs although sometimes in short chains and occasionally in long chains (see fig. 29a). It has often been described under the name Bacterium lactis acidi, but when claimed to be a Streptococcus, the name Streptococcus lacticus was suggested for it, and at present the latter name seems to be replacing the older. It would probably be better to call it Streptococcus lactis thus reviving Lister's original specific term but putting it in the genus Streptococcus where it probably belongs. All these names- and in Germany the name Bacterium Guntheri as well-refer to the same organism. (Bacterium acidi lactici, on the other hand, is a distinctly different species.) Fig. 29. Types of Bacteria Found in Milk a,The lactic acid organism; b, streptococci, probably from udder; c, cocci, probably from udder; d, Bad. coli; e, Ladobacillus bulgaricus While it seems proper to call this organism a streptococcus, the substitution of the new name for the old one has produced an un- fortunate misunderstanding, since it has led frequently to the con- fusing of this common milk organism with the pathogenic streptococ- cus mentioned above. One or two investigators, indeed, have been strongly inclined to believe that the two forms are identical; although it is definitely shown that there are very great differences between the dairy streptococccus and that which is associated with pus formation, udder troubles, and septic sore throat. The pres- ence of this latter organism in milk is always a matter of suspicion, but the presence of the lactic acid type is perfectly normal. While these two organisms, therefore, seem to be absolutely distinct and to have a totally different significance in milk, the fact that both are commonly called Streptococcus has caused unfortunate confusion MILK BACTERIA AND THEIR ACTIVITIES 123 in speaking of bacteria in milk; for while some bacteriologists insist that the presence of a comparatively few thousand streptococci in milk is enough to condemn the milk, others declare that the presence of a streptococcus in hundreds of thousands or even in millions is perfectly normal in ordinary milk. The difference in statement in these cases is due to the fact that reference is made to the two differ- ent types of streptococci. To keep matters clear, it is therefore neces- sary in speaking of streptococci in milk, to distinguish sharply as to which of the two types is meant. This lactic acid streptococcus is a facultative anaerobe, growing better without much oxygen. It grows very scantily, therefore, upon the surface of various culture media, but grows well in the deeper layers, where oxygen is lacking. It is a very difficult organism to cultivate in the laboratory on the ordinary agar media, for its growth is so sparse as to be scarcely visible. Inoculated into milk, however, it grows with great rapidity. It speedily attacks the milk sugar, producing lactic acid therefrom, which in a short time curdles the milk. The intensity of this power varies with different strains of the organism. Some cultures are so active that they curdle milk in the course of six hours at 37°C. (98°F.), while others require twenty-four to thirty-six hours, and others are so weak that they do not curdle the milk at all. They all appear, however, to be the same organism, differing simply in the intensity of their action upon milk. It is often possible to convert one of the weaker strains into a vigorous acid producer by transferring it from one tube of milk to another after but a few hours in each tube, taking care not to let the milk in any culture tube reach the curdling point. Too much acid seems to be distinctly detrimental to the organism, so if allowed to grow in the same batch of milk too long, the organism does itself harm. The lactic acid organism grows readily at temperatures from 60 to 100°F., although temperatures between 60 and 70° seem to be most favorable to its growth. At this temperature it is so much more vigorous than the other bacteria in milk that it is pretty sure to outgrow them all, and milk kept at such a temperature is usually found after a short time to contain an almost pure culture of this organism. When growing alone in milk, this organism produces a clear solid curd, without whey or gas bubbles, and without strong odor. In these respects it is quite different from the next type of organism to be considered. 124 BACTERIOLOGY The fact that this organism has the power of checking the growth of other organisms is one of great practical importance. There is little doubt that the reason it has this inhibitive action is largely because of the production of acids, for as is well known, most bacteria are incapable of growing in the presence of any considerable quantity of acid. This fact makes this species the dairyman's friend. So long, of course, as the dairyman wishes to keep his milk fresh and sweet, no microorganism can be a friend. But in the preparation of dairy products like butter and cheese, where preservation is the aim, the product must be protected from putrefaction. The lactic acid bacteria effectually prevent the putrefying organisms from growing and thus constitute a necessary adjuct to the preparation of dairy products. The coli-aerogenes group. Besides the last mentioned type of organism, there are others in milk which produce lactic acid, thus souring and curdling the milk (fig. 29d). Their action, however, is quite different from that of the typical lactic acid organism, in that they produce gas as well as acid from the milk sugar. The curd, therefore, is not a smooth hard curd, but is full of gas bubbles. It is a softer curd, as a rule, and in connection with it unpleasant odors and tastes develop. For these reasons, organisms of this group are never friends of the dairyman. They not only sour his milk, but are likely to ruin either cheese or butter. There is known to be more than a single species in this group. The two of especial prominence are the true colon organism, generally called Bacterium coli, and a type called Bad. aerogenes. The former is probably the same as the organism so abundant in the human intestines, and comes chiefly from the cow's manure, a little of which is always likely to get into milk during the milking. A very similar organism has been found on grain, so there is a possibility of its get- ting into milk from this source; but if such is the case, it is certainly a rare occurrence. Bad. aerogenes differs from the colon organism chiefly in the amount and kind of gas it produces and in showing less frequent motility, but it also shows other points of distinction that are evident upon careful laboratory tests. In its relation to dairy problems it must be classed with B. coli as an undesirable type. It probably comes from manure and causes similar unpleasant fer- mentations in milk. In a sample of milk containing gas-producers, it is never certain what proportion belongs to either of these two types. MILK BACTERIA AND THEIR ACTIVITIES 125 Together they may be called the coli-aerogenes group, and looked Upon as undesirable. Their presence ordinarily indicates contamina- tion with manure, while the presence of the lactic acid type suggests at the worst nothing more than unclean utensils. Lactobacillus group. There is a third type of organism producing lactic acid in milk which differs considerably from either of the above. It had been observed by a few European investigators without exciting much attention until fairly recently Metchnikoff found it Ui special sour milk drinks very commonly prepared in Bulgaria. The organism first observed is a long, very slender rod, showing a Quite characteristic granular staining (fig. 29e), very different in appearance from either of the other lactic acid types. Its growth is favored by a higher temperature than bacteria in general and it Produces the highest acidity in milk of any known organism. At first it was not thought to occur in ordinary milk; but search for it showed that it is really a common form outside of Bulgaria. It does not grow well in milk that has been kept cool nor does it thrive °n ordinary media and hence had escaped the notice of bacteriolo- gists until their attention was drawn to it and new methods were Used to find it. This organism was named Bacillus bulgaricus. The name Lactobacillus bulgaricus is probably to be preferred. The chief interest in this organism comes from Metchnikoff's l(leas concerning its health-giving properties. Finding that the Bulgarians are a very vigorous race with a large percentage of cen- tenarians, he associated this fact with their habit of drinking this Peculiar form of soured milk. His theory was based on the assump- tion that these organisms in the intestine prevent the putrefaction °f the intestinal contents. The putrefaction of the food in the in- testine is probably undesirable since it would be likely to lead to the absorption of poisonous putrefactive products. Metchnikoff be- lieved that the absorption of such products causes a slow poisoning of the system, which is responsible for much of the illness of mankind, aild that the prevention of this putrefaction would diminish illness aud give greater length to life. There is, of course, some probability that since putrefaction of dairy products can be prevented by the growth of lactic acid organisms, the same thing might happen in the lr>testine. Upon this theory Metchnikoff based the advice to drink s°Ur milk extensively, especially milk soured with this Bulgarian organism. This suggestion, for some reason, struck a very respon- 126 BACTERIOLOGY sive chord in the public imagination and there began a rapidly- growing demand for cultures of this organism for the purpose sug- gested. The demand for it has continued to increase until it has become a very large one and quite an industry has sprung up to furnish cultures for this purpose. Many different brands have been placed on the market, some of which contain the Bulgarian organism in nearly pure cultures, some mixed with other species and others which do not contain the desired organism at all. The question whether the use of such cultures accomplishes what Metchnikoff claimed has been the subject of much experiment. Manifestly if these organisms are to act in the intestine so as to check putrefaction, they must grow in the intestine. It was at first thought that this was the case, but it has subsequently been found that this conclusion was due to confusion with a similar organism now recog- nized under the name of Lactobacillus acidophilus. It has been quite definitely shown that the true bulgaricus type disappears rap- idly from the intestine even after heavy feeding with such cultures. These observations render it doubtful whether the use of Metchnikoff's cultures accomplishes anything along the line claimed by him. On the other hand, their rapidly increasing use, the confidence the public is beginning to have in them, and the fact that they are widely recom- mended by many physicians indicate that some advantage has followed their use. Perhaps this advantage comes from the fact that they are generally used in milk, and that a milk diet always stimulates the growth of the acidophilus type in the intestine, which organism may have the beneficial effects claimed by Mechnikoff for his bacterium. Some enterprising concerns are now putting up acidophilus instead of bulgaricus cultures; but in view of their natural development in the intestines of milk-fed animals or human beings, it is doubtful how much good the consumption of the organisms does. Recent investigations have shown organisms of this Lactobacillus group to be abundant in some kinds of cheese. There is reason to believe, indeed, that they are among the important organisms con- cerned in the ripening of ordinary American (Cheddar) cheese. Less common types In addition to the species already mentioned there are numerous others which are found often in milk more or less abundantly, but MILK BACTERIA AND THEIR ACTIVITIES 127 which do not commonly play a part in dairy processes. Among them may be mentioned the following: Peptonizing or digesting bacteria. Many species of bacteria secrete an enzyme which attacks protein and converts it into a soluble pro- duct. This action is similar to that of the digestive juices on protein and is consequently frequently spoken of as digestion. Apparently, indeed, it is the means by which the bacteria prepare the protein for their own absorption; and the process is therefore properly described as digestion. The bacteria which have this action commonly cause liquefaction of gelatin and are therefore spoken of as liquefiers. Upon milk they commonly have two actions: first coagulation, then di- gestion. The coagulation, however, is not due to the formation of acid, as these organisms commonly produce alkalinity or else cause no change in the reaction, and the few which do produce acid generally do not do so until after the milk is curdled. The curdling is due to an enzyme closely resembling the rennin produced in the stomach of mammals. The digestion is due to the conversion of the curdled casein into a soluble product so that the final product is liquid again. The digestion is also due to an enzyme produced by the bacteria, apparently a different enzyme from the one which coagulates the casein, having an action similar to that of the trypsin obtained from the intestines of animals. The result of this action is to convert the casein into simpler compounds known as peptones. Some species have slow powers of digestion others rapid, sometimes so rapid that the preliminary coagulation is not evident to the eye. The conversion into peptone may be completed in two days or may require three or four weeks. The bacteria that produce this action are quite varied. Among them are some large motile spore-bearing rods (as shown in fig. 21) as well as other small bacteria of a quite different type. They are fairly common in milk, but are in general looked upon as undesirable contaminations coming from some form of dirt, and having an action Mich renders the milk unfit to drink. They play but small part in ordinary dairy processes although they sometimes damage cheese, for if not held in check by the lactic acid bacteria they may grow Until they have softened the cheese and caused tastes and odors which ruin it. Market milk rarely shows any effect of this class of organ- sms, largely because their action is commonly overshadowed by the Uiore rapid growth of the lactic acid organisms. In earlier years a sweet curdling" was sometimes mentioned, which was due to this 128 BACTERIOLOGY class of bacteria; but with the improvement of dairy methods, so as to keep the bacteria under control, such phenomena have practically disappeared. Bacteria producing "milk faults." Not very long ago certain dairy troubles were common, which proved to be due to unusual milk bacteria. With better methods these "faults" have so largely dis- appeared that an enumeration of them here is all that is necessary. Slimy or ropy milk is the most common of these faults, and still causes trouble at times not only in single dairies but sometimes in the supply of a whole community. Although in some countries milk of this nature is relished, ropiness makes a milk unfit for the market in this country. The bacteria causing this trouble have been isolated and studied. They are thought to come from the water sup- ply used in washing milk utensils, although there may possibly be other sources as well. Bitter milk is another trouble reported in the past, although rare today. Sometimes this is caused merely by some article in the cow's diet, but it has also been found due to bacteria. Colored milk of various sorts has been observed, such as blue, yellow or red milk. All of these colors have proved to be due to certain unusual bacteria developing in the milk. In the laboratory, by the choice of the proper bacteria it is possible to obtain milk of prac- tically any color; but the predominance of these pigment-forming bacteria in market milk is rare. These facts are of scientific interest but of no practical importance today, since the greater care given to milk production is sufficient to prevent their occurrence. Much the same statement can be made concerning other milk faults-such as tainted milk, soapy milk, turnip-tasting milk and a non-curdling milk, i.e., milk which will not curdle even when old-all of which are the result of bacterial growth in milk. Each of these has been found as a dairy trouble, but they are very rare. Alcoholic fermentation. Milk does not readily undergo an alcoholic fermentation and pure cultures of yeast have little effect upon it. When however, a mixture of yeasts and certain bacteria are intro- duced, it may undergo such fermentation. Certain beverages are produced in this way, kefir, for example. Kefir is made by adding "kefir grains" to milk. These grains prove to be a mixture of bac- teria and yeasts, the bacteria apparently converting the lactose into a form which the yeasts can ferment. Other forms of fermented milks are produced by adding a little cane sugar and then fermenting by the action of yeast. Some of these fermented milks, known under various names such as kumys, matzoon, bacillac, etc., are quite popular in some places. MILK BACTERIA AND THEIR ACTIVITIES 129 PHASES OF GROWTH OF NORMAL BACTERIA IN MILK Milk is so sure to become inoculated with a variety of bacteria, that every ordinary sample of milk must be looked upon as a fertile field planted with a large number of different kinds of organisms, all of which are endeavoring to feed upon it and to multiply. It becomes, therefore, a matter of interest to note what changes such milk will undergo under the influence of the many kinds of organisms which are contending within it for mastery. These changes depend more or less upon external conditions, primarily upon the temperature at which the milk is stored. We have seen that some species grow readily at low temperatures, others at moderate temperatures, while still others prefer or even require high temperatures. If the milk is kept for several days below 50°F. there will be a slow increase in the numbers of bacteria until in time they are very numerous. The bacteria that grow at these temperatures, however, are not the forms which cause the ordinary souring, but are more likely to be the peptonizing forms. Their products of growth are more likely to be harmful than those of the lactic acid types. Old milk, therefore, even if kept cool, may be Unwholesome, although not soured, because of the development of the bacteria that grow at lower temperatures. When kept at moder- ate temperatures-60-80°F.-the type of bacteria that multiplies uiost rapidly is the ordinary lactic acid type, which quickly sour and curdle the milk. These organisms are so vigorous and so well adapted to growth in milk that at these temperatures thay rapidly out-distance all others. At temperatures still higher-80-100°F.- the gas producing organisms (the coli-aerogenes group) seem to be uiore distinctly favored than the lactic acid type. At these higher temperatures, it is true, the ordinary lactic acid type grows readily enough, but as the coli-aerogenes forms also develop with great rapidity, the milk is not only soured quickly but as a rule is filled with gas bubbles. In this type of souring, unpleasant tastes and °dors develop to such an extent as to spoil the product completely. This is the reason why a dairyman tries to keep his milk at a tempera- ture between 60 and 70°F. when he wishes it to sour normally either t°r butter-making or for cheese-making. At temperatures over 100°F. there is a tendency for the bulgaricus type to predominate. Such an occurrence is uncommon in milk; but it is sometimes ob- served in whey which has been heated to kill disease germs (i.e., Pasteurized, see p. 150). If the pasteurization has been incomplete aud the whey is then stored in vats without cooling, the temperature 130 BACTERIOLOGY is likely to favor this type of organism which may then overgrow everything else. In spite of all such differences in the flora the study of a large num- ber of normal samples of milk has shown that ordinarily the bacterial life in milk passes through several very distinct phases, each of which is due to the predominating growth of certain types of microorgan- isms. These phases are as follows: Phase I. For a short period after the milk is drawn from the cow there is no increase in the total number of bacteria in it. In fact, if the number of bacteria is counted immediately after milking and then after one to three hours, the number is frequently found to have diminished during this period. This is often spoken of as the germicidal action of fresh milk, although it may not be correctly so- called. Certain species, apparently, that are in milk at the outset are unable to live there and die out, while others get in that live and shortly begin to develop. It is not at all impossible that the forms which die out are udder bacteria which find conditions unfavorable outside the body, and that numbers do not begin to increase until the ordinary milk bacteria have found entrance. The length of this first period varies with the temperature of the milk. It is usually from three to four hours, although if the milk is cold it may be longer. Phase II. The second phase is characterized by the gradual increase in numbers of bacteria. For a period of several hours there is a gradual multiplication, the various species that are present grow- ing with different rates of rapidity according to their special natures. This period may last from four or five to twenty-four hours or more, according to the temperature at which the milk is kept, the time being much longer in cold milk than in milk that is allowed to become warm. It is likely to last until the bacteria become numerous enough to be counted by millions. Phase III. Lactic acid bacteria prove to be much more vigorous in milk kept at ordinary temperatures than any of the other mis- cellaneous types; as a consequence they multiply more rapidly than the others, and examinations made hour by hour show a constantly increasing percentage of the lactic acid type. With the increase in the lactic acid bacteria, the amount of acid in the milk is also increased, and this increasing acid gradually checks the growth of the other bacteria and eventually even the growth of the lactic acid bacteria themselves. Before their growth is entirely stopped, however, the milk becomes so acid that it curdles. Sour milk may become almost a pure culture of the lactic acid bacteria, or of this organism together MILK BACTERIA AND THEIR ACTIVITIES 131 with some of the coli-aerogenes type. Their numbers increase into the billions at the time of curdling and then decline, probably because the milk becomes too acid for their growth; but they may continue to predominate for some time in the curdled milk. The length of this period is sure to be several days, and it may be many days, according to circumstances. Phase IV. The acidity of the curdled milk gradually becomes less. Several different causes commonly contribute to decrease the acidity, but in all cases it is apparently due to the growth of some kind of microorganism. In many cases it is the growth of molds, which are capable of growing in a more acid medium than bacteria can endure, and which secrete substances that partly or wholly neu- tralize the acid. Important among these molds is one peculiar to milk known as Oidium lactis. Yeasts also frequently grow in the curdled milk. As the growth of all these organisms tends to lessen the acidity, in time the acid curd acquires a neutral or even alkaline reaction. When this occurs, there appears the next stage. Phase V. After the acid has been neutralized, a variety of bacteria are able to act upon the protein. Many putrefying and decomposing organisms attack the casein and disintegrate it. These are either present in the milk before curdling or find their way into it from some extraneous source after the curdling. In the end, through their agency, the milk turns into a vile smelling, putrefying mass, in which the casein as well as all the other ingredients are decomposed. If this is allowed to continue to its ultimate end, nearly all the organic ingredients are converted into water or gas; but under natural conditions the process never continues to quite such complete disintegration. As long as the milk remains in the first three stages, it is still valu- able and can be utilized as a food. After it passes into the fourth stage, however, it is of little or no value, unless the process has been artificially controlled as in cheese making. Certain forms of cheese, indeed, (such as Limburger) are marketed when they are in the beginning of the last stage, decomposition having begun under con- trolled conditions, but not having progressed far enough to ruin the product. REFERENCES H- W. Conn. Practical Dairy Bacteriology. Orange Judd Co., New York, 1908. Orla-Jensen. Dairy Bacteriology. Transl. by P. S. Arup. Blakiston & Co., Philadelphia, 1921. ft- L. Russell and E. G. Hastings. Experimental Dairy Bacteriology. Ginn & Co., Boston, 1909. CHAPTER II Market Problems Nearly all the problems that have arisen in connection with the handling of milk are due to bacteria and their activities. The methods of handling it and the large quantities in which it is con- sumed raw make it a very important medium in the spread of disease. Furthermore the rapidity with which non-pathogenic bac- teria multiply in milk and their power of decomposing and spoiling it, make great care necessary on the part of both producer and dis- tributor. The public has a right to demand that the milk which it purchases shall not be dirty, stale, or dangerous to health, inasmuch as anyone of these conditions makes it less valuable or even harm- ful. The nature of milk is such that we cannot determine any one of these three matters at a glance. Even the solid dirt in it cannot be seen, because milk is opaque, and much of the most objectional forms of dirt are either liquid or else composed of too small particles to be revealed by any method of sedimentation or straining. These conditions, however, are all closely associated, either as cause or as effect, with the bacterial content of the milk, when it is put on the market; and the control of market milk therefore aims primarily at regulating the bacterial content. Fresh milk from healthy cows contains but small numbers of bacteria. Hence it follows that any lot of market milk containing bacteria in large numbers must have been drawn from cows with diseased udders, or contaminated with considerable filth during the milking, or collected in improperly cleaned pails or milking machines, or else stored at a high tempera- ture or kept long enough to allow great multiplication of the bacteria. Any one of these factors may, and generally does injure the quality of the milk. Hence, although some dangerous conditions such as the presence of disease germs bear no relation to the total number of bacteria present, the bacterial content is generally taken as a fair indication of the value of the milk to the consumer. For these reasons there is a constant effort made to control the number of bacteria, keeping them below certain more or less definite figures. In earlier years milk inspection was directed almost wholly MARKET PROBLEMS 133 toward the chemical nature of the milk; but in recent years, as a result of the stress laid upon the sanitary side of the matter, the greatest weight is given to the bacteriological analysis. In most places the bacterial content is looked upon as indicating the whole- someness of milk more certainly than the chemical composition. Exactly what interpretation to place upon the bacterial analysis, however,- is somewhat uncertain. In raw milk the presence of but small numbers of bacteria is an inevitable indication that the milk has been produced under proper conditions of cleanliness and is fresh. When the numbers are large, we are unable to say whether the trouble in any case has been due to dairy conditions and an Unusual amount of filth, or to improperly washed utensils, or to age of the milk, or to insufficient refrigeration. Inasmuch, however, as any of these factors is undersirable, the general feeling is that such uiilk should be condemned, even though we are unable to determine which of these factors causes the high bacterial count. Upon these grounds, therefore, those concerned in controlling the public milk supply have felt justified in insisting upon the adoption of bacterial standards; and the adoption of such standards has been becoming uiore and more common during recent years all over the country. FACTORS PREVENTING HIGH BACTERIAL COUNTS The factors which are chiefly concerned in producing a low bac- terial content in milk are now fairly well known, although it has taken a good many years of experimentation to determine them. Public authorities are endeavoring to emphasize in the minds of aU handling milk the importance of carefully controlling the factors that determine the bacterial content. These have been found to be chiefly as follows: Healthy cows. Cows which have udder diseases are practically certain to give milk contaminated with the bacteria which cause these diseases. Such organisms are the Streptococci or pus-forming O1'ganisms, the tubercle bacilli, and possible some others. Inasmuch as these organisms are likely to be a source of trouble to man, it is fcot necessary to emphasize the fact that the cow producing milk tor the public should be in health and especially should be free from aoy type of udder disease. There has been quite a decided improve- ment in this respect since these facts have been known, for whereas 111 former years little attention was paid to the matter, at present 134 BACTERIOLOGY there is quite a general tendency to exclude infected cows from milk- producing herds. It should be noted here that it is difficult, and indeed sometimes quite impossible, by any present known bacteriolog- ical methods to detect disease germs in milk. No routine method has yet been devised which is accurate enough and quick enough to detect the tubercle bacilli in milk for the purpose of keeping such milk from the market. The detection of the pathogenic cocci is probably a little more simple, for there is considerable evidence that long-chain-forming streptococci in milk are pathogenic forms. Certain types of streptococci, like the lactic acid organism, are sure to be in all samples of normal milk; but those which form long chains (see fig. 29b) are not found in normal milk from healthy udders, and their presence almost invariably indicates mastitis or some milder form of udder infection. When milk from any herd is found to contain appreciable numbers of long-chained streptococci, it is advisable to examine the cows for the purpose of isolating any that may be infected, and generally one rr two cows with infected udders can be thus located. Discarding the milk from these cows keeps the streptococci out of the milk from the herd. Cleanliness in dairy methods. This resolves itself into two phases, first, keeping dirt and filth from entering the milk during the milking process, and second, the thorough cleansing of the milk utensils into which the milk is to be received. To keep dirt out of the milk during the milking process it is neces- sary to remember the source of such dirt, namely; the cow's tail, flanks, and udder; the milker's hands and clothes; and to a less extent, dust particles in the air. Of these three sources, the first is the largest, as anyone can understand by examining the condition of the cow's flanks in any dairy where scrupulous cleanliness is not observed. In the case of hand-milking, the two factors which do most to prevent the primary contamination of the milk are small- topped milk-pails and intelligent milkers. There are various designs of small-topped milk pails which have been used quite widely in milking, all so designed as to expose only a small opening for the entrance of material from the cow or the air, sometimes with this opening covered by a cotton or cloth strainer so as to filter out the large particles of dirt. Figure 30 shows one of the most commonly used small-topped pails. Such pails are very practical and cut down the number of bacteria appreciably. Milking machines also MARKET PROBLEMS 135 keep out dirt from the air and from cow; but for the reasons we have already discussed, they are likely to increase the number of bacteria unless intelligently manipulated. Intelligence and interest on the part of the milkers are probably more important than any matter of equipment in the production of clean milk. It might seem that methods adopted for keeping dirt out of the milk would work as well in the hands of one milker as in Fig. 30. A Small Topped Milk Pail those of another, but such does not prove to be the case. It has been conclusively shown that the number of bacteria in milk drawn by an intelligent milker, who is interested in results, is decidedly less than in that drawn under identical conditions by one who lacks intelligence and interest in the work. This was well demonstrated m one quite famous experiment, in which ten dairymen, all of whom knew how to produce clean milk, went to various farms in a neigh- boring community from which the milk was regularly dirty, and, without making a single change in the equipment they found, drew milk as free from bacteria as they were accustomed to obtain at home. For this reason it is not possible to predict the quality of the milk from an inspection of the physical condition and the equip- ment of a dairy. 136 BACTERIOLOGY Probably the greatest source of the large numbers of bacteria frequently found in milk before it leaves the farm is from unclean milk utensils. This is due partly to the carelessness of the farmer or his wife, and partly also to the actual difficulty of the task. It is fairly easy to wash milk pails clean; but to get them bacteriologically clean is quite difficult, and as we have already seen, the organisms not removed by the washing multiply unless the cans are thoroughly dried. Cans in which milk is to be shipped to market are some- times in a frightful condition when the milk is put in them. Some- times they have merely been rinsed in cold water and have very strong odors of decomposition when the milk is poured into them. Under such conditions, no care that the dairyman can exercise will enable him to furnish milk to the neighboring city without large numbers of bacteria in it. Sometimes the dairyman and sometimes the milk company that receives the milk is responsible for these conditions; and the carelessness is not uncommonly due to the fact that the farmer and the milk dealer each relies on the other to do the washing and sterilizing of the cans. When milking machines are used the danger of this kind of con- tamination is greatly multiplied. We have seen that considerable intelligence is necessary to produce clean milk with a machine. It often happens, indeed, that when a large part of the milk supply comes from dairies with milking machines, the only way to improve the quality of the supply is to give the dairymen painstaking instruc- tion in the care of the machine. Their extensive use at present is bringing about many special problems that are perplexing our boards of health and sanitary authorities. Cooling the milk. When drawn from the cow, milk is at a tempera- ture at which bacteria grow with the greatest possible rapidity. If, however, milk can be cooled at once to a temperature down close to freezing, it may be kept for very many hours, and indeed for several days, with no appreciable increase in the number of bacteria. The realization of this fact has slowly been growing on the part of all who handle milk. It has resulted in many devices for cooling the milk promptly on the farm and it has also resulted in the modifica- tion of the methods of transportation. The refrigerator milk car, which is now used almost universally in certain sections of the northern part of our country, is the result of the same realization. Unfortunately, even up to the present time, there are many sections MARKET PROBLEMS 137 of the country that have not adopted the cooling system in trans- portation, but depend upon rushing the milk promptly into the city, so as to deliver it to the customer within a few hours from the time it is milked. Under these circumstances, the bacteria multiply with great rapidity, and the result, so far as concerns number of bacteria, is surprisingly unsatisfactory. Years of examinations in the larger cities (such as Washington, D. C.) which thus receive their milk without cooling have shown that the average bacterial content of the milk is far over a million, and in warm weather samples of milk distributed to the consumer containing fifty millions of bacteria (as determined by plate count) are fairly common. It will, of course, be evident that the distance over which the milk has to be transported before it is delivered to the customer will have a large bearing upon the amount of multiplication. The milk supply of our communities, indeed, may be roughly grouped into two classes, that with long hauls and that with short hauls. By short haul milk is meant that which is produced so close to the customer that it can be carried promptly into the city and delivered within a few hours of the time it is milked from the cow, morning's milk being delivered in the forenoon. By long haul we mean the condition in larger cities whose milk supply comes from a greater distance so that it has to be transported by railroads, requiring many hours for the journey. In such cases the milk is rarely less than twenty-four hours old when it is delivered to the con- sumer, and more commonly it may be thirty-six to forty-eight hours old, and not infrequently older still. Under these circum- stances, of course, the problem of temperature plays an extremely great part in the keeping quality of the milk and the number of bacteria it contains. Actual study of the condition of the milk m our larger cities, however, shows that those cities what are supplied with milk by the long haul method are frequently better served as to freshness of their milk, measured by its bacterial content, than cities obtaining it by the short haul method. This is because re- frigeration in transportation is generally employed in the former case. When the haul is a short one, the dairyman does not appre- ciate the need of cooling and is likely to rush the milk into the city Quickly without cooling. Under these conditions, the warmth of the milk as drawn from the cow keeps it for some time in just the condition for rapid growth of the bacteria, so that when delivered a 138 BACTERIOLOGY few hours later the milk is actually in a worse condition than the cooled milk sent to the larger city which does not reach its destination until forty-eight hours old. Three distinct methods have been adopted to control the quality of milk for the benefit of the public: 1. Farm and dairy inspection. This is an attempt to improve the quality of the milk by inspection of the sources from which it is obtained, and insuring good conditions in the dairy and in the methods of handling the milk. This method was taken up first by some large dairy firms in order to control their product, and has come to be quite widely adopted by cities for the purpose of protecting the public. In order to make it as efficient as possible, dairy score cards have been devised, so arranged as to give a numerical value to each of the factors noticed in the dairy inspection, the total amounting to one hundred, so that each dairy can be distinctly graded on a percentage scale. A number of these score cards have been devised differing from each other in various details, but all having the same general purpose. The value of such dairy inspection can hardly be questioned, but the score cards have certain limitations that decidedly affect their usefulness. Inspection, in the first place, is very expensive, for it costs a great deal to keep a corps of inspectors composed of men trained to do their work satisfactorily and to have enough inspectors to make frequent visits to the dairies possible. Experience has shown that a single visit of a dairy inspector has very little influence upon the condition of a dairy, inasmuch as the dairyman is very likely to go back to his old methods immediately after the inspection, paying no attention to the advice he has been given. To be really efficient, the inspector should have frequent enough contact with the dairy to give thorough instruction as to proper methods. Fur- thermore the inspector must be a man who has had sufficient practi- cal experience in dairying to inspire confidence in his advice. The dairy score card, too, while pointing out certain factors that the dairyman should give especial attention to, does not give much indication as to the quality of the milk produced in any dairy; for, judged by chemical and bacteriological tests, a dairy having a high score is just about as likely to produce poor milk as one having a low CONTROLLING THE QUALITY OF MARKET MILK MARKET PROBLEMS 139 score; The limitations thus set to the efficiency of dairy inspection is, however, no argument against inspection, but only indicates that this method can not be relied upon as sufficient to control the market milk supply. 2. Legal standards, accompanied by chemical and bacteriological analysis of the milk. Chemical standards for market milk have been recognized for a long time, and are found on the statute books of most states. The possibility of a bacteriological standard as a method of controlling sanitary factors and cleanliness has only recently been recognized. Laboratories for the bacteriological testing of market milk, however, have been multiplying rapidly in recent years, and each year sees a larger number of our cities recog- nizing a bacteriological standard in some form. In some cases a limit is set as to the number of bacteria that are permissible in milk to be sold in the market. In other cases a bacteriological count is used only as a method of getting a general notion of the character of the various dairies, as part of a more careful survey of the sanitary conditions under which the milk is produced. Bacteriological standards have very great utility in controlling the market milk supply, but they too have their limitations. When properly used, they will give a very emphatic statement, for reasons already indi- cated, concerning the cleanliness and freshness of the milk samples, but no bacteriological analysis would enable one to state positively that a given sample of milk is or is not suitable for consumption. Wholesomeness and safety are dependent on other factors besides bacterial numbers. Unfortunately, up to the present time, no one has devised any analytical method to detect disease germs in milk, even in cases when such disease germs are positively known to be present. Such analyses, therefore, can only be used to indicate the general nature of some particular milk supply, and not for approving °r condemning any samples of milk that are on the market. The difficulty of enforcing a legal standard is great, and especially great in the case of bacterial standards. While bacterial standards are proving more and more useful each year and are more and more widely used as a method of determining the freshness and cleanliness of market milk, they have not proved adequate to solve completely the problem of putting a safe milk on the market. 3. The grading of milk. Milk is almost the only food commonly sold in the market without being graded according to quality. In 140 BACTERIOLOGY past years it has been customary in nearly all communities to have just one grade of milk, selling always at the same price, and, provided it comes within the limits of certain relatively low chemical standards the milk of poor quality receives the same price as that of higher qual- ities. The practical result of this has been to force out of the market all milks except those that just barely conform to the legal standard. For a number of years now both the public and the dairymen have been recognizing this condition as unsatisfactory, and there have been more or less tentative efforts to grade the market milk. Certain dairymen, wishing to appeal to a high class custom, advertise milk of a special quality upon which they put particular names, and not a few milk dealers have developed a somewhat extensive and lucrative business in selling a high grade milk at a high price. The most general movement of this kind has been the production of "certified milk." This is a grade of milk the whole- some nature of which is certified by a commission largely composed of medical men who have looked into the conditions surrounding its production and its distribution, have made chemical and bacterio- logical analyses of the milk produced by individual dairymen, and then have given to such dairymen the privilege of putting the label "certified" upon their milk for a given period of time. The value of such a label on a milk bottle depends upon the thoroughness with which the inspection has been made by the certifying board, and this varies in efficiency in different communities. It must always be remembered that no individual sample of milk can be certified, but that the certifying board can only guarantee that the conditions under which the milk was produced are of a high character which they can endorse. There is no doubt that in general such certified milk is superior to the general market product from the standpoint of wholesomeness and that the plan of furnishing certified milk to the market has been one of the means of notably improving the average character of this food product. Certified milk, however, always demands a very high price, for the safeguards which the certifying board insists upon are very expensive, making it impossible to sell the product at a low price. Even at the high prices such milk commands, its production is not a very profitable undertaking, because of the comparatively limited demand, the consequent high cost of delivery, as well as the excessive cost of production. For these reasons certified milk has never been produced or sold in very large quantities and has hardly affected the general market problem. MARKET PROBLEMS 141 More recently a plan for grading all milk sold in any community has been suggested, and it is being somewhat rapidly adopted in various localities. In this grading it has been recognized that there are three general grades of milk desired by the public: first, a very high grade suited for the use of babies, invalids, and others who are satisfied with nothing short of the best; second, a milk for general table use, safe and satisfactory enough for healthy adults to drink, although not sufficiently safeguarded to be recommended for babies or invalids; and third, a milk for cooking purposes which may be perfectly satisfactory although on the point of souring or even unsafe to drink raw. Of these three grades, the demand for the second is the largest. It has been proposed to recognize these three grades Under the names A, B, and C. Where a grading system of selling milk is in practice, it is necessary to have the grading done by public officials, and not by private dairies or milk companies, on account of the danger of dishonest labeling. The grades must be controlled by public inspection. Just What shall constitute the limits of milk to be sold in each of these three grades is not, and probably cannot be, specificially stated. Where the grading system has been introduced., the grading has been based partly upon the conditions of the dairy which produces the milk and hence upon the score card rating, and partly upon the chemical and bacteriological analysis of the milk at the time it reaches the consumer. Certain minimum requirements are always Necessary in order that a dairy may sell milk with the grade A, B, °r C on the label. Where such a grading system is in force, there is a tendency to require pasteurization of the milk (i.e., partial sterili- zation; see p. 150) for all except grade A milk, on the ground that this is the only method of protecting the public from possible danger to health. This plan of grading the public milk supply is rapidly coming into Use. Provided the standards for the different grades are properly selected and some method of inspection is adopted to see that all milk is correctly labeled, this plan not only enables the public to hnow the quality of the milk it is purchasing but also enables the dairyman that produces a good product to obtain a better price for Attempts to enforce a legal standard by prosecuting dairymen Whose milk does not come up to that standard have always been difficult, expensive and commonly futile. Experience has shown 142 BACTERIOLOGY that a very much greater success in improving the product is ob- tained by putting the stimulus in the form of an extra price for milk with a higher quality. The fundamental key to the successful application of a grading system is the bacterial analysis of the milk, for this is the only test by which the analyst at the city end of the line can get any idea as to the character of the milk, of the methods by which it has been handled, its general cleanliness or freshness. A grading system, therefore becomes efficient just in so far as the bacteriological analysis of milk can be applied. The dependence of milk standards upon bacterial analysis makes it very important that there be satisfactory methods of counting the bacteria in milk. The ordinary method by which this is done is by means of the plate technic already described (p. 54). It has long been recognized by bacteriologists that this technic does not give a count of all the bacteria for the following reasons: bacteria occur in milk in groups of two or more and at times even in large clumps that do not break apart during the plating process, giving rise each to but a single colony on the plates; no medium has yet been found upon which all milk bacteria can grow; the rapid growth of some colonies on the plates often prevents the development of other bacteria less adapted to the conditions; in ordinary analyses, plates are counted after about two days incubation, which does not allow the development of the more slowly growing bacteria. With so many sources of error it is natural that results vary in the hands of different workers.1 Extensive experiments indeed, in which the same sample of milk has been sent to different laboratories and plated by many different workers, have shown variations at times of several hundred per cent. It is plain therefore that the plate count is at best a very rough approximation and never represents the entire bacterial content of the milk. In spite of this fact, there is an un- fortunate tendency upon the part of health officials and even bacteriol- ogists themselves to say categorically that such and such a milk contains so many bacteria per cubic centimeter. Such definite BACTERIAL ANALYSIS OF MILK 1 To avoid some of these errors official methods for making plate counts have been established. By securing uniformity in technic, this tends to make the results of different laboratories comparable. Even with the official methods, however, results vary greatly in different hands. MARKET PROBLEMS 143 statements based upon an imperfect technic have led to many- misconceptions. Such statements are particularly unfortunate because there is now a technic for counting more nearly the total number of bacteria in milk. This technic is as follows: With a special capillary pipette, 0.01 cc. of milk is measured out and placed on a microscopic slide. It is then spread over an area of exactly 1 sq. cm. with the use of guide lines placed beneath the slide. It is then dried on the slide, the fat removed by immersion in xylol, the smear fixed to the slide with alcohol, stained with methylen blue, and examined under a microscope. The microscope used should have a field of measured size so that it is possible to compute the number of bacteria per cubic centimeter of the milk from the number seen with the microscope. This technic, to be sure, does not give absolutely accurate counts, and indeed gives considerable variation when determinations are made separately by different men upon the same sample of milk; but it does give a more nearly correct idea of the total number of bacteria present than does the plate method. Each individual or- ganism seen can be counted, even though in a clump or unable to grow on the plates, so the microscopic counts are almost invariably higher than the plate counts. Presumably the higher counts are the more correct ones, although there is a possibility that the micro- scope causes some dead bacteria to be included with the living. All evidence, however, indicates that bacteria quickly lose their staining Powers after death; so the microscopic count is not likely to be much affected by this source of error, except in the case of recently pas- teurized milk. The microscope does not furnish accurate counts of Hulk under a 10,000 plate count (i.e., such milk as can be sold as certified"), but it is adapted to making the distinction between the grades A, B, and C of the ordinary supply. It is particularly well adapted to the examination of milk as delivered at the pasteurizing Plant; and on account of the rapidity with which samples may be examined under the microscope,2 it is possible to make a report upon the milk of a large number of different dairymen within a few hours 2 This rapidity is possible only by making rough estimates. Exact counts Under the microscope are very time-consuming on account of the number of fields that must be examined. A glance at a few fields, however, generally suffices to show in which grade to class the milk, provided only the three grades of ordinary market milk are considered. 144 BACTERIOLOGY of the time the milk is delivered. It has been found possible to make the examination quick enough under some circumstances to return an undesirable can of milk to the dairyman before he leaves the milk receiving station. Although this rapidity in securing results allows a more efficient control of milk supplies, the micro- scopic method will probably never entirely supplant the plate method. As these two methods of counting bacteria are in use, any statement as to the numbers of bacteria in a sample of milk must be accepted critically until it is known whether the count is based on the use of culture plate or microscope. REFERENCES Standard Methods for the Bacteriological Examination of Milk. 4th edition. Published by the Amer. Public Health Assn., New York, 1923. R. S. Breed and J. D. Brew. Counting bacteria by means of the microscope. N. Y. Agric. Exper. Station, Tech. Bui. 49. 1916. CHAPTER HI The Relation of Milk Bacteria to Public Health SANITARY SIGNIFICANCE OF BACTERIA IN MILK The question as to whether the bacteria in milk have any distinct relation to public health is rather a complicated one; when we re- member that the mouth, stomach and intestines of all people are filled with bacteria in predigious numbers, it becomes evident that the comparatively few bacteria introduced by drinking milk may not have any significance at all upon the health of the consumer. On the other hand, it is perfectly well known that under some circum- stances, the bacteria in milk are unwholesome and even deadly. There are two phases of the subject that may be properly considered. 1. Injury to the milk as a food As the bacteria in milk multiply, they cause the decomposition of the various ingredients, so that in the end the milk would be de- stroyed as a food by being completely decomposed. The first un- desirable change is the common souring of milk, which means that the lactose is being converted into lactic acid. Further changes in the milk ingredients such as the peptonization of the casein, decom- position of the fat, and finally complete putrefaction, are the later results of the unlimited growth of bacteria in milk. It is generally recognized that decomposed food is not a satisfactory material to Use, and cases of milk or food poisoning well testify to this fact; but at what point the decomposition of milk has progressed far enough to affect materially the value of the milk it is impossible to Say. All we can say is that when but a few thousand bacteria are to be found per cubic centimeter, the decomposition is so slight as to be negligible; but when many millions are found it must be re- garded with some suspicion. We must also recognize that the pres- ence of lactic acid or of the lactic acid bacteria in milk is in no sense mjurious, and that sour milk, with its billions of bacteria per cubic centimeter is for some purposes a very valuable food. Market containing unusually large numbers of lactic acid bacteria is to 146 BACTERIOLOGY be condemned merely because the public desires sweet milk for drinking purposes, and the presence of these bacteria will shortly cause it to become sour. 2. Diseases distributed by milk Abundant evidence is now at hand indicating that certain micro- organisms in milk may be the source of specific disease. This is so well established that some scientists have even declared that milk, when consumed in the raw state, is the most dangerous of all food products. The specific diseases known to be distributed through milk are: Tuberculosis. The milk of cows infected with tuberculosis, espe- cially if the disease is located in the udder, is known to contain tubercle bacilli in considerable numbers and in an active condition. While not a very large number of cattle have tubercular udders, the number is sufficient to be a possible source of danger, and it is a demonstrated fact that 10 per cent or more of market milk does con- tain active, virulent, tuberculosis germs. It is known, furthermore, by experiment that such milk can produce the disease in small animals. Presumably it is a source of danger to children consuming the milk, for the presence of bovine tuberculosis in considerable numbers of children fed on cow's milk has been demonstrated. The danger, apparently is confined to a large extent, if not wholly, to children; for there is little evidence as yet of adults contracting the disease from milk. The extent of this danger cannot be stated in figures, but it seems perhaps to be safe to say that about a quarter of the cases of tuberculosis among children are contracted from milk of tuberculous cattle. Typhoid fever. Numerous epidemics of typhoid fever have now been definitely traced to the milk supply. Inasmuch as cows do not have typhoid fever, the contamination of the milk with typhoid bacilli must be a secondary one, and not primary as in the case of tuber- culosis. On the other hand, while the tubercle organism does not multiply in milk, the typhoid bacillus is well adapted to growth in it, and if a few contaminating organisms get into a milk supply they may multiply rapidly before the time of distribution, causing a widespread infection from a small original contamination. The methods by which milk becomes contaminated with typhoid bacilli are somewhat varied, but they are apparently of three general sorts: RELATION OF MILK BACTERIA TO PUBLIC HEALTH 147 (1) by the use of contaminated water in the washing or rinsing of milk utensils; (2) by the employment in the dairy of persons who have some contact with typhoid patients; (3) by the employment in the dairy of typhoid bacillus carriers. The last of these three sources has in recent years attracted considerable attention. It is definitely known that after a person has recovered from an attack of typhoid fever he may carry the typhoid bacilli with him in his body for a varying length of time. In some cases they disappear in the course of a few weeks, in other cases they may remain months and occa- sionally even years. A chronic bacillus carrier, as such a person is called, is constantly shedding typhoid fever germs, and his employ- ment in a dairy is a decided source of menace to the public. In tracing typhoid epidemics due to milk, in recent years, this has appeared to be one of the most prominent methods by which the dairy becomes infected. Unfortunately it is one extremely difficult to guard against, for typhoid carriers are not easy to detect. The employment of a single such carrier in a dairy will be a constant source of danger to the public, and the difficulty of detecting such carriers has made it quite impractical hitherto to prevent them from handling the milk. As a consequence, the public is in constant danger of typhoid fever from this source, and there seems to be no method of protection except pasteurization of milk, which practice has been extended rapidly in recent years. Scarlet fever. The cause of this contagious disease is not as yet definitely known, but we do know that occasionally it is distributed through the milk supply. Apparently in these cases the contamina- tion of the milk is a secondary one: that is, the milk gets its contamina- tion not from the cow but from someone handling the milk. While scarlet fever epidemics distributed in this way have not been com- mon, enough instances have been observed to indicate that this is °ne source of danger arising from the use of raw milk. Diphtheria. The definite cases in which diphtheria has been distributed by milk have been rather few, and while certain examples seem to be beyond question, the facts in our possession at present mdicate that milk is one of the smaller means by which this disease !S distributed. It is always regarded, however, as one of the possible sources of danger from the milk supply. Septic sore throat. In recent years septic sore throat has come to be regarded as one of the more important diseases distributed by 148 BACTERIOLOGY milk. Although quite a serious disease, it has been recognized only within a comparatively few years, and in an epidemical form has but very recently attracted the attention of public health authorities. It is characterized by the presence of a somewhat peculiar long- chained streptococcus, growing in the throat of the persons infected. In many respects the disease is closely related to scarlet fever, and indeed is regarded by some as merely a different manifestation of this well-known disease. Several epidemics of this disease have now been studied and the distribution of the cases suggests that milk is the medium through which they have been spread. In certain cases, indeed, suspicion has pointed to certain cows with strepto- coccus infection of the udders, a fact which adds new significance to the presence in milk of long-chained streptococci derived from the udder. There seems to be no practical method of excluding such occasional infections from the milk, for with present methods it is almost impossible to guard against the possibility of some animal with occult udder infection furnishing dangerous milk for public consumption, and no ordinary medical inspection would be sufficient to keep from the dairy persons suffering from the disease. Here again, pasteurization is the one really effective protection for the public. Diarrhoeal troubles. As is well known, much illness and many deaths among children, during summer weather especially, are due to intestinal troubles, most of which are characterized by diarrhoea. These troubles do not appear to be caused by any specific disease, but show considerable variety. It has not been possible to trace them to any specific bacteria, but the general feeling at the present time is that the large numbers of bacteria in summer milk are re- sponsible for them. Experience has shown that they are very largely prevented if the milk is properly pasteurized. At the present time we are inclined to look upon them not as infectious disease but as a form of poisoning due to the ingestion of certain decomposition products from decaying foods. If this is the case they are naturally the most common in summer when the food is most likely to undergo bacterial decomposition, and it is also evident that milk is one of the most likely sources of such troubles, inasmuch as it is a large part of the diet of children and is almost sure to undergo decomposi- tion, in hot weather, from bacterial growth. Data have been col- lected, in fact, showing that the summer illness and deaths among RELATION OF MILK BACTERIA TO PUBLIC HEALTH 149 children are more common among families living upon milk of high bacterial content than among those consuming fresh milk with few bacteria. Many methods more or less successful have been devised for pro- tecting milk from the action of bacteria, either to destroy the bacteria in the milk or to prevent their growth. Some of these are chemical, and consist in the addition of more or less harmless chemicals to the milk. Borax, formalin, and hydrogen peroxide are the most com- monly used chemicals. Of these formalin has been used the most widely, although it is forbidden by public statutes. Hydrogen peroxide has the advantage of rapidly destroying the bacteria in the milk and then itself being converted promptly into ordinary water and thus becoming harmless. All of these methods of disinfecting milk are at present illegal, however. Other methods of producing the same result are the treatment of milk by physical means. Electricity and ultra-violet light have been applied for this purpose, and although considerable has been claimed for them they have not as yet proved to be practical. There is a chance that the future may bring some practical results from this type of treatment, but at present it is merely in an experi- mental stage. The most widely used physical agent for this purpose is heat, and this has been employed even in days preceding bacteriological knowl- edge. One method of employing heat is complete sterilization. Inasmuch, however, as milk is almost sure to contain certain resistant bacterial spores, it requires a heat greater than that of boiling water produce perfect sterilization, which can be accomplished only by beating the milk under pressure in specially devised apparatus. Simple boiling of the milk has been frequently adopted, and while this does not heat it sufficiently to destroy all spores, it does destroy dangerous organisms and protects the milk for some time from the deleterious action of bacteria. This process, also, is sometimes Called sterilizing the milk, although it is not a complete sterilization. ■Against both of these methods of heating milk serious objections have been raised, on the ground that they lower the food value of the milk. The application of such high heat produces profound changes in the nature of the milk. It coagulates the albumen, PROTECTION OF MILK AGAINST BACTERIA 150 BACTERIOLOGY destroys the enzymes, partly caramelizes the sugar, and apparently also has a deleterious action on the so-called "vitamines," the active principles of diet which are not well understood but seem to be ab- solutely necessary elements in nutrition. These changes in the nature of the milk appear to be sufficient to affect its value notice- ably as a food. Children brought up upon boiled milk as a sole article of diet do not thrive, and manifestly suffer from malnutrition. While, therefore, boiling milk is a process that is still somewhat common in special emergencies, it is not a process that is recommended anywhere as a method of treating the public milk supply. Pasteurization. Pasteur first suggested the use of a moderate amount of heat for the protection of wines against troubles that were likely to occur during its fermentation; and this method, under the name of pasteurization, has come to be of very wide application in the milk industry. The essential feature of the process consists of heating the milk to a temperature high enough to destroy the disease germs, but not high enough to produce the changes in milk above mentioned, which are thought to be responsible for the de- creased nutritive value of the sterilized milk. It has taken many years to determine the temperature which is most satisfactory for this purpose. When first applied to milk, the custom was to heat the milk to about 195°F. (80°C.), to leave it at this temperature for a few minutes, and then to cool it rapidly. This temperature is widely adopted in European countries, and was used in this country at the outset. Accumulating experience, however, soon showed that this temperature does not avoid all undesirable chemical changes. A taste characteristic of boiled milk is developed and such milk is not satisfactory as a sole article of diet for children. Used alone, such milk is believed to produce a mild form of scurvy and some other diseases in children. Experiments have been made therefore to learn whether the temperature can be reduced and still kill the pathogenic organisms that might be present. It proves that the agents of diphtheria, scarlet fever, and infectious sore throat are comparatively easily killed by a moderate heat. The tubercle bacillus, on the other hand, does not succumb quite so easily, and of course the spore-producing pathogenic organisms are not killed by any method of pasteurization. This last factor, however, does not need to be taken into consideration, because no spore-producing pathogens are known to be distributed by milk. RELATION OF MILK BACTERIA TO PUBLIC HEALTH 151 The tendency in recent years has been to use the temperature of 145°F. (62°C.), maintaining this temperature about half an hour. This proves sufficient for destroying all the pathogenic organisms of milk with the possible exception of the tubercle organism. Some experiments have seemed to indicate that milk thus heated may still contain living tubercle bacilli, especially if heated in a quiet condition so that a scum forms on the surface; but with the methods ordinarily used the organism seems to be either killed at this tem- perature or so greatly reduced in virulence that milk thus treated is no longer suspected of carrying tuberculosis. Up to the present time no case of tuberculosis has been attributed to milk properly pasteurized at this temperature; and the method is considered to render the milk entirely safe. This temperature is so low that the chemical changes above mentioned do not occur in the milk; and so far as concerns taste or nutritive value, the milk is left in practically the same condition as before pasteurization. There has been and still remains in places a prejudice against the pasteurization of milk. This is partly traceable to the fact that as originally used pasteurization heated the milk to temperatures which lessen its nutritive value, and partly to the feeling that pasteuriza- tion may be used as a substitute for cleanliness in the dairy, dairymen Perhaps taking less pains to keep the milk clean when they know is to be pasteurized before consumption. Furthermore, pasteuri- zation has often been found to be carelessly and inefficiently done, so that a false sense of security is obtained when the milk may stiH be dangerous to drink. The answers to these objections are as follows: It is still uncertain whether pasteurization at low temperatures seriously injures the "vitamine" content of the milk; but if it does, it applies only in the case of young children fed on milk as a sole article of diet, and its deficiency can be overcome by the use of vegetable or fruit juices containing the lacking elements. The tendency toward carelessness m the dairy can be prevented by the plan of grading milk and re- quiring milk for pasteurization to meet a certain bacterial standard before as well as after pasteurizing. The unreliability of commercial Pasteurization can be remedied by official inspection or by requiring a bacterial standard for the pasteurized milk. In recent years the prejudice has been decreasing and the practice °f pasteurization has been rapidly extending. The fundamental 152 BACTERIOLOGY reason is the growing realization that in no other way can the public be protected against the infectious 'diseases distributed by milk. Experience has shown that no amount of care, official or private, given to the conditions in the dairy can insure the milk against an occasional tuberculous cow in the herd, an obscure case of udder trouble, or the employment in the dairy of an unsuspected typhoid bacillus carrier. Epidemics have been traced to such sources in the highest class of dairies where the greatest care is taken to protect the milk. Certified milk, though surely protected more than other milk, is not free from these dangers; and the managers of certified dairies feel that any day some infectious disease, against which they have no adequate protection, may be proved to have come from their milk. Under these conditions, while the total percentage .of infec- tious diseases attributable to milk is not a large one, the feeling is rapidly growing that the public should be protected from this danger when it can be done so simply as by pasteurization. When we further bear in mind that the United States is the only country consuming much of its milk raw, it seems manifest that the practice of pasteurization is likely to become well nigh universal for the public milk supplies of this country. REFERENCES L. A. Klein. Principles and Practice of Milk Hygiene. Lippincott, Phila- delphia, 1917. H. N. Parker. City Milk Supply. McGraw-Hill, New York, 1917. J. Race. The Examination of Milk for Public Health Purposes. Wiley & Sons, New York, 1918. CHAPTER IV Manufactured Milk Products The problems connected with the manufacture of ice cream are primarily physical and chemical and need not concern us here. Bacteria play no part in the process and the only concern the manu- facturer has in regard to them is to keep them from souring his cream before freezing. As the cream is sometimes stored for a while before freezing, it often has quite a high bacterial content when it is used, even though not yet sour, and freezing does not kill the bacteria. Ice cream is sometimes kept in cold storage for a con- siderable time before selling, and until recently it was supposed that the bacteria it contained would die out or certainly not grow during this period. Apparently they do decrease in numbers for a while; but recent experiments indicate that after a month or more of storage there is a marked increase in bacterial content. This is a little surprising, although there is other evidence that bacteria can multiply at the temperature of freezing water (see p. 173). Whether the bacteria in ice cream do any harm is still unknown. If wholly of the lactic acid type on account of the age of the cream before freezing, they probably do no harm provided the cream was still sweet when frozen. But the lactic acid organisms do not grow at low temperatures and the bacteria developing in stored ice cream are of other types. Possibly they may produce harmful decomposi- tion products. Cases of ice cream poisoning are not unknown; but whether they are due to the growth of bacteria in the ice cream 18 still an unanswered question. On general principles an ice cream With a reasonably low bacterial content seems most desirable; but beyond doubt, much of the ice cream consumed by the public with- °ut any ill effects contains enormous numbers of bacteria. ICE CREAM BUTTER Origin of cream ripening. Cream is rarely churned while it is fresh, but is allowed to remain for a length of time at a moderate temperature, to undergo a process known as ripening. The origin 154 BACTERIOLOGY of the practice of ripening is doubtless the fact that as butter was made originally on the farm, where the dairies in many cases had only a few cows, it was necessary to keep the milk two or three days or even more before a sufficient amount of cream would accumulate for a churning. During this process the bacteria that were in the cream would inevitably grow and produce the phenomenon which became known as ripening or souring. When, later, butter began to be made in creameries, where large amounts of cream were deliv- ered each day, the necessity for keeping the cream did not occur. But experience soon showed that the butter made in these creameries had a somewhat different quality from that made upon the farm, and in order to get a product equally acceptable to the public, it was necessary to allow it to ripen in the creamery, although not necessarily to the extent usually practiced on the farm. Gradually as experience accumulated, the adoption of a period of ripening became practically universal in the creameries, so that at the pres- ent time nearly all butter is obtained from cream that has been kept warm for several hours at least to undergo the changes called ripening. Purposes of ripening. Although at the outset the effect of the ripening of the cream upon the quality of the butter was not under- stood, the purposes of cream ripening are now fairly well known. During this ripening, bacteria are multiplying rapidly in the cream, producing important chemical changes in it. The milk sugar is partly converted into lactic acid, so that the cream becomes some- what sour, and certain other decomposition changes are being brought about by other bacteria that grow along with the lactic acid organism. When subsequently churned, it is found that the churning is easier than it would be in fresh cream, that a somewhat larger yield of butter is obtained, but most important of all, the decomposition products that have arisen during the ripening add certain flavors and aromas to the butter, which give it its special character. Control of ripening. Experience and experiment alike have shown three facts: (1) The character of the butter, and therefore its market value, is quite variable with different types of cream ripening. The value of butter in the market changes about 33 per cent according to the nature of the ripening that takes place in the cream. (2) Cream, as ordinarily collected, contains very many different kinds MANUFACTURED MILK PRODUCTS 155 of bacteria, some samples having a few and some many species. (3) The nature of the ripening is very different in accordance with the bacteria that produce this change. Some species of bacteria produce good-flavored and others bad-flavored butter. It is evi- dently desirable that the ripening of the cream should be carried on by species of bacteria giving favorable results. The process as formerly carried out, however, made no attempt to control the nature of the ripening except as to controlling temperature. If the cream was kept at a temperature of about 65°F. it was found that favorable results could generally be obtained, due to the stimulation of the favorable organisms and the repression of the others. But even at the best, the method of ripening was a haphazard one, which fre- quently resulted in undesirable flavors and tastes. Early in the study of dairy bacteriology it became evident, there- fore, that some method of controlling the process was to be desired, for the sake of uniformity. The great revolution that had been produced in brewing by the application of pure yeast cultures was fully understood, and the attempt was made to apply the same general principles to cream ripening. For this purpose, several bacteriologists began the study of the types of bacteria in ripening cream, and having found the species producing favorable results, began plans for utilizing pure cultures of such organisms in the control of cream ripening. These cultures were called "starters" and their use has become well-nigh universal in all the higher class creameries. The starters that have come into general use have been of two different types. First there are the artificial starters, or commercial cultures, that is, cultures of bacteria prepared in labora- tories for this special purpose. Sometimes these are pure cultures of a single species, sometimes they are a mixture of more than one species; but always they consist of active cultures of organisms shown by experience to produce a favorable type of ripening. In practi- cally all cases the important organisms in these starters is the or- dinary lactic acid type. The cultures are prepared in commercial laboratories and placed on the market under various names. The other type of starter is known as the natural starter, prepared by the dairyman himself. It is nothing more than naturally soured oadk, prepared with special precautions. For the reasons already shown, milk kept at a temperature from 60 to 70°F., will after a few days show a very great predominance of the lactic acid type, if not 156 BACTERIOLOGY a pure culture of this organism. If this type of organism is present alone or almost alone the milk will become a sour smooth curd, without gas bubbles, and the experienced dairyman can tell from the general appearance and taste of the milk whether it has been soured by the normal type of bacteria or by some of the abnormal forms. If he finds that such a milk shows the proper type of curd, he uses it as a starter for producing a normal cream ripening in butter making. Both of these types of starters are extensively used by butter makers, the former being more reliable, but somewhat expensive, the latter being less reliable, but practically without expense to the butter maker. In the use of such starters, also, two different methods are em- ployed. When first recommended, a quarter century ago, pasteuriza- tion of the cream was considered necessary before adding the starter. This was to get rid of the bacteria already present and thus to give a free field for the growth of those inoculated in the starter. This requirement of pasteurization very decidedly limited the use of starters, because in most butter making communities proper machin- ery and facilities for pasteurizing were lacking. Experiments were therefore made to determine whether the inoculation of such a starter into cream without pasteurization might not produce equally useful results. While it seemed theoretically to be necessary to get rid of the bacteria already present in the cream, experience has shown that the lactic acid bacteria grow so much more rapidly in milk than any other species, as to overcome the effects of all other types present in the milk to which the starter is added. Hence their use in unpasteurized cream is about as efficient as after pasteurization. The discovery of this fact has made the use of starters practical, and has resulted in a very rapid extension of this method of con- trolling cream ripening. It is now nearly universal in the civilized world. Within very recent years the use of pasteurization has been extend- ing in this country, for a different reason. Just as there is a growing demand for pasteurized milk to protect the public from possible danger so there is a growing demand for the pasteurization of cream. After such pasteurization it is absolutely necessary to use a bacterial culture to start the ripening, for pasteurized cream does not ripen spontaneously. It is uncertain whether the demand for the pas- teurization of cream used in butter making will grow or not. MANUFACTURED MILK PRODUCTS 157 The use of artificial cultures in the ripening of cream is the butter manufacturer's chief defense against "butter faults." This term is used to apply to a variety of troubles sometimes occurring in butter, producing undesirable flavors. The butter-maker has found that by the use of artificial cultures, or even by a natural starter, he can to a large measure prevent these faults. If the cream reaches the creamery in a moderately fresh condition, the use of a starter is an entire preventive of such faults, and even though the cream be somewhat old, the addition of a starter so modifies the bacterial activities in it that fairly good butter may be obtained from cream otherwise likely to yield a very poor product. In practice this has been one of the large causes leading to the extensive use of butter starters. Application to butter substitutes. The utilization of this power of bacteria to produce flavors of butter has been applied to the manu- facture of other market products resenjbling butter. In the manu- facture of oleomargarine, various fats are mixed together in a melted condition, and subsequently cooled rapidly, thus becoming of the consistency of butter. The product when colored by a yellow color- mg matter (generally anatto) has a great similarity to butter in appearance but not in taste. The manufacturer has found it neces- sary, therefore, to introduce into this artificial mixture some flavors resembling those of true butter. For this purpose he ripens a lot °f cream or milk, sometimes naturally or more commonly by the addition of artificial cultures, then adds a certain proportion of this soured milk or cream to the product, thus impregnating the mixture of oils with the flavors developed during the ripening. By this oieans a product so closely resembling butter is sometimes obtained that it is difficult to distinguish it from true butter. In the manufacture of what is known as "process butter" or "ren- ovated butter," the same general plan is adopted. These are made from old butter that has become rancid and spoiled for table use. The old butter is melted and washed, the dirt and any soluble prod- ucts washed out of it, so that clean, and rather pure butter-fat is obtained. This is then mixed with ripened cream or ripened milk 111 the same way as in the case of oleo manufacture, and the result is a product strongly resembling fresh butter in taste and in appearance and indeed, in its chemical nature. 158 BACTERIOLOGY CHEESE The ripening of cheese. The value of cheese is even more inti- mately associated with its flavor than is the value of butter. While cheese is a most valuable food from the standpoint of nutrition, the primary reason for its popularity and a large part of the reason for its value in our diet is due to its flavor. Flavor, it is true, has no food value; but a proper flavor is so necessary for controlling the secretion of digestive juices and the proper digestion of foods that properly flavored foods have a decided physiological importance. The characteristic flavors develop in the cheese during the process of ripening. Cheese consists of a curd of milk, separated from the liquid part, as a rule, by the action of rennet, being subsequently drained or even pressed to exclude a large part of the water. The solid curd thus obtained is shaped into one form or another according to the variety of cheese being manufactured. This partly dried and shaped curd is the green cheese. The green cheese is set aside for a certain period of time at some particular temperature for a change known as ripening. During the ripening the cheese very greatly changes in nature, chemically as well as in appearance and taste. Curdled casein undergoes chemical changes rendering it more or less soluble, and far more easily digested. As a result of these changes, it becomes softer, in some cases ("soft cheeses") being converted into a semi- liquid, creamy mass. While this change in texture is taking place, the flavors develop which are characteristic of the ripened product. It is impossible at the present time to write the whole story of the ripening of cheese. The phenomenon, indeed, is an extremely varied one. There are some hundreds of different kinds of cheeses on the market, each with its own special history in ripening. Each of these cheeses is therefore a special problem for science to study, and any general statement concerning cheese ripening would have to be very general indeed to cover such a variety of special cases. It can be stated, however, that the processes going on during the ripening are of two general natures, one apparently purely chemical, the other associated with the growth of microorganisms. The chemical process is due to the enzymes present in the green curd, of which there are at least three. There is the rennet used to pro- duce the curdling; with the rennet there is pepsin, derived from the same stomach glands that produced the rennet; and there is a third 159 MANUFACTURED MILK PRODUCTS enzyme always present in normal milk, known as lactase, which has the power of slowly changing casein into soluble products. These three enzymes have opportunity of acting slowly upon the cheese during the long ripening period; and it is probable, if not certain, that a part of the softening of the casein is due to the slow but con- tinued action of one or more of these enzymes. Both the lactase and the pepsin have the power of changing the casein into a soluble material. Although in certain types of cheeses the changes that occur may be due largely to the action of these enzymes, this is by no means the whole of the ripening process. The flavors of the cheese, upon which its value largely depends, make their appearance toward the close of the ripening and apparently are not produced by enzyme action. In all types of cheese, various kinds of microorganisms- molds, yeasts, and bacteria-grow vigorously during the ripening, although the kinds developing in the different cheeses differ greatly from one another. By placing cheeses in an atmosphere of chloro- form to ripen, it has been found possible to prevent the growth of the normal cheese microorganisms without stopping enzyme action. Such cheeses show a fairly normal softening of the curd, but do not develop normal cheese flavors. Evidently the growth of certain microorganisms is necessary in normal cheese ripening. The exact Part each one of these agents plays in cheese ripening appears to vary very much with the different types of cheese, and each type of cheese is therefore a problem in itself. A few of the general facts may be briefly summarized as follows. Hard Cheeses. In this class of cheeses, after the milk has been curdled and drained of as much whey as will naturally separate from lt, the final curd is placed under heavy pressure to squeeze out as much liquid as possible. The result is a hard, tough curd, which ftever, even after complete ripening, becomes very soft. This type mcludes the American cheeses, the Cheshire cheeses, the Swiss cheeses, and some others. The ripening of such cheeses is a slow Process, requiring many weeks, and indeed a number of months for lts satisfactory completion. During all this time the enzymes Present are probably acting slowly upon the casein, resulting finally m its partial conversion into soluble compounds. The development bacteria, meanwhile, shows a series of more or less definite phases. ■First, there is a somewhat rapid multiplication of the lactic acid 160 BACTERIOLOGY bacteria, until after a few days they become very numerous. Later the number of bacteria declines, and finally in the late ripening the number may be very much smaller than at first. It is still uncertain just what organisms actually bring about the flavors which develop during the ripening, but recent work points to the agency of certain streptococci other than the lactic acid type. Organisms of the Lactobacillus type are active in ordinary cheese ripening, but it seems possible to dispense w'ith them provided the ordinary lactic acid Fig. 31. Three Types of Cheese Center, Cheddar (an unusually gassy sample); left, Swiss; right, Roquefort. type is present, although there seems to be a slight difference in the product according to which of the lactic types causes the souring. While much still remains to be discovered on this subject, it is certain that various types of faulty cheese owe their spoiling to the growth of undesirable microorganisms. Cheese faults are numerous in cheese making, sometimes infecting large lots of cheeses and spoil- ing them for market. They are of many kinds, sometimes affecting the taste, and sometimes the appearance. Among the most common may be mentioned swelled cheese, a condition characterized by the formation of gas bubbles within the cheese which grow and cause MANUFACTURED MILK PRODUCTS 161 the cheese to swell until it may actually burst; sweet flavored cheese, spotted cheese, black cheese, tainted cheese, etc., represent other types of more or less common troubles. Many of these faults have been studied by bacteriologists and found to be due to the growth of either yeasts or bacteria in the cheese, which do not occur in normally ripening cheese. In the case of cheese ripening, as in the case of cream ripening, these faults may be to a large extent pre- vented by the use of proper starters at the outset. When the milk is provided with normal lactic acid bacteria and they are allowed to develop until the milk is sufficiently acid, the cheese faults are not liable to occur. When, however, the milk is not souring in the proper manner when the rennet is added, there is a much greater likelihood of unfavorable fermentation in the cheese. Cheese makers have, therefore, been learning that, provided the milk is not too old when it reaches them, a means of controlling the ripening and pre- venting some of these faults is within their reach. Artificial cul- tures for this purpose were first used in making Edam cheese in Holland, where a form of slimy milk is added to the milk to be made into cheese, in order to control the ripening. Later the use of lactic bacteria has been much more widely extended. An even more complete control of the ripening process has been put into practice by the Department of Agriculture in recent years. The use of pasteurized milk for cheese making has been almost im- possible because it does not ripen properly; but the Department has recently found that the inoculation of pasteurized milk with the proper pure cultures results in an almost complete control of the ripening process. The cultures used are lactic acid organisms and the streptococci above mentioned. The cheeses thus pro- duced are better than many commercial cheeses. How they compare with the best cheeses is a matter of taste. In some mar- kets the flavor of certain commercial cheeses would be preferred, but to some palates the flavors of the pure culture cheeses are better than those of any commercial cheese. Whether the use of pure cultures will revolutionize the manufacture of hard cheese as it has in the case of bread-making and butter-making remains to be seen. Soft cheeses. In the soft type of cheese the whey is simply allowed to drain from the curd without any special pressure, the result being n much wetter curd from which the cheese is made. In the different types of soft cheeses there is considerable variation in the amount 162 BACTERIOLOGY of whey that is retained in the curd, but they all have much more water than is present in the hard cheeses. Under these circum- stances, a very different type of ripening occurs, inasmuch as such wet curd offers a very favorable medium for the growth of micro- organisms. While the enzymes that may have been in the original milk may have something to do with the ripening of this type of cheese as well as with that of the hard cheeses, it is beyond question that the chief portion of the ripening in soft cheeses is due to the action of microorganisms. In some of the cheeses so made, molds play a very large part in the ripening, in others it is carried on largely by bacteria, and yeasts are not infrequently present either as an accidental or a necessary factor in the ripening. In practically all these soft cheeses the first change to take place is a souring of the curd under the influence and growth of lactic acid bacteria; but the subsequent changes depend upon the conditions and the method of manipulation. In certain types, like the Camem- bert, the surface of the cheese is kept somewhat dry and furnishes a favorable opportunity for the growth of molds, a special species known as Penicillium camembertii being characteristic of this cheese. This mold secretes an enzyme which acts upon the surface of the cheese, gradually liquefying the curd, the cheese becoming softer and softer, the softening beginning at the surface and gradually progressing toward the interior, until the whole is converted into a creamy mass when the ripening is completed. At the same time, other microorganisms, possibly Oidium lactis, develop upon the ripening cheese and cause its peculiar flavor. In other types like Roquefort and Gorgonzola, the chief ripening agent is also a Peni- cillium, but of a different character, being a blue mold in this case. In the manufacture of Roquefort cheese this mold is intentionally inoculated into the curd, and the subsequent treatment of this cheese is such as to stimulate the growth of these molds. They produce not only enzymes which soften the casein, but also certain by-prod- ucts that cause the piquant taste characteristic of this cheese. In other cheeses like the Limburger, the surface of the cheese is kept constantly wet, so as to prevent the growth of molds and stimulate the growth of bacteria. In this case, therefore, the cheese is ripened primarily by the action of decomposition bacteria, and the resulting product is a combination of the action of the enzymes originally present and the decomposing action of the bacteria. Limburger MANUFACTURED MILK PRODUCTS 163 cheese is, therefore, very close to a putrefying cheese, as anyone can readily appreciate from the odor of a thoroughly ripened cheese of this type. The satisfactory manufacture of soft cheeses depends upon con- trolling the conditions which regulate the growth of microorganisms, and it has proved a rather difficult task to control these conditions artificially. Sometimes it has been necessary to import the desired microorganisms from a country where they are abundant to another country where they are absent and where they are desired for the ripening of cheese. This has been done in the manufacture of Roquefort cheese in the United States, and also to a certain extent in the case of Camembert cheese. In the natural home of these cheeses, however, the dairies have become so impregnated with the microorganisms in question that the cheese-maker has to take no special pains to insure the inoculation of the cheeses with them, and needs only to control the conditions so as to stimulate the organisms desired and to check the growth of those which would be harmful. Speaking in general, we must look upon the ripening of soft cheeses as almost wholly a phenomenon of microbial action, in which the natural milk enzymes play little part. Their action in the case of the different cheeses has, however, been only partially explained at the present time. The immense variety of cheeses, the varying con- ditions and varying microorganisms concerned have rendered the subject a very complex one, and the problem has been solved only m the case of a few special types of cheese. It is probable that in the future very much more practical results will be obtained from studies of this sort. Economic value of cheese-making. It is rather interesting to note that cheeses represent a highly nutritious product which under ordinary circumstances would undergo putrefaction and be ruined, but which is preserved by the action of microorganisms upon it. The results obtained have been purely empirical, and in the manu- facture of cheeses there has been no thought of the problems or the Principles concerned. Dairymen for centuries have been accus- tomed to put aside milk to keep for a future day, and Jong experience has shown them that if curdled and the curd placed under certain conditions of temperature and moisture, instead of putrefying, it will undergo changes which enhance rather than injure the flavor. 164 BACTERIOLOGY Different conditions and different microorganisms in various parts of the world have resulted in the development of a large variety of cheeses. It is only in recent years that the bacteriological study of cheese has shown that the conditions adopted have been such as to favor the growth of organisms that keep down the putrefactive forms and thus preserve the curd in an edible condition. In the end, all cheeses tend to undergo undesirable putrefactive changes, if kept moist; but this may not occur for many months, as the conditions of their manufacture protect them for a considerable time against putrefactive decomposition. 2. MICROORGANISMS IN RELATION TO THE FERTILITY OF THE SOIL CHAPTER V Soil Microorganisms in General IMPORTANCE OF MICROORGANISMS IN THE SOIL Whenever a ditch is dug into the soil, it is plain at a glance that the surface soil is quite different from the deeper layers. The sur- face soil may be rich and loamy, having the deep brown color that is generally characteristic of soil; but the change into subsoil begins within 6 to 10 inches of the surface. Gradually, as one digs deeper into the subsoil, the soil is found to become lighter and less loamy. Laboratory analysis shows that this subsoil is different chemically from the surface soil, the chief difference being that it is very poor in organic matter. The surface soil alone is fertile. Anyone realizes this who has tried to start a lawn around a new house oh land filled in with the soil from the excavation. Unless such soil is specially fertilized it is often two or three years before the grass can get a good footing. Undoubtedly when the first soil was formed it was much like sub- soil, but, if anything, even less adapted to plant growth; so the phenomena that take place in the soil excavated from a cellar re- semble in a small way those that must have taken place when soil was first formed on the surface of the earth. The fault with the subsoil is that it does not contain plant nutrients in available form. As a result only the hardiest plants and the ones that need but little food material can live upon it. Such plants soon begin to grow in the exposed subsoil, obtain some of their nutrient materials from the rneager supply in the soil and the rest from the air. These first plants are sometimes unable to live till maturity; but when they die their remains decompose and enrich the soil to a slight extent, making it that much easier for the next plants to live upon it. The Process continues until the former subsoil is as rich as ordinary surface soil. 166 BACTERIOLOGY The greater richness of the surface soil is due to the accumulation of plant residues throughout countless ages. The decomposing residues of the plants furnish the nutrient materials needed that other plants may live. This decomposition is brought about by the agency of microorganisms. It is one part of the general decomposition processes we have already discussed. This is a further illustration of the importance of these breaking down processes in nature. Soil fertility is dependent upon them. Plant growth is dependent upon soil fertility; and animal life upon plant life. Therefore all life on earth is dependent upon the activities of these microorganisms in the soil. For this reason the study of these organisms is important. Con- sciously or unconsciously every farmer cultivates bacteria as well as the plants that he harvests. A good yield of his crops depends upon the proper handling of the invisible organisms within the soil. Any new knowledge, therefore, that teaches how to give to the benefi- cial soil bacteria the proper conditions for growth is not only of practi- cal value to the farmer but will eventually prove of benefit to all mankind. Nearly all of this knowledge that we so far possess has been ob- tained by experience rather than by study. For ages the soil has been under cultivation and new methods have been tried out one after another. Farmers in general have used the same methods as employed by their fathers; but occasionally some bright man has experimented with new methods and perhaps has found some new way of doing something that gives better results than the old way. His neighbors have followed his example after that, and later the farmers of other districts have done likewise; until at last this new method becomes part of the traditional farm practices handed down from father to son. As such things have happened repeatedly throughput the ages gradually the methods of handling the soil have been more and more perfected; but it is very seldom that the reasons for any particular practice have been known by the man first adopting it. During the middle ages and early centuries of modern time, speculations were rife as to why the soil was fertile. Nearly every thing conceivable was thought to be the food of plants except the materials that modern investigation has shown to be the true nu- trients. The theories were alike in nothing except their apparent SOIL MICROORGANISMS IN GENERAL 167 ridiculousness as judged from the modern point of view; and naturally they did not go far toward changing agricultural methods. Real agricultural science is less than a century old, and as yet has not greatly influenced soil management. Dairy methods as we have seen, have been revolutionized within a generation; but the soil is still being cultivated by essentially the same methods as were used centuries ago. Science is showing us the reasons behind many ancient practices, and is teaching some modifications of these prac- tices which do give better results; but taken all in all the most ap- proved methods of today are not so very different from the prac- tice of our ancestors. Nevertheless, in some particulars the scientific study of soil has led to distinctly better practice and there is good reason to hope that its contributions to practical agriculture will become greater and greater. With the beginnings of agricultural science, it was thought that the problem of soil fertility belonged wholly to chemis- try, and certain chemical theories were proposed that have been largely refuted in practice. Later the complex structure of soil and its complicated physical properties were appreciated, and the study of soil physics was expected to accomplish much of practical value. But it has proved that neither physics nor chemistry alone can solve the problems of soil management. The chemical nature of the soil, m fact, is so dependent upon the activities of microorganisms that it cannot be understood without a knowledge of soil biology. It is not impossible that eventually soil practice as well as dairy practice niay be revolutionized by bacteriology. SOIL AS A HABITAT FOR BACTERIA We have seen that the bacteria of milk generally come from out- side sources such as diseased udders or dirty utensils; and that almost any organism that gets into milk may grow there provided it is not crowded out by the ones already present. Soil, unlike milk, has a bacterial flora of its own. Although soil is constantly being con- taminated from without-through the air, by animals walking over it °r by implements used in cultivating it-the kinds of microorganisms within it show surprisingly few changes. This seems strange at brst thought; but it is not difficult to explain. The soil has remained essentially the same composition for ages, and gradually certain strains of microorganisms have developed that are specially adapted 168 BACTERIOLOGY to live under such conditions. At the present day, only those forms that have become especially adapted to soil can survive and other forms introduced from without are quickly overcome in the struggle for existence. Thus soil has come to have a microscopic flora of its own; and very similar kinds of bacteria are found in soils in widely separated localities. It is plain, then, that the structure and composition of the soil have determined the kinds of bacteria that live within it. For this reason some understanding of the nature of soil is necessary to the student of soil bacteriology. Soil is very different from any other natural habitat of bacteria, as it is composed mostly of solid imper- vious material in which bacteria cannot live. Soil might be spoken of as a mixture of soil grains, organic debris, water, and air. Bacteria cannot live in soil grains or in air and must therefore live in the soil water or water-saturated organic matter. The soil water, of course, is not pure water, but contains much material in solution. Its composition and its relation to the soil grains and the soil air are matters of much importance in contributing to an understanding of soil bacteria and their activities. The soil grains are of various sizes and shapes. The largest are known as sand particles; for when the majority of the soil particles are of this large size, the soil has the characteristics by which we recognize sand. The medium-sized grains are known as silt particles; and when they predominate in a certain soil, it is usually spoken of as a silt or a silt loam. The smallest particles of all are scarcely larger than the bacteria themselves, they are called clay parti- cles; because when they predominate in a soil it has the plasticity when moist and the tenacity when dry that characterize clay soils. No soil is ever made up wholly of either sand, silt or clay particles; but is composed of mixtures of all three. The proportion of each size present determines the texture of the soil; and to the various combinations of sand, silt and clay particles such names are given as: coarse sands, fine sands, coarse sandy loams, fine sandy loams, silts, silty loams, silty clay loams, clay loams and clays. The smallest soil particles are of greatest importance for the bac- teriologist, because in part they are the debris of organic material. These together with some larger undecomposed bits of organic matter form a highly absorptive mass that holds large amounts of water and furnishes admirable conditions for the growth of bacteria. This SOIL MICROORGANISMS IN GENERAL 169 organic matter, although important, composes a comparatively small part of the total bulk of the soil. The arrangement of the soil particles is no less important than their size. Anyone who has lived in a region of clay soils knows how entirely different the same soil may be when well cultivated or when allowed to become compact and sun-baked. In the former case it is said to be in good tilth. The difference between good and poor tilth is a difference in structure, due to differences in the arrange- ment of the soil particles. Sand particles, because of their large size, do not cling much to one another and always tend to lie evenly somewhat like apples in a barrel. The smaller soil particles on the other hand, are inclined to stick together forming large masses. Between these masses of soil particles there are large air-spaces; the larger the masses, the larger the air-spaces, and the better the structure of the soil. If a clay soil is handled much while wet, there is danger of breaking up the masses of soil particles, and then the individual clay particles settle down much as though they were sand- grains; but because of their small size, the air-spaces between them are very small. A clay soil in this condition is said to be puddled, and because of the small air-spaces within it is compact and not suited to plant growth. In considering the life of bacteria in the soil, the relation between the soil particles and soil-moisture is even more important than the structure. Each soil particle is surrounded by a tiny film of water- or strictly speaking, is partly surrounded, for the particle is always in contact with other particles on one side or another. When several particles are bound together in a mass, sometimes the whole mass is surrounded by water, or sometimes the film is still thinner, fitting rnore closely around the individual particles so as to leave air-spaces within the mass. The dryer the soil, the thinner the moisture films. The wetter the soil becomes the thicker these films grow till at last they fill up the air-spaces entirely. Such a water.-logged soil is suit- able neither for plant nor bacterial growth. It is in this film of moisture that the bacteria live. Sometimes they live singly, or sometimes cling together in sheets; but the thinness °f the water-film undoubtedly keeps them from forming very thick Masses or moving about extensively. As a matter of fact, the pre- dominating bacteria in soil are immotile. When the soil becomes too dry these films become so thin that the bacteria cannot get the 170 BACTERIOLOGY necessary water; when it becomes too wet, the air-spaces become so filled up with water that the bacteria cannot get the necessary air. Between these two extremes, however, conditions are favorable to their growth. Inasmuch as the moisture films under such conditions are thin they are always in close contact with the soil air, and the interchange of gases between air and solution can go on very easily. For this reason the bacteria in soil can obtain their oxygen quite easily and soil proves an especially well adapted medium for the growth of aerobic bacteria. KINDS OF MICROORGANISMS ADAPTED TO THESE CONDITIONS It is very difficult to learn the number of different kinds of bacteria that live in the soil, or the number of individuals of each kind that may be present in any particular soil. When soil is plated in the ordinary culture media and incubated for a few days at fairly high temperatures, as is generally done in studying milk or water, only a small part of the bacteria develop colonies. This is because the tem- perature is unfavorable to some, the chemical composition of the medium unfavorable to others, while others, although able to grow under such conditions, are crowded out by the more rapid growth of those organisms that are particularly favored by such an environ- ment. In milk, also, there are bacteria that do not produce colonies on culture plates; but it is fairly easy to make a microscopic prepara- tion of milk which shows the bacteria and gives a good idea as to the actual number present. Microscopic preparations showing the bacteria in soil do not give accurate counts of the organisms present, and it has never proved possible to make more than a rough estimate as to the number of different kinds of bacteria in soil that do not produce colonies on culture plates. It is quite probable that the majority of soil bacteria are overlooked by means of the plate method. If the media used in the culture plates do not contain a great deal of organic matter and if the plates are incubated for a week or ten days at comparatively low temperatures (18 to 25CC.), a great many more colonies develop than appear on ordinary media at higher temperatures. This is probably because the organisms that grow rapidly under the latter conditions and prevent the growth of the more numerous bacteria grow more slowly on the simpler media at low temperatures. When the plating technic is thus adapted fro soil work, it is found that the majority of organisms developing on the SOIL MICROORGANISMS IN GENERAL 171 plates are very small short rods, mostly immotile, but sometimes with polar flagella. A few of these bacteria liquefy gelatin quite rapidly, like the well-known Ps. fluorescens, which decomposes or- ganic matter and is distinguished by the production of a greenish fluorescence in ordinary bacteriological media. Most of them, however, liquefy gelatin very slowly, and some do not liquefy it at all. They differ quite sharply from the characteristic bacteria of milk which ordinarily require large amounts of organic matter. Next to these short rods, the most abundant group of organisms developing upon plates made from soil is the group of Actinomycetes. They were discussed in the first part of this book when it was men- tioned that they seem to be intermediate between bacteria and molds. There is great diversity in the kinds of Actinomycetes to be found in soil and undoubtedly the different kinds have different functions. It has been shown that some of them are capable of carrying on certain stages in the decomposition of organic matter; but the functions of the greater number of them are entirely unknown. When grown in the laboratory the most striking characteristic of the Actinomycetes is their production of pigment. The colors produced by some of them are nothing short of beautiful. Their chromogene- sis, however, is scarcely to be considered of practical importance. In the soil they probably do not produce pigment as the presence of much organic matter greatly limits their power in this line. One other characteristic of many Actinomycetes deserves to be mentioned, because of their relation to the odor of soil. Many cultures of Actinomycetes give off a musty odor that is very striking, and quite peculiar to this group of organisms. It is sometimes described as an earthy odor; but it would be more correct to say that s°il has an Actinomyces-odor, as it is undoubtedly the growth of these organisms in soil that gives it its characteristic odor. This odor is more striking in sod soil than in fallow soil, as can be proved by simple experiment; and it has been shown that Actinomycetes are more abundant in sod than in fallow soil. Another well-known group of bacteria is almost universally present ln soil, although in considerably less abundance. These are the spore- forming bacteria, of which B. cereus and B. mycoides are two of the best known members. They are described as occurring in numbers between 100,000 and 1,000,000 per gram; but only a little work has been done to see whether these are primarily spores or vegetative 172 BACTERIOLOGY forms. This is an important point because where they occur as spores they cannot be active. There is some reason to believe that they occur in soil normally as spores but occasionally find conditions favorable and then multiply for a short time. Nevertheless their constant presence in soil has led many soil bacteriologists to study them and they are frequently mentioned in the literature as among the important soil bacteria. There are also a good number of other kinds of bacteria to be found in soil. In fact soil becomes inoculated through the air with all kinds of bacteria conceivable and many of them are able to grow slightly; but the numbers of most of these kinds are small compared with the numbers of those just mentioned. Cocci, for instance, are relatively scarce. The acid forming bacteria so typical of milk occur in but very small numbers, if at all; and the same is true of the gas-forming bacteria typical of fecal matter and sewage. Bacteria are not the only microorganisms that live in soil. Higher fungi are always present, and their spores are very abundant in soil. They are undoubtedly important, and take part in certain phases of the decomposition of organic matter; although to a large extent their functions remain unknown. Algae sometimes live in the soil; but their activities must be limited to the uppermost layer, where there is light, as algae cannot live in the dark. Also, protozoa have been found. It is claimed by some that the protozoa are very important because they prey upon the beneficial bacteria and in that way lessen the fertility of the soil. By others, however, it is insisted that protozoa cannot be active in soil unless it is very wet so that the moisture films are quite thick and that at other times they must exist in the form of inactive cysts. This dispute is not settled, so it cannot yet be said that protozoa have been proved to be important in soil. All this shows that the soil is inhabited by a complex population of microorganisms. Each different kind of microorganism has its own activities and requires conditions especially adapted to its growth. Some are mutual rivals, others can live together without influence upon each other, wiiile still others actually benefit one another. Obviously under such circumstances, if anything happens to harm or to benefit one kind of microorganism, many other kinds will be indirectly affected. It would seem as though there would be constant fluctuations in both kinds and numbers of the microbrgan- SOIL MICROORGANISMS IN GENERAL 173 isms in the soil; but as already stated such changes are surprisingly few. The reason for this is that during the long periods of time that bacteria have been living in the soil, an equilibrium has been established. Each kind of microorganism keeps others from de- veloping more than to a certain definite extent, and on the other hand its own development beyond a certain point is prevented by the activities of rivals of its own. Thus the hostile influence of the different kinds on each other keeps the whole microscopic popula- tion quite constant, both in kinds and in numbers. Sometimes, however, something happens which disturbs the equilibrium. Thus, for instance, if some antiseptic is mixed with the soil those kinds of microorganisms most susceptible to its action are killed or largely killed. Then after the antiseptic is removed by evaporation if volatile, or by decomposition if not volatile, it is a long time before conditions become as they were originally. The killing of certain kinds of microorganisms has destroyed the equilib- rium; hence other kinds, freed from the hostile action of those that succumbed to the antiseptic, can increase to unusually high numbers. As a result, such treated soil contains an abnormal number of bacteria for months afterwards and it is only gradually, as the types that were destroyed succeed in reestablishing themselves, that the normal kinds and usual numbers of microorganisms are to be found. There is some evidence to indicate that freezing of the soil in the winter similarly disturbs the equilibrium and allows certain kinds of bacteria to multiply excessively. This may explain the peculiar fact that higher plate counts are ordinarily obtained from frozen soil than from unfrozen soil. REFERENCE H. J. Conn. Soil Flora Studies. N. Y. Agric. Exper. Station, Tech. Buis. 57- 60. 1917. CHAPTER VI Effect of Bacteria on Plant Nutrients BACTERIA IN RELATION TO ORGANIC MATTER Plant food. In studying bacteria and their activities in soil it is necessary to consider first certain points in regard to the physiology of higher plants, such as the kinds of food they require and the sources from which they obtain them. Plants, like animals, are composed largely of the four chemical elements, nitrogen, carbon, hydrogen, and oxygen, combined in various different manners with smaller amounts of potassium, phosphorus, iron, sulfur and a few others. In part, these four elements-together with others in smaller amounts- are combined to form highly complex substances known as proteins; in part, carbon, hydrogen and oxygen, alone, are combined to form less complex bodies such as wood, cellulose, starches and sugars. It is evident that plant food must contain these four elements in abundance and in some form that can be converted by plants into proteins, wood, cellulose, and so forth. The two elements, hydrogen and oxygen, plants can obtain in large quantities from water. Of carbon and nitrogen, however, they do not have an inexhaustible supply. The only form of carbon available to green plants is carbon dioxide. This gas is never present in the air in large quantities, and unless the supply of it were constantly renewed, it would soon become exhausted. The renewal of the carbon dioxide supply is brought about largely by the respiration of animals, as it is one of the waste products they eliminate. As a result there is never any carbon starvation of green plants, provided animal life is sufficiently abundant and the animals are provided with plenty of carbon in their food to supply their needs and to be oxidized eventually into carbon dioxide. Animals are ordinarily provided with their carbon in the form of plant tissue; so if all forms of vegetable carbonaceous matter were foods for animals, both plants and animals would never lack this important element. Certain plant tissues, however, such as wood, contain much carbon, and yet are not food for animals. It is conceivable that the world's EFFECT OF BACTERIA ON PLANT NUTRIENTS 175 supply of carbon might in course of time become converted entirely into some such form and be available neither to green plants, nor to animals. It is the action of microorganisms, as we shall see later, that prevents the universal carbon starvation that would result from such a condition. Nitrogen, however, is the element of plant food of which the source is most likely to become exhausted. Such a remark sounds strange considering that air is four-fifths nitrogen; but gaseous nitrogen is not available to most plants. Ordinary green plants require, or at least prefer, to have their nitrogen in the form of nitrate (i.e., some salt of nitric acid). There is evidence that some plants, if not all, nre able to use some other form of nitrogen, such as ammonium salts or perhaps even organic matter; but apparently most of their nitrogen is obtained from nitrate. Now nitrates are present in soil in very small quantities, and they are not produced as waste products cither by plants or by animals, .so it is really a critical question how the supply of nitrates is maintained. As we shall see, here again, bacteria are of extreme importance. End-products of metabolism. Plants and animals convert these food elements into end-products of a very different nature. As already stated, plants consist primarily of protein, wood, cellulose, and carbohydrates (i.e., starches and sugars); and so far as plants are concerned, these are probably the only important end-products that contain either carbon or nitrogen. We do not know whether Plants excrete any waste products through their roots; but we can state definitely that the amount of carbon and nitrogen excreted by plants, either through their leaves or through their roots, is comparatively small. It is evident, therefore, that the greater part of the carbon and nitrogen assimilated by plants accumulates in their bodies and is not released until their death, except as it is given °ff in fruit or falling leaves. In brief, plants leave the carbon, which they obtain from carbon dioxide, and the nitrogen, which they ob- tain from nitrates, in the form of highly complex organic material. These organic forms of carbon and nitrogen are eaten by animals aod are converted by them into other material almost, if not fully, as complex. Animal bodies consist of proteins', somewhat different from plant protein, of fats, bony tissue and so forth. When the flesh of one animal is eaten by another, these materials enter into new combinations, but forming similar and equally complex com- 176 BACTERIOLOGY pounds. In the course of this process, however, much carbon is given off as carbon dioxide, much hydrogen and oxygen as water, and much nitrogen as urea or some body much like urea in chemical composition. Carbon dioxide and water are immediately available to plants; but urea is not, nor are the organic substances of which animal bodies are composed. Animals, therefore, make most of the carbon available again to plants, but leave the nitrogen in some form of organic matter that cannot be used by plants. The importance of soil microorganisms is because they are able to utilize these end-products of plant and animal metabolism and leave the carbon and nitrogen they contain in forms that are again available to plants. Thus microorganisms complete the cycle through which these two elements pass. The carbon and nitrogen cycles are quite different, the latter being much more complex than the former; but the agency of bacteria plays an important part in each cycle. We will study these two.cycles in detail in the following pages. Humus. Nearly all of the changes which take place in these or- ganic materials while they are being made available again for plants take place in the soil. Plant and animal bodies and animal excreta generally accumulate in or upon the ground and, as they decompose, the soil absorbs the products of decomposition. It is plain that this partially decomposed organic matter must form a considerable part of the soil. This material is called humus. Obviously the humus must be a very important soil constituent since it contains the mate- rials from which plants eventually obtain all of their nitrogen and much of their carbon, and for this reason it has been extensively studied. Much work has been done to determine its chemical nature. Originally it was thought to be a definite chemical com- pound, a salt of the so-called humic acid; but upon more careful investigation, no such substance as humic acid was found to exist. Instead it was then claimed that humic acid was merely a mixture of a number of other acids, to which names and even chemical formulae were assigned. Later investigators, however, proved these acids to be as far from definite chemical compounds as was humic acid. Not until very recent years have substances been isolated from humus possessing crystalline structure and proving upon careful investigation to have definite chemical composition. These sub- stances prove to be very varied, some of them new compounds, others EFFECT OF BACTERIA ON PLANT NUTRIENTS 177 compounds already known to chemists. As a result it is well estab- lished today that the "humus" is really a complex mixture of organic compounds. It may contain any organic substance capable of being produced by the decomposition of plant or animal tissues or animal excreta that may find their way into the soil. Whatever may be the composition of humus, there can be no ques- tion as to its importance from the standpoint of soil bacteria. Ob- viously bacteria cannot live on the insoluble mineral matter of the soil and few of them are able to live on mineral matter alone, even though it is soluble. Hence the majority of soil bacteria must ob- tain their nourishment from the organic matter in the soil. THE CARBON CYCLE Green plants, as we have seen, obtain their carbon in the form of carbon dioxide, and the carbon dioxide supply of the atmosphere is kept up primarily by the respiration of animals. In other words the carbon cycle is ordinarily a very simple one: CO2 is built up by green plants into starches and sugars, which are eaten by animals and oxi- dized within their bodies for the sake of the energy thus produced, the chief product of oxidation being carbon dioxide. The simplicity of the cycle, however, is diminished by the fact that both plants and animals produce certain substances not directly available to the other. Thus, plants convert some of the carbon into wood and cellulose, which animals cannot use as food; and animals leave considerable of the carbon in such forms as protein, fat, and sugars, none of which are available to plants. TTood. One of the most important plant products which contains carbon in a form unavailable to animals is wood. The quantity of wood produced in a forest is great enough to make it unnecessary to dwell upon the extent of the loss this would mean, provided the carbon remained permanently locked up in this form. It is equally evident, however, that it does not remain permanently in the form of Wood. The trunk of a fallen tree remains apparently unchanged for but a year or two and then slowly begins to soften and eventually to crumble, until at last it cannot be distinguished from the soil around it. This action is due to the growth of microorganisms. As soon as the wood dies, it is penetrated by the mycelium of fungi, which gradually softens the wood by means of enzymes they produce. Some °f these fungi are entirely microscopic, while others produce large 178 BACTERIOLOGY spore-bearing bodies, such as the mushrooms that are always abun- dant on decaying wood. After these fungi have acted on the wood for a while, certain bacteria are able to make use of the material. As a result of these various microscopic agencies, a large number of differ- ent carbon compounds are formed, all of them simpler than wood. Each of these compounds serves in turn as food for a new kind of microorganism, by which it is converted into a still simpler form, until at last all the carbon of the wood is converted into CO2 and escapes into the air. Thus it becomes available to plants once more.1 Cellulose. That part of the carbon which is converted into cellu- lose is also unavailable to animals. Cellulose composes the bulk of leaves, and hence a large quantity of it is produced by plants, al- though its bulk is not so striking as that of the wood produced by trees. The fate of cellulose was not understood for some time, although it was well known that some agency caused the decomposi- tion of leaves. Finally, experiments showed that filter paper, which is almost pure cellulose, would disappear entirely if left in soil for a number of months. Investigation was then made to find out what kinds of microorganisms could cause this decomposition. As a result, certain organisms have been shown to have the power. It has been a matter of dispute whether it is an ability possessed by a very few bacteria or one belonging to a considerable number of differ- ent bacteria and fungi; but it is now believed to be a fairly common property among microorganisms. The cellulose-decomposing or- ganisms convert the cellulose partly into CO2 and partly into methane (CH4); but as methane is easily oxidized by purely chemical agencies into CO2 and H2O, the final result is to convert all the carbon into the form which is available to plants. It is probable that some cellulose is also decomposed in the intes- tines of animals, especially herbivorous animals. It is possible that these animals secrete some cellulose-digesting enzyme; but it is also possible, as believed by some, that the digestion of cellulose is due to the acton of bacteria inhabiting the intestines of these animals. There is no question but that such bacteria have been found in their 1 These same processes are sometimes quite undesirable. The rotting of timber is caused by these microorganisms, wherever there is sufficient moisture for their growth, such as in contact with soil. Nearly all methods of preserving wood, such as seasoning, painting, oiling, impregnating, are' designed to prevent the growth of microorganisms by the exclusion of water. EFFECT OF BACTERIA ON PLANT NUTRIENTS 179 intestines, but it has not been proved that the animals are unable to digest the cellulose without their help. If so, this is a case of symbio- sis, the association of two kinds of organisms, both of which benefit from the association. Animal tissue. Animals do not oxidize all the carbon which they eat into carbon dioxide. Much of it, of course, is built up into their body tissue, in various combinations with other elements. Most of it is in the form of protein or fat, but small amounts are in the form of sugar. Proteins, as we have already seen, contain nitrogen as well as carbon, while fats and sugars contain merely carbon, oxygen and hydrogen; but none of them are of any immediate value to plants. In part, animal tissue is eaten by other animals and thus the carbon in it is used over again; but not all dead animals serve as meat for others, so the agency of microorganisms is necessary in order to com- plete the cycle. Proteins are acted upon by the decomposition bacteria, which we shall study more carefully when we take up the nitrogen cycle; sugars and fats are decomposed by other bacteria especially adapted to them. By their activity various decomposition products are formed, of which the most important carbonaceous ones are certain organic acids; but eventually these are all completely oxidized by bacteria and the carbon converted into CO2 again. Nature's carbon economy. Thus we have seen that the element carbon is handed back and forth between green plants and animals, as it is given off by animals as CO2, in which form it can be used by green plants, and is left by green plants in forms which can be used by animals as food. The irregularities in this cycle arise from the fact that the bodies of dead animals contain carbon in forms unavail- able to plants, and certain kinds of plant tissue do not serve as food for animals. In both of these cases, the cycle is completed by the activities of microorganisms which are able to use these materials as food and to convert the carbon into CO2 and thus put it within the reach of plants again. Thus the carbon economy of Nature is complete. REFERENCE E. B. Vorhees and J. G. Lipman. A Review of Investigations in Soil Bac- teriology. U. S. Dept. Agric., Off. of Exper. Stations, Bui. 194. 1907. CHAPTER VII Effect of Bacteria on Plant Nutrients {Continued) THE NITROGEN CYCLE It has just been stated that green plants ordinarily obtain their nitrogen from nitrates and build it up into protein forms that are food for animals, but that animals do not transform the protein nitro- gen into any form that is directly available to plants. As the cycle is completed by the agency of microorganisms is it necessary to take up the steps by means of which protein is converted into nitrate and thus made available to plants again. Decomposition. As shown in the first part of this book, decomposi- tion is the process by means of which organic material is broken down into simpler compounds. The term is a very general one and covers activities of most of the organisms that make carbon again available to plants as well as of those that break down nitrogenous compounds. The decomposition bacteria are of many different kinds and each kind possibly has different activities from every other. Plainly it is impossible to discuss them all here; but of these activities, the one most important in the nitrogen cycle is called ammonification. Ammonification. This process as its name suggests, results in the formation of ammonia. The ammonifying bacteria act upon the complex nitrogenous material produced by plants and animals, con- verting it into various simpler compounds, eventually leaving the nitrogen it contains in the form of ammonia or some ammonium salt. The chemical changes involved are very complicated, more compli- cated than those involved in the breaking-down of carbohydrates, cellulose, and wood, because proteins are much more complex bodies than the non-nitrogenous organic matter. No one has attempted to make a complete study of the process, but certain classes of inter- mediate products are known, and have been given names such as peptides, amino acids, and amides. The amides and amino acids are compounds containing the -NH2 group, which forms ammonia (NH3) when broken off from the rest of the compound. This break- EFFECT OF BACTERIA ON PLANT NUTRIENTS 181 ing off of ammonia from the amides and amino compounds is very easily accomplished by bacteria; and the ammonia given off becomes ammonium hydroxide (NH4OH) if water is present and in the pres- ence of acids combines with them to form ammonium salts. Thus whatever the intermediate products may be, the decomposition gen- erally ends, so far as nitrogen is concerned, in the production of am- monia or ammonium salts. The whole process, therefore, is spoken of as ammonification, although some of the bacteria that take part in it are capable merely of carrying on the first stages of it and do not produce ammonia themselves. One form of ammonification that deserves special notice is the decomposition of urea. Such a large quantity of nitrogen is eliminated by animals in this form that its conversion into simpler compounds is a matter of great importance, and its decomposition has been made the subject of quite careful study. The chemical formula of urea, CO(NH2)2, shows that it contains the amide (NH2) group, and suggests that ammonia should be readily split off from it. In fact, the mere combination of one molecule of urea with two mol- ecules of water (H2O) converts it into ammonium carbonate (NH4)2COS. A considerable number of bacteria have been found that utilize the nitrogen of urea and convert it into ammonium salts, Perhaps according to this formula, but probably by some more com- plicated process. They are known as the urea bacteria. One of the places where these different forms of ammonification go °n most extensively is in the manure pile. The manure pile is very pich in nitrogen, most of which is contained in the liquid part of it. "I he urea bacteria, as well as the other ammonifiers, are sure to be Present in the manure, so the nitrogen is rapidly converted to am- monia. This ammonia is given off in gaseous form provided there is r*° acid present to combine with it and to retain it in the form of some ammonium salt. As the manure pile is seldom acid, much ammonia escapes from it as gas and is thus lost, so far as its immediate Use by plants is concerned. This loss of ammonia is very evident from the odor of a manure pile. If manure is mixed with soil instead °I being allowed to accumulate in piles, more ammonia is fixed and less given off into the air. Because of the great value of nitrogen as a fertilizer, immediate application of the manure to soil is plainly the more economical procedure. 182 BACTERIOLOGY When ammonification is complete a good share of the work is done in rendering nitrogen again available to plants; but still more remains to be done. Some plants, it is true, can obtain their nitrogen from ammonium salts, and probably if there were no natural means by which nitrates could be formed, plants would long ago have ac- quired the ability of living wholly upon them; but the fact remains that green plants in general obtain their nitrogen from nitrates and seem to obtain it more readily from that source than from salts of ammonia. Hence it becomes a matter of much interest to see how the ammonium salts are converted into nitrates. Nitrification. It has been known for a long time that in some way nitrates are formed in nature. Neither is it a matter of very recent discovery that ammonium salts are fairly rapidly converted into ni- trates. For a long time, however, it was supposed that this was a spon- taneous chemical process. It was not until the late seventies that the process was discovered to be a biological one, a discovery based upon the observation that no nitrates were formed if all life activity was stopped by means of an antiseptic. This observation led to numerous experiments which pointed to bacteria as being the causal agents; but for a long time all attempts to isolate these bacteria were failures. After a large number of unsuccessful attempts to isolate the organ- isms, it became evident that they must be bacteria unable to grow on the ordinary laboratory media. This fact was demonstrated almost simultaneously by two men, Winogradsky and Warrington, the former working in Russia, the latter in England. Winogradsky was the first to obtain these organisms in pure culture, although War- rington apparently accomplished the same thing before Winogradsky's results were published. The two men used entirely different methods. Winogradsky inoculated soil into a solution containing some ammonium salt, and when nitrates were formed, inoculated a second flask of the solution from the first. This was repeated until almost all but the nitrifying organisms were excluded. Then he made gelatin plates from the material, and found very few colonies on the plates. Having learned that none of these colonies were the bacteria he sought, he concluded that the organisms in question must be on his plates between the colonies. Accordingly he inoculated media with gelatin picked up where there were no visible colonies, and at last obtained the organisms he sought. Warrington, on the other hand, did not use culture plates. After he had obtained a flask EFFECT OF BACTERIA ON PLANT NUTRIENTS 183 of vigorously nitrifying material in much the same manner as Wino- gradsky, he diluted it to such an extent that each drop contained on the average just one bacterium. Then he placed one drop each in a series of tubes containing nitrifiable material, in some of which he found, after incubation, pure cultures of the correct organism. Even with these methods, however, the results were at first rather inconsistent. Winogradsky at last showed what was the trouble. There was apparently no one organism capable of converting ammonia into nitrate, for the process takes place in two distinct steps, each step carried on by a different organism. The first step is the oxidation of ammonia into nitrite and water, thus : NH, + 30 = HN02 + H2O The second step is the oxidation of nitrite into nitrate, thus: HN02 4- 0 = HN08 The first step is carried on by bacteria often called the nitrous fer- ments. Winogradsky found two of them, one a round form which he named Nitrosococcus, the other a rod form which he named Nitrosomonas. The second step is carried on by bacteria known as the nitric ferments. Winogradsky gave the name Nitrobacter to the form which he observed. There is still some question as to whether these organisms of Winogradsky's are the only nitrifiers. It has been claimed, although the work has never been confirmed, that an organism has been found which can convert ammonia directly into nitrate, and it has also been suggested on the basis of purely indirect evidence, that some organism exists which converts organic matter into nitrates without the intermediate action of other organisms. But all this is pure speculation; and at the present time the only known nitrifiers are those described by Winogradsky. Because of the importance of nitrification, these organisms deserve further study. In their food requirements the nitrifying bacteria are distinctly different from the ordinary bacteria. They belong to the group of autotrophic bacteria already mentioned (p. 69); that is, they can obtain their nitrogen and carbon entirely from inorganic sources. When Winogradsky made this discovery, it was scarcely believed at first; for it had not been supposed that any organism could live on inorganic material alone unless it contained chlorophyll like the green 184 BACTERIOLOGY plants and could thus use sunlight as a source of energy. The nitri- fiers not only have no chlorophyll, but can live in utter darkness. It is plain, therefore, that they must have some other source of energy. It has been found on investigation that this source of energy is the oxidation of ammonia to nitrite and water or of nitrite to nitrate, both of which chemical changes liberate energy. Recently quite a number of other organisms have been found that can obtain energy from chemical changes; but at the time nitrifiers were discovered they were thought to stand in utter contrast to all other forms of living matter. One of the other peculiarities of nitrifiers in their relation to organic matter is that besides being able to live without it, they seem to thrive better in its absence. Winogradsky discovered that the reason ordinary bacteriological media were not adapted to their growth was that they contained organic matter. Investigation showed that in ordinary laboratory culture, organic matter, even in extremely high dilution, is very toxic to them. This surprising discovery led to many speculations as to how they could grow in nature, where organic matter is almost universal, as for example in the manure pile, where nitrification is known to occur. The generally accepted theory, and one which found its way into many textbooks, was that they could not become active until the decomposition bacteria had destroyed all the organic matter. This theory was not supported by observation, because nitrates appear in the manure pile long be- fore all organic matter is decomposed, and in fact it has been definitely proved that nitrifiers are living under such conditions. It has further been shown that nitrifiers can grow vigorously in soil to which considerable organic matter has been added, and it has been dem- onstrated that if they are cultivated under conditions affording good aeration, such as in blocks of hardened gypsum, certain kinds of organic matter actually stimulate their growth. This matter of good aeration, in fact, seems to be the most impor- tant physical factor upon which their growth depends. In soil, they live in the very thin moisture films, in close contact with the soil air; but in laboratory media such good aeration is not easily brought about. In some way not yet explained, close contact with air enables them not only to resist the toxic effect of organic matter, but perhaps even to derive some benefit from such material, which is harmful under conditions of poor aeration. EFFECT OF BACTERIA ON PLANT NUTRIENTS 185 Besides air, these bacteria have need of moisture, and nitrification stops in fairly dry soil. Too much moisture, however, is harmful. The optimum moisture content cannot be stated definitely because it varies in different soils, but in general the nitrifiers thrive best at that moisture content which is also best adapted to plant growth. It is also necessary that the soil should not be too acid. Acids develop in soil as a result of various decompositions, and sometimes the reac- tion becomes too acid for the growth of nitrifiers. Another factor influencing their growth, although one less thoroughly understood, is the action of higher plants. It has been shown, for instance, that while corn is growing in soil, more nitrates are present around it at the height of its growing season than at any other time. This may be partly explained if we assume that corn can use organic matter directly; but it strongly suggests that the growth of corn must stimu- late nitrification. In short, much still remains to be learned as to the conditions best adapted to their growth. Significance of nitrification. It is evident that nitrification com- pletes the cycle of nitrogen by rendering it again available for plant use. The important stages of this cycle are: nitrates converted to proteins by green plants; plant protein eaten by animals and con- verted in large part to urea; urea and protein destroyed by the de- composition bacteria and finally converted to ammonia; ammonia converted to nitrate by the nitrifiers. All these stages are illustrated in the diagram (fig. 30). In this diagram the forms of nitrogen from which energy can be obtained are at the top, those without energy at the bottom. In converting any form into any other form below it on the diagram, energy is liberated; while on the other hand it requires energy from some other source to build up any form of nitro- gen to one higher in the diagram. Thus green plants utilize the sun's energy to convert nitrate into protein, while the decomposition bac- teria in general require no outside source of energy because energy is liberated in converting protein or urea into ammonia. Similarly, the nitrous bacteria obtain their energy by oxidizing ammonia, and the nitric bacteria theirs by oxidizing nitrite. Denitrification. Nitrification being beneficial, we must regard any process which destroys nitrates as harmful. Such a process is denitrification, that is, the partial or complete reduction of nitrates by bacteria. There are really two separate processes known by this uame. The first, partial denitrification, results in the formation of 186 BACTERIOLOGY nitrite and ammonia from nitrate; the second, complete denitrifica- tion, converts nitrate and nitrite into free nitrogen. The first process is exactly the reverse of nitrification. It will be seen from figure 31 that this is not an energy-liberating process. Hence denitri- fiers of this sort require organic matter as a source of energy. Such organisms are very common in nature, many of the decomposition Fig. 32. The Nitrogen Cycle A diagram showing the transformations nitrogen undergoes in nature bacteria having the ability to convert nitrates into nitrites or am- monia. It is evident that although this process undoes the work of the nitrifiers, it does not result in permanent harm, provided the nitrifying organisms are present to build up again the nitrates that have been reduced. Complete denitrification, however, is more harmful. The free nitrogen it produces is of course given off into the air, and is not available for immediate use by plants. This would be a permanent 187 EFFECT OF BACTERIA ON PLANT NUTRIENTS loss, provided there were no way of returning the lost nitrogen to the soil. When this process was first discovered it was thought to be a very common one, a constant menace to agriculture; but it has since been shown that in ordinary soils it occurs very rarely It is a matter for concern only in heavily manured soils such as in the greenhouse or in market gardening. "Leaks" in the nitrogen cycle. It will be seen from the above discussion that there are two points in the nitrogen cycle-leaks as it were-where nitrogen is given off into the atmosphere and thus becomes lost to the soil. One of these "leaks" is the result of am- monification, which causes the formation of gaseous ammonia under certain conditions. Ordinarily the ammonia produced is fixed by the acids present or is absorbed physically in the soil; but when am- monification is very active, such as in a loosely packed manure pile, some ammonia is actually lost. The other "leak" is the result of complete denitrification, which as we have seen is not a very common process in nature, but which is responsible for enough loss of nitrogen to amount to considerable in course of time. It is easy to under- stand that if there were no way of recovering the nitrogen lost through these "leaks," the soil would soon become exhausted of this impor- tant element, and life would be impossible. Besides these biological processes by which nitrogen is lost there are certain other agencies carrying nitrogen out of reach of plants. Human food, of course, contains much nitrogen, most of which eventually finds its way into the sewage and is emptied into the streams and thus poured into the ocean. Similarly, rains wash nitrates and other soluble forms of nitrogen out of the soil, and these too go into the ocean. Theoretically, what nitrogen accumulates at the bottom of the ocean may eventually become available to plants again; but practically it is lost. Another loss of nitrogen is from direct chemical decomposition, nearly all explosives, for instance, liberating free nitrogen. Recovering lost nitrogen. Of course it is evident that this loss is made good in some way. Plants and animals have been using the earth's supply of nitrogen for ages without exhausting it. Not only that, but the location of nitrogen, wholly in the very crust of the earth's surface, has convinced scientists that it has all been derived from the air. In other words, plant and animal life have not ex- hausted the earth's nitrogen, but there has, in some way, been an 188 BACTERIOLOGY actual increase in the stock of nitrogen throughout the ages that life has been in existence. The source of this nitrogen was a matter of dispute for a long time. At first it was taken almost for granted that plants used atmospheric nitrogen. When it became evident that this source was not ordinarily available to them, the theory was proposed that they could use the traces of ammonia present in the air. It was then shown that this theory did not agree with facts, and the matter remained as much in dispute as ever. Meanwhile, however, it had long been known in practice that plants of the legume family can thrive in very poor soil, and flourished independent of nitrogenous fertilizers. It was even shown that the soil upon which legumes were growing became richer in nitrogen rather than poorer. Finally it was discovered that under certain conditions, soil exposed to the air, without any plants growing upon it, slowly gains in nitrogen. Obviously this increase in nitrogen must come from the air, as there is no other source. In other words, atmospheric nitrogen can in some way be "fixed" in the soil. The method by which this fixation of nitrogen takes place remained un- known for some time; but at last the classic investigations of Berthe- lot, Hellriegel, Beijerinck and Winogradsky threw light on the subject. Symbiotic nitrogen-fixation. The first form of nitrogen-fixation to be carefully studied was that which takes place through the agency of legumes. It was known even back in Roman times that certain legumes enriched the soil and acted almost like manures. Gradually their use for this purpose came to be an accepted agricultural practice; but practical farmers knew no reason for it, and scientists, even if they accepted the belief themselves, were able to offer no explanation. It was not until 1886 that it was finally shown, by Hellriegel and Wilfarth, that this power of fixing nitrogen was associated with the nodules that are generally found on the roots of legumes, and that these nodules contained microorganisms. The agency of microor- ganisms they showed to be necessary to enable the legumes to use the nitrogen of the air. Typical legume nodules are shown in figure 33. They are extremely interesting little bodies now that we know how important they are. For centuries, however, no one paid much attention to them. The first written mention of them, so far as we know, was made in the seventeenth century. Then they w'ere regarded as root galls. EFFECT OF BACTERIA ON PLANT NUTRIENTS 189 Gradually more and more was learned about them until at last their full significance was realized. They may be found on the roots of almost any legume if the soil is washed out from around them. They vary from the size of pin-heads up to the size of small peas. Under the microscope they show fairly long filaments of a gelatinous mate- rial, in which tiny microorganisms are imbedded. These microorgan- isms resemble bacteria in size, but are irregular in shape, generally occurring in the formof club-shaped bodies: or as Y's or T's. If one of the nodules is crushed in a sterile plate and an agar medium containing sugar but little or no nitrogen is poured over it and allowed to harden, after a number of days colonies of a certain kind of bacteria develop. These bacteria on ordinary media are simple rods. Sometimes they appear peritrichic, but cultures are so often found with only one flagellum to a cell that many claims have been made that they are monotrichic, and the matter of their flagellation cannot be considered settled. Under certain conditions these rods develop into irregular forms similar to those observed in the nodules (see fig. 34). These irregular bodies are called bacteroids and are quite characteristic of the legume nodule organism. The organism was first obtained in pure culture in 1888 by Beijerinck, who named it Bacillus radicicola. Today it is sometimes called by this name, often by the name Pseudomonas radicicola by those who believe it monotrichic, and often Rhizobium radidcola by those who consider it sufficiently different from other bacteria to justify making a separate genus of it. This organism can live alone in the soil, and may possibly be able to utilize atmospheric nitrogen when growing under such conditions; but from the practical standpoint, its free-living existence is the least important part of its life. As soon as a legume seedling sends down roots into the soil, these bacteria, if present, penetrate the young roots, and spread from cell to cell by means of the gelatinous fila- ments just mentioned. Meanwhile the plant tissue is stimulated to grow abnormally and form the characteristic nodule, within which the bacteria live. While they live there, the plant is able to use the atmospheric nitrogen. It is still uncertain just how this result is brought about. It has been claimed by some that the plant itself ls able to assimilate the free nitrogen, stimulated or aided in some way by enzymes that the bacteria secrete; while others have insisted that 190 BACTERIOLOGY the bacteria utilize atmospheric nitrogen, converting it into some soluble form that can be assimilated by the plant. Today the latter view is generally accepted because the bacteria have been shown to utilize free nitrogen when living alone, but the legumes do not seem to have the ability under any conditions except when the proper bacteria are present. Fig. 33. Nodules on the Roots of a Legume Fig. 34. The Legume Nodule Organism This relation between the legumes and the nodule bacteria is quite unusual. Many kinds of bacteria are known to penetrate the tissue of living plants and live there, sometimes producing nodules or other swellings not unlike those on legume roots; but they are always parasites causing injury to the plant and obtaining benefit only for themselves. In this case, however, both organisms benefit from the association: the bacteria by securing a good place to live where they are free from the hostile activities of rival organisms, and the plants by securing an abundant supply of nitrogen. Some writers insist that this is virtually a case of parasitism; and, indeed, there is much similarity between the invasion of the tissues by this 191 EFFECT OF BACTERIA ON PLANT NUTRIENTS organism and that by true parasites. But in the ordinary sense of the word, these bacteria are not parasites. Since both organisms derive benefit from the association, it seems more fitting to call it a case of symbiosis-that is, a relation between two organisms that is to the advantage of both. After this relation had been learned, certain other facts were observed that seemed at first to be hard to explain. It was found that a soil might support a vigorous growth of one legume, allowing an abundant production of nodules, while a different legume might grow but poorly with an entire lack of nodules. Plainly the soil contained the right kind of bacteria for the one legume, but did not seem to for the other. This raised the question whether there might not be more than one. species of legume organism. This question has been a matter of dispute for a long time, and the dispute is not yet entirely settled, largely because we do not know what constitutes a species among bacteria. But this much is known: an organism isolated from the nodule of a certain legume is not capable of pro- ducing nodules on all other legumes indiscriminately; there is evi- dence that in course of time it may become adapted to other kinds of legumes from those to which it was originally adapted, but this is not quite certain. About fourteen of these types or varieties have been described, but only six are adapted to the cultivated varieties of legumes. These six are: 1. Adapted to beans 2. Adapted to peas, and vetch 3. Adapted to alfalfa, sweet clover, and bur clover 4. Adapted to the other clovers 5. Adapted to cowpea, peanut, trefoil and acacia 6. Adapted to soy bean. Whether to call these separate species or merely varieties of one sPecies is largely an academic question. The practical side of the matter is that a soil may contain the right kind of bacteria for one legume but not for another. A7on-symbiotic nitrogen-fixation. Before the action of bacteria in the legume nodules was understood, it had been shown that under certain conditions uncropped soil might gain in nitrogen, but the method by which the gain took place was unknown. Finally in 1885 Berthelot showed that this nitrogen-fixation took place only in Unsterilized soils, and hence was probably due to the action of some 192 BACTERIOLOGY microorganism. About ten years later Winogradsky succeeded in obtaining pure cultures of anaerobic bacteria capable of fixing nitro- gen when grown in the laboratory. Since then, numerous bacteria and fungi have been found to have the power to a greater or less extent. Of them, the most frequently mentioned belong to the group generally called by the generic name Azotobacter, because they are able to use comparatively large quantities of atmospheric nitrogen, while the other organisms do so to a considerably less extent. The first members of this group were isolated by Beijerinck in 1901 and called by him A. chroococcum and A. agilis. They are oval organisms considerably larger than ordinary bacteria. There can be little doubt but that some of these organisms are responsible for the increase of nitrogen that takes place in uncropped soil. The Azotobacter forms are generally considered to be the chief agents in this process, because of their greater ability to fix nitrogen under laboratory conditions, although it has never been shown how great their ability in that respect may be in the field. In fact, it has been claimed by some that the importance of non-symbiotic nitrogen-fixation has been greatly overestimated and that under soil conditions it does not take place to any great extent. Neverthe- less there seems good reason to believe that throughout long periods of time much nitrogen has been added to the soil in this way. Significance of nitrogen-fixation. A glance at the diagram on p. 185 shows that nitrogen-fixation closes up the gap in the nitrogen cycle caused by denitrification. It counterbalances the loss caused by this process, and as denitrification is undoubtedly of small extent it also counterbalances that caused by the evolution of free ammonia. The diagram also shows that the fixation of nitrogen is a process that requires energy from some other source. The legume bacteria un- doubtedly obtain their energy from some products of the plants upon which they live. The free-living nitrogen-fixing bacteria can grow in laboratory media containing no organic matter except sugar, a fact which shows that they can obtain the necessary energy from the decomposition of sugar. In soil, sugar is seldom present; so evidently in nature these organisms obtain their energy from the decomposition of some other sort of organic matter. Nature's nitrogen economy. It may be well to sum up the nitrogen transformations and observe just how nature's nitrogen supply is conserved and used over and over again. Green plants, with the EFFECT OF BACTERIA ON PLANT NUTRIENTS 193 aid of sunlight as a source of energy, are able to utilize simple inor- ganic forms of nitrogen, primarily nitrates, and build them up into complex proteins. Plant proteins are eaten by animals and con- verted into proteins of other sorts, little energy being required in the process, any necessary energy being derived from the oxidation of other organic material. Eventually all nitrogen eaten by animals, with the exception of that in their body tissue, is eliminated in the form of urea, a fairly simple compound, but too complex to be as- similated by plants. Urea, as well as such nitrogen as is left in pro- tein form upon the death of plants or animals, is attacked by decom- position bacteria and eventually converted into ammonia (ammoni- fication), a process from which the bacteria are able to obtain energy. In this process some ammonia escapes into the air in gaseous form and is lost, but most of it remains in the soil in the form of ammonium salts. Some plants can use ammonium salts as a source of nitrogen, but to what extent is unknown. In general, plants are thought to obtain their nitrogen from nitrates. Nitrates are formed from ammonium salts by the action of the nitrifying bacteria, which can obtain their energy from purely inorganic sources, the oxidation of ammonia to nitrite and water, and of nitrite to nitrate. These proc- esses would complete the cycle except for the denitrifiers which de- compose nitrate to nitrite, to ammonia, or even to free nitrogen, thus Undoing the work of the nitrifiers and causing a partial loss of nitro- gen from the soil. To counteract the effects of the denitrifiers and the partial loss of ammonia in ammonification, recovery of nitrogen from the air is necessary. This is performed largely by the bacteria that live in the nodules on legume roots, and also to some extent by certain free-living bacteria and fungi, these organisms obtaining the Necessary energy by the decomposition of some form of organic Platter. Thus the permanent loss of any nitrogen is prevented. ORGANIC MATTER IN THE SOIL Now that the transformations of carbon and nitrogen, the two important elements in organic matter have been discussed, it will be well to review the facts learned about organic material in the soil. The organic part of the soil it will be remembered, is spoken of as humus. Now we see why humus cannot possibly be a single chemical compound or even a mixture of a few different compounds. The organic materials that get into the soil are extremely varied, 194 BACTERIOLOGY derived from all sorts of plant and animal sources. Once in the soil, they undergo decomposition of different kinds, depending upon the material to decompose and upon the microorganisms present. At any given time the humus in the soil consists of a complex mixture of all these materials in various different states of decomposition, and it stands to reason that an almost infinite number of different chemical compounds must be present. If there were any way of learning just what compounds were pres- ent in any soil, much information could be obtained as to the produc- tiveness of that soil, and many attempts have been made to obtain such information. An actual analysis is at present impossible. The ordinary soil analysis merely shows the abundance of the different elements without showing whether they are in readily available forms. At first it was thought that these analyses would prove of much practical value; but the more the chemistry of the soil was studied, the more difficult it became to show any correlation between the soil analyses and fertility. What we would like to know is the extent to which the organic matter of the soil has decomposed and its availability to plants. It has been noticed, however, in the course of such studies, that the more completely decomposed humus is, the smaller the amount of carbon it contains in proportion to the amount of nitrogen. This can be readily understood after what we have just learned about these two elements. As carbon decomposes, it is converted more and more into carbon dioxide, which escapes into the atmos- phere; but only a small part of the nitrogen is converted into gaseous compounds. Thus in a soil containing fairly fresh organic matter, the carbon content may be fifteen times the nitrogen content, but in the same soil after the organic matter is largely decomposed the carbon content may be less than ten times the nitrogen content. Everything else being equal, the former soil can bear better crops than the latter. But as so many other factors are involved in fer- tility, it is still uncertain just how much help this determination may be in learning the productiveness of any soil. BACTERIA IN RELATION TO MINERAL MATTER There are certain elements of plant food that are necessary to plants in small amounts, but which are never present in organic matter in abundance. Most important among these elements are EFFECT OF BACTERIA ON PLANT NUTRIENTS 195 phosphorus, potassium, sulfur and iron. They are generally pres- ent in so much greater abundance in the soil minerals than in the organic matter of soil, that plants ordinarily obtain them from mineral rather than from organic sources. They are less often than nitrogen the limiting factors in fertility, although it is frequently of value to add either phosphorus or potassium to a soil. There is much less to be said about bacteria in relation to these elements; but plants cannot live on undecomposed rock, so that even in this particular the bacteria are of value. Furthermore, in spite of the small amounts of these elements used by plants, it is self-evident that in course of time Nature's supply would be exhausted unless in some way they might be again rendered available for plant use. Bacteria, then, help in two different ways to supply plants with an available source of these elements: They break down organic matter in which these elements exist and free them in simpler forms; they help decompose rock and the mineral matter in the soil, con- verting these elements into soluble form. The former action is merely part of the general decomposition processes already discussed. In the course of the decomposition the phosphorus, sulfur, potassium, and iron contained in the organic matter become detached from the rest of the molecules and are converted into simpler forms, even though the bacteria that accomplish this have no special relation to the elements in question. It is not yet entirely understood just what happens to these elements in the course of this process, as the activity is a complex one, depending upon the organisms that take part in it and upon the organic matter attacked. But it is known that the phosphorus is ordinarily converted into phosphates and much of the sulphur into hydrogen sulphide. The influence of bacteria in decomposing rock and rendering mineral materials soluble has been more thoroughly studied. Rock is first broken up by frost action and the other processes of weather- ing; but as soon as microscopic life is able to get a foot-hold upon it the carbon dioxide and organic acids produced by the microorganisms assist in its decomposition. Much of the phosphorus in rock and in soil occurs in the form of insoluble phosphates and much of the Potassium in the form of silicates, which are also insoluble. In part these elements may be made available by acids or other solvents secreted by plant roots; but undoubtedly bacteria play an even larger part in the process. Bacteria are known to produce carbon 196 BACTERIOLOGY dioxide and organic acids, agents perfectly capable of dissolving the insoluble phosphorous and potassium compounds. Bacteria are therefore thought to be quite important in rendering these two elements available to plants. The relation of bacteria to iron is less understood, although there are known to be bacteria that make use of iron in their metabolism; so in the transformations of this element they evidently have a part to play. Their relation to sulfur is more important. The sulfur of mineral origin in the soil generally exists in the form of sulfates which are ordinarily soluble and available to plants; and therefore bacteria are of comparatively little significance in rendering mineral sulfur available. But organic sulfur, in the course of its decomposition, is converted largely into hydrogen sulfide, a gas which escapes into the air and cannot be used by plants as a source of sulfur. It is inter- esting, therefore, to find that there are bacteria which can use hydro- gen sulfide, converting it into free sulfur, and other bacteria which can oxidize free sulfur into sulfates. Such bacteria are not very numerous; but undoubtedly they are abundant enough to keep plants supplied with the small amount of sulfur they need. These mineral elements are needed not only in the metabolism of higher plants, but in that of animals as well. Phosphorus, more- over, seems to be an especially important element in bacterial nutri- tion. Some kinds of bacteria seem to have a greater need of phos- phates than of nitrogen compounds in the medium in which they live. Hence it follows that while bacteria are important in rendering phosphorus available to plants, they also need a good supply of this element themselves. This interaction of bacteria upon fertilizers and of fertilizers upon bacterial growth is one that must be constantly remembered, although as yet it is not fully understood. An especially complicated interaction of this sort occurs between bacteria and the bases calcium and magnesium. These two ele- ments are of very little importance as plant nutrients; but their car- bonates are of great significance in soil because of their influence upon acidity. The growth of bacteria and of plants tends to cause the accumulation of acids to such an extent that the soil would in a comparatively short time be unable to support plant growth, unless neutralized. Lime and magnesium carbonates are quite strongly basic and can combine with these acids, thus neutralizing them. EFFECT OF BACTERIA ON PLANT NUTRIENTS 197 Hence the presence of these carbonates in sufficient abundance to neutralize the acids is a matter of great importance to plants, al- though the bases themselves are of small value to the plants as nutrients. For this same reason these two bases are important to bacteria, as bacterial growth is retarded if the soil becomes acid. But on the other hand, bacteria act on lime and magnesium carbonates. The carbonates are soluble only in water that contains carbon dioxide or other acid. These acids are produced by bacteria, and as a result the carbonates are dissolved and tend to be washed out of the soil. Bacteria, therefore, are responsible for a large part of the loss of lime and magnesia constantly occurring in soil. This loss, however, is partly counteracted by the action of bacteria upon organic matter and upon certain lime-containing rocks, which may result in the production of carbonate. In other words, the relation of bacteria to calcium and magnesium is a complex one, not wholly understood, but one which is of considerable importance in maintaining the Productivity of soil. REFERENCES E. F. Smith. Bacteria in Relation to Plant Diseases. Vol. II. Carnegie Inst., Washington, 1911. See section on Root Nodules of Leguminosae, pp. 97-146. T. J. Bttrrill and R. Hansen. Is Symbiosis Possible Between Legume Bacteria and Non-legume Plants? Illinois Agric. Exper. Station Bui. 202. 1917. CHAPTER VIII Practical Lessons from Soil Bacteriology CONTROL OF BACTERIA IN THE SOIL As yet the practical results obtained from our knowledge of soil bacteria are few. There is no reason why eventually soil practice may not be as much modified by knowledge of the bacteria as dairy practice has been. After it is known just what conditions favor both the beneficial and the harmful bacteria in the soil, it should be possible to control the bacterial flora of the soil so as to stimulate the growth of the one and to suppress the other. Certain results along this line have been obtained and they probably point in the direction in which practical soil bacteriology will develop in the future. Four different methods of controlling the soil bacteria have been found of practical value: (1) by inoculation, (2) by modifying the soil, (3) by partial sterilization of the soil, and (4) by crop selection. By inoculation. One of the earliest means by which it was hoped to make bacteriology of practical use in soil management was by inoculation. As soon as it was learned that certain bacteria were more beneficial to vegetation than others, it was thought that it might be of practical value to transplant these bacteria from soils in which they were present to other soils in which they did not occur naturally. Great hopes were attached to this method of improving soil conditions, and various different kinds of bacteria were experi- mented with. Best known among them was a preparation put upon the market under the name of Alinit. Alinit was a culture of a species of bacteria which a German investigator, Caron, had found unusually abundant in a certain especially productive soil. He claimed that this organism enriched the soil by utilizing atmospheric nitrogen. It was sold for a while; but careful experiments showed that it was of no benefit to the soil beyond the fertilizing value of the material upon which the bacteria in the cultures were living. Subsequent work showed it to be an organism (Bacillus cercus) which is widely distributed in nature and is very common in all soils. It is not ordinarily a very active organism in soil; but its PRACTICAL LESSONS FROM SOIL BACTERIOLOGY 199 spores are practically always present, and the futility of inoculating with Alinit is therefore manifest. Various other attempts have been made to improve soil by adding bacteria that are known to produce beneficial nitrogen transforma- tions-nitrogen-fixation or nitrification, for example. In general they have failed. Ammonifiers, nitrifiers and nitrogen-fixing bacteria are almost universally distributed, and if they do not grow in any soil it is ordinarily because conditions do not favor their growth. Every soil, moreover, is stocked with its own flora; and as we have seen, the organisms composing that flora have gradually adapted them- selves throughout the ages to the conditions in that soil and have reached a state of equilibrium. Under such circumstances there is little chance that any foreign microorganism could flourish in rivalry with the natural flora and establish itself in sufficient numbers to be of practical importance. In studying dairy bacteriology we have seen that it is possible to control such processes as cream-ripening by means of bacterial inoculation, even without previous steriliza- tion; but in that case an organism is added which is especially adapted to growth in milk or cream. In soil, however, there is little chance that any microorganism especially fitted to live in that particular environment should have escaped being introduced into it spontaneously sometime during the course of the ages since the soil was first formed. For this reason it is illogical to attempt soil inoculation unless the soil has first been sterilized or has been so modified as to adapt it to the organism one desires to introduce. Sterilization of field soil is impractical; but there is no reason why, as our knowledge of the subject increases, we cannot modify soil conditions in such a way as to adapt them to an organism not nor- mally present. As yet, it has proved practical to do this only in the case of the legume organism. When any variety of the legume organism is inoculated into a soil not previously stocked with it, seeds of the legume to which it is adapted are sown at the same time. The roots of the young seedlings, as they develop, offer conditions suited to the freshly introduced bacteria. For this reason legume inoculation, properly practiced, is a success. The relation of the legume bacteria to nitrogen-fixation was dis- cussed in the last chapter. The practical application of their powers is nothing new. Legumes were used to enrich the soil long before it 200 BACTERIOLOGY was known why they enriched it. Now that their value is under- stood, farmers are definitely instructed to use a legume in all their rotations, and so far as possible to see that all of their land has some legume growing on it every few years. There are two ways of returning to the soil the nitrogen thus obtained from the air. The first is called green-manuring, and consists simply in plowing the leguminous crop into the soil. There it decomposes and the nitrogen is eventually converted in large part into nitrates. The second method is to feed the crop to the stock, plowing in only the roots, and then using the manure on the field. By this latter method rather more of the nitrogen is lost, for as we have already seen, nitrogen losses are likely to be high in decomposing manure; but nevertheless the method is often more satisfactory than green-manuring. By letting the cattle convert the crop into manure, the fixed nitrogen is used twice, first as food for the animals and then as fertilizer for the crops. Farm manures, moreover, are more easily converted into humus than green-manures, and have a more beneficial effect upon the physical condition of the soil. In some soils, indeed, decomposi- tion takes place so very slowly that green-manuring is out of the question, although animal manures can be used quite satisfactorily. Naturally, the successful utilization of atmospheric nitrogen by means of legume cultures is possible only if the legume selected finds the bacteria adapted to it in the soil in which it is grown. Sometimes it will happen that a legume which grows luxuriantly in one locality makes a very poor growth in another. In such a case it will or- dinarily be found that in the latter locality the roots of this legume do not produce nodules. This means either that the right kind of bacteria is not present in the soil or that conditions are not right for their growth. In such cases it has been found that inoculation of the soil with the proper kind of legume bacteria is often of great practical value. This was realized as long ago as 1896, when a prep- aration known under the trade name of Nitragin was brought out by some German investigators and placed upon the market. Nitra- gin was a culture of the legume organism, and under proper condi- tions it did secure the inoculation of the legumes and the fixation of atmospheric nitrogen. The first cultures, however, were used with but indifferent success, partly because it was not then realized that different varieties of the organism were needed for different legumes and partly because of the difficulty in keeping the bacteria alive for 201 PRACTICAL LESSONS FROM SOIL BACTERIOLOGY any length of time in artificial culture. As these difficulties were understood the original cultures were modified. Agricultural Ex- periment Stations took up the matter, as did the United States Department of Agriculture; and at the present time there are not only a number of different commercial establishments that prepare these cultures, but the Department of Agriculture and many of the experiment stations distribute them. The cultures prepared today are ordinarily more satisfactory than those prepared at first. They are furnished with the name of the legume to which they are adapted written on the label, and they are generally put up in such a form- sometimes in boxes containing sterilized soil-that they do not die out so quickly. Another method of inoculation for legumes which has been used with rather more uniform success is by transporting soil from some field in which the legume in question makes a good growth to the field in which it does not bear nodules. There is no question about the success of this method, as it has been put into practical use by farmers in many parts of the world. The objections to it are the freight charges necessary to transport large bulks of soil and the danger of transporting troublesome weeds or plant diseases. For this reason there is no question but that pure culture inoculation will be more practical than inoculation with soil, just as soon as the cultures put upon the market are uniformly reliable. Sometimes a farmer uses a culture of the legume bacteria without success and blames the culture when the fault actually lies in his soil. It may be either that the soil was already stocked with the necessary bacteria before inoculating or else that the soil conditions did not favor the growth of the organisms with which he inoculated it. In the former case the farmer could have found nodules on the roots of his legumes before inoculation, had he searched for them, and would have known that some treatment such as fertilization, rather than inoculation, was necessary in order to give him a good crop. In the latter case, inoculation should be accompanied with the proper soil treatment (liming, for instance, if the soil is too acid) to assure a good growth of the bacteria. Naturally, if the bacteria are already present, or if the soil conditions are such that they can- not grow, failure to obtain an improved crop after inoculation is not due to any fault of the culture used. For this reason no one is advised to go to any great expense in inoculating his fields until he has tested the treatment in an experimental way on a small area. 202 BACTERIOLOGY By modifying the soil. The dependence of the bacteria upon physi- cal and chemical conditions in the soil, although it does make inocu- lation difficult, has its beneficial aspect. By modifying the soil conditions the flora can be modified, without introducing new organisms. By cultivation, for instance, it is possible to stimulate the bacterial activities that go on most rapidly in the presence of a good supply of oxygen, and as the beneficial activities are generally aerobic, soil conditions are improved. Probably this explains in part the good effects of cultivation. Of course it is not to be for- gotten that cultivation improves the structure of the soil and helps to conserve the moisture, both of which have direct effects upon the crops; but undoubtedly a large part of the benefit is indirect, resulting from the stimulation to the aerobic activities of bacteria. The same thing may be said of the effects of lime. The beneficial activities cannot go on in the presence of an appreciable amount of acid. Certain microorganisms, such as yeasts, molds, and a few special bacteria can live in the presence of considerable acid or per- haps even grow better under such conditions than in the absence of acid, but these organisms do not carry on the important transforma- tions in organic matter. Correcting the acidity of soil is therefore a means of stimulating desirable bacterial activities. Liming has some direct influence upon plants; but perhaps its greatest benefit is due to its effect upon bacteria. Fertilizers probably have a similar influence, although less is known about it. Their direct action upon plants is so striking as to overshadow completely their indirect action due to their effects upon bacteria. Nevertheless they must have some such indirect action. All the forms of nitrogen, phosphorus and potassium used as fertilizers have food value for bacteria as well as for plants. Their addition to the soil probably stimulates some kinds of bacteria, and may depress others, resulting in changed bacterial activities. Little is known on the subject, however, and it is generally consid- ered that the principal use of fertilizers is as plant foods. By partial sterilization. This is really a method of controlling the soil flora by modifying soil conditions, although it is so different from the methods just mentioned that it deserves separate discussion. When (about 1893) the experiment was first tried of treating the soil of vineyards in France with carbon disulphide, it was not sup- posed that the treatment would have any beneficial effect beyond PRACTICAL LESSONS FROM SOIL BACTERIOLOGY 203 killing the nematodes that were infesting the roots of the grape- vines. The effect, however, was so much better than expected and was so evident even where nematodes had been causing no trouble, that there was evidently some other benefit from the treatment besides the killing of nematodes. It was discovered elsewhere, at about this same time, that if soil was heated and then used for plants, their growth was better than in unheated soil; and later it was ob- served that other volatile antiseptics besides carbon disulphide often had the same effect upon plant growth. Naturally, it was supposed at first that the benefit to plant growth must come from the effect of sterilization upon the soil bacteria; but this idea was hard to accept, because at just about that time, scientists were beginning to appreciate the extreme importance of soil bacteria and the dependence of plants upon their activities. How could plant growth be improved by a treatment which would presumably kill some or all of these bacteria? This ques- tion was one of so much interest that it was immediately attacked by many different investigators. It was found that partial steri- lization decreases the number of bacteria in soil only for a very short period, after which they increase to much above their original numbers. There have been many theories proposed to explain this great increase. It has been suggested that partial sterilization in- creases the food for bacteria by killing the larger organisms in the soil and thus making them available. Another theory, rather more generally accepted, is that the treatment kills certain harmful or- ganisms, thus destroying the normal equilibrium and allowing the remaining microorganisms to increase to an abnormal extent. A special form of this theory, held by some, is that normal soils contain large numbers of Protozoa, which ordinarily prey upon the bacteria and thus prevent them from reaching as high numbers as is possible after the protozoa have been killed off by partial sterilization. Others believe that the results are not due to the killing of any microorgan- isms but rather to the direct stimulating effect of antiseptics used in small quantities, or to the increase in availability of soil organic matter after heating. The probability is that no one of these theories is the sole explanation, but that they are all true in Part. All these theories are mentioned merely to show that here is one of the obscure points in soil bacteriology that still remain to be investigated. 204 BACTERIOLOGY Whatever the explanation, the fact remains that partial sterili- zation is ordinarily a benefit to soil, and the problem immediately arises as to whether the treatment has any practical value. Small quantities of soil can be heated quite easily; and occasionally this method has been used in greenhouses with good results. One of the drawbacks has been found to be that the soil seems to be slightly toxic immediately after heating, causing poor germination of seeds and slow growth of seedlings. With some soils the recovery from this toxic stage is very rapid, with others it lasts a week or two; and therefore it is generally advisable to wait awhile after heating soil for a seedbed before planting seeds in it. With this precaution the treatment can be used in the greenhouse; but the immense diffi- culties in the way of heating field soil make it of little or no practical value in the field. Methods have been devised for running steam into the soil; but if they are of any real value it is only under special conditions. Under some conditions it is possible to heat the soil by burning off brush or grass-a procedure which has long been known to be of benefit to the soil, although its advantages were previously supposed to be due solely to the fertilizing value of the ashes. Under field conditions the only practical way of applying partial sterilization is with volatile antiseptics. It has been found possible to improve soil by boring holes a few inches deep, pouring carbon disulphide into them and filling them up again. This has resulted in increased crop yields, although it has never been shown that the method is financially profitable. By crop selection. Undoubtedly one of the most practical means of controlling the soil flora will be found to be by the use of proper crop rotations; but at present only a very few applications of this method are known. Best known is the use of legumes. The prac- tice of including a legume in every rotation is really one means of modifying the soil flora. Undoubtedly, when the matter has been more thoroughly studied, it will be found that certain rotations are advantageous and others disadvantageous because of the influence of crops in these rotations upon the soil bacteria. It has been found, for instance, that nitrates accumulate under certain crops and are exhausted rapidly under others. The explanation of this fact may be that different crops utilize different forms of nitrogen; but the probability is that certain crops stimulate nitrification and hence cause an increase in the soil nitrate. In this case the ideal procedure PRACTICAL LESSONS FROM SOIL BACTERIOLOGY 205 would be to have some such crop always precede any crop which makes an especial drain upon nitrate nitrogen. It is especially important that, under ordinary conditions, some crop be kept growing on the soil during the whole of the growing season. This is not because of the influence of the crop upon the bacteria, but because the nitrates produced by the bacteria are soluble and will drain away unless some crop is growing on the soil to make use of them. In former times it used to be thought that the soil should have a rest after a number of years and for one growing season should be kept fallow. Fallowing is still practiced in some localities, but never to give the soil a rest, in the strict sense of the word. The chief if not the only need of fallowing is in dry regions where there is not sufficient rainfall to support a crop each year, although by saving the moisture in the soil one year a good crop can be grown the next. In all but arid regions fallowing is no longer considered good practice. Instead it is recommended that farmers go to the other extreme and use the soil for some minor crop after harvesting the major crop, thus letting the soil stay idle only in winter. Such minor crops are known as cover crops or catch crops. Generally a legume is used for this purpose, and is plowed into the soil as a green manure. Methods designed to stimulate nitrification, ammonification, and so forth, are valuable because they render available the material already in the soil; but it will be seen that they do not add anything to the permanent fertility of the soil. Each crop removes a certain amount of nutrient material, and although such practices as liming and cultivation cause a higher yield of the following crop, they also cause that crop to remove more of the nutrient material in the soil than it would have used otherwise, and thus to bring so much nearer the final exhaustion of the soil. The great value of the nitrogen fixing bacteria, as we have already seen, lies in the fact that they the supply of nutrient material in the soil rather than render available that with which it is already supplied. But there are °ther necessary elements besides nitrogen, and each crop removes some of them. In some way the supply of all food elements must he kept up if we would maintain the soil in permanent fertility. MAINTAINING PERMANENT FERTILITY 206 BACTERIOLOGY Except for the flood-plains of the Nile, where the overflowing river annually fertilizes the soil, there is only one country where fields have been kept under cultivation, without the addition of mineral fertilizers, for so many centuries that they may well be said to have been kept in permanent fertility. That country is China. The Chinese have maintained the fertility of their soil by allowing no product of the soil to go to waste and by returning to it eventually everything that comes from it. They have no sewers, and have developed a very elaborate system of saving, collecting, and distrib- uting over the fields, all forms of animal and human excreta. By this means they prevent the loss of almost everything except nitro- gen; and by means of nitrogen-fixing bacteria and legumes, Nature undoubtedly makes up for the loss of nitrogen. The Chinese system hardly appeals to. western ideals and indeed has decided objections from the sanitary point of view. Under western conditions, even with the greatest possible conservation, there is bound to be a loss of nitrogen in large amounts and of ap- preciable amounts of potassium and phosphorus, which are daily emptied into the ocean in the form of sewage. We have already seen how the nitrogen supply may be maintained by the use of legumes; and obviously they must be used much more extensively under our conditions than with the Chinese, who save all their nitrogen except that which escapes into the atmosphere in the course of decomposition. The elements phosphorus and potassium, however, cannot be replaced by natural means. The only way we can replace the phosphorus and potassium thrown away in the sewage is by the use of artificial fertilizers. Of these two elements, phosphorus is the most necessary, because in most soils potassium is moderately abundant. Fortunately phosphorous fertilizers are much cheaper than those containing nitrogen. The principal means, therefore, of maintaining the permanent fertility of the soil are: (1) the intelligent use of legumes, together with green-manuring or live-stock farming, according to which is most practical under local conditions; (2) the use of mineral ferti- lizers, especially phosphates. Such a practice, of course, does not appeal to the tenant farmer, who rents his land for a short time only. He desires the greatest possible immediate money returns, which can often be obtained by some readily available fertilizer like potassium nitrate. But the landowner, who is interested in keeping his land PRACTICAL LESSONS FROM SOIL BACTERIOLOGY 207 from deteriorating, should endeavor to return to the soil as much nutrient material as is removed from the farm. HANDLING MANURE In preventing all unnecessary removal of nutrient material from "fhe farm, it is especially important that none of it be lost from the pianure. Manure is particularly rich in nitrogen, and nothing fesens its value as a fertilizer so much as the loss of this element -from it. As we have already seen, the processes, ammonification, denitrification and nitrification, go on very vigorously in the manure pile, and as there is always some loss of nitrogen before these proc- esses are complete, the danger of losing part of the value of the ir/anure is no small one. Outside of the dairy industry, bacteriology has made no greater practical contribution to agriculture than in Reaching the necessity for careful handling of manure. Not many years ago it was a common sight to see large piles of manure outside of stables, entirely unprotected from the rain, through which the water would leach and carry much of the soluble nutrients into a neighboring stream or into some pigsty or yard not used for vegetation. Liquid manure would sometimes be saved, but often large amounts of it were allowed to soak into earth floors and were never recovered. Such bad management is still found today in unprogressive communities, but it is much more uncommon than it was. Under good management it has been found possible to save all but about 15 per cent of the nitrogen in manure; while under poor management almost half of it is often lost. The important principles to be kept in mind in handling manure are: (1) the bulk of the nitrogen is in the urine, which must not be lost but must be absorbed in soil or some form of bedding which can be applied to the field; (2) the longer manure stands before being applied to the field, the greater the nitrogen loss; (3) protection from the weather prevents loss by leaching; (4) storing upon an impervious floor saves the liquid portions of the manure; (5) exclusion of air by compacting and occasional moistening with water or liquid ma- nure lessens the amount of ammonification and prevents some loss of ammonia. The realization of these facts has led to a number of improvements in methods of handling the manure. It is now a very common practice to spread it on the field at very frequent intervals, without allowing it to accumulate in piles. This is a procedure to 208 BACTERIOLOGY be recommended whenever weather conditions and farm practices permit, although fresh manure is sometimes slightly harmful to plants especially if it contains much straw. In places where im mediate spreading does not prove best, covered manure she< with concrete floors have proved advisable; and often it is customa to let the cattle have their exercise in these sheds, thus keeping manure thoroughly compacted. Another method of conserving manure, dependent upon an eny tirely different principle, has also been proposed. This is by addijwi some preservative. Some of the preservatives suggested for manner are germicides; but in general germicides are unsatisfactory they stop the desirable as well as the undesirable fermentation. More satisfactory are chemicals that combine with the ammojfy, that is formed and prevent it from escaping into the air. NeatfHf any acid will do this; but most acids are either too expensive, tUfr caustic, or undesirable for other reasons. The addition of aOU phosphate (rock phosphate treated with sulphuric acid) to however, has proved quite practical, not only because it fixes th ammonia, but because the addition of phosphorus to manure make it a more complete fertilizer. A preservative very commonly use(j in Europe is dried peat, which is employed in the place of straw 01 sawdust as a litter. In some way not yet understood, peat keep, down odors, prevents the loss of nitrogen, and acts as an especially fine absorbent for liquids; hence its use is very practical, where available. Besides being a source of nitrogen, manure is very valuable be- cause of its organic matter, which increases the humus in the soil. As manure stands the organic matter decomposes, so that the bulk of rotted manure may be but a quarter of that of fresh manure. For some purposes this rotting seems to be advantageous, greenhouse men always preferring rotted manure for their pots and seed beds. Nevertheless the loss during the rotting is tremendous, and it is perfectly possible that much humus-forming material is thus dissi- pated. The changes during the decomposition are so complex, how- ever, that little advice can be given as yet as to how to get the most humus into the soil from a given quantity of manure. . BACTERIA IN RELATION TO MISCELLANEOUS INDUSTRIES CHAPTER IX The Preservation of Food One of the oldest practical applications of bacteriology is in the after of food preservation. It has been known from time im- * smorial that moist food undergoes changes that generally render offensive and finally unfit for use. It has also been known for a J ng time that the spoiling of food can be delayed by cooling and ■evented by drying or sometimes by salting and smoking; and tore recently-although before the beginnings of bacteriology-the ' -rocess of canning was found to preserve certain kinds of food that 3ould not be kept by any of the older methods. The preservation )f food has always had a great practical value, because so many kinds of food are abundant at certain seasons and scarce at others; so it is natural that information accumulated on this subject long before it had a scientific basis to rest upon. But the discovery of microorganisms and their agency in decomposition has opened up new methods that have completely revolutionized certain industries connected with food production and distribution. Everyone is familiar with the souring of milk, the molding of bread, the decaying of meat, the rotting of fruit, the fermenting of fruit-juices and other similar phenomena. Anyone who has ever thought at all on the subject realizes that different kinds of food undergo different kinds of decomposition; and the student who has followed the discussions in this book will understand that the differ- ent kinds of decomposition are due to different kinds of micro- organisms. But the practical value of this information is not obtained until it is learned what kinds of microorganisms cause which types of decomposition and which kinds of food are liable to attack by each. THE SPOILING OF FOOD 210 BACTERIOLOGY Bacteria attack any kind of food suited to their growth. Yeasts and molds are ordinarily present and can grow in nearly any kind of food; but the bacteria are able to grow so much more rapidly that when conditions are favorable to them they prevent the growth of other microorganisms. The conditions that favor the growth of bacteria are: (1) plenty of moisture; (2) a neutral reaction, as bac- teria are very susceptible to the harmful influence of acidity or alkalinity, especially the former; (3) absence of growth-inhibiting substances, even including such common household preservatives as salt and sugar (if used in sufficient quantity). Foods, therefore, that are liable to bacterial decomposition are: fresh meat, eggs, milk, and moist vegetables. Yeasts grow under much the same conditions as bacteria except that they can stand more acid and more sugar. In fact yeasts seem to prefer materials that are distinctly acid and contain large amounts of sugar. They will grow in the most acid fruit juices and in solu- tions containing so much sugar that bacterial growth is impossible. Yeasts require the presence of considerable oxygen; whereas many kinds of bacteria are able to grow in the complete absence of air. Molds are like yeasts in tolerating acid and sugar and in requiring the presence of oxygen; but they differ from bacteria and yeasts in preferring less moisture, growing, in fact, in materials that are sur- prisingly dry. Molds, therefore, grow not only with yeasts in fruit juices and jellies, but also in fairly dry foodstuffs like bread and flour. The practical value of knowing whether a food is liable to decom- position by bacteria, by yeasts or by molds is because of the dif- ferent methods necessary to protect food from the action of these three groups of microorganisms. The principles to remember are: yeasts and molds are easily killed by heat, mere boiling or even the use of still lower temperatures being sufficient, while some bacteria produce spores that are able to resist temperatures above that of boiling water; exclusion of air prevents the growth of molds and yeasts but not that of certain kinds of bacteria; addition of sugar (in sufficient quantity) prevents the growth of bacteria but not that of molds or of yeasts. It is evident that the problem of food pres- ervation vaiies with the group of microorganisms which causes the spoiling; so the foods that are liable to bacterial decomposition and those that are not subject to attack from bacteria, can be considered separately. PRESERVATION OF FOOD 211 PRESERVATION OF FOOD LIABLE TO BACTERIAL DECOMPOSITION Food that is subject to attack by bacteria must either be so treated that these organisms cannot grow in it, or else all the bacteria in it must be killed and contamination with others prevented-a process which is more difficult than killing yeasts or molds because of the very resistant bacterial spores. The methods commonly used are drying, cooling, adding preservatives, and heating. Drying. Perhaps the most effective means of preventing bacterial growth is by drying, although this process is not applicable to those foods that are spoiled by drying. Drying does not kill the bacteria present, but they cannot multiply without water. Molds require less water than bacteria; so when dried foods become slightly moist, they are likely to be spoiled by molds rather than by bacteria. Methods of drying depend upon the kind of food. Some kinds of food, such as grains, peas, and beans, dry naturally; while others require artificial heating. Meats are sometimes dried naturally by placing in the sun in hot dry climates; but in ordinary temperate regions decomposition would set in before drying had progressed far enough to preserve the food. For that reason meats are ordinarily dried by the use of artificial heat. Nearly all forms of dried meat and fish with which we are familiar are smoked and salted as well as dried, this curing not only adding a pleasant flavor to the product but helping to preserve the meat because of the mild antiseptic properties of the salt and the materials absorbed from the wood smoke. It has recently been found that even such very moist foods as milk and eggs can be dried without being spoiled. The usual method °f drying them is to spray them into a warm chamber from which the air has been partly exhausted, where they are almost immediately converted into a fine powder. Dried milk and eggs are of use princi- pally in cooking, although good dried milk is not unpleasant to drink when dissolved in the proper amount of water. Of course dried foods are never like fresh foods even though enough water is added to bring them up to their original moisture content; but modern ingenuity has in many cases come near to making a product that tastes like the original. Cooling. Like drying, cooling does not kill the bacteria, but merely prevents or retards their growth. In this respect, cooling is 212 BACTERIOLOGY not as efficient as drying, for cooled foods cannot be kept indefinitely. The length of time cooled food can be kept depends upon the extent of cooling. Ordinary cooling in an ice-box merely preserves the food for a few days; while freezing preserves food for months. The chief advantage of cooling over drying is that the nature of the food is not changed unless it is actually frozen, and some foods can even be frozen without change. Refrigeration by ice has been known for a long time, but its use does not make it possible to keep food for a very long period. Com- paratively recently, however, the practice known as "cold storage" has been introduced and has revolutionized the distribution of food. Some kinds of food vary so greatly in their abundance and hence in their market value at different seasons of the year that any method by means of which they can be kept from the season of plenty till the season of scarcity is a matter of much practical importance. Not long ago we ate chickens only in winter because they spoiled so quickly in summer, and eggs only in summer because hens did not lay in winter. Now we eat chickens and eggs all the year-even though we do have to pay fancy prices at times for the latter commodity. The process of cold storage, which has brought about this revolution, is nothing more than keeping food at or near the freezing point. Some kinds of food, such as meat and fish, can be frozen solid and kept in that state for long periods of time. Butter also may be fro- zen. Eggs and vegetables, on the other hand, are spoiled by freezing and must be kept at higher temperatures. Eggs are generally kept at temperatures only a degree or two above freezing. Vegetables keep well at such temperatures; but as nearly all vegetables that can be stored keep fairly well at somewhat higher temperatures, artificial cooling is not so often used for them. The cold storage industry has become very important in recent years, and has conferred a great service upon society. This method of keeping food has been abused and has fallen somewhat into disrepute; but the folks who decry it do not realize how dependent they are upon the system. Cold storage eggs, to be sure, are not as good as fresh ones, and meat cannot be kept indefinitely frozen without de- teriorating; but cold storage results in saving much food that would otherwise be lost and is therefore of great economic importance. Because of the popular prejudice against cold storage, various legal restrictions have been adopted, some wise, and some unwise, so that PRESERVATION OF FOOD 213 it is now illegal at most places to keep food more than a certain length of time. But in general cold storage food is not objectionable if sold tor what it is and not under the guise of fresh food. Chemical preservatives. Another means of preventing the growth of bacteria in food is by adding to it some chemical that holds them m check. If the preservatives are powerful enough to kill the bacteria, however, they are likely to be dangerous to the health of those eating the food. In fact, the serious objection to the use of preservatives in food is that most of them are poisons and may make the food unwholesome. This is particularly true of strong germi- cides like formalin-which is sometimes used in milk, although illegally. Other chemicals, like benzoic and salicylic acids, have been widely used because they are efficient preservatives and do not affect the taste of food; and it is rather more doubtful whether they are harmful to health. They have been so widely used that they cannot cause much harm in the small amounts ordinarily employed. Nevertheless they are known to be poisonous in large enough quan- tity, and popular prejudice has been so strong against them that their use today is surrounded by many legal restrictions. Quite in a different class are certain time-honored preservatives that are added not only to save the food, but to impart a desirable flavor. The curing of meats is an example of this sort of preserving. So also is the household use of sugar, salt or vinegar to preserve foods. Vinegar (as in pickles) and salt can each be used in sufficient Quantity to prevent the growth of all microorganisms; but sugar is an effective preservative only against bacteria. It is often necessary, therefore, to give further treatment to food to which sugar has been added, or else it will be spoiled by fungi or yeasts. Certain vege- tables can be covered with salt and thus preserved, sometimes using enough salt to prevent all bacterial growth, or sometimes using less salt and allowing certain bacterial changes to take place which give the food distinctive flavors (e.g., sauerkraut). A similar process, although probably depending upon a different Principle is the preservation of eggs in water glass (sodium silicate). If eggs-strictly fresh-are covered with a solution of sodium silicate, they can be kept for months without bacterial action taking Place. Of course they do not retain their fresh taste for more than a month or two, but they are usable for a long time and are practically as good as cold storage eggs. 214 BACTERIOLOGY Heating. When heat is used in preserving food, the microorgan- isms are actually killed; and if the process is thorough and the food well sealed it can be kept indefinitely. This method of preserving food is much older than cold storage and in its day*worked as great a revolution as the newer method has in recent years. We have seen (p. 19) how Appert in 1810 devised the process of canning, even before it was known that fermentation and decay were caused by microorganisms. The first foods to be canned were fruits and Fig. 35. Glass Fruit Jars, as Used for the Home Canning of Fruits and Vegetables other products in which bacteria do not grow well; so the matter of heating and preserving them was fairly simple. When the method was first applied to vegetables like beans, peas and corn, difficulty was encountered at once. These products are favorable media for bacterial growth, and indeed are generally laden with spores of bac- teria capable of resisting high temperature for a considerable period of time. Plain boiling is not enough to kill these bacteria and the vegetables canned in that way could not be kept without spoiling. PRESERVATION OF FOOD 215 Later work has shown that it is possible to sterilize such foods, but that simple boiling is not enough. The method ordinarily used is practically the same as the bacteriologist uses in sterilizing his media; that is, heating under steam pressure to temperatures higher than the boiling point of water. By this method of "processing," to use the technical term, even the most difficult vegetables can be pre- served. It has also been found, however, that longer periods at lower temperatures are almost as successful, and in home canning this is the method beginning to be largely used. The vegetables are packed in the cans cold, then covered with water and brought to a boil, in a closed receptical, and kept at boiling temperature for the required length of time. Table 1 shows the length of time necessary for various vegetables, both with the use of steam pressure and in boiling water. This method of home processing, however, although it has been frequently used and although it has been recommended by the Depart- ment of Agriculture, is open to serious criticism. One organism in- deed, which is responsible for the fatal disease botulism (see pages 220 and 357) has been shown to be able to resist these temperatures for the times recommended, and to be able to produce its toxins while the cans are stored. For this reason home-canned vegetables are open to suspicion, except in the case of such as tomatoes whose acid content prevents the growth of the organism; and it is always well to boil the contents of such cans five or ten minutes before even tasting. Boiling destroys the toxin of botulism. Some vegetables, although liable to bacterial decomposition, can be preserved without the prolonged use of high temperatures. Tomatoes are the best known example. It will be seen from table 1 that twenty-two minutes at boiling is sufficient. Tomatoes are also very easily canned by simply stewing in an open kettle for a few minutes and then pouring into boiling hot cans. The ease with which they can be canned is because of the acid naturally present in them. The acid is not enough to prevent the growth of all bacteria; but in the presence of acid, bacteria are killed at lower temperatures than in neutral solutions, and therefore tomatoes are much easier to can than vegetables that are not acid. Fruits, although ordinarily subject to decomposition by yeasts or molds, will support the growth of bacteria in the absence of other microorganisms. The acid and the sugar present in fruits are more 216 BACTERIOLOGY than bacteria prefer, but not too much to stop their growth entirely. Hence the ordinary method of canning fruit is to add enough sugar to prevent the growth of bacteria and then to treat it as a food not subject to bacterial attack. The canning of fruits will therefore be taken up in the next section. Meats and eggs are not preserved by heating because of the changes produced in them by heat. Milk can be sterilized by heating; but as we have already seen (p. 150), the usual method of heating milk is pasteurization (that is, heating at low temperatures) which merely delays its spoiling. Condensed milk, with sugar added, is not a TABLE 1 VEGETABLE BLANCHING* STERILIZING Hot water Steam pressure Pressure Time minutes minutes pounds minutes Beets 3 to 8 90 20 35 Tomatoes (To loosen skin) 22 20 10 Corn 5 to 10 180 20 35 Beans (string or lima) .. 2 to 5 120 20 ' 40 Peas 2 to 5 120 20 40 Squash or pumpkin 10 90 15 35 * Blanching is immersing in hot water for the time specified, and then dipping briefly in cold water. Length of time for blanching* and sterilizing vegetables in cans favorable food for bacteria and can easily be preserved by heating. Some brands of cheese that decompose readily can be preserved by heat and are often sold canned; in which condition they are satis- factory, although their flavors and consistency are somewhat changed by heat. Preventing bacterial decomposition. Summing up, it seems that of the foods liable to bacterial decomposition (i.e., meats, eggs, milk and vegetables), meat is preserved for short periods by cooling, for long periods by freezing, curing, or drying; eggs by holding just above the freezing point, by keeping immersed in a solution of sodium silicate, or by drying; milk by cooling, by pasteurizing and then cooling, or by drying; vegetables by drying, salting, pickling, or canning. PRESERVATION OF FOOD 217 PRESERVATION OF FOOD NOT LIABLE TO BACTERIAL DECOMPOSITION The foods not liable to bacterial decomposition, it must be remem- bered, are those that are too acid, too sweet, or too dry for bacterial growth-such as jellies, some fruits and fruit juices, stewed fruits and vegetables to which sugar has been added, breads, pastry, and flour. The same general methods of preservation are used as when guarding against bacterial growth, but a few slightly different principles must be borne in mind. Drying. Many fruits can be dried and kept indefinitely, while flour is sufficiently dry for preservation unless kept in a moist place. Dried foods seldom become moist enough to allow bacterial growth; but they do often become moldy if not kept dry. Cooling. Refrigeration can be used for fruits as well as for meats, but if they are kept in cold storage, care must be taken not to let the temperature go below freezing, as fruits are injured if frozen. Not all molds are held in check even by temperatures almost at the freezing point, and as fruits are quite favorable media for fungi, it sometimes happens that fruits rot even in cold storage. Adding preservatives. Nearly all the preservatives applicable to fruit products are illegal. Salt injures the flavor, and sugar does not prevent the growth of molds and yeasts. Some can be put in vinegar and pickled, a process which is essentially equivalent to the addition °f a preservative. Various more powerful preservatives, such as borax and even formalin have been used commercially; but this practice is now forbidden. Heating. The process of canning fruits was developed considerably before heat could be successfully used as a means of preserving foods liable to bacterial decomposition. Simple boiling was quite early discovered to be sufficient to sterilize fruits, fruit juices and so forth. The reasons for this are easy to understand after what has been stated above of the affect of acids and sugar on bacterial growth. This is especially true of jellies, with their high sugar content, as anyone will realize who notices how long a tumbler of jelly may be kept exposed to all sorts of contamination without any sign of spoiling. The simplicity of preserving fruit products has lead to the common household method of canning which is essentially as follows: Fruit 218 BACTERIOLOGY and sugar are boiled together for a short time, thus stewing the fruit and killing all yeasts and molds present. Then they are poured hot into cans that have been boiled in order to kill the same kinds of organisms. The cans are sealed while hot, with the exclu- sion of practically all the air. When the material is poured into the cans, some contamination is bound to occur, bacteria and mold spores being almost universal. The bacteria do not find a suitable environment for growth, while the mold spores do not grow in the absence of air. Occasionally a small mold colony will appear in such a jar; but if well sealed, the mold seldom grows enough to do damage. TABLE 2 FRUIT BLANCHING STERILIZING Boiling water Steam pressure Pressure Time Strawberries None None None None 1 minute 1 minute lj minutes lj minutes minutes 16 16 16 16 16 16 20 20 pounds 10 10 10 10 15 15 15 15 minutes 5 5 5 5 5 5 6 6 Blackberries Sweet cherries Peaches Gooseberries Sour cherries Apples Pears Length of time for blanching and sterilizing fruits in cans Jellies are preserved in a very similar way; only in this case the containers are often left open until the jelly has hardened, and their contamination with mold spores is inevitable. Their subsequent sealing with paraffine or other air-tight seal prevents mold growth and ordinarily there is no danger of spoiling. Commercial canning of fruits, however, is more like that already described as applicable to vegetables, except that less heat is used. This method is also applicable to household use and has several advantages over the older method. The method is essentially to place the fruit in the cans cold, immersed in a syrup of sugar and water. Then the cans are kept in boiling water or steam for the length of time found best for the particular fruit being canned, after which they are removed and sealed. Table 2 shows the length of heating necessary for different fruits. PRESERVATION OF FOOD 219 FOOD POISONING The preservation of food for any length of time always opens up the possibility that undesirable or harmful decomposition products may develop in it. There are frequent cases on record of extensive poisoning resulting from the use of canned food or cold storage products. Fish, and especially shellfish, are thought to be particu- larly liable to changes of a harmful nature. The use of oysters, for instance, more than a few hours old, in the warmer months of the year is so likely to be harmful that in most markets these shellfish are not obtainable between April and September. The exact nature of the poisonous products produced in stored food, the organisms that produce them, and the illness caused by them are in most cases obscure, but they are generally thought to be of bacterial origin. Types of food poisoning. To understand this class of disorders it must be recognized that there are several ways in which sickness may be caused by food. (1) The food may be indigestible due to faulty handling or cooking. (2) The food may have been partly decomposed by the action of bacteria, producing poisonous sub- stances. (3) The food may contain pathogenic bacteria. (4) The food may in some little understood way disagree with the person °r persons eating it so as to cause the production of toxins in the intestinal tract. (5) The food may be actually poisinous to certain individuals, due to certain immunological reactions between its proteins and those of the person's body; i.e., anaphylaxis, a phenom- enon discussed in Chapter III of Part III of this book. Of these five types of disorders it is plain that the first bears no relation to spoilage of the food, and the last two probably not; while the last two are generally associated with peculiarities of different individuals so that they cannot be controlled by any improvement in methods of handling food. This leaves the third and fourth types of food poisoning as of special interest to the bacteriologist. Food poisoning due to pathogenic bacteria. Ordinarily when patho- genic bacteria are taken into the system with food, the sickness which follows is not spoken of as food poisoning. This is because in most cases considerable time elapses between the entiance of such bacteria into the body and the appearance of the disease, so that it is difficult, if not impossible, to associate the disease with the food from which the infection occurred. Sometimes, however, such a 220 BACTERIOLOGY violent epidemic arises in this way, occurring among people who have had some one common source of food that it is natural to suspect this food as being to blame. Typhoid fever is thus very often as- cribed to some food infection. The connection is also plain in the case of some less violent diseases when they have very short incuba- tion periods. It is quite possible, in fact, that some of the digestive troubles brought about by the use of some particular kind of food and generally spoken of as food poisoning may be actual infections by organisms that are able to produce their effects in the body very promptly. Food poisoning due to the action of bacteria on the focd. The effects most commonly referred to as food poisoning are those brought about by the partial decomposition of food through the action of microorganisms. This type of food poisoning is often called "pto- main poisoning," due to the rather mistaken impression of the early physiological chemists that it is due to the presence of a class of chemical compounds known as ptomains. This impression arose from the fact that compounds of this group had been found in spoiled food and had been found to be toxic. Subsequent investi- gation, however, has shown that food may be toxic from the action of decomposing bacteria without the presence of ptomains, and that even when the ptomains are present they are often not the truly toxic products. Nevertheless, these compounds have achieved a bad reputation and to the popular mind almost any form of digestive disturbance thought to be due to food is "ptomain poisoning." Especially prominent in the popular mind at present is a food poisoning known as botulism. This is really a distinct disease pro- duced by a definite microorganism, although the organism in ques- tion is a saprophyte and unable to grow in the human body. The disease is caused by definite chemical poison which this micro- organism produces when growing in certain food products. The organism is called Bacillus botulinus (or sometimes Clostridium botuminum). It is a spore-bearing organism which grows in the entire absence of air, its spores being so resistant to heat that they may survive if the heat of canning is not great enough or applied for a sufficient length of time. In fact, the recent publications by the United States Department of Agriculture on methods of canning (from which the tables 1 and 2 have been prepared) have lead to a considerable dispute, not yet wholly settled, as to whether this PRESERVATION OF FOOD 221 organism is killed by prolonged heating at the boiling temperature. Its growth in canned food does not cause such a noticeable decomposi- tion as that of some other organisms, and hence the food sometimes finds its way to the table-with disastrous results. The toxin produced by the organism, however, is destroyed by boiling; so the simple boiling of all canned food before use (for at least five minutes) removes the danger from this source. Tracing the cause of an epidemic. When an outbreak of disease occurs anywhere that is thought to be due either to water or to food, whether caused by pathogenic microorganisms or by toxins in the food, it is important that the cause of the outbreak be determined as soon as possible so as to prevent more cases. It is sometimes rather difficult to trace such an epidemic to its cause and it requires a man trained in this particular kind of work. As a result, public health work has developed a few specialists in epidemiology who are ln a way detectives skilled in locating the agents of disease instead of those of crime. In investigating such an epidemic the data is sometimes quite Puzzling, but the method of attack is really quite simple. The first step is always to obtain information from the patients, if possible, or if not, from those who are closely associated with them, as to the date of the attack and the food eaten on the day or days when infec- tion or poisoning may have occurred. Each statement of this sort obtained from a patient generally eliminates certain possible sources of the trouble and after several such statements have been obtained xt is often possible to find just one common food or drink that has been consumed on the day when the trouble apparently occurred. Then the rest of the investigation consists of determining whether all the other patients partook of this particular food and trying to account for the few cases that did not do so. As soon as the food or drink partaken by all the patients is learned is necessary to find how it became infected if the disease is an in- fectious disease, or to find out what was the cause of spoilage if ffie disease is a poisoning due to the products of decomposition. In fact the case against any article of food or drink is not complete Until this last mentioned information is at hand; and naturally this information is necessary in order to learn how to protect the public against another outbreak. CHAPTER X The Practical Use of Fermentations In the preservation of food methods are employed to prevent undesirable activities of microorganisms. Certain other fermenta- tions, however, are beneficial, and have been brought more or less under man's control so as to serve important practical ends. The problem here is to encourage rather than to destroy the micro- organisms. Foremost among the fermentations that have been put to practical use is the activity of yeasts in producing both alcohol and carbon dioxide, the former of which is used in the manufacture of alcoholic beverages, the latter in the raising of bread. Other practical processes in which microorganisms are known, or at least suspected, to play some part are: vinegar manufacture, tobacco curing, manufacture of sauerkraut and other salt pickles, the retting of flax and hemp, as well as the production of ensilage and other cured fodders. ALCOHOLIC BEVERAGES All alcoholic beverages owe their alcohol to the activity of yeasts, although in some the amount of alcohol is increased by artificial means. There are a very large number of different types of liquors, and even in times of prohibition, it is of interest to the bacteriologist to know the differences between the different kinds, although the knowledge may be of no practical importance. Various different materials, as is well known, undergo alcoholic fermentation and the various different beverages bear different names according to the material from which they are produced. Thus wines are made from grapes, beers from malt, and cider from apples. These plain fer- mented beverages are all distinctly different, moreover, from the distilled liquors (rum, whiskey, etc.) which have had the amount of alcohol greatly increased by the process of distillation. Wines are made by allowing grape juice to ferment. Ordinarily if left to itself the grape juice undergoes the desired fermentation- It is a very favorable medium for yeasts and not especially adapted to the growth of bacteria. It is almost sure to become inoculated PRACTICAL USE OF FERMENTATIONS 223 with yeasts because these are universally distributed and are almost always on the skins of the grapes. Sometimes, however, the fermen- tation does not proceed in the typical manner and the result is un- desirable. This is due to the growth of microorganisms other than the desired yeasts, and many methods have been proposed to rid the ripening wine of them. Heating has not proved practical because of the chemical changes it produces, and the method in most common use depends primarily on sedimentation. When grape juice has been partly freed of its microorganisms, its fermentation must be hastened by the use of a "starter" exactly as when butter is produced from pasteurized cream. The use of these starters has also been found to give improved results even in untreated grape juice. The starters are prepared by allowing ripe grapes to ferment and, if the fermenta- tion is desirable, the material is added to the unfermented wine. Pure cultures of the proper yeasts can be obtained commercially and give satisfactory results. Wines differ greatly from each other; but they all fall into certain general types. White and red wines differ according to the color of the grapes used and according to whether or not skins are removed before crushing. Dry wines have had their sugar entirely consumed by the yeasts, while sweet wines still contain sugar either because the fermentation has been stopped before its completion or because extra sugar has been added. Fortified wines have had their alcohol content increased by the addition of spirit obtained by distilling wine. Sweet wines are generally fortified, either because their alcohol content is Undesirably low or because the remaining sugar would be consumed by the yeasts but for the additional alcohol. The addition of alcohol stops further fermentation because yeasts cannot grow if more than a certain amount is present. Fortified wines are generally of too high alcohol content to support the growth of any microorganisms. Beers are made from grain. Grain, however, does not contain any sugar in the first place, and sugar is necessary for the production of alcohol. But it has been found that when seeds germinate a ferment {diastase} is produced, which converts the starch into sugar. Ger- minated grain in which the starch has been changed to sugar is known as malt. Malt is ordinarily made from barley although occasionally some other grain is used. In the process of malting, the grain is soaked in water and kept wet for between two and three weeks at a constant and fairly low temperature. The sprouted seeds 224 BACTERIOLOGY are then dried with mild but constantly increasing heat, so as to kill the seedlings without destroying the enzymes. The radicles are removed from the seeds by friction and the malt is ground and mixed with water, under which conditions the enzymes act rapidly and produce sugar (maltose) from the starch. The extract of the malt is known as wort. The wort is boiled to kill the microorganisms and destroy the enzymes; and hops are added for the sake of the flavor they give and the antiseptic effect of certain resins in the hops. Then the wort is allowed to ferment. Beers differ according to the kind of yeast that causes the fermen- tation. Some yeasts attack nothing but the maltose and as a result do not produce much alcohol. Others produce alcohol not only from the maltose but from certain other products of diastatic action on starch, known as dextrins, which are intermediate between sugar and starch; and as a result the beer has a high alcohol content. If wort is allowed to ferment spontaneously there is no certainty as to which kind of fermentation will take place, and as a brewer likes a dependable process, he generally starts the fermentation with a pure culture of the desired yeast. Large brewers often prepare their own pure cultures of yeast. It must be remembered that yeast produces not only alcohol, but also carbon dioxide, from the fermentation of sugar. In wines the latter is unnecessary and is allowed to escape into the air; but in beer a certain amount of carbon dioxide improves the taste and keep- ing qualities. In order to retain this gas, the beer is kept in tightly bunged casks after the fermentation has reached a certain stage, and no further carbon dioxide escapes. This very same fermentation as explained below, is used in bread-making for the sake of the carbon dioxide it produces, not for the sake of the alcohol. Sometimes abnormal flavors develop in beer either during or after the fermentation. They are generally spoken of as "diseases" of beer. It is subject to many different "diseases," but they are practically always due to the growth of undesirable microorganisms. Some are caused by undesirable yeasts, others by bacteria, and the abnormal flavor produced varies with the kind of organism causing the trouble. These troubles are practically prevented in modern breweries by cleanliness to avoid contamination as far as possible, and by pasteurization of the beer after bottling-a process which, it will be remembered, originated with Pasteur. PRACTICAL USE OF FERMENTATIONS 225 Various other fermented beverages are made, of which the best known in this country is cider. Cider is made from apples much as wine is made from grapes. Similarly perry is made from pears, and various other less common drinks are made by allowing other fruit juices to ferment. An alcoholic drink is made from honey known as hydromel or mead. In Japan, sake is made from rice, the starch having been previously converted to sugar by diastatic enzymes produced by a mold. Distilled spirits. Strong alcoholic drinks and commercial alcohol are both prepared by essentially the same method. Some material containing an abundance of starch or sugar is allowed to undergo alcoholic fermentation (preceded by the action of diastase, in the case of starchy substances) and then the alcohol is concentrated and purified by distillation. This method of purification depends upon the fact that alcohol has a much lower boiling point than water and will distill at a temperature so low that very little water is given off and the condensed fumes contain mostly alcohol. In preparing pure alcohol the process of distillation is repeated as often as neces- sary for complete purification. In preparing distilled liquors the first distillate is ordinarily used, and as this is not pure alcohol the character of the beverage varies with the fermented material. Thus brandy is distilled from fermented grapes, whiskey from fermented grain, and rum from fermented sugar cane. Alcohol is also prepared from sugar beets, from apples and from some other fruits. BREAD-RAISING The rising of bread is due to the same microbial activity that causes the fermentation of wines and beers, only in this case it is the carbon dioxide produced by the yeast that is desired instead of the alcohol. The gas produced by the yeast cells, when mixed with the dough, blows it full of innumerable small holes, thus causing it to "rise," and the subsequent baking volatilizes the alcohol and drives off the gases formed, leaving the bread light and spongy instead of tough and heavy as it would be if the dough had been baked before rising. The methods used are essentially the same whether a few loaves are made in the home or many in the large bakery. The first bread baked by mankind bore little resemblance to that which we ordinarily see on our tables today. It was probably discovered in the first place that pulverized grain forms a sticky mass 226 BACTERIOLOGY when mixed with water, which becomes edible upon heating. Such a bread has to be baked in thin slices or else it becomes entirely unmasticable. It is hard and brittle, and would never have appealed to the human taste sufficiently to become "the staff of life" unless methods of improving it had been learned. The first step in convert- ing it into modern bread was taken when it was discovered that if the dough were allowed to stand a day before baking it would or- dinarily swell and become lighter, and then if baked would be much Fig. 36. Samples of Dough Incubated Over Night Left, without yeast; right, with yeast added. more easy to chew. It was observed that sometimes the dough rose more rapidly than at others, sometimes the bread made from it tasted good and at other times was sour. The reasons for these differences were not understood; but they troubled the early cooks, who undoubtedly took as much-or perhaps more-pride in the uniformity and tastefulness of their cooking as their descendants do today. It was probably some such dissatisfied cook-one of the nameless inventors to whom we moderns owe so much-who dis- covered leaven. Perhaps some day she had unusual luck with her dough, which had risen very rapidly and was entirely free from sour- PRACTICAL USE OF FERMENTATIONS 227 ness. She wished she could repeat it another day, and the idea occurred to her that if she saved a little of this risen dough and added it to the next lot, her good luck might continue. Her success was so great that she repeated the experiment, and soon made a practice of saving a little of one lot of dough to leaven the next batch. Her friends soon copied her scheme, and so the practice spread. It was not for centuries afterward, when microscopes were in use, that yeasts were discovered in the leaven; and their isolation and commercial distribution is a very modern practice. Today, leaven has almost gone out of use in civilized communities, both the housewife and the baker having pure cultures at their disposal; and as a result, they can depend upon a fairly uniform rising of their dough. When dough is allowed to rise naturally or when leaven made from old dough is added, both bacteria and yeasts take part in the process. The actual production of gas which raises the dough is due mostly or wholly to yeasts acting upon the sugar. To hasten the process, sugar is sometimes added to the dough artificially; but its addition is not necessary, for dough ordinarily contains sufficient diastase to con- vert a small amount of starch into sugar and thus to furnish the yeast with all they require. The action of the bacteria in the rising dough is less understood, except that the undesirable souring is due to them. Bacteria also have some action, poorly understood, upon the gluten in the dough; and it is thought that the bread holds its moisture better if there has been some bacterial action during the rising, but not enough to cause a noticeable sour taste. For this reason, in some Places-especially southern Europe-leaven is still preferred by some People to commercial yeast. It is evident, therefore, that bread-raising is a more or less compli- cated process, in which bacteria and yeasts each play a part. It is possible by various means to stimulate one group of microorganisms at the expense of the other; and this has lead to different methods of bread making, each of which has its advantages. At the one extreme is bread of southern Europe, leavened with old dough, which has Very good keeping qualities, but is somewhat dark colored and is likely to be too sour to suit the taste of one who is not used to this sort of bread. At the other extreme is the bakers' white bread in America, made with a large inoculation of commercial yeast and with such a short period of rising that bacteria have no time to develop, a bread which is almost colorless and is free from sourness, but dries 228 BACTERIOLOGY out so quickly as to become stale in a day or two. Between these extremes are other methods, varying in the amount of yeast used and the length of time the dough is allowed to rise, which produce bread of intermediate quality. Perhaps the last word in bread-raising still remains to be heard. Bread-raising is one of those processes where mankind has learned to control microbial activities, at first without understanding the nature of the forces involved and later with more success after the discovery of the microorganisms themselves. The primitive man who first ate baked bread would not recognize modern bread. Part of its changed character is due, to be sure, to modern methods of milling wheat which give a flour containing the lighter and most starchy portions of the grain; but the greatest change from primitive bread is due to modern methods of controlling the growth of yeast. Faults of bread. Bread is not as liable to be spoiled by the growth of undesirable microorganisms as are beer and wine, largely because bread-raising is such a brief process that few bacteria have a chance to develop. Sour bread, as we have seen, is a common fault of bread raised with leaven, due to the growth of acid-forming bacteria, but is practically unknown when large enough quantities of yeast are used for inoculation to give these organisms an ascendancy over any bacteria that may be present. Another fault of bread, very rare, has been ascribed to bacteria and is called slimy bread. This trouble develops a few hours after baking, at which time it is eharacterized by a cobwebby appearance of the bread when broken. Definite bacteria causing this trouble have been described, which sometimes infect a bakery and cause trouble day after day, and can be eliminated by such methods as careful sterilization of utensils and the use of reliable cultures of yeasts. VINEGAR MAKING The fermentation involved. The manufacture of vinegar is another industry of some importance dependent directly upon the activities of microorganisms. There are various different methods of making vinegar, differing from each other not only in the scale of manufac- ture but also in the kind of raw material used and in the rapidity with which the process takes place; but whatever the method, and whether made on a large scale for commercial purposes or on a small scale for home consumption, the principle involved is the same. There are PRACTICAL USE OF FERMENTATIONS 229 certain bacteria which feed upon alcohol and convert it largely into acetic acid, and vinegar making is the result of their growth. Any starchy or sugary material that cannot be used more profitably in some other way may serve as the raw material, the starch or sugar being first converted into alcohol. There are thus two or even three steps in vinegar making: the conversion of starch (if starchy material is used) into sugar by the action of diastase; the conversion of sugar into alcohol by the action of yeasts; and the conversion of alcohol into acetic acid by the action of the acetic acid bacteria. As the first two steps are the same as in the manufacture of alcoholic drinks, it is the third step which concerns us here. Fig. 37. Types of Acetic Acid Bacteria When an alcoholic liquid is exposed to the air a film popularly known as the mother of vinegar forms on its surface; and following the appearance of this film, the alcohol is slowly converted into acetic acid. This film contains the acetic acid bacteria. There are various kinds of acetic bacteria, some of which have the power of growing out into long and occasionally enlarged filaments (see fig. 37). They are all very dependent upon the presence of oxygen and grow well only °n the surface of liquids. They all produce acetic acid from ethyl alcohol and propionicjacid from propyl alcohol according to the equations: C2H8 • OH 4- O2 -> CH, • COOH + H2O ethyl alcohol acetic acid C3H7 • OH 4- O2 -> C2H6 • COOH 4- H2O propyl alcohol propionic acid 230 BACTERIOLOGY This reaction does not take place if more than 14 per cent of alcohol is present, as alcohol seems to have an antiseptic action on these organisms if present in greater concentration than this. Besides the acetic acid there are certain ethers produced in smaller quantities which give a desirable flavor to the vinegar; but after the bacteria have used up the greater part of the alcohol and its concentration falls to 1 or 2 per cent, they begin to decompose these ethers and finally even the acetic acid they have produced, converting it into CO2 and H2O. It will thus be seen that if the process is allowed to continue too long there will be a loss in strength of the vinegar, a result which is avoided in practice by adding more alcohol as soon as its concentration has fallen to about 2 per cent. Orleans method. The oldest method of manufacturing vinegar is very simple. Large barrels or casks are used which are kept lying on their side so that the surface of the contained liquid will be as great as possible. Holes in the heads of the barrel above the surface of the liquid allow the circulation of air. To start the process the barrel is first filled one-third full of good vinegar, which is bound to contain the necessary bacteria and then wine or cider added in small amounts once a week until the barrel is about two-thirds full. By this time enough new vinegar will have formed so that an amount about equal to the amount of wine or cider added can be drawn off through the bottom bung hole (acetic acid being heavier than alcohol and settling to the bottom). More of the alcoholic liquid may then be added and the process continued, drawing off vinegar and adding more wine or cider at intervals. This process is slow, but is said to produce the best flavored vinegar. The method employed for producing vinegar on a small scale for home consumption is essentially the same. In its simplest form, any water-tight barrel is used of such a size that the amount of liquid to be put in it will have a good exposure to the air, the cider made from surplus apples is poured in, and allowed to stand until it tastes like good vinegar. Sometimes a little vinegar is added at the start to hasten the process, and rather better results can be obtained if the cider is added little by little as in the commercial process. A simple arrangement for this is shown in fig. 38. The heads of this barrel are provided with two holes (which should be screened to keep out flies) one near the surface of the liquid, the other near the top. The top bung hole is provided with a funnel leading to the bottom of the PRACTICAL USE OF FERMENTATIONS 231 barrel by means of a glass tube; the lower bung hole with a bent glass tube to serve as a gauge. At intervals vinegar can be withdrawn through the gauge tube and fresh cider added through the funnel without disturbing the film of ''mother" on the surface. Pasteur, as a result of his studies on the acetic fermentation, Pointed out the weaknesses of this method. The process is slow, Material is lost through evaporation, aeration of the surface is not Perfect, the bacterial film is likely to be broken and submerged, thus causing undesirable anaerobic fermentations, and the quality of the Fig. 38. Cross Section of a Vinegar Barrel P', Funnel for introduction of wine or cider; 0, 0, screened openings for aeration; L, level of liquid in the barrel; E, glass tube used as a gauge and to draw off vinegar. (After Bioletti.) vinegar is variable. His suggestions led to certain recent modifica- tions of the old method. This modified method, sometimes called the Pasteur method, differs principally from the Orleans method ln that shallow vats are used with special arrangements to assure circulation of the air above the surface. Only pasteurized wine (mixed with vinegar in definite proportions) is used; and light wooden gratings floated on its surface keep the "mother" from breaking and sinking. This process is more rapid than the old method and produces eflually good vinegar. 232 BACTERIOLOGY The "quick," method. Even the latest modifications of the Orleans method all have the disadvantage of requiring considerable time and of being therefore rather expensive. A method has been devised, known as the German or "quick" process, which considerably lessens the time required. A tall cylinder is filled with shavings (preferably of beech) and is so arranged with perforated top and bottom that air passes up through the shavings and liquid trickles down over them. The operation is generally started by soaking the shavings with vinegar, after which the alcoholic liquid is poured through slowly and intermittently. By the time the liquid reaches the bottom of the cylinder, its alcohol is largely converted into acetic acid; but ordinarily it must pass through several such cylinders in succession or through the same one several times before the process is complete, The efficiency of the apparatus depends upon various factors in the way it is set up and operated, of which the most important is tempera- ture, the results proving best at about 30°C. (90°F.). At its best, the process is so very rapid that it resembles a purely chemical change rather than a biological activity. Nevertheless it is depend- ent upon temperature, upon preliminary inoculation with vinegar, and does not take place if everything is sterilized and bacteria are excluded-all of which facts show the biological nature of the process. The same bacteria, indeed, are found when this method is used as are found in the vinegar barrel. Evidently their activities are greatly increased by the free access to the air furnished by the arrangement of shavings in the apparatus. The quick method is not applicable to wine or cider, which quickly coat the shavings and clog up the passages through them. As the best vinegar is obtained from wine or cider, that made by the German process is not of the best quality. Rotating barrels. A further modification of the method which is sometimes used is claimed to combine the speed of the German process with the quality obtained by the Orleans method. In this case, the barrels used are filled wholly or partly with beech chips, and are set up in such a way that they can be rotated. Frequent rotation causes the liquid to trickle over the chips and gives it good aeration, but there are no air passages to clog, even if wine or cider is used. Different grades of vinegar. A consideration of these various methods of manufacture shows plainly that vinegar is not all alike and that some kinds are of considerably better quality than others. PRACTICAL USE OF FERMENTATIONS 233 •Differences in quality are principally dependent upon: raw materials, kinds of bacteria causing the fermentation, method of manufacture, presence of undesirable organisms. The best raw materials are wine and cider, but malt, honey or various fruit juices may be used with good results. Commercially, however, the greatest quantity of "vinegar" is made from distilled spirits obtained from grain, potato or molasses. Such "vinegar" is really an imitation of the true prod- uct, as it is nothing but dilute acetic acid, free from flavoring mate- rial or color-except as color is added artificially to make it resemble true vinegar. The bacteria causing the desired fermentation are practically bound to be present provided a small amount of vinegar is mixed with the alcoholic liquid to start the process. As this is the common practice, there is ordinarily so little trouble in getting the proper fermentation that there has been no call for pure cultures of these bacteria. In case all the material used is pasteurized and it is there- fore necessary to use a starter, one can be obtained very simply with- out the services of a bacteriologist. If a liquid containing about 4 per cent of alcohol and about 2 per cent of acetic acid and a small amount of nitrogenous matter is exposed to the air in a warm place for several days, the acetic bacteria will appear in a film on its sur- face. Beer, or wine, mixed with vinegar and with filtered, boiled Yeast extract gives good results. The film forming on its surface can be floated off onto the surface of the liquid to be acetified. Ordina- ry, however, this is not considered to give enough improvement to be worth the trouble. Of undesirable organisms, the chief trouble arises from "vinegar eels," small nematodes that often develop in vinegar. If they are allowed to accumulate, putrefaction results. They can be kept under control by frequent cleaning, by pasteurizing the liquids used, and by other similar methods. These differences in quality are seldom taken into account by the manufacturer. The desire generally is to produce saleable vinegar as cheaply as possible, and the public is not sufficiently Well acquainted with the best vinegar to demand it. The manufac- turer is more concerned if the process is too slow or if the vinegar is n°t strong enough in acetic acid, results likely to be obtained if the conditions of the fermentation are not the most favorable. The recent modifications in methods of manufacture, therefore, are gen- 234 BACTERIOLOGY erally intended to increase the speed of the process or the strength of the product. When vinegar is made for domestic use, however, speed is of little consequence, nor does strength of the product matter greatly, inasmuch as the vinegar is generally made from fruit that would otherwise go to waste. For this purpose, therefore, an ordinary vinegar barrel is used, and little attention is paid to the conditions controlling the fermentation; as a result the product may vary considerably in strength and flavor, but has a good chance of being of better flavor than commercial vinegar. TREATMENT OF FODDER Treatment of hay. In handling fodder of any kind, the effort is ordinarily made to prevent or delay bacterial action. This is the object in drying grass to make hay. The drying of hay presents no great difficulties in a dry climate as exposure to the sun is ordinarily sufficient. The real difficulties are encountered in regions where rains are so frequent that it does not dry promptly; for in moist hay many undesirable changes take place. When wilted grass is gathered in piles and allowed to stand somewhat moist the biochemical ac- tivities are very active, so active that the temperature within the mass rises surprisingly. At times such a pile may become hot enough to cause spontaneous combustion. There is considerable dispute as to the causes of the self-heating of hay. There are three possible causes: (1) The respiratory processes of the grass cells. The life activities of the plants do not stop for some time after mowing, and they might easily account for some of the changes in the moist hay pile. (2) Enzyme action. All liv- ing cells secrete enzymes, and there must be many different kinds in a pile of freshly mown grass. Their activities undoubtedly go on after death of the cells and may explain much of the heating. (3) Bacterial action. Bacteria are sure to be present in such a mass, and their activities must be quite appreciable. The chief reason for thinking that bacteria are not entirely responsible for the phenomena is that the temperature of the hay may become so high as to kill bacteria, and microorganisms are not believed to be able to cause such high temperatures. Inoculation of sterile hay with cultures of bacteria does cause heating, but not ordinarily higher than 160°F. For this reason, it is commonly supposed that all three of the above- mentioned processes take part in causing the self-heating of hay. PRACTICAL USE OF FERMENTATIONS 235 Brown hay. In some countries of considerable rainfall, where drying of the hay is impractical, this fermentation is utilized to pre- pare a fodder which is said to be very palatable to cattle, although quite different from ordinary hay. The wilted grass is piled in a stack 13 to 16 feet high and 16 to 24 feet in diameter. The grass is trodden down to some extent and the stack is covered to protect it from rain. The temperature rises, but not over 160°F., and there is no danger of spontaneous combustion. The hay remains in this pile until it is used. In the mean time the hay has become converted into a firm dry mass, brown, or almost black if heating has been too great. It has become acid and has a rather pleasant aromatic odor. There is considerable loss of nitrogen, but in regions where this is the only practical way of curing hay, such loss is not a matter of consequence. This loss of nitrogen makes this kind of "fodder (brown hay, so called) an uneconomical proposition in places where hay can be dried. Burnt hay. Another similar way of utilizing the self-heating of hay is employed in some places. The grass is thrown up into piles 10 to 13 feet high, and is packed down as tightly as possible, so as to exclude the admission of air. After about two days the tempera- ture of the mass is taken by means of a thermometer and as soon as it reaches 75°C. (157°F.) the grass is spread out in thin layers on the ground. Its own heat soon dries it, and after a single turning it is ready to be stored. Such hay has a more aromatic flavor than sun- dried hay and is known as burnt hay. Silage. In the case of corn, there is more reason for utilizing these fermentations than in the case of grass. Corn contains con- siderable water and loses much of its value for fodder when it is dried. If kept moist, various fermentative changes may take place, some desirable, other undesirable. The silo has been devised as a means of controlling these changes so that the resulting product loses as little as possible of its original nutrient value and gains in- stead of loses in palatability. During this process there is always some loss of food material. The greatest loss is in carbohydrates; but some nitrogenous material ls also destroyed. The amount of loss varies from 4 per cent to 40 Per cent, depending largely upon the efficiency with which air is excluded from the silo. This loss should of course be prevented as much as possible, and it follows therefore that the most efficient 236 BACTERIOLOGY silo is the one so constructed as to allow the greatest possible exclusion of air, and that the more tightly the contents are packed the better will be the results. The silo is a tall air-tight compartment, with an opening at the top to use in filling, and openings at the side from which to withdraw the silage as it is used. The corn is harvested when the plants are mature, generally after the first frosts in the fall. The entire plants are chopped into moderately small pieces and packed into the silo. Sometimes considerable pressure is used in packing. Only the top layers in the silo have free access to air, so the resulting fermentations are practically anaerobic. During the first few days after filling, the temperature rises to some extent although not as high as it does in the process of preparing brown hay. After a few days more, the material slowly cools, and its nature changes. It becomes acid with an aromatic odor and flavor. Off-flavors, such as due to putrefac- tion or mustiness sometimes develop, but do not appear if the process is properly controlled. If properly prepared, silage keeps good for months, but after once opening the silo the contents must be quickly used up, for the exposure to air causes undesirable fermentations. The same three factors as in the case of self-heated hay, respiratory changes, enzyme action, and bacterial activities, have been suggested as possible causes for the fermentation of ensilage. It is quite likely that in this case also all three factors play their part. There has been considerable disagreement as to whether or not bacteria are important in the process, the common opinion at one time being that it was due almost entirely to respiration of the living plant cells. Recent work, however, shows the great abundance in silage of certain acid-forming bacteria much like the so-called Bulgaricus group (see p. 125), and suggests that these organisms play an im- portant part in the process. Silage of a sort may be produced in experimental jars in which the growth of bacteria is prevented by the presence of chloroform vapor; but the typical silage fermentation seems to take place only in the presence of microorganisms. MISCELLANEOUS FERMENTATIONS Tobacco. The preparation of tobacco is another process in which fermentation is put to practical use. The leaves when gathered are partially dried, generally in specially constructed drying sheds. They are then cured, the method of curing varying in different climates. The leaves are sometimes packed in boxes for this process PRACTICAL USE OF FERMENTATIONS 237 and are allowed to stay unopened for several months; or sometimes they are heaped in piles with occasional turning and repiling until the proper stage of curing is reached. During this process, considerable heat is produced and the nature of the leaves is greatly changed. The most striking changes are the change in texture and flavor and the production of the final brown color; but these changes are accom- panied by a decrease in nicotin, a disappearance of sugar, an increase in ammonia and nitrate, as well as a loss of water. In general these changes are due to oxidation. The process is apparently a fermen- tation of some sort, and to the tobacco-grower a very important fer- mentation; for the leaves as gathered are valueless, and their marketa- bility depends entirely upon the success of the curing. Theories to explain the curing of tobacco are very similar to those proposed to account for the silage fermentation and the self-heating hay. To some, enzymes secreted by the tobacco leaves appear the principal factor; to others, bacteria seem mainly responsible; while a third theory is that after the initial growth of bacteria has caused the production of heat, the rest of the process is purely a chemical oxidation. Bacteria are numerous upon the leaves of fer- menting tobacco, and some have been isolated and named. Some of them are claimed to be responsible for the curing and attempts have even been made to control the process by means of pure cultures. It ls doubtful if such attempts have been completely successful; al- though the care with which the secrets of tobacco curing are guarded makes it impossible to be certain that no one is using bacteriological methods successfully. Salt pickles. Some kinds of green vegetables, such as string beans, cucumbers and cabbage, can be preserved by allowing them to Undergo a certain form of fermentation which protects them against further decomposition. This is done by packing them with 1 to 3 Per cent of salt in some form of air-tight container, and subjecting them to pressure by means of weights placed upon them. The pres- sure and the salt combined, draw out the juices from the vegetables aud an anaerobic fermentation takes place in these juices. Con- siderable lactic acid is produced and the mass becomes so acid that the putrefactive bacteria are unable to grow. During the process, various changes occur, and not only lactic acid but other organic acids, alcohol and other fermentative end-products are produced. The resulting product is quite acid and has a very characteristic flavor. It can be kept for some time, but eventually organisms ap- 238 BACTERIOLOGY pear in it that consume the lactic acid, and when it has become alkaline it undergoes putrefaction. The best known products of this class are dill pickles and sauer kraut. Sauer kraut is made on a large scale at present. Originally it was a home-made product, eaten mostly in Europe; but now it is quite popular in this country, and is made in factories as well as in the home. The process is essentially the same whether it is made in barrels at home or in large tanks or vats in the factory. Cabbage is sliced so that the leaves are cut into shreds and is then packed into the containers with salt, covered with boards and weights placed on the boards. Quite a little time is allowed for the fermentation to take place. In sauer kraut factories the material is then canned in brine, and the cans processed with heat. Sauer kraut can be kept for some months without canning, but the canned product ordinarily keeps indefinitely. Various bacteria undoubtedly take part in this fermentation, but the most important ones seem to be lactic acid bacteria, although different from those which are characteristic of milk. Some of these bacteria have been isolated and have been utilized experimentally for preparing sauer kraut. With the use of these pure cultures, the process can be considerably hastened, but it still remains to be shown whether they are practical on a large scale. They would appeal only to the manufacturer, because when sauer kraut is made at home, a little time saved is of small consequence; and it has yet to be shown that the product is better when made with pure cultures than when left to ferment naturally. Retting of flax and hemp. Linen and hemp are prepared from the fibres of the flax and hemp plants respectively. In the plants these fibres are firmly bound together with the wood and bark by means of a glue-like substance known as pectin, and it is necessary to remove the pectin before the fibres can be used. This has been accomplished from time immemorial by means of bacterial action. The plants are either exposed to alternate drying and wetting on the surface of meadows, or else are bound together in bundles and im- mersed in water. Under such conditions, certain bacteria become active and dissolve the pectin, freeing the fibres from each other, so that they can be combed out and used for spinning or weaving. Tanning. Bacteria are concerned in various ways in the tanning of hides, although their action is not fully understood. The first step necessary in treating the hides is to remove the hair, which is PRACTICAL USE OF FERMENTATIONS 239 accomplished by soaking in a solution of lime. Bacteria grow in this solution and are thought to assist in loosening the hair, because °ld solutions, which contain large numbers of bacteria, are more efficient than new solutions. The combined action of the lime and bacteria so loosen the hair that it can be scraped off the hide by hand. In order to soften the hides that are to be made into soft leather, further treatment is given them in which bacteria are prominent. They are placed in wooden tanks containing pigeon, chicken or dog excrement in water. They are kept in motion in these tanks by Cleans of a large revolving wheel. The bacteria partly decompose the hides and thus soften them, and care must be taken to remove the skins before they are too much decomposed. In the later stages of the process when the hides are soaked in tanning solutions, bacteria play play some part, for a lactic acid fermentation is known to occur 111 the tan-pits, and may have its influence on the tanning of the leather. APPLICATION TO NEW PROCESSES The practical possibilities of fermentations are far from exhausted. Nearly every year sees some new process proposed or perfected depending upon the action of some microorganism. The unusual stimulation that the war gave to invention has led to much investiga- tion of bacteria and their activities; and far-seeing men are beginning to realize what an important part they may play in the industries of e future. The rubber industry is already beginning to wonder if acteria can produce cheaply some of the raw materials they need, he possibility of producing glycerin, sugar, or even other important c emicals by bacteria is being considered; and there is no telling what be brought out next by new inventions in this line. One of the most interesting developments in this line is the pro- oction of diastase. We have already seen that diastase is ordinarily obtained as malt, in other words from sprouting grain. Now it has cen known for some time that certain fungi produce diastase, but 1 is only recently that the idea has arisen of looking to them for its commercial production. Recently, however, methods have been evised for obtaining the diastase produced by certain molds, rain is valuable and malt is therefore expensive; molds can grow on Waste material of no value for other purposes, and diastase can be Prepared from them much more cheaply than malt can be manu- actured. As a result, the takadiastase as it is called, although . n°Wn for but a short time, is already of considerable commercial lmportance. CHAPTER XI The Manufacture of Pure Cultures and Sera Of recent years there has been growing up a bacterial industry of an entirely different kind from any of those we have been consider- ing. There is coming to be quite a demand for pure cultures of certain bacteria, and for preparations containing them, their toxins or their antitoxins. The industry to supply this demand began with the production of pure cultures of yeasts. The original yeast was always obtained by using a little of the last batch of dough or sedi- ment from some fermented liquor. Such material was of course a very impure culture of the desired organism, and as the advantages of special kinds of yeasts began to be learned, it began to be realized that pure cultures of the best varieties would be more satisfactory. Pure cultures could not be prepared to advantage by the average baker or liquor manufacturer, so laboratories began undertaking to supply them. The first development of this industry on a large scale was in the laboratories of Dr. Hansen of Copenhagen. As brewers and bakers came to understand the advantages of pure cul- tures, the demand for these products grew rapidly, and commercial laboratories producing them multiplied. Later other pure cultures came to be demanded on a commercial scale, and they were prepared sometimes by these same laboratories, sometimes by others. A slightly different industrial development along this same line is the production of bacterial sera. When Pasteur discovered that a vaccine could prevent anthrax and that rabies could be warded off by an attenuated virus, there was no way that such material could be supplied in any quantity. The demand for the Pasteur treatment for rabies was so great, however, and the preparation of the inoculating material so difficult that special laboratories were established. These laboratories were accompanied with facilities for treating patients and were known as Pasteur Institutes. They were soon to be found all over Europe and later at various places in America; but they did not prepare any sera except the material for rabies. So, as new diseases were one by one conquered with vaccines, antitoxins, or attenuated viruses, there arose such a demand for these preparations MANUFACTURE OF PURE CULTURES AND SERA 241 that commercial laboratories have been started in various places. There is now quite a large industry that is concerned wholly with the preparation of bacterial sera and related products. The principles underlying the production of these sera is to be taken up in a later part of this book; but here we can consider briefly some of the practical phases of their manufacture. The subject must be treated lightly, however; to discuss it fully would require a book devoted to nothing else. A few illustrations will help to show what complicated methods are necessary in the manufacture of these products and what careful controls must be maintained throughout. SMALLPOX VACCINE The first step necessary in the production of this vaccine is to obtain healthy young cattle. They must be selected with the greatest care to avoid any possible contagious disease, and after their selection they must be kept under observation for a while in a detention stable. There they are inspected by a veterinarian, and tested for tuberculo- sis. If no signs of any disease appear after they have been quaran- tined for several weeks, they are considered fit to use. Then their hair is clipped and their entire bodies are thoroughly scrubbed with soap and water and some disinfectant solution, after which they are ready to be admitted to the laboratory. The laboratory in which they are inoculated is as scrupulously clean as the operating room of a hospital. No one but the operator and a few qualified attendants are allowed in the room, visitors, if present, having to stand outside and watch through a window. The walls, floor, and equipment of the room are frequently washed with disinfecting solutions. The animal to be inoculated is placed upon an operating table, so secured that his struggles will not interfere with the work. The skin over the abdomen is then shaved, cleaned and disinfected, and then is scarified with a sterile instrument. The scarified area is then inoculated with a stock virus which is kept on hand in the laboratory, after which the animal is placed in another room for propagation of the virus. In this room the animal lives for a week or more in absolute cleanliness. An attendant is con- stantly present to remove droppings and other dirt, and the room is kept as free as possible from bacterial contamination. After a week or so, vesicles develop on the inoculated surface which contain the lymph that is used for vaccine. The animal is 242 BACTERIOLOGY then put on the operating table again, the surface of the vesicles carefully washed and sterilized, and the lymph removed with a sterile instrument. It is required by the government that the animal be killed before removing the lymph and that later a careful autopsy be performed. The discovery of any disease in the animal is enough to condemn that lot of vaccine. The vaccine is very carefully handled to avoid contamination, and is mixed with glycerin, which prevents the development of the ordinary contaminating bacteria. It is inoculated into guinea pigs to see if it contains disease germs and into some other animal to determine its potency. Inoculations are also made into culture media under aerobic and anaerobic conditions. Anaerobic cultures are injected into guinea pigs to test for the presence of tetanus toxin. If all these tests are favorable, the vaccine is considered satisfactory and is placed in sterile capillary tubes or on sterile ivory points, ready for distribution. Under proper conditions it remains active for about a year. OTHER VACCINES The smallpox vaccine is assumed to be a culture of the smallpox organism attenuated by its sojourn in the calf, although this has never been absolutely proved. Accordingly any attenuated virus that is used to produce immunity is called a vaccine. There are not very many diseases for which this method of establishing immunity proves to be practical, and most important of them is rabies. In this case the vaccine is used to ward off the effects of the bite of a mad dog. The virus is attenuated by removing the spinal cord of a rabbit dead of rabies, and drying over caustic potash at a tempera- ture of 23°C. The material is ready to use after a certain number of days' drying, depending upon whether it is to be used as a first dose or a later dose. The patient is first inoculated with a greatly at- tenuated preparation, then with one which has dried for a shorter period, and so on until at last the powerful virus itself can be used. The preparation of this vaccine presents great difficulties, for material in all stages of drying must be kept constantly on hand and it cannot be kept after the period of drying is over. For this reason this vac- cine is not handled by commercial houses, and is administered only in connection with the Pasteur Institutes, where facilities are at hand for keeping all strengths of the vaccine constantly on hand. MANUFACTURE OF PURE CULTURES AND SERA 243 Attenuated viruses are used in combating certain animal diseases, namely blackleg, hog cholera and anthrax. The blackleg virus is attenuated by drying at temperatures of 85 to 100°C., and the an- thrax virus by growing at a temperature above its optimum. The method used in the case of hog cholera is quite different; for in this case the active virus is inoculated into the animal together with a protective serum obtained by special immunization of some suscep- tible animal. Bacterial vaccines, or bacterins as they are sometimes, and perhaps more properly called, are somewhat different as in this case the virus is not attenuated but actually killed. The killing is ordinarily accomplished by heat, but sometimes by chemicals. The best known disease which is combatted in this way is typhoid fever. The technical preparation of bacterins is fairly simple as it is merely necessary to kill the organisms at as low a temperature as possible, then to count under the microscope the number of dead organisms present and to prepare infusions containing definite numbers. DIPHTHERIA ANTITOXIN The first step in the preparation of antitoxin for diphtheria is obtain a pure culture of the diphtheria organism. The organism Can easily be obtained from the throat of a diphtheria patient, but to purify the culture it is generally considered necessary to inoculate into a guinea pig and to re-isolate the organism from its tissues. This culture is then inoculated into large flasks of beef broth and incubated at 37°C. for about two weeks at the end of which time the broth contains the diphtheria toxin in great concentration. The organisms are removed from the broth by passing through a porcelain biter, and the filtrate, preserved with some germicide, is kept in a refrigerator until used. The antitoxin is prepared by injecting the toxin into a horse. The horses used for this purpose are submitted to as rigid an inspec- tion as the calves used in preparing vaccine. Any horse that proves io the slightest abnormal is rejected, and nothing but absolutely ealthy animals are used. The antitoxin horses are kept in sanitary stables capable of being kept extremely clean and everything possible is done to promote their welfare. The toxin is injected periodically in increasing doses. The first dose is generally a fraction of a cubic centimeter. The effects of this dose are slight and soon pass off. 244 BACTERIOLOGY Then a second and stronger dose is given which is followed by a third as soon as the reaction from the second dose is over. Increas- ing the dose in this way, it is finally possible to inject 300 cc., or more, at once without harmful effects. This immunity is due to the antitoxin produced in the horse's body. This treatment requires perhaps two or three months and after it is over, a rest of a few weeks must be given the horse to allow the toxin to be completely absorbed. Then for about two years, the horse is able to furnish antitoxin. At regular intervals the horse is bled through the jugular vein under aseptic precautions in a room specially designed for this purpose. A sterile tube is inserted into the vein and the blood is allowed to run out through a rubber tube into a sterile vessel. Ten or 15 liters can be obtained from a horse at a single bleeding without causing any apparent inconvenience to the horse. The blood is allowed to clot and the serum filtered through a porcelain filter after the addition of some preservative. The serum is then tested thoroughly as to its potency and freedom from bacterial contamination and placed in glass containers for distribution. Finally a few of the containers ready for distribution are selected at random and again examined for contamination, before the material is considered safe for use. Each tube in which the antitoxin is distributed is marked as to the number of units it contains. One unit is the smallest amount of the antitoxin capable of protecting a guinea pig of 250 grams weight from the effects of a lethal dose of toxin. The lethal dose of toxin is the smallest amount necessary to cause death in the absence of antitoxin. In this way it is possible to determine the strength of any lot of antitoxin and to calculate how much to use in treatment. OTHER ANTITOXINS By exactly the same methods as those used in the case of diphtheria, fit is possible to obtain an antitoxin specific against any soluble 'toxin. The best known of these is the antitetanic serum, but there .are others of considerable importance. Among them is the antiserum against the toxin produced by B. botulinus (the organism responsible for the worst type of food poisoning). Similarly there have been produced antitoxins against snake and spider venom and against three plant toxins, abrin, ricin and crotin. None of these antitoxins compare with diphtheria antitoxin in their importance as commercial products. MANUFACTURE OF PURE CULTURES AND SERA 245 TUBERCULIN AND MALLEIN One other preparation of great importance, although for a slightly different purpose, is tuberculin. This material has no effect upon healthy animals but is slightly toxic for tubercular ones, causing a slight fever if injected beneath the skin or causing inflammation if injected into the skin (or, in the case of human beings, if applied upon the surface of the skin). This property causes it to be of great diagnostic value. It is prepared by inoculating the tubercle organism into flasks of beef bouillon containing 5 per cent glycerin. The cultures are incubated at body temperature for two months or more without being disturbed. During that period they must have good access to air and an even temperature. After growth is complete, the flasks are sterilized in flowing steam, and the contents then evaporated over a water-bath to one-tenth the original volume. The niaterial is then filtered through a porcelain filter to remove the bac- teria, and is ordinarily diluted eight times with weak carbolic acid solution. In this form it is ready for distribution. Mallein is an exactly similar material prepared from the glanders organism, the method of preparation being essentially the same as in the case of tuberculin. It is similarly diagnostic for glanders, the temperature reaction being accompanied in this case by a local swelling which is probably of as much diagnostic value as the rise in temperature. PART III PATHOGENIC ORGANISMS 1. HUMAN AND ANIMAL DISEASES CHAPTER I Microorganisms as the Cause of Disease In the preceding division of this book we were concerned primarily with the saprophytic microorganisms. Now we are to study the parasitic organisms, which as we have already seen are generally pathogenic. They are those organisms that have the power of over- coming the resistance of living tissue and are thus able to grow in the living bodies of animals or plants. It is interesting to learn that when a microorganism does have this power, it ordinarily loses its ability to live a free and independent life, apart from its host. This is generally true both of large parasites like the tapeworm and of micro- scopic parasites like bacteria. They have become adapted to the parasitic life, and do not ordinarily live long if removed from their hosts. Even the typhoid organism, which is frequently found in water, is incapable of living a non-parasitic life for more than a comparatively few days, unless cultivated under special laboratory conditions. The influence of the parasite upon its host has been the primary stimulus in developing the science of bacteriology. It is hardly to be expected that any parasite can live upon or within a living animal or plant without producing some effect upon it; but the effect may vary greatly in nature. In some cases the effect is so slight as to be hardly noticeable, while in others it produces serious derangements of the physiological processes and results finally in the death of the host. We generally speak of such a derangement as disease. The cause of disease is a subject of acute interest to everyone, a fact which has greatly encouraged the development of pathogenic bacteriology. Fortunately the kinds of microorganisms capable of living a parasi- tic life in the human body are few in number. Hundreds of kinds of bacteria have been described by bacteriologists in the last generation, but only a few of them have been found capable of living a parasitic life. The number definitely known to cause trouble as human para- sites is not much over a score; and, while others may be found by 250 BACTERIOLOGY later studies, it is almost certain that we are nearly at the end of such discoveries. The same thing is even more true in regard to other groups of microorganisms-yeasts, fungi, and protozoa. Parasites belonging to these classes are known and some of them-especially the parasitic protozoa-are extremely important, but compared to the saprophytic members of these groups they are very uncommon. As already pointed out, it was suggested some centuries ago that microscopic organisms might be the cause of disease, an idea which kept cropping out from time to time during the period of obscure knowledge which followed. There was, however, no evidence for its support, and the idea was really nothing more than a guess on the part of a few who were interested in the study of microscopic life. The spread of epidemics, especially those of cholera in Europe, always stimulated the study of disease and its causes; so every time that cholera made its appearance there was a multiplication of guesses as to its cause and its relation to microscopic germs. About the middle of the nineteenth century the idea was brought into public notice in a much more definite manner, when Henle pointed out (see p. 27) the logical method by which the question might be settled; but it was really not until 1865 that the germ theory of disease was put on a scientific basis. This was not in connection with any human disease, but with a disease of the silk worm. Pebrine. We have already seen (p. 28) how this disease in the early sixties became so widespread as to threaten the very existence of the silk industry in France, and how Pasteur was called upon to study it and to discover some method of control. Pasteur, as we have seen, was successful in this, but he did not carry out the logical procedure outlined by Henle. Pebrine is not caused by bacteria but by organisms much more difficult to obtain in pure culture than bac- teria, and in these early days even bacteria could not be isolated. Naturally, therefore, Henle's procedure, which required the isolation of the suspected organism, could not be then carried out in full, and the germ theory was not absolutely proven. Nevertheless, the fact that Pasteur made a practical application of this theory came near to proving it correct. This work of Pasteur really inaugurated the modern germ theory of disease, as well as establishing the genius of Pasteur. It is true THE GERM THEORY OF DISEASE MICROORGANISMS AS CAUSE OF DISEASE 251 that for some years further development of the idea was delayed, and science was still uncertain as to whether the principle established by Pasteur could be applied to the study of other diseases. But the triumphant solution of this problem was the first step in proving that the growth of microscopic organisms as parasites in animals may be the cause of animal diseases. Anthrax or splenic fever. It was some fifteen years after the studies upon pebrine before another disease was traced to its parasitic source. In this case a true bacterium was found to be the cause. The disease was anthrax, which had been known since the time of Moses, and had long been one of the greatest menaces to agriculture. It was known all over the world, affecting cattle and sheep primarily, although attacking some other animals, including human beings- bearing the name of wool-sorter's disease or malignant pustule in uian. That it is likely to spread through a herd of animals after ■once making its appearance has been long known, and many have been the attempts to study its cause and to find some method of cure or prevention. The use of the microscope very early revealed, in the blood and tissues of anthrax victims, small immotile, rod-shaped objects present in great numbers, which were suggested by Davaine (as early as 1868) as*possible causes of the disease. Very extensive studies, as we have already seen, were made by Pasteur, by Koch, and by other experimenters upon anthrax in its various relations, ffs methods of distribution, its destruction by heat and disinfection, and numerous other questions concerning it. A monumental mass of data, indeed, has accumulated concerning this disease. Since it readily attacks many different animals, small as well as large, it offers an opportunity for experimentation that is not furnished by strictly human disease, and it has been one of the most fruitful subjects of investigation in connection with the germ theory of disease. It has furnished the key to many of the most important Problems associated with the control of bacterial disease, and it ls hardly an exaggeration to say that the early development of bacteriology centered around anthrax. Wider application of the germ theory of disease. As soon as the fundamental principle of disease germs was established, the next Question was simply to what extent it explained diseases of animals and man in general. A complete answer to such a question, of course, could not be made, for the individual diseases had to be in- 252 BACTERIOLOGY vestigated and traced, one after another, to their origins; but gradually the necessary information accumulated. The greatest impetus given to the subject came from the plate method devised by Koch. By this method, it will be remembered, it was first possible for the average investigator to obtain a pure culture of a microorganism. Pasteur and Koch, to be sure, had already obtained pure cultures of the anthrax organism without the use of solid media and culture plates; but the ordinary investigator was not a genius like these two men, and was generally at a loss to know how to isolate an organism- With no methods of isolation, rigid proof of causal agency could not be obtained. Koch's plate method made possible the general application of Henle's principles for determining the cause of a disease. It was fitting, therefore, that Koch should restate the principles of Henle in the form generally known as Koch's postulates. These principles we have already reviewed (p. 27). Stated briefly they required proof of the following points: (1) constant presence of the organism in animals suffering from the disease, (2) its isolation, (3) production of the disease upon experimental inoculation, and (4) recovery of the same organism from experimentally inoculated animals. Koch's plate method opened up such possibilities that*these postulates were soon carried out by various investigators and many diseases of man and animals were traced, one after another, to their source in micro- scopic life. INFECTIOUS DISEASES In general, the results of the generation following the introduction of Koch's new method have been to show that all of a certain class of diseases, which we now call infectious diseases, are produced by the parasitic life of microorganisms of one kind or another living within the tissues of the diseased animal. But this extended study has shown that the organisms living as parasites are by no means alike and do not belong to the same group of living beings. Diseases of man and animals due to parasites may, so far as concerns their causes, be arranged in a few groups, as follows: Diseases produced by animals of some size. The study of parasitic worms like trichina and tape worm does not belong within the scope of this book, beyond calling attention to the fact that these parasites of considerable size are agents in producing certain types of disease. MICROORGANISMS AS CAUSE OF DISEASE 253 Among them perhaps the most widely known, in general, is the organism causing the hook worm disease. Diseases produced by microscopic animals. The unicellular animals known as Protozoa were not at first suspected to be associated with diseases, but we know now that not a few of the most serious and fatal human and animal diseases are produced by animal rather than by vegetable parasites. These include, beside the pebrine disease of the silk worm above mentioned, the sleeping sickness of Africa, malaria, and some others to be mentioned later (p. 342). Diseases produced by plants of some size. Some fungi too large to be called microscopic occasionally grow in or upon living tissue, and give rise to well-known infection. There are very few of these that attack human beings, the best known being ring worm and favus. In these cases the fungus produces a mycelial growth that penetrates the skin, breaking through to the surface for the purpose of producing spores. Diseases produced by bacteria and yeasts. Yeasts only occasionally Produce disease in human beings or animals, but there are some which *nay be found living attached to the surface membranes. It is very common, for example, to find yeasts in great numbers in the niouth along with the other mouth organisms. But these do no lnJury and are not in any sense pathogenic. One or two obscure and rare diseases have been attributed to yeast. They are little known, physicians alone-and in fact not all physicians-being familiar with their names. Bacteria, on the other hand, are the Jttost common parasites, and pathogens. So closely have bacteria been associated with disease that to the public mind, bacteria and disease germs are frequently considered identical. Diseases produced by ultra-microscopic organisms. Some diseases are now found to be produced by organisms too small to be seen even with the highest powers of the microscope. It may of course be Questioned as to what grounds we have for saying that they are organisms if they cannot be seen; but the evidence obtained, although not absolute proof, is fairly conclusive. It is not possible, even with the highest powered microscope, to see objects smaller than the Wave-length of light, about 0.1 micron (0.0001 mm.); but there are known bacteria of just about this size and there seems to be no theo- retical reason why smaller ones should not exist. Discharges or °°d from certain diseases are infectious even after being passed 254 BACTERIOLOGY through porcelain filters so fine as to remove all microorganism visible under the microscope. A small amount of this filtered material causes the disease in an inoculated animal and large amounts of the infectious material may be obtained from this animal. The disease virus may thus be carried through a long succession of animals with repeated filtration between inoculations. This ap- parently means that the virus increases in quantity, and has prob- ably been growing and multiplying just as if the infectious material were filled with organisms too small to be seen by the microscope. It is possible, moreover, to make porcelain filters fine enough to exclude the virus, so it evidently is not a liquid. If it is a solid material capable of growing and multiplying, we are justified in calling it living, and hence in speaking of it as an organism, even though the microscope does not disclose it. We refer to these unseen infectious agents as ultramicroscopic organisms. Among the diseases probably caused by them are: foot-and-mouth disease, pleuropneumonia, cattle plague, swamp fever of horses, sheep pox, horse sickness, and perhaps infantile paralysis, measles, whooping cough, and hog cholera. THE TRANSMISSION OF DISEASE The gradual accumulation of facts concerning these different classes of disease organisms has led finally to a clear understanding of what is meant by the term infectious. This term has been of long-standing use, and has been sometimes confused with the term contagious, sometimes used differently, and has not had any very clear meaning. With our present recognition of the parasitic nature of certain diseases we are in a position to understand clearly what is meant by infectious. Infectious diseases are those that are pro- duced by some living parasite, which is capable of multiplying, and which can be carried, directly or indirectly, from person to person, always redeveloping the disease in the new host to which it is carried. The infectious nature of a disease, therefore, is dependent upon its being produced by a living parasite which multiplies within the body. Hence the term infectious has nothing to do with the method of distribution of the disease from person to person, but only with its being caused by a microscopic parasite. All infectious diseases are alike, nevertheless, in that they may be carried, directly or indirectly, from one individual to another- MICROORGANISMS AS CAUSE OF DISEASE 255 The readiness with which they can be thus transmitted, however, varies very much with different infectious diseases. Sometimes the method of transmission is direct, so that a second person in contact with the patient, or even in contact with articles that he has handled, may acquire the disease. In this case the disease is commonly called contagious, a word evidently derived from "contact." In other cases the method of transmission is roundabout, and so indirect that one never acquires it directly from another. In these cases, though the diseases are infectious, they are not contagious. Clearly the readiness with which microorganisms may be transferred from one patient to another will depend upon several factors, primarily the following: Elimination from the patient. To produce the diseases, micro- organisms must multiply within the body of the patient, and if they are subsequently to produce trouble in another person they must be eliminated from the patient's body. The methods of elimina- tion vary. Sometimes they are excreted from the intestines, some- times from the mouth, or from the nose, sometimes from eruptions °f the skin, and sometimes by other methods of exit. There is Usually little difficulty in determining the method of elimination of the infectious agents, because such diseases are likely to be ac- companied by unusual discharges, and when this occurs it is always a matter of extreme probability that the discharges contain the in- fective agents. In a few cases, like malaria, there are no such excretions, and the method of elimination is very different. Transportation. The parasitic organisms are not capable of any extensive locomotion. While it is true that some of them are endowed with powers of motion, it is confined to liquids, and they have no active methods of distributing themselves through the air over dry land. They must, therefore, depend upon some second- ly method of being carried to and fro. It is the study of these secondary methods that has constituted modern sanitation, for sanitation consists largely of discovering the methods by which infective material is distributed through the community, and then endeavoring to check such distribution. Knowledge of these facts has enabled our modern sanitarians very largely to check the distribution of epidemics which in former years ran riot through communities. 256 BACTERIOLOGY Invasion. After reaching their new victim, the microorganisms must find some means of entering the body, and here again the means are various. As a method of protecting ourselves against the attack of these invaders, it is evidently a matter of wisdom to learn the means of invasion in the case of each disease, for then we are in a measure prepared to resist such invasion. We have learned, indeed, that the human body has a number of efficient protections against the ordinary invasion of microorganisms. The outer skin of our body is an impervious series of layers of cells, tightly packed upon each other in such a way that microscopic organisms cannot pass through, except in case of injury. The external parts of our body are therefore fairly well protected, although possibly around the hair follicles there may be an occasional chance for invasion. To a less extent also the lining of the alimentary tract is capable of resistance against microorganisms, although the resistance is not as efficient and is more easily and commonly broken down. In regard to some of the invading parasites, moreover, such as cholera and diphtheria, for instance, the organisms grow within the alimentary tract and apparently do their injury without even entering the tissues at all. The accumulation of knowledge along these lines has been one of the largest factors in the reduction of the ravages of infectious dis- eases in our communities. THE SCIENCE OF PUBLIC HEALTH The factors above mentioned constitute the foundation stones upon which has been built in the last generation the science of sanitation and public health. That there has resulted an extensive reduction in the death rate has been made very manifest. A careful study of the conditions, however, shows that this reduction has been due very largely to the diminution of the amount of infectious dis- eases, and that it has been especially marked in connection with the death rate of children. There has been little reduction of the non-infective diseases, and the effects upon the adult community have been far less than upon childhood. In general, the method by which this has been accomplished has been through the discovery of the methods by which the infectious material is eliminated from the diseased patient, the method by which it is transported from person to person, and the method by which it finally finds its way into a new MICROORGANISMS AS CAUSE OF DISEASE 257 individual; and then by devising some practical means to stop its spread at some point. Each disease has to be handled separately m such a campaign, for there are no two of them amenable to exactly the same methods of protection. One further factor upon which the struggle for improved public health is dependent is the method of rendering individuals immune against some of these infectious diseases. To understand this we must first learn the methods by which disease is produced. HOW MICROORGANISMS PRODUCE DISEASE Even in the early days of bacteriology the question was often asked as to how diseases can be produced by microorganisms, and in the years since then various tentative answers have been given. Many phases of this problem are still obscure; but it proves that in practically all cases the actual injury to the animal is caused by certain poisonous substances that the parasites produce. These Poisons, or toxins, act on the host in such a way as to injure some of its bodily functions. This was pointed out by Pasteur long ago, when he was working on anthrax. He found that when the anthrax bacilli were growing in a liquid in a tall jar they would eventually settle to the bottom, but that the clear supernatant liquid when injected into animals produced the symptoms of anthrax although it contained no microorganisms. The illness produced, however, did not develop as an ordinary attack of anthrax, but it appeared unusually quickly, reached its maximum almost at once and, unless death occurred, immediately disappeared; while if a small quantity the precipitated bacteria were inoculated into a healthy animal, the disease came on slowly and progressed until death ensued. The °nly possible interpretation of this phenomenon is that anthrax bacteria produce certain poisons, which accumulate in the culture, and that inoculation of the animal with the liquid alone results immediately in poisoning by these excreted products, whereas the inoculation with the bacteria themselves produces no effects until they have a chance to grow and develop more poisons. The study °f other diseases has shown that in some cases the action of micro- organisms is exactly similar to that of anthrax. Diphtheria and tetanus bacilli, for example, produce intensely violent poisons, known as diphtheria toxin and tetanotoxin, and it is these poisons that produce the actual symptoms of the diseases. In some other diseases 258 BACTERIOLOGY no toxin is directly excreted from the bacilli, but probably in all cases the action of the organism is something the same. The study of these toxins and the method by which they act on the body has developed into a very extensive branch of the science, known as immunology, which has become extremely complicated, and difficult to understand. The toxic products produced by these pathogenic organisms appear to be of at least three types. First, the soluble toxins. These are excreted from the bodies of the growing bacteria, accumulate in the liquid in which the bacteria are growing, and may be isolated from these liquids. The second type are the so-called "endotoxins." In this case the toxic products are not excreted from the bodies of the bacteria, but are retained within the cell itself. They may be, however, obtained in solution for testing by crushing the body of the cell, so as to liberate them, and then by dissolving the product from the crushed mass. The third type of toxic products are the toxic proteins. Little is known about these, except that when bacteria are broken to pieces it is found that certain substances that are not soluble, but which are poisonous in nature, remain in the solid precipitate. They are thought to be of a protein nature, and are insoluble in ordinary liquids. The nature of different infective diseases, and especially the methods that have to be adopted for combatting them, vary quite decidedly in accordance with which of these three types of toxins are produced by the parasites and give rise to the symptoms. CHAPTER II Resistance and Immunity It is evident that the body of a living animal has some power of resisting the invasion of microscopic organisms. This is shown by the fact that if the germs of ordinary putrefaction be injected into the flesh of a living animal, they do not succeed in getting a foothold, and being unable to grow, produce no particular trouble and are soon absorbed or ejected from the body. If, however, these same organisms are injected within or even smeared on the surface of the flesh of the same animal after death, they find it a good nutriment, begin to grow and rapidly cause its putrefaction. There is clearly some factor in the living tissue that resists the action of the ordinary non-pathogenic organisms. Furthermore it is found that if certain bacteria are inoculated into freshly drawn blood, they at first not only fail to grow but actually decrease in numbers, showing that something in the blood destroys them; while later this same blood proves to be a good nutriment for them and they develop rapidly. These facts show that the living body has powers of resistance against the invasion of non-pathogenic organisms. Now if, on the other hand, pathogenic bacteria are inoculated into a living body, they seem to be able to overcome these powers of resistance, they get a foothold, grow and develop, producing in the end the diseases for which they are known. It would seem thus that the primary difference between the pathogenic and the non-pathogenic micro- organisms is in the fact that the former have the power of over- coming the body's resistance, while the latter do not. Fortunately for us, among the many hundreds of kinds of bacteria that have been discovered, a comparatively few seem to be able to overcome the resistance of the human body, and there are therefore but small lumbers of pathogens. The actual number of pathogenic bacteria ls still unknown, but there are not many over thirty known today to have the power of causing human disease. Immunity. By the term "immunity" is meant the power possessed by an organism of resisting the invasion of a microscopic parasite. Sometimes a whole race of animals is immune to a disease that 260 BACTERIOLOGY attacks other kinds of animals; sometimes individuals of a species are born with the power of resisting some disease to which the rest of their race are susceptible; sometimes an individual acquires im- munity to a disease to which it is naturally susceptible. There are, in other words, different types of immunity, which we must consider separately. TYPES OF IMMUNITY Race immunity. Microorganisms that are capable of producing disease in one race of animals are frequently quite unable to attack another species. No lower animal, for example, is attacked by the typhoid fever germs; while on the other hand hog cholera (as well as many other animal diseases) does not attack mankind. Sometimes this resistance may be due to physical peculiarities of the immune race-the high body temperature of birds, for example, may be the cause of their immunity to anthrax, as it is too high for the good growth of the organism. In most cases, however, we are quite unable to find any simple reason why an organism that attacks one race cannot attack another. Such race immunity is nevertheless perfectly well established, and is one of the prominent factors in controlling public health; for as a rule, the species of parasitic micro- organisms that attack lower animals are able to do little if any injury to man. While there are some striking exceptions to this rule, like anthrax and tuberculosis, it is so generally true that it is the basis of many sanitary regulations. We have learned, for example, that the various excretions from the human body are far more likely to be dangerous to our health than similar excretions from other animals. Human sewage, containing as it does the mixed excreta from thou- sands of men, some of whom are likely to be suffering from contagious diseases, is to man one of the most dangerous materials in existence; but it is not particularly dangerous for lower animals. On the other hand, the excreta from domestic animals, while surely far from attractive, are of no importance in the spread of human disease. In general it is found that a microscopic parasite which attacks one species of animal is more likely to attack other animals closely related to it in the zoological system than those more distant from it. Some of the diseases, for example, that are common in the human being cannot be given to any lower animal except monkeys. This is true of syphilis. Occasionally race immunity seems to show no RESISTANCE AND IMMUNITY 261 connection with biological relationship, since animals as closely related as the domestic mouse and the field mouse are very dif- ferently affected by mouse plague, which attacks one and leaves the other entirely unaffected. Within the limits of the human race, also, the principle applies to a certain extent, as certain races of men are more subject to some diseases than other races. The negro race, for example, is almost immune to malaria, a disease that is extremely common among the white race, while the negro is more subject to smallpox than is the white race. Similarly the Japanese are almost immune to scarlet fever. Individual resistance. Among a race of animals subject to an infectious disease, considerable variation is found in the ability of different individuals to resist its attack. Some members of the species will be found to have a high power of resistance while others will succumb readily to it. It is not uncommon to find a family in which one child escapes a disease that has attacked every other member, or vice versa, one child alone will contract a disease to which all its brothers and sisters have been equally exposed. Such in- stances cannot always be due to accident, but plainly indicate great differences in the susceptibility of different individuals in the same species to the same disease. Sometimes this individual resistance is due to certain factors that are rather superficial. An uninjured skin, for example, is a protec- tion against many diseases where the infection is through the skin; hence a person with an uninjured skin has a resistance against these diseases that one with a broken skin does not have. Sometimes, too, similar factors are found in connection with the diseases whose germs enter through the mouth or nose. The epithelial lining of the nose, the mouth, the throat and alimentary canal has some resisting power against the invasion of bacteria-due perhaps in part to the cilia (microscopic hairs) with which its cells are provided and which are constantly in motion; and the breaking down of this resistance makes a person liable to attack by organisms that he can ordinarily resist. A cold, for example, by producing inflammation in some of the air passages may so break down their resistance that the organ- isms of tuberculosis or pneumonia may get an opportunity to enter the body. This fact in a measure undoubtedly explains why a cold 18 liable to develop into consumption or into pneumonia. The digestive juices of the stomach also form a partial protection against 262 BACTERIOLOGY germs swallowed, as they are acid and nearly all bacteria are unable to grow in the presence of acid. Hence persons whose stomachs are decidedly acid might resist the invasion of such diseases as typhoid or cholera whereas others whose digestive juices are less acid might succumb to such an attack. Variations in all these factors explain some particular cases in which persons are more or less immune against certain diseases; but there are other factors concerned in this personal resistance that are more complex and more fundamental in problems of immunity. These more complex factors are the ones which form the basis of the new science of immunology. Acquired immunity. Some individuals are immune to certain diseases from birth; others acquire immunity sometime later in life. In the former case we speak of natural immunity, in the latter of acquired immunity. Acquired immunity plays a very large part in the control of infectious diseases. In has long been known that after an attack from an infectious disease a person is likely to be more or less thoroughly protected for some time against a second attack of the same disease, even though he may be repeatedly ex- posed to infection. As a rule, people do not have such diseases as smallpox, scarlet fever, measles, and so forth, a second time. The completeness of such immunity varies greatly with different diseases and different individuals. With some diseases it is almost absolute, while with others it is slight; with some it may last for years or even for life, while with others it may pass off in a few months. The possibility of utilizing this principle of acquired immunity has been dimly perceived for some centuries. Warding off a severe attack of smallpox by means of an artificially acquired mild attack is a very ancient practice and is apparently similar in principle to modern vaccination, as introduced some years ago by Jenner; but this principle was not recognized and consciously utilized until Pasteur (p. 57) brought out his anthrax vaccine. The principle concerned in this discovery was a very fundamental one, for it seemed to indicate that by merely weakening the virulence of a pathogenic organism cultures may be obtained that will protect an individual against attacks of the unweakened organism. There seemed to be practically no limit to the possibilities of this method, and bacteri- ologists began to hope that they might conquer all infectious diseases by the inoculation method. RESISTANCE AND IMMUNITY 263 Acquired immunity may be either natural-such as after the re- covery from disease-or artificial-such as after vaccination. Arti- ficial immunity is of two distinct types, known as active immunity and passive immunity. In the former case the individual is made to develop immunity by the active power of his own body in driving out the weakened organisms introduced as a vaccine. In the latter case, however, the cells of his body take no active part in the process. With diphtheria, for example, it is possible to bring about a rapid cure by the injection of antitoxin. The recovery in this case, how- ever, does not mean that the individual has acquired for himself the power of resisting the disease, but simply that he has been enabled to resist it temporarily by means of the artificial substances injected into his body. The immunity thus produced is fleeting, lasting only as long as these immunizing materials (in this case antitoxin) are retained within the body and disappearing as soon as they are eliminated. Passive immunity is therefore of short duration, while active immunity is more persistent. TO WHAT IS IMMUNITY DUE? This question, what causes immunity, has been raised almost from the beginning of the study of the germ theory of disease, and has been subject to an immense amount of experimental work. Trom theoretical grounds it would seem that any parasite capable of living in the body of an animal would be able to continue to live there, to multiply, and to become finally numerous enough to pro- duce the death of the host. With infectious disease, however, the history of the case generally shows a very different ending. After the animal is inoculated or infected with the disease, there is a period of varying length of time, known as the incubation period, before any symptoms make their appearance; then comes the onset °f the disease, marked by the characteristic symptoms, which con- tinue to increase in severity until either death occurs or a crisis is Cached; if the crisis is passed there is a gradual recovery, the symp- toms subside, and then the individual is protected for a considerable time against a second attack of the same microorganism. It is evident, therefore, that the infecting organism does not continue indefinitely to find conditions in the body favorable to its growth; and several questions force themselves upon us. Why should an animal be able to resist the attack of any of the microorganisms? 264 BACTERIOLOGY If parasites can grow in the body, why should they not continue in all cases to multiply until they are so numerous as to produce death? The recovery from the disease and the subsequent immunity are plainly closely related; but what forces produce recovery, and what forces continue to protect the individual from a further attack of the same organism? Why is this immunity effective only against the particular disease from which the animal has recovered and not against diseases produced by other microorganisms? We have by no means complete and satisfactory answers to these questions as yet; but in the last generation many important facts bearing on them have been learned, which are rapidly yielding practical results and have virtually Introduced a new phase of the science of medicine. There have been two types of answers to questions of this sort, one based upon biological and the other upon chemical conceptions. We have already seen how one theory has been developed by a French school, the other by a German school, and that a dispute has waged over the facts for many years. The dispute between the two has to a large extent ended at the present time, and the question has been settled by means of a sort of compromise; for it proves that each of these two schools of bacteriology has reached an important truth and that the real facts embrace a combination of both theories. We have already briefly discussed these two theories; but we must now take them up in more detail. THE BIOLOGICAL THEORY OF IMMUNITY, OR PHAGOCYTOSIS In the early days when the ideas of germ diseases were beginning to develop, Metchnikoff, whose name has been so closely associated with phagocytosis in recent years, was working not upon bacterio- logical problems but upon ordinary egg embryology. In 1880 he made the observation that in some cases among higher animals certain amoeba-like cells are capable of engulfing in their body small particles, something in the same way as the protozoan, Amoeba, engulfs its food. This intracellular digestion, as it was called, was demonstrated by him in a number of instances, and by others as well, and it was thought at the time to have special interest in its bearing upon the origin of the multicellular animals from the unicel- lular forms. In later years Metchnikoff, working at the Pasteur Institute, developed these early observations into his now famous phagocytosis theory. This theory is based upon the demonstrable RESISTANCE AND IMMUNITY 265 fact that the white blood corpuscles, or leucocytes, have the power of taking into their body any irritative, invading substance like microorganisms or even lifeless matter. As developed by Metchni- koff, the essential principles of this theory are: 1. The leucocytes of the blood collect at the point of invasion when the body is attacked by microorganisms. This is a general law and has been well proved for some germ diseases. 2. With some extremely violent and usually fatal diseases, the leucocytes instead of being attracted to the point of invasion, are repelled. Fig. 39. Leucocytes (White Blood Corpuscles) At the upper right hand corner is one laden with presumably dead bacteria. 3. The leucocytes when thus attracted to the point of invasion certainly engulf within their bodies the invading bacteria. This has been proved by numerous observations, and is now accepted as demonstrated. As Metchnikoff developed his theory, he assumed that the leucocytes thus engulf living bacteria and destroy them. Hence the final conclusion: 4. Phagocytes, as Metchnikoff named the leucocytes acting in this manner, are a prominent defense against the invasion of the body by microscopic organisms. The last of these four points is really the crucial one; for while lt was admitted even at the outset that microorganisms are engulfed by the leucocytes, it was denied that living bacteria could be thus engulfed. The opponents of the theory claimed that the bacteria were first killed by other agencies and that the leucocytes simply engulfed their dead bodies, thus acting as scavengers rather than as 266 BACTERIOLOGY policemen. This question was long debated, Metchnikoff and his school pointing out many facts that seemed to imply the truth of their theory. If the anthrax organism, for example, is inoculated into an immune animal, such as a dog, the invading bacteria are quickly taken up by the phagocytes, whereas if inoculated into a susceptible animal, such as a sheep, they are not engulfed by the lecuocytes and develop rapidly in the body. Such facts as these certainly suggest that the leucocytes are actively concerned in bring- ing about immunity. One of the best illustrations of this principle of phagocytosis is the phenomenon of inflammation. This process may generally be looked upon as a healing process, athough it does not of course always result in healing. When irritating bodies, like invading parasites, get into active tissues, there is a general tendency for the accumulation of blood and lymph in the region of the invaded area, the area as a result becoming more or less red and swollen, and tender to the touch. The leucocytes especially accumulate in large numbers and may be found by microscopic study to be actively engaged in attacking the invading parasites. Meanwhile around the inflamed area there is an increased multiplication of the body cells to form a sort of connective tissue wall to separate the invaded area from the rest of the body, and thus to protect the rest of the body from further excursions of the invading parasites. Within this walled-off area the leucocytes are carrying on their contest with the microscopic organisms, and eventually by their action in combination with the tissue cells that have produced the limiting wall, the invasion is ordinarily checked. The leucocytes within the walled-off space ordinarily break through the surface of the skin, after the infection is checked and are discharged in the form of pus, which is found to be made up largely of lecucocytes containing the invading parasites in large numbers. When this process is entirely effective, the in- vading organism may be confined to small areas, and produce no general trouble. This not infrequently happens, when the body is invaded by some blood-poisoning organisms, which produce boils, abscesses and carbuncles. Even in the more serious troubles, like tuberculosis, the organisms are frequently walled off from the rest of the body, in the tubercles that are produced in the lungs or glands or elsewhere. In all these cases, the leucocytes are undoubtedly active factors in repressing the invading parasite. RESISTANCE AND IMMUNITY 267 The theory that immunity is largely due to these leucocytes was extremely popular when it first made its appearance, being the type of theory that is sure to appeal to the imagination. References were even made to the education of the white blood corpuscles, the assump- tion being that a corps of veteran guards is more valuable than a similar number of guards that have had no experience. Hence it was stated that after the white blood corpuscles had once learned to handle any particular kind of invading organism, like those of smallpox, for example, they were more capable of doing so a second time, and hence the person was protected against a second attack. According to this theory, therefore, acquired immunity is due to the fact that the white corpuscles can learn by experience the proper methods of attacking the invading parasites. A large amount of data bearing upon the subject was accumulated by Metchnikoff and his students, who succeeded in proving that the leucocytes certainly do engulf the bacteria while they are alive, and that they certainly play some part in protecting the body against bacterial attack. During the time that this theory was developing, however, another very different series of facts had been gathered, leading to a totally different method of explaining immunity. THE CHEMICAL THEORY OF IMMUNITY The term "chemical theory" while not entirely satisfactory as a designation for the hypothesis to be described, will serve to indicate the contrast between this and the biological theory, inasmuch as immunity in accordance with this theory is due to the presence in the living tissues of certain chemical substances which have a dele- terious action upon the invading organism. The first evidence obtained to this effect was the discovery that living blood has what has been called a germicidal property, bacteria dying off at first in freshly drawn blood although able to grow well in blood that has stood for a number of hours outside the body. This property of freshly drawn blood is destroyed by moderate heat, and is evidently due to some substance or substances in blood that are destroyed by heat. The discovery of such substances, naturally suggested the idea that the reason bacteria cannot successfully invade an immune animal is because these germicidal substances destroy the bacteria that find entrance. 268 BACTERIOLOGY The next confirmation of the chemical theory was the discovery of diphtheria antitoxin by Behring and Kitasato. Antitoxin could readily be explained in terms of the chemical theory, while its rela- tion to phagocytosis was very doubtful. ,Its discovery, together with other similar facts that soon appeared, placed the chemical theory upon a strong footing and led scores of bacteriologists to carry on experiments in accordance with the terms of this theory. It did not appeal to the imagination as much as the theory of phagocy- tosis, but on the other hand it proved a much better basis for experi- mentation. Metchnikoff's theory left one in the dark as to how to improve the "education" of the phagocytes to make them more efficient, but the chemical theory almost immediately opened up the way to innumerable practical lines of investigation. There is today no question but that the recovery from an infec- tious disease commonly takes place as the result of the development within the body of certain protective substances produced by the activity of the living tissues. The presence of the invading parasites influences the host in some unexplained manner so that its tissues give origin to substances which eventually check the ravages of the parasites. These protecting substances, called antibodies, are of an endless variety of different kinds, but may be grouped in two general classes: antitoxins and anti-bacterial substances. Antitoxins. Some pathogenic bacteria secrete violent poisons (toxins') which are absorbed by the body and cause the symptoms of the disease. In defense, the body produces antitoxins-a specific antitoxin for each toxin-which counteract the effects of the toxins and render them harmless. Thus we have diphtheria antitoxin and tetano- antitoxin produced in defense against diphtheria and tetanus re- spectively. Such antitoxins do not act upon the bacteria but simply upon the toxins they produce and by preventing the poisoning action of the toxins enable the individual to recover. This power of the living tissues of producing antitoxins is a very broad one and is effective not only against bacterial poisons, but against all other soluble toxins as well. By inoculating an animal, for example, with small but gradually increasing quantities of snake venom, the animal can be made to produce an antidote to the poison-called anti- venine (a substance which is obtained on a large scale from a horse and is used as a protection against the effects of snake-bite). The same thing happens in the case of the poison of spiders, and even in the case of certain vegetable poisons. RESISTANCE AND IMMUNITY 269 Anti-bacterial substances. With some diseases, the protective substances do not act upon the poisons but upon the bacteria them- selves. They are called anti-bacterial substances or lysins. This seems to be the case when the toxins are endo-cellular-that is, are not excreted from the bacterial cell-because then it is apparently impossible for the body to produce antitoxins to counteract them. These antibodies attack the bacteria directly and by some means either injure or destroy them. They are very different in their action and very different in practical value from the antitoxins, for while the latter have been found very useful in curing diseases, the former are of little use as a cure but of great value in preventing disease. The side-chain theory. What the actual nature of these antibodies is and how they are produced is a subject that has been very exten- sively studied and concerning which an enormous amount of data has accumulated during recent years. The topic, however, is ex- tremely complex, and is as yet very largely in the region of theory only, with few demonstrated facts. The theory which has been most fruitful of results is the one advanced by Ehrlich; but it is so com- plicated that an attempt to consider it in detail would be too long an excursion for us to take here. It is generally known as the side- chain theory because of an analogy which Ehrlich considered to exist between immune bodies and certain parts of organic compounds which chemists call side-chains. The molecules of certain organic compounds are believed to consist each of a complex, central body to which is attached one or more simpler combinations of atoms known as side-chains. Ehrlich's conception of a toxin was that it could become attached to the more complex compounds of the body much as a side-chain is to the central part of a chemical molecule, aml he considered that the anti-bacterial substances in the blood acted in a somewhat similar manner upon invading microorganisms. •Antitoxins he considered to be substances produced by the body when stimulated by the presence of toxin, that combine with the f°xins in such a manner that the toxins can no longer become at- tached to the body proteids and are therefore neutralized. Ehrlich and his followers thus continued to expand the theory until they had named a long series of hypothetical substances each having its own Part, although sometimes but a small part, in the general phenomena °f immunity. This theory may not be true, but it has been extremely 270 BACTERIOLOGY fruitful in results, and has brought about very many important discoveries, together with extremely useful practical applications in the way of controlling infectious diseases and augmenting the resisting power of the individual. It seemed at first that the biological and chemical theories of immunity were totally inconsistent with each other; for if the body be protected against the invasion of microorganisms by the produc- tion of chemical antibodies there would seem to be no need of phago- cytic activity. The two theories in question-as we learned when studying the history of bacteriology-were developed in German and French laboratories, respectively, and a rivalry between their adherents developed as soon as the two theories were distinctly formulated. For a number of years, data were collected in profusion by both schools, and the evidence for the truth of both theories rapidly accumulated. It has finally been recognized that both theories must be in a measure correct and that the true explanation of immunity must be a compromise or a combination of the two. That the living tissues contain chemical substances inimical to the growth of bacteria has been amply demonstrated; but there is considerable question whether these substances alone are capable of protecting the body from all diseases. That phagocytes are capable of engulfing live bacteria, not simply dead ones, has also been dem- onstrated; but it has been shown, in the case of some diseases, that the phagocytes are unable to dispose of the invading bacteria until the bacteria have been previously weakened by sojourn in the living tissues of the host. In other words, certain substances present in the blood and other living tissues of animals seem to have the power of injuring the vitality of invading bacteria so that the phagocytes can destroy them. These substances alone cannot de- stroy the parasites and therefore are not in themselves sufficient to account for immunity; but after they have acted upon the parasites these latter organisms can be engulfed and destroyed by the phago- cytes. The name opsonins has been given to these substances. As a result of such discoveries, it is apparent that in many cases of immunity the phenomenon is explained by a combination of the two rival theories, as it is a two-fold process, due partly to the injurious action of certain chemical substances upon the invading COMPROMISE THEORIES RESISTANCE AND IMMUNITY 271 parasite, and partly to the active power of the phagocytes. While it cannot be claimed at present that such an explanation holds for all cases of immunity, this statement illustrates the fact that neither the biological nor the chemical theory is a complete explanation. As a further illustration of this same fact, it is interesting to notice that the rat and the dog are both immune against anthrax, but the immunity of the dog is due to the activity of its leucocytes in de- stroying the invading organism, while that of the rat is due to anti- bacterial substances in its blood. It is evident, therefore, that im- munity is not always due to the same sort of causes. There are at least two types of immunity, one distinctly biological, due to the activity of the leucocytes, and the other, partly chemical, due to the presence of antibodies. Thus both the biological and the chemical theory have a large basis of truth in them. BACTERIOPHAGE During recent years some entirely new theories concerning immu- nity have been proposed. These new theories, however, are not rival theories to those just mentioned, but are considered rather to supple- ment them. They relate to entirely different phenomena. A histor- ical discussion of the subject affords a good explanation of these phenomena. i The first observation of this sort had nothing to do with immunity. I[t was noticed in India about 1896 that the water of the river Jumma h!ad a noticeable antiseptic effect upon bacteria, especially upon the cholera vibrio. Filtering through porcelain did not destroy this a ntiseptic property, but boiling did. No explanation of the phenom- e non was given at the time. A more striking observation was made by Twort in 1915. He W carrying on experiments with vaccine virus obtained as usual h;om the pustules on a calf, and found one lot contaminated with a h.ucrococcus. He isolated this micrococcus, and found that the film o. f growth in some cases was marked with translucent spots that slowly iread over the entire culture, while the cocci were transformed into fi) ae granules. Other strains of his culture did not show this appear- ance, but if one of these normal strains was touched with a platinum w ire, which had previously been in contact with the translucent gi 'owth, a spot of the same sort appeared in the growth touched with and this spot spread over the whole culture. This translucent 272 BACTERIOLOGY growth could be mixed with sterile water and filtered through porcelain and yet still had the power of causing normal growth to become translucent even though only a minute amount of the filtrate were transferred to it. Heating to 60°C. destroyed this property of the filtrate. A year or two later the more striking experiments of d'Herelle were published and the term "bacteriophage" introduced into the language of bacteriology. The phenomena of d'Herelle were slightly different; and he insists, indeed, that they are entirely different ih nature and cause from those of Twort. The first observation of d'Herelle was in the case of a dysentery patient. An examination ojf the feces was made each day by inoculating bouillon, incubating ovey night and then filtering through porcelain, and adding twelve drops of the filtrate to a fresh culture of the dysentery organism in bouillon. Ordinarily the growth in this second tube remained normal. Ono day, however, the original tube showed no growth; and when a culture of the dysentery organism was treated as usual with a few drops of the bouillon, or with a few drops of the same material filtered, the culture cleared completely, and the bacteria apparently disappeared from it entirely. Apparently some material had been obtained froip the feces of the patient which could dissolve the dysentery organism. This principle remained present in the feces from that time on. Meanwhile the patient recovered, the improvement beginning the day when this phenomenon was first observed in the broth inoculated with the feces. It was further found that if a drop of one of the cultures killed by this principle from the feces was added to a young bouillon culture of the dysentery organism, this in turn became dissolved, and another drop of it could have the same action on another culture. This was found to continue through several passages from one culture to another; and instead of losing in potency, the dissolving action became more rapid with each transfer. A very small amount of one of these dissolved broth cultures (0.00001 cc.) would have this action- If a culture treated with the minute quantity was smeared im- mediately over agar and incubated, it was found that the resulting growth showed two or three small circular areas entirely free from bacteria. If before smearing over the agar the broth culture wa£ incubated two hours, there would be perhaps six or eight of these clear areas in the growth on agar; if incubated two hours before RESISTANCE AND IMMUNITY 273 smearing over agar, one hundred or more of the clear areas would be observed, rendering the whole growth nearly transparent; while if incubated three hours, no growth at all could be obtained upon agar incubated from it. Now there seemed to d'Herelle to be only one explanation of these phenomena. Something was present in the intestines of the patient after the disease began to mend which dissolved the dysentery organism. It was so small that it could pass through a porcelain filter, and it was capable of multiplication, as proved by the fact that a drop of the cultures several transfers removed from the original still had the power of destroying the organism. Nothing but an ultra-microscopic living being could have all these properties, and such d'Herelle concluded it to be. Just as large organisms are preyed upon by bacteria, so he concluded bacteria to have their ultra-microscopic parasites. One of these parasites on bacteria he called a bacteriophage. When a person's intestines become inhabited with this bacteriophage, he concluded that person would be protected to some extent against the bacteria which are destroyed by this ultra-microscopic organism. D'Herelle has made quite extensive investigations on the subject and claims that there is only one species of bacteriophage. This species may become especially active against some particular type of bacteria, but it is not specific against that type alone, as it can also destroy other kinds. It will be seen that if this is true, there is still another type of immunity besides the biological and chemical forms discussed above. It is possible to be immune to a disease at least temporarily because of being infected with the bacteriophage. It must be understood that those who hold this theory do not claim that it explains any of the phenomena of immunity discussed above, which are already ex- plained either by phagocytosis or by the chemical theories; it merely explains new phenomena of immunity which we must now add to the ones previously observed. Meanwhile there have been other theories to explain these new phenomena. Everyone admits the truth of the observations of Twort and of d'Herelle; but not all are of d'Herelle's school in interpreting them. Of other theories, the most commonly accepted one is that cultures under certain conditions break down to form a substance toxic to themselves. This toxic substance acts on fresh 274 BACTERIOLOGY bacteria in such a way as to destroy them and to produce more of the toxin. In this way the quantity of the toxic substance is constantly being increased without its having any powers of multiplication in itself. The exponents of this theory claim that certain facts can be thus explained that are not accounted for by the bacteriophage theory. The matter is undergoing considerable discussion at present, and is not likely to be settled for some time. The practical side of the question is also being much discussed. If the phenomenon is due merely to a substance produced by the bacteria themselves, the principle may not have much value in fighting disease. If, however, it is due to a living bacteriophage, it may be possible to inoculate individuals with this ultra-microscopic organism and thus to protect them against disease. Efforts to produce immunity in this way have not yest been successful, but the possibility is so promising it is likely to be extensively investigated in the near future. REFERENCES E. O. Jordan. A text book on General Bacteriology. 7th edition. Saunders, Philadelphia, 1922. See Chapter 8. W. Park and Anna W. Williams. Pathogenic Microorganisms. 6th edition. Lea and Febiger, Philadelphia, 1917. F. d'Herelle. The Bacteriophage: Its Role in Immunity. Translated by G. H. Smith. Williams & Wilkins Co., Baltimore, 1922. CHAPTER III The Practical Value of the Phenomena of Immunity THE ARTIFICIAL PRODUCTION OF IMMUNITY Pasteur's discovery of the method of protecting animals against anthrax prepared scientific medicine to expect that similar methods would be devised for protection against various other germ diseases. This anticipation has been realized, but not in all cases along exactly the line pointed out by Pasteur. In the investigations of the forty years since Pasteur's discovery, bacteriologists have been busily engaged not only in searching for the cause of different germ diseases, but in studying their nature and possible methods of protection against them. Greatest progress along this line has been obtained by learning the methods of producing artificial immunity. This line of investigation has proved of such practical value that today nearly all the infectious diseases of domestic animals have been mastered by learning how to render animals immune. This has not yet proved possible in the case of all human diseases, because of the greater difficulty of experimentation, and the consequent slower progress; but several of the worst of man's diseases are now con- trolled by methods of this very sort, and their mastery is regarded as one of the greatest recent triumphs in medicine. Both the chemical and the biological theories of immunity have led to practical results in the control of disease. In fact, the rivalry between the adherents of the two theories has proved of considerable benefit to mankind, because of the additional stimulus which it has given to investigations along this line. It is interesting, however, to notice that the same disease has never been conquered by both lines of attack. Some diseases can be controlled by means of anti- bodies, that is, according to the chemical theory; others by means of the powers of phagocytosis, that is, in accordance with the biological theory. PRACTICAL RESULTS FROM THE CHEMICAL THEORY OF IMMUNITY It was the chemical theory from which results were first obtained that attracted the public eye. We have already seen how diph- 276 BACTERIOLOGY theria antitoxin was first obtained from the blood of rabbits by Behring and Kitasato, and was subsequently obtained on a large scale by Roux from the blood of a horse. The result of this dis- covery was the complete mastery of diphtheria. The method by which the material was originally obtained, and essentially the same method used today, is briefly as follows. Diphtheria bacteria are cultivated in a bouillon in which they grow readily, and after a few days the bouillon is found to be filled with a considerable quantity of diphtheria toxin, the same substance which, when produced by the organisms growing in the throat, poisons the individual and produces the symptoms of the disease. This toxin is next injected in small quantities into the body of a horse, certain definite intervals being allowed to elapse between successive inoculations. The injection of the first lot of toxin has no noticeable effect on the horse, only it renders him capable of standing a second inoculation of a larger amount, and this protects the animal from a still larger amount at a third inoculation made later. These inoculations of toxin are kept up for a number of weeks until, as experience has shown, the maximum result is obtained. In the blood of the horse, during this period, a quantity of antitoxin has accumulated. There is no theoretical reason why antitoxin cannot be produced naturally in the human body by this same method, that is, by grad- ually increased injections of toxin. In this way lasting immunity against diphtheria could be brought about. Practically, however, the method is out of the question, as the danger of contracting diphtheria is hardly great enough to justify the disagreeable effects of the treatment. Recently it has been learned that by mixing the toxin and antitoxin in exactly the right proportions a neutralized toxin can be obtained that causes no ill effects upon injection, but which stimulates the body to produce antitoxin much as would the toxin alone. The reason for this must be that the artificially in- troduced antitoxin gradually loses its strength, leaving free toxin which as it regains its toxic powers stimulates the production of antitoxin. In this way active immunity is produced without ex- periencing any disagreeable effects from the toxin injected. The method of treatment is quite new, and it is not known yet how last- ing is the immunity it confers, but there is good reason to believe that it is as permanent as the immunity of the horses that are im- munized by the injection of toxin. On this assumption the method PRACTICAL VALUE OF PHENOMENA OF IMMUNITY 277 is now being used to immunize school children in some of our large cities, and it is certainly reducing the percentage of diphtheria cases that occur. The discovery of the method of procuring antitoxin revealed a principle which, it was thought at first, might have a wide applica- tion. A further study of the various types of germ diseases, however, has shown that this particular principle does not apply to very many of them. It applies only to those diseases that are fought in the human body by the production of antitoxins, such as diphtheria and tetanus. In the case of those diseases which the body naturally resists in this way, it is possible to assist nature by means of the artificial production of the needed antitoxin, as has been done in the case of the two diseases just mentioned. In the case of most diseases, however, the body protects itself not by antitoxins but by means of anti-bacterial substances. These differ from antitoxins in the fact that instead of counteracting the toxic effect of the poison, they act as partial germicides upon the bacteria themselves, warding off the disease, or producing its cure, by actually destroying the invading germs. This apparently is true in the case of smallpox, for example. Up to the present time no satisfactory method of producing these anti-bacterial substances artificially has been devised, so that the principle discovered in the case of diphtheria does not apply to small- pox or to other diseases similarly combatted by the body. Hence there are but few diseases to which the principle discovered in the case of diphtheria can be applied. Instances where its application has been possible and practical will be mentioned later in the dis- cussion of the different diseases. Attenuated viruses. It has just been said that the artificial pro- duction of anti-bacterial substances is not possible. By this is meant merely that it is not possible to produce them in one animal for artificial introduction into another. It is possible, however, to stimulate artificially the production of these antibodies within an animal so as to render that animal immune, even though its blood does not thereby become of value in curing or preventing the disease m other individuals. Thus Pasteur, in the days before there was any theory to explain the results, injected weakened cultures of anthrax mto sheep or cattle and caused them to produce such large amounts of the specific anthrax antibodies that they became immune to the disease. The control of smallpox by vaccination is undoubtedly 278 BACTERIOLOGY due to the same principle (i.e., inoculation with an attenuated virus) although it has never been proved for certain that the cowpox virus used for vaccination is actually a weakened culture of the smallpox organism. In both these cases it will be seen that the resulting im- munity is active, instead of passive as in the case where antitoxins are used. The same principle is used in rabies and in a few animal diseases, with the production of artificial active immunity in all cases, presumably through the stimulation of the body to produce anti-bacterial substances. Antitoxins and anti-bacterial substances are very different in their practical value. An antitoxin can be produced in abundance, kept on hand, and can be introduced into the blood very quickly whenever needed; but the individual thus treated produces no new antitoxin and the supply injected is soon eliminated. Anti-bacterial substances, on the other hand, must be produced by the individual it is desired to protect, a process which requires some time; but once immunized in this manner, the individual continues to produce the antibodies for months, for years, or perhaps even for life. Therefore antitoxins are of great aid in producing a cure and of little value in bringing about lasting immunity; while the anti-bacterial substances are of little value as a cure but confer an immunity which does not quickly pass off. Serum therapy. The extensive use of antitoxins and vaccines in curing and in preventing disease has caused the introduction of the term serum therapy. This term, strictly applied, refers to the use of antitoxins obtained in the serum of some animal's blood, and it might apply equally well to smallpox vaccination, where the material used is the lymph from the cowpox pustules on a calf. Commonly, however, the term is used to refer to the control of disease by any means of inoculation, vaccination, or injection of any material that increases the natural immunity of the individual so treated. Serum sickness. In the use of antitoxin there occasionally has occurred an immediate effect of the injection which has been called serum sickness. Some persons appear to be extremely sensitive to the injection of the serum of the horse, so that such an injection at once produces trouble. The effect is due simply to the serum and not to the antitoxin, and will be brought about if pure horse serum is injected. This sensitiveness is sometimes increased by a previous injection, so that after a person has received a little antitoxin PRACTICAL VALUE OF PHENOMENA OF IMMUNITY 279 without bad effects, he may become supersensitive to the serum so that a second injection will cause illness. This extra sensitive condi- tion is called anaphylaxis. To reduce this danger the antitoxin now in use is concentrated so that only very small amounts of serum are necessary to furnish the desired amounts of antitoxin. With the modern antitoxin preparations, the cases of serum sickness are rare, and they are not serious enough to form any argument against anti- toxin in cases where there are strong reasons for its use, either as a cure or as a protection when exposure to diphtheria is known to have occurred. PRACTICAL RESULTS FROM THE THEORY OF PHAGOCYTOSIS The practical results arising from the study of immunity as a chemical phenomenon brought the chemical theory into very great prominence during the twenty years following the first production of antitoxin. The biological theory, although of great theoretical interest, did not at first seem capable of yielding results of practical value. Recently, however, practical application has been made of discoveries arising from Metchnikoff's theories, and there seems to be promise of results of as great, if not greater, value than those that arose from the chemical conception of immunity. These results have come from what are called bacterial vaccines or bacterins. Their extensive use in recent years has led to the introduction of the term vaccine therapy. These vaccines are not sera, like the smallpox vaccine, nor attenuated viruses, like the anthrax and rabies vaccines. Bacterial vaccines. As already noticed, phagocytes are commonly unable to attack invading microbes until they have been weakened by some substances, called opsonins or bacteriotropins, which are Present in the blood of the invaded animals. Careful testing shows that the quantity or power of these opsonins varies markedly in dif- ferent individuals, and indeed varies at different times in the blood of the same individual. Manifestly if some method could be found of increasing the power of the opsonins in any individual, this would be a means of increasing his resistance against microorganisms, inasmuch as this increased opsonic power would tend to insure the destruction of the invading bacteria by the phagocytes. The method of accomplishing this was discovered by accident. It was noticed that if the dead bodies of some kinds of bacteria, killed by low heat, are injected beneath the skin, the phagocytic activity 280 BACTERIOLOGY against the particular kind of bacteria injected is increased. This discovery was confirmed by further investigation, and was the basis of the opsonic theory, first advanced by Wright. In Wright's hands this theory developed finally into a practical method for increasing the opsonic power of individuals so as to give them increased resist- ance against invading parasites. This method, in brief, is the injection beneath the skin of a definite number of dead bacteria of the particular kind against which it is desired to establish immunity. The preparations of dead bacteria are the bacterial vaccines. These vaccines are widely used today and have resulted in very great improvements in the treatment of certain diseases. The diseases where they are applied will be noticed in later pages. The preparation of bacterial vaccines is very simple, much simpler than the preparation of antisera. A culture of the organism produc- ing the disease is allowed to grow at its optimum temperature for a short period, which varies slightly according to the particular species in question. At the end of this time, the growth is mixed with a sterile salt solution of such a strength as to be of the same density as the blood and other body fluids (i.e., a physiologically normal salt solution, as it is called). This liquid, containing great numbers of the disease germs in question, is subjected to low heat (60°C. or a little less) for a period found by practice to be sufficient to destroy the life of the bacteria. Care must be taken in choosing the temperature and the time, because too much heat destroys the value of the vaccine, and too little does not kill all the organisms. After this sterilization by heat, careful testing must be made to determine with certainty that the organisms are all killed. This is usually done by inoculating some of the heated material upon a fresh culture tube of agar, to see whether anything is left alive that is capable of growing. The next step is to count, by certain micro- scopic methods, the number of dead bodies of bacteria that are present. The product, when diluted to contain the proper number of dead bacteria, and preserved with carbolic acid or some other disin- fectant, is a bacterial vaccine. The number that are injected at a single dose varies with different kinds of bacteria; sometimes it is as low as fifty million, and sometimes it may be several hundreds of millions, the number being determined by experience. As a rule, also, to produce a perfect result with such a vaccine, more than one injection is made, two or three following each other at intervals of a PRACTICAL VALUE OF PHENOMENA OF IMMUNITY 281 few days, generally with a stronger dose at each successive injection. The result of this treatment is to increase the opsonic power of the individual against this specific infection, and thus to increase phago- cytosis and to give him a greater protection against this particular disease. There are two different sorts of bacterial vaccines, known as autogenous and stock vaccines, respectively. To prepare an autog- enous vaccine a culture is isolated from the patient of the particular strain causing his trouble. The vaccine is prepared from this culture as above described, and is then used to protect the individual in question from further trouble from this same organism. In this case it is certain that the vaccine will be made from the particular kind of organism that is attacking the patient in question. Some- times, however, it is impossible to take the time necessary to prepare an autogenous vaccine, a process which requires several days at the shortest. In such cases a vaccine is used which has already been prepared from bacteria isolated from some other individual. Vac- cines of this sort are called stock vaccines, because they can be made in large quantities and can be kept on hand almost indefinitely ready for use at the moment they are needed. The disadvantage of stock vaccines is that they are not definitely known to be made from the same kind of bacteria that are attacking the patient. Although cultures of a specific disease germ obtained from different individuals are essentially alike, different strains do differ in some respects. Some, for instance, are more virulent than others. It might there- fore follow that a stock vaccine produced from a certain strain of a particular organism might not be the one required in some case where the patient was infected with a slightly different strain of this same kind of bacteria. To meet this objection as far as possible, stock vaccines are frequently made from several different strains mixed together, with the expectation that such a mixture will be more likely to produce results than would a vaccine made from any single strain. Practical value of bacterial vaccines. The value of bacterial vac- cines is best illustrated by one special application of them-namely, in the case of typhoid fever. A few years ago experiments seemed to indicate that injections of dead typhoid bacteria conferred a certain amount of immunity against this disease. Absolute proof was diffi- cult to obtain because no lower animal is susceptible to typhoid and 282 BACTERIOLOGY human inoculation is out of the question; but the method of vaccina- tion was repeatedly tested out in the case of people likely to be ex- posed to typhoid, until the data showing the truth of this extra- ordinary discovery have become very extensive. The conclusion has been reached that an almost perfect immunity is established, which lasts for one or two, or perhaps sometimes for three years. The vaccination has proved to be harmless, although there may be a feeling of malaise for a while after the injection. The first extensive application of this method of vaccination was in the case of soldiers who, because of the peculiarities of camp life, are more subject to typhoid exposure than any other class of people. The success in such cases was so immediate and so great as to lead to a wide adoption of the method, until at che present time nearly all soldiers in all the army camps of the world are thus protected against this former scourge of military life. The practice is extending widely among civilians as well, particularly among those who are obliged to travel and are thus more likely to be exposed to infection. There has hardly been any discovery in the history of medicine as reliable and efficient as this anti-typhoid vaccination. It was at first thought that immediately after vaccination a person was some- what more susceptible to the disease for a few days; hence at the out- set the advice was given not to use anti-typhoid vaccination if a person was believed to have been exposed to the disease. Further study, however, has shown that this is not the case, and at the present time persons are very commonly thus vaccinated when they think they have been exposed and wish to be protected from the disease. One can hardly conceive of a more surprising discovery than that an efficient method of protecting a person from the attack of an invading parasite is to fill his blood with many millions of the dead bodies of this parasite. But strange as the method is, its success is beyond question, and while it was somewhat doubted when first intro- duced, it is now one of the universally acknowledged methods of protection against typhoid fever. Each year now sees this same method of attack used against more and more different diseases. In some, like typhoid, its value is proven, but in others it is of more doubtful value. Next to typhoid, greatest success has been had in the case of the pyogenic infections, that is, the organisms that cause pus-producing sores. PRACTICAL VALUE OF PHENOMENA OF IMMUNITY 283 METHODS OF PRODUCING IMMUNITY Slimming up the matter of immunity, we find that up to the present time there have developed five primary methods by which immunity may be produced among animals. They are as follows: 1. Recovery from an attack. If an infection is not serious enough to produce death, and the individual attacked recovers, he is found in the case of certain diseases, to have a more or less permanent im- munity. This is nature's method, and has only rarely been used artificially-such ms formerly in the case of smallpox, when the only method of protection was exposure to attack from a mild case. 2. Inoculation with an attenuated virus. By growing the disease organisms under conditions that weaken them it is possible to secure a strain that produces a very mild type of the disease and yet brings about immunity. Vaccination against smallpox was the first application of this principle, but was the result of an accidental dis- covery. Pasteur's method of inoculating against anthrax was the first conscious application of the principle to disease control. 3. Injection of toxin. In diseases that are caused by soluble toxins secreted by the infecting parasites, injection of a very small quantity of the toxin into an animal, followed a few days later by a little larger quantity, and then later by a still larger quantity, slowly establishes an immunity against that disease. This is the principle by which diphtheria antitoxin is obtained, for by injection of the toxin into the horse at intervals, the horse finally becomes absolutely immune against diphtheria, even though infected by the most viru- lent organisms. This principle has proved practical in the case of human beings only when the toxin is mixed with antitoxin, as in producing immunity against diphtheria. 4- Injection of antitoxin. The antitoxin produced within an ani- mal thus immunized by injection of toxin confers brief, passive, immunity if introduced into some other animal. 5. Injection of bacterial vaccines. Cultures of disease bacteria, killed at low temperatures, are sometimes, as we have just seen, of value in stimulating the body to produce certain anti-bacterial substances that increase the efficiency of the phagocytes. Typhoid fever is the best illustration of this method of producing immunity. It is not always possible to apply any of these five methods to the fighting of human diseases. Human life is too valuable for experi- mentation, and some of the methods mentioned are more or less 284 BACTERIOLOGY dangerous, so that they are not practical to apply to man. In the applicability of these methods to the human race, it has to be borne in mind first, that such inoculations are always a source of some danger, unless extreme care be used. Any injection of extraneous material into the body-even a scratch of a needle-is liable to produce blood poisoning, while the introduction of foreign protein, even though entirely sterile, may often show toxic effects. There have undoubtedly been unfortunate results from these causes that have laid the foundation of a prejudice against protective inocula- tions. These dangers are becoming less and less each year, as the methods are perfected, and they are infinitely less than the good accomplished. Protective inoculations are also troublesome, cost some money, and sometimes considerable inconvenience. Even though protective methods might be developed against all kinds of infectious diseases, it is hardly to be expected that mankind will submit to being inoculated against all diseases. Hence protective inoculation against disease has thus far been applied to mankind only in emergencies or for certain types of very serious diseases that are widely distributed. The following list contains the only instances where they have been to any extent applied in protecting mankind: 1. Vaccination against smallpox. 2. Use of antitoxin against diphtheria. 3. Use of toxin-antitoxin mixtures in immunizing against diph- theria. 4. Use of antitoxin against tetanus. This is a successful method of prevention, and occasionally of cure. 5. Use of antivenin as a cure for the bite of poisonous snakes. 6. Use of a spinal meningitis serum. Has been fairly successful. 7. Protective inoculation against rabies has been very successful. 8. Bacterial vaccines have been used in the case of blood poisoning, typhoid fever, whooping cough, bubonic plague, pneumonia, with varying success. In some cases the results have been very satisfac- tory, in others of doubtful value. 9. Certain types of dysentery have been cured by a special serum made from a horse. 10. Asiatic cholera has been treated by weakened cultures, and also by bacterial vaccines. The results are claimed to be fairly successful. 11. Carbuncles, abscesses, and boils have been successfully treated with bacterial vaccines. PRACTICAL VALUE OF PHENOMENA OF IMMUNITY 285 THE DIAGNOSIS OF DISEASE WITH THE AID OF THE PHENOMENA OF IMMUNITY One of the most important of modern-developments in medicine is the rapid and accurate diagnosis of disease. Failure to apply the proper treatment is generally the result of incorrect diagnosis, and Physicians have long been struggling with the problem of how to find out correctly what disease a patient is suffering from before it becomes too late to treat it. The bacteriologist has been a great help to the physician in this matter, and there are today, scattered all over civilized communities, bacteriological laboratories where physicians may send specimens for diagnosis. In the case of certain diseases, like diphtheria and tuberculosis (see pp. 90 and 341), the causal organisms are sufficiently characteristic in appearance so that microscopic examination of the discharges by a trained bacteri- ologist is sufficient for diagnosis; but such diseases are few, and if they were the only ones the bacteriologist could diagnose, these laboratories would not be of great value. Fortunately, however, Modern ingenuity has taught the bacteriologist to use the phenomena of immunity we have just been studying as a means of diagnosing disease with almost absolute certainty. When Pasteur first investi- gated the matter of immunity, no one supposed that he was opening the way not only to improved methods of treating disease but also to better methods of diagnosing it. That such proved to be the case ls just one of the many surprises that has developed from the study of bacteriology. Tuberculin. One of the oldest means of diagnosis by methods of this general nature was devised by Koch, and was originally sup- posed to be a cure, not merely a means of diagnosis, for tuberculosis. Koch grew the tubercle organism in bouillon containing glycerin tor a period of several weeks at body temperature, then killed the bacteria in flowing steam and filtered the product through a por- celain filter to remove the dead bodies of the bacteria. This material seemed at first to have a curative effect upon consumption and was Put on the market under the name of Tuberculin. It was widely bailed as another of Koch's triumphs; but as the evidence gradually accumulated, it proved that his claims for it were unfounded. Con- sumption is a disease of such slow development that considerable time is necessary before one can tell whether any treatment has actually been efficacious-a fact which is so well known by quack 286 BACTERIOLOGY doctors that consumptives are among their favorite victims. It was undoubtedly for this reason that Koch was deceived as to the value of tuberculin, and that it took some time for the world to discover that he wras mistaken. At the time, Koch's reputation suffered considerably; but today tuberculin has proved so valuable for diagnosis that it is all but forgotten that Koch's predictions in regard to it were not fulfilled. When a healthy animal or human being has a dose of tuberculin injected beneath his skin there is no effect; but if the individual is suffering with tuberculosis-no matter how mild the case-he has a slight rise in body temperature during the next twenty-four hours. This test is particularly valuable in selecting dairy cattle that are free from tuberculosis, its only drawback being that it is so very delicate that an animal with a very light temporary attack reacts just as strongly as one in the last stages of the disease. The exact nature of this reaction is not known, but it is believed to be one of the general class of phenomena that we have been studying. In this case no immunity is involved; but the bacteria evidently produce some substance in the bouillon which reacts in some way with other substances produced in the body on an individual infected by these bacteria, and as a result a mild fever is produced. (Certain other tuberculin preparations, made somewhat differently/ from the above, were brought out later by Koch and are claimed to have some value in treating consumption. Just how useful they an? has not yet been determined.) Mallein. Cultures of the organism causing glanders are treated in practically the same way as tubercule cultures are in preparing tuberculin, and the product is known as mallein. It is used for the diagnosis of glanders much as is tuberculin in the case of tuberculosis. A positive mallein reaction is shown not only by a rise in temperature, but also by a swelling at the point of injection. (For another method of diagnosing glanders, see a few paragraphs below.) Agglutination. When an individual is infected with certain diseases his blood becomes filled with substances that cause the bacteria of the particular infection in question to draw together in clumps or, as we say, to agglutinate. This seems to be a preliminary step in the establishment of immunity; but the substances causing agglutination are apparently distinct from lysins, opsonins, or any other immune body we have yet studied. They are called aggluti- PRACTICAL VALUE OF PHENOMENA OF IMMUNITY 287 nins. Whatever their function may be in the body, they have cer- tainly proved of great value to the diagnostician. It happens that the agglutinin produced in the body in the case of any disease is specific, that is, it agglutinates the particular bacteria causing that disease and no others. It also happens that the agglutinins are produced very early in the disease, even before the symptoms are Well developed. As a result, it is a fairly simple matter to take a drop of the blood from a person who is suspected to have one of these diseases, to mix it with some of the bacteria causing the disease and to observe whether agglutination takes place. This observation may be made with or without the use of a microscope. An illustration of an application of each method will show how the test is made. Typhoid fever is commonly diagnosed by a microscopic agglutina- tion test. A drop of blood from the patient is dried on a small sheet of metal and sent to the laboratory where the test is to be made. There it is dissolved in a little water and mixed on a cover glass with a drop of a typhoid culture. The cover glass with the drop on it is then inverted over a concavity on a microscopic slide, sealed to prevent drying and is allowed to stand for a few hours. Then it is examined under the microscope, and if the bacteria occur in clumps, a positive diagnosis of typhoid is made; if, however, they remain separate and motile, the diagnosis is negative. This is known as the Widal test, and is made in large numbers in some laboratories. The macroscopic method of determining agglutination may also be used for typhoid; but it is not so convenient and is less often used. We will therefore use a different illustration for the macroscopic method of making the test. Glanders is generally diagnosed by the macroscopic agglutination method. To make this test it is necessary to have on hand a fluid containing the dead glanders organisms in suspension. This sus- pension is made by growing the organisms on agar, then removing the growth and mixing it with sterile physiologic salt solution (i.e., a 0.9 per cent solution of NaCl in water), and killing the bacteria by heating for half an hour at 60°C. This fluid can be preserved by the addition of some germicide, usually carbolic acid. The blood from the horse suspected of having glanders is allowed to coagulate, a small amount of the serum is removed and greatly diluted. If not diluted enough the reaction is meaningless, because normal serum will cause agglutination if undiluted. Ordinarily it is diluted so that 288 BACTERIOLOGY a series of tubes will contain different dilutions varying from 1:200 up to 1:2000. A definite amount of the suspension is added to each of these tubes and allowed to stand for a few hours. Then agglutina- tion can be easily recognized by the presence of a flocculent precipi- tate of bacteria at the bottom of the tube. If the horse from whom the blood was taken has glanders the stronger dilutions will be sure to cause agglutination and sometimes even the weaker ones. If glanders is present, therefore, it can always be detected by the pre- cipitate in some or all of the tubes. Glanders can also be diagnosed by the same method as typhoid; but as it is generally easy to obtain a considerable quantity of blood from the horse, the macroscopic method is preferred as the more convenient. The Wassermann reaction. In the case of one disease, namely syphilis, it has been found possible to make a diagnosis on the basis of these phenomena of immunity, but by a different method. The syphilis diagnosis is called the Wassermann reaction, after the man who first applied the technic to this particular disease. It is a very ingenious procedure, but its details are too complicated to go into here. A few words, however, will show something of the nature of the technic. If the blood of some animal (say a sheep) is injected into the veins of a different kind of an animal (a rabbit, for instance), the blood of the latter is subsequently found to contain certain bodies known as haemolysins that destroy the corpuscles of the for- mer's blood. A small amount of the rabbit's blood containing these haemolysins added to fresh sheep's blood converts the latter into a clear red liquid. One of the substances in the blood helping in this reaction is called "complement" and is of enzyme-like nature, readily destroyed by low heat. If the rabbit's blood is heated so as to de- stroy the complement, it no longer haemolyses the sheep's blood; but this complement is present in all blood, so if a small amount of some other animal's blood (say a guinea pig's) is added to the mixture of rabbit's blood (heated) and sheep's blood, hemolysis does take place. It has been found possible, however, to combine the comple- ment of the guinea pig's blood with other immune bodies so that it is no longer free to act upon the sheep's blood. This is spoken of as "fixing the complement." Now the Wassermann reaction depends upon a method, that need not detain us here, of fixing this comple- ment in the case of syphilis by the use of serum (or spinal fluid) from a syphilitic patient. In this test the various factors are so PRACTICAL VALUE OF PHENOMENA OF IMMUNITY 289 delicately balanced that no fixation occurs unless the person has syphilis. If, therefore, the serum is treated in the proper way and mixed with the various necessary preparations, and is then added with the heated rabbit's blood to sheep's blood, the corpuscles of the sheep's blood will be destroyed if the patient does not have syphilis, but the blood remains normal if the patient is a syphilitic. Complicated as this sounds, the test is very accurate if made carefully, and although it requires skillful manipulation, it is possible to make a large number of tests at the same time, and the reaction has great practical value in diagnosis. The extreme ingenuity of the technic and the amount of patience that must have been necessary to devise it in the first place have won the admiration of the entire scientific world. The Schick test. One very interesting application of the principles we have been considering is in the case of diphtheria. Some people are naturally immune to diphtheria while others are susceptible. When this disease appears in a large family or in a school it is of great value to know which of the well members are susceptible and which are not, so as not to waste antitoxin in immunizing those that are already immune. The susceptibility to diphtheria is determined by the Schick test. To make this test some diphtheria toxin is prepared and the smallest amount capable of killing a guinea pig is determined. Then the toxin is diluted so that each dose will contain sff of that amount. This very small dose is injected into the skin of the person to be tested. If redness appears around the point of injection within one or two days, the individual is susceptible; if no redness appears he may be considered immune. The greatest value of this test has proved to be when diphtheria breaks out in a school. By examining cultures from the throats of the scholars it is possible to pick out all those who are already infected; but until the Schick test was devised there was no way of telling which of the uninfected scholars might come down with the disease as the result of exposure before the sick scholars were ex- cluded from school. As a result, in severe epidemics, it was often necessary to close the schools entirely until time enough had passed for those exposed to develop the symptoms. Now, however, by the simple injection of this small amount of toxin, it is possible to select the scholars that are susceptible, and all the rest may be allowed to attend school as usual without danger of developing the disease and spreading it. CHAPTER IV Sanitation In the control of disease there are three different methods of accomplishing results: sanitation, development of individual resist- ance, and medical 'treatment. The last of these has been practiced since the days of the ancients, and although it is a far cry from the superstitions of the priests, healers, and medicine men of early times to the serum therapy introduced by bacteriologists, medical treat- ment has always had the same object-the cure of disease. But, as has long been realized, "an ounce of prevention is worth a pound of cure," and for some time physiologists have had in mind the pro- tection of the individual against disease by increasing his powers of resistance. Today the emphasis put on this method of attack is much greater than ever before; the bacteriologist with his studies in regard to immunity and the physiologist with his studies on nutrition have both made important contributions. It is now realized, how- ever, that if prevention of disease is to be thorough it must go behind the individual and must deal with the very habits of society. That is the object of sanitation. The cure of disease is purely a medical subject and does not con- cern us here except in so far as it relates to the treatment of disease with bacteriologic sera. The development of personal resistance is largely a physiological problem, and we have already discussed its bacteriological aspect in connection with immunity. Sanitation needs more detailed discussion. CONTROL OF DISEASE IN ITS RELATION TO THE METHOD OF TRANSMISSION The spread of disease. Of fundamental importance in sanitation is the knowledge as to how disease is spread. The sanitarian can well dispense with the knowledge as to what microorganism causes any particular disease he is fighting; but he must know how it is spread. Yellow fever was just as truly conquered while we did not know its cause; pneumonia still baffles us, although we know organisms that can cause it. SANITATION 291 Different infectious diseases are spread in very different ways. Some are directly contagious, others are only indirectly contagious, while still others do not seem to be contagious in any degree. It is plain that those parasites with no habitat outside the human body must pass directly from one man to another, while those that live elsewhere, either as saprophytes or as parasites on other animals may take such indirect routes of transmission that they hardly seem to be infectious. Such knowledge is very important to the sanitarian. Direct contagion is generally the easiest to prevent; for when a person is sick it is comparatively easy to induce him to stay indoors and to quarantine the household. The indirect forms of contagion, however, are more insidious and much more difficult to handle. The object of the sanitarian in studying any disease is to find the one most vulnerable point in its transmission and to direct special effort against that. If at any point in the cycle of the organism in passing from man to man, its progress can be stopped, the disease can quickly be brought under control. The sanitarian is always looking for oppor- tunities of this sort. Direct contagion. All those parasites that Ijave no habitat outside the human body must be directly contagious, unless they are capable of living for a long time in a dormant condition outside the body. Now, so far as we know, none of the diseases of this class (see Chapter V) are caused by organisms capable of resisting adverse conditions for any length of time, and as a result we find that they are eliminated from the body in some form which makes direct contagion easy. Some are skin diseases like smallpox and scarlet fever; some are diseases of the respiratory tract, such as diphtheria; others, like syphilis and gonorrhea produce virulent discharges through abscesses or excretions from the body. Some other parasites, like the tubercle organism, although capable of living outside the human body, often Pass from man to man by direct contagion and must be treated by the sanitarian much like those that are peculiar to man. In preventing the spread of directly contagious diseases, the im- portant points are the early recognition of the disease and the isola- tion of cases. Sometimes, as with diphtheria, a rigid quarantine is necessary. With other diseases less strict measures suffice, provided the patient is kept from that particular form of contact with others which allows the transmission of the parasite in question. With other diseases like infantile paralysis, the exact method of elimination of 292 BACTERIOLOGY the parasite is not known, and strict quarantine is generally recom- mended on the theory that some one of the measures used will be the right one to prevent the spread of the organism. In all such measures of isolation, it is necessary to destroy the infectious material given off by the patient. Hence the contaminated discharges should always be caught in cloths or vessels that can be burned or sterilized. If such measures are rigidly enforced, it is possible to control those diseases that pass from one man to another only by direct contagion. Distribution through food and water. Some diseases like typhoid and cholera are largely spread through drinking water. The spread of typhoid through food is also common; and, indeed, quite a number of diseases may be food-borne. In the epidemics of such diseases, the different victims may often have nothing to do with each other, while the folks in closest contact with the patients may escape. The most serious epidemics are likely to occur in the case of water- borne diseases. Sometimes, indeed, the water supply of some city may remain contaminated with the typhoid organism for years, causing a constantly high typhoid rate among those who use the water. In most cases, however, the water becomes suddenly con- taminated, a severe epidemic occurs, after which the community takes steps to clean up the water supply, and no more new cases ap- pear until the town becomes careless about its water again. One of the best examples of a typical water-borne epidemic is the cholera epidemic of Hamburg in 1892 (see p. 331). A typhoid epi- demic, which illustrates equally well how disease may be spread by drinking water, occurred at Plymouth, Pennsylvania, in 1885. The disease first made its appearance very suddenly in April as an epi- demic of 700 cases. After a few weeks few new cases developed and the epidemic quickly died out. Water was suspected to be the cause and an investigation of the water supply was made. The usual supply of the city was a mountain stream, but during the winter while the stream was frozen, water was taken from the Susquehanna river. Upon investigation it was found that about two weeks before the epidemic began, the small stream had thawed and its water was again turned into the city mains. Then carrying the investigation further it was found that in a house near one of the reservoirs on this mountain stream there had been a case of typhoid during the preceding winter. The excreta from the patient had been allowed to accumulate on the slopes of the stream w'here they had remained SANITATION 293 frozen until the thaw. The thaw, therefore, resulted in a tremendous pollution of the water supply all at once. Hence the typhoid out- break was very sudden and violent, and as there was no further source of contamination, the epidemic stopped almost as suddenly as it began. Fig. 40. The Water Supply of Plymouth, Pennsylvania, at the Time of its Typhoid Epidemic It is seldom, however, that a water-borne epidemic can be traced to a single case. The most serious outbreaks of typhoid, for instance, occur in cities that are drinking water from some body of water that receives the city sewage. In some cities this condition has continued for so long that typhoid cases are regularly present in considerable numbers. Their total number at any one time is never so high (in 294 BACTERIOLOGY proportion to the population) as was the case at Plymouth, because after an attack a person becomes immune for a number of years; so in a city with constantly polluted water many people are temporar- ily immune to the disease. Nevertheless the total typhoid rate is always high if the water is constantly polluted; and it is roughly possible to tell how pure the water supply of any city is by computing the number of deaths from typhoid per year per 10,000 inhabitants. Thus an annual typhoid death rate of over 20 per 10,000 (except possibly in cities with a high percentage of negroes) indicates that something is wrong with the water supply. When it reaches 100 per 10,000, as it has at times in certain American cities, it means that the inhabitants are virtually drinking dilute sewage! Food-borne epidemics seldom have quite such a wide-spread dis- tribution of cases as when the diseases are spread by water. A con- taminated food is seldom distributed among as many people as may drink from a single contaminated source of water. Milk is the most widely distributed of all foods that are consumed raw, and more epidemics are traceable to milk than to any other food. Sometimes it is possible to follow out some particular milk route through a city and to show that all the cases of an epidemic occurred on that one route. Occasionally such an epidemic has been traced back even further, to a single dairy and a single case of typhoid on that farm. Besides milk, no food is considered to have caused widespread out- breaks of disease except shellfish, which are so often raised or fattened in polluted water as to be an appreciable source of danger. Almost any kind of food, however, that is eaten raw may spread disease, although not to sufficient extent to demand the attention of the sanitarian. The control of diseases spread by water or food presents many and varied problems. Perhaps the simplest of such problems is presented by the community that is using contaminated water but has a source of pure water available. In such a case, if the outbreak is severe enough, it is generally possible to convince the inhabitants of the wisdom of changing their source of water, even though considera- ble expense must be incurred in so doing. To free a city of water- borne infection is not so easy when no better source of water is at hand. Some cities, faced with this situation have built aqueducts over a hundred miles long to obtain pure water; but the more common way is to use the contaminated water after treating it in some way so SANITATION 295 as to rid it of the dangerous organisms. This may be discussed more fully under the bacteriology of water. But most complex of all are the problems raised by food-borne diseases. The production of sanitary milk, for example, is almost a science in itself. Although distinctly a sanitary problem, milk sanitation is so intimately con- cerned with farm practice that it has already been discussed under the heading of dairy bacteriology (pp. 145 to 152). The various methods introduced in recent years by boards of health to control the production of sanitary milk are of more or less efficiency and do tend to make milk a safer product; but they do not make it absolutely safe. Pasteurization is more efficient; and if all milk were well pasteurized, the spread of disease by this food could be entirely pre- vented. The spread of disease by means of shellfish presents its own problems; although by controlling the shellfish industry, this danger is being lessened. When a disease is spread by some other food, however, it is seldom possible to find out how to prevent it before the danger is over; but disease is so seldom spread in this way that little attention is given to the matter, except in efforts to improve the sanitary conditions of establishments where food is handled. Distribution by means of insects. Insects may be agents in the spread of disease, either by being intermediate hosts themselves for the causal organism, or by purely mechanical transfer from one man to another. The best example of the latter is perhaps the house- fly in the case of typhoid. The agency of this insect in carrying the typhoid germs from privies to food seems to have been proved beyond Question, and there are undoubtedly many other diseases spread in the same way, although not perhaps to the same extent, by this filthy little companion of human beings. Fleas and lice act similarly, °ne of the most important diseases thus spread being typhus. The agency of insects as intermediate hosts in the spread of disease is another important factor in distribution, as shown by those diseases in which the parasite spends one stage of its existence in the body °f the insect. Such human diseases as malaria and yellow fever are spread in this way. Before the agency of insects in such diseases was learned, their spread was very mysterious. They plainly were not spread by con- tact, but they seemed to abound in certain localities. The prevalence °f malaria in swampy regions was known long before the reason has been suggested. It was even supposed that some poisonous gas 296 BACTERIOLOGY (or "miasm") was given off from the swamps and produced the disease. Fighting "miasms" was a difficult proposition, but when the agency of insects was discovered sanitarians had something tangible to attack. Insect-borne diseases, indeed, are now regarded as among the most easy to control, for insects are easier to fight than microorganisms. In the life history of these parasites, the so- journ in the insect host proves the point which offers the least re- sistance to attack; and eradication of the insect host is therefore the logical method of sanitation. Fig. 41. The Yellow Fever Mosquito (Stegonia) Left, male; right, female Control measures in the case of insect-borne diseases have led to some of the greatest feats in sanitary engineering the world has ever known. Most remarkable of them is the conquest of yellow fever in the Panama Canal Zone. Without exaggeration, it can be said that the French effort to dig a canal through the Isthmus was defeated by the mosquito. To be sure, no one knew that this little insect was their chief enemy at the time, but it was well recognized that yellow fever was an unsurmountable obstacle. Deaths from this disease were unbelievably common and the attempt had to be abandoned, without even taking the trouble to remove the digging machinery that had been carried to the Isthmus. Later the agency of the mos- quito in spreading yellow fever was learned and when the United States undertook the canal project, our engineers recognized their SANITATION 297 enemy. So a large force of sanitary engineers was sent ahead of the diggers. These men lived at first within mosquito netting as far as possible, and thus kept free of the disease. But before the main construction work began they had cleared out the underbrush in which the insects lived, drained the swamps where they bred, until at last there was scarcely a mosquito to be found in the entire zone. The result was that before the laborers arrived, yellow fever was conquered. Today the canal zone is said to be one of the healthiest places in the world. Less effort has been made to control other insect carriers of disease. The fly is still with us in spite of its typhoid reputation. "Swatting" it does little good, when there are countless manure piles in which it may breed, and protecting manure piles from flies is not at present a practical measure in the country. In the city, flies are fewer today than formerly, largely because the automobile is replacing the horse, and flies breed only in manure, and other similar filth; but although the sanitarian has been lecturing against the fly for years, the decrease m numbers is probably due less to his teachings than to the natural trend of the times. Distribution by means of larger animals. In the case of a few dis- eases some larger animal causes their spread from man to man. Thus rabies is spread by dogs; and plague is ordinarily spread by rodents, fleas carrying the infection from the rats or squirrels to man. In such cases the attack of the sanitarian is directed towards the animal distributing the disease. To control rabies, as it is impossible to destroy all dogs, stray dogs are killed and owners are required to keep their dogs muzzled. To control the plague, it has been found very effective to kill rats and squirrels. Thus when it was discovered that the plague had become common in San Francisco, the first efforts of sanitarians were to eradicate rats. All houses were made rat-proof, that is sealed around the ground so that rats could not enter, and all possible living places for rats were either destroyed or sealed. This treed the city of rats; but it was then found that the disease had mfested the ground squirrels in the surrounding country, and the danger of their spreading the plague to distant communities was at once recognized. The fight against squirrels was much more difficult than that against rats; but it has been carried on with great success. It was found that the squirrels in leaving the district around San Francisco must pass through certain valleys. In each of these passes 298 BACTERIOLOGY a narrow zone was cleared of all wood and underbrush, apd a constant watch was kept upon it. In this way practically no squirrels were allowed to leave the region of San Francisco, and gradually they have been killed off within the region; so that now the plague situation in California is regarded as being under control. Distribution by means of healthy human beings. One of the most baffling problems to the sanitarian is that of bacillus "carriers"- human beings who carry disease germs without being sick themselves. Many healthy individuals carry diphtheria germs in the throat, for example, and are probably as great a danger to those near them as if they were suffering from the disease. The same thing may happen in the case of various other diseases, and the problem presented by these "carriers" is no simple one. To locate such an individual is by no means easy, for there is seldom the slightest reason for suspecting that he harbors disease germs; and if located, it is nearly as difficult to confine him and keep him from spreading the disease. The difficulties in solving this problem are well illustrated by a case that attracted a good deal of attention all over the country. A number of cases of typhoid had occurred in a few families which had employed one after the other a certain cook, and in each family the cases of typhoid occurred about two or three weeks after the cook came to them. This aroused suspicion, and upon examination she was found to have virulent typhoid bacilli in her intestines, although she herself was perfectly healthy. The ease with which a cook harboring typhoid germs might spread the disease to others can be well understood. When she was found to be the distributor of ty- phoid, the health authorities hardly knew what to do with her. To let the family last employing her discharge her would do little good, as she could easily get work elsewhere. At first there was talk of confining her; but to imprison a woman just because she was carrying disease germs through no fault of hers was so unjust that less severe measures were taken. It was decided that she was no danger to the community except as a cook, so at last she was released from the surveillance of the health department upon giving her word not to serve again in that capacity. Meanwhile her case had become rather notorious and she was known the country over as "typhoid Mary." This sobriquet must have been rather distasteful to her, and un- doubtedly prevented her from getting employment as a cook or in any other line of work, so it is not surprising that she soon disappeared. SANITATION 299 A number of years later some other cases of typhoid were laid to a cook, the board of health investigated, and there was "typhoid Mary" again, back at her old calling and still distributing virulent bacilli. When a "carrier" can continue to spread disease germs for as long a time as this, it is easy to understand why sanitarians are at their Wits' ends to know how to handle the problem. One other difficult problem of this sort, which is even more serious because of the much greater number of cases, is presented by the large number of negroes in some of our cities. The white and black races are not equally susceptible to all diseases, and to some of them, such as malaria and typhoid, the negroes are very resistant. They contract these diseases, but often so lightly that they go about their Work as if nothing were the matter, and spread the germs among the more susceptible whites. In the case of tuberculosis and syphilis, on the other hand, the relations are the reverse; and although the rapid spread of these diseases among the negroes is generally blamed to the unsanitary conditions under which they live, there can be little doubt that in part they are infected by white "carriers." Sanitarians have been more fortunate, however, in solving the problem of diphtheria "carriers" in schools. The weakness of all quarantine measurers in regard to this disease at first was that many healthy children carried the germs in their throats but were allowed to go to school. The modern method of overcoming this difficulty is to have the throats of all scholars examined whenever diphtheria breaks out in a school. Then all the scholars harboring the germs are quarantined whether well or sick, and none of them are allowed to return until microscopic preparations from their throats no longer show the diphtheria organism. This works a little hardship and causes considerable exasperation in the families of those children that continue to have the germs in their throats for weeks or months without a sign of sickness; but it proves efficient in preventing wide- spread epidemics and causes much less hardship than an epidemic would. Other means of distribution. There are various other means by which diseases may be carried from one individual to another, some of which are important while others are of so little importance as to be of sbght concern to the sanitarian. One of the most important means ls through the saprophytic life of an organism in between periods of parasitism. Certain kinds of bacteria, for instance, that cause in- 300 BACTERIOLOGY flammation and pus-formation when they invade wounds seem to be capable of leading a saprophytic life. Similar if not identical bacteria (cocci) are nearly always present on the skin. Whether these forms are the same as the pathogenic forms or not, and if they are the same, whether they have temporarily lost their virulence or are merely waiting an opportunity to attack is unknown; but it is certain that many of the pus-producing organisms do live outside the body and become parasitic when they find means of entrance through a wound or at some other point of lowered resistance. Such organisms as these are so nearly universal that sanitation of the ordinary sort is quite inefficient as a defense against them. Protection against them is more along the line of cleanliness, thorough disinfection of wounds, and increasing the individual resistance. Another class of diseases has a similar although slightly different method of spread. These are the diseases like anthrax, tetanus, and so forth, that are caused by spore-forming bacteria which can live over in the spore condition until another chance for parasitism occurs. The peculiar problem presented by these diseases is partly because the spores may remain alive for many years although the conditions are most unfavorable for their growth, and partly because spores resist drying, boiling, and the action of some disinfectants. For- tunately, human diseases of this class are few and comparatively rare. When they do occur, the sanitarian must insist on unusually thorough disinfection. Cremating or deep burial of bodies, and burning all contaminated effects that can possibly be burned are commonly recommended. THE RESULTS OF SANITATION It is hard for a modern to picture the misery and prevalence of diseases among the people in the Middle Ages. Even the historian can give us little idea, because statistics in those days were very incomplete; but the few facts we have show us that disease rather than good health was considered the common condition of man. The change from such conditions to those of today is nothing short of marvelous; and by far the greatest change has occurred during the last hundred years. Surgery, medicine, and serum therapy have all helped in bringing about this progress; but more of it is thanks to sanitation than to any other cause. This can easily be realized when we see what sanitation accomplished in the Panama Canal Zone or in SANITATION 301 "'ar-devastated countries where sanitarians have been given a free hand. Such cases of the complete triumph of sanitation are rare °nly because it is seldom in civilized communities that sanitarians have a clear field. Their rules cause so many restrictions upon men's habits as to be burdensome; and the objects of sanitation have to be accomplished only by degrees, one step at a time, so as to meet as little opposition as possible. Yet even under these difficulties much has been accomplished. Life insurance authorities say that the tables of life-expectancy made up twenty-five years ago are now obsolete. Life insurance rates have not gone up, in spite of the general increase in almost all expenses, but have tended to go down. There seems to be little question that the average life today is longer than it was a hundred or even fifty years ago; and if the sanitarian claims the lion's share of the credit, is he claiming more than his due? CHAPTER V Strictly Human Diseases In the following chapters will be given in brief outline the facts that have accumulated during the last fifty years concerning the most common diseases that are known to be produced by parasitic organisms. The small amount of space which can be given this subject makes it impossible to do more than state in briefest form the most important facts that have been discovered concerning each of them. The primary value of these facts to the public is in their relation to the problem of public health, and the methods thay may be adopted for preventing the distribution of such infectious diseases. For this reason, their methods of distribution will be emphasized in the following discussion rather than the specific causes of the diseases. It is a matter of more significance to know that malaria and yellow fever are both distributed from patient to patient by mosquitoes than it is to know that one of them is produced by a microscopic animal and the other by a spiral microscopic organism, the nature of which is not known. Where the infectious agent is incapable of living outside the human body, it is evident that the infective material must pass directly from person to person. Where, however, the parasitic organism is capable of living for a greater or less length of time outside the human body, perhaps in the body of other animals, the problem of contagion and methods of distribution become very much altered. The order in which the infectious diseases will be taken up in the following pages has therefore close parallel with the ability of their causal agents to live a non-parasitic as well as & parasitic life. DISEASES CAUSED BY PARASITES WITH NO IMPORTANT NATURAL HABITAT OUTSIDE THE HUMAN BODY This disease has been handed down to us from past centuries, having been first recorded in Europe in the sixth century, and froifl that time on never disappearing entirely from the western world- Smallpox STRICTLY HUMAN DISEASES 303 We who are living in the twentieth century have no conception of the ravages of this disease in former times. Today it seems to be a comparatively mild sort of sickness, rarely producing death, so mild that those who do not understand the situation think vaccination is Worse. This is very different from the history of earlier days. The 17th century saw some 10,000,000 deaths from it in Europe, and there is no means of estimating the amount of suffering and dis- figurement caused by it in the non-fatal cases. Its milder character today is undoubtedly to be explained by the modern method- vaccination-of combating this formerly dreaded scourge. There are really two fairly distinct types of smallpox: the severe type of former days, known as variola, and the mild type, as modified by vaccination, which is called varioloid. True variola is a very loathsome as well as one of the most fatal diseases of mankind. In this type of smallpox, the characteristic pocks keep appearing day after day until certain parts of the body are literally covered with them. They seem to have a special predilection for the face, and the features become so bloated and covered with sores as to be entirely unrecognizable. The sores generally break, and the discharge is offensive both to sight and to smell. The disease is accompanied by a very high fever, and often results in death. Even after recovery, the skin generally remains permanently pitted. Varioloid, on the other hand, is a very mild disease. The rash appears promptly, the pocks are generally few and scattered, no new ones appearing after the first day or so. A slight fever is generally present while the patient is breaking out, but it goes down promptly, and after that the temperature is normal. The pocks seldom become discharging Pustules, and seldom leave permanent scars. This mild type of the disease is the form practically always observed in individuals who have been vaccinated but whose immunity has worn off; and indeed, it is by far the most common form of smallpox known today. The cause of smallpox is as yet unknown, in spite of many extensive studies on the subject. There has been found living in the epithelial cells of smallpox patients certain bodies which have been thought to be very minute protozoa. Some of those who have studied them have even given careful descriptions of a somewhat complicated metamor- phosis, through which these organisms go in the cells and nuclei of the epithelium of the skin, and they are convinced that the organisms ln question are the causal agents of smallpox. It has never been 304 BACTERIOLOGY possible, however, to cultivate them outside the human body, and no knowledge is at hand at the present time as to whether they are the active agents of infection. No bacterium has been found that can be called the cause of the disease, although it is extremely common to find some of the ordinary pyogenic cocci located in smallpox pus- tules, and it is believed that they contribute very largely to producing the severe symptoms of the worst cases. Whatever be the actual cause of the disease, there has been as yet no evidence that true smallpox exists outside the human being, with the possible exception of some closely allied animals like monkeys. The infective material has no habitat in nature, and the only method of distribution seems to be from person to person. Smallpox, indeed, is one of the most contagious of all known infective diseases, passing with the greatest readiness from person to person. Even in this matter, howrever, our knowledge is rather limited; for, although in- fectious matter is certainly present in the lymph of the pustules, we do not know whether the disease is spread normally by contact, by the breath, or otherwise. Neither do we know how the infectious material enters its new victim. No method of acquiring the disease, is known, however, except direct contagion. The successful combat against this formerly dreaded plague dates back now to a little more than a century. The extremely fatal and disagreeable nature of the disease led the public to be willing to adopt almost any method of protection that might be devised. The discovery that a person who had recovered was immune from small' pox led to a general feeling that people who had it in childhood were to be envied rather than otherwise, since they were protected io later life from the disease. Hence there arose, first in the east, and later in Europe, the method of controlling the ravages of the disease by artificially inoculating people with a mild type of smallpox, thus giving them protection. This method of protection was quite widely followed for many years, in spite of the great dangers connected with it, and in spite of the fact that some persons thus inoculated actually suffered death from the inoculation. The discovery of Jenner, that cowpox, which is now generally considered to be a very mild form of smallpox, might be readily given to persons without injury to them and would give them protection against smallpox, revolu- tionized the method of fighting this dreaded plague. STRICTLY HUMAN DISEASES 305 The exact relation between smallpox and cowpox is not known. It is ordinarily impossible to infect cattle with the smallpox virus, and if any infection takes place the result is not true cowpox. But it has been found that if a monkey is inoculated with smallpox and another monkey from the first, and this process continued through perhaps a few more monkeys, the virus becomes changed in its nature, is no longer so virulent for man but can infect cattle, producing a disease like cowpox. Although the exact identity of the two diseases has not been proved, the facts suggest that the history of smallpox, has been something like this. When men first began protective inoculation against it, they naturally selected mild cases from which to inoculate themselves and they were therefore seldom seriously sick. The doctors used lymph from these inoculated cases to inocu- late others who desired protection. In the course of time the virus was modified in the same way modern investigators have modified it by passage through monkeys. By careful selection of mild cases and old pustules in obtaining their infective material, the early Physicians became quite skillful in producing light attacks in those inoculated, sometimes very similar to the effects of vaccination today. This mild form of smallpox was consciously spread abroad and in- troduced into rural communities as well as into cities. This form of the disease being presumably infectious for cattle, it was inevitable that cows became inoculated from the men handling them. This caused the appearance of cowpox. Subsequently other milkers became inoculated from the cowpox sores on the udders; and then it was noticed among the country people that individuals so infected did not contract smallpox. This observation, repeated to Jenner, gave him the idea of vaccination. Now that vaccination is general, the severe type of the disease does not have a chance to get a foot- bold, and has almost disappeared. Although it has never been proved that this is the actual relation between cowpox and the vari- ous types of smallpox, there is considerable evidence to support the theory. At first the practice of for lack of a thorough under- standing of its nature, gave rise to many serious troubles. It was customary in the early days to have the virus taken directly from a cow or from some vaccinated person and inoculated into another, the result being that other diseases were not infrequently inoculated 306 BACTERIOLOGY into the new individual from the old. In later years, however, this method has been discarded for the one of carefully cultivating the virus in the body of a perfectly healthy calf. The vaccine thus obtained is guarded by methods that have developed in recent years, and practically all the dangers have been removed. At the present time, although serious troubles occasionally follow vaccination, they may be traced in practically all cases to improper methods on the part of the physician, or more often to carelessness on the part of the person vaccinated, rather than to any trouble with the vaccine. For if the vaccination wound is allowed to become infected with microorganisms from external sources like soiled clothing, trouble is likely to arise just as when any wound becomes infected with disease germs. The evil consequences following vaccination have been growing less and less as the methods have improved, until at the present time they have very nearly disappeared. So far as concerns the protection afforded by vaccination, no one who faces the actual facts has any question as to the results. Vaccination undoubtedly gives a protection to the person who is successfully vaccinated which lasts for some time. The exact length of time is unknown, but it is undoubtedly several years, and if a person after the lapse of several years is revaccinated he is then com- monly protected for life against this disease. The value of vaccina- tion is shown by the fact that those countries that have adopted it have uniformly been less subject to this disease than those that have not adopted vaccination, and that the amount of smallpox is in inverse proportion to the rigidity of the vaccination laws. Germany, with its compulsory double vaccination, has practically driven small- pox from its territory, while other countries that have not adopted such a rigid regime still suffer from the disease more or less, the extent of the suffering depending on the looseness of the vaccination laws or of their application. Vaccination has driven smallpox from some countries, reduced it in others, and has everywhere lessened its severity-for a person once vaccinated, even if he does subsequently loose his immunity and contract the disease, is quite sure to have a much more mild case than one who has not been previously vac- cinated. So efficient is this method of protecting against the disease that public health officials now recognize that the public has the remedy against this disease wholly in its own hands. STRICTLY HUMAN DISEASES 307 Leprosy Leprosy, known for so many centuries in the Orient, has been in recent years subjected to extensive studies by the new methods. The disease seems at last to be under control, but it has been con- quered not through the efforts of bacteriologists, but by the applica- tion of a drug, chaulmoogra oil, the medicinal properties of which seem to have been discovered by accident. Although bacteriology has not assisted in controlling the disease, it has discovered its cause. The causal organism is a bacterium very closely resembling in micro- scopic appearance the organism that produces tuberculosis, and it also resembles it in being "acid fast," that is, in absorbing certain stains and holding them so firmly that it cannot be decolorized by acids. The leprosy organism, however, will not grow in any of our culture media, and all attempts to cultivate it artificially have failed. The evidence that it is the cause of leprosy is based primarily upon the fact that it is always found in leprous patients, especially in the Parts of the body where the disease is localized, and that it is not found in other people. No inoculation experiments have been possible; but at present it is practically agreed that this bacillus is the cause of leprosy, although conclusive proof is lacking. The method by which the disease is distributed is also unknown. It has always been regarded as contagious, and yet the evidence for conta- gion is extremely slight. The historic method of contending against the disease is by isolating the leprous patients, and this has been very generally adopted the world over where leprosy has been found,, although some leper colonies are being abandoned as a result of the newer methods of treating the disease. It is not settled beyond question that leprosy of today is identical with the biblical disease. It differs from the account in the Bible sufficiently to make some scientists doubt if they are the same dis- ease. While leprosy is an extremely serious disease, and usually fatal, it is slow to progress, and is one of the rarer diseases that attack mankind. Scarlet fever This very common disease of childhood must at the present time be classed with those whose cause is unknown, for in spite of careful study by bacteriologists, no specific organism has been found that can be regarded as the active agent in producing scarlet fever. 308 BACTERIOLOGY Certain minute microscopical bodies have been found in some of the epithelial cells of the skin which have been regarded by some as being protozoa and the cause of the disease. This, however, is not at the present time generally credited. It is also found that in nearly all, if not all, cases of scarlet fever certain streptococci are extremely common in the throat. Some bacteriologists have insisted that they are really the cause of scarlet fever, or at least are closely associated with it; but this is not yet generally admitted. Whatever be the cause of scarlet fever, it is quite certain that its chief habitat is in the human being, and up to the present time there is no indication that it exists elsewhere in nature, unless possibly in cows. The primary method of acquiring the disease is by contact; but there is evidence that under some circumstances epidemics of a disease so closely like scarlet fever as to be perhaps identical with it may be distributed by some other means. Several epidemics of this nature have been traced to milk, in some instances the indication being that the milk was infected with the active agent by coming in contact with a convalescent patient, though in other cases the trouble may have been derived from cows suffering from some udder infection. Whether all these instances are to be regarded as true scarlet fever cannot be settled at the present time. Closely associated with scarlet fever, frequently occurring with it, and sometimes regarded as identical with it, is a somewhat newlv recognized infection called septic sore throat. This has come to public notice particularly in the last few years, a number of severe epidemics having been recently recorded. While it has never been as greatly feared as scarlet fever, the mortality is fairly high, rivaling scarlet fever itself in that respect. It is not, however, like scarlet fever, primarily a children's disease, but is likely to attack adults. Microscopic study of the patients has disclosed the practically universal presence of a streptococcus, resembling, if not identical with, the one found in scarlet fever patients. This streptococcus, when isolated and purified, and tested on animals, is found to be pathogenic. Septic sore throat is probably, at least in some cases, distributed by the milk supply. The most serious epidemics that have been recorded have been traced to the milk supply of certain dairies, in some cases the milk apparently being infected by patients who are suffering from this trouble coming in contact with the milk in the dairy, and in other cases the trouble being apparently traced STRICTLY HUMAN DISEASES 309 to cows having inflamed udders. The udders of the cows so infected are found to have in them an organism practically identical with that which is found in the throats of patients suffering from septic sore throat. The evidence, therefore, suggests that both the udder infection and septic sore throat are produced by the streptococcus m question, which thus has a habitat both in mankind and in milch cows. The public has apparently no protection against this particu- lar type of epidemic except the pasteurization of milk, for no practical method has been suggested for keeping the milk supply from being contaminated with this dangerous organism. Cows might develop the trouble even in the most carefully watched herds. Scarlet fever and septic sore throat not infrequently occur together, and so close is the association in some epidemics that some who have studied these outbreaks are convinced that the two are actually the same disease, attacking people in different fashions and producing Quite different symptoms according to the method of attacking the patient and the power of resistance on the patient. This has by no means been proved at the present time, and is not in general credited. It is merely known that the two diseases are often associated with each other and have many symptoms in common, both showing the presence of similar streptococci in the throat. Gonorrhoea This disease of the sex organs has been known for many centuries, historical records going back at least to the time of the Roman empire. It has been claimed by somewhat this disease was largely responsible for the downfall of Rome and the eventual extinction of the Roman race. It is still producing great havoc in all sections of civilized countries. It is very common, a large percentage of men being infected with it. It is very hard to get a true idea as to its prevalence nnd seriousness, because its nature is such as to lead its victims to conceal it and to ascribe subsequent troubles to any other cause rather than to gonorrhoea; but popular opinion to the contrary, Physicians are generally agreed that it is one of the most common and serious diseases of modern life. The disease is produced by a well-known bacterium, often called the gonococcus (fig. 42), which is found in the infected areas, and is commonly to be detected in the pus cells which are discharged from 310 BACTERIOLOGY the patient. Its detection by microscopic examination of the pus is very easy, so that by microscopic aid the diagnosis of the disease is extremely simple. The organism in question is one of very slight resistance to adverse conditions. It dies promptly upon drying, and is not known to exist normally in nature outside of the human body. The fact that the coccus dies so quickly after removal from the environment of the living human being makes it a disease whose distribution is practically confined to contact. Articles of clothing, or any other article that may become infected with the discharges containing the gonococcus will remain infectious for only an extremely short time, the organisms dying promptly; hence the danger of dis- tribution in this way is small. As a result, protection against the disease may be most efficiently brought about by avoiding contact with persons suspected of being infected. As elimination of the disease germs is through the urethra in the male and the vagina in Fig. 42. The Organism of Gonorrhoea the female, the most common method of transmission is through sexual intercourse. Sometimes, however, other parts of the body become infected, the most serious site of infection being the eyes, as the gonococcus can cause the complete loss of sight in but a few days. The eyes of adults seldom become infected, but a baby born of a mother suffering from gonorrhoea is almost sure to have its eyes infected at birth. This danger is so great and has resulted in such a large number of cases of blindness that it is now the practice of all good doctors to place a drop of some silver salt in the eyes of each new-born child, which kills the gonococcus, if present, without harming the eyes. No vaccine or serum has yet been devised that proves to be of much value in treating or controlling the disease. Syphilis, or lues This disease, although not confined to the sexual organs, is generally spread in the same way as gonorrhoea. Although not so common as STRICTLY HUMAN DISEASES 311 gonorrhoea, it is more serious in its effects. It has not been known so long as gonorrhoea, having been apparently introduced into Europe after the discovery of America, brought to Europe from the new World. This origin of syphilis has not been definitely proved; but there is no record of it in Europe until 1493 or 1494, while there is some evidence that American Indians knew the disease in a mild form. Its outbreak in Europe shortly after the discovery of America Was very violent, so violent as to suggest that it was attacking a race of people that had never known it before. Serious as it is today, ffs effects are rather long drawn-out, while this first outbreak in Europe saw many cases with death resulting in a few days. Follow- lng its introduction into Europe it spread widely through all civilized races, and not until fairly recently has its origin in the New World been suspected. Fig. 43. Treponema pallidum, the Cause of Syphillis The cause of the disease is definitely known now to be an organism Earned Treponema pallidum, a spiral organism (fig. 43), belonging to a group which at present we do not know whether to class with the bacteria or with the protozoa, although the present tendency is to regard it as allied to the animal rather than to the vegetable kingdom. ■Practically, the decision of this matter is of no importance, however. The organism is very slightly resistant to external conditions, cannot resist drying and dies very quickly when removed from the living body. It is not known to exist normally in any other animal except plan, although it may be inoculated artificially into monkeys and also into a few other animals. The fact that it does not occur normally ln any animal but man, and that it dies almost immediately upon being removed from the human body, produces a condition much the Same as with gonorrhoea. The organisms are eliminated through the syphilitic sores, wherever they may occur, and as the sores are often °n the face the disease may be spread by kissing, by use of common 312 BACTERIOLOGY drinking cups, and so forth; infection may also take place under some circumstances through soiled clothing; but such cases are exceptional, and the transmission is almost wholly confined to intimate personal contact. The disease could be very effectually checked if sexual irregularities could be stopped. Unlike gonorrhoea, syphilis is recognized by the public in general as a very serious disease. As it is always regarded as a disgrace, its victims will generally deny its presence as long as possible; but the nature of the disease makes concealment more difficult than in the case of gonorrhoea. The first sore, which appears at the site of infection, is a small matter, but after that sore heals the infection spreads and often the whole body becomes covered with sores (the secondary stage). Later the widespread sores disappear and the disease is likely to enter its tertiary stage in which it attacks flesh, bone, and even nervous tissue, taking on many loathsome forms and causing some very severe cases of insanity. No serum or vaccine has yet been devised for this disease, but recently there has been prepared a drug by synthetic methods, called salvarsan or arspheno- benzene, which is very effective in the treatment of the disease. Although not an absolute specific against syphilis, as at first thought, this drug properly used is a remarkably good curative agent. Pneumonia This disease, characterized by inflammation of the lungs and in- filtration of fibrous matter into the air passages, has become the largest cause of death in temperature regions and one that has not been diminishing with the reduction in the general death rate-thus showing that we have not learned how to fight it. The control of this disease has proved quite a baffling problem, but quite recently facts have been discovered that may lead eventually to its solution. That it is an infectious disease has been recognized for some time, even though it appears to be only very slightly contagious. Oc- casionally small epidemics have been recorded, and there was a world-wide epidemic in 1918-1919 associated with influenza (see p. 319) but as a rule the cases are sporadic, and one case does not come directly from another. The difficulties in studying it have arisen partly from the fact that the disease is not always caused by the same microorganism, and further because some of the bacteria responsible for it are quite common inhabitants of the normal mouth and throat- STRICTLY HUMAN DISEASES 313 The majority of cases are produced by a coccus, often called the pneumococcus, but a certain number of cases are caused by totally different bacteria. Even the cases incited by the pneumococcus are not all alike, three different types of pneumococcus having been detected, enough alike to be classed together but so different that they cannot be treated in the same way. Bacterial vaccines have been prepared which sometimes have proved useful and sometimes useless, a fact now perhaps explained when we recognize the three types of pneumococcus and the fact that a vaccine to have curative powers must be of the same type of organism as that producing the particular case of the disease in question. But the laboratory determination of the specific type of organism found in any particular case of pneu- monia is now pointing the way toward a more intelligent use of vac- cines. Sera are also being experimented with, and there is some promise that we may eventually get the disease under control by means of some of these methods. The fact that the pneumonia organisms are fairly common in- habitants of the mouths and throats of normal persons explains some puzzles about the disease, but raises other new ones. It shows that the average individual has been exposed to the bacteria at some time in his life; but it raises the question why such persons continue well in spite of this infection and why under some circumstances they develop the disease. It shows clearly that the normal person must have an efficient protection against the organisms in question and that this protection may sometimes be broken down. We can under- stand why a cold in the lungs may be followed by pneumonia, since the cold located in the air passages may break down their resistance. We can also understand why it is not ordinarily contagious, for if the germs are in the mouth normally, it would clearly make little difference if a few more reached the throat from a patient. The occasional epidemics may be explained by the appearance of some specially violent strain which has unusual powers of breaking down body resistance; or perhaps, the epidemic may be due to the wide- spread presence of some other very contagious disease, like influenza m the recent epidemic, which lowers the resistance of the body and makes the victim susceptible to the attack of organisms generally present but ordinarily harmless. It is manifest that considering the complex nature of the exciting causes, no one method of fighting pneumonia could be expected, and 314 BACTERIOLOGY certainly no protective vaccine could be expected, to be efficient in all cases. There are at present on the market certain mixed vaccines purporting to contain all the organisms capable of causing pneu- monia. Conceivably their use might be effective whatever the cause of the attack in any particular case; but there is little evidence to show that they are of greater value than any other form of treatment. Conservative physicians have not yet begun to use them. Diphtheria The almost complete mastery over this disease, formerly so dreaded for children, has been one of the great triumphs of modern bacteriology. A generation ago this disease was more dreaded than any other children's disease, and the death rate was very high, ranging from 50 to 75 per cent of the cases in ordinary epidemics. At the present time, not only has this high death rate been cut down Fig. 44. The Diphtheria Organism to a very low figure, much below 10 per cent (almost to a vanishing quantity, indeed, if the disease is properly treated), but we have also learned methods of controlling the epidemics and preventing their spread, thus reducing the number of cases. The first step taken to- ward this end was the discovery of the bacterium (fig. 44) that causes the disease, first found by Klebs and afterward especially studied by Loeffler, now often called the Klebs-Loeffler bacillus, in honor of its discoverers. The organism is generally named Bacterium diphtheriae. A new name is Corynebacterium diphtheriae. This organism has now been most thoroughly studied, and its general relations are well understood. When growing in any favorable medium it secretes a soluble toxin of intense violence, known as the diphtheria toxin. The organism does not actually enter the body, but in ordinary cases of diphtheria simply grows on the surface of the throat, forming masses there in the so-called "false membrane." The toxins here STKiCTLY HUMAN DISEASES 315 produced are absorbed into the circulatory system, producing a direct poisoning of the tissues. It has been learned as the result of a vast amount of study and ob- servation that the method of distribution of the disease is practically always by contact with persons who are either suffering from the disease or who carry the organisms in their throat, although they may not be sick themselves. Occasionally the diphtheria germ is distributed by milk, which becomes infected from the handling of the milk or the milk vessels by convalescents or others that have the germs in their mouth, and it is possibly distributed at times by other indirect methods, as by domestic animals. But in the vast majority of cases, it is either by direct or indirect contact that diphtheria passes from person to person. It is primarily through the schools that this organism gets distrib- uted among the children of a community, because in the schools they are brought in close contact with each other, and many of the thoughtless habits of school children are such as to facilitate the dis- tribution of the germs from person to person. Many children, moreover are immune against this disease and may carry the violent germs in their mouth without themselves being in the slightest degree affected by them; but the bacilli they carry are as virulent as those that are in the mouth of a person that is suffering from a severe case °f the disease. Hence such individuals are just as truly a source of danger to their playmates as would be a frank case of diphtheria; indeed, they are likely to be more dangerous in the school, because an actual case of diphtheria is speedily detected and removed from contact with other scholars, whereas one of these so-called "carriers" °f the diphtheria bacillus may remain in school for a long time. The presence of a carrier in a school is now believed to be the primary cause of case after case cropping out among school children. The detection of such carriers is a very simple matter, for it only requires the microscopic examination of cultures from the throat to detect which scholars are thus a source of danger to their playmates. The exclusion of these carriers from school for long enough to let their throats clear up-that is, to get rid of the diphtheria germs-is usually quite sufficient to prevent new cases of diphtheria from de- veloping in the schools. This method is now very widely applied, and efficient health officers, when they find diphtheria breaking out in the school, immediately have the throats of all children examined, and then exclude the carriers from school. Such a plan is far more 316 BACTERIOLOGY efficient than to close the schools when an epidemic appears. Prompt measures along these lines are now successful in preventing the ex- tensive diphtheria epidemics that were formerly so common. The diphtheria organism is not a resistant one, and dies quite quickly after it is removed from the tissues of the human body, although it is somewhat more resistant than the gonococcus. Ap- parently it is never distributed through the air, unless it may be in the moisture particles that are coughed from the mouth of a patient, and in this case the danger is confined to the immediate vicinity of the patient, such particles soon sinking to the ground, drying, and the diphtheria germs promptly dying. The discovery of diphtheria antitoxin, which was such an epoch- making event in bacteriology, has been mentioned on another page and need not be repeated here. Since the introduction of this mate- rial as a method of treating diphtheria, in 1896, there has been a more or less constant decline in the death rate. At the outset there was considerable opposition to its use, and some question as to its effi- ciency. But as the years have passed and there has been a steady decline in the fatality of the disease as well as in the number of cases, and as this diminution has continued without variation, the opposi- tion has almost disappeared, and the use of antitoxin is now the stand- ard method of combating the disease. So efficient is the diphtheria antitoxin that the modern doctor tells us that if he can obtain access to the case in the first two or three days, there is practically no failure to cure. Checking epidemics by the detection of carriers, treating cases by antitoxin, and more recently the immunization of school children by means of toxin-antitoxin have proved such efficient methods of combating the disease that it has been practically mas- tered. Its control represents one of the greatest triumphs of bacteriology. Measles Although beyond doubt a germ disease, the cause of measles has not been discovered. The contagious material seems to be discharged through the mouth and nose, especially during early stages of the disease, even before the eruption appears on the skin. Measles is one of the most contagious of all diseases, and one that is very difficult to control. This is doubtless due in part to the fact that the con- tagion is at its height even before the disease is recognized, so that it is readily spread by persons who are coming down with the disease STRICTLY HUMAN DISEASES 317 but are not yet recognized as patients. Methods of obtaining pro- tection against the disease by vaccines or sera have not hitherto1 been successful. There is almost no natural immunity against the disease; but the immunity following recovery is almost complete And very lasting. For this reason it is primarily a child's dis- ease, although adults that have escaped in childhood are equally subject to it. Whooping cough In 1906 there was discovered a bacillus which may be the cause of this well-known children's disease. It is a non-motile rod which has been called Bacterium pertussis, and may be found in considerable abundance in the discharges from the mouths of patients. It so closely resembles other bacteria, however, that are normal in the sPutum that it cannot be detected by simple microscopic examina- tion, nothing except cultural methods serving to identify it. The organism apparently grows in the air passages, and may be found in the cells of the epithelial lining. It is in the tissues between cilia, and difficult to dislodge, remaining there for a considerable time, and thus. Apparently causing the protracted course of the disease. This organ- lsm has not been absolutely demonstrated as the cause of whooping p°ugh, although it apparently produces somewhat similar troubles inoculated into animals. Bacterial vaccines have been made from and have been used with at least partial success, although at the Present time this success is too indefinite to justify far-reaching claims as to their efficiency. Claims have also been made that the disease is caused by a filterable virus. Meningitis Meningitis is a general name for certain kinds of troubles due to of the meninges of the spinal cord and brain. It is n°t, therefore, a specific disease, and the different forms are produced by different agents. For example, the pneumococcus, Streptococcus 'Pyogenes, the influenza bacillus, the typhoid and the colon bacillus, the organism of bubonic plague, the glanders and the tubercle organ- 1Srns, as well as others, are known to produce meningitis when at- tacking the meninges of the spinal system. In addition to all these, however, there is one organism especially associated with the epi- demic form. This organism has been called the meningococcus, 318 BACTERIOLOGY and is found in the spinal fluid in the large majority of cases of men- ingitis, especially of the epidemic variety. It is pathogenic for some of the lower animals as well as for man. In the human race the mortality of the disease is very high, ranging from 50 to 80 per cent. Bacteriologists had hoped for some time that a serum might be procured which would enable physicians to handle the disease; and after various experimenting, a serum was obtained from a horse by injecting into the horse increasing doses of killed meningococcus cultures. This serum has been somewhat extensively used, since it was discovered in 1906, and while it is by no means as successful aS diphtheria antitoxin it has considerable value. Manifestly this serum is of no value except in the particular type of meningitis caused by the meningococcus, and in the use of the serum it is first necessary, therefore, to demonstrate microscopically that the case in question is caused by this organism. Statistics show that there has been a marked reduction in the mortality of meningitis, following the use of this serum. It is not an absolute cure, however, the death rate among young children especially being still quite high. Even here, nevertheless, the mortality has been reduced from about 80 per cent to less than 50 per cent, and in the later years of life to a considerable lower percentage. Its average mortality has been reduced to about 30 per cent. Influenza This disease, which dates back certainly to the fifteenth century and probably much earlier, has attracted special notice in the past thirty years, particular attention having been called to it by a serious epidemic in 1889. Under the name of influenza there are un- doubtedly a variety of different types of infection, inasmuch as any severe form of nose and throat or lung infection is likely to be called influenza at a time when this disease is common. But in addition to these miscellaneous disorders, there is undoubtedly a specific influenza infection, though it is not always easy to detect it. In 1889 there was discovered a bacillus present in great abundance in the secretions of patients suffering from this disease, and it is generally known as the influenza bacillus, although bacteriologists are by no means agreed that it is the cause of the disease. The organism has been found somewhat pathogenic for animals when inoculated. It is STRICTLY HUMAN DISEASES 319 Undoubtedly contagious, and is widely distributed at the time of epidemics, although many people are evidently quite immune against it, either temporarily or permanently. Persons who are perfectly well may harbor the organism in their lungs or throats for a long time, and possibly communicate the disease to others. Such facts may explain some of the peculiar facts in regard to the epidemics. The recent very severe epidemic (1918-1919) has increased the general interest in the disease, but it has not greatly clarified our knowledge as to its cause and method of spread. The so-called influenza organism, although present in a large number of cases, has not been found universally, and the best authorities consider that the causal agent of the disease is still unknown. The occur- rence of large numbers of deaths in this epidemic with symptoms apparently identical with pneumonia has raised the question whether influenza may have a pneumonia-like phase or whether in the present epidemic if may have been associated with pneumonia, giving rise to conditions making pneumonia contagious and epidemic. Al- though scientists the world over have been giving their attention to the disease, these questions have not been definitely answered. Nor is there agreement as to how to avoid infection. Some cities adopted rigid closing measures before the disease reached them, while others went to the extreme of requiring everyone to wear a gauze mask over mouth and nose while in public places. Closing public meeting places was found to delay the onset of epidemic; but neither that nor the compulsory wearing of face masks was found to diminish the number of cases eventually breaking out in a community. Nearly every large city had about the same percentage of cases, regardless of measures taken to avoid the spread of contagion. Such facts have confirmed the former belief that the disease is very contagious, but that many people are naturally immune to it. Beyond that, the epidemic has not taught us much. It has even raised the question as to whether influenza is all the same disease and whether the recent epidemic was actually caused by the same organism as that which incites the milder form of the disease which is with us more of the time. Attempts have been made to produce a protective serum, and also bacterial vaccines, in the endeavor to control influenza. These have not been as yet of much use. During the recent epidemic great claims were made for some of these preparations, due largely to 320 BACTERIOLOGY the fact that they were seldom employed in any institution until majority of the susceptible individuals had already succumbed, and the following decrease in the number of cases, really caused by the exhaustion of susceptible material, was ascribed to the effect of treatment. Whenever carefully controlled experiments were planned with one group of people treated and another similar group untreated, no real benefit from the treatment was discovered. At the present time these vaccines and sera cannot be considered a success. Poliomyelitis; infantile paralysis This is one of the still mysterious diseases, which has specially come to the public attention in recent years. It has been known in a somewhat indefinite fashion for nearly a century, but simply as sporadic cases. It was first noticed as an epidemic in 1881, and since then epidemics have been common. In the first decade of the twentieth century many epidemics of varying severity were studied, and the disease attracted wide attention from the public as well as from the medical profession. It has certainly been in- creasing in extent during recent years. It is largely confined to children, though not exclusively so. It is a disease of widely varying intensity, ranging from an attack so mild that it may not be noticed at all to one so severe as to produce death in two or three days. The percentage of fatalities cannot be given since there are clearly many people attacked in such a mild form that the disease in not recognized. Among the clearly recognized cases the fatality is frequently about 25 per cent. The disease is dreaded largely on account of its after-effects. The infectious agent attacks the nervous system, especially the spinal cord, producing quickly an infiltration into the nerve tissues from the blood, causing the temporary paraly- sis of the motor cells. In many cases, after the acute stage is over, the inflammation disappears and a recovery of functions takes place. But in many others the motor cells are destroyed by the virus, and when once destroyed they are never replaced, so that permanent paralysis ensues, the extent of which is dependent upon the amount of destruction of the motor cells. Sometimes only a weakening of the action of certain muscles remains, but in other cases whole muscles or groups of muscles lose their functions permanently. That this is a germ disease is evident from its general nature as well as from the fact that it can be carried from the human being to STRICTLY HUMAN DISEASES 321 monkeys by inoculation. Its rapid extension as an epidemic also proves its parasitic nature. The agent which causes it is not yet known. Flexner has found in infectious material an extremely minute body which he has thought to be a living agent and the cause of the disease, although others have not confirmed this conclusion. Some have thought it to be caused by a streptococcus. The infec- tious material is abundant in the spinal cord and brain, also in some of the discharges from the patient. It is found in the nasal discharge and also in the intestinal contents. It is not present, unless in minute quantity, in the blood. The method by which this virus is carried from person to person is also unknown. That it directly or indirectly passes from person to person is shown by the spread of epidemics, there being abundant instances where one person has carried the disease into hitherto Uninfected districts. Some believe it to be directly contagious from one individual to another, while others think there may be an inter- mediate carrier, possibly some insect. It is known that well persons may be carriers, since active virus has been obtained from the nasal passages of people who have been in contact with patients but have not had the disease themselves. It has been found, however, that during the epidemics it is unusual to have two cases in the same family. This indicates either a very slight contagiousness or a general immunity against the disease, so general that in most families only a single member is subject to it. How these puzzling and seem- ingly contradictory facts are to be explained does not yet appear. The fact that it is confined to children has led to an attempt to pre- vent its distribution by establishing a quarantine against those under sixteen from infected districts. But this has been a failure, as would naturally follow from the fact that adults as well as children may be carriers, even though not subject to the disease. No great success has yet attended the use of sera or vaccines in combating the disease. A person who has recovered from an at- tack is immune from a second attack and his blood serum contains some antibodies. Such serum in the few cases where it has been used seems to be of some value in helping recover. But the serum is not available in any quantity. Most animals are quite immune against poliomyelitis and are not available as serum producers. The monkey can be given the disease, but is not a source of serum in quantity. Up to the present we are without any efficient means of combating or curing this dreaded disease of childhood. 322 BACTERIOLOGY Lethargic encephalitis Recent years have seen the appearance of what looks like a new disease, the cause of which is still unknown. It is generally con- sidered an infectious disease, but it is apparently in no sense con- tagious. It is a brain disease, the most characteristic symptom being the development of coma, from which has come the popular name, sleeping sickness. Although often called by this name the disease must be distinguished from the true sleeping sickness of Africa, to be described on a later page. The frequency of this disease has been increasing since the war, but the reasons for its spread as well as its cause remain a mystery. It is generally believed to be one of the disorders following in the wake of the influenza epidemic, and possibly in some way brought about by the epidemic. The disease is not ordinarily fatal, although death is the outcome in an appreciable percentage of the cases. DISEASES CAUSED BY PARASITES THAT CAN ALSO LIVE A NON- PARASITIC LIFE The organisms producing the infectious diseases discussed above are found in nature only as parasites in human beings, and conse- quently the only source from which they are to be acquired by the human being is some human source. The types of pathogenic or- ganisms that can live a non-parasitic life may have other important modes of distribution, and may be acquired by human beings by methods quite different from those of the diseases just described- Hence totally different methods must be adopted for fighting them- Typhoid fever Typhoid is an intestinal disease, characterized by a long run of fever, often resulting in death, or in case of recovery in an extremely long period of convalescence. It is generally many months before the patient has recovered his original health. It is a very serious disease to the community, both because of its great prevalence and because of the large amount of temporary incapacity it produces- Its prevalence is shown by the fact that there are each year in the United States 300,000 to 500,000 cases and about 35,000 deaths- Because of the large number of deaths each year and the still larger number of people disabled for a considerable period, the direct loss STRICTLY HUMAN DISEASES 323 to the community from this disease is very great. There is a seasonal distribution in the number of cases. In the early fall the number of cases rises for a few months and then falls off, but there remain a considerable number all through the winter, spring, and summer to show another rise the next fall. In addition to this regular curve there occur frequent sporadic outbreaks, when an extremely large number of cases may develop within a few days of each other. To explain the typhoid of our communities we have then three conditions to take into account: the constant presence of a certain amount of the disease at all seasons; the periodic rise and fall every autumn; and the occasional severe but brief epidemics. It appears-as will be seen presently-that these three modes of occurrence are probably due to different methods of distribution. Fig. 45. The Typhoid Organism The causal organism. The cause of typhoid, Bacterium typhosus, was first discovered in 1880, and was subsequently obtained in pure culture. It was difficult to prove it to be the cause of the disease because animals are immune to it and experimental inoculation of human beings is out of the question. Proof has been obtained by accident, however, when cases have developed in laboratory assist- ants who have handled typhoid cultures without taking care to keep the material from their mouths. There is even one instance of a person who swallowed a typhoid culture for the purpose of commit- ting suicide, and he also developed a typical case. Further proof has been obtained from the use of the cultures to make bacterial vaccines that are specific against typhoid, and strong evidence from the agglutination test (see p. 287). This organism is found in the glands, the intestines and to a slight extent in the blood of typhoid patients. The contents of the intestines and frequently the urine 324 BACTERIOLOGY contain enormous numbers of the bacilli (3,000,000,000, per cubic centimeter having been observed in the urine). In the body they produce various toxic substances which cause the symptoms of the disease. Neither medicine nor bacteriology has found any cure for typhoid. Certain antibodies are gradually produced in the blood of a patient during the disease, which slowly bring about recovery, and all the physician can do is to develop the patient's vitality, by nursing, proper diet, and so forth, to the greatest extent, and thus to stimu- late the production of antibodies. Bacteriologists have made many attempts to obtain a typhoid antitoxin similar in its effect to the diphtheria antitoxin, but without success. The antibodies developed in the patient have not as yet proved of use in treating the disease artificially. It is found, however, that one attack conveys to the patient a certain amount of immunity, and for several years he is not subject to another attack. Although no antitoxin has been produced, it has proved possible to confer immunity artificially by the use of bacterial vaccines. These have already been described ■(p. 281). The immunity thus produced apparently does not last much more than two years; but while it does last, it seems to be very •efficient. Methods of distribution. The typhoid bacteria, although capa- ble of living a non-parasitic life under some conditions, and growing readily in a variety of media, provided no other bacteria are present, have no natural habitat outside the human body. They live for perhaps six weeks in water, and for longer periods in ice; but no- where in nature do they live long enough to render any material a source of danger for any length of time unless it is freshly infected. Their presence in water always indicates fairly recent contamina- tion with human sewage, as it is primarily in the urine and feces that they are eliminated from the body. Any other material besides water that may possibly become contaminated with sewage is also liable to contain the typhoid organism; but to be of danger it must be something that is taken into the human mouth, as no source of inoculation except through the alimentary canal seems to cause the disease. From these facts we can understand the common methods of distribution of typhoid, and thus obtain the knowledge necessary for an effective campaign against it. The primary methods of distribution are as follows: STRICTLY HUMAN DISEASES 325 1. Direct contagion. Direct contagion is not very common in the case of typhoid, but it is known to occur. A patient's bedding and eating utensils are almost bound to become contaminated with germ-containing discharges, from which a nurse or other attendant may easily get them on his hands and from them into his mouth. In this way the few cases of direct contagion are probably easily explained. 2. Milk. It is easy for milk to become infected with typhoid, either by washing the milk utensils in contaminated water or by direct contamination from some milker who has a case of so-called "walking" typhoid. Typhoid germs can multiply in milk, so by the time the contaminated milk reaches the consumer there is a ehance of its containing considerably more than when it leaves the dairy. Within the few years that the subject has been studied, some three hundred epidemics have been traced with practical certainty to the milk supply, and there are doubtless many others that have not been detected, through lack of proper study. All these milk- borne epidemics are of an explosive type, that is, many cases appear within a comparatively few days of each other, after which practically no more new cases appear. The reason for this is simple. Milk is but rarely contaminated with typhoid germs, and ordinarily the epidemic arises from the milk of a single dairy on one day or perhaps two or three days in succession. After this, the germs disappear from the milk, but they have done their harm, and after the usual incubation period of about two weeks, cases of typhoid develop one after another; yet, inasmuch as the milk is by this time no longer contaminated, the epidemic dies out as rapidly as it developed, bhis explosive character is quite typical of milk epidemics and in itself is enough to make one suspect milk as the cause of a particular epidemic. When milk is suspected as the cause, it is not possible to obtain proof by making a bacteriological study of the milk from the suspected dairy; for by the time the disease has been traced to an individual dairy, all of the contaminated milk will have been consumed and the milk from the same source will probably be free from all contamination. The evidence, therefore, for the distribu- tion of typhoid fever through milk has never consisted of the dis- covery of typhoid germs in milk, but has always been of an indirect nature, such as showing that the cases have all developed on certain particular milk routes. 326 BACTERIOLOGY There is no sure method of protecting milk from contamination with typhoid germs. Excessive care and cleanliness lessen the danger; but cases of "walking" typhoid and typhoid "carriers" in a dairy form a danger that cannot be avoided by the greatest diligence. The only sure method of protecting the public from this danger is to pasteurize the milk, a practice which is becoming more and more common, and promises to become soon practically universal. 3. Oysters. That raw oysters are occasionally a means of typhoid distribution has been abundantly demonstrated. They sometimes become contaminated in the harbors where they grow, through the sewage that is poured into the water and gradually extends further and further from shore. Still more often they are contaminated by the process of "floating," that is, placing them in fresh water to make them grow plumper and become more marketable. Floating does not really increase the value of the oysters and as they are often placed for this purpose in contaminated creeks, it is a dangerous practice. Typhoid is not very often caused by eating oysters, but a few epidemics have been definitely traced to this source. The danger seems to be mostly in the fall months-although this has not been definitely proved-because the oysters hibernate during the winter and do not then absorb water within their shell to any con- siderable extent, and they are not eaten in spring or summer. Pro- tection against this source of danger is accomplished by: (1) for- bidding the raising of oysters in sewage-contaminated waters; (2) forbidding the floating of oysters in fresh water; and (3) by cooking them before eating. The last method is the most efficient; but it destroys the flavor of the raw oyster. 4. Raw vegetables. There is a chance that typhoid is sometimes distributed by raw vegetables such as lettuce, radishes, watercress, and celery, but it is doubtful if the danger is serious enough to be considered. The only danger of this sort comes from "sewage farms" where sewage is used for water and fertilizing the fields; but sewage farms are not very common in this country. 5. The fly. During the Spanish-American war, typhoid fever became excessively abundant in the American camps, and the com- mon fly was soon suspected as causing its spread. As is well known, flies breed upon manure and upon human excreta when the latter are available. It is equally well known that flies are attracted by open STRICTLY HUMAN DISEASES 327 food; so it is easy to put two and two together and conclude that flies are important agents in the spread of typhoid wherever there are unscreened privies or latrines. Fairly conclusive evidence has been obtained that this is the case. It has been shown, for instance that cases of typhoid and the number of flies rise and fall together; in fact, the annual fall increase in typhoid occurs at the time when flies are most numerous, and may well be caused by them. This agency of the fly in distributing typhoid has caused it to be called the "typhoid fly," and has inaugurated the campaign against the fly which has been prosecuted vigorously in the last few years. In this campaign, the least effective method of destroying the fly has proved to be the killing of adult flies. Flies breed so rapidly that it is hardly possible to diminish their numbers by destroying the adults. The breeding place, rather than the flies, should be de- stroyed, as this prevents the hatching of the young. Inasmuch as flies breed primarily in garbage pails, manure piles, and privies, the proper methods of attacking flies as distributors of typhoid is to screen all places that contain such material. A little attention paid to this matter will produce rapid and valuable results. 6. "Carriers" All typhoid fever patients retain the bacilli of this disease in their alimentary canal for some little time after re- covery. Many patients retain them for weeks or months and some for many years. In these latter cases, the organism seems to be- come colonized somewhere in the individual-in the bile duct., for instance-and to grow and multiply for years, being constantly shed from the body either in the feces or in the urine or both. Such persons are known as typhoid "carriers." It has been calculated that possibly 4 per cent of the typhoid patients become carriers, and this of course means that there are a large number in our communities at all times. Such individuals are naturally a source of danger to all with whom they come in contact. If a carrier is employed in a dairy, the community supplied with milk from this dairy is pretty sure to suffer from an abundance of typhoid. If employed as a cook, those consuming the food are likely to become infected. Unfor- tunately there is as yet no satisfactory method of detecting such individuals. The only known method is to examine the excreta for typhoid germs, which is a long task, not always very certain, and one which manifestly cannot be applied to all the members of a commu- nity indiscriminately. Even more unfortunate is it that at present 328 BACTERIOLOGY we have no method of treating a typhoid carrier so as to rid him of the bacteria. Various methods have been tried but without great success. Having no adequate means of protecting ourselves against these individuals, their presence in our communities undoubtedly explains in a measure the constant presence of typhoid in spite of all attempts to get rid of the other more evident sources of infection. 7. Water. Drinking water is perhaps the greatest, certainly one of the greatest, sources of typhoid. It is responsible for its constant oc- currence month after month as well as for many epidemics. Numer- ous epidemics have been traced beyond question to the water supplies. The first famous case was in the town of Plymouth, Pennsylvania, in 1885 (see p. 378), where in a small town there occurred 1104 cases with 104 deaths, all of which were plainly traced to a water supply contaminated by a single case of the disease. Many other similar epidemics have since been traced to water. In general they differ from milk-borne epidemics in that they are of a less explosive charac- ter, reaching their maximum more slowly and subsiding more slowly. The methods by which drinking water is contaminated are too well known to need any explanation. Running streams and wells in any inhabited country may be easily contaminated; any reservoirs or any other source of supply is liable to contamination unless pro- tected with great care. Fortunately the organisms scarcely multiply in water; but they may remain alive five or six weeks. Accumulated experience has shown that the amount of typhoid fever in a city rises and falls with sewage contamination of the water supply. The typhoid death rate for cities with uncontaminated water supplies averages 20 or under per hundred thousand population; while cities with badly contaminated drinking water may have 100 or even 150 per hundred thousand. So close is this connection that the typhoid death rate of any city may be regarded as a measure of the purity of its water supply. This fact has been strikingly illustrated again and again by the immediate and striking reduction in the number of typhoid cases which always follows the purification of a water supply, by filtration or other means. 8. Ice. The question as to whether typhoid fever is likely to be distributed by ice is one over which there has been much discussion, considerable experimenting, and concerning which there is as yet no consensus of opinion. One or two small epidemics have been claimed to be due to this cause, but the evidence is inconclusive. It is known STRICTLY HUMAN DISEASES 329 that typhoid bacilli can be found alive in ice two or three months, and perhaps longer, after the ice has been frozen, a fact which has been used as a basis for the claim that ice cut from polluted waters is unsafe. But when quantitative tests of this same ice are made, it proves that only a very few of the organisms have survived, and it is claimed that they have lost much of their virulence. Some therefore insist that polluted water is as safe frozen as it is filtered; for even filtration does not kill all the organisms and yet removes practically all danger from typhoid? Perhaps a fair statement of the matter is that clear ice, after storage for three months or more, is practically free from virulent typhoid germs; but that snow-ice does not become purified so quickly and may be a source of danger even after storage for three nionths. The danger from ice is probably not very great, but it cannot be entirely overlooked. Fig. 46. The Cholera Organism In general. From the facts above given, the following general statement seems to be warranted: The constant continuation of typhoid month after month in any community is probably to be attributed to bacillus carriers, or a contaminated water supply, or both. The annual rise in the number of cases, which comes in the early fall months, is probably due to distribution by insects. The sporadic outbreaks of violent epidemics, which may occur at any season of the year, are probably due to milk, oysters, or to some Unusually heavy contamination of drinking water. Asiatic cholera Asiatic cholera has always been a disease of terror. Endemic (that is, a large number of cases always present) in eastern countries, has during the last century swept many times over western Europe, and produced raging epidemics, with a large percentage of fatalities. Progressing as it does with extreme rapidity, producing fatalities ln a comparatively few hours, and spreading with extreme rapidity °ver a community, the disease has always given rise to terror, 330 BACTERIOLOGY bordering on panic. It is an extremely violent gastro-intestinal disease, with vomiting and purging, accompanied by severe cramps. The mortality is about 40 or 45 per cent of the cases; and after death, marked tissue destruction of the alimentary tract is found. Until about 1890 no methods of fighting the disease had been devised. The first step toward a control of the disease was under- taken by the German cholera commission, with Koch at its head, who succeeded in discovering, by studying the cholera in Alexandria, where it was endemic, that it is caused by a comma-shaped organism, figure 46, that grows in large numbers in the intestines. Animals do not take cholera under any circumstances, and it has therefore proved impossible to inoculate animals and observe whether or not a typical case of cholera developed. Accidental observation has been made, however, which renders it almost beyond dispute that this so-called "comma bacillus," Microspira comma, is the real cause of the disease. There have been at least three cases of people who have swallowed cholera cultures, intentionally or unintentionally, and in every case the result has been the development of a typical case of cholera. Attempts to combat the disease by forming sera and vaccines have been numerous, and more or less success has attended some of the experiments. The most successful have been vaccines made of pure cultures of the cholera bacillus, the use of which is claimed to produce a marked reduction in the number of cases and also a notice- able reduction in the severity of the cases that do occur. This vaccine, however, has not been very generally used. The practical control of cholera has come through a knowledge of the means by which the bacilli are distributed from person to person. Living as it does, practically confined to the lower intestine, the vio- lent cholera bacilli are sure to pass from the body in the feces, and the problem of protecting other persons from attack is merely to protect the community at large from being infected from fecal dis- charges. The discharges of both patients and of bacillus carriers may be dangerous, for it has been found that healthy persons some- times carry virulent cholera bacilli in their intestines. The problem is essentially the same as preventing the distribution of typhoid, but it is rendered somewhat easier by the fact that the cholera or- ganism is not resistant to external conditions. It dies very quickly upon drying, cannot stand the least amount of acid, and is quickly STRICTLY HUMAN DISEASES 331 killed if ordinary saprophytic bacteria are growing in the same culture with it. In water it is found to be able to live for a varying length of time, from a very few days to several weeks, though in most bodies of water it cannot be found longer than six or eight days after inoculation. The most useful piece of information that has been learned con- cerning cholera is its method of distribution in epidemics. The famous cholera epidemic of Hamburg in 1889 gave the real solution of the problem. In that epidemic it was shown that the phenomenon was dependent upon the purity of the public water supply. The city of Hamburg using the unfiltered water of the river Elbe, suffered very extensively, some twenty thousand cases of cholera developing m a few weeks. The city of Altona, which is contiguous with Ham- burg (a street forming the main part of the boundary between the two cities), used the same drinking water, but had taken the precaution to build large filters, so that no raw water was consumed by its in- habitants. In this city there were almost no cases of cholera that had not been imported from the city of Hamburg. This fact, together with various other pieces of information, demonstrated that the raging epidemics of cholera which the European countries had before experienced were to be attributed to contaminated water supplies. This was indeed a very natural assumption, considering the method in which the epidemics developed, always appearing almost simul- taneously over a whole city at once, without any contact between patient and patient, and also when it was further remembered that the cholera organisms voided by the patients may live for at least a number of days in ordinary water. As soon as this fact was fully realized the spread of cholera was controlled. Previous to that date, epidemics were of frequent oc- currence in Europe, and whenever it appeared, the spread was rapid, deaths occurred in thousands, and in all cases terror and panic resulted. Since this discovery at Hamburg, no such conditions have occurred in Europe. While cholera has appeared several times, and in a few cases has become somewhat prevalent in certain localities, no raging epidemic such as previously visited Europe has been known. The public authorities have found that the method of preventing such epidemics is to guard the water supply, and the moment cholera niakes its appearance, endeavors are made to insure the purity of the water used by the population. This has always been successful, 332 BACTERIOLOGY since 1889, in preventing the development of the raging epidemics. We may confidently hope and expect, therefore, that there will be no serious outbreak of Asiatic cholera in the future, except in a few cases where carelessness has been allowed to develop in the handling of water supplies. It is not to be inferred that drinking water is the only method by which the cholera germ is distributed. There are certainly others that occur occasionally; inasmuch as the infectious germs are in the excrement, any method by which these discharges may find their way into the body of another person may be a means of distribution. But all other methods of distribution of the bacilli are incidental and unusual, and the protection of the water supplies amply safe- guards the public from cholera epidemics. One other matter in connection with the control of cholera has been completely changed in recent years. Formerly, when an immigrant ship arrived, coming from a port known to have cholera, the ship wras put in quarantine for some weeks to wait for cases to develop. To- day the much more efficient and less oppressive method is adopted of submitting all persons aboard the ship to a laboratory examination. The cholera germ, if present, will be in the fecal matter, and cultures therefore are made from the feces of all suspects and are carried to a laboratory for examination. By inoculation onto special media it is practically possible in all cases to determine whether or not cholera germs are present in the intestines of each individual. Only those individuals showing the cholera bacillus need be quarantined. As a result the long quarantining of ships is no longer necessary; but inside of two or three days practically all the passengers can be released without danger to the community. Dysentery does not seem to be a specific disease, but a general name that is applied to a variety of intestinal troubles characterized by somewhat similar symptoms, the most prominent of which is a violent diarrhoea. There appear to be several types of dysentery, two of which at least, are fairly well known. One is the so-called "amoebic dysentery," which is due to the development of an amoeba in the intestines, where it multiples in great numbers, and gives rise to the inflammatory condition of the disease. The other is produced by a bacillus belonging to the same group as the colon and typhoid bacilli, Dysentery STRICTLY HUMAN DISEASES 333 but different from either of them. In most cases of chronic dysen- tery it is the amoebae that are the primary cause. In cases of acute dysentery, especially in temperate climates, the amoeba are rarely the cause, and it is usually the above-mentioned bacillus that is responsible for the trouble. Dysentery appears to a certain extent to be contagious, and mild cases or carriers are regarded as responsi- ble for distributing the infection in a large majority of cases. Water may become infected, or food, or any article of clothing used by these carriers or by patients. The organism doubtless finds its way into the new host through the mouth. Laboratory methods of diagnosis by means of agglutination have been developed, but they are not very extensively used at the present time. No protective vaccination bas been perfected, although the use of immune sera in treating cases has given some considerable success. Dysentery is distributed all over the world, the type produced by the bacillus being more common than that produced by amoebae. REFERENCES A Text-book on General Bacteriology. 7th edition. Saunders, Philadelphia, 1922. • Park and Anna W. Williams. Pathogenic Microorganisms. 6th edition. Lea & Febiger, Philadelphia, 1917. •A- I. Kendall. Bacteriology: General, Pathological, and Intestinal. Lea & Febiger, Philadelphia, 1916. CHAPTER VI Diseases Caused by Parasites Attacking Both Human Beings and Lower Animals SPREAD THROUGH LOWER ANIMALS AS INTERMEDIATE HOSTS A certain group of human diseases do not ordinarily pass directly from man to man, but have to be carried from one human being to another through the agency of some lower animal that is also subject to the disease. We have seen how typhoid is often spread by means of flies; but in this case the flies do not take the disease, merely carry' ing the germs mechanically. In the case of the following diseases, however, the causal organism lives a parasitic life not only in matt but also in the animal agents that spread the disease from one person to another. Animals that thus contract the disease and carry fl from one man to another are spoken of as intermediate hosts. The diseases thus spread are not ordinarily directly contagious. In one -the plague-contagion is possible, although not the usual method of spread; in others, such as malaria, contagion is impossible because the parasite completes its life history only in the intermediate host and cannot infect another human being until its life history fl complete. The practical importance of these facts is great. The intermediate hosts are large enough to be seen with the naked eye and are mud1 more easily eradicated than the disease germs; yet, if the disease must pass through these hosts in passing from man to man, tion of the animal host is as efficient in conquering the disease would be the complete destruction of the parasites themselves- Obviously this gives a hint as to the most effective way of controlling this class of diseases. The bubonic plague The plague used to be one of the most serious pests of mankind) and its history is intensely interesting. It was the "black death or "black plague" of past centuries, which at one time caused utter devastation in Europe. It has several times swept over DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 335 countries, and the number of deaths it has produced is to us in the twentieth century almost incredible. It is reported, for example, that in the fourteenth century one quarter of the inhabitants of Europe died from it. As late as 1665 an epidemic in London became so violent as to cause the cessation of all business, the inhabitants fleeing in panic to the country. It killed 70,000 people in one season ln the city of London alone (out of less than 200,000 population) and did not wholly die out until a large conflagration, the following year, burned out a considerable portion of London and apparently cleaned out the plague. The disease has been less extensive in recent years, but has made its appearance periodically in different Parts of the world. In 1893 it started in Hongkong, spreading Rapidly over the East. It found favorable conditions in India, and ln ten years caused several millions of deaths. It has continued up to the present time, with varying degrees of intensity, and has spread paore or less over the world, having reached the new world in several delated localities, but never producing such havoc in the new world as in the old. The plague is a disease produced by a specific bacterium, Bacterium pestis, that has been easily found and proved to be the causal agent. This organism has at least two quite different methods of attacking the human body. It sometimes attacks the glands, especially those around the groin, and causes them to swell, producing what are known as buboes. Hence the name bubonic plague. In other cases it attacks the lungs, and produces a rapidly fatal disease, the so-called Pneumonic plague. The organism that produces these two types, however, is the same in both cases. The bubonic form of the plague does not seem to be especially contagious, but it is believed that the pneumonic form is violently So- The material that is coughed from the mouth or blown from the aose of the infected patient is found to contain living virulent or- ganisms, and it is thought that other persons inhaling these organ- 1Srns may themselves acquire the disease in the pneumonic form. Attempts to combat the bubonic plague by the production of vaccines and sera have been numerous, and some of them have been rePorted to be thoroughly successful. A bacterial vaccine has been used, and it has been claimed that the inoculation of people with it gives a certain amount of protection against the plague. In dis- tricts where the inoculations have been carried on, at all events, there 336 BACTERIOLOGY seems to be a smaller proportion of victims among the inoculated than among uninoculated people. Our chief protection against the disease, however, does not lie in any form of treatment applied to the individual, but in sanitation. To modern bacteriology we owe the knowledge as to how to check the ravages of the plague. For a long time the methods by which it is distributed remained mysterious; but eventually it was noticed that in times and localities where epidemics of it developed there were found many dead rats-even lying around in the streets-before the plague broke out. Later it was shown that these dead rats were infected with B. pestis; but the method by which human beings be' come infected from the rats was not at first evident. It was finally shown, however, that the bacilli are carried from rat to rat, from rat to man, and doubtless from man back to rats again, through the bites of fleas. The actual distributor of the plague proves to be $ particular kind of flea which normally infests rats, squirrels and a few other rodents, but will bite human beings on some occasions- perhaps when none of these natural hosts are available. These fleas, biting the infected rats, become themselves infected with the germs, and transfer them to the next animal they infest. This discovery has shown the line of defense against the plague to be the extermination of rats and squirrels, and the "rat-proofing" of build' ings, that is closing all openings through which rats might enter. With the information now at hand, there is no especial need t° fear the wide extension of the bubonic plague in civilized countries, where there is efficient health control. This is well illustrated by the experience of recent years; because this disease has two or three times invaded the ports of the United States and has been promptly controlled. The most difficult task has been in California; for when it first made its appearance in San Fransisco its presence was kept secret, from a mistaken notion that it would be better for the city tf outsiders did not know that it harbored the dreaded bubonic plague- The disease spread rapidly, however, until it could be no longer concealed. Then an active campaign was undertaken to exter- minate the rats, which was accomplished with the expense of con' siderable money. It was then found that the disease had spread beyond San Fransisco, having infected a certain species of squirrel, and the campaign had to be extended to include squirrels in the sur- rounding country-a much more difficult task. At present the DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 337 disease is at least under control, and infected rats or squirrels are a rarity. Watch is kept now at all seaports, for it is introduced into a new country by the rats aboard ships. If dead rats are found commonly on the wharves or in a sea-coast city, it is a suspicious matter and should lead to an investigation. Laboratory examina- tion of these rats shows whether they are infected with the plague, and if so a vigorous campaign should be begun against them. Ex- perience has shown that by proper vigilance along these lines our shores can be protected from this most dreaded disease. Malaria Malaria is one of the longest known of human diseases, for we can trace it back at least to the beginning of the Christian era, and some claim that there is still earlier evidence of it. It has long been as- sociated with damp and swampy regions, and has been commonly supposed to be due to the breathing of poisonous air. In tropical climates it is one of the most serious diseases, far more deaths arising from malaria in such regions than from any other cause. In the development of modern theories of germ disease it was inevitable that this one should also be carefully studied, and a microscopic protozoon was discovered living in the red corpuscles of malaria patients. The life history of this parasite has now been carefully studied and it is found to undergo a complete metamorphosis with considerable regularity. As shown in figure 47, the organism for a number of hours increases in size within the corpuscle, eventually breaking up into a number of spores, which soon break out of the corpuscles, enter new corpuscles and then repeat the cycle. At the time when the spores burst out into the blood, there seems to be liberated certain poisonous excretions which give rise to the peculiar symptoms of the disease, chills followed by fever. While the organisms are growing in the corpuscles, the patient is quite com- fortable, but periodically the spores are discharged and the chills and fever appear. The period between attacks is the same in any one type of the disease; but there have been found three different types of malarial organism, each of which has a different length life cycle. The first produces the common type of the disease, in which the chills and fever appear every other day, the second a less common type in which they appear every three days, and the third produces the so-called autumnal fever, in which there is no periodicity but a 338 BACTERIOLOGY continual fevci. The last form of disease is the most serious and the one that most commonly ends fatally. Malaria is one of the few diseases for which medicine has furnished a specific cure. The organism is very easily killed by quinine, and if the dose of quinine is taken at such a time as to be absorbed into the blood and to be present there in considerable quantity when the spores break out of the corpuscles, this one dose of quinine is likely to kill them all and to produce a quick recovery. As it cannot al- Fig. 47. Life Cycle of the Malaria Organism, in Man and in the Mosquito ways be timed exactly right, frequent repetition of the dose is gener- ally necessary. While other methods of treatment are adopted, none is so efficient as the use of quinine. It was quite a number of years after the discovery of this organism before its method of distribution and invasion into the human body was discovered. But eventually, in 1898, as a result of the work of Manson and Ross, followed by numerous investigators in various other parts of the world, it was triumphantly demonstrated that a certain kind of mosquito is responsible for the distribution of malaria, and apparently solely responsible. If a mosquito of the kind known DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 339 as Anopheles1 bites a patient suffering with malaria, the malarial organisms enter the body of the mosquito, and undergo a cycle dif- ferent from that which they go through in the human blood, even- tually becoming sexually mature, the two sex cells mating with each other, the fertilized cell breaking up into numerous minute spores, Fig. 48. The Malarial Mosquito Anopheles Left, normal position of larva at the surface of quiet water; right, normal Position of adult when at rest on a wall. Fig. 49. The Common Mosquito, Culex Left normal position of larva at the surface of quiet water; right, normal position of adult when at rest on a wall. which find their way eventually into the salivary glands. If sub- sequently this infected mosquito should bite an uninfected person, the spores are pretty sure to be inoculated into the person, and he will probably come down, after the proper length of time, with an attack of malaria. When this method of distribution was first sug- 1 The common mosquito is known by the name of Culex. This form has no relation to malaria. 340 BACTERIOLOGY gested, it received a great amount of criticism, and many objections were raised to it from various standpoints. All of these objections, however, have been removed by the further careful study of the conditions surrounding the disease, and it is now accepted beyond peradventure that to the Anopheles alone we owe the distribution of malaria. The value of this discovery can hardly be overrated. Malaria has sometimes been said to produce the death of more than half of the human race; and while this is probably an exaggeration, it is certainly true that there is no other disease so serious in tropical climates as malaria. Recognizing this mosquito as the means of distribution has put into our hands the methods of controlling the disease, and the result has been an organized campaign against the mosquito. Mosquitoes breed only in quiet water, and it is by no means an impossible task to see that no water is allowed to stand in puddles, empty tin cans, or elsewhere for long enough to allow their larvae to mature. Whenever this form of campaign has been effectively carried out, malaria has diminished and even disappeared. Few other discoveries have led so quickly to such beneficial results. Yellow fever This disease is primarily one of the new world, and has been to a large extent confined to the tropical regions. Occasionally it has invaded the semi-tropical, and even temperate climes, and when it has done so has produced immense devastations. People of the present generation know very little of the ravages produced by yellow fever in the past. Many have been the investigations made by bacteriologists to determine the cause of the disease, but until very recently the infectious agent was still uncertain. It is now believed to be due to a very small, spiral organism, probably a Spiro- chaeta, to which the name Leptospira icteroides has been given. Yellow fever has been practically mastered in recent years by the discoveries of the American Yellow Fever Commission that went to Cuba in 1899, and demonstrated conclusively that the disease is distributed from patient to patient by the bite of a very small mosquito known as Stegonia. The triumphal conquest over yellow fever that followed this discovery is well known, and needs no de- scription at this point. It may be briefly summarized by stating that in tropical climes, where sufficient care is taken to prevent the DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 341 breeding of mosquitoes, and especially to protect patients from the bite of mosquitoes, yellow fever can be rapidly brought under con- trol and even be made to disappear. The result of this sort of campaign in the Panama belt during the building of the Panama Canal is a matter of public knowledge, and certainly represents one of the greatest triumphs of sanitation. This, however, was no greater success than the crushing out of an epidemic of yellow fever that appeared in New Orleans in midsummer; for in all previous history a summer epidemic was sure to spread until the frosts in the winter checked its further development. So successful has been the campaign against yellow fever that it is no longer a disease to be dreaded, even in tropical climates; for proper sanitary precautions furnish absolute protection against it. Sleeping sickness This disease is named from one of its symptoms, as in the later stages of the disease, the patient goes into a condition of lethargy, from which he cannot be awakened, and which finally ends in death. 1 he disease is distinctly an African disease, and is far more common ln the negro race than in the white. So serious is it that some dis- tricts in Africa have been practically depopulated in the last two °r three decades by its ravages. The study of its cause has been largely in the hands of the English, because it has been in the British possessions in Africa that the disease has been most common. Its inciting cause has been proved to be a protozoon called a Try- panosome (fig. 51). This type of protozoa has an elongated body, with a flagellum at one end, a red eye spot, and a special membrane- like sail running from one end of the body to the other. Finding entrance into the human body, they develop slowly, and produce a disease which is extremely lasting, two or three years being commonly necessary to bring it to its final culmination in death. The organ- lsms are found in abundance in the spinal fluid, especially in the later stages of the disease. The method by which the human being becomes inoculated has been determined to be through the bite of a Ay, Glossina palpita, commonly known as the tsetse fly. Apparently ths parasite lives also on some of the larger animals of Africa, and the fly becomes inoculated by sucking the blood of some of the native animals; afterwards its bite may infect a human being. No very successful methods of fighting this disease have yet been devised, 342 BACTERIOLOGY but manifestly the solution lies along the line of controlling the insect carrier. Measures in this direction already adopted are more or less successful in places where they can be satisfactorily carried out. The group of protozoa-trypanosomes-to which the organism of sleeping sickness belongs contains quite a number of varieties that are parasitic on various animals. The first one of these diseases to be known was long ago recognized to be spread by an insect and was called the tsetse fly disease. Early African explorers found that the natives believed the bite of the tsetse fly (a different species from the one just mentioned) to be fatal to certain domestic animals. Fig. 50. Glossina palpita, the Tsetse Fly Spreading Sleeping Sickness, Before and After a Meal This belief of the natives proved to be essentially correct. African explorers found it impossible to take their cattle or their horses through certain districts, for a single bite of one of these tsetse flies was practically sure to produce sickness and death. The disease produced was known as nagana. For a long time its cause was not understood; but was later found to be a parasitic trypanosome, one that is distributed by this species of tsetse fly. The fly apparently becomes infected from biting some of the wild herbivorous animals that are themselves infected with the disease, and then transmitting it to domestic animals. The disease is not so common in these regions now as in former years, and this is attributed to the gradual disappearance of the larger wild herbivorous animals. DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 343 A recent German discovery seems to promise the control of these diseases. This discovery is not a serum or a vaccine, but a synthetic drug similar to the one used in treatment of syphilis (see p. 312). This drug is still of secret composition and is known only under the trade name ''Bayer 205." Very encouraging results are reported in the case of sleeping sickness; while in the case of nagana it promises to be useful although it is not a complete cure. Fig. 51. The Trypanosome Causing Sleeping Sickness Rabies This is another one of the diseases which modern bacteriology has largely mastered, even though it has not definitely discovered its cause. While primarily a disease of dogs, it also attacks cats, rabbits, cattle, swine, and indeed quite a number of other animals may be artificially inoculated with it. It is also one of the most dreaded diseases of mankind, and is generally contracted through the bite of an infected dog. It has different symptoms in different animals, the one from which it is named hydrophobia (i.e., dread of water) being noticeable as a rule.in no animal but man. Certain bodies have been found in the brain cells of rabid animals (see fig. 52), which are called Negri bodies, after their discoverer, Negri. There is much controversy as to what they are. Some have claimed them to be protozoa, the actual cause of rabies. Others have held that they are not living organisms at all, but are results of the disease rather than its cause. Whatever their nature may be, they are found only in animals that are suffering from this disease, and are therefore diagnostic of rabies. It is possible to make a very rapid diagnosis of rabies in a dead animal by examining microscopic preparations, properly stained, made from 344 BACTERIOLOGY the animal's brain. Their presence is a sure indication of rabies, and their absence an almost sure indication of the absence of rabies, although there is a small percentage of error. Before the Negri bodies were discovered, the only way to tell whether a dog that had bitten a person was rabid or not was to inoculate animals from its brain, a test requiring so much time as to be of no value to the person bitten. Rapid diagnosis is really very important, because the method of preventing the disease is expensive and troublesome, so that no one desires to submit to the treatment unless he knows the dog which has bitten him was actually suffering from rabies. At the present time many laboratories all over the civilized world are prepared to make this diagnosis upon receipt of the head of any dog that has bitten a human being. Fig. 52. a, A Normal Brain Cell; b, A Brain Cell with Negri Bodies as in Rabies The treatment for the prevention of rabies which was developed by Pasteur (see p. 59) has now come to be very widely used through- out the civilized world, and it is one of the most efficient of modern methods of conquering diseases. Pasteur discovered that the spinal cord of an animal that had died of rabies was intensely infectious at first, but gradually lost its power of producing the disease as it dried. To attenuate this virus, therefore, rabbits are given rabies, and after their death their spinal cords are removed and placed in chambers where they dry slowly. Laboratories where the material is made keep on hand at all times spinal cords in different stages of the drying process. When a person has been bitten and desires treatment, an emulsion is first made of one of the oldest spinal cords and inoculated into the patient; then after a few days an inoculation is made from a somewhat fresher cord and at proper intervals stronger DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 345 and stronger material, the whole treatment being so gauged that it is complete in about two weeks. If this treatment is begun im- mediately after the person has been bitten, his protection from the disease is practically sure. Typhus fever Typhus has been preeminently a disease of filth. It has been common in prisons, in barracks and especially in army trenches where cleanliness is difficult or impossible. Its actual cause is not yet discovered though a bacterium has been seen that has been claimed without very good evidence as its causal agent. The last few years have, however, disclosed its method of distribution and have thus shown the line of attack against it. It seems to be distributed by the bites of the body louse, and apparently in no other way. This discovery has been followed by efficient means of fighting the disease, so that while in earlier wars it was a menace to soldiers, in the latter years of the last war it was held in check. The fact that the louse is the distributing agent explains perfectly its close association with prisons, trenches, and filth in general. No means of protective in- oculation has yet been perfected. WeiVs Disease (infectious jaundice') This is principally a war disease, as it has almost never been noticed except in the camp life during warfare. The trenches of modern warfare have proved particularly favorable to its spread, so it has been brought to the attention of the medical profession partic- ularly during the recent years of war. The disease is recognized by a high fever, accompanied by intense muscular pains followed by jaundice. It is believed to be caused by a Spirochaeta, which has also been found in the kidneys of wild rats. The rats seem to be able to harbor the organism for a long time without suffering any special harm. Such being the case, the soil of the trenches would naturally become contaminated with the infectious excreta of the rats, and it is easy to see why the disease is common in the trenches. It has been recognized on almost every battle front, especially in the south of Europe and in Japan. 346 BACTERIOLOGY Trench fever Trench fever is an unusually interesting disease, because it was unknown until after the outbreak of war in 1914. The name given it is somewhat inappropriate, because it attacks many persons who have not been in the trenches. It spreads rapidly throughout the camps in epidemic form. The disease is characterized by a fever, sometimes of a relapsing nature, accompanied by severe and per- sistent shin pains that may last for months. It is not a fatal disease, but it brings about much suffering and keeps men on the sick list for a considerable length of time. There is no question but that trench fever is a distinct disease different from any previously known. It is also recognized to be an infectious disease, for it has been transmitted by injecting a patient's blood into healthy men who have volunteered for the experiment; but its cause is unknown and so is its natural method of spread. Lice are under suspicion, but they are such common creatures under the conditions of trench life that it is difficult to get the necessary evidence to incriminate them. Of course, the question arises as to whether it is really a new disease or was simply unknown before the war. No disease has ever been known to spring suddenly into existence, so the probabilities are that it was present before the war in some remote quarter, not well explored by modern physicians and that the war with its gathering of men from all quarters of the globe into rather unsanitary conditions has given the disease a chance to spread. Another equally interest- ing question is whether the disease will disappear completely now that hostilities are over. There is at least no evidence that the returning armies have carried it home with them and spread it widely over the world. SPREAD PRIMARILY FROM MAN TO MAN WITHOUT INTERMEDIATE HOSTS The few following diseases, as well as those we have just considered attack lower animals as well as human beings; but they differ from the preceding in passing ordinarily from one man to another. They do not use animals as intermediate hosts. The importance of this is evident at once, for it makes it impossible to prevent their spread by eradicating some lower animal. The line of attack in the case of these diseases has to be from a very different direction. DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 347 Pyogenic diseases One of the first and most striking results of the germ theory of disease was in learning to control various forms of infection that follow surgical operations and other wounds. Surgeons had long recognized that there are two dangers to be feared in operations. The first is the shock that comes from the operation itself. But the second danger was a more serious one in former years; for if the per- son recovered from the shock he was likely to develop a few days later the so-called surgical fever, the wound becoming inflamed and filling with pus (suppuration). No one could predict to what extent inflammation and suppuration would continue, and frequently this condition extended until it produced death. During the civil war in the United States it was found that patients who were operated upon in hospitals were more likely to suffer from this inflammation than those thus treated on the battle field. It was not then under- stood; but today it is realized that the unsanitary hospitals of those days became contaminated with virulent pus-forming (pyogenic) bacteria. It was Lister who first recognized the striking similarity between these inflammatory troubles and putrefaction. The similarity led him to enquire whether it were not possible to find methods of pre- venting the entrance of microorganisms into the wound; for he was convinced that inflammation and putrefaction were due to similar causes. Out of this conception arose antiseptic surgery, in which the world owes a great debt to Lister. Antiseptic surgery later developed into aseptic surgery, which has been adopted by all modern surgeons. In the antiseptic surgery of Lister, the operation was performed under a spray of carbolic acid, upon the assumption that the organisms causing the trouble came primarily from the air. Further study and experiment showed that the danger was not so much the organisms in the atmosphere as those that were attached to the skin of the patient, to his clothing, to the hand of the surgeon, or to the imple- ments used in making the operation. Consequently the surgeons learned to adopt aseptic methods, that is to sterilize thoroughly all objects coming in contact with the wound; and when this was done they found that no further attention needed to be paid to .the use of a disinfectant sprayed in the air. The triumphs of modern surgery are too well known to need any description here, but it may be per- haps properly stated that they are based wholly upon the plan of keeping pyogenic organisms from infecting surgical wounds. 348 BACTERIOLOGY During all of Lister's work on the subject he failed to find the bacteria that caused these troubles. They are now w'ell known, however, and have been carefully studied. One of the difficulties preventing their discovery at first was the fact that they are very widely distributed in nature, so widely distributed, indeed, that at first it seems hardly possible for them to be the cause of specific disease in human beings. Furthermore, there appear to be three or four different types of bacteria associated with pyogenic infections, any one of which may cause quite a number of different types of disease. Such human infections as boils, abscesses, carbuncles, erysipelas, septicemia, pyaemia, all types of blood poisoning, etc., characterized in general by the formation of pus, appear to be very closely associated with each other, and to be produced by one of these few organisms. Fig. 53. Streptococcus pyogenes The organisms concerned are all spherical, that is cocci, and there appear to be at least three somewhat different ones. There are the Staphylococci, bacteria of the micrococcus type, one of which pro- duces a white growth, and one an orange yellow growth, known respectively as the albus and aureus types. There is also a Strepto- coccus, generally known as S. pyogenes (fig. 53), which is rather more violently pathogenic than the Staphylococcus type. The forms of human infection above enumerated apparently may be produced by any one of these various types of organisms, except that the more severe diseases seem to be caused in general by the Strepto- coccus, the less severe ones by the Staphylococcus type. There is no constant association of any one type of organism with any one type of pyogenic disease. All of them have the power of multiplying in the tissues, producing inflammation, which causes the congrega- tion of leucocytes in great numbers, and eventually brings about the accumulation of pus. Besides these ordinary pus-producing infections, there is another, less common, but important enough to deserve mention, caused by a rod-shaped organism instead of by a coccus. This is the blue pus DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 349 °r green pus observed sometimes in suppurating wounds. The organisms causing it, Ps. aeruginosa (more often called Ps. pyo- cyanea'), grows readily on artificial media, producing upon them the same characteristic blue-green pigment that is observed in wounds. It is not a very common parasite, and its attack is not as serious as that of the ordinary pyogenic organisms. In the last few years, fairly successful methods of combating some of these infections have been found in the use of bacterial vaccines. Bacterial vaccines made from the Staphylococci or the Streptococci are extremely easy to produce, and are now prepared in quantities and are used very extensively by the medical profession. In many cases the results have been satisfactory. Such vaccines have been very efficient in the cure of carbuncles and are often of help in septicemia. There is some question as to whether or not they give rise to a lasting immunity. Tuberculosis A more monumental mass of data has been collected concerning, tuberculosis than any other human disease. Tuberculosis has been known for centuries, its origin being wholly lost in the dim light of antiquity. It is distributed all over the world, attacks all races of uiankind, although some races are more susceptible to it than are others. It is more common in the temperate and colder climates than in warmer regions, a fact that is attributed to a large extent to the indoor life of people in the colder climates. It has been until recently the largest cause of human disease in the temperate climates, though in recent years in localities where active campaigns have been carried on against it, it has fallen below pneumonia as a cause °f fatality. The causal organism Bacterium tuberculosis or Myco- bacterium tuberculosis is "acid-fast" like that of leprosy (see p. 307). Characteristics of the disease. One of the noticeable features about the distribution of tuberculosis is its tendency to be handed down from generation to generation in certain families. Some families have one or more consumptives in every generation, while other families are entirely free from the disease. This fact, at first thought, seems to mean that tuberculosis is inherited. In the past, indeed, such was generally assumed to be the case, and the freedom with which healthy people can mingle with consumptives without con- tracting the disease was taken to indicate that it was a constitutional rather than an infectious disease. Finally, however, as we have 350 BACTERIOLOGY already seen, Koch succeeded in isolating the tubercle organism and in demonstrating it to be the cause of the disease. This discovery has completely changed the attitude of the public toward the dis- ease. It proved that, whether hereditary or not, it must be infec- tious. Further studies have shown that the disease is not in the ordinary sense hereditary, but is always due to infection. When the children in consumptive families are found to develop the disease it is generally because they have been infected from their parents. If they develop the disease in later years, when living separately from their parents, it is probably because they have inherited a constitu- tion that renders them particularly liable to tubercular infection. In other words the tendency to tuberculosis is inherited rather than the disease itself. From the time of Koch's discovery, therefore, the fight against tuberculosis has been based upon the two assump' tions that the disease is contagious, and that a tendency to the Fig. 54. The Tubercle Organism disease may be inherited. These assumptions have resulted in a totally different method of attack from that of earlier days. The result of this new attitude toward tuberculosis is strikingly shown in the curve of mortality from human tuberculosis. While for some time the death rate from the disease has been slowly de* dining, thanks to the general improvement in sanitary conditions, there was a very sharp break in the curve of decline shortly following the discovery of Koch, and from that time on the reduction in fatali' ties has been more rapid than before, until as we have seen, it has ceased to be the leading cause of deaths in our advanced communities- This reduction in the death rate has been due partly to the adoption of methods for preventing the distribution of the tubercle bacillus, partly to the development of methods of resistance, and partly to better knowledge as to treatment of incipient cases, which can noW be quickly discovered by microscopic examination. That individuals can acquire tuberculosis from each other is abundantly proved, but there has been a greater dispute for the last diseases attacking human beings and lower animals 351 thirty years as to whether the disease may be acquired from other animals. Diseases in cattle and other domestic animals, character- ized by the development of nodules in the diseased tissues, which we n°w call tubercles, had been known before the time of Koch, and had been called by various names. But until the tubercle bacillus was discovered, there was no realization that these diseases were essentially identical with human tuberculosis. Koch, however, found that they were caused by an organism which seemed to be in all essential respects identical with that which causes the disease in mankind. This discovery naturally led to the question whether human beings may not acquire tuberculosis from cattle, either from eating their flesh or from drinking their milk; and a dispute arose which continued for at least twenty-five years. For some fifteen years or more after Koch's discovery it was generally assumed that the tubercle bacillus was identical in cattle and in men, but it was Koch himself who first made public any doubts as to this con- clusion, when he asserted in a famous address given in 1900 that the bacillus found in cattle is not identical with that found in human beings. This led to extensive study and experiments all over the world, and in the course of the next fifteen years data had accumu- lated to such an extent that the following conclusions were reached: There is a slight difference between the tubercle bacillus found in cattle and in human beings. This difference can be detected both by microscopic examination and by inoculation experiments, for it is found that the bovine bacillus is more virulent for animals than is the human bacillus. Either type of this bacillus seems to be capable °f producing tuberculosis in the human being, although the human bacillus is less capable of producing the disease in cattle. It has also been ascertained that milk from tuberculous cattle is likely to contain virulent bacilli, especially if the disease is located in the udder. If the disease is not located in the udder, the chance of the milk containing the bacillus is considerably less, though appar- ently in some cases the milk of such animals becomes infected. Market milk in most countries, obtained from miscellaneous herds, is sure to contain the tubercle bacillus in a considerable percentage of any lot of random samples taken. The proportion of samples thus infected is very variable under different circumstances (10 to 30 per cent), but it is so common that practically anyone who drinks market nulk will occasionally or perhaps frequently swallow virulent tubercle bacilli. 352 BACTERIOLOGY Such infected milk under some circumstances undoubtedly pro- duces tuberculosis among those that drink it. There is good evidence that tuberculosis among children that live largely upon cow's milk is frequently attributable to this cause. Various tests seem to show that between 20 and 30 per cent of the cases of children's tuberculosis are due to the milk supply. The adult appears to be more resistant against the disease, or to be less frequently exposed, for the evidence that tuberculosis may be given to adults by milk is very scanty, and only a few cases have been reported that seem to indicate this source. Practically, then, the danger from tuberculous milk is confine'd to childhood. For children, however, the danger is a definite positive one, and should be avoided by all possible means. Method of distribution. The ordinary method of distribution of this disease depends upon the fact that, although it may attack almost any part of the body, it nearly always invades the lung tissues, producing the condition known as consumption. The lungs have a tendency to break down with the formation of pus, and the broken- down tissue is discharged into the air passages and is eliminated through the throat and mouth. Consequently the sputum and nasal discharges of a consumptive are sure to contain active tubercle organisms in large numbers. Sputum, indeed, appears to be the primary means of distribution of tuberculosis among human beings- The organisms can live for some time after being dried, and dried sputum may be distributed with the dust and thus be a constant source of danger. The coughing patient, moreover, ejects large numbers of these organisms in the small particles of moisture that are blown from his mouth, and while they ordinarily fall directly to the ground, they may easily be breathed into the lungs of anyone who is close to the patient. Perhaps a still greater source of danger comes from the common habit of placing the fingers in the mouth and thus transferring sputum to others' hands or to objects handled. The fact that sputum is the chief means of distribution has led to the protec- tion of the public primarily by guarding against danger from the sputum of consumptives. This is done by disinfection, by burning, by giving the patient directions to collect his sputum in easily de- stroyed receptacles, to avoid coughing or sneezing without a handker- chief before his mouth, and in every way possible to prevent other people from coming in contact with the discharges from mouth or nose. diseases attacking human beings and lower animals 353 Resistance. Human beings are endowed with a large amount of resistance against the attack of the tubercle organism. While the organism is capable of growing in almost any part of the body, if it once gets a foothold, the resisting powers of the body against it are so great that it seldom succeeds in getting a foothold. This has been one of the surprising and hopeful discoveries connected with tuberculosis. In former years an attack of tuberculosis was con- sidered to be almost a verdict of death. This was to a large extent because such an attack was not recognized until it had reached a some- what advanced stage, when it was beyond hope of recovery. Modern bacteriological methods have enabled us to discover the incipient cases, and experience has shown that a large proportion of the cases are able to recover completely under proper treatment. More sur- prising still is the discovery that nearly all persons have had mild attacks of tuberculosis at some time during their life. This has been learned from the postmortem examinations of persons who have died °f various diseases, the examinations showing in a large percentage of the cases healed tubercular lesions on some organ of the body. This discovery, together with the knowledge that cases of recognizable tuberculosis are widely scattered through our communities, has led to the conclusion that probably all people are frequently exposed to tuberculosis, but that the majority of them are so resistant that they cither do not suffer from the invasion or else soon develop sufficient unmunity to prevent the further development of the parasites. This puts a very different aspect on the whole tuberculosis problem, in- dicating that in combating the disease great emphasis should be Placed upon the means of increasing resistance, and not simply uPon the methods of preventing the spread of the germs. Increased resistance is found to be brought about by fresh air, sunlight, and proper food, or in short, with conditions that lead to general personal health. Tuberculosis among cattle. Whatever may be the conclusion as to the dangers that the human race is subject to in using milk and meat from tuberculous cattle, there is no question that tuberculosis constitutes one of the great menaces to the agricultural and dairy mdustries. This disease has become extremely common among domestic cattle, and also among swine. It is very fatal, and while some animals undoubtedly recover from a mild attack of the disease, Just as men do, nevertheless if tuberculosis finds its way into a herd 354 BACTERIOLOGY of cows, it is a matter of extreme difficulty to get rid of it. I* seems, moreover, to be a growing menace, because statistics at the present time show a very much larger percentage of tuberculous cattle than a generation ago. To what extent this larger percentage is due to more careful observation and more thorough record we cannot say, but apparently the disease has increased in the last generation. Controlling tuberculosis among cattle would be a simple matte! if all tubercular animals might be slaughtered; but because of the financial loss involved, this has proved impracticable. The first practical plan was proposed by a Dane, Dr. Bang. The essentials of plan of were: keeping the tubercular and non-tubecular members herdhisa absolutely separate; and removing the calves of tubercular mothers from their mother immediately after birth. Slight modi' fications of Bang's plan have been used in other countries; and d has been well demonstrated that some such method as this is sufficient to keep bovine tuberculosis well under control. The first requisite in such methods of control is to be able to diagnose the disease in an early stage, before the affected animals have become a menace tn the others with which they are in contact. To recognize tuberculosis in an early stage in cattle by clinical means is not possible; but it haS been found possible to do this by means of tuberculin. Koch, as we have already seen, discovered that by allowing th6 tubercle bacilli to grow for a considerable length of time in a glycef' ine bouillon certain excreted products are produced which have some peculiar relations to the disease. He found that when this material freed from the active bacilli, either by filtration or sterilization, was injected in small quantities into the body of a healthy individual, produced no reaction, but when injected into one already suffering from tuberculosis, the material proved quite poisonous, even in veO small quantities, promptly giving rise to a temperature reaction' This tuberculin has proved most useful in determining the presence of tuberculosis in suspected or even in unsuspected animals. Treat' ing a herd of cattle with a definite but rather small quantity of tube!' culin will at once separate the animals that are healthy from those that are tubercular, the latter showing a rise in temperature, and the former failing to show it. The use of tuberculin for this purpose has been very extensive, inasmuch as it has proved to be the onb' reliable method within our reach of recognizing tuberculosis among DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 355 animals that are only slightly affected. The reliability of the test has naturally been brought into question and extensive studies of it have been made. It has been found to be very sensitive in detecting all except some very advanced cases of the disease-a matter of no practical importance, since advanced cases can always be recognized by a veterinarian. In fact, tuberculin is almost too sensitive for practical purposes, as the reaction is exactly as prompt and definite with incipient cases as with those that have progressed further, and animals with incipient tuberculosis often recover, remaining of value to their owner for years. It does not, therefore, furnish a means of telling which of the reacting animals are so slightly affected that they are neither a source of danger to other animals nor likely to develop severe cases themselves, and which are already so far progressed as to be a constant menace to the other members of the herd; but it is extremely accurate in separating tuberculous from non-tuberculous cattle. The only exception to the latter statement is that if a tu- berculous animal has been given one injection of tuberculin, it will not react again for several days. This fact unfortunately has been learned by unscrupulous cattle dealers, who treat cattle with tubercu- lin just before selling them so that a prospective buyer cannot dis- cover that any of them are tubercular. But even with these ob- jections, tuberculin has been of great value and enormous quantities of it are made now in all civilized countries and are used by dairymen and veterinarians everywhere in testing the herds to pick out cases of tuberculosis. In some places the sale of milk from reacting animals is forbidden. One of the great problems in connection with bovine tuberculosis is to devise effective legislation aiming to control it. The first at- tempts were to condemn without compensation all tubercular animals; but as tuberculin picks out many animals presenting no other evidence of tuberculosis, the dairymen would not stand for such legis- lation and inspectors trying to enforce the law were met with shot- guns. As a result, modern legislation is less drastic. Some states condemn tubercular cattle but agree to compensate their owners for their loss-a plan all right in theory, but one which, through lack of funds to pay for the condemned cattle promptly, often results in unwillingness on the part of the farmers to employ tuberculin and thus to disclose any possible tuberculosis in their herds. In other states a fairer but more difficult plan is attempted of encouraging 356 BACTERIOLOGY the use of tuberculin and cooperating with the farmers in building up sound herds without immediate slaughter of all reacting animals. The problem is a difficult one, and important purely from the economic standpoint, with the added importance of possible danger to health through the use of milk from tubercular animals. Tetanus or lock-jaw That dirty wounds are sometimes followed by lock-jaw, and that this disease is practically always fatal, has been known for a long time- Bacteriologists have discovered that the cause of this trouble is the so-called tetanus bacillus (Bacillus tetani'), a spore-bearing organism, which has been found in soil and some other places (see fig. 55). It is more common in some parts of the world than in others. As it produces spores it can live for a long time even under adverse con- ditions, and remains a source of danger long after the death of the animal in whose tissues it last grew. It may possibly be able to live an active life in the soil at times. At all events objects lying on or in the soil may become contaminated with it and a wound made with such an object may inoculate the tetanus organism into one's flesh- Fortunately it seems that tetanus spores alone rarely cause disease, as they are quickly engulfed by phagocytes; but if the tissues are badly macerated by the wound, if other organisms are present to lower the phagocytic activity, or possibly if other conditions are present to protect the spores until they have developed into active bacilli, infection may take place. The organisms do not invade the tissues extensively, and produce very little trouble at the site of in- fection; but growing there, they produce an intensively violent poison, tetano-toxin, which causes muscular spasms, with other symp- toms of severe poisoning and eventually death. The tetano-toxin is a soluble poison, and therefore yields to the same method of treatment as diphtheria toxin. A tetano-antitoxin has been produced somewhat similar to diphtheria antitoxin. This substance, however, does not prove of as much value as diphtheria antitoxin, at least in the way of cure. After the patient has once Fig. 55. The Tetanus Organism DISEASES ATTACKING HUMAN BEINGS AND LOWER ANIMALS 357 shown signs of tetanus, it is very difficult to produce any recovery, even by the use of large doses of tetano-antitoxin, although this has been done in some few cases by injecting the material directly into the brain. As a method of preventing the disease, however, it is very efficient. It was used in enormous quantities in the armies in Europe during the war, for a certain quantity of it inoculated into a wounded man prevents the development of tetanus. Used thus, it has proved extremely efficient, and practically no dirty wounds now result in tetanus in persons who are given a dose of tetano-antitoxin. Gaseous gangrene There is a certain organism, or perhaps a group of organisms, known by various technical names but possibly more concisely denoted by the popular term "gas bacillus," causing a gangrene characterized by frothing and bloating of the tissues, due to the production of gas. Although known for some time, this infection has come into special prominence during the war, because of its frequent occurrence in wounds. Like the tetanus organism the "gas bacillus" is a spore- forming anaerobe, and its spores seem to occur in soil, so that wounds contracted under trench conditions are likely to be infected with cither of them. Its effects cannot be warded off by means of any antitoxin yet produced; a fact which undoubtedly explains why it caused more trouble in the recent war than the tetanus organism. One method of treatment developed during the war was to introduce another organism, known as the Reading bacillus, into the infected wounds, the claim having been made that this organism destroys the one causing the infection. This method of making one organism fight another for us is extremely interesting if actually efficacious; but the treatment has not yet had sufficient trial to show whether the claims are justified. Botulism Still another human disease due to a spore-forming anaerobe differs quite distinctly from any we have yet considered in that the toxins are produced outside the body, the disease being a pure case of poisoning without any growth of microorganisms in the tissues. It has been observed that poisoning sometimes occurs after eating Partly spoiled food products, and that sometimes certain violent symptoms occur that often result in death. This violent form of food-poisoning is known as botulism and the organism that produces 358 BACTERIOLOGY the poison in the food is called Bacillus botulinus. This organism has been cultivated in the laboratory and is found to produce violent poisons upon ordinary laboratory media. These poisons, however, are destroyed by heating, a fact which shows that cooked food is safe from this danger. Considerable attention has been given to botulism recently in connection with the home canning of vegetables, that was stimulated by the food shortage during and following the war. Some of the methods recommended depend upon the fact that nearly all bacterial spores are killed by sufficiently prolonged exposure to the temperature of boiling water. Now, the spores of B. botulinus are very resistant and have been shown to resist the amount of heat generally used in the home. This fact has caused home canning of vegetables to be viewed with a little suspicion, although there seems to be little evi- dence of botulism from this particular cause. There is, indeed, no danger from this source provided all canned vegetables, particularly if there is the least suspicion of spoiling, be brought to a boil before they are even tasted. Rather more excitement over botulism has arisen recently from a series of outbreaks at widely scattered places in the United States, all definitely traced to the use of canned ripe olives. The method of curing ripe olives before canning and the temperatures frequently used in sterilizing are such as to allow a good opportunity for the growth of this organism without certainty of killing it in the canning process. The occurrence of these cases of botulism from this source has created a popular prejudice against ripe olives; but there is every reason to believe that the manufacturers are now thoroughly awake to the danger and are doing everything possible to make their prod- uct as safe as any other canned food. Besides botulism, there are other more indefinite and less thoroughly understood forms of food poisoning, popularly called ptomaine poisoning, inasmuch as some of them are thought to be due to chemical compounds known as ptomaines that are produced by bacteria growing in foods. This type of poisoning is less violent and is generally confined to digestive disorders. Cooking does not bring protection in this case, some outbreaks of food poisoning having been traced to the use of food-especially fish-that had been cooked after undergoing slight decomposition. CHAPTER VII Animal Diseases That Do Not Ordinarily Attack Human Beings There are undoubtedly many more pathogenic organisms which do not attack human beings than there are which cause disease to man- kind. Perhaps every kind of animal has its microscopic parasites, and although many of these parasites may attack man, great numbers of them do not. The largest number of purely animal diseases have never been studied, because they are of no practical importance. Animal diseases are of more than theoretical interest to us only when the species of animal attacked is utilized by mankind for practical purposes. As a result, our knowledge of animal diseases is at all complete only in the case of horses, cattle, swine, sheep, poultry, and possibly also bees. The milder and non-contagious diseases of these domesticated animals have been given scant attention; but their serious contagious diseases, involving, as they often do, the annual loss of many thousands of dollars, have been given more thorough study than some human diseases. The following discussion of these diseases is not designed to be complete, but is intended merely to give a general idea of the more important infectious diseases of animals. The first three diseases mentioned on the following pages, anthrax, symptomatic anthrax, and malignant oedema, like the last three in the preceding chapter, are caused by spore-forming bacteria. These six diseases might easily be classed together on that account, for they have several practical points in common. Bacterial spores, as we have seen, are so resistant to heat and drying that disinfection is very difficult in the case of these diseases. Anthrax and tetanus spores may remain alive for years and be capable of producing in- fection under the proper conditions. Very deep burial and cremation are the only safe methods of disposing of bodies of victims of these diseases; and because of the possibility that these organisms may become active in soil and spread through it in the moisture films, it is not absolutely certain that deep burial is perfectly safe. 360 BACTERIOLOGY DISEASES OF MAMMALS Anthrax This is one of the most serious diseases of cattle. It is very con- tagious and very fatal among animals and in Europe has resulted in enormous losses of cattle. The disease has a very rapid course, cattle dying in two to five days and sheep in twenty-four or thirty- six hours. The preliminary symptoms of the disease are not very distinctive, and it is chiefly to be recognized by its very rapid de- velopment and by the appearance of the internal organs after death. One very characteristic feature is the enlargement of the spleen, a fact which has given the disease the name of splenic fever when it Fig. 56. Bacillus anthracis, the Cause of Anthrax occurs in animals. In man it is quite rare, and is more often local, being confined to a few boils or bad sores, although it may be serious and is not infrequently fatal. The name malignant pustule is often given to it when it attacks human beings. As we have already seen, anthrax was one of the first diseases to be traced to its microscopic cause, and also the first important disease, after smallpox, to be conquered by a vaccine. The organism w'hich causes the disease, Bacillus anthracis, is a spore-forming rod, and for this reason the disease is one of the hardest to control by disinfection. The spores are eliminated with the feces, and may contaminate the grass eaten by other animals. They may contaminate the soil when bodies of anthrax victims are buried and later may be brought to the surface by earth worms. Man's chief interest in this disease-aside from the historic point of view-is economic, due to the great loss of cattle. But at times human beings become infected. In recent years there has been a good deal of suspicion attached to shaving brushes. A few fatal cases of anthrax have occurred among men who have not touched animals or their hides; and upon investigation the anthrax organisms have been found in the bristles of shaving brushes purchased by the ANIMAL DISEASES 361 victims shortly before their death. Presumably the bristles were made of the hair of some animal that had died of anthrax. Although a few such cases have been reported, the danger is undoubtedly slight. The chief source of infection to man is through the handling of hides and hair. Hence one name for human anthrax is wool- sorters' disease. The anthrax vaccine, already described, is a very efficient preven- tive against the disease. Among animals its use is often very practi- cal when the disease is widespread, although it is impractical for human use, because of the rarity of the disease in man. Symptomatic anthrax This disease, also known by the names quarter evil and blackleg, has apparently been common among cattle for a long time, although until recently it has been confused with true anthrax. Its most striking characteristic is the presence of much swelling on the upper part of the leg, but not below the knee. The portions thus affected turn black from the accumulation of blood (hence the name blackleg) and the hair is likely to fall off. The disease is not confined to the legs, however, for serious constitutional symptoms appear and it is generally fatal. It sometimes develops into a serious menace to the owner of cattle. Until recently it was rather uncommon in America, although it has been common in Europe for some time. The cause of this disease is a spore-forming anaerobe bearing some resemblance to the "gas bacillus." It is toxic for many animals but there is no evidence of its causing disease in man. It is probably spread from animal to animal by contact, but infection may occur through eating infected tissue. Like all other spore-forming bacteria, it may remain alive for a long time, although not active in the tissues long after the animal's death. The same methods of disinfection are necessary as in the case of anthrax, including the burning or deep burial of all carcasses. A vaccine has been prepared which is successful in protecting animals from the disease. It is prepared by drying the infected tissue of animals at a low temperature, grinding into a powder, mix- lng with sterile water to form a paste and heating at temperatures high enough to kill the vegetative forms of the organism. The vaccine for the first dose is heated at 100°C. (212°F.) for six hours, a treatment which so weakens the organism that it does not kill the 362 BACTERIOLOGY animals inoculated with it. The vaccine for the second dose is heated at 94°C. (201°F.) for four hours, a treatment which leaves the organ- ism considerably weakened but virulent enough to kill small animals like guinea pigs. The use of these two vaccines or modifications of them is very common in protecting cattle against blackleg. Malignant oedema This is one of the less common infections of cattle, horses, and other domestic animals, caused by a spore-forming anaerobe much like those of gaseous gangrene and symptomatic anthrax. It finds entrance through wounds. As in the case of tetanus, infection is much more likely to take place if the wound is deep, badly macerated, or if other infectious organisms are present. The disease is quite serious, and is characterized by swelling and gas-formation at the point of infection. Glanders This disease is primarily a disease of horses, but it also attacks man occasionally. While quite rare in mankind, it is one of the serious diseases, since it is nearly always fatal. It is somewhat widespread among horses, and in this animal assumes varying forms of different degrees of severity. It is frequently fatal, and in most cases is so contagious as to menace the health of a stable; and glanders victims are commonly slaughtered upon discovery of the disease, to protect the animals with which it might come in contact. The cause of glanders is a well-known organism known as Bacterium mallei, which is eliminated from the various running sores and from the nasal discharges. The organisms may apparently enter the new host either through the mucous membrane of the mouth or nose or through an abraded skin surface. No satisfactory cure has been discovered, and the only method of combating it is by the somewhat extreme method of slaughtering all infected animals. The organism is pathogenic for many animals, including asses, guinea pigs, cats, dogs, moles, field mice, and mankind. Since the fight against this disease must be based primarily upon the removal of infected animals from the proximity of others, it is very important to have some satisfactory method of diagnosing glanders in its early stages. The methods by which this is now ac- complished depend upon immunity reactions and have been briefly discussed on a previous page. In the first place there is the use of ANIMAL DISEASES 363 mallein (see p. 286), which gives a temperature reaction like tuber- culin in tuberculous animals or if injected into the eye produces a local reaction which indicates quite plainly the presence of the disease. In the second place there is a complement fixation test; and in the third place an agglutination test much like the Widal test for typhoid fever, which can be made by either the microscopic or the macro- scopic method (see p. 287). Each of these methods is in use at the present time, and each of them has certain particular advantages. Having all three methods at our disposal there is practically no diffi- culty in diagnosing glanders in any stage of its development, and in picking out quickly the animals that should be slaughtered to pro- tect the others. Contagious abortion A certain pathologic condition causing considerable trouble in some dairy herds is known as contagious or infectious abortion. It ls a peculiar disease in that the health of the cow is scarcely affected, and its effects become noticeable only during pregnancy. It seems to affect the fetus and its membranes rather than the cow herself, and generally results in premature birth of the calf. If carried to term, the calf is generally feeble and unable to survive. This disease, of course, is not the only cause of abortion among cattle; but dairy- men have recognized it for some time as distinct from accidental abortion, as it is plainly contagious and may spread through an entire herd. It is a widely distributed disease, known in nearly all agricul- tural communities, and a similar if not identical disease has been reported in sheep, horses, pigs, and goats. The parasite, Bacterium abortus, is a short, non-motile rod, that does not form spores but is quite resistant to drying. Its most peculiar characteristic is that it does not seem to thrive either in the open atmosphere or in the entire absence of oxygen. In stable cultures of serum-gelatin-agar, incubated in the ordinary atmosphere, the growth appears only in a certain zone about 5 mm. beneath the surface. In a somewhat rarefied atmosphere, the organism is able to grow on the surface of the media. The organisms escape at the time of the abortion with the fetus and the uterine contents. This material may easily infect other animals unless the attendants are scrupulously cleanly. A cow that has once aborted from this cause is likely to abort a few times more, although after a while she seems to overcome the infection and to 364 BACTERIOLOGY become immune. The disease can be controlled by isolating the affected cows, burning all materials discharged at the time of the abortion, and by allowing no diseased cow to be covered by any bull that also serves healthy cows. Hemorrhagic septicemia There is a group of diseases occurring in various animals, all quite similar and caused by organisms much alike. One of the chief charac- teristics of these diseases is the occurrence of hemorrhages in different organs throughout the body. The most important of these diseases is human plague, which we have already considered. Then there is fowl cholera, the disease upon which Pasteur made his classic studies concerning immunity. The plague is apparently a disease of rodents capable of attacking human beings as well; but the other diseases of this group are strictly animal diseases. Among them are swine plague, rabbit septicemia, and similar diseases of cattle and horses. They are caused by short, non-motile bacteria, characterized by deeper staining at the ends than in the middle of the rod. The organ- isms, if actually all distinct species, are so much alike that they plainly form a compact group and are regarded by some bacteriologists as forming a genus by themselves, which has been named Pasteurella. There is little agreement, however, as to how many species or varieties there are in this group. Actinomycosis A few diseases are believed to be due to Actinomycetes, that group of organisms, mentioned on p. 101, which seems to be intermediate between bacteria and fungi. Definite proof of causal action is generally lacking; but one of these diseases is fairly well known and the agency of a certain Actinomyces in causing it is generally recog- nized. The disease is called lumpy jaw of cattle, or bovine Actinomy- cosis. It causes hard abscesses and other swellings generally on the tongue and at other places in the head; but internal organs may be affected. The disease becomes chronic in the infected individuals and does not seem to be contagious. The exact method of infection is unknown. The infective organism is called Actinomyces bovis. Hog cholera One of the most serious diseases of swine is known as hog cholera. It is so widespread and serious, indeed, as to be of decided economic ANIMAL DISEASES 365 importance in all parts of the world where hogs are raised. It pro- duces a severe fever, is generally fatal and can be recognized after death by characteristic red spots on various organs and by button- shaped ulcers in the large intestine. It is extremely contagious and is so common that nearly all stock yards and stock cars are sources of infection. There has been much dispute as to the cause of the disease. A bacterium, B. suipestifer, was first described; and because of its common, if not universal, presence in cases of the disease, it was generally accepted as the cause. It is readily pathogenic to cabbits and guinea pigs, and will infect hogs if sufficiently large doses are used. Such animals seem to have the typical disease, even to the characteristic lesions on the various organs; but the disease thus produced is not contagious, even though the blood of the infected animals be injected into healthy hogs, and animals that recover are not immune against natural hog cholera. The blood of animals having the natural disease has long been known to be very infectious, and recent investigations show that it does not lose its virulence by being passed through very fine porcelain filters. As a result of this discovery the disease is now considered to be due to an ultramicro- scopic organism, the exact nature of which is not known because no °ne has succeeded in cultivating it. Some investigators claim that the disease may be caused by a protozoan, but to account for all the facts °n this theory they have to assume that some stage of the protozoan's life history is of ultramicroscopic size. The role actually played by suipestifer is still unknown, but it is generally considered to be a common secondary invader which may greatly influence the course of the disease. The disease has proved impractical to control by means of disin- fection and quarantine, but fortunately it proves possible to protect against it by means of a serum treatment. Immune hogs are given Jarge doses of blood from diseased hogs, and they then become "hyper- immune." Injection of serum from these hyperimmunized animals causes passive immunity which becomes an active immunity if small Quantities of the virus are injected at the same time. This serum is used to considerable extent by hog raisers when they have reason to fear an outbreak of hog cholera. Foot-and-mouth disease One very serious disease of cattle, although it sometimes attacks other domestic animals and even man, is known as foot-and-mouth 366 BACTERIOLOGY disease. The name is due to the characteristic eruption which ordi- narily appears inside the mouth and on the skin around the hoofs of the cattle, although lesions are also found on other parts of the body- It is quite contagious and spreads rapidly. It has been common in Europe for some time. In this country there have been a few out- breaks, one of them only a few years ago, which have caused tempo- rary alarm; but federal authorities have always taken vigorous steps to keep the disease within bounds, so it has never become as prevalent here as in Europe. The cause of this disease is quite generally re- garded to be an ultramicroscopic organism not quite so small as the virus of hog cholera but able to pass through moderately fine porce- lain filters. No method of artificial immunization has proved satis- factory. In fact natural immunity lasts for but a short period. DISEASES OF POULTRY Except for the larger domestic animals, no group of animals have had more attention given to their diseases than have poultry. As a result, there are several infectious diseases of chickens that have been studied and are known today to be due to definite micro- organisms. TT/ifte diarrhoea of chicks This disease, often called bacillary white diarrhoea to distinguish it from a similar disorder thought to be due to amoebae, is quite common in some portions of the country and causes considerable loss. It has been quite thoroughly studied in recent years. Chicks show signs of the disease when they are four or five weeks old, when they appear listless and emaciated, with a high mortality. The whitish discharge from the bowels is not a universal symptom, but is present often enough to justify the name of the disease. The organism known as Bacterium pullorum, is present in many of the organs of the diseased chicks. Those chicks that recover often retain the organism in their ovaries and later transmit it through their eggs to the next generation. As it may be spread thus through the adult hen and through food contaminated with infectious discharges, it is very easy for the entire flock to become infected. It is controlled by taking care to use no eggs for hatching that have been layed by infected hens. ANIMAL DISEASES 367 Chicken cholera This is a common disease in Europe, although rather rare on this side of the Atlantic. It is a diarrhoeal disease, with intestinal dis- charges of varying color, reddish-brown to greenish. It is almost always fatal. The chief interest in this disease is due to the fact that it is the first disease for which Pasteur prepared a vaccine from an attenuated culture (see p. 57). The organism causing it has been given various names, but it plainly belongs to the same group as those causing hemorrhagic septicemia of rabbits and cattle, to which the generic name Pasteurella is often assigned. Fowl diphtheria This condition does not seem to be caused by a specific micro- organism. It is characterized by a filmy secretion over the eyes or some of the breathing passages, known as a false membrane. The disease is popularly known as roup, or canker in its later stages. Various microorganisms have been described as causing this condi- tion, among the best known of which is Ps. aeruginosa (synonym, Ps. pyocyaneus), which causes blue pus in man. DISEASES OF INSECTS Like all other forms of life, insects have their parasitic diseases. Most of them we know nothing about; but a few of them are of suffi- cient practical importance to have been studied. This is especially true of the diseases of silk worms and bees, both of which insects are cultivated by man and have direct money value. Those insect diseases which have been studied are not nearly so definitely under- stood as the diseases of higher animals. It is ordinarily impossible to inoculate an insect and then to keep that individual insect under observation unless it be confined under such unnatural conditions that the experiment has little value. A further difficulty lies in the fact that many of the bacteria found in the intestines of insects, some of which are undoubtedly pathogenic, fail to grow upon laboratory media. As a result, there has been much confusion in ascribing these msect diseases to definite bacteria, and our knowledge on the subject is very imperfect. Silk worm diseases are interesting because of their relation to the history of bacteriology. We have already seen how Pasteur left his 368 BACTERIOLOGY chemical studies to investigate a serious condition among the silk worms of France, which proved to be a combination of two diseases, known as pebrine and flacherie. Pebrine is considered to be due to a protozoan and flacherie to a streptococcus. Both diseases attack the larvae and the insects thus attacked seldom spin cocoons; but their symptoms are sufficiently distinct to be evident to anyone who is familiar with silk worms. Control in both cases is obtained by the microscopic examination of worms coming from known stock, and if they prove uninfected, building up a race free from the disease by keeping insects from this healthy strain under strictly sanitary conditions. Several bee diseases are known, three of which, known as American foul brood, European foul brood, and sac brood, are especially im- portant. It is very difficult to determine their causal organisms. American foul brood is believed to be due to a spore-forming bacillus known as B. larvae. European foul brood has been ascribed in the past to one or the other of two different spore-formers called re- spectively B. alvei and B. orpheus, although they are now considered to be merely secondary invaders. A non-spore-forming organism, called B. pluton, is regarded as the actual cause of the disease, al- though it has never been isolated and studied and cannot therefore be fed to healthy bees to see if it causes the disease. Sac brood has been claimed to be due to a filterable virus. But the extreme diffi- culty in carrying out inoculation experiments in the hive has pre- vented the establishment of good proof in any of these cases except that of B. larvae. The control of these diseases is cultural. A good strong hive of vigorous bees is seldom subject to serious attack of disease; and if one of these diseases gets a foothold in any hive, the bee keeper should always strive to increase the strength of the bee colony. In the case of American foul brood, however, the disease cannot be driven out of a hive when once infested, and generally the hive has to be burned. REFERENCE R. E. Buchanan. Veterinary Bacteriology. Saunders, Philadelphia, 1911. 2. BACTERIOLOGY OF WATER AND SEWAGE CHAPTER VIII Number and Kinds of Bacteria in Water One of the greatest concerns of the ancients was to furnish their cities with an abundant supply of water. The remains of the ancient aqueducts and cisterns are among the most remarkable of the ruins of those days. Scarcely a structure of the ancient world still in exist- ence is more striking than the long rows of stately arches of the Roman aqueducts which brought water to the city from sources long distances away. Such ambitious structures make it appear as though the Romans were willing to pay a good deal to obtain pure water; and yet that may not have been the case. Pumps and pipes tight enough to be used as syphons were unknown in those days, and the only way a large supply of water could be brought to the city was to conduct it m artificial channels from some distant source higher than the city. Physical limitations of this sort forced the ancients to use water from purer sources than the near-by-rivers; and yet they probably were not wholly ignorant even in those days of the value of pure water. We find Hippocrates, indeed, (about 400 B.C.) advising that a polluted water be boiled and filtered before drinking. Nevertheless the real dangers from contaminated waters were not appreciated; and of course no one suspected such water to be teeming with living creatures too small to be seen with the naked eye. Water was one of the first substances to be examined with the mi- croscope. We have seen how Leeuwenhoek, the father of micro- scopy, looked at water, among other things, and found it teeming with 'animalculae." In a drop of water from a pool which had a scum on its surface he found such large numbers that it was impossible to count them. This immediately suggested to him that we must drink myriads of these tiny creatures, some of which we swallow while others remain in our mouths. He had already observed similar objects between the teeth, so now he concluded that these micro- organisms on the teeth must come from drinking water. He even considered the possibility of their entering the blood stream, but 370 BACTERIOLOGY concluded that it would need even more minute creatures than these to pass into the blood vessels. Later microscopists continued the study of water and described many microscopic animals and plants- protozoa and algae in abundance. But for some time the bacteria in water were overlooked or ignored. It was not shown definitely until 1892 that disease germs might be spread through water. The Hamburg cholera epidemic es- tablished this fact. From then on bacteriologists became intensely interested in the bacteria of water, and new phases of the problem are continually manifesting themselves. Bacteriologists have hunted for disease germs in water, studied the normal bacteria in water from various sources, learned to distinguish between the bacteria of pure water and those of sewage contaminated water, besides working out practical methods for the sanitary control of water. Such a wealth of facts has accumulated that they cannot all be taken up here. We must begin with a study of general considerations; but we must always remember that the interest of bacteriologists in water has centered around the sanitary side of the problem. They have studied water not because it is a good habitat for bacteria in general, but because we drink water and our drinking water may contain disease germs. SOURCE OF BACTERIA IN WATER Rain and snow. So few bacteria are present in pure rain or snow that we may consider water to be almost sterile as it falls from the heavens. Rain water has been reported to contain 20 bacteria per cubic centimeter, but ordinarily much fewer. The denser the habita- tion the more bacteria found in the rain, a fact which at once suggests that those present in rain drops are actually washed out of the air in falling. Probably rain-drops are sterile when the moisture of the atmosphere condenses to form them. Snow contains many more bacteria than rain by the time it reaches the earth, hundreds perhaps per cubic centimeter of melted snow; but this is easy to understand. A good-sized rain drop would make many snow flakes and each snow flake would present as much surface to the air in falling as the rain- drop and would pick up as many bacteria. That the source of these bacteria is the air is shown by the fact that snow falling on high moun- tains is practically sterile. Hail may contain many bacteria, for some unexplained reason; but hail contributes such a small part to the waters of the earth that as a source of bacteria it is of no importance. NUMBER AND KINDS OF BACTERIA IN WATER 371 The air. As just shown, the source of nearly all, if not all, of the bacteria in rain and snow is the atmosphere. In this way the air contributes its quota of bacteria to the surface waters of the earth; and as bacteria are constantly settling out of the air they must be showered gradually into all bodies of water. Nevertheless their numbers are small compared to the bacteria that get into water from other sources; and if water came into contact with nothing containing more bacteria than the air it would be almost sterile after standing long enough for these few organisms to settle out of it. Surface soil. The upper 6 inches of soil, as we have seen, contain very large numbers of bacteria, probably many more, indeed, then the few millions per gram shown by the plate count. Nearly all rain-water falls on the soil, and either runs over the surface or trickles through its upper layers; so it is to be expected that brooks and ponds will contain many soil bacteria. Similarly, shallow wells receive washings from the surface of the soil and contain bacteria washed out of the soil. The bacteria that get into water in this way are much more numerous than those that come from the air. Thus, shallow well water has sometimes been found to contain several thousands of bacteria per cubic centimeter, even though unpolluted, and the great- est number of them must come from the soil. We know, too, that considerable numbers of the bacteria in streams come from the soil, although streams so often receive sewage or other animal pollution which contains bacteria in large numbers that in most cases the majority of bacteria in such water do not come from the soil. Not all soil bacteria seem to thrive in water. We have seen, for instance, that soils practically always contain many spores of Actino- mycetes; but Actinomycetes are rarely found in water that has been running over the surface of the soil. When such water is plated it gives rise to many colonies of typical soil bacteria, but to few if any Actinomycetes. Whether they die in water or whether they simply are not washed out of the soil is not known; but the fact shows that only certain kinds of soil bacteria are found in surface waters. These kinds, however, are quite characteristic; and if water is found con- taining certain spore-formers, such as Bacillus mycoides, and B. cereus, their source may be almost unhesitatingly pronounced to be soil. This is sometimes a matter of practical importance when water is being analyzed that comes from a spring or deep well; for surface contamination in such water may easily mean pollution with sewage or other undesirable matter. 372 BACTERIOLOGY Underground sources. Although the original source of all water is snow or rain, some of it passes underground for so long a time and makes such long subterranean journeys that when it finally comes to the surface again in springs or deep wells, it is hard to think of it as coming from clouds in the first place. The question naturally arises whether its bacteria are removed or whether more are added to it during its long journey underground. Analyses of spring water and of water from deep wells uncontaminated by surface washings, show comparatively few bacteria, plate counts ordinarily running less than 50 per cubic centimeter. This is more than rain-water usually con- tains; but as this water has generally been in contact with the surface soil for some time before going underground, it has probably had its bacterial content much reduced by the underground passage. Some bacteria may be washed out from the deep layers of the soil, a fact suggested by observations showing different kinds of bacteria in deep waters from those occurring in surface waters; but the number thus added is so few compared with the ones filtered out that we may consider water to derive no bacteria from underground sources. Animal manures. Water not infrequently receives direct drainage from pig-sties, manure piles, and so forth. Manures teem with bacterial life, and this drainage water may add bacteria to the nearby brooks. Fresh manure contains intestinal bacteria; but these organ- isms soon die out and are replaced by ordinary decomposition bac- teria. Hence barnyard drainage does not add intestinal bacteria to water unless it comes directly from fresh droppings of the animals. If, moreover such water passes through the soil on its way to the streams, almost all of the bacteria are removed. Hence waters in general receive but a small percentage of their bacterial flora from this source. Sewage. The most dangerous source of water bacteria is sewage. Sewage contains refuse of all sorts from human habitations; but most harmful are the human excreta. Human excreta contain two differ- ent classes of bacteria: the ordinary intestinal bacteria which are commonly harmless, and the less common pathogenic bacteria. The ordinary intestinal bacteria are so numerous that they can be easily detected in sewage polluted water, and their presence in water is one of the common tests used to determine whether water is polluted in this way. The pathogenic bacteria are never numerous, and are rarely common enough in water to be detected by laboratory NUMBER AND KINDS OF BACTERIA IN WATER 373 methods; but their presence in water, even in infinitesimal numbers, is a menace to health if that water be used for drinking purposes. In- deed, it is the possible presence of these few pathogenic bacteria that makes the study of water bacteria necessary. If water were never contaminated with sewage, or if sewage never contained pathogenic bacteria, bacteriologists would have had but a theoretical interest in water bacteriology and our knowledge of the subject would still be quite meager. The enormous number of bacteria contained in sewage is well illustrated by a few figures showing the number of bacteria in river water above and below certain cities. Thus certain analyses of water from the river Isar at Munich gave plate counts of 300 per cubic centimeter above the city and about 13,500 below the city; and the drainage canal which receives Chicago's sewage was found to have a count of 1,200,000 just beyond Chicago, only 3700 per cubic centimeter 159 miles below the city, but 760,000 a little further down after receiving the sewage from Peoria. Of course, only a small percentage of these are pathogenic; but these large figures show what a problem is presented by the sewage pollution of water. There are many different sources of water on the face of the earth; and with the number of bacteria from the different sources varying to the extent we have just seen, it is easy to understand that some kinds of water may contain very few and others very many bacteria. Mountain streams and springs may sometimes be nearly sterile, but rivers that have passed large cities are sure to contain enormous numbers of the microorganisms. As we have just seen, sewage con- tributes bacteria to water in the largest numbers, and, hence, in a very rough way, the number of bacteria in water is proportional to the extent of sewage pollution. Water with a large bacterial count is generally regarded with suspicion, and knowledge of this fact has led the public to think that the bacteria in water are dangerous in them- selves. This is not true. An unpolluted stream may have thousands of bacteria per cubic centimeter after a rain and be entirely fit to drink; while water from a spring or well is suspicious if the count goes into the hundreds. The number of bacteria present is merely a guide as to the possibility of pollution and cannot be interpreted correctly unless the source of the water is known. VARIATIONS IN NUMBERS 374 BACTERIOLOGY It has frequently been noticed that when a stream has been con- taminated by the sewage from a city and its bacterial content has been greatly increased, the numbers constantly decrease as the water flows down-stream below the city. This has led to much discussion as to the self-purification of streams. There is no question as to the decrease in numbers, but there has been much dispute as to what causes it. Of course, as water flows down-stream more water from tributary streams is constantly being added to it; and as this water is generally less polluted, the number of bacteria per cubic centimeter is often lowered from this cause alone. But this is not the whole explanation. It has been found that stagnant water also purifies itself. In fact, the common opinion to the contrary notwithstanding, bacteria have been found to die more rapidly in still water than in flowing water. Investigation has shown that various factors are involved, all of which undoubtedly explain the decrease in part: the gradual settling of bacteria to the bottom; their destruction by the action of sunlight; their consumption by protozoa, many of which live almost exclusively on bacteria; and the gradual exhaustion by the bacteria of their food supply and of the oxygen dissolved in the water, thus making it impossible for many of them to live. Perhaps one kind of bacteria destroys another. At all events, it is certain that the typhoid organism dies more quickly in unsterilized water than in sterilized water. This diminution of numbers is a matter of great importance. It renders fit to drink by natural methods some waters which have once been contaminated; and the same principle has been made use of by sanitary engineers in some of their schemes for purifying water. ACTION OF BACTERIA UPON WATER Although all water, unless muddy or highly colored, has a very similar appearance, different waters vary considerably in chemical composition, and chemical changes are constantly going on in any body of water. Water which contains sewage, farmyard, or even woodland drainage, always contains much organic matter. This organic matter is a good food for bacteria and the microorganisms that live upon it change its nature. The changes that take place in it are quite similar to those we have already studied in connection with soil. It is first attacked by decomposition bacteria which con- vert it eventually into ammonia, a process which is completed in NUMBER AND KINDS OF BACTERIA IN WATER 375 water much more quickly than in soil. The ammonia is then nitrified. Nitrates, in fact, are so significant as an end product of these chemical changes that many water bacteriologists speak of the whole process as nitrification, although only the last stage is true nitrification. The nitrifying bacteria in water are probably the same kinds as those that act in soil, although they have been less studied. If so, un- doubtedly, nitrite is an intermediate stage between ammonia and nitrates, although nitrite is rarely present in water except in the merest traces; when present in appreciable quantity, indeed, nitrite indicates very extensive organic contamination, generally sewage pollution. The nitrates are subsequently denitrified much as in soil, and the nitrogen passes into the air as free nitrogen. In soil, as we have seen, this is undesirable; but in water it is just the opposite. The more rapidly the nitrogen is reduced to a gaseous state and given off into the air, the more satisfactory the water. Other changes in the organic matter also occur. Under certain conditions, for instance, there is an evolution of methane (CH4) or hydrogen. But the most important changes are the nitrogen transformations. KINDS OF BACTERIA IN WATER It is customary to classify the bacteria of water into three groups, true water bacteria, soil bacteria, and intestinal bacteria. This is a purely artificial classification, however, as shown by the fact that such organisms as the nitrifying bacteria are equally characteristic of both water and soil. Nevertheless, it is a very practical classifica- tion. If the typical water bacteria are present alone, the water is known to come from a deep source, and its purity can scarcely be questioned. If soil bacteria are present, it is known to be a surface water, or a deep water contaminated from surface drainage, and dangerous contamination is a possibility. If it contains intestinal bacteria, it may be unquestionably condemned, because it is bound at some time to become infected with disease germs. The natural water bacteria are less known than the other sorts. Because they are not dangerous, do not indicate possible danger, and have not been found to have any practical significance, they have not been given a very careful study. Some of them are cocci, some small rods. Nearly all except the cocci are motile, generally with polar flagella. Some are chromogenic, red, yellow, orange, or violet, 376 BACTERIOLOGY while others are non-chromogenic. One of the most striking groups is the fluorescent group, of which Pseudomonas fluorescens is the best known member. This particular organism is claimed by water bacteriologists to be one of the natural water bacteria; but soil bacteriologists also claim it, and it is probably equally characteristic of water and of soil. The group is quite striking because of the greenish fluorescence these organisms produce in ordinary media. Ps. fluorescens itself is a very rapid liquefier of gelatin : although others of the group do not liquefy. These fluorescent organisms of water and soil seem to be closely related to the organism causing blue pus (see p. 349). The most striking soil bacteria that get into water are the liquefying spore-forming bacteria: Bacillus mycoides, B. megatherium, B. cereus, and others. These bacteria are very interesting. They do not seem to be natural water bacteria, nor do they seem to be very active in soil; but their spores are always present in soil in fairly large and very constant numbers, and when water washes through soil these seem to be the organisms that predominate in the water. The colonies of these bacteria in either agar or gelatin are quite striking, and in the examination of deep waters they furnish a very good indication as to whether there has been any contamination with sur- face drainage. When colonies of Actinomycetes are found on plates from water, they may also be ascribed to soil; but they are never as numerous in surface water as their abundance in soil would lead one to expect-perhaps because their spores do not wet easily with water, on account of an apparent greasiness, and are not therefore easily washed of the soil. Various other soil bacteria, of the non-spore- forming type, without characteristics striking enough to identify them, undoubtedly occur in surface water; but there are true water bacteria so nearly like them that bacteriologists do not attempt to distinguish between the soil and water types in this group. The intestinal bacteria are of the greatest significance. Nearly all of them are perfectly harmless, but others are so very dangerous that water may always be condemned if any intestinal organisms are present. Of the pathogenic organisms, the bacillus of typhoid is by far the most common and the best known. The cholera organism is less common, but more dreaded when present. The organisms of various other intestinal diseases, less serious and less understood, may also be present in water. But far more numerous than these are the NUMBER AND KINDS OF BACTERIA IN WATER 377 non-pathogenic intestinal organisms which in themselves are of no special significance. The chief importance of these non-pathogenic forms is that on account of their abundance they are easier to find in water than the dangerous bacteria. One or two of them are quite easy to recognize and thus become of importance as an indication of sewage pollution. The best known of these is Bacterium coli, an organism which is fairly easy to recognize because of the kind of fermentation it produces in sugars. This organism is very abundant in the discharges from intestines and is rarely found elsewhere in any abundance. It is occasionally pathogenic; but the normal man apparently has such resistance against it that it is not one of the dangerous bacteria in water. As an index of sewage pollution, how- ever, it is of great importance. How it is thus used we shall see in the following chapter. CHAPTER IX Water in Its Relation to Disease At one time the public was willing to drink any water that tasted good and did not look dirty. Now, however, they are beginning to demand "pure" water-"good, pure, and wholesome," as it is often written in water contracts. In this case, as in so many others, the public knows about what it wants, but expresses its desires in too indefinite terms. Pure water, strictly interpreted, is never found except in a chemical laboratory, where it is extremely difficult to obtain and even more difficult to maintain pure. Such water is hardly what the public desires. Ordinary distilled water, moreover, which is the nearest approach to pure water that can be obtained in quantity, has a peculiar taste which makes it objectionable to the public. What the public really desires can be more clearly stated by the phrase "safe to drink and inoffensive to the senses." Naturally no such long expression is in common use; but it is well to remember that such are the desirable qualities of a good supply of drinking water. How completely any water fulfills these conditions depends upon its source. Rain water is as nearly pure as natural water can be, and it is free from disease germs; but to collect it by artificial means in large enough quantities for use is so difficult that it is rarely at- tempted. As ordinarily collected, in fact, rain water is anything but pure, for it is generally allowed to run over some roof and is stored in unprotected cisterns. Spring water and deep well water are ordinar- ily the safest for drinking, although in collecting them for use they are exposed to some danger of contamination. Water from ponds, lakes, brooks and rivers may be safe or unsafe, depending upon the distance it has travelled from its original source (rain or ground water) and the amount of human habitation it has passed by in the meantime. Hence brooks and ponds are much more likely to be fit to drink than lakes and rivers. River water is almost always dangerous. Standing water is generally safer than running water, because the bacteria have had a chance to settle. WATER IN ITS RELATION TO DISEASE 379 This relation of drinking water to disease has given bacteriologists many problems to solve. Water as it passes over the land carries everything with it, good or bad, that is soluble or small enough to be transported. Human habitations always have large quantities of waste that must be disposed of and the most convenient way of doing so is to dump it into the nearest body of water. Human beings often have contagipus diseases; so it is inevitable that most of the water in thickly settled regions contains disease germs. Hence with the increasing denseness of the population the difficulty in obtaining safe drinking water becomes greater and greater. This has given the sanitary engineer some enormous problems which he some- times solves by disposing of sewage in such a way as to make it harmless and sometimes by carrying drinking water for great dis- tances, as for instance in the case of the Ashokan Reservoir in the Catskills which supplies New York City about 100 miles away. It is possible that some kinds of water can produce disease although unpolluted. Certain peaty waters, for instance, that are dark colored on account of the organic matter they contain, are claimed to pro- duce diarrhoea in folks who are not accustomed to their use. It has also been claimed that the water in regions where goitre is prevalent has some relation to that disease, probably because it lacks the iodides present in water near the sea-coast. The statement has even been made that such water when given to monkeys causes their thy- roid glands to enlarge. This relationship, however, cannot be regarded as definitely established. Certain diseases are especially likely to be transported by water. They are naturally those diseases that are contracted through the mouth and eliminated through the discharges that pass into the sew- age. Pre-eminent among these water-borne diseases are typhoid and cholera, both of which are caused by bacteria capable of living for some time in water. Certain other diseases of less importance are spread through water, nearly all of which are intestinal and are characterized by diarrhoea. So often are these diseases spread by water that they are often used as an index of the purity of some par- ticular water supply. Typhoid, in particular, is so wide-spread that sanitarians often judge a water supply by the typhoid death rate of the community it supplies. An annual typhoid death rate over WrATER-BORNE DISEASES 380 BACTERIOLOGY 16 per 100,000 is considered suspicious, and if it be over 20 per 100,000 (unless there be reason for suspecting flies or some of the other means of spread discussed above, p.325), the water supply can be almost unhesitatingly condemned. Cholera is not common enough and diarrhoeal complaints are too common to serve as an indication of water pollution. Nevertheless it has frequently been observed that when a city puts in filter plants or obtains its water from some new uncontaminated source, the miscellaneous diarrhoeal diseases, as well as typhoid, greatly diminish; and a great prevalence of these obscure diseases in any community casts suspicion on the water, even if no typhoid be present. Sometimes a water supply may become so badly contaminated with either cholera or typhoid that an epidemic of the disease breaks out. In this country, at least, cholera epidemics are rare, but typhoid epidemics are not uncommon even yet, in spite of the great progress in sanitation during recent years. Sudden outbreaks of typhoid throughout any community are generally due to contaminated milk or to contaminated water; and when the cases are particularly wide- spread, new cases appearing each day for a considerable length of time, water can be unquestionably concluded to be the means of spread. One of the early epidemics in America to be traced to water in- fection was the typhoid outbreak at Plymouth, Pennsylvania, in 1885 which has already been mentioned (p. 292). As shown by the sketch map (fig. 40), the water supply of this town came from a brook, which had been dammed at four separate places to make small reser- voirs for storage. It was ordinarily a very safe source of water; but this particular winter a man came to the house shown near reservoir no. 4 who had contracted typhoid in Philadelphia. At this time the stream was frozen so deeply that the water supply of the town had to be taken temporarily from the neighboring river, the Susquehanna. Also on account of the cold weather, the patient's excreta remained undecomposed in the deep snow on the hillside where his nurse had thrown them. In March came the thaw, the town began using the water from this brook again; and at the same time the collection of typhoid excreta was washed down into the brook. The result was over a thousand cases of typhoid during the spring and summer. The outbreak was one of the most violent ever known, probably because the water was contaminated all at once with such a large WATER IN ITS RELATION TO DISEASE 381 number of typhoid organisms which had remained alive throughout the cold weather. Another very interesting typhoid epidemic which shows the method by which water-borne epidemics are spread was the Hudson-Mohawk epidemic in 1890-1891. Seven towns are clustered around the junction of the Mohawk and Hudson Rivers, as shown by the ac- companying map (fig. 57). All of these towns except Lansingburg, which uses water from the hills, took their drinking water at this Fig. 57. Map of the Region Around the Mohawk-Hudson Junction Showing the source of each city's water at the time of the typhoid epidemic. Water intakes indicated with an X. Waterford, Troy and Lansingsburgh escaped the epidemic. time directly from one of the rivers without filtration. Schenectady Cohoes, and West Troy (now Watervliet) drank Mohawk water; Troy and Waterford the Hudson water taken from above the junc- tion; while Albany used the Hudson river water after it had already received the waters of the Mohawk. The epidemic began at Schenec- tady in the summer of 1890; it began at Cohoes in October and at West Troy in November, and at Albany the very last of December. The other three towns, Waterford, Lansingburg, and Troy, which did not use water contaminated by the Schenectady sewage, escaped 382 BACTERIOLOGY entirely, except for isolated cases imported from one of the towns where the disease was epidemic. The epidemic showed clearly how one town may infect a neighboring town down stream by discharging its sewage into the river which the other uses for drinking purposes. As a result of this object lesson, all these towns now either filter their water or have sought other sources. MEANS OF DETECTING POLLUTION OF WATER It is frequently a matter of practical importance to know whether some particular water supply is likely to spread disease, and the water analyst is called upon to answer this question more often than any other. Hence many methods have been devised to detect the con- tamination of water. The methods seldom involve, the actual de- tection of the typhoid organism, for in spite of the frequency with which this contaminates water, it is rarely present in large enough numbers to be detected in the laboratory. The typhoid organism has been isolated from contaminated water for certain special pur- poses, but it is not practical to hunt for it when testing the purity of any water supply. As a matter of fact it is not the actual presence of typhoid bacteria, so much as the possibility of their presence in the future that interests us. Even if we knew that some particular tumblerful of water was free from the typhoid organism and perfectly safe to drink, we would hardly recommend continuous use of water from the same source provided we knew that this water supply was constantly receiving sewage and might at any time become contami- nated with typhoid or some other dangerous organisms. The im- portant thing is to detect sewage pollution rather than actual con- tamination with disease bacteria; for sewage pollution always means the possibility of disease. It is, moreover, much easier to detect sewage pollution than to discover the presence of disease bacteria. The methods used are partly bacteriological and partly chemical. Bacteriological methods-Quantitative. As we have just seen, the greater the pollution, the greater the number of bacteria in water. For this reason it is possible to get a rough idea as to the healthful- ness of water by determining the number of bacteria in it. Attempts have been made to assign limits beyond which the numbers of bacteria cannot rise without branding the wrater unsafe; but this has not proved possible. A count which w'ould condemn spring water wrnuld be perfectly normal for unpolluted surface water. Water with a WATER IN ITS RELATION TO DISEASE 383 count of 1000 per cubic centimeter is always suspicious whatever its source, but water may often be dangerous with fewer bacteria. One method often used to increase the significance of the counts is to incubate the plates at body temperature (37°C.). At this tempera- ture few ordinary water bacteria are able to grow, but the intestinal bacteria grow readily. Hence a high count at this temperature has much more sanitary significance than if a lower temperature of incubation be used. A good surface water, indeed, should not give a count of much over 100 per cubic centimeter at this temperature, or a ground water much over 10 per cubic centimeter. These quantitative tests, therefore, are of real value, but only when the source of the water is known. A quantitative bacteriologi- cal analysis of miscellaneous water samples from unknown sources tells almost nothing about the sanitary quality of the water. But if the source of a water sample is known, it is possible by incubating one set of plates at about room temperature another at body tempera- ture and comparing the two counts thus obtained, to get a fair idea as to the quality of the water. Water with a count higher than normal for waters of that particular class should always be regarded as suspicious. Qualitative. Much more information can be obtained by qualita- tive methods than by quantitative. Certain kinds of bacteria, for example, are never known to occur in water unless it has received washings from surface soil. Some of these organisms produce colonies that are very striking on either agar or gelatin plates; and if colonies of this kind are found on plates made from well or spring water it is safe to conclude that it has been contaminated with sur- face water. Now, deep water is generally free from pollution, while surface water in the neighborhood of habitation often contains disease bacteria. Hence ground water that contains these bacteria from surface soil can never be drunk with quite as much confidence as though there were no evidence of surface contamination. Even more valuable information can be obtained by testing for intestinal bacteria. There are a few bacteria that live normally in animal intestines which do not die out if put in water and which can be recognized by fairly simple tests. Most important of these is the form known as B. coli. This organism is characterized by vigorous gas production from sugars and is quite easy to recognize. The tests commonly employed to detect it are simple enough to give good re- 384 BACTERIOLOG £ suits even in the hands of an inexperienced bacteriologist. The media used to detect it are commonly inoculated with 1, 5, and 10 cubic centimeter samples of the water to be tested. The occasional pres- ence of B. coli in 10 cc. samples is a matter of little significance but if it is found regularly in 1 cc. samples the healthfulness of the water is decidedly questionable. Some badly contaminated waters show the presence of this organism in samples much smaller than 1 cc. There is hardly a single subject in the whole field of bacteriology that has seen more discussion than the significance of B. coli in drink- ing water. When its relation to sewage contamination was first discovered, this organism was hailed as an almost sure test for danger- ous drinking water. Soon, however, bacteriologists began to find B. coli in other places. They quickly realized that it is as abundant in animal intestines as in those of human beings; and before long it was also found on grass, grain, and at other places where intestinal contamination was very unlikely. Such discoveries for awhile caused the B. coli test to fall somewhat into disrepute. But eventually it was shown that a roughly quantitative B. coli test has great value. If water is entirely free from B. coli we may feel confident that the water is safe to drink; if present only at the rate of one individual to 10 cc. or less, the water need not be condemned unless other tests point to pollution; but if present in every 5 cc. or especially in every 1 cc., the water may be regarded with decided suspicion. Chemical methods. While bacteriologists have been devising these tests to determine the healthfulness of drinking water, chemists have been equally awake to the importance of the problem and have devised tests one after another that are sometimes more valuable than the bacteriological tests in detecting sewage pollution. There has, indeed, grown up a branch of chemistry known as sanitary chemistry, which is to a large extent concerned with the analysis of water for the purpose of determining its purity. Although the methods are not bacteriological, they have such a decided bearing on our subject that we must at least study the principles that underly them. There are two entirely different sorts of water analysis. One is the complete analysis, which takes into account all the material in solution. This sort of analysis is rarely of practical value, because the greatest part of the dissolved material is of mineral origin and en- tirely unoffensive. Such an analysis is of value if water is to be used in boilers and may contain mineral matter in solution that will precipi- WATER IN ITS RELATION TO DISEASE 385 tate in the boiler and in the pipes; but from the sanitary point of view the results have little meaning. The other sort of water analy- sis is known as the sanitary analysis, and takes into account only those determinations that are of sanitary significance. Those mate- rials that are generally of intestinal origin are recorded even though they occur in but a few parts per million, while other materials occur- ring in much greater quantity are entirely disregarded. One of the most important tests in the sanitary analysis of water is the test for chlorine. All water contains chlorine, to be sure, varying in amount according to the distance from the sea of from salt-bearing rocks; but chemists know to within a few parts per million how much chlorine there should be in the water in any particular locality, and any over this normal amount they regard as suspicious. It is suspicious because human beings alone of all animals eat so much salt that it can be detected in water polluted with their excreta. In short, an excess of chlorine generally means human pollution. Another series of tests are those for the various stages in the decom- position of nitrogenous matter, which is finally converted into ammonia, then into nitrite and subsequently into nitrate. The presence of any of these three forms of nitrogen in quantity is suspi- cious ; but the presence of nitrite in particular is suggestive of sewage pollution, probably because the nitrite does not accumulate unless there is a very large amount of organic matter present but is im- mediately converted into nitrate. Still another significant test is to determine the amount of oxygen dissolved in the water. The growth of bacteria in water always uses up oxygen. When sewage is de- composing in water, the oxygen is used up quite rapidly until there is practically none left in solution. This exhaustion of the oxygen may go on to such an extent that fishes cannot live in it. Now it is very easy, by simple chemical tests, to learn how much oxygen is in solution; and in this way very important indications of sewage pollu- tion may be obtained. Sometimes a single one of these chemical tests will be sufficient to show whether water is safe to drink; but more often they must all be made and the results carefully compared with what is known about the source of the water. If to the chemical analysis are added the results of bacterial tests, the sanitary quality of any water can be very accurately determined. CHAPTER X Purification of Drinking Water If every community could be supplied with water from an uncon- taminated source like mountain springs or deep wells, water-borne diseases would be practically eliminated. As this ideal is out of the question in any thickly settled region, it becomes a matter of great importance to treat water so as to destroy the dangerous bacteria within it. If water is so treated, even grossly contaminated rivers and lakes may supply city reservoirs without endangering the health of those who drink them. In this way a large number of our cities get safe water to drink, although so situated that no uncon- taminated water is available. The most effective method of killing the bacteria in contaminated water is to boil it. None of the water-borne disease germs can resist boiling for any great length of time, five minutes being usually enough to kill them. This method of purifying water can be used to great advantage in the household when there is reason to suspect the water supply. Boiling the water does not harm it in any way as a drink, and renders it safe as water can be made. This method is of little use, however, in protecting a community. The method is impractical to employ on a large scale, and to boil and cool w'ater in the home without giving it a disagreeable taste is so much work that the average housekeeper cannot be expected to make use of it. Even more effective is distillation, a method which is employed under some conditions; but besides being expensive to produce, distilled water has a peculiar taste which is often disagreeable to one unaccustomed to it. Another method of purifying water, theoretically satisfactory, is to pass the water through porcelain filters. Unglazed porcelain has such fine pores that it can filter out all bacteria. Filters of this sort have been made to fit onto faucets, and at one time they wTere much advertised for purifying water in the home; but they are hardly to be recommended. After a few days' use, the bacteria grow through the pores of the filter and can then pass into the water PURIFICATION OF DRINKING WATER 387 in larger numbers than were present before filtering. As a result, these filters are worse than useless and give their users a false sense of security. They can be used successfully only if removed every few days and baked, an operation which the housekeeper will seldom perform. Like boiling, this method of filtration cannot be entrusted to the individual housekeeper; and both methods are impractical to adopt on a large scale in purifying the water for an entire com- munity. As a result, other methods have been adopted in practice,, theoretically less ideal, but actually proving much more satisfactory than boiling, distilling, or filtering through porcelain. SEDIMENTATION One of the simplest methods of purifying water is to allow the impurities to settle out by the action of gravity. The visible tur- bidity can be almost entirely removed in this way, as shown by the greater clearness of water flowing out of a lake than of that which flows into it. Even bacteria, tiny as they are, can be largely re- moved in this way. An interesting example of this was shown by the typhoid epidemic at Ithaca, New York. This city has two sources of water, one supplying the lower part of the town, the other supplying the hill where the university is located. At the time of the typhoid outbreak there were practically no cases among the people using the latter supply although there were a few cases on the shores of the creek which contributes to this supply and some folks drinking directly from the creek above the reservoir contracted the disease. The reservoir is a small pond artificially enlarged, and near the entrance is a submerged mound which breaks up the cur- rents and allows the water in the reservoir to be fairly quiet. Un- doubtedly it was the sedimentation taking place that protected the consumers of the water from this source. Any lake or pond through which the water flows quietly acts as a natural sedimentation basin in this same way and partially protects the consumers of the water. Artificial sedimentation basins are often built, which consist of large areas into which water can flow, currents being stopped by baffles near the entrance. Sometimes this method assures sufficient puri- fication, but more often it has to be combined with some of the other methods in order to secure water which is safe to drink. 388 BACTERIOLOGY FILTRATION Filtration of water, in the sense the term is ordinarily used, refers to an entirely different process from the filtration through porcelain which actually strains out the bacteria. The filtration of water supplies, as ordinarily practiced, is to allow water to trickle through large beds of sand and gravel with drains at the bottom to collect the purified water. The spaces between the sand grains in these beds are so large that they can act as true filters only for the rather coarse material suspended in the water; but as a matter of fact, passage through these beds removes nearly all the bacteria. Bac- teria are much smaller than the spaces between the sand grains, and the question naturally arises, what removes the bacteria if they are not filtered out. In this connection it is important to notice that during the action of a filter bed a gelatinous scum forms on its surface, composed of bacteria and the products of their growth. A filter bed is not efficient until this scum has formed, the bacteria passing through a newly laid bed in large numbers. Evidently the scum plays an important part in removing the bacteria. Its action has never been fully explained, but certain facts are known. It is known, for instance, that as the water trickles through this mass of living bacteria, its organic matter and its oxygen are rapidly con- sumed so that when it passes out of the filter bed it cannot support bacterial life. Undoubtedly many bacteria die on this account, as they pass through the filter. Possibly also the bacteria living in the scum have a repressing action on the bacteria already in the water. Whatever the true explanation, the phenomenon is undoubtedly biological rather than mechanical. Such filters as this are called slow sand filters in distinction from the type to be described in the following paragraph. The water flows through the sand so slowly that large beds are needed to supply enough water for a fair sized community. In practice, moreover, at least two filter beds are necessary, large enough so that the entire supply can be handled when one bed is out of commission; for the beds have to be cleaned occasionally and for some time after cleaning their efficiency is lowered, so there are times when the water must be filtered with one bed out of use. The loss in efficiency after clean- ing, in fact, is so great that it is no exaggeration to say that sand filters work best when dirtiest! Such being the case, it seems as though cleaning were unnecessary-worse than useless, indeed. PURIFICATION OF DRINKING WATER 389 Unfortunately, however, after a certain length of time, a filter bed becomes so clogged with dirt and the products of bacterial growth as to be almost impervious to water. Naturally such a filter has to be cleaned, however much it may lose in efficiency. Filter beds are often used in connection with a sedimentation basin into which the water flows and remains quiet for some time. A very large number of the bacteria as well as much dirt settle out in this basin and the filter does not clog so quickly. Less efficiency in the filter is required when sedimentation is first allowed to take place. If lake water is used, this sedimentation takes place naturally and no artificial basin is needed. Rapid filters depend upon a somewhat different principle. The slow type of filter was developed in Europe; but when they were first employed in America they were found to be less satisfactory because of the large quantities of suspended matter carried by many waters in this country. In an attempt to get rid of this material engineers devised the scheme of precipitating it by means of some chemical flocculating agent like alum. Then it was found that the water could be readily passed through a sand filter and much more rapidly. Not only that, but the bacteria were found to be caught in the flocks of precipitating material so that they were filtered out also, even though the water was passed rapidly through the filters. The advantage of this sort of filtration is that small filter beds are sufficient and less land has to be given up to them, while they are, if anything, more efficient than the large beds of the slow type. As the water passes through these filters so rapidly and is laden with the precipitated masses of alum (or whatever flocculating agent is used), the filters clog very quickly. Daily cleaning is ordinarily needed, and special mechanical devices for accomplishing it must be installed, such as arrangements for stirring up the surface layers while passing a stream of water over the beds, or for inverting the direction of the water flow so as to clean out the sand by the upward passage of the water. CHEMICAL TREATMENT A still more modern method of purifying water is to treat it with some chemical which kills the bacteria but is not poisonous to human beings. The first successful method of treating water in this way was not to kill bacteria, but algae. In some reservoirs 390 BACTERIOLOGY certain algae develop which decompose and give the water very disagreeable tastes. At first the only way known to prevent their growth was to cover the reservoir to exclude the light-a rather expensive undertaking. Then it was discovered that copper sulfate is very poisonous to algae while not very harmful to man. So the practice was adopted of dissolving it in the water of an algae-in- fested reservoir. The method proved very successful; and although some folks are afraid to drink water thus treated, it has been shown to contain too little copper to cause the slightest ill effects to man. Later this same principle has been applied to bacteria in water supplies. The first method was to treat the water with ozone. Ozone is a very efficient germicide and as it breaks up into ordinary oxygen it is absolutely harmless to man. The only difficulty in its use is that it is a gas and is difficult to bring into intimate contact with the water. Methods were devised of spraying water into tall cylinders filled with ozone, and considerable destruction of the bacteria was accomplished. But the practical difficulties in the method and its expense have prevented it from coming into general use. Meanwhile another method of chemical treatment was devised which closely resembles the copper sulfate treatment for algae. This consists of adding small quantities of chlorinated lime to water. This compound contains what s known by chemists as "nascent oxygen," that is, loosely bound oxygen atoms which are very easily freed from the other atoms (calcium and chlorine). The great affinity of such oxygen atoms for other combinations causes them to be great purifying agents. It is to the nascent oxygen that the germicidal power of this compound is due. It is harmless to man, and indeed quickly decomposes in water into inactive compounds. Its use in water supplies has been found practical when it is desired to purify a contaminated water without filtration. For purifying water in swimming tanks it has been found even more practical. Still more recently the use of liquid chlorine has been proposed for this same purpose, and it has proved rather more satisfactory. To generate the chlorine and apply it to the water requires certain special apparatus, which because of patent rights is at present quite expensive; but the treatment itself is not expensive, and proves much easier to control than the dosing with chloride of lime. Chlorination is coming to be used more and more. Some communities adopt it PURIFICATION OF DRINKING WATER 391 instead of filtration. Some cities that ordinarily filter their water keep a chlorination outfit in readiness to use when the filters are temporarily out of use. Ocean steamships are coming to adopt this method of treating water, for the apparatus is compact, and a ship with this outfit aboard can pick up its drinking water at any port without question as to whether it comes from a pure source or not. REFERENCES W. P. Mason. Water Supply. 4th edition. Wiley & Sons, New York, 1916. S. C. Prescott. Elements of Water Bacteriology. 4th edition. Wiley & Sons, New York, 1924. Allen Hazen. The Filtration of Public Water Supplies. 3rd edition. Wiley & Sons, New York, 1910. CHAPTER XI The Bacteriology of Sewage The sewage discharged by any community contains in its final form nearly everything that goes into the city. It contains not only the excreta from human beings but the wastes from factories, rain water, and various other miscellaneous matters. It naturally varies in its composition in different communities; but the one com- mon constituent, and indeed the most dangerous, is the material eliminated from human bodies. It is dangerous because of the great probability that it contain organisms pathogenic to man. The wastes from isolated habitations can be dumped into streams or buried in the soil without causing nuisances; those from small com- munities may easily be carried away in rivers; but the disposal of sewage from large cities is one of the hardest problems engineers have to solve. Left to itself sewage undergoes certain changes of fer- mentative nature. At first it putrefies and becomes more offensive than the original material. Later the organic matter is oxidized and the material gradually loses its offensive nature, being converted eventually to large extent into simple compounds like ammonia, nitrate, carbon dioxide, and water. When sewage is poured into a body of water, these same changes go on, so that unless new sewage is added, it gradually disappears. As we shall see later, modern methods of sewage disposal make use of the natural changes, by causing them to take place more rapidly. KINDS OF BACTERIA Very little is known as to the kinds of bacteria in sewage. This is partly because many of them do not grow on ordinary bac- teriological media and for that reason are difficult to study, but it is also because the interest of bacteriologists has been in the activities of the sewage organisms rather than in determining their species. About the only exception to this statement is B. coli, which is of interest not so much in sewage, where its presence is universal, as in water where its presence generally indicates sewage contamination. BACTERIOLOGY OF SEWAGE 393 Except for this organism, very few sewage bacteria are known by name. The others are classified as to whether their activities are aerobic or anaerobic, oxidative or reducing. It is a matter of much practical importance that some of the bacteria produce anaerobic, putrefactive changes, which cause a liquefaction of the solids in the sewage, while others cause oxidative changes which result in the production of gaseous products. There is little definite knowledge, however, as to what kinds of bacteria in the sewage actually produce which of these changes. SEWAGE PURIFICATION The difficulty of sewage disposal has been increasing with the growth of large cities. If the amount of sewage dumped into a stream is very large, it is not a sufficient safeguard to take care not to drink the water of that stream unfiltered. Filtration does not remove all the bacteria, and if the contamination is great enough some of the dangerous forms are likely to pass through the filters. Besides this actual danger, the public often objects to the nuisance caused by the large quantities of sewage a city may pour into the stream. In fact the offensive odors were objected to before the danger was realized; and the first attempts to purify sewage were designed to meet certain laws passed in England as to the prevention of nuisances. Screening and sedimentation. The simplest means of purifying sewage is purely mechanical. One mechanical means of accomplish- ing partial purification is by screening. The screens remove the larger solid particles and the liquid that passes through is less offen- sive than before screening. The screens very quickly clog, however, and unless some means of cleaning them is devised, the sewage is soon unable to pass through them. The most satisfactory sewage screens, therefore, are provided with mechanical devices for cleaning. Some- times there are scrapers on endless chains that pass across the screen; in other outfits the screens themselves are in the form of endless belts or revolving wheels and, after going through the sewage stream, pass over scrapers that remove the sludge. A rather more effective, although slower, means of removing the sludge from sewage is by sedimentation. The sewage is allowed to stand for a certain length of time in one of a series of tanks. By a system of valves, each tank is allowed to fill and then remain undis- 394 BACTERIOLOGY turbed while the remaining tanks are filling. Just before the last tank is full, the supernatant liquid in the first tank is withdrawn, so that there is room to fill it with a new dose of sewage. Mechanical purification of this sort is not complete. In some cases it is sufficient to prevent nuisance if the sewage is then poured into a body of water; but in other cases further purification by other methods are necessary. Chemical precipitation. After some experience with mechanical purification of sewage, it was found that much more material could be removed from it by chemical means. The addition of chemical flocculating agents like alum or iron hydroxide causes a heavy pre- cipitation, bringing down much of the finest material in suspension. The liquid after this treatment is very clear. When this method was first devised it was supposed that the sludge would be valuable fertilizer and by selling it the cost of the purification could be counter- balanced. Unfortunately, however, this sludge is so low in nitrog- enous matter that it is of little use as a fertilizer and generally has to be disposed of in other ways. This disposal of sludge is a great problem in connection with all methods of sewage purification; but the problem is greatest when chemical precipitation is used because the volume of the sludge obtained is the greatest under this method of treatment. The sludge is too wet to burn readily and is so pu- trescible that unless burned, dumped into water, or carried to places distant from habitation, it becomes a serious nuisance. Septic tanks. An improved method of purifying sewage was obtained when it was observed that sewage kept in a large tank from which air is excluded gradually decomposes, much of the solid matter liquefying and considerable material passing off in the form of gas. It was found that if sewage is allowed to run into a large covered tank and to pass through so slowly that no currents are formed and considerable time is required for the passage, it flows out of the tank much less offensive than untreated sewage. The purification in such a ''septic tank" (as it is called) is not complete, however, and the effluent is not entirely free from bad odors. After treatment in a septic tank sewage can be poured into a stream and cause much less nuisance than if it were untreated or treated by mechanical means alone; but such streams can hardly be regarded as safe to drink, and there is a limit to the amount of such treated sewage that can be added to water without causing offensive odors. BACTERIOLOGY OF SEWAGE 395 A recent modification of the septic tank, known as the Imhoff tank is a decided improvement. Figure 58 shows a cross section of a tank built on the Imhoff principle. Such a tank has two chambers, one within the other, the sewage entering the inner chamber and remaining almost entirely within this inner chamber as it passes from one end of the tank to the other. The only connection between the two chambers is at the bottom, which is composed of two converging sides that do not quite meet, thus leaving a narrow opening the Fig. 58. Cross Section of an Imhoff Tank entire length of the tank. Through this opening the sludge settles, collecting at the bottom of the outer chamber, from which it can be withdrawn by a pipe line. As the sludge decomposes in an ordinary septic tank, bubbles of gas arise which keep the liquid agitated and interfere with the digestion of the sewage. In the Imhoff tank all the bubbles from the sludge pass up through the outer chamber and do not disturb the sewage. This arrangement allows better de- composition of both sludge and liquid portions of the sewage than takes place in the old type of tank. 396 BACTERIOLOGY In any such tank the process is largely anaerobic and the decom- position of the sewage is not complete. The odors from the tanks are likely to be quite disagreeable. Large quantities of sludge are pro- duced in the tank which must be removed periodically. The sludge is valueless and is very difficult to dispose of without creating a nuisance. For all of which reasons, the anaerobic tank, except as a preliminary treatment, is not a very satisfactory way of handling sewage. To purify the sewage completely some method of inducing oxidation is necessary either alone or in addition to the anaerobic tank. Broad irrigation. One of the early methods of sewage disposal in England was to pour it over large areas of land. It was found that if the soil was sufficiently sandy it could dispose of large quantities of sewage without causing offensive odors. Moreover crops might be grown on the land and derive considerable nourishment from the sewage. This method of sewage disposal, however, proved imprac- tical on heavy soils; and even the sandy soils are likely in time to become "sewage sick," as the English expressively call it, draining poorly and having no further agricultural value. Sand filtration. Later, the principles involved in broad irrigation were put into use in a more scientific way. Large beds of sand were used, composed of sand of carefully determined size and with a sys- tem of tile drains underneath so that the water from the sewage would run off easily. Each bed was given intermittently a dose of a definite amount of sewage and then allowed a certain length of time for the sewage to soak in and drain off. It was found that the effluent of a filter bed of this type may be very pure. This was the first type of sewage disposal plant to prove successful in America. It was very thoroughly studied at Lawrence, Massachusetts, and the action of the filter beds was found to be bacteriological. Oxidiz- ing bacteria of various sorts act on the sewage as it passes over the sand grains, converting the nitrogen eventually almost wholly into nitrate. These intermittent sand filters are so efficient that they are used today in many places where a supply of sand is easily obtained. They require a large amount of land, however, and if sand has to be obtained from any distance they are very expensive. Their efficiency can be increased, and consequently the amount of land necessary can be decreased by using a settling tank to pass the sewage through BACTERIOLOGY OF SEWAGE 397 before running onto the filter bed; but even at its best, this type of sewage disposal is impractical for large cities, simply because of the large area needed. Contact beds. In the attempt to improve the sand filters, a new type of sewage purification plant was devised which is known as the contact bed. This is designed to be used in localities where there is not a large supply of sand available. Such a bed is composed of fairly coarse material like coal, slag, or pieces of broken pottery. The sewage is poured on and allowed to stand a while, then drawn off and a new dose of sewage run in. The action in this case is largely if not wholly bacteriological, but the action of the bacteria is slightly different from that in the sand filter. In sand there is always suffi- cient air to allow aerobic growth; but in the contact bed there is very little air when full of sewage. As a result, aerobic and anaerobic action follow each other in succession, an arrangement which may have some advantages, although it tends to prevent either process from becoming well established. Nevertheless the contact bed is generally considered more economical than the sand filter unless sand occurs naturally at the location of the plant; and as contact beds do not need to be as large, they lend themselves to a more compact installation. Trickling beds. Still another type of filter bed has been developed, which is generally considered to be quite an improvement over the contact beds. Coarse material is used in the trickling beds, but instead of being filled completely full and then being allowed to empty, the sewage is sprinkled over them and is thus constantly run- ning through the open spaces in the bed. The sprinkling is some- times effected by a revolving or travelling tube or trough, and some- times by a stationary nozzle. Each method of sprinkling has its advantages, the object being to obtain an even distribution of the sewage over the entire bed by the use of some device that does not clog and is simple enough in operation not to need constant attention. A bed of this kind furnishes aerobic conditions around the sewage at all times, and anaerobic action does not occur. This method proves under ordinary conditions to be the most economic and ef- ficient method of purifying sewage. Trickling beds, however, give off bad odors and furnish a breeding place for flies, as a result of which they cannot be located near habitation. 398 BACTERIOLOGY Most approved methods. None of these methods of sewage purifi- cation is perfect. Septic tanks, or even the improved Imhoff form, are unsatisfactory as a final treatment, because the purification is not complete. The sand filters are objectionable because of the large amount of land and the cost of getting sand if it has to be brought from a distance. Contact beds are not dependable, and frequent cleaning is necessary. Sprinkling filters cause objectionable odors and breed insects, while their effluent contains considerable suspended matter. It has generally proved necessary to combine some sort of tank with some sort of oxidizing bed, and the combina- tion generally recommended today is the Imhoff tank with the sprinkling filter. The effluent after this combined treatment is clear and not subject to further putrefaction, and can therefore be poured into a stream without causing a nuisance. But even this treatment is not wholly satisfactory. One of the greatest objections to all these forms of sewage treat- ment is the production of sludge. There is much solid matter discharged from our sewers that is not liquefied in an Imhoff tank, and this material collects quite rapidly in tanks and filter beds. Eventually it has to be disposed of, and its disposal is no simple matter. It is very putrescible and cannot be left long near human habitations. If there is a large body of water handy, its disposal is simply a matter of dumping it off boats; but inland cities often have to carry it many miles before an unobjectionable place for dumping it may be found. Drying and burning is sometimes attempted, but it is an expensive process. To make the matter still worse, it is realized that sludge contains much material that might be valuable as a fertilizer. There have been many methods suggested for utilizing it. When the chemical treat- ment of sewage was first devised, it was thought that the sludge could be used as a fertilizer, but the large amount of worthless material in it made this impractical. The same thing is true to a less extent of all forms of sludge. Large quantities of valuable food elements are constantly being thrown away in the sludge; but as yet no efficient method has been devised for making these elements available. So the economic loss of this fertilizing material has to be added to the cost of disposing of the sludge. Activated sludge. One of the most recent methods of sewage disposal is an effort to produce a usable sludge. Although the BACTERIOLOGY OF SEWAGE 399 method has been used in only one or two places, it has been pretty well demonstrated to be a practical method and is promising enough to deserve special mention. This newest method of treatment depends upon aeration. The raw sewage, with no preliminary treatment of any kind, is run into a tank which has a porous bottom. Through this bottom, a stream of air is kept constantly flowing, so that it passes up through the sewage in numerous small bubbles. After several days the sewage is almost completely oxidized into inoffensive material, and is thoroughly liquefied so that little sludge accumulates. The purified sewage can then be drawn off and more raw sewage introduced. This second lot purifies much more quickly, due undoubtedly to the accumulation of bacteria in the tank. A third lot is purified still more rapidly, and at last complete purification occurs in only a few hours from the time the sewage is run into the tank. Meanwhile an appreciable amount of sludge has collected, although not nearly as much as in any of the older methods of purification. If a small quantity of this sludge is transferred to a new tank constructed on the same principle as the first, purification of the sewage is rapid from the start. This sludge, containing large numbers of the oxidizing bacteria that cause the purification, is known as "activated sludge." The advantages claimed for this method are: (1) Organic material is converted almost wholly into nitrates and other inoffensive soluble materials and odorless gases; (2) the quantity of sludge produced is small; (3) the sludge is a good fertilizer. This last point has been shown by numerous experiments conducted by the advocates of the method. There seems to be no question as to its value, but the objection to using it comes from its high water content and the difficulty in drying it. At the present time the activated sludge method of treating sewage is being used in a few places on a large enough scale to put it to a practical test. Its users are already claim- ing the treatment to be economically sound, and if some satisfactory method of handling the sludge so as to dispose of it as a fertilizer can be found, the method promises to solve some of the most perplexing sewage disposal problems. REFERENCES L. P. Kinnicutt, C.-E. A. Winslow and II. W. Pratt. Sewage Disposal. 2d edition. Wiley & Sons, New York, 1919. H. N. Ogden and H. B. Cleveland. Practical Methods of Sewage Disposal. 1st edition. Wiley & Sons, New York, 1912. A. M. Buswell and others. Activated Sludge Studies. Illinois State Water Survey, Bui. 18. 1923. 3. BACTERIA IN RELATION TO PLANT DISEASES CHAPTER XII The Science of Plant Pathology HISTORY The possibility of diseases of plants being caused by fungi was realized a century ago, very much as the germ theory of animal disease was conceived before bacteria were known. Fungi were known long before bacteria, and as they were often observed grow- ing on plants, it is natural that the idea of their causing plant dis- eases should have arisen early. There were books on plant dis- eases, in these early days, indeed, but these books were hardly what we should call scientific treatises today. Like all early scientific works, they were little more than catalogs of the various theories that had been held as to the nature of plant disease, and none of their authors thought to obtain experimental evidence in support of any theory. The first real science of plant pathology started at about the same time as the science of bacteriology. In 1858, when Pasteur was doing some of his early work, Kuhn published a book on plant diseases in which for the first time scientific facts on the subject were carefully presented. At about the same time the botanist, DeBary, who had been studying fungi, became interested in their possible relation to disease among plants and soon became the recognized leader in this line of investigation. From this time on the develop- ment of the subject has been rapid, almost if not quite as rapid as the development of knowledge regarding bacteria and animal diseases. The greatest factor in stimulating this development was the discovery of what could be done in controlling plant diseases. In 1883 the now famous Bordeaux mixture (copper sulphate, lime, and water) was discovered and proved to have such a striking effect in controlling disease among plants that its use soon became very general. This discovery led to much investigation as to the best means of control- ling the diseases, and it was found, just as in the case of animal SCIENCE OF PLANT PATHOLOGY 401 disease, that each agent of infection had its own peculiarities and had to be attacked in some particular way. To learn the best method of attack, a thorough investigation of the causal agent, its life history, and methods of invasion, was necessary; so this led to the accumula- tion of much information in regard to plant pathology. The earliest known diseases of plants were all caused by fungi. The discovery of one bacterial disease of animals after another naturally raised the question whether bacteria might not also cause some plant diseases; but for some time no conclusive evidence of such diseases was obtained. One botanist, indeed, (Albert Fischer) became convinced that bacterial diseases of plants were impossible, partly on account of the acid nature of plant juices and partly be- cause bacteria were not known to produce cellulose-digesting en- zymes. About 1880, however, Burrill, in America, obtained very con- clusive evidence that pear blight (fire blight) is caused by a species of bacteria; and when Fischer still continued to deny the possibility of bacterial diseases of plants, Erwin Smith took up the cudgils and presented still more evidence to show that bacteria can be the causal agents. Erwin Smith was very particular to fulfill all of Koch's postulates, and he is generally regarded, indeed, as the one who has been most insistent that they be applied to plant pathology as well as to the diseases of animals. His evidence was so conclusive that after his dispute with Fischer no one further questioned the existence of bacterial diseases among plants. The present day work on plant diseases is of a very practical nature. In America, work on this line has been largely confined to agricultural experiment stations, and their efforts have been directed chiefly to- ward the control of the diseases. The problems are increasing all the time, because of the rapid spread of little known diseases over larger areas and into new countries; but in general the control measures are proving effective and are preventing the plant disease situation from becoming more serious. So rapid has been the development that the subject today is a large one. Not as many books, to be sure, have been written on it as in regard to animal diseases, but that is largely because of the in- complete and rather unstable nature of present-day knowledge on the subject. The mass of accumulated facts is as great and the number of known diseases even greater. The subject is too large to be treated adequately in a book on general bacteriology. In the 402 BACTERIOLOGY present pages it is treated in a rather small space, not because it is unimportant, but for the same reason that animal diseases (exclusive of human) are given little space, namely because although of great economic importance it does not touch human welfare as intimately as do such subjects as human disease, sanitation, or milk control, nor does it deal with such fundamental facts as does the study of bacteria in the soil. TERMINOLOGY One of the most striking things to the student of animal disease when he first turns his attention to plant pathology is the sort of names applied to the diseases. Occasionally one finds a name like anthracnose or necrosis, which has a latin or greek origin; but in general the diseases are called rots, blights, mildews, smuts, and so forth. The popular names, indeed, are used by scientists as well as by the laymen in speaking about plant diseases. Physicians long ago, instead of adopting the popular names of human and animal disease, gave them new and more definite names of classical derivation. Plant pathologists have never done this. They have generally been men with a practical message to deliver to farmers and, next to securing knowledge of the diseases, their chief concern has been to put this knowledge in such language that farmers could understand it. Naturally this would have been almost impossible if they had renamed the diseases with latin names.- Instead, they have adopted the popular names and, when naming a new disease, they have always tried to use terms familiar to farmers. This popular terminology sometimes tends to make medical men look askance at plant pathology. But, although the subject has developed along practical lines, there is no reason for regarding it as unscientific. The most important plant diseases have been studied in a thoroughly scientific manner. Athough the use of popular terms has sometimes caused looseness in terminology, plant pathol- ogists are doing their best to stabilize the names they use, fixing each of the popular names to some definite disease. Perhaps the names are not quite so definite as are medical terms; but they are more definitely understood by laymen. As a result, farmers are gradually getting into the habit of using the names in the most ap- proved scientific manner, and actually call plant diseases by their correct names to a somewhat greater extent than the people in general SCIENCE OF PLANT PATHOLOGY 403 do human ailments. The popular terminology of plant patholo- gists, therefore, has its advantages. CONTROL The control of plant diseases is not yet on such a scientific basis as that of animal and plant diseases; but during the last ten years great progress has been made. Generally the object sought is not cure but rather prevention. Occasionally it may be worth while to cure a disease in some large plant, but ordinarily it is more important to prevent the parasite from spreading. Various methods are adopted, such as destroying all infected plants, pruning off in- fected tissue, and spraying with germicides. The last mentioned method is an important one, but for one large class of diseases, those caused by bacteria, it is of no avail. The object of spraying is to cover the plants with a layer of dried poison which will be dissolved by the excretions of the germinating spore and will then kill the young fungus. The oldest effective spray is Bordeaux mixture. Although not used so generally now as formerly, it is still a very important method of preventing fungus diseases. It is made up in various different proportions of copper suphate, lime, and water, some having been found best for some diseases, others for others. It is now being re- placed in many cases of lime sulphur. This is prepared by dissolving sulphur in a solution of quick lime in water. It is generally more efficient than Bordeaux mixture, although in the case of one disease, bitter rot of apple, it does not seem to be effective. It is not used in the case of potato diseases as it harms the foliage. But for fruit trees in general it is now the most commonly used spray. CHAPTER XIII Bacterial Diseases Although there are by no means as many diseases of plants caused by bacteria as by fungi, quite a good number are now definitely known to be of bacterial origin. They are quite varied in their effects upon the plants. Some clog the vessels of the plant and cause it to wilt; others soften and destroy the tissues; others cause swellings of the trunk, branches or roots; while still others attack the plant generally, invading all or nearly all the tissues. In general, these four types of disease are spoken of as of wilts, rots, galls, and blights, respectively; although the names commonly given to the different diseases do not always conform with this classification. Bacterial diseases are in general more difficult to control than fungus diseases. Sprays are not effective because the bacteria live quite deep in the tissues and are not spread by spores. With fungi, sprays take effect because the germinated spore on the surface of a new host plant is very susceptible to poisons and can be easily killed. Bacteria do not have this specially critical stage in their life history, and ordinarily cannot be killed by any poison not harmful to the plant. Ordinarily a plant once infected with a bacterial disease cannot be cured. The best that can be done is by selection of good seed or by proper rotation of crops to prevent the disease from getting started another year. The chief exception to this statement is fire blight, a disease which can often be successfully treated by cutting jout the diseased parts of the tree. CERTAIN WELL KNOWN DISEASES Fire 'blight. This disease is caused by an organism called Bacillus amylovorous. The organism has various hosts, but it is most com- mon and disastrous on pear; from which fact it is often called pear blight. It is also common on apple and has been found on quince, plum and hawthorn. It is believed to be spread wholly or almost wholly by insects. Bees carry the bacteria to the blossoms in the spring. From the blossom it grows down into the softer tissues of the twigs and generally reaches the branches during the fall. There BACTERIAL DISEASES 405 it winters over, and the next season passes down into the large branches and eventually reaches the trunk. During this progress it causes death to the tissues it invades. The flower tips wilt and blacken, finally drying, the twigs and even the small branches may be killed. The larger branches or the trunk, if infected, are seldom killed outright; but large infected areas appear on the surface. It is noticed at first by a water-soaked appearance of the bark which later blackens and shrivels. Eventually the dead tissue is separated from the surrounding tissue by a very sharp line in the bark, which generally appears as a deep crack. The infected area is ordinarily marked by the presence of numerous gummy drops of exudate. In these drops the causal organism is present in large numbers, and splendid opportunity for its spread by insects is thus afforded. Un- der the right conditions, the disease spreads very rapidly, sometimes killing a pear tree before its presence has been realized. The leaves wither and dry, looking much as if the tree had been fire-swept, a symptom which has given rise to the name fire blight. Surgery has proved a very effective means of controlling the disease. If an orchard known to be infected is gone over carefully in the winter with saw and pruning knife, cutting out all diseased areas, the trees can generally be saved. Careful sterilization of the tools is very important in this work, as the disease is extremely infectious. Winter is the most effective time for the pruning, be- cause in the absence of insects the disease is not being spread at the same time that the work is being done; but as it is much more diffi- cult to recognize diseased areas in winter than in the growing season, the work is often done in the spring, although at this season the disease cannot be so quickly eradicated. Black rot of cabbage. The cause of this disease is Pseudomonas campestris. It is practically confined to cabbage, and is the most serious disease to which this plant is subject. Although known as black rot, it is not a rot in the true sense of the word, bearing more resemblance to the blights, although having some peculiarities all its own. The organism is thought to winter over in the soil of fields that have been infested, and is thus in a position to infect new plants the next summer. It enters through the pores in the leaves, causing dead areas to appear around the edges. These infected areas are yellow, but with deep brown or black veins. From the edges of the leaf the infection passes down through the veins to the midrib, 406 BACTERIOLOGY killing the leaf as it progresses, and then down through the fibro- vascular bundles to the stem. As it goes, it blackens the fibro- vascular bundles, so that if the petiole of an infected leaf be cut across, the cross section shows each fibro-vascular bundle plainly marked as a black dot. These blackened fibro-vascular bundles are filled with the infecting organism, which can be obtained from them in practically pure culture. Sometimes when the affected bundles are cut, the bacterial growth oozes out in yellow drops, yellow being the characteristic color which this organism produces on nearly all media. In the advanced stages of the disease, the tissues killed by Ps. campestris fall ready prey to saprophytic organ- isms, so that an offensive decay sets in, this secondary phenomenon giving rise to the name black rot. Control of the disease is very difficult. Discarding infected seed- beds and practicing rotation of crops are the most effective measures if the disease once becomes established. No satisfactory method has been found for saving the plants after they have become infected. Soft rot. The organism causing this disease has been called by various names, the most approved being Bacillus caratovorous. A great many vegetables are subject to this type of rotting, and al- though at first different organisms were thought to attack different plants, they are now all believed to be the same. The rotting generally appears during storage, but may appear while the vege- tables are still in the ground. It is a very common disease, and at times causes serious losses. Bean blight. Caused by Pseudomonas phaseoli. This is a very common and sometimes serious disease of beans. It may attack any part of the bean plant, but is especially noticeable on the leaves and pods. The leaves show irregular water-soaked patches, which later turn brown. The pods show round spots which extend through to the seeds themselves after the disease is somewhat advanced. The progress of the disease is slow, not ordinarily becoming noticeable until the pods have formed. It is quite easy to confuse this infection with anthracnose (see p. 412) an entirely different disease, caused by a fungus; but they can be distinguished by anyone who has had a little experience. Control of this disease depends almost wholly upon seed selection. Seed should never be taken from pods that show signs of the blight, even though the seeds themselves are not spotted. Seed should not be taken from an infected field; and if any BACTERIAL DISEASES 407 infected beans are found in a lot of loose seed, the whole lot should be condemned for seed purposes. Bacterial wilt of melons and cucumbers. The cause of this disease is known as Bacillus tracheiphilus. Although melons and cucum- bers are most susceptible, other cucurbits, such as squashes and pumpkins, may be attacked. The organism seems to be spread almost wholly, if not wholly, by the agency of biting insects. It enters the vessels of the plants, clogging them and causing the plant to wilt. If the infection is in the central stem, the whole plant wilts at once; if nearer the tip of some branch the wilting is local at first but gradually extends toward the center. In the later stages of the disease, cavities may be formed in the stems and even in the fruit. Crown gall. Pseudomonas tumefaciens, the cause of this disease, attacks nearly all fruit trees and several other plants. Its presence is characterized by the production of galls or tumors on the roots of the plants near the surface of the ground. The progress of the disease may be quite slow, but in time the surrounding tissues are affected, and the plant is cut off from its roots and dies. Sometimes the disease causes serious losses. The demonstration of its bacterial origin was quite difficult. It has long been known to be infectious, but no causal organism could be found. At last, however, the presence of Ps. tumefaciens was discovered, but the organism was observed to occur in the galls in but very small numbers. Nevertheless, successful inoculation experi- ments were performed with it, and there is no longer any serious question but that it is the cause of the disease. In this connection, a very interesting theory has been proposed by Erwin Smith, the most prominent investigator of this disease. He has pointed out certain very striking analogies between crown galls and cancerous tumors in man. In his mind, this similarity suggests the possibility that cancer may be also a morbid growth produced by the body under the stimulus of some microorganism as yet undis- covered. The reason why it has not yet been discovered he thinks may be the same as that which caused the difficulty in discovering Ps. tumefaciens, namely the fact that the growth of tumors may be produced without great multiplication of the invading organism. This theory remains pure speculation, for no one has succeeded in demonstrating any such fact in the case of cancer; but its mere sug- gestion makes this disease of much more interest to the medical pro- fession than any other plant disease. 408 BACTERIOLOGY The control of crown gall presents difficulties similar to those in the case of other bacterial diseases of plants. Removing the tumors seldom does good, as the infection does not seem to be localized in the swelling. Fruit growers must be cautious in selecting nursery stock to see that galls are not already present. It is also possible to prevent some cases by being careful not to injure the trees near the roots, such injuries often leading to infection. Potato scab. This disease is not produced by a true bacterium, but by one of the intermediate forms named Actinomycetes. The species causing the disease is Actinomyces scabies. In most respects it is like a very simple fungus; but as bacteriologists so often speak of this group as belonging with the bacteria, we will consider it in the present chapter rather than in the following. Potato is the only known host of this parasite, although certain similar and possibly identical Actinomycetes have been observed in the roots of other plants. It is a very common disease, scabby potatoes being quite a familiar sight on the market. It has no symp- toms except the irregular dark colored "scabs" on the tubers. A light case of the disease is of no special consequence, but if the pota- toes are very scabby, their market value is lowered. The plant itself is never appreciably injured by the disease. Control of the disease has presented considerable difficulty. The organism lives in the soil, and if a field becomes infested the best plan is to grow some other crop. Rotation of crops generally keeps the disease under control. The organism is favored by a neutral or alkaline soil, and is especially likely to give trouble after liming. Hence the application of lime just before planting potatoes is not recommended. Selection of clean seed is always advisable; and treatment of suspicious seed in formalin has been recommended. A very recently proposed method of control is to treat the soil with a certain definite amount of sulfur. Bacteria live on the sulfur and convert it into sulfuric acid, causing the soil to become so acid as to prevent the growth of the parasite. In this acid soil, the potatoes grow free from scab. After harvesting the potatoes the soil can be neutralized for the following crop by adding lime. This treatment, however, requires such careful planning and adjustment of the reac- tion that it is not recommended for general use. Even in some of the experiment stations it has not proved satisfactory. REFERENCE E. F. Smith. An Introduction to Bacterial Diseases of Plants. Saunders, Philadelphia, 1920. CHAPTER XIV Fungus Diseases Certain members of nearly every class of fungi are parasitic upon plants. As a result, the fungus diseases of plants are almost innu- merable. To describe them all would require more space than could be given them in a book on bacteriology; and yet the subject is so akin to animal pathology that many of them must be mentioned and even briefly described. The list given here will hardly give the student an idea of plant pathology as a science, but will serve to show some of the varied aspects of parasitism of microorganisms upon plants, to serve as a comparison with the affects of animal parasites, as described in the preceding sections of the book. GROUP OF FUNGI CONCERNED The classification of fungi does not concern us to any great extent, but in order to comprehend the diseases they produce, it is necessary to review briefly the main groups into which they are ordinarily classified by botanists. They are divided first into Phycomycetes (algae-like fungi) and Eumycetes (true fungi). The Phycomycetes are characterized by the absence of septae, or dividing walls, separat- ing one cell from the next. In general they are fairly simple fungi, although this is not true of all members of the group. The true fungi have their hyphae (i.e., the threads of which they are composed) divided into separate cells by septae, and are classified by their method of fructification. The fungi ordinarily produce two types of spores one of which is formed by simple division of the hyphae, while the other is borne in some special manner. The former is known as the imperfect form, the second as the perfect. On this basis they are classified as follows: Fungi imperfecti: A more or less artificial group in which are placed those fungi that are not yet known to produce perfect forms. Ascomycetes. In the perfect form of this group of fungi, little spore sacs (known as asci) are produced, each of which contains a definite number (generally eight) of spores. 410 BACTERIOLOGY Basidiomycetes. The spores in the perfect form of this group are borne on little finger-like processes known as basidia. PHYCOMYCETES Among the various diseases caused by Phycomycetes, the follow- ing are probably the most important. Damping off. Parasite: Pythium deBaryanum. This is a fairly common disease which attacks various hosts. It is most likely to occur on seedlings grown in the greenhouse, plants being especially susceptible to it when grown in a crowded condition in a warm moist atmosphere. Sometimes the symptoms appear only a few days after germination of the seeds. The stems are generally attacked near the surface of the ground, the tissues becoming soft and appearing water-soaked. The stem finally gives way entirely and the seedling collapses. Brown rot of lemon. Parasite: Pythiacystis citrophthora. This disease is very serious in regions where lemons are grown and handled. It occurs in the orchard, the packing house and in storage. It occurs on the fruit alone, where it appears first as a brownish or purplish discoloration which rapidly spreads over the lemon and from one lemon to another causing the tissues to soften and producing a pecu- liar striking odor. Although attacking the fruit alone, it is easily distinguished from ordinary decay organisms. The most effective means found to control the brown rot is to pre- vent its spread from one lemon to another when they are washed, by putting some mild antiseptic in the wash water. White "Rust." Parasite: Cystopus candidus. This is a very com- mon disease, but not one of great economic importance, for the plant most often attacked is the ubiquitous shepherds purse. Other members of the same family (Cruciferi) such as radish, horse radish, cress, turnip, and mustard, are also attacked. It generally causes a swelling of the infected tissues, which become white with the spores of the fungus. Downey mildews. K series of diseases, all very similar and pro- duced by closely related species of fungi, are known as mildews or downy mildews. These fungi produce brownish or yellowish spots on the leaves or other parts of the plants infested, and upon these spots (generally from the under surface of the leaf) a very fine hairy growth is borne. Downy mildew of grapes is the most important FUNGUS DISEASES 411 of these diseases. Nearly all of them can be controlled by proper spraying with lime sulphur. The various parasites and their respective hosts are as follows: Grape Plasmopara viticola Cucumber Plasmopara cubensis Crucifers Peronospora parasitica Onion Peronospora schleideniana Lettuce Bremia lactucae Lima bean Phytophthora phaseoli Late blight and rot of potato. Parasite: Phytophthora infestans. This is the most serious malady of potatoes, and in many places is known simply as the "potato disease." It can be very disastrous, especially in warm, moist weather, and in 1845 caused a famine in Ireland by bringing about the failure of that country's principal crop. It is characterized by large, irregular, diseased areas on the leaf, accompanied by wilting. It also attacks the tubers, producing the well-known dry rot. The dry rot often escapes notice until the potatoes have been in storage for some time, when it is followed by the action of ordinary decay organisms that bring about a wet rot. The disease can be very effectively controlled by spraying with Bordeaux mixture, beginning when the plants are about 6 inches high. This should be accompanied by a system of rotation and by careful selection of the seed from fields free from the disease. FUNGI IMPERFECTI One by one the various diseases produced by organisms that used to be ascribed to the Fungi imperfecti are being placed in other groups as their perfect forms are being discovered. Naturally, the most important diseases have had the most study, and their perfect forms have been first found. As a result, the diseases now ascribed to this group of fungi are to a large extent the less important diseases. There are, however, two of sufficient importance to be mentioned, early blight of potato, and anthracnose of beans. Early blight of potato. Parasite: Macrosporium solani. This disease is quite common wherever potatoes are grown, although it is not one of the most serious potato diseases. It is a leaf blight marked by brown, circular, or elliptical spots on the leaves. The establishment of the disease is encouraged by injury to the leaves 412 BACTERIOLOGY such as insect bites. If the disease progresses, the leaves dry up and there may be considerable loss in the crop. It proves possible to control this disease quite effectively by seed selection and by spraying with Bordeaux mixture. Anthracnose of beans. Parasite: Colletotrichum lindemuthianum. This disease and the blight, above described, are the two most serious diseases of beans. Anthracnose is very common and sometimes quite disastrous. The pods, stems and leaves are attacked, somewhat as in the case of blight, but most characteristic are the spots on the pods. These are dark, sunken spots, which often show a pinkish tint due to the pink color of the spores of the fungus. Control is best effected by selecting seeds from healthy pods, burning infected material, and rotating other crops with beans. ASCOMYCETES Peach leaf curl. Parasite: Exoascus deformans. This is quite a serious disease that is especially common, following damp weather in the spring. The most characteristic symptom is the deformity of the leaves, a very pronounced arching and curling of the blades. Twigs are also attacked and become swollen at their tips. Flowers and fruit are occasionally affected. Sometimes it is serious enough to destroy nearly all of the leaves and under such conditions the tree of course bears little fruit. The parasite seems to gain entrance as the first buds are opening in the spring, nearly all the damage result- ing from this early infection. Spraying with Bordeaux mixture or lime sulfur a little before the buds open has proved very effective in controlling the disease. Brown rot of peaches, plums, etc. Parasite: Sclerotinia fructigena. This quite serious disease attacks nearly all varieties of stone fruits. It generally appears first as a brown decaying spot on the fruit, which rapidly involves the surrounding tissue until the whole fruit decays. Then the fungus appears in the form of little tufts of spores on the surface of the fruit. Later the decayed fruit may fall to the ground or may cling to the branches, gradually drying until it be- comes "mummied." In the year following a severe attack the flowers and twigs may also be affected. It frequently attacks the fruit after picking, a fine lot of fruit sometimes being made entirely worthless by the time it reaches the market. Spread of the disease can be FUNGUS DISEASES 413 diminished by removing the mummied plums and diseased twigs during the winter and by an early spraying to destroy the free spores that may be on the branches. Root rot of tobacco. Parasite: Thielavia basicola. This disease, quite serious at times to tobacco, is also known to attack less severely various legumes. It is most likely to attack the plants in an alkaline soil under conditions of considerable moisture. Roots of diseased plants are stunted and, as a result, the plants ordinarily make poor growth or even die. The disease has been found to manifest itself chiefly in the seed beds and can frequently be eradicated by sterili- zation of the soil used in the beds. The use of acid phosphate to prevent alkalinity seems to be of some advantage. Wilt of cotton and watermelon. Parasite: N eocosmosporti vasin- fecta. This is a very serious disease of cotton, also observed in water- melon and cowpea, although it is not absolutely certain that all are caused by the same parasite. It is typically a wilt disease, the dam- age resulting from the cutting off of the water supply of the plant after the parasite has clogged the vessels. Some varieties of cotton are susceptible and others resistant to the disease, and the planting of resistant varieties offers one of the most promising methods of controlling it. Ergot. Parasite: Claviceps purpurea. This fungus attacks grains and grasses without causing any serious disease to the plants infected. Its chief interest comes from the drug ergotine produced by one of its stages. It causes certain grains in a head to become abnormally large and finally replaces them entirely with a large, thick-walled body, which serves as a resting stage to carry the fungus over to the next year. These ergot grains contain a toxic principle which is valuable for certain therapeutic purposes, but is quite poisonous to stock that may eat them with the grass in a field infested with the fungus. Black knot of plums and cherries. Parasite: Plowrightia morbosa. This is one of the most common and most unsightly diseases of fruit trees, although not as serious as some of the others we have considered. It is characterized by large black warts or excresences that appear on the sides of the twigs and branches in the early spring and grow in size until summer. Thorough pruning of the diseased branches is the best method of control, although eradication is practically im- possible, for all wild plums and cherries are affected. 414 BACTERIOLOGY Black rot of grapes. Parasite: Guignardia bidwellii. This is a very serious and widespread disease of the grape. It appears first in the form of brown spots on the leaves, and then a little later a purplish or brown spot appears on the unripe fruit, which ordinarily spreads over the whole of the grape, causing it to shrivel. Control is effected by spraying once before the growing season begins, then as soon as the buds open and four or five later applications every two weeks or so. Strawberry leaf spot. Parasite: Mycosphoerella fragariae. This is an extremely common disease of the strawberry, nearly all beds being affected to a greater or less extent. It is characterized by spots on the leaves that are red at first and then fade into a pale brown or even whitish area bounded by a ring of red or purple. Apple scab. Parasite: Venturia pomi. Pear scab. Parasite: Venturia pyrina. These two diseases are produced by very closely related fungi and are much alike in symptoms. Both are serious diseases under the right conditions. Most frequently the fruit and leaves are attacked. Spots are formed on the leaves, which generally show only on the under surface, although covering both surfaces in severe cases. The first appearance of disease on the fruit is in the form of small round spots which spread until the familiar scabs appear, which may even render the fruit unmarketable. Spraying before the fruit buds open is the generally recommended method of control. Bitter rot of apple. Parasite: Glomerella rufomaculans. This is probably the most destructive apple disease. Warm, moist weather especially favors its spread, so that enormous damage may be done by it during a single week under the proper conditions in midsummer. It occurs also on other plants, although on none so severely as on the apple. It attacks chiefly the fruit, where it first appears as a small brown spot beneath the skin. This spot rapidly spreads and becomes sunken, gradually involving the greater part of the apple, the diseased part decaying. The portions of the fruit near the de- caying areas have a bitter taste. Control can be effected by spray- ing with Bordeaux mixture (not lime sulphur) making the first application about forty days after the petals fall and continuing the applications every two weeks for a couple of months. FUNGUS DISEASES 415 BASIDIOMYCETES Loose smut of wheat. Parasite: Ustilago tritici. Loose smut of oats. Parasite: Ustilago avenae. Two very similar diseases, of the type known as smuts, produced by closely related parasites, attack wheat and oats respectively, and are of considerable economic im- portance. They infect the seeds at the time of germination and the mycelium penetrates the tissue of the growing plant, maturing at the same time that the grain matures. By this time the grain kernels have been entirely consumed by the fungus and nothing but a black dusty mass of spores is found where the kernel should be. The control of these two diseases is based principally on seed treatment. It has been found possible to use such a strength of formalin as to kill the smut on the seeds without harming the seed; or it is possible to use hot water, heating it to a certain definite temperature and emersing the seeds for a definite length of time. Corn smut. Parasite: Ustilago zece. This common disease is characterized by peculiar swellings on various parts of the corn plant, which at maturity break open and disclose large masses of sooty spores. Frequently kernels in the ear will be infected and swell to large size, and the tassels are often attacked. Control consists simply in removing the diseased portions before the spores are formed and in burning the material thus removed from the plants, for the parasite can live in the soil or compost pile and be ready to attack the next crop unless destroyed. Bunt, or stinking smut of wheat. Parasite: Tilletia foetens. This disease is not as serious as the loose smut, but often causes much damage. It differs from the loose smut in that the smutted kernels remain closed by the bracts around them and are not visible. Al- though it is possible to pick out the smutted heads of wheat from their general appearance, they often escape observation and get into the bins and are even milled. At this time, they give off a dis- agreeable odor which explains the popular name of the disease. Formalin treatment of the seed has been found to secure good control. Rusts. There are a very large number of plant diseases known as rusts, produced by members of the group Uredinales. There are probably two thousand species of fungi in this group and they are all obligate parasites, so it is easy to see what a large number of 416 BACTERIOLOGY diseases they must cause. This group is especially characterized by the great variety in spore formation which any one species may show. There are five distinct types of spores that may be produced by one species in the course of its life history, although some species do not produce more than four, three, or even two of these forms. Some- times a species will produce all of its stages on a single host plant; while in other cases an alternation of hosts is necessary. In the latter case, it produces some of its stages on one host, the rest on the other, both kinds of plants being necessary in order for the fungus to continue its existence. Sometimes it may assume such a different appearance on the two hosts that no one would suspect the two diseases to be caused by the same parasite; and mycologists have had extreme difficulty in tracing out the connection/ One of the best known examples of this is the apple rust, which is caused by the same organism as causes "cedar apples," certain peculiar large tumors on the branches of the cedar. The most common manifestation of these fungi is well described by the popular term rust, as the leaves may become covered with rusty colored spots containing the spores. By far the greatest number of the rusts are of little practical significance; but the following are important: Asparagus rust. Parasite: Puccinia asparagi. Black rust of grain. Parasite: Puccinia graminis. Alternate host: barberry. Brown rust of wheat and rye. Parasite: Puccinia rubigo-vera. Alternate host: borage. Apple rust. Parasite: Gymnosporantium macropus. Alternate host: cedar. Orange rust of raspberry and blackberry. Parasite: Gymnoconia peckiana. Control of these diseases is sometimes partly effected by spraying; but wherever the parasite in question requires an alternate host, the most effective control measure is eradication of this other host. Thus, barberry is very undesireable in the vicinity of grain fields; and in its absence, black rust gives practically no trouble. True Basidiomycetes. Rusts and smuts are caused by organisms that differ from the true members of this group and are often put in other groups by botanists. The true Basidiomycetes are mushrooms and toadstools, and but few of them are parasitic. The most im- portant parasite is Corticum vagum, which produces a serious root FUNGUS DISEASES 417 and stem rot on various plants, including sugar beets, beans, carrots, cabbage, cotton, lettuce, potato, radish, sweet potato, pumpkin, watermelon and pea. It is often called Rhizoctonia disease, that being the name given to the fungus before its perfect form (i.e., the basidiospore stage) was discovered. Various other Basidiomycetes cause decay of wood. These wood fungi are not true parasites, in that they seldom if ever attack live tissue. Their appearance upon a living tree indicates the existence of wood which is already dead, as they seldom attack a healthy tree. Their chief importance is due to the destruction of timber which they may cause. MYXOMYCETES Club root of cabbage. Parasite: Plasmodiophora brassicae This disease is very interesting because caused neither by a bacterium nor by a true fungus. The parasite is a slime mold, a member of that very interesting group of organisms which resemble protozoa in some respects and fungi in others. It attacks the roots of cabbage plants, causing the roots to swell to very large size, and sometimes brings about considerable damage to the plants. REFERENCES B. M. Dvggar. Fungous Diseases of Plants. Ginn, Boston, 1909. L. R. Hesler and H. H. Whetzel. Manual of Fruit Diseases. Macmillan, New York, 1917. W. H. Rankin. Manual of Tree Diseases. Macmillan, New York, 1918. F. L. Stevens and J. G. Hall. Diseases of Economic Plants. Revised by F. L. Stevens. Macmillan, New York, 1921. PART IV APPENDIX APPENDIX I. MEDIA-MAKING BOUILLON Mix the following: Water, 1000 cc.; peptone, 5 grams; Liebig's beef extract, 3 grains. Boil over an open flame for about an hour, or heat in a flask in an autoclav at 15 pounds for about forty-five minutes. Then filter through filter paper in a funnel. Add enough water to bring up to 1 litre again, if it has been boiled over a flame. If cooked in an autoclav it is best to add no water, for if loss occurs in the autoclav it is ordinarily due to spilling over, not to evaporation. Next adjust the reaction, as follows: Remove a small portion of the medium and add to it a few drops of a 0.04 per cent brom thymol blue solution. If too alkaline, the medium turns this indicator blue, if too acid it remains yellow, while if of the correct acidity it turns it green-grass green if the right amount of the indicator is used. The medium is not likely to be too alkaline, but may be neutral or acid according to the kind of peptone used. If too acid, pro- ceed as follows: Measure out 10 cc. of the medium, place in an evaporating dish and dilute with considerable water. Add 1 cc. of the brom thymol blue solution. Place the evaporating dish under a burette containing Ar normal NaOH. (If NaOH of exactly this strength cannot be readily obtained a solution of approx- imately this strength, i.e., 0.4 per cent, may be used.) Take the reading on the burette. Allow the solution to fall drop by drop into the evaporating dish, stirring between drops. As long as the material remains acid the color is yellow. A distinct green indicates neutrality. Then take the burette read- ing again and determine the difference between the two readings. Multiply this difference by 100 in order to obtain the amount of alkali required to the litre of medium. To add this amount would dilute the medium too much, so it is better to add a smaller amount of a stronger alkali. For this purpose use alkali exactly 10 times as strong as that used in titration, that is normal NaOH if Ay normal was used in the burette. To the 1000 cc. of medium add 10 times as much of the stronger alkali as was used of the weaker in titration. The bouillon should be placed in a flask or in test tubes, plugged with cotton and sterilized in an autoclav for twenty minutes at 15 pounds pressure. While sterilizing take care to see that a small stream of steam is coming out of the vent of the autoclav; otherwise there is danger that the temperature inside will not be sufficient to sterilize. SUGAR BROTHS Prepare bouillon according to the directions just given, but after adjusting the reaction add 1 per cent (10 grams per litre) of the desired sugar. The 422 BACTERIOLOGY sugars used are ordinarily dextrose, lactose, or sucrose. It is often advisable to add an indicator to the broth to detect acid production from the sugar; in which case the most satisfactory indicator is brom cresol purple. Two cubic centimeters of a saturated aqueous solution of this indicator should be added per litre of medium. Sugar broths are ordinarily used in fermentation tubes in order to detect gas production from the sugar. In which case, the media should be distributed in the fermentation tubes after dissolving the sugar, plugged with cotton and sterilized for twenty minutes at 15 pounds pressure. NUTRIENT AGAR Measure out the same ingredients listed above for bouillon. Place them in a litre flask together with 15 grams of agar. If the agar used is in the form of shreds it may be found most convenient to cut it into small pieces. Some agar is furnished in powdered or granular form. The flask containing these ingredients should be placed in an autoclav and heated for forty to fifty minutes at 15 pounds pressure. During this process care should be taken to prevent the safety valve from blowing off suddenly, as a sudden change in the pressure stirs up the contents of the flask and inter- feres with sedimentation. For the same reason, the autoclav should be allowed to cool slowly without opening any valve to let the steam escape until the pressure is down to zero. Then remove the flask and pour carefully, with as little disturbance of the sediment as possible, through absorbent cotton in a large funnel, catching the filtered medium in another flask. The reaction may then be adjusted by the same method described above. It is sometimes preferred to have the reaction already adjusted before heat- ing. This can be done after a little experience with any lot of the ingredients used; for it can readily be determined how much alkali is needed to neutralize and the required amount can be added before dissolving the agar. In such a case it is always advisable to test the reaction with brom thymol blue after the heating. The agar should be sterilized in the autoclav either in flasks or tubes as desired. If the agar is melted when placed in the autoclav allow twenty minutes at 15 pounds; but if it is solid, enough extra time should be allowed to give it a chance to melt. In tubes, the agar melts in about ten minutes at 15 pounds; while a litre of it in a flask requires about thirty minutes for melting. It is ordinarily desirable to use the tubes for agar slants. These are obtained by letting the agar harden while the tubes are lying in a slanted position so as to give a large oblique face of the agar on which to inoculate bacteria. Prepare agar exactly as above, but before tubing add 2 cc. per litre of saturated aqueous brom cresol purple and 10 grams of the sugar to be tested (ordinarily dextrose, lactose or sucrose). Sterilize in test tubes twenty minutes at 15 pounds pressure, and let the tubes harden in a slanted position. SUGAR AGAR, WITH BROM CRESOL PURPLE APPENDIX 423 NITRATE BROTH AND NITRATE AGAR These are prepared by adding 1 gram per litre of KN03 to bouillon and to nutrient agar respectively before tubing. NUTRIENT GELATIN Use the same ingredients as for agar except that the 15 grams of agar should be replaced by 120 grams of gelatin. The ingredients should be dissolved with the use of less heat than in the case of agar. This may be accomplished in several ways: heating in a flask in flowing steam (with no pressure) in an autoclav; heating in a dish over a steam bath; or heating in a double boiler with water, or preferably a strong salt solution in the lower part. If heating is done in a dish, dish and contents should be weighed before heating. Heat for about half an hour. Weigh (if cooked in a dish), and add water to bring up to original weight. Then adjust the reaction to neutrality to brom thymol blue, as directed in the case of agar. In the case of gelatin the reaction will always prove acid before adjustment, so that added alkali is always needed. After adjusting the reaction, heat again for about half an hour. Then cool by standing in water until the gelatin is no longer hot to the touch, or to be exact until its temperature falls below 50°C. Separate the white of an egg carefully from the yolk, and mix the white with a little water. Add the solution of white of egg to the gelatin, and stir in thoroughly. Cook for an hour, and restore lost water. (If heated in a flask in a sterilizer, no water may have been lost). The object of the egg is to clarify the medium, and by the end of an hour's heating the egg-white should have coagulated and be lying on the surface of the medium. The contents should now be strained through absorbent cotton in a funnel, preferably pouring them into the funnel through a gauze strainer to remove the bulk of the egg, taking pains not to break up the coagulum. The medium should then be put in test tubes and plugged, and sterilized for fifteen minutes at 15 pounds pressure. If the best grades of bacteriologic gelatin and peptone be used, it is not necessary to use the white of egg in clarifying. In this case, the directions in the last paragraph may be omitted, the medium may be filtered directly through cotton, tubed and sterilized. PLAIN GELATIN Mix 60 grams of gelatin with 500 cc. of water. If good grade gelatin be used, clarification is never necessary and the procedure is simply as follows: Dissolve by heating in steam or in a double boiler. Adjust reaction as above described. Heat fifteen minutes longer, filter through cotton and tube. Sterilize fifteen minutes at 15 pounds pressure. II. CHARACTERIZATION OF BACTERIA As mentioned in one of the early chapters of this book, bacteria are de- scribed partly by morphological and partly by cultural or physiological charac- teristics. Characteristics of the latter sort are of so many different sorts and can be recorded in such varied ways that it often becomes difficult to com- pare the cultures described by different workers. To prevent this difficulty it is important that a uniform system of description be used. The Society of American Bacteriologists has been working for a number of years to draw up a system of uniform characterization, and has placed at the disposal of bacteri- ologists a chart known as the Descriptive Chart. Several editions of this chart have been published, each a slight improvement of the preceding. The latest chart at the time this book goes to press is given in the plate following this page. This chart is printed on 8 by 12-inch cards, and it is intended that one chart be used for each culture studied. These charts can be obtained from the Society of American Bacteriologists, ordinarily through the chairman of its Committee on Bacteriological Technic. It is possible to obtain from the same source copies of a manual of methods to be used with the chart. Anyone who is studying a large number of bacteria should consult this manual; but for the benefit of those students who do not have the manual at hand, the most important methods of pure culture study are given here. PURIFICATION OF CULTURES The cultures used for study are ordinarily obtained by the plating technic described on p. 46. Such cultures often come from contaminated colonies and it is very important that the cultures to be described are pure. Hence purification is ordinarily necessary. The procedure for purification is as follows: With a flamed platinum needle remove a minute portion of the growth from an agar slant culture and transfer it to a tube of sterile water. Mix thoroughly. Sterilize a platinum loop in the flame and transfer two loopfuls to a melted and partly cooled tube of agar or gelatin (preferably the same medium as that from which the culture was first isolated). Transfer two loopfuls from this tube to a second tube of the same medium. Then pour the contents of each tube into a sterile petri dish and let it harden. Incubate and notice the colonies that develop. If the culture was pure, all the colonies should be practically alike. Make a fresh agar slant culture from a colony like the original one. This should be without question a pure culture, but it is always advisable to ex- amine it under the microscope by the staining technic described below to be sure that the organisms present all look alike; and during the study, new microscopic preparations should be made and examined critically from time to time. Preoared byjH. I. Conn, \ K. N. Atkins, f Committee on Endorsed by Society of American Bacteriologists DESCRIPTIVE CHART LF. Norton, ST** at the annual meeting Dec. <50, 1920 -w q e. Harmon ) Name of organism. Source Date >f isolation Studied by Culture No Invigoration of culture: Date Medium used Temperature Number of transfers Length of each incubation.. . .days. Series No . . MORPHOLOGY Index No.* BRIEF CHARACTERIZATION As each of the following characteristics is determined, indicate in proper marginal square by means of figure, as designated below: Note-Underscore required terms. Sketched Vegetative Cells, Medium used temp age days. Form, spheres, short rods, long rods, filaments, com- mas, short spirals, long spirals, curved. Arrangement, single, pairs, chains, fours, clusters, cubical packets. Limits of Size Size of Majority Ends, rounded, truncate, concave.. Capsules, present on How stained PRIMARY CHARACTERISTICS Microscopic Features Form: 1, streptococci; 2, diplococci; 3, micrococci; 4, sarcime; 5. rods; 6, commas; 7, spirals; 8, branched rods; 9, filamentous Spores: 1, central; 2, polar; 3, absent Flagella: 1, peritrichic; 2, polar; 3, absent Gram stain: 1, positive; 2, negative • - Sporangia, present, absent. Medium used temp age days. Form, elliptical, short rods, spindled, clavate, drum- sticks. Limits of Size Size of Majority Endospores, present, absent. Location of Endospores, central, polar. Form, spherical, elliptical, elongated. Limits of Size Size of Majority Wall, thick, thin. Sporangium wall, adherent, not adherent. Miscellaneous Biochemical Reactions Pathogenicity, etc. : 1, for manj 2, tor animalsj 3, for plantsj 4, parasitic but not pathogenic; 5, saprophytic; 6, autotrophic Relation to oxygen: 1, strict aerobe; 2, facultative anaerobe; 3, strict anaerobe Gelatin liquefaction: 1, positive; 2, negative In nitrate media: 1, nitrite and gas; 2, nitrite but no gas; 3, neither nitrite nor gas Chromogenesis: 1, flourescent; 2, violet; 3, blue; 4, green; 5, yellow; 6, orange; 7, red; 8, brown; 9, pink; 0, none - Motilty In broth. On agar Flagella, No Attachment, polar, bipolar, peritrichiaie. How stained Carbohydrate Reactions Diastatic action: 1. positive; 2, negative From dextrose: 1, acid and gas; 2, acid without gas; 3, no acid Irregular Forms, Present on in.... daysjat "C. Form spindled, cuneate, filamentous, branched, or From lactose: 1, acid and gas; 2, acid witnout gas; 3, no acid From sucrose: 1, acid and gas; 2, acid without gas; 3, no acid - Staining Reactions. Gram Acid fast Special stains SECONDARY CHARACTERISTICS Vegetative Cells Diameter: 1, under 0.5M; 2, between 0.5M and IM; 3, over 1/* Length: 1, less than 2 diameters; 2, more than 2 diameters Chains (4 or more cells): 1, present; 2, absent - CULTURAL CHARACTERISTICS Capsules: 1, present; 2, absent - Spores Shape: 1, round; 2, oval to cylindrical Diameter: 1, less than diameter of rod; 2, greater than diameter of rod Underscore required terms. Sketches - Underscore required terms. Agar Stroke Incubation Temperature °C Age d Growth, scanty, moderate, abundant, none. Form of growth, filiform, echinulate, beaded, spread- ing, arborescent, rhizoid. Elevation of growth, flat, effuse, raised, convex. Lustre, glistening, dull. Topography, smooth, contoured, rugose. Optical Characters, opaque, translucent, opalescent, iridescent. Chromogenesis Photogenic. Fluorescent. Odor, absent, decided, resembling............. Consistency, butyrous, viscid, membranous, brittle. Medium, grayed, browned, reddened, blued, greened. Nutrient Broth Temperature °C Age d Surface growth, ring, pellicle, flocculent membranous, none. Clouding, slight, moderate, strong, tran- sient, persistent, none, fluid turbid. Odor, absent, decided, resembling Sediment, compact, flocculent, granular fiaky, viscid on agitation, abundant scant, none. / Cultural Features w Agar Stroke Abundance: 1, abundant; 2, moderate; 3, slight; 4, absent Lustre: 1, glistening; 2, dull Surface: 1, smooth; 2, contoured; 3, rugose colonies: 1, punctiform; 2, round (over 1 mm. diameter); 3, rhizoid; 4, filamentous; 5, curled - Gelatin Stab Temperature _ .»C Age .d Growth, uniform, best at top, best at bottom. Line of puncture, filiform, beaded, papillate, villous, arborescent. Liquefaction, none, craleriform, napiform, infundi- buliform, saccate, stratiform; begins in d' complete in d. Depth of liquefaction in tube of 10 mm. diameter evenly inoculated at 20° C. for 30 days mm. Medium, fluorescent, browned. Medium (liquid) Temperature °C Age d Gelat 5 in colonies: 1, punctiform; 2, round (over 1 mm.); 3, irregular; 4, fila- nentous Acid: 1, sufficient for curdling; 2, insufficient for curdling; 3, no acid Rennet curd: 1, present; 2, absent Peptonization: 1. present; 2, absent Medium ! (solid) Temperature .'C Age Medium Temperature °C Age d 'Recording the ''Index Number" here is optional; but its use will be found convenient if the charts are to be filed according to the salient characteristics of the organisms. The Index Number consists of the first thirteen figures from the margin (primary characteristics) copied down in the order of their occurrence in the margin, placing a dash wherever a heavy nds occurs, in the margin. Thus, B. coli belongs to the group 5312-41220-1111. Agar Colonies Temperature °C Age d Growth, slow, rapid.. Form, puncti/orm, circular, irregular, mycelioid, filamentous, rhizoid. Surface, smooth, rough, concentrically ringed, radiate. Elevation, fiat, effuse, raised, convex, pulvinate, umbonate. Edge, entire, undulate, lobate, erose, filamentous, curled. Internal structure, amorphous, finely-, coarsely-granu- lar, filamentous, curled concentric. Sketches Gelatin Colonies Temperature °C Age d Growth, slow, rapid. Form, punctiform, circular, irregular, mycelioid. filamentous. Elevation, flat, raised, convex, pulvinate, crateriform {liquefying). Edge, entire, undulate, lobate, erase, filamentous, floccose, curled. Liauefaction, cup, saucer, spreading. Internal structure, amorphous, finely-, coarsely- granular. filamentous, curled, concentric. Sketches PHYSIOLOGY TEMPERATURE RELATIONS Optimum temperature for growth °C. Maximum temperature for growth °C. RELATION TO OXYGEN Method used Medium Temperature Aerobic growth: absent present, better than anaerobic growth. Anaerobic growth: absent, occurs in presence of dextrose, oi sucrose, of lactose, of °C. itrate; better than FERMENTATION Temperature . °C Minimum temperature tor grovnn CHROMOGENESIS Nutrient broth aerooic growtn. DIASTATIC ACTION Breadth of clear zone on starch agar plates. in days: Additional data .. Medium containing a w o tn O a tn o O z S g Nutrient gelatin MILK Temperature .... °C. .... and: a < o cn o iNuciicnu agar. Potato Reaction: 1 day. .2 days 4 days .7 days .10 days Gas Acid curd: 1 day Rennet ctird: 1 day .2 days 4 days .2 days 4 days .7 days .7 days .7 days .10 days -.... .10 days .10 days days. First appearance of acid PRODUCTION OF INDOI repionizaviou; i way x ways uays Reduction of litmus in days: of methylene blue in First appearance of alkali Indo! absent Present in ................. days NITRATE REDUCTION Reaction after ...... days Nitrite: 1 day. Medium. .2 days 4 days _. Temperature.. .7 days °C. .10 days Reaction after days PRODUCTION OF HYDROGEN Medium: H S absent, present m SULFIDE Gas. Nitrite: Gas: 1 day. 1 day. 1 day. Medium .2 days 4 days .2 days 4 days .7 days Temperature .7 days .7 days .10 days 6C. .10 days .10 days Max. H-ion Cone days SPECIAL TESTS (e. g. PATHOGENICITY) APPENDIX 425 MICROSCOPIC STUDY OF BACTERIA Motility. Put a fair-sized drop of water on a cover glass, and mix with it a small amount of a culture not over twenty-four hours old. Drop a cover glass on it. If the drop of water is too small the cover is likely to be so close to the slide that the bacteria are unable to move. Even better results can be obtained by placing two pieces of broken cover glass on the slide with the drop of water between them so that they hold the cover glass slightly above the slide. Another way of accomplishing the same purpose is to examine a hanging drop, prepared by placing the drop on a cover glass, mixing the bacteria with it, and then inverting it into a cavity on a special microscopic slide made for this kind of work. Put this preparation on the microscope stage and examine it with a | inch objective, using an electric light placed close to the mirror. If not ex- perienced in this kind of work, the student will be likely to find some difficulty in distinguishing the bacteria, for they are very hard to see when unstained. When they are found they will be seen as colorless objects, visible only because of greater refraction than the water around them. Notice whether they are motile or immotile. If motile they will be seen to dart back and forth, some- times crossing the field and moving out of it. Often, however, the smaller bacteria will be found to move very slightly without real locomotion, merely oscillating around a fixed point. This oscillation is a purely physical phenom- enon, known as Brownian movement, which any tiny object, living or dead, undergoes when suspended in a liquid. So the student must not call a culture motile unless the bacteria actually move back and forth in the microscopic field. STAINING SOLUTIONS In the microscopic work described below, the following staining solutions will be necessary: Loeffler's methylen blue Saturated alcoholic solution of methylen blue, 15 cc.; potassium or sodium hydrate, 1:10,000 solution,1 50 cc. Carbol-fuchsin Saturated alcoholic solution of fuchsin, 5 cc.; 5 per cent solution of car- bolic acid, 45 cc. Crystal (or gentian) violet solution For this stain, gentian violet was always called for until recently. At present, however, so many different brands of gentian violet are on the market, all varying considerably from one another, that it seems best to use crystal violet which is more constant and seems to have the same staining reactions. Either of the two following formulae may be used. The anilin formula is most commonly used, but is very unstable and must be used while it is only a ' Prepared by adding 1 cc. of a 1 per cent solution to 99 cc. of water. 426 BACTERIOLOGY few days old. The ammonium oxalate formula is permanent and seems to stain exactly the same as the anilin formula. Anilin formula. Saturated alcoholic solution crystal (or gentian) violet, 6 cc. ; anilin water (i.e., 1 cc. anilin oil shaken with 49 cc. water and then filtered), 50 cc.; 95 per cent alcohol, 5 cc. Oxalate formula. Saturated alcoholic crystal (or gentian) violet, 10 cc.; 1 per cent ammonium oxalate solution, 40 cc. Safranin solution Saturated alcoholic solution of safranin, 1 cc.; water, 9 cc. Bismarck brown solution Bismarck brown, 0.2 grams; hot water, 10. cc. Filter after dissolving. Pyronin solution Saturated alcoholic solution of pyronin, 1 cc.; water, 9 cc. Lugol's iodine solution Iodine, 1 gram; potassium iodide, 2 grams; water, 300 cc. STAINING DRIED BACTERIA In the middle of a clean glass slide place a drop of water, and mix with it a very small amount of growth taken from an agar slant culture, not over twenty-four hours old. This growth should be removed from the agar slant with a flamed platinum needle. Dry the slide with moderate heat, as over a steam radiator or on a water bath. Then cover the bacteria on the slide with a drop of one of the staining solutions, methylene blue generally giving the best results although some bacteria stain better with fuchsin. Let the stain stay on between ten seconds and one minute. Then wash off in running water and dry the slide in gentle heat. Place a drop of immersion oil on the stained bacteria and examine under a -pj inch oil immersion objective. If the student has never used an immersion lens on a microscope he should get help from his instructor or at least read very carefully the instructions furnished with the microscope. The bacteria should be examined with bright daylight or with an electric light filtered through "daylight" glass. The diaphragm beneath the condenser should be wide open. Examine the bacteria and sketch. Then make similar preparations from the other cultures when about twenty-four hours old. Notice how the different cultures differ in size and shape. Spores. Make similar preparations from all the cultures when several days old, to see if spores are present. Spores will show as oval bodies which do not take the stain. Sometimes a culture this old consists only of spores. If both rods and spores are found in the same preparation notice whether the spores are thicker or thinner than the rods. Sometimes rods will be observed with spores in them. Sketch the spores, and the spore-bearing rods if the latter are present. APPENDIX 427 Irregular forms. The above preparations several days old will often show, in the case of cultures that do not produce spores, various forms of different shape or size from the one day cultures. Sometimes these will be club-shaped, sometimes filamentous, and sometimes very small spheres. In case any such forms are observed, prepare a fresh agar slant and make smears from it every day for a week, preferably all on one slide. Stain them at the same time and examine under the microscope making sketches of any changes in morphology observed from day to day. Before doing this it is necessary to be positive that the culture is pure; and it is ordinarily a good plan to replate the culture after this study and see if the same irregular forms occur in the repurified culture. Gram stain. Prepare a dried smear on a slide from a young agar slant culture just as directed three paragraphs above. Stain it one minute with either of the crystal (or gentian) violet solutions given above. If the anilin formula is used pour off the stain after it has stood exactly a minute, and blot the slide with filter paper, but do not wash; if the oxalate formula is used, rather better results are obtained by washing the slide briefly before blotting. Then treat with Lugol's iodine solution for one minute. Pour off the iodine solution; do not wash, but carefully blot until practically dry. Place the slide in absolute or 95 per cent alcohol for thirty seconds, agitating constantly; during this time the alcohol will dissolve out much of the stain. Then counter- stain for thirty seconds in safranin, Bismarck brown or pyronin, using the solutions given above. Wash and dry; then examine under a microscope. The bacteria will be found either blue from the crystal (or gentian) violet and iodine, or else colored by the counter-stain. Different species differ in this respect, the "Gram-positive" organisms retaining the blue color, and the "Gram-negative" organisms losing it. All cultures should be tested by this staining method; but it is well to stain at least three different slides from each culture, because some organisms are variable in this respect and no culture can be called unquestionably either Gram-positive or negative unless at least three preparations agree. RECORDING MORPHOLOGY In making records of the cultures studied a separate page of the note-book should be used for each culture. The first heading on each page should be Morphology; and under this should be entered those of the following charac- teristics which apply. (For their meaning see glossary, p. 431.) Form: Spheres, short rods, long rods, filaments, commas, short spirals, long spirals, curved. Arrangement: Single, pairs, chains, fours. Ends rounded, truncate, concave. Spores present, absent. If spores are present record: Location: Central, polar. Form: Spherical, elliptical, elongated. Wall: Thick, thin. 428 BACTERIOLOGY Size: Larger than vegetative cell, smaller than vegetative cell. Sprangium wall adherent, or not adherent. If irregularly shaped forms are present, make a note to that effect, and sketch. Record whether the culture is motile or not. Record whether Gram-positive or Gram-negative. STUDY OF PHYSIOLOGICAL AND CULTURAL CHARACTERISTICS For this work, besides the media listed above, tubes of plain milk, litmus- milk, and brom-cresol-purple milk should be prepared. Prepare these as follows: Plain milk. Place fresh skim milk in test tubes, about 10 cc. to the tube. Milk must be very carefully selected for this purpose, ordinary market milk being often too old. Test the milk by adding a drop of brom-cresol-purple to a little of it; if not acid, the milk takes a blue color. Acid milk can be neutral- ized with NaOH, but it is best to use fresh milk which requires no neutraliza- tion. The tubes should be plugged with cotton and sterilized in an autoclav at 15 pounds pressure for thirty minutes. Litmus milk. Before tubing, add enough litmus solution to the milk to give it a distinct blue color. The litmus solution is prepared by soaking 50 grams of dry litmus cubes with 250 cc. of water for twenty-four hours, and then filter- ing through filter paper. The tubes should be plugged and sterilized the same as the tubes of plain milk. Brom-cresol-purple milk. Make a saturated solution of brom-cresol-purple- in water. Add enough to make the milk a distinct blue, which will require perhaps 2 cc. to the litre of milk. Then tube and sterilize as usual. Inoculations. When all the necessary media are ready, the cultures selected for study should be transferred into fresh agar slants. When these fresh cul- tures are twenty-four hours old they should be inoculated into the following: Four agar slants of nitrate agar (or nitrate broth if the organism grows better in liquid media than on agar slants). Two gelatin stabs, (made by dipping a straight platinum needle into the mass of bacteria and then thrusting it straight into the gelatin hardened in a tube, carefully withdrawing it without disturbing the gelatin). Two fermentation tubes of each of the three sugars. Two agar slants of each of the three sugar agars with brom-cresol-purple. Two milk tubes. Two litmus milk tubes. Two brom-cresol-purple milk tubes. Two tubes of broth. Two tubes of peptone solution (1 per cent dissolved by boiling, then filtered). Incubate at room temperature, or at 25°C. if an incubator at that tempera- ture is available, except the gelatin stabs, which should be kept at 20°C. or lower. Then study as follows: Growth on plain agar slant. This should be studied when twenty-four hours old and again when three days old. The points to be noticed are: abundance, APPENDIX 429 form, elevation, lustre, topography, optical characters, chromogenesis, con- sistency, color of medium. On the page in the note book for each culture, and under the heading, agar slant, record those of the following terms which apply. (The meaning of the terms can be found in the glossary on p. 431.) Abundance: scanty, moderate, abundant, none. Form: Filiform, echinulate, beaded, spreading, arborescent, rhizoid. Elevation: Flat, effuse, raised, convex. Topography: Smooth, contoured, rugose. Optical characters: Opaque, translucent, opalescent, iridescent. Chromogenesis: Record color. Consistency: Butyrous, viscid, membranous, brittle. Color of medium: Medium grayed, browned, reddened, blued, greened. Gelatin stabs: Examine on first, second, fourth and seventh day after inoculation, and then keep two weeks longer and examine again. Record the form of growth along the line of puncture and the type of lique- faction, if any occurs, using those of the terms below which apply. Line of puncture: Filiform, beaded, papillate, villous, aborescent. Liquefaction: None, crateriform, napiform, infundibuliform, saccate, stratiform. Record day on which liquefaction begins. Growth in broth. Notice form of surface growth, presence of clouding, and nature of sediment. Record as follows: Surface growth: ring, pellicle, flocculent, membranous, none. Clouding: slight, moderate, or strong; transient, persistent or none; fluid turbid. Sediment: compact, flocculent, granular, flaky, viscid on agitation; abun- dant, scant, or none. Fermentation of sugars, a. Production of acid. Examine the tubes of the different sugar agars on the 1st, 2nd, 4th and 7th days, recording the acid as none, weak, moderate, or strong, according to the extent to which the purple of the indicator has changed to yellow. Notice whether all sugars are equally acidified, and if not which is the most so. b. Production of gas. There are two ways of observing whether a culture produces gas from any sugar: noticing the presence or absence of bubbles or cracks in the agar slant; and by noticing whether gas collects in the closed arm of the fermentation tube. Record which sugars give gas with each organism and whether in agar or in broth, or both. If gas is present in the fermentation tube, figure out whether the'closed arm is jV, |, |, |, or | full of gas. Action on milk. Study the tubes of milk on the first, second, fourth, seventh and tenth days, recording the day on which coagulation occurs (if the milk curdles), and the day on which peptonization begins, if the milk peptonizes (i.e., the casein digests, resulting in a clear yellow liquid). If neither coagu- lation nor peptonization takes place, record that fact. From the tubes of litmus milk and brom-cresol-purple milk, record whether acid is present on each of the above days. Notice whether the two indicators agree as to the reaction, and if not make a note of the difference. From the litmus milk, 430 BACTERIOLOGY notice whether the litmus is reduced (i.e., decolorized) and the day on which the reduction becomes noticeable; if the color subsequently reappears, make a note of that fact. Reduction of nitrate. Of the four tubes of nitrate agar (or broth) inoculated with each culture, one should be tested for nitrate reduction on the first day after the inoculation, one on the second, one on the fourth and one on the seventh. Notice whether gas bubbles or cracks are present in the agar. To test for nitrate prepare the following solutions. A. Sulphanilic acid, 8 grams; dilute sulphuric acid (1:20), 1000 cc. B. a-Naphthylamine hydrochloride, 5 grams; dilute sulphuric acid (1:125) 1000 cc. Place two or three drops of each reagent in each tube of broth or on the surface of each agar slant to be tested allowing the liquid to run down to the base of the slant. If nitrite is present a deep red zone will soon show at the base of the slant, which varies in depth with the quantity of nitrite produced. In the notes record whether each culture produces gas (probably free nitrogen) and whether nitrite is strong, weak or absent. The presence of either nitrite or gas indicates reduction of nitrate. A negative test, however, is not conclusive, as nitrite may be produced and converted into some other form so rapidly as to escape observation. So in case of negative results, merely state: Neither nitrite nor gas observed. Production of indol. On the fourth day test the tubes of peptone solution for the presence of indol by the following tests: Vanillin test. To 5 cc. of the culture add 5 drops of a 5 per cent solution of vanillin in 95 per cent alcohol and 2 cc. of concentrated sulphuric acid. Indol gives a clear orange by this test, which reaches its greatest depth in two or three minutes. A reddish violet (produced by tryptophane) should be dis- regarded. This test is not quite specific for indol, as one of the closely related com- pounds, produced as an intermediate product by the bacteria gives the same color as indol. So to make the test more definite the positive cultures should be tested by the more specific Ehrlich test. Ehrlich test. The reagent is a 2 per cent solution of paradimethyl-amino- benzaldehyde in 95 per cent alcohol. One cubic centimeter of this reagent is added to the culture, then drop by drop concentrated hydrochloric acid is added until a red zone appears between the alcohol and peptone solution. On standing the zone deepens and widens. The red color is soluble in chloro- form and the test may be confirmed by shaking the culture with chloroform to see if the pigment dissolves. If it proves soluble the test is considered positive. RECORDING THE CHARACTERISTICS After completing the above tests, the student should have a description of each organism on a sheet in his note book. If more than five or six cultures are studied it is always best to have these descriptions recorded on the stand- ard descriptive chart above mentioned (see plate facing p. 424). The APPENDIX 431 records from the note book can easily be transferred to the charts by under- lining on the chart those terms already recorded in the note books. Ordi- narily the bacteriologist makes his records directly onto the chart; but it is best for the student to record the characteristics of his first cultures in a note book before entering them on the charts, so as to familiarize himself with the terminology. GLOSSARY OF TERMS USED IN CHARACTERIZING BACTERIA Adherent, applied to sporangium wall, indicates that remnants of sporangium remain attached to endospore for some time. Aerobic, growing in the presence of free oxygen; strictly aerobic, growing only in the presence of free oxygen. Anaerobic, growing in the absence of free oxygen; strictly anaerobic, growing only in the presence of free oxygen; facultative anaerobic, growing both in presence and in absence of free oxygen. Arborescent, branched, tree-like growth. Beaded, in stab or stroke culture, disjointed or semi-confluent colonies along the line of inoculation. Bipolar, at both poles or ends of the bacterial cell. Brittle, growth dry, friable under the platinum needle. Butyrous, growth of butter-like consistency. Chains, four or more bacterial cells attached end to end. Chromogenesis, the production of color. Ciliate, having fine, hair-like extensions, resembling cilia, sometimes not visible to the naked eye. Clavate, club-shaped. Coagulation, the separation of casein from whey in milk. Contoured, an irregular, smoothly undulating surface, like that of a relief map. Convex, surface the segment of a sphere. Crateriform, a saucer-shaped liquefaction of the medium. Echinulate, a growth along line of inoculation with toothed or pointed margins. Effuse, growth thin, veily, unusually spreading. Endospores, thick-walled spores formed within the bacterial cell; i.e., typical bacterial spores like those of B. anthracis or B. subtilis. Filaments, applied to morphology of bacteria, refers to thread-like forms, generally unsegmented; if segmented, to be distinguished from chains (q. v.) by the absence of constrictions between the segments. Filamentous, growth composed of long, irregularly placed or interwoven threads. Filiform, in stroke or stab cultures, a uniform growth along line of inoculation. Flocculent, containing small adherent masses of bacteria of various shapes floating in the culture fluid. Fluorescent, having one color by transmitted light and another by reflected light. Granular, composed of small granules. Infundibuliform, form of a funnel or inverted cone. Iridescent, exhibiting changing rainbow colors in reflected light. . Membranous, growth thin, coherent, like a membrane. 432 BACTERIOLOGY Napiform, liquefaction in form of a turnip. Opalescent, resembling the color of an opal. Papillate, growth beset with small nipple-like processes. Pellicle, bacterial growth forming either a continuous or an interrupted sheet over the culture fluid. Peptonization, rendering curdled milk soluble by the action of trypsin. Peritrichiate, covered with flagella over the entire surface. Persistent, lasting many weeks or months. Plumose, a fleecy or feathery growth. Polar, at the end or pole of the bacterial cell. Radiate, showing ray-structure. Raised, growth thick, with abrupt or terraced edges. Reduction, removing oxygen from a chemical compound. Refers to the conversion of nitrate to nitrite, ammonia, or free nitrogen, and to the decolorization of litmus. Rhizoid, growth of an irregular branched or root-like character, as in B. mycoides. Ring, growth at the upper margin of a liquid culture, adhering to the glass. Rapid, developing in twenty-four to forty-eight hours. Rugose, wrinkled. Saccate, liquefaction in form of an elongated sac, tubular, cylindrical. Slow, requiring five or six days for development. Sporangia, cells containing endospores. Spreading, growth extending much beyond the line of inoculation, i.e., several millimeters or more. Stratiform, liquefying to the walls of the tube at the top and then proceeding downwards horizontally. Transient, lasting a few days. Truncate, ends abrupt, square. Turbid, cloudy with flocculent particles, i.e., cloudy plus flocculence. Viscid, growth follows the needle when touched and withdrawn; sediment on shaking rises as a coherent swirl. REFERENCE Manual of Methods for Pure Culture Study of Bacteria. Prepared by the Committee on Bacteriological Technic of the Society of American Bacteriologists. Published by the Society, Geneva, New York, 1923. (With revised folders of later date.) HI. REACTION OF MEDIA AND HYDROGEN-ION CONCENTRATION This chapter is added for the benefit of those students who have not had a thorough training in chemistry. It can be omitted if the course is not accom- panied by laboratory work, or if the laboratory work is purely of the most ele- mentary nature; but to complete all the laboratory work given in the last chapter, a knowledge of hydrogen-ion concentration and its relation to bac- teriological media is very helpful. WHAT IS HYDROGEN-ION CONCENTRATION? One of the theories which is most helpful in interpreting chemical phenom- ena is the ionic theory. According to this theory, every salt, acid, or base (i.e., alkali), when dissolved in water, breaks up to some extent into "ions." The ions in a solution of an acid are hydrogen on the one hand the acid radical (e.g., SO4) on the other; in a solution of a base they are the OH radical on the one hand and the metal on the other; in a solution of a salt, metal-ions and acid-ions are present. Even pure water (H2O) dissociates to a very small extent into H-ions and OH-ions. The H-ions bear a positive electric charge, the OH-ions a negative charge. Free H-ions are acid, free OH-ions are basic. In pure water both are present in equal amounts, thus neutralizing each other. Acids and bases dissociate to a much larger extent than pure water, the amount of dissociation depending ordinarily upon their strength. The stronger acids (sulphuric and nitric, for instance) ionize to a greater extent than the weaker (acetic, for instance and other organic acids). As all acids, no matter what their acid radical may be, yield H-ions upon disso- ciation, hence the more completely an acid ionizes in solution, the greater the H-ion concentration of that solution. Therefore, if different acids are dissolved in the same ratio of acid to water, the H-ion concentration is an index of the strength of the acid. Similarly, if different bases are dissolved in water in the same proportions, the OH-concentration is an index of the strength of the base. If to a quantity of pure water (containing H and OH-ions) a drop of sul- phuric acid be added, the mixture will contain H-ions, OH-ions and SOd-ions. The number of free H-ions will be greatly increased because of the large number added in the drop of acid; but the number of hydroxyl-ions will be decreased, because the change in ionic equilibrium will cause many of them to combine with free H-ions to form water. In other words, the H-ion con- centration will be increased, the OH-ion concentration decreased. If, further, a larger quantity of the acid be added, the H-ion concentration will be still further increased, the OH-ion concentration still further decreased. It is evident, therefore, that H-ion concentration increases and OH-ion concen- tration decreases as the acidity increases. Either may be used as an index of acid reaction; but H-ion concentration is preferred because it increases with increasing acidity. 434 BACTERIOLOGY When a base is added to water or to a neutral solution, the relation is exactly the reverse. The concentration of OH-ions is increased, that of H-ions is decreased. Either may be used as an index of alkalinity in terms of the ionic theory; but H-ion concentration is ordinarily used, for the sake of uniformity. Hydrogen-ion concentration is inversely proportional to the dissociation of the base and therefore to the true alkaline reaction of the solution. Obviously a saturated solution of a strong base such as KOH has an exceedingly small H-ion concentration, but it is nevertheless measurable. When an acid or base is mixed with water containing other materials in solution, the matter is more complex. Various combinations are possible between the acid or base and the other materials in solution which prevent the H-ion concentration from being increased or decreased as much as it would have been if the same amount of acid or base, respectively, had been added to pure water. Materials which thus repress the H-ion concentration of a solution upon the addition of acid, or the OH-ion concentration upon the addition of base, are called "buffers." Organic matter in solution is es- pecially likely to exert a buffer action. When buffers are present, the H-ion concentration of any acid solution will obviously be lower than it would have been with the same amount of acid present but no buffers in solution. The actual reaction of such a solution is also lower, for only the ionized portion of an acid exhibits acid properties. The H-ion concentration, therefore, is a more correct index of the acid or alkaline reaction in a case like this than is the quantity of acid added. All ordinary bacteriological media are highly buffered. This is really a very important fact; for in the absence of buffer, the slightest addition of excess acid either by bacterial growth or by careless adjustment of the reac- tion would render the media too acid to support life. As a result bacteria can hardly be cultivated in an unbuffered solution. The peptone generally added to bacteriological media is fully as valuable because of its buffer effect as because of the food it contains. BUFFERS THE SYMBOL pH After deciding to use H-ion concentration as a measure of reaction, the first necessity is to obtain some simple and concise method of expression. For this purpose the figures actually expressing H-ion concentration are quite unwieldy because 'of its extreme minuteness in weakly acid and in basic solu- tions. The H-ion concentration of pure water, for example, has been shown to be 0.000,000,1 gram per litre. A simpler method of indicating this small quantity is by the logarithmic form expression, i.e., 10-7 or log-7. This method of expression has been quite generally adopted and the symbol pH is used to represent the logarithm, with the minus sign omitted; or using mathematical language, pH is the logarithm of the reciprocal of the H-ion concentration expressed in grams per litre. This symbol is now quite APPENDIX 435 generally used to signify H-ion concentration. By this method of expression, the H-ion concentration of pure water is indicated by the formula pH = 7.0. Because pH is an invert logarithm, it decreases with increasing H-ion con- centration. Therefore, acid solutions have a pH-value smaller than 7.0, basic solutions a pH-value greater than 7.0. It has been found, for instance, that the pH-value for tenth normal HC1 is 1.05, for normal acetic acid 2.4, while for tenth molecular NH40H it is 11.2. If desired the OH-concentration of a solution may be expressed by the similar symbol pOH, which is also an invert logarithm. For any solution the sum of the pH-value and the pOH -value is 14, and hence if one is known the other can be computed. Thus for normal acetic acid (pH = 2.4) thepOH; value is 14 - 2.4 = 11.6. There are two different methods by which the H-ion concentration of a solution may be determined: the electrometric method and the colorimetric method. The electrometric method is generally the more accurate; but it is time-consuming and requires quite complicated apparatus. The colorimetric method is much simpler, is accurate enough for all ordinary bacteriological work and can be applied easily to any ordinary bacteriological culture medium. Hence it is the only method we will consider here. This method depends upon color changes produced in certain substances (indicators) by varying reaction. Each indicator changes from one color to another between quite narrow limits of H-ion concentration, and between these two limits every shade of the indicator corresponds to a definite pH-value. Bacteriologists have long used litmus, which is valuable for certain purposes, but does not give accurate H-ion measurements. We have now in place of litmus quite a series of very accurate indicators whose sensitive ranges meet or even overlap and extend from pH = 1 to pH = 10. The relations of these indicators to H-ion concentration is shown in figure 59, and in the table on p. 438. Of special interest to bacteriologists are the three indicators nearest to the neutral point (pH = 7.0), i.e., brom cresol purple, brom thymol blue, and phenol red. Of these brom thymol blue is most useful in adjusting the reac- tion of media to neutrality, because it is yellow in acid solutions and blue in basic solutions, passing through various stages of green between the pH-values 6.0 and 7.6. At the neutral point it is grass-green, a shade that can be easily be recognized by the eye after a little practice. As a result it is possible to obtain media that are essentially neutral, no matter what their composition may be, merely by adding sufficient acid or base so that brom thymol blue becomes a grass-green when added to them (see p. 421). It is plain that a rough idea can be obtained as to the H-ion concentration of any solution by simply finding which indicators give their acid color in it and which give their alkaline color. After a little experience with these indi- cators it is possible to do even better than this by inspection of the shade of color produced by whichever indicator or indicators are sensitive at the pH- vahie of the solution in question. Accuracy, however, can best be obtained by MEASUREMENT OF H-ION CONCENTRATION 436 BACTERIOLOGY TABLE 3 INDICATOR CONCEN- TRATION RECOM- MENDED FULL ACID COLOR FULL ALKALINE COLOR SENSITIVE RANGE Thymol blue (acid range) per cent 0.04 Red Yellow pH 1.2 - 2.8 Brom phenol blue 0.04 Yellow Blue 3.0 - 4.6 Methyl red 0.02 Red Yellow 4.4 - 6.0 China blue 0.06 Blue Colorless 5.0 - 7.0 Brom cresol purple 0.04 Yellow Purple 5.2 - 6.8 Brom thymol blue 0.04 Yellow Blue 6.0 - 7.6 Phenol red 0.02 Yellow Red 6.8 - 8.4 Cresol red 0.02 Yellow Red 7.2 - 8.8 Rosolic acid 0.12 Colorless Red 7.0 - 9.0 Thymol blue (alkaline range).. 0.04 Yellow Blue 8.0 - 9.6 Phenolphthalein 0.02 Colorless Red 8.0 - 9.6 Cresolphthalein 0.02 Colorless Red 8.2 - 9.8 Color changes of certain indicators actual comparison with the colors produced by the indicators in solutions of known H-ion concentration. Such standards can be prepared quite easily in the chemical laboratory; but for the bacteriological laboratory sufficient accuracy is best procured by a different method. This method can be best understood if the student will perform the following exercise. Color standards for H-ion determinations Prepare a dilute solution of some acid (e.g., 1 cc. concentrated HC1 in 120 cc. water, i.e., about 0.1 normal) and a dilute solution of some alkali (e.g., a 0.4 per cent solution of NaOH or about 0.1 normal). Select eighteen test tubes about half an inch in diameter, being careful to see that they are all of about the same bore. Place 10 cc. of the distilled water in each; to nine of them add one drop of the dilute acid, and one drop of the dilute alkali to each of the other nine. Then arrange in two rows in a test tube rack, the alkali in front, and the acid behind, thus obtaining nine pairs of tubes, an acid and an alkali tube in each pair. Then select any one of the indicators for which it is desired to obtain color standards, brom thymol blue for instance. This should be used in an alcoholic solution of the strength given in table 3. With a 1 cc. pipette graduated into tenths, measure out 0.1 cc. and place in the left hand tube of alkali, measure out 0.2 cc. for the second tube of alkali, 0.3 cc. for the third and so on, putting 0.9 cc. in the last. Then start with the left hand tube of acid and add 0.9 cc. of indicator to it, place 0.8 cc. in the second, and so on, adding 0.1 cc. to the last. It will be seen then that the first pair of tubes (pair no. 1) has 0.1 cc. in the alkali and 0.9 cc. in the acid, the second (pair no. 2) 0.1 in the alkali and 0.8 cc. in the acid, and so on, each pair of tubes containing between them just 1 cc. of the indicator. APPENDIX 437 Fig. 59. Color Changes in Clark and Lubs' Indicators Compared with the pH-Values of Certain Important Solutions 438 BACTERIOLOGY Now look at a source of light (daylight) through each pair of tubes in succession. The first pair of tubes gives a shade of the indicator very near its acid color, the next a little less so while the sixth is very near its alkaline color. Each of these shades of color corresponds to that obtained by adding the indicator in the proportion of 1:10 to a solution of some definite H-ion concentration. The pH-value corresponding to each pair in the case of the various indicators can be determined from table 4. These color standards give accurate results only when the solution to be tested is colorless. Nearly all bacteriological media are somewhat colored, and in using the colorimetric method, allowance for this has to be made. It is done as follows: Fill two tubes with the medium to be tested, to one of these tubes adding the proper indicator in the proportion of 1:10. Leave the other TABLE 4 pH values corresponding to the successive pairs of tubes in the color standards described on p. 437 BROM* PHENOL BLUE METHYL RED BROM CRESOL PURPLE BROM THYMOL BLUE PHENOL RED CRESOL RED thymol! BLUE Pair no. 1 3.1 4.05 5.3 6.15 6.75 7.15 7.85 Pair no. 2 3.5 4.4 5.7 6.5 7.1 7.5 8.2 Pair no. 3 3.7 4.6 5.9 6.7 7.3 7.7 8.4 Pair no. 4 3.9 4.8 6.1 6.9 7.5 7.9 8.6 Pair no. 5 4.1 5.0 6.3 7.1 7.7 8.1 8.8 Pair no. 6 4.3 5.2 6.5 7.3 7.9 8.3 9.0 Pair no. 7 4.5 5.4 5.7 7.5 8.1 8.5 9.2 Pair no. 8 4.7 5.6 6.9 7.7 8.3 8.7 9.4 Pair no. 9 5.0 5.95 7.2 8.05 8.65 9.05 9.75 * In preparing the standards for this indicator, use more acid in the acid tubes-at least 1 cc. of 1:120 HC1. f The alkali tubes in the case of this indicator must contain at least two drops of 0.4 per cent NaOH. tube without addition. Place the tube of plain media behind the twTo tubes of the color standard with which it is desired to compare the medium, and see whether the shade obtained by looking at the light through all three tubes is the same as that of the medium containing the indicator. In doing this it is generally desirable to look at the medium containing the indicator through two tubes of plain water in order to get a depth corresponding to that of the color standards. WHY THE TITRATION METHOD IS ILLOGICAL Until recently the reaction of bacteriological media has always been ad- justed by titration to phenolphthalein. A computation was made of the per cent of normal alkali necessary to neutralize the solution and the acidity was stated as equivalent to this per cent of normal acid; then acid or alkali was APPENDIX 439 added so as to make the "reaction" 1 per cent normal acid. The assumption upon which this procedure is based is that the reaction of the solution is measured by the amount of base necessary to neutralize. Ten cubic centi- meters of N/10 HC1 and 10 cc. of N /10 acetic acid each require 10 cc. of N/10 NaOH to be neutralized, but the HC1 is much more acid than the acetic acid. In bacteriological media, moreover, there are nearly always present certain materials which have a buffer effect. Although but weakly acid, they combine to a marked extent with the base used in titration and thus prevent the neutral- ization of the solution as rapidly as would be the case if no buffer were present. Peptone, for example, has a very strong buffer effect upon solutions in which it is present. As a result, in peptone solutions w'hich are actually neutral, the titrable acidity may be quite high, because the NaOH used in titration com- bines with the peptone without greatly lowering the reaction of the solution. Hence titration is a very illogical method of determining the reaction of solu- tions containing peptone. Some brands of peptone are neutral, as determined by brom thymol blue, and yet titrate 1 per cent acid to phenolphthalein in 1 per cent solutions, because of their high buffer content; others are acid to brom thymol blue, and yet titrate considerably under 1 per cent acid in 1 per cent solutions, because of a lower buffer content. Furthermore, any peptone solution has practically the same H-ion concentration no matter what its dilution may be; but the titrable acidity is lower the more it is diluted. Hence the titration method leads to the error of adding acid to a 0.1 per cent solution of peptone to make its "reaction" the same as that of a 1 per cent solution. A further fault of the titration method is that there is no sharp phenolphtha- lein neutral point. The indicator begins turning red (see table 3) at about pH = 8.0 but does not reach its full alkaline color until about pH = 10.0. Some bacteriologists have used its full color as an end point, others have stopped at the first appearance of pink, while others have tried to use a mid- point. The result has been considerable variation in the hands of different men. The titration method is still in use in some laboratories, and is still neces- sary for some purposes, but it is plainly quite illogical to employ it for adjusting the reaction of media or for determining the reaction caused by bacterial growth. The colorimetric method of determining H-ion concentration is more logical, is of more significance, and is more simple to use, after the prin- ciples of the technic are once learned. REFERENCES W. M. Clark and H. A. Lubs. The Colorimetric Determination of Hydrogen- ion Concentration. Jour. Bact., ii, 1-34, 109-136, 191-236. 1917. W. M. Clark. The Determination of Hydrogen-ions. 2d edition. Williams & Wilkins Co., Baltimore, 1922. INDEX The principal references are given in bold-faced type Abortion, contagious, 357 Acetic acid bacteria, 229 Actinomyces, 101 bovis, 367 scabies, 408 Actinomycetes, 91, 93, 171, 364 Actinomycosis, 364 Aerobacter, 99 Agar, 47, slant, 53 Agglutination, 286 Alcoholic beverages, 222-225 fermentation, 73, 128 Alinit, 198 Ammonification, 180-182 Anaphylaxis, 219, 279 Anopheles, 339 Anthracnose of beans, 412 Anthrax, 31,251, 354 355 mastery of, 57-59 organism, 33, 360 vaccine, 59 Anti-bacterial substances, 269, 278 Antibodies, 268 Antiseptic surgery, 30 Antitoxin, 61, 243, 268, 278, 283 diphtheria, 61-63, 64, 243-244 tetanus, 356 Appert, 19, 215 Apple, bitter rot of, 414 rust, 416 scab, 414 Asci, 409 Ascomycetes, 409 Asparagus rust, 416 Attenuated viruses, 58, 277-278, 283 Autoclav, 49 Autotrophic bacteria 69, 106 Azotobacter, 99, 192, agilis, 192 chroococcum, 192 Bacillus, 36, 98 alvei, 368 amylovorous, 404 anthracis, 360 botulinus, 220, 244, 358 bulgaricus, 125 caratovorous, 406 cereus, 171, 198, 376 larvae, 368 megatherium, 376 mycoides, 171, 376 orpheus, 368 radicicola, 189 subtilis, 37 tetani, 356 tracheiphilus, 407 Bacteria, chromogenic, 35 colonies of, 45, 46 effect of light upon, 102 effect of moisture on, 103 effect of temperature on, 102- first observations of, 13 first use of term, 36 in relation to mineral matter, 197 kinds of, in soil, 170 microscopic study of, 425 numbers of, in milk, 120 in soil, 171 in water, 373 physiology of, 102-109 relation to carbon, 177-179 relation to lime, 197 relation to nitrogen, 180-194 relation to phosphorous, 195 relation to sulfur, 196 reproduction of, 87-89 size and shape, 85-87 spores of, 20, 88 with fat drops, 90 Bacteriaceae, 98 442 INDEX Bacterial association, 104-105 Bacterial wilt of melons and cucum- bers, 407 Bacteriophage, 271-274 Bacteriotropins, 279 Bacterins, 243, 279 Bacterium, 17, 36, 99 abortus, 363 acidi lactici, 122 aerogenes, 124, coli, 122, 124, 377, 383, 392 diphtheriae, 314 Guntheri, 122 lactis, 121 lactis acidi, 122 mallei, 362 pertussis, 317 pestis, 335 pluton, 368 pullorum, 366 suipestifer, 365 tuberculosis, 349 typhosus, 323 Basidia, 410 Basidiomycetes, 410 Bean blight, 416 Beans, anthracnose of, 412 Beer, 223 Beggiatoa, 100 Behring, 62, 268 Beijerinck, 192 Bismarck brown, 426 Bitter rot of apple, 414 Black death, 334 Black knot of plums and cherries, 413 Black plague, 334 Black rot of cabbage, 405 of grapes, 414 Black rust of wheat and rye, 416 Bordeaux mixture, 400, 403 Botulism, 357-358 Bouillon, 51, 421 Bread, faults of, 228 -raising, 225-228 Breaking-down processes in nature, 69-71 Bremia lactucae, 411 Broad irrigation, 396 Brom-cresol-purple, 422, 428, 435 milk, 428 Brom-thymol-blue, 421, 435 Broth, 51, 421 Brown rot, of lemon, 410 of peaches, plums, etc., 412 Bubonic plague, 297, 334-337 Budding, 36 Buffers, 434 Building-up processes in nature, 68- 69 Bunt (or stinking smut) of wheat, 415 Burrill, 64, 401 Butter, 153, 154 faults, 157 substitutes, 157 Butyric acid, 25 fermentation, 25 Cabbage, black rot of, 405 club root of, 417 used for sauerkraut, 238 Camembert cheese, 162, 163 Canal Zone, sanitation of, 296 Canning, 19, 215, 217 fruits, 217 vegetables, 215 Carbon cycle, 177-179 Caron, 198 "Carriers," of disease organisms, 298, 315, 327 Catalysts, 77 Cedar apples, 416 Cellulose, 178-179 -decomposing organisms, 178 Cheese, 158-164 American, 159 Camembert, 162 Cheshire, 159 faults, 160 Gorganzola, 162 hard, 159 Limburger, 162 ripening of, 158 INDEX 443 Roquefort, 160, 162 soft, 161 Swiss, 159,160 Cherries, black knot of, 413 Chicken cholera, 57, 366-367 Chlamydobacteriaceae, 93 Chlorophyl, 81 Cholera, 329 epidemic at Hamburg, 292, 331 Chromogenesis, 35 Cilia, 80 Clay soil, 168 Coccaceae, 97 Cohn, 17, 36 Cold storage, 212 Coli-aerogenes group, 124 Colletotrichum lindemuthianum, 412 Colonies of bacteria, 45, 46 Complement, 288 -fixation, 288 Condenser, Abbe, 40 Conidia, 82 Contact beds, 397 Contagion, 291-292 Contagious abortion, 363 Contagious diseases, 255 Corn smut, 415 Corynebacterium diphtherias, 314 Cotton, wilt of, 413 Counting bacteria, by plate method, 54, 142, 380 under microscope, 43, 54, 143 Cowpox, 57, 304 Cladothrix, 100 Classification of bacteria, 16, 34-37, 92-101 Claviceps purpurea, 413 Clay loam, 168 Clostridium, 98 botulinum, 221 Club root of cabbage, 417 Cream ripening, 153 Crop selection, 204-205 Crown gall, 407 Crystal violet, 425 Cucumbers, bacterial wilt of, 407 Cultivation, 202 Cultural characteristics, 95 Cultures, isolation and study of, 52-53 Curdling, acid, 51, 130 sweet, 127 Cyst, 81 Cystopus candidus, 410 Dairy inspection, 138-139 Damping off, 410 "Darwinizing," 42 Davaine, 31, 251 Decay, 72-73 Decomposition, 71 of food,210 Denitrification, 185-187 Density of solution, effect on bac- teria, 104 Descriptive chart, 424, 430 Diagnosis of disease, 285-289 Diastase, 74, 76, 223, 239 Dilution method of obtaining pure cultures, 43-44 Diphtheria, 147, 314 antitoxin, 61-63, 64, 243-244 organism, 90, 314 Diseases of poultry, 366 Distilled spirits, 225 Downy mildews, 410 Drying food, 211, 217 Dyes, 40 Dysentery, 332 Early blight of potato, 411 Ehrenberg, 17, Ehrlich, 63, 269 Elimination of disease germs, 255 Enzymes, 73-79, 158 Epidemics, food-borne, 294 milk-borne, 294, 325 tracing the cause of, 221 water-borne, 292, 328 Ergot, 413 Eubacteria, 97 Eumycetes, 409 Exoascus deformans, 412 444 INDEX Fermentation, 21, 71, 73-79 alcoholic, 73, 128 butyric, 25 Liebig's theory of, 22 Pasteur's theory of, 25 Ferments, organized, 76 unorganized, 76 Fertility, permanent, 205-207 Filters, slow sand, 388 porcelain, 254, 386 rapid, 389 Filtration, of sewage, 396-397 of water, 388-389 Fire blight, 404 Fischer, Alfred, 65, 401 Fission, 36, 80, 87 Flagella, 80, 86, 98 Fleas, in relation to plague, 336 Flies, in relation to typhoid, 297, 399 Foot and mouth disease, 365-366 Food poisoning, 219-221 Food, preservation of, 209 spoiling of, 209-210 Fowl diphtheria, 367 Fracastoria, 15 Fuchsin, carbol, 425 Fungi, 81-84,253, 409 "Gas bacillus," 351 Gaseous gangrene, 351 Gelatin, 45 media, 423 Gentian violet, 425 Germicides, effect of on bacteria, 104 Germ theory of disease, 27, 30, 34 250-258 Glanders, 287, 362-363 Glomerella rufomaculans, 414 Gonococcus, 309 Gonorrhea, 309-310 Gorgonzola cheese, 162 Gram stain, 427 Grapes, black rot of, 414 Green-manuring, 200 Guignardia bidwellii, 414 Gymnoconia pekiana, 416 Gymnosporantium macropus, 416 Hemorrhagic septicemia, 364 Hamburg, cholera epidemic of, 272, 331 Hansen, 240 Haplobacteria, 97 Hay, brown, 235 burnt, 235 Hellriegel, 188 Henle, 27, 250 Henle's principles for determining the cause of a disease, 56, 252 d'Herelle, 272 phenomenon, 272 Hoffman, 20 Hog cholera, 364r-365 Hudson-Mohawk epidemic, 381 Humus, 176-177 Hydrogen-ion concentration, 433-139 measurement of, 435-438 Hydrophobia, 59, 343-344 Ice cream, 153 Ice in relation to typhoid, 328 Imhoff tank, 375 Immunity, 259-271 active, 263 acquired, 262-263 artificial production of, 275-289 biological theory of, 264r-267 chemical theory of, 267-270 Ehrlich's theory of, 63, 269 Metchnikoff's theory of, 63, 264-267 due to individual resistance, 261- 262 natural, 262 passive, 263 side-chain theory of, 269 of a race, 260 theories of, 63-64 Indicators, 52, 422, 435-438 Indol, production of, 430 Infantile paralysis, 320-321 Infectious diseases, 254 Infectious jaundice, 345 INDEX 445 Influenza, 318-320 Insect-borne diseases, 296 Insects, diseases of, 367-368 distribution of disease by, 295-297 Invasion by disease organisms, 362 Involution forms, 87 Jaundice, infectious, 345 Jenner, 57, 304 Kefir, 128 grains, 128 Kircher, 13 Kitasato, 61, 268 Klebs, 42, 314 Koch, 27, 32, 44, 55, 251, 285, 350 Koch's postulates, 27, 56, 252 Kuhn, 400 Lactic acid, 25 organism, 42, 44, 121, 155 Lactobacillus, 99, 125, 160 acidophilus, 126 bulgaricus, 121, 125 Late blight of potato, 411 Latour, 22 Leaven, 226 Leavened bread, 21, 226 Leeuwenhoek, 13 Legume nodules, 188 organism, 188-191, 199 Legumes, 188, 199, 204 Lemon, brown rot of, 410 Leprosy, 307 Leptospira icteroides, 340 Lethargic encephalitis, 322 Leucocytes, 265 Liebig, 22, 79 Limburger cheese, 162 Lime, 196 Lister, 30, 43, 121, 347 Litmus, 52 milk, 428 Lock-jaw, 356-357 Loeffler, 314 Loeffler's methylen blue, 425 Loose smut of wheat, 415 of oats, 415 Lophotrichic flagella, 98 Lugol's iodine solution, 425 Lysins, 269 Macrosporium solani, 411 Malaria, 337-340 organism, 338 Malignant oedema, 362 Mallein, 245, 286 Malt, 223 Manure, handling of, 207-208 Measles, 316-317 Media, culture, 51-52, 421-423 reaction of, 52, 433 solid, 44-47 Melons, bacterial wilt of, 407 Meningitis, 317-318 Meningococcus, 317 Metabolism, end products of, 175-176 of bacteria, 105-106 Metachromatic granules, 90 Metchnikoff, 62, 125, 264 Methods, development of, 39-54 Methylen blue, Loeffler's solution, 425 Microbiology, 71 Micrococcus, 36, 97 Micrococci, 87, 121 Microscopes, 39 Microspira, 100 comma, 330 Milk, bacterial analysis of, 139, 142- 144 bacteriological standards for, 139 bitter, 128 certified, 140 chemical analysis of, 139 colored, 128 cooling of, 136-138 diseases distributed by, 146-147 faults, 128 grading of, 139-142 growth of bacteria in, 119-120 kinds of bacteria in, 121-128 legal standards for, 139 market problems, 132-144 446 INDEX numbers of bacteria in, 115, 120 sanitary significance of bacteria in, 145-152 sources of bacteria in, 115-116 Milking machines, 116-118, 120, 136 Milking utensils, 116 Milk pails, small-topped, 135 Molds, 82, 210 Monas, 36 Monotrichic flagella, 98 Muller, 16 Multiplication of bacteria, 87 prodigious rate of, 37 Mushrooms, 81 Mycelium, 82 Mycobacterium tuberculosis, 349 Mycosphoerella fragariae, 414 Naegeli, 17 Needham, 18 Negri, 343 bodies, 343 Neocosmospora vasinfecta, 413 Nitragin, 200 Nitrate agar, 423 broth,423 reduction of, 430 Nitrification, 182-185 Nitrobacter, 99, 183 Nitrogen, recovering loss of, 187, 188 Nitrogen cycle, 180-197 diagram of, 186 N itrogen-fixation, non-symbiotic, 191-192 significance of, 192 symbiotic, 188-191 Nitrosococcus, 183 Nitrosomonas, 99, 183 Nutrient agar, 422 gelatin, 423 Oats, loose smut of, 40 Oidium lactis, 162 OH-concentration, 433 Opsonins, 270, 279 Origin of microscopic life, 18 Oxygen, relation of bacteria to, 103 Oysters in relation to typhoid, 326 Parasites, 107 Pasteur, 20, 23, 28-29, 32, 38, 56, 57 59, 77, 250 and fermentation, 24-26 Institutes, 60, 240, 242 Pasteurization, 26,150-152,156, 224 Peach leafcurl, 412 Pear scab, 414 Pebrine, 28,250-251, 367 Penicillium camembertii, 162 Pepsin, 76, 158 Peptone, 51, 421 Peptonizing bacteria, 127 Peritrichic flagella, 98 Peronospora parasitica, 411 schleideniana, 411 Perty, 17, 92 Petri dishes, 46 Phagocytes, 264-267 Phenolphthalein, 438 Phenol red, 435 Phosphorus, 195 Phycomycetes, 409 Physiological groups of bacteria 106-109 Physiology of bacteria, 102-109 Phytophthor a infestans, 411 phaseoli, 411 Plague, 297, Plant food, 174-175 Plant pathology, history of, 400 Plasmodiophora brassicae, 417 Plasmopara cubensis, 411 viticola, 411 Plate method, 44-48, 142, 252 Platinum needles, 53 Plectridium, 99 Pleomorphism, 34 Plenciz, 15 Plowrightia morbosa, 413 Plums, black knot of, 413 Plymouth epidemic, 292, 380 Poliomyelitis, 320-321 Potato, early blight of, 411 INDEX 447 Potato, late blight and rot of, 411 scab, 408 Poultry, diseases of, 366 Pneumococcus, 313 Pneumonia, 312-314 Preservatives, 213, 217 Protective inoculation, 56-57 Protista, 92 Protozoa, 80-81 disease production by, 253 in soil, 203 Pseudomonas, 99 aeruginosa, 349, 367 campestris, 405 fluorescens, 171, 376 phaseoli, 406 pyocyanea, 349, 367 radicicola, 189 tumefaciens, 407 Ptomain poisoning, 220 Public health, science of, 362-363 Puccinia asparagi, 416 rubigo-vera, 416 Pure cultures, 41-43 Putrefaction, 24, 72 Pythiacystis citrophthora, 410 Pythium de Baryanum, 410 Pyogenic diseases, 347-349 Pyronin, 426 Rabies, 59, 343 344 vaccine, 242 Rats, in relation to plague, 297, 336 Reaction of media, 52, 433-439 Redi, 18 Refrigeration, 212-213, 217 Rennet, 158 Reproduction, 87-89 Retting of flax and hemp, 238 Rhizobium, 99 radicicola, 189 Rhizoctonia, 417 Rivalry between French and Ger- mans, 60-61 Root rot of tobacco, 413 Ropy milk, 128 Roquefort cheese, 163 Roux, 61, 62 Rusts, 415 Saccharomycetes, 84 Safranin, 426 Salt pickles, 237 Sand, 168 Sanitation, 290-301 of canal zone, 296 Saprophytes, 107 Sarcinae, 87, 98 Sauerkraut, 213, 238 Scarlet fever, 147, 307-308 Schick test, 289 Schizomycetes, 18, 85 Schroeder, 20 Schroeter, 35, 44 Schulze, 19 Schwann, 19, 22 Sclerotinia fructigena, 412 Septic sore throat, 147-148, 308-311 Septic tanks, 394-396 Serum, sickness, 278-279 therapy, 278 Sewage, 372-373, 392-399 chemical precipitation of, 384 filtration of, 396 purification of, 393-399 sedimentation of, 393-394 Silage, 235-236 Silo, 235 Silt, 168 loam, 168 Sleeping sickness, 341-343 Slimy milk, 128 Sludge, activated, 398-399 disposal of, 394 Smallpox, 57, 302-306 vaccine, 57, 241, 304 Smith, Erwin, 401 Smut, loose, 415 stinking, 415 Society of American' Bacteriologists, 96, 424 Soft rot, 406 448 INDEX Soil, as habitat for bacteria, 167-170 control of bacteria in, 198 inoculation, 198 partial sterilization of, 202-204 structure and composition of, 168 texture of, 168 Spalanzini, 19 Species of bacteria, 94 Spirillaceae, 94, 100 Spirillum, 17, 36, 100 Spirochaeta, 17, 36 Spirochaetae, 94 Spirosoma, 100 Spontaneous generation, 18 Spores of bacteria, 20 discovery of, 37 germination of, 89 Squirrels in relation to plague, 297, 336 Staining bacteria, 40 Staining solutions, 425 Staphylococcus, 97 pyogenes, 121 Starters, 155 Sterilization, 49 Stinking smut of wheat, 415 Strawberry leafspot, 414 Streptococci, 87, 121, 134, 148 Streptococcus, 97 lacticus, 122 lactis, 122 pyogenes, 121, 310, 340 Subsoil, 165 Sugar broths, 421 Sulfur, 196 Sun, as source of energy, 68 Surface soil, 165 Symbiosis, 179, 191 Symptomatic anthrax, 361 Syphilis, 310-312 Taka-diastase, 239 Tanning, 238 Temperature, relation of bacteria to, 103 Tetano-antitoxin, 356 Tetano-toxin, 356 Tetanus, 356-357 Thermal death point, 103 Thielvaia basicola, 413 Thunder storms, in relation to the souring of milk, 120 Tilletia foetens, 415 Tilth of soil, 169 Titration, 438 Tobacco, curing of, 236-237 root rot of, 413 Toxins, 257, 268, 283 Transmission of disease, 254r-255 Transportation of disease, 255 Trench fever, 346 Treponema pallidum, 311 Trichobacteria, 97 Trickling beds, 397 Trypanosomes, 342 Tsetse fly, 341, 342 Tubercle organism, 56, 349-350 Tuberculin, 245, 285, 354 Tuberculosis, 146, 349-356 among cattle, 353 distribution of, 352 resistance to, 353 Twort 272 Tyndall, 20 Typhoid fever, 146, 287, 322-332 bacillus carriers, 147 Hudson-Mohawk epidemic, 381 Plymouth epidemic, 292, 380 Typhus fever, 345 Udder, bacteria in, 115 diseases of, 115, 133 Ultra-microscopic organisms, 91, 253 Urea, 181 Ustilago avenae, 415 tritici, 415 zeoe, 415 Vaccination, 57, 262, 283, 305 Vaccines, anthrax, 59 autogenous, 281 bacterial, 243, 279, 283 rabies, 242 smallpox, 57, 241-242, 304 INDEX 449 Vaccine therapy, 279 Venturia pomi, 414 pyrina, 414 Vinegar, barrel, 231 making, 228-234 Orleans method, 230 quick method, 232, mother of, 229 Vibrio, 36 Vitamines, 151 Warrington, 182 Wassermann reaction, 288-289 Watermelon, wilt of, 413 Water, action of bacteria upon, 374 bacteriological analysis of, 382 chemical analysis of, 384 chemical treatment of, 389-391 detecting pollution of, 382-385 filtration of, 388-389 kinds of bacteria in, 375-377 numbers of bacteria in, 373 pure, 378 sanitary analysis of, 385 sedimentation in, 387 source of bacteria in, 370-373 Water-borne diseases, 879-382 Water glass for preserving eggs, 213 Weils disease, 345 Wheat, black rust of, 415 bunt or stinking smut of, 415 loose smut of, 415 White diarrhoea of chicks, 366 White rust, 410 Whooping cough, 317 Wilfarth, 188 Wilt of cotton and watermelon, 413 Wines, 222 Winogradski, 182, 192 Wood, decomposition of, 177-178 Wright, 280 Yeasts, 21, 36, 74, 78, 84, 210, 253 Yellow fever, 296, 340-341 Yersin, 61 Zymase, 78