Z.6- UNIVERSITY OF CALIFORNIA LIBRARY THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW j\J MAR DEC JAN* 19B hn3V4cEC ' Xn 5m-10,'22 A TEXTBOOK OF BACTERIOLOGY A TEXTBOOK OF B ACT E R I OLOG Y A PRACTICAL TREATISE FOR STUDENTS AND PRACTITIONERS OF MEDICINE AND PUBLIC HEALTH BY HANS ZINSSER, M.D. PROFESSOR OF BACTERIOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNI- VERSITY, NEW YORK CITY; BACTERIOLOGIST TO THE PRESBYTERIAN HOSPITAL; FORMERLY PROFESSOR OF BACTERIOLOGY AND IMMUNITY, STANFORD UNIVERSITY, CALIFORNIA; COLONEL MEDICAL OFFICERS' ' ' RESERVE CORPS, U. S. A. • • Y » *( WITH A SECTION ON PATHOGENIC PROTOZOA BY FREDERICK P. RUSSELL, M.D. BRIGADIER GENERAL, MEDICAL OFFICERS' RESERVE CORPS, U. 8. A., FORMERLY PROFESSOR OF BACTERIOLOGY AND PATHOLOGY, ARMY MEDICAL SCHOOL AND GEORGE WASHINGTON UNIVERSITY (Completely revised and rewritten from the original text of Hiss and Zinsser) WITH ONE HUNDRED AND SIXTY-FOUR ILLUSTRATIONS IN THE TEXT FIFTH EDITION D. APPLETON AND COMPANY NEW YORK :: 1922 :; LONDON COPYIUGHT, 1910, 191-1, 1916, 1918, and 1922, BY D. APPLETON AND COMPANY our ITY T<1-' PRINTED IN THE UNITED STATES OF AMERICA PREFACE TO THE FIRST EDITION THE volume here presented is primarily a treatise on the funda- mental laws and technique of Bacteriology, as illustrated by their application to the study of pathogenic bacteria. So ubiquitous are the bacteria and so manifold their activities that Bacteriology, although one of the youngest of sciences, has already been divided into special fields — Medical, Sanitary, Agricul- tural, and Industrial — having little in common, except problems of general bacterial physiology and certain fundamental technical procedures. From no other point of approach, however, is such a breadth of conception attainable, as through the study of bacteria in their relation to disease processes in man and animals. Through such a study one must become familiar not only with the growth character- istics and products of the bacteria apart from the animal body, thus gaining a knowledge of methods and procedures common to the study of pathogenic and non-pathogenic organisms, but also with those complicated reactions taking place between the bacteria and their products on the one hand and the cells and fluids of the animal body on the other — reactions which often manifest themselves as symptoms and lesions of disease or by visible changes in the test tube. Through a study and comprehension of the processes underlying these reactions, our knowledge of cell physiology has been broadened, and facts of inestimable value have been discovered, which have thrown light upon some of the most obscure problems of infection and immunity and have led to hitherto unsuspected methods of treatment and diagnosis. Thus, through Medical Bacteriology — that highly specialized offshoot of General Biology and Pathology — have been given back to the parent sciences and to Medicine in general methods and knowledge of the widest application. It has been our endeavor, therefore, to present this phase of our subject in as broad and critical a manner as possible in the sections dealing with infection and immunity and with methods of biological diagnosis and treatment of disease, so that the student and practi- 47T0203 vi PREFACE TO THE FIRST EDITION tioner of medicine, by becoming familiar with underlying laws and principles, may not only be in a position to realize the meaning and scope of some of these newer discoveries and methods, but may be in better position to decide for themselves their proper application and limitations. We have not hesitated, whenever necessary for a proper under- standing of processes of bacterial nutrition or physiology, or for breadth of view in considering problems of the relation of bacteria to our food supply and environment, to make free use of illustrations from the more special fields of agricultural and sanitary bacteriology, and some special methods of the bacteriology of sanitation are given in the last division of the book, dealing with the bacteria in relation to our food and environment. In conclusion it may be said that the scope and arrangement of subjects treated of in this book are the direct outcome of many years of experience in the instruction of students in medical and in advanced university courses in bacteriology, and that it is our hope that this volume may not only meet the needs of such students but may prove of value to the practitioner of medicine for whom it has also been written. It is a pleasure to acknowledge the courtesy of those who furnished us with illustrations for use in the text, and our indebted- ness to Dr. Gardner Hopkins and Professor Francis Carter Wood for a number of the photomicrographs taken especially for this work. P. H. H., JR., H. Z. PREFACE TO THE FIFTH EDITION THE present or Fifth Edition of the TEXTBOOK OF BACTERIOLOGY represents an almost complete rewriting of the book. The four preceding editions were in each case altered and brought up to date from time to time, but in all of them the original plan of presen- tation, conceived at the first writing, was preserved. In the present edition many and important additions of material and changes in manner of presentation have been made. Bacteriology and the reasoning based on bacteriological and im- munological discoveries have become more and more closely interwoven with the clinical and public health aspects of infectious diseases. Indeed, if this had not been sufficiently apparent before 1914, the experiences of the late war have demonstrated, conclusively, how im- possible it is to organize either hospitals for infectious diseases or organizations for the control of epidemics without the intimate participation of men trained in bacteriology. It seems to us, also, to have become apparent that the bacteriologist who takes an active part in the work of a hospital or in directing sanitary undertakings, must have a very thorough understanding of the clinical and public health aspects of the problem as a whole. The conception upon which the preparation of the new edition of the book has been based, therefore, is the belief that no thorough understanding of the clinical problems of infectious disease or of larger public health measures can be attained without thorough familiarity with the bacteriological and immunological facts upon which clinical and sanitary reasoning must be based. The book represents, therefore, in a brief way, an attempt to correlate a labora- tory knowledge with the branches of medicine and prophylaxis to which it is most directly applicable. We have felt that a Textbook of Bacteriology, primarily aimed at the needs of medical students and physicians, may be regarded as neglecting a great opportunity or, perhaps, even an obligation, if it omits emphasis upon prevention. In order to accomplish this purpose it has been necessary to add brief clinical data in the case of each vii vin PREFACE TO THE FIFTH EDITION variety of infection and to incorporate in the more important chapters, brief discussions of the principles underlying sanitary procedure. In the sections on technique we have eliminated many methods which we have ceased to use ourselves. At the laboratory of the Bacteriological Department of the Medical School of Columbia Uni- versity we have had the opportunity of having advanced students and staff try out a great many bacteriological procedures and have, in consequence, been able to eliminate a number of methods that, as matters of routine, have been practically dropped from our practice. We have considerably simplified the section on media. The newer methods of titration have been added. The immunological section has been considerably changed and we think simplified. It is not the purpose of a book of this kind to pre- sent a critical thesis on theoretical immunity. We have, therefore, re- stricted ourselves to the exposition of the more important principles and practical methods needed by routine laboratory workers. Short sections on the normal bacterial flora of the human body have been, added, with particular consideration of the important work done in recent years by Herter, Kendall, Rettger and others. Most of the sections dealing with the pathogenic microorganisms themselves have been completely rewritten and the order of presentation consid- erably altered in order to bring together, more logically, infections which are usually considered together from the clinical point of view. The diagnostic and therapeutic principles in which bacteri- ological and immunological reasoning and technique are involved have been thoroughly dealt with. The section on Protozoa has been completely revised by one of us along the same general lines adopted for the revision of the bacteri- ological section. The writers realize that the inclusion of clinical and epidemio- logical data in a Textbook of Bacteriology is considerably at variance with the usual treatment given to the subject in books of this kind. But it is hoped that this manner of treatment will add considerably to the value of the book for those who are interested in microorganisms particularly in their relationship to clinical and preventive medicine. In conclusion great ful acknowledgment is made to a number of our associates in the Department of Bacteriology at Columbia for valuable aid in the preparation of this Edition. Dr. J. 0. Hopkins, Associate in the Department who lias boon working with the patho- genic molds, has rewritten the section dealing with these organisms. PREFACE TO THE FIFTH EDITION ix Dr. J. Howard Mueller, Assistant Professor of Bacteriology, has critically revised the sections dealing with the chemical metabolism of bacteria, and Miss Ann KuttniT, Instructor in the department, has elaborated, and revised the chapter dealing with the anaerobic- in- fections which have gained such an important place in the study of traumatic injuries. We are indebted to Dr. Oscar Tcague for valuable suggestions in connection with the chapters on cholera and plague. HANS ZINSSER FREDERICK F. RUSSELL CONTENTS SECTION I THE GENERAL BIOLOGY OF BACTERIA AND THE TECHNIQUE OF BACTERIOLOGICAL STUDY CHAPTER PAGE I. THE DEVELOPMENT AND SCOPE OF BACTERIOLOGY 1 II. GENERAL MORPHOLOGY, REPRODUCTION, AND CHEMICAL AND PHYSICAL PROPERTIES OF THE BACTERIA 9 III. THE RELATION OF BACTERIA TO ENVIRONMENT, AND THEIR CLASSI- FICATION 27 IV. THE BIOLOGICAL ACTIVITIES OF BACTERIA 45 V. 'THE DESTRUCTION OF BACTERIA 76 VI. METHODS USED IN THE MICROSCOPIC STUDY AND STAINING OF BAC- TERIA Ill VII. THE PREPARATION OF CULTURE MEDIA 133 VIII. METHODS USED IN THE CULTIVATION OF BACTERIA . . . . . . 172 IX. METHODS DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA, ANIMAL EXPERIMENTATION 196 X. THE BACTERIOLOGICAL EXAMINATION OF MATERIAL FROM PATIENTS AND OUTLINE OF FLORA OF THE NORMAL HUMAN BODY . . 206 SECTION II INFECTION AND IMMUNITY CHAPTER PAGE XI. FUNDAMENTAL FACTORS OF PATHOGENICITY AND INFECTION . . . 230 XII. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 240 XIII. TOXINS AND ANTITOXINS 255 XIV. PRODUCTION AND TESTING OF ANTITOXINS 269 XV. SENSITIZING ANTIBODIES (PHENOMENA OF LYSIS, AGGLUTINATION, PRECIPITATION, ETC.) 277 XVI. THE TECHNIQUE OF SERUM REACTIONS 301 XVII. PHAGOCYTOSIS . . 330 xiv CONTENTS SECTION VI BACTERIA IN AIR, SOIL, WATER AND MILK CHAPTER PAGE LI. BACTERIA IN THE AIR AND SOIL 1010 III. BACTERIA IN WATER 1016 LIII. BACTERIA IN MILK AND MILK PRODUCTS. BACTERIA IN THE INDUSTRIES . 1027 SECTION VII PATHOGENIC PROTOZOA CHAPTER PAGE LIV. THE AMCEB^J 1050 LV. MASTIGOPHORA 1073 LVI. SPOROZOA 1098 LVII. INFUSORIA 1137 LVIII. TECHNIQUE OF BLOOD EXAMINATIONS FOR PROTOZOA .... 1140 INDEX OF AUTHORS 1145 INDEX OF SUBJECTS . .1159 SECTION I THE GENERAL BIOLOGY OF BACTERIA AND THE TECHNIQUE OF BACTERIOLOGICAL STUDY CHAPTER I THE DEVELOPMENT AND SCOPE OF BACTERIOLOGY As we trace back to their ultimate origins the lines of develop- ment of living beings of the animal and plant kingdoms, we find them converging toward a common type, represented by a large group of unicellular organisms, so simple in structure, so unspecialized in function, that their classification in either the realm of plants or that of animals becomes little more than an academic question. How- ever, even such microorganisms, in which the functions of nutrition, respiration, locomotion, and reproduction are concentrated within the confines of a single cell, and in which adaptation to special con- ditions more readily brings about modifications leading to the pro- duction of a multitude of delicately graded transitional forms, fall into groups which, either in structure or in biological attributes show evidence of a tendency toward one or the other of the great kingdoms. Most important of these unicellular forms, for the student of medical science, are the bacteria and the protozoa. The former, by reason of their undifferentiated protoplasm, their occasional possession of cellulose membranes, their biological ten- dency to synthetize, as well as to break down organic compounds, and because of the transitional forms which seem to connect them directly with the lower plants, are generally placed in the plant kingdom. The latter, chiefly on the basis of metabolism, are classi- fied with the animals. Knowledge of the existence of microorganisms as minute as the ones under discussion was of necessity forced to await the perfec- tion of instruments of magnification. It was not until the latter half of the seventeenth century, therefore, that the Jesuit, Kircher, in .*> BIOLOGY AND TECHNIQUE 1659, and the Dutch linen-draper, van Leeuwenhoek, in 1875, actually saw and described living beings too small to be seen with the naked eye. There can be no doubt that the small bodies seen by these men and their many immediate successors were, at least in part, bacteria. And indeed the descriptions and illustrations of several of the earliest workers correspond with many of the forms which are well known to us at the present day. During the century following the work of these pioneers, the efforts of investigators lay chiefly in the more exact morphological description of some of the forms of unicellular life, already known. Conspicuous among the work of this period is that of Otto Friedrich Muller. In the generation following Miiller's work, however, a marked advance in the study of these forms was made by Ehren- berg,1 who established a classification which, in some of its cardinal divisions, is retained until the present day. Meanwhile the regularity with which these ' ' animalcula " or "infusion animalcula" were demonstrable in tartar from the teeth, in intestinal contents, in well-water, etc., had begun to arouse in the minds of the more advanced physicians of the time a suspicion as to a possible relationship of these minute forms with disease. The con- ception of * ' contagion, ' ' or transmission of a disease from one human being to another, was, however, even at this time, centuries old. The fact had been recognized by Aristotle, had been reiterated by medieval philosophers, and had led, in 1546, to the division of con- tagious diseases by Fracastor, into those transmitted "per contac- tum," and those conveyed indirectly "per fomitem." It was for these mysterious facts of the transmissibility of disease, that clini- cians of the eighteenth century, with remarkable insight, saw an explanation in the microorganisms discovered by Leeuwenhoek and his followers. In fact, Plenciz of Vienna, writing in 1762, not only expressed a belief in the direct etiological connection between microorganisms and some diseases, but was the first to advance the opinion that each malady had its own specific causal agent, which multiplied enor- mously in the diseased body. The opinions of this author, if trans- lated into the language of our modern knowledge of the subject, came remarkably near to the truth, not only as regards etiology and transmission, but also in their suggestion of specific therapy. The conception of a "contagium vivum" was thus practically 1"Die Infusionstierchen, " etc., Leipzig, 1838. DEVELOPMENT AND SCOPE OF BACTERIOLOGY 3 established with the work of Plenciz and many others who followed in his train, but the astonishingly slight impression which the acute reasoning of these men left upon the medical thought of their day is illustrative of the futility of the most penetrating speculation when unsupported by experimental data. The real advancement in the scientific development of the subject was achieved along entirely different lines. In 1837, Schwann, a botanist, showed that the yeasts, found in fermenting substances, were living beings, which bore a causal relationship to the process of fermentation. At almost the same time, similar observations were made by a French physicist, Cagniard-Latour. The opinions ad- vanced by these men on the nature of fermentation aroused much interest and discussion, since, at that time and for a long period thereafter, fermentation was ascribed universally to protein decom- position, a process which was entirely obscure but firmly believed to be of a purely chemical nature. Although belief in the discovery of Schwann did not master the field until after Pasteur had completed his classical studies upon the fermentations occurring in beer and wine, yet the conception of a "fermentum vivum" aroused much speculation, and the attention of physicians and scientists was attracted to the many analogies exist- ing between phenomena of fermentation and those of disease. The conception of such an analogy, however, was not a new thought in the philosophy of the time. Long before Schwann and Cagniard-Latour, the philosopher Robert Boyle, working in the seventeenth century, had prophesied that the mystery of infectious diseases would be solved by him who should succeed in elucidating the nature of fermentation. Nevertheless, the diligent search for microorganisms in relation to various diseases which followed led to few results, and the suc- cesses which were attained were limited to the diseases caused by some of the larger fungi, favus (1839), thrush (1839), and pityriasis versicolor (1846). During this time of ardent but often poorly con- trolled etiological research, it was Henle who formulated the postu- lates of conservatism, almost as rigid as the later postulates of Koch, requiring that proof of the etiological relationship of a microorgan- ism to a disease could not be brought merely by finding it in a lesion of the disease, but that constant presence in such lesions must be proven and isolation and study of the microorganism away from the diseased body must be carried out. 4 BIOLOGY AND TECHNIQUE It was during this period also that one of the most fundamental questions, namely, that of the origin of these minute living beings, was being discussed with much passion by the scientific world. It was held by the conservative majority that the microorganisms de- scribed by Leeuwenhoek and others after him were produced by spontaneous generation. The doctrine of spontaneous generation, in fact, was solidly established and sanctified by tradition, and had been applied in the past not alone to microorganisms.2 And it must not be forgotten that without the aid of our modern methods of study, satis- factory proof for or against such a process was not easily brought. Needham, who published in 1749, had spent much time in fortify- ing his opinions in favor of spontaneous generation by extensive experimentation. He had placed putrefying material and vegetable infusions in sealed flasks, exposing them for a short time to heat, by immersing them in a vessel of boiling water, and had later shown them to be teeming with microorganisms. He was supported in his views by no less an authority than Buffon. The work of Needham, however, showed a number of experimental inaccuracies which were thoroughly sifted by the Abbe Spallanzani. This investigator re- peated the experiments of Needham, employing, however, greater care in sealing his flasks, and subjecting them 'to a more thorough exposure to heat. His results did not support the views of Needham, but were answered by the latter with the argument that by excessive heating he had produced chemical changes in his solutions which had made spontaneous generation impossible. The experiments of Schulze, in 1836, who failed to find living organisms in infusions which had been boiled, and to which air had been admitted only after passage through strongly acid solutions, and similar results obtained by Schwann, who had passed the air through highly heated tubes, were open to criticism by their oppo- nents, who claimed that chemical alteration of the air subjected to such drastic influences had been responsible for the absence of bac- teria in the infusion. Similar experiments by Schroeder and Dusch, who had stoppered their flasks with cotton plugs, were not open to 2 Valleri-Radot, in his life of Pasteur, states that Van Helmont, in the six- teenth century, had given a celebrated prescription for the creation of mice from dirty linen and a few grains of wheat or pieces of cheese. During the centu- ries following, although, of course, such remarkable and amusing beliefs no longer held sway, nevertheless the question of spontaneous generation of minute and structureless bodies, like the bacteria, still found learned and thoughtful partisans. DEVELOPMENT AND SCOPE OF BACTERIOLOGY 5 this objection, but had also failed to convince. The question was not definitely settled until the years immediately following 1860, when Pasteur conducted a series of experiments which were not only im- portant in incontrovertibly refuting the doctrine of spontaneous generation, but in establishing the principles of scientific investiga- tion which have influenced bacteriological research since his time.3 Pasteur attacked the problem from two points of view. In the first place he demonstrated that when air was filtered through cotton- wool innumerable microorganisms were deposited upon the filter. A single shred of such a contaminated filter dropped into a flask of pre- viously sterilized nutritive fluid sufficed to bring about a rapid and luxuriant growth of microorganisms. In the second place, he suc- ceeded in showing that similar, sterilized ' ' putrescible " liquids, if left in contact with air, would remain uncontaminated provided that the entrance of dust particles were prohibited. This he succeeded in doing by devising flasks, the necks of which had been drawn out into fine tubes bent in the form of a U. The ends of these U-tubes, being left open, permitted the sedimentation of dust from the air as far as the lowest angle of the tube, but, in the absence of an air current, no dust was carried up the second arm into the liquid. In such flasks, he showed that no contamination took place but could be immedi- ately induced by slanting the entire apparatus until the liquid was allowed to run into the bent arm of the U-tube. Finally, by exposing a series of flasks containing sterile yeast infusion, at different atmos- pheric levels, in places in which the air was subject to varying degrees of dust contamination, he showed an inverse relationship between the purity of the air and the contamination of his flasks with microorganisms. The doctrine of spontaneous generation had thus received its final refutation, except in one particular. It was not yet clear why complete sterility was not always obtained by the application of definite degrees of heat. This final link in the chain of evidence was supplied, some ten years later, by Cohn, who, in 1871, was the first to 3 In a letter to his foremost opponent, at this period, Pasteur writes: "In experimental science, it is always a mistake not to doubt when facts do not compel affirmation. ' ' The critical spirit pervading the scientific thought of that time in France is also well expressed by Oliver Wendell Holmes, who said that he had learned three things in Paris: "Not to take authority when I can have facts, not to guess when I can know, and not to think that a man must take physic because he is sick. ' ' 6 BIOLOGY AND TECHNIQUE observe and correctly interpret bacterial spores and to demonstrate their high powers of resistance against heat and other deleterious influences. Meanwhile, Pasteur, parallel with his researches upon sponta- neous generation, had been carrying on experiments upon the subject of fermentation along the lines suggested by Cagniard-Latour. As a consequence of these experiments, he not only confirmed the opinions both of this author and of Schwann concerning the fermentation of beer and wine by yeasts, but was able to show that a number of other fermentations, such as those of lactic and butyric acid, as well as the decomposition of organic matter by putrefaction, were directly due to the action of microorganisms. It was the discovery of the living agents underlying putrefaction, especially, which exerted the most active influence upon the medical research of the day. This is illustrated by Lister's work. The suppurative processes occurring in infected wounds had long been regarded as a species of putrefac- tion, and Lord Lister, working directly upon the premises supplied by Pasteur, introduced into both the active and prophylactic treat- ment of surgical wounds the antiseptic principles which alone have made modern surgery possible. There now followed a period in which bacteriological investiga- tion was concentrated upon problems of etiology. Stimulated by Pasteur's successes, the long-cherished hope of finding some specific microorganism as the causal agent in each infectious disease was revived. Pollender, in 1855, had reported the presence of rod-shaped bodies in the blood and spleen of animals dead of anthrax. Brauell, several years later, had made similar observations and had expressed definite opinions as to the causative relationship of these rods to the disease. Convincing proof, however, had not been brought by either of these observers. Finally, in 1863, Davaine, in a series of brilliant investi- gations, not only confirmed the observations of the two authors mentioned above, but succeeded in demonstrating that the disease could be transmitted by means of blood containing these rods and could never be transmitted by blood from which these rods were absent. Anthrax, thus, is the first disease in which definite proof of bacterial causation was brought. Speaking before the French Academy of Medicine at this time, Davaine suggested that the manifestations of the disease might in reality represent the results of a specific fermentation produced by DEVELOPMENT AND SCOPE OF BACTERIOLOGY 7 the bacilli he had found. This, in a crude way, expresses the modern conception of infectious disease. Within a few years after this, 1868, the adherents of the parasitic theory of infectious diseases were further encouraged by the dis- covery, by Obermeier, of a spirillum in the blood of patients suffering from relapsing fever. It is not surprising that the successes attained in these diseases, fostering hope of analogous results in all other similar conditions, but without the aid of adequate experimental methods, should have led to many unjustified claims and to much fantastic theorizing. Thus Hallier, at about this time, advanced a theory as to the etiology of infectious diseases, in which he attrib- uted all such conditions to the molds or hyphomycetes, regarding the smaller form or bacteria as developmental stages of .these more complicated forms. Extravagant conjectures of this kind, however, did not maintain themselves for any length of time in the light of the critical attitude which was already pervading bacteriological re- search. Progress was made during the years immediately following, chiefly in the elucidation of suppurative processes. Rindfleisch, von Recklinghausen, and Waldeyer, almost simultaneously, described bodies which they observed in sections of tissue containing abscesses, and which they believed to be microorganisms. Notable support was given to their opinion by similar observations made upon pus by Klebs, in 1870. In view, however, of the purely morphological nature of their studies, the opinions of these observers did not entirely pre- vail. Satisfactory methods of cultivation and isolation had not yet been developed, and Billroth and his followers, with a conservatism entirely justified under existing conditions, while admitting the con- stant presence of bacteria in purulent lesions, denied their etiological significance. The controversy that followed was rich in suggestions which greatly facilitated the work of later investigators, but could not be definitely settled until 1880, when Koch introduced the tech- nical methods which have made bacteriology an exact science. By the use of solid nutritive media, the isolation of bacteria and their biological study in pure culture were made possible. At about the same time the use of anilin dyes, developed by Weigert, Koch, and Ehrlich, was introduced into morphological study and facilitated the observation of the finer structural details which had been unnoticed while only the grosser methods employed for tissue staining had been available. 8 BIOLOGY AND TECHNIQUE With the publication of Koch's work, there began an era un- usually rich in results held in leash heretofore by inadequate tech- nical methods. The discovery of the typhoid bacillus in 1880, of the bacillus of fowl cholera and the pneumococcus in the same year, and of the tubercle bacillus in 1882, initiated a series of etiological dis- coveries which, extending over not more than fifteen years, eluci- dated the causation of a majority of the infectious diseases. Coincident with the elucidation of etiological facts began the inquiry into that field which is now spoken of as the science of immunity. The phenomena which accompany the development of insusceptibility to bacterial infections in man and in animals, first studied by Pasteur, have become the subject of innumerable re- searches and have led to results of the utmost practical value. The problems which were encountered were first studied from a purely bacteriological point of view, but their solution has shed light upon biological principles of the broadest application. Investigations into the properties of immune sera, while making bacteriology one of the most important branches of diagnostic and therapeutic medicine, have, at the same time, inseparably linked it with physiology and experimental pathology. By the revelations of etiological research, and by the study of the biological properties of pathogenic bacteria, contagion, an enemy hitherto unseen and mysterious, was unmasked, and rational cam- paigns of public sanitation and personal hygiene were made possible. Upon the same elucidations has depended the development of modern surgery — a science which without asepsis and antisepsis would have been doomed to remain in its medieval condition. Apart from its importance in the purely medical sciences, the study of the bacteria has shed beneficial light, moreover, upon many other fields of human activity. In their relationship to decomposi- tion, the conditions of the soil, and to diseases of plants, the bacteria have been found to occupy a condition of great importance in agri- culture. Knowledge of bacterial and yeast ferments, furthermore, has become the scientific basis of many industries, chiefly those con- cerned in the production of wine, beer, and dairy products. The scope of bacteriology is thus a wide one, and none of its various fields has, as yet, been fully explored. The future of the science is rich in allurement of interest, in promise of result, and in possible benefit to mankind. CHAPTER II GENERAL MORPHOLOGY, REPRODUCTION, AND CHEMICAL AND PHYSICAL PROPERTIES OF THE BACTERIA BACTERIA are exceedingly minute unicellular organisms which may occur perfectly free and singular, or in larger or smaller aggregations, thus forming multicellular groups or colonies, the individuals of which are, however, physiologically independent. The cells themselves have a number of basic or ground shapes which may be roughly considered in three main classes : The cocci or spheres, the bacilli or straight rods, and the spirilla or curved rod forms. The cocci are, when fully developed and free, perfectly spherical. When two or more are in apposition, they may be slightly flattened along the tangential surface, giving an oval appearance. The bacilli, or rod-shaped forms, consist of elongated cells whose long diameter may be from two to ten times as great as their width, with ends squarely cut off, as in the case of bacillus anthracis, or gently rounded as in the case of the typhoid bacillus. The spirilla may vary from small comma-shaped microorganisms, containing but a single curve, to longer or more sinuous forms which may roughly be compared to a corkscrew, being made up of five, six or more curves. The turns in the typical microorganisms of this class are always in three planes and are spiral rather than simply curved. Among the known microorganisms, the bacilli by far outnumber other forms, and are probably the most common variety of bacteria in existence. Many variations from these fundamental types may occur even under normal conditions, but contrary to earlier opinions it is now positively known that cocci regularly reproduce cocci, bacilli bacilli, and spirilla spirilla, there being, as far as we know, no. mutation from one form into another. The size of bacteria is subject to considerable variation. Cocci may vary from .15 ;u to 2. ju in diameter. The average size of the ordinary pus coccus varies from .8 p. to 1.2 /x in diameter. Fischer has given a graphic illustration of the size of a staphyloccocus by calculating that one billion micrococci could easily be contained in a drop of water hav- 9 10 BIOLOGY AND TECHNIQUE ing a volume of one cubic millimeter. Among the bacilli the range of size is subject to even greater variations. Probably the smallest of the common bacilli is the bacillus of influenza, which measures about .5 fji in length by .2 /* in thickness. The limit o!' the optical possi- bilities of the modern microscope is almost reached by some of the known microorganisms, and there are some diseases which are caused by organisms so small as to be invisible by any of our present methods. In fact, the virus causing the peripneumonia of cattle has been shown to pass through the pores of a Berkefeld filter, which are impenetrable to the smallest of the known bacteria.1 The coc- coid or globoid bodies grown by Noguchi from poliomyelitis virus are small enough to pass through such filters, but are still visible with the highest lens magnifications. Whether or not these minute bodies should be regarded as bacteria is questionable. It seems likely that they represent an entirely different class of organisms. It is worth FIG. 1. — TYPES OF BACTERIAL MORPHOLOGY. mentioning, also, that organisms like streptococci may show minute forms, especially when grown under anaerobic conditions, which are almost as small as the globoid bodies. It seems a general rule that anaerobically grown cocci may assume smaller forms. MORPHOLOGY OF THE BACTERIAL CELL. — When unstained, most bacteria are transparent, colorless, and apparently homogeneous bodies with a low refractive index. The cells themselves consist of a mass of protoplasm, surrounded, in most instances, by a delicate cell membrane. The presence of a nucleus2 in bacterial cells, though denied by the earlier writers, has been demonstrated beyond question by Zett- now, Nakanishi,3 and others. The original opinion of Zettnow was that the entire bacterial body consisted of nuclear material inti- 1 Nocard and Eoux, Ann. Past., 12, 1898. 2 A. Fischer, Jahrbiicher f . wissen. Botanik, xxvii. 3 Nakatnishi, Munch, med. Woch., vi, 1900. MORPHOLOGY, REPRODUCTION, ETC. 11 matcly intermingled with the cytoplasm. The opinion now held by most observers who have studied this phase of the subject favors the existence of an ectoplasmic zone which includes cell membrane and flagella, but is definitely a part of the cytoplasm, and an ento- plasm in which is concentrated the nuclear material. Biitschli* claims to have demonstrated within this entoplasmic substance a reticular meshwork, between the spaces of which lie granules of chromophilic or nuclear material. Confirmation of this opinion has been brought by Zettnow5 and others. Nakanishi, working with a .special staining method, asserts that some microorganisms show within the entoplasmic zone a well-defined, minute, round or oval nucleus, which possesses a definitely characteristic staining reaction.6 In the bodies of a large number of bacteria, notably in those of the diphtheria group, Ernst,7 Babes,8 and others have demonstrated granular, deeply staining bodies now spoken of as met achromatic granules, or Babes-Ernst granules, or, because of their frequent posi- tion at the ends of bacilli, as polar bodies. These structures are irregular in size and number, and have a strong affinity for dyes. They are stained distinctly dark in contrast to the rest of the bac- terial cell with methylene blue, and may be demonstrated by the special methods of Neisser and of Roux.9 Their interpretation has been a matter of much difficulty and of varied opinion. Those who first observed them held that they were a part of the nuclear material of the cell. Others have regarded them as an early stage in spore formation, or as arthrospores.10 Again, they have been interpreted as structures comparable to the centrosomes of other unicellular *Biitschli, «Bau der Bakterien," Leipzig, 1890. 5 Zettnow, Zeit. f. Hyg., xxiv, 1897. 'The method of Nakanishi is carried out as follows: Thoroughly cleansed slides are covered with a saturated aqueous solution of methylene blue. This is spread over the slide in an even film and allowed to dry. After drying, the slide should be of a transparent, sky-blue color. The microorganisms to be examined are then emulsified in warm water, or are taken from the fluid media, and dropped upon a cover slip. This is placed, face downward, upon the blue ground of the slide. In this way, bacteria are stained without fixation. Nakanishi claims that by this method the entoplasm is stained blue, while the nuclear material appears of a red- dish or purplish hue. 1 Ernxt, Zeit. f. Hyg., iv, 1888. 8 See section on stains, p. — . 9 Babes, Zeit. f . Hyg., v, 1889. 10 See section on sporulation, p. 16. 12 BIOLOGY AND TECHNIQUE forms. As a matter of fact, the true nature of these bodies is by no means certain. They are present most regularly in microorganisms taken from young and vigorous cultures or in those taken directly from the lesions of disease. It is unlikely that they represent struc- tures in any way comparable to spores, since cultures containing individuals showing metachromatic granules are not more resistant to deleterious influences than are others. Their abundant presence in young vigorous cultures may indicate a relationship between them and the growth energy of the microorganisms. There is no proof, however, that these bodies affect the virulence of the bacteria. Cell Membrane and Capsule. — Actual proof of the existence of a cell membrane has been brought in the cases of some of the larger y^_. ., ; violet 1 c.c. Saturated solution ammonia alum 10 c.c. Filter the stain directly upon the preparation and heat for three or four minutes. Wash in water, dry, and mount in balsam. Differential Stains. — GRAM'S METHOD.IS — By this method of staining, which is extremely important in bacterial differentiation, bacteria are divided into those which retain the initial stain and those which are subsequently decolorized and take the counterstain. The former are often spoken of as the Gram-positive, the latter as Gram-negative bacteria. The reasons for the differential value of Gram's method are not entirely clear, but must, of course, depend upon peculiarities of the chemical constituents of the bacteria themselves. A considerable amount of work has been done on the subject which has not been entirely conclusive. A discussion of the subject may be found in Wells' "Chemical Pathology," Second Edition, page 105. Wells states that the results of Gram's method may be ascribed to the formation of an iodin-pararosanilin-protein compound in the Gram- positive bacteria. Only dyes of this group, namely, gentian violet, methyl violet, etc., will form such combination. He quotes Burgers16 as stating that trypsin will digest Gram-negative, but not Gram- positive bacteria, and the gastric juice affects only a few of the Gram-positive species. It has also been suggested that the fatty substances in the bacterial bodies may have some relationship to the Gram-stain, and that the bacterial cell wall is the part most directly involved in the staining reaction. Benians17 has found that when Gram-positive bacteria are artificially disintegrated they lose their characteristic reaction and become Gram-negative, and this has been confirmed for tubercle bacilli in Wells' laboratory by 15 Gram, Fortschr. d. Med., ii, 1884. 18 Burgers and collaborators, Zeit. f. Hyg., 1911, 70'. 17 Benians, Jour, of Pathol. and Bacter., 17, 1912, 199. MICROSCOPIC STUDY AND STAINING 121 Sherman.18 Wells' suggestion is that the iodin may render the cell membrane impermeable to alcohol. Preparations are made on cover-slips or slides in the usual way. The preparation is then covered with an anilin gentian-violet solution which is best made up freshly before use. The staining fluid is made up, according to Gram's original direc- tions,19 as follows: Five c.c. of anilin oil are shaken up thoroughly with 125 c.c. of distilled water. This solution is then filtered through a moist filter paper. To 108 c.c. of this anilin water, add 12 c.c. of a saturated alcoholic solution of gentian-violet. The stain acts best when twelve to twenty-four hours old, but may be used at once. It lasts, if well stoppered, for three to five days. A more convenient and simple method of making up the stain is as follows: To 10 c.c. of distilled water in a test tube add anilin oil until on shaking the emulsion is opaque ; roughly, one to ten. Filter this through a wet paper until the filtrate is clear. To this add saturated alcoholic solution of gentian- violet until the mixture is no longer transparent, and a metallic film on the surface indicates saturation. One part of alcoholic saturated gentian- violet to nine parts of the anilin water will give this result. This mixture may be used immediately and lasts two to five days if kept in a stoppered bottle. Cover the preparation with this; leave on for 5 minutes. Pour off excess stain and cover with Gram's iodin solution for 2 to 3 minutes. Iodin 1 gm. Potassium iodid 2 gm. Distilled water 300 c.c. Decolorize with ninety-seven per cent alcohol until no further traces of the stain can be washed out of the preparation. This takes usually thirty seconds to two minutes, according to thinness of preparation. Wash in water. Counterstain with an aqueous contrast stain, preferably Bismarck brown,20 dilute fuchsin or safranin. PALTAUF'S MODIFICATION. OF GRAM'S STAIN. 21 — The staining fluid as prepared by this modification possesses the advantage of retaining 18 Sherman, Jour, of Infec. Dis., 12, 1913, 249. 19 Gram, loc. cit. 20 To make up Bismarck brown solution, prepare a saturated aqueous solution of the powdered dye by heating. Cool and filter. Dilute 1 to 10 with distilled water. 21Sharnosky, Proc. N. Y. Pathol. Soc., Oct., 1909, n. s., ix, 5, 122 BIOLOGY AND TECHNIQUE its staining power for a longer period than does the anilin-water gentian-violet described in the original method. The staining fluid is prepared as follows: 3-5 c.c. anilin oil are added to 90 c.c, distilled water and 7 c.c. absolute alcohol. This mixture is thoroughly shaken and filtered through a moist filter paper until clear. Then add: Gruebler's gentian-violet 2 gm. The fluid should stand twenty-four hours, during which a precipitate forms. This is filtered before use. This gentian-violet solution retains its staining power for from 4 to 6 weeks. It is good only when a metallic luster develops on the surface. It is used in the following way: Spreads on cover-slips or slides are dried and fixed as usual. Then apply: Anilin-water gentian-violet (as above), three minutes'. Gram's iodin solution, two minutes. Absolute alcohol (with stirring), thirty seconds. Counterstain, without washing in water, in aqueous fuchsin or in weak carbol-fuchsin. Jensen's Modification of the Gram Stain*2 — Jensen prepares his smears in the ordinary way. He uses a 5 per cent solution of methyl violet, "6B." This solution is stable. It is poured on the fixed smear for *4 to l/2 minute. The methyl violet solution is poured off and, without washing, is covered with LugoPs solution, and this is poured off and fresh Lugol added and left on for 1/2 minute. The iodine solution is now poured off and the preparation washed with 98% alcohol. When decolorized, the 98% alcohol is washed off with a few drops of absolute alcohol, and the preparation counterstained with a solution of neutral red consisting of 1 gram of neutral red in a liter of water to which 2 c.c. glacial acetic acid has been added. Apparently the important point in Jensen's stain is that the preparation is never washed with water, and that the final washing off of the 98° alcohol is done with a few drops of absolute. We have not used this stain as a routine and do not know how reliable it is. STERLING'S MODIFICATION OF 0 RAM'S METHOD. — 2 c.c. anilin oil + 10 c.c. 95% alcohol. Shake ami add 88 c.c. distilled water. 5 grams 23 Jensen, Berl. klin. Woch., 49, 1912, 1163. MICROSCOPIC STUDY AND STAINING 123 gentian violet are ground in a mortar and the anilin solution added slowly while grinding. Filter. This solution keeps, and stains in one-half to one minute.23 CLASSIFICATION OF THE MORE IMPORTANT PATHOGENIC BACTERIA ACCORDING TO GRAM'S STAIN. Gram-positive. (Retain the Gentian-violet.) Micrococcus pyogenes aureus Micrococcus pyogenes albus Streptococcus pyogenes Micrococcus tetragenus Pneumococcus Bacillus subtilis Bacillus anthracis Bacillus diphtherias Bacillus tetanus Bacillus tuberculosis and other acid-fast bacilli Bacillus aerogenes capsulatus Bacillus botulinus Gram-negative. (Take Counter stain.) Meningococcus Gonococcus Micrococcus catarrhalis Bacillus coli Bacillus dysenteriae Bacillus typhosus Bacillus paratyphosus Bacillus fecalis alkaligenes Bacillus enteritidis Bacillus proteus (proteus) Bacillus mallei Bacillus pyocyaneus Bacillus influenzas Bacillus mucosus capsulatus Bacillus pestis Bacillus maligni oedematis Spirillum choleras Bacillus Koch- Weeks Bacillus Morax-Axenfeld Stains for Acid-Fast Bacteria. — These methods of staining are chiefly useful in the demonstration of tubercle bacilli. These bacteria because of their waxy cell membranes are not easily stained by any but the most intensified dyes, but when once stained, retain the color in spite of energetic decolorization with acid. For this reason they are known as acid-fast bacilli. The first method devised for the staining of tubercle and allied bacilli was that of Ehrlich. 23 This is the routine method employed in our laboratory at present. In using Sterling's stain the time of staining can be abbreviated as follows: Sterling's Gentian Violet one minute lodin thirty seconds to one minute 124 BIOLOGY AND TECHNIQUE EHRLICH METHOD.24 — This method is now rarely used. Cover-slip preparations are prepared as usual and fixed by heat. Stain with anilin water gentian-violet, hot, three to five minutes, or twenty-four hours at room temperature. Decolorize with thirty-three per cent nitric acid one-half to one minute. Treat with sixty per cent alcohol, until no color can be seen to come off. Counterstain with aqueous methylene-blue. Rinse in water, dry, and mount. ZIEHL-NEELSON METHOD.25 — Thin smears are made upon cover- slips or slides. Fix by heat. Stain in carbol-fuchsin solution as given on page 115. The slide or cover- slip may be flooded with the stain, and this gently heated with the flame until it steams, or else the cover-slip may be inverted upon the surface of the staining fluid, in a porcelain dish or watch-glass, and this heated until it steams. This is continued for three to five minutes. Decolorize with either five per cent nitric acid, five per cent sulphuric acid, or one per cent hydrochloric acid for three to five seconds. The treatment with the acid is continued until subsequent washing with water will give only a faint pink color to the preparation. Wash with ninety per cent alcohol until no further color can be removed. If, after prolonged washing with alcohol, a red color still remains in very thick places upon the smear, while the thin areas appear entirely decolorized, this may be disregarded. Wash in water and counterstain in aqueous methylene-blue for one-half to one minute. Rinse in water, dry, and mount. By this method the tubercle bacilli are colored red, other bacteria and cellular elements which may be present are stained blue. GABBET'S METHOD. 26— Gabbet has devised a rapid method in which the decolorization and counterstaming are accomplished by one solution. The specimen is prepared and stained with carbol- fuchsin as in the preceding method. It is then immersed for one minute directly in the following solution : Methylene-blue 2 gnis. Sulphuric acid 25 per cent (sp. gr. 1018) 100 c.c. MEhrlich, Deut. med. Woch., 1882. 28 Ziehl, Deut. med. Woch., 1882 • Neelson, Deut. med. Woch., 1883. 26 Goblet, Lancet, 1887. MICROSCOPIC STUDY AND STAINING 125 Then rinse in water, dry, and mount. This method, while rapid and very convenient, is not so reliable as the Ziehl-Neelson method. PAPPENHEIM 's METHOD.27 — The method of Pappenheim is devised for the purpose of differentiating between the tubercle bacillus and the smegma bacillus. Confusion may occasionally arise between these two microorganisms, especially in the examination of urine where smegma bacilli arc derived from the genitals, and less fre- quently in the examination of sputum where smegma bacilli may occasionally be mixed with the secretions of the pharynx and throat. Preparations are smeared and fixed by heat in the usual way. Stain with hot carbol-fuchsin solution for two minutes. Pour off dye without washing and cover with the following mixture: Corallin (rosolic acid) 1 gm. Absolute alcohol 100 c.c. Methylene-blue added to saturation Add glycerin28 20 c.c. This mixture is poured on and drained off slowly, the procedure being repeated four or five times, and finally the preparation is washed in water. The combination of alcohol and rosolic acid decolorizes the smegma bacilli, but leaves the tubercle bacilli stained bright red. BAUMGARTEN'S METHOD.29 — This method is recommended by the author for differentiation between the bacillus of tuberculosis and the bacillus of leprosy and depends upon the fact that the tubercle bacillus is less easily stained than Bacillus leprse. Smears are prepared and fixed by heat in the usual way. Stain in dilute alcoholic fuchsin for five minutes. Decolorize for twenty seconds in alcohol, ninety-five per cent, ten parts, nitric acid one part. Wash in water. Counterstain in methylene-blue. Wash in water, dry, and mount. 27 Pappenheim, Bcrl. klin. Woch., 1898. 28 The glycerin is added after the other constituents have been mixe2X10-4 = log 5000 = 3.7. Another way of getting at this is as follows: Supposing that, [H] = 2X10-6, Pj?/ = log. of reciprocal, or, Now, log. 1 = 0, log. 2 = 0.3, log. 10~6=-6. Therefore : or, P7/ = 0-[+0.3-6] or 5.7. The following table gives the relationship of the old method of expression to the new: 5 Sorensen, Biochem. Zeit., 1909, 21, 131, 201. 140 BIOLOGY AND TECHNIQUE HYDROGEN ION CONCENTRATION PH VALUE ixio-1 1 IXIO-2 2 IXIO-3 3 IXIO-4 4 1X10~5 5 ixio-6 6 IXIO"7 7 1X10~8 8 ixio-9 9 1X10~10 10 Indicators are substances, usually weak organic acids or which change in color when subjected to changes of reaction, that is, to changes of hydrogen or hydroxyl ion concentrations. A table which we take from a pamphlet prepared by the Army Medical School, will give the color reactions at various P# measurements for a number of common indicators. COLOR REACTIONS IN SOLUTIONS OF DIFFERENT HYDROGEN ION CONCENTRATION (2) True acidity 10~3 10~4 10~5 10~6 10~7 10~8 10~9 10-io Methyl orange Rose red Orange red Yellow < — <- «- ^ Rosolic acid Yellow - -» -> Rose Red - - Congo red Blue Violet Reddish violet Orange Orange red «- *- *^ Litmus Red -* -> Reddish violet Violet Blue '*- <— Phenolphthalein Color- less -> 7» r» -» -* Red < — Clark and Lubs particularly have been responsible for introducing the method at present in use in bacteriological laboratories, based upon the preparation of solutions which can be used as standards for the colorimetric measurements of culture media. The solutions must be very carefully prepared, and should, whenever possible, be con- trolled by potentiometer measurements; however, with proper care THE PREPARATION OF CULTURE MEDIA 141 and the use of pure substances by a reliable worker, this may, with reasonable safety, be omitted. The principle of making the dilutions is that carefully measured amounts of molecular solutions of acids and alkalis are mixed in series so that each successive tube shall con- tain a definite hydrogen ion concentration. These tubes are the standard. Clark and Lubs, in the first of the series of papers noted above, cite tables of mixtures for such purposes, with a range of PH extending, from 1 to 10. For the exact composition of all of these mixtures, the reader is referred to the original paper of Clark and Lubs, page 25. For the routine of the ordinary laboratory bacteriology, only the fourth section of this table is necessary. This is as follows: KH2PO4— NaOH 5.8 50 c.c. M/5 KH2PO4 3. 72 c.c. M/5 NaOH Dilute to 200 c.c. 6.0 50 c.c. M/5 KH2PO4 5. 70 c.c. M/5 NaOH Dilute to 200 c.c. 6.2 50 c.c. M/5 KH2PO4 8. 60 c.c. M/5 NaOH Dilute to 200 c.c. 6.4 50 c.c. M/5 KH2PO4 12. 60 c.c. M/5 NaOH Dilute to 200 c.c. 6.6 50 c.c. M/5 KH2PO4 17. 80 c.c. M/5 NaOH Dilute to 200 c.c. 6.8 50 c.c. M/5 KH2PO4 23. 65 c.c. M/5 NaOH Dilute to 200 c.c. 7.0 50 c.c. M/5 KH2PO4 29. 63 c.c. M/5 NaOH Dilute to 200 c.c. 7.2 50 c.c. M/5 KH2PO4 35. 00 c.c. M/5 NaOH Dilute to 200 c.c. 7.4 50 c.c. M/5 KH2PO4 39.50 c.c. M/5 NaOH Dilute to 200 c.c. 7.6 50 c.c. M/5 KH2PO4 42. 80 c.c. M/5 NaOH Dilute to 200 c.c. 7.8 50 c.c. M/5 KH2PO4 45. 20 c.c. M/5 NaOH Dilute to 200 c.c. 8.0 50 c.c. M/5 KH2PO4 46.80 c.c. M/5 NaOH Dilute to 200 c.c. The method of procedure in using these solutions is as follows: From the solutions made above a colorimetric scale is prepared. All glassware must be very carefully cleansed, and it should be remem- bered that some of the cheaper glassware obtained in laboratories at the present time often seems to give off considerable amounts of alkali. Whatever method of cleaning is used, the final thorough rinsing must be done thoroughly with redistilled water. 10 c.c. each of the respective series of standard mixtures is placed into each test tube, and to it 10 drops of the indicator are added. For the range from 6.8 to 8.4, which is sufficient for all ordinary pathogenic work, the indicator used is phenol-sulfon-phthalein, or phenol red in con- centration of 0.02 per cent aqueous solution. If ranges from 6 to 7.6 are desired, brom-thymol blue in a concentration of 0.04 per cent may be used, and when ranges just above 8 are desired, cresol red in concentration of 0.02 per cent is recommended. 142 BIOLOGY AND TECHNIQUE A series of tubes so prepared, each of which contains 10 c.c. of each of the graded mixtures, with indicator added, represents a colorimetric scale against which the media can be standardized. This standardization is now carried out as follows, and can be utilized for any media which are not too highly colored and not turbid: Into a thoroughly cleaned test tube 2 c.c. of the medium are measured and this diluted with 8 c.c. of redistilled water. 10 drops of indicator are then added, and after thoroughly mixing, a color reading is taken against the scale. If the reaction is too acid, as is usually the case, N add =77 NaOH from a burette or graduated pipette, a drop at a time, until the color matches that of the standard tube. By calculating from the amount of weak alkali added, the total quantity of media N is then brought to the desired P# with — NaOH. In the titration of agar, Clark and Lubs recommend that the broth be titrated and adjusted before the addition of the agar, in order to avoid possible colloidal changes between the agar and the indicator, and that the agar be added after the adjustment is made. It is possible, however, without excessive error, to carry out the titration of media containing agar in the same way as outlined above, adding cold water to the hot agar, and making the comparison at once at a temperature of 35° to 40° before the mixture has jelled. In solutions like bacterial media, which haw a certain amount of color or some turbidity, a so-called " comparator " may be used in the form of a wooden box painted black with four holes for test tubes and a slit in front and behind, so that it can be looked through against a source of light. The arrangement of this is given in the following cut: LIGHT SOURCE EYE Arrangement for Reading Titration. THE PREPARATION OF CULTURE MEDIA 143 Methods of Clearing Media. — Clearing with Eggs. — When culture media" are prepared from substances containing no coagulable pro- tein, it is often necessary, for purposes of clearing, to add the whites of eggs, and then to heat for forty-five minutes in the Arnold sterilizer. In the following detailed descriptions, the direction "clear with egg" has been given whenever such a step is deemed necessary. The exact technique of such a procedure is as follows: In a small pot or pan, the whites of several eggs (one or two eggs to each liter of medium) are beaten up thoroughly with a little water (20 c.c.). This egg white is then poured into the medium, which, if hot, as in the case of melted agar or gelatin, must first be cooled to about 50° to 55° C. The mixture is then thoroughly shaken and steamed in the Arnold sterilizer for thirty minutes. At the end of this time, the flask containing the medium is removed from the sterilizer and thoroughly shaken so as completely to break up the coagulum which has formed. It is then replaced and allowed to steam for another fifteen minutes. At the end of this time the medium between the coagula should be clear. It is now ready for filtration through cotton. Filtering Media through Cotton. — The filtration of media after clearing, either by the addition of eggs or by the coagulation of the proteins originally contained in it, is best done through absorbent cotton. A small spiral, improvised of copper wire is placed as a support in the bottom of a large glass funnel. A square piece of absorbent cotton is then split horizontally giving two squares of equal size. Ragged edges and incisures should be avoided. These two layers of cotton are then placed in the funnel, one piece above the other in such a way that the direction of the fibers of the two layers is at right angles one to the other. They are then gently depressed "into the filter with the closed fist. The edges of the cotton are made to adhere to the sides of the funnel by allowing a thin stream of tap water to run over them, while smoothing them against the glass with the hand. The medium, when poured into such a filter, should be poured along a glass rod at first, to avoid running down the sides or burst- ing the filter. After filtration has begun, the filter should be kept as full as possible. The first liter or so which comes through may not be clear, but the filter gains in efficiency as the coagulum settles into the fibers of the cotton, and the first yield may be sent through a second time. Filtration of agar or gelatin is best done in a warm 144 BIOLOGY AND TECHNIQUE room with windows and doors closed, and the filter covered with a lid, to avoid too rapid cooling. The funnel and filter should be warmed just before use. Filtering through Paper. — Many media may be efficiently cleared by filtration through close filter paper without the aid of coagula. The Tubing of Media. — Most of the media described in the fore- going section are used in test tubes. In order to fill these tubes, the media are best poured into a large glass funnel to which a glass discharging tube has been fitted by means of a short piece of rubber tubing. Upon this is placed a thumb cock. The plug is then removed from the test tube by catching it between the small and ring fingers of the right hand and the glass outlet is thrust deeply into the test tube, in order to prevent the medium from touching the upper portion of the test tube where the cotton plug will be lodged. About 7 to 8 c.c. are put in each test tube. Sterilization of Media. — By Heat. — Media which contain neither sugars, gelatin, glycerin, nor animal serum may be sterilized in the autoclave at fifteen pounds pressure for fifteen minutes to half an hour. Media which contain these or other substances subject to injury from the high temperature, must be sterilized by the frac- tional method, i.e., by twenty minutes' exposure in the live steam sterilizer (Arnold, Fig. 8, page 83) on each of three consecutive days. During the intervals between sterilizations, they should be kept at room temperature or in the incubator, to permit the germination of spores which may be present. Media containing animal serum or other albuminous solutions which are to be sterilized without coagulation, may be sterilized in water baths, or in hot-air chambers, at temperatures varying from 60° to 70° C., by the fractional method. In such cases five or six exposures of one hour on succeeding days should be employed. By Filtration. — It is often desirable in bacteriological work to free fluid from bacteria. This is frequently necessary for the sterili- zation of blood-serum or exudate fluids, or fOr obtaining toxins free from bacteria. For these purposes a large variety of filters are in use. Those most commonly employed are of the Chamberland6 or Berkefeld type, which consist of hollow candles made of unglazed porcelain or diatomaceous earth. Both these types are made in various grades of fineness, upon which depend both the speed of • Pasteur and Chamberland, Compt. rend, de 1'acad. des sci., 1884. THE PREPARATION OF CULTURE MEDIA 145 filtration and the efficiency. They are made in various forms and models, some of which are shown in the accompanying figures. In JIG. 10. — BERKEFELD FILTER. FIG. 11.— REICHEL FILTER. most of the methods of filtration commonly employed the fluid which is to be filtered is sucked through the walls of the filter, either by 146 BIOLOGY AND TECHNIQUE a hand suction-pump or by some form of vacuum-pump attached to an ordinary water-tap. The hollow candle-filter may either be firmly fitted into a cylin- drical glass chimney and surrounded by the fluid which is to be filtered, or else the candle may be connected to the collecting flask with sterile tubing and suspended freely in the fluid. Perfect filters of these types will hold back any of the bacteria known to us at present. Filters before use must be sterilized. The candles themselves are subjected to 150° C. in the hot-air sterilizer for one hour. The glassware and washers necessary for setting up the apparatus may be sterilized by boiling. In order that filters may be repeatedly used with good result, it is necessary that they should be carefully cleaned from time to time. This is best done in the following way : Filters through which fluids from living cultures have passed are first sterilized in the Arnold steam sterilizer. Their exterior is then carefully cleaned with a fine brush. Following this a five- tenths per cent solution of potassium permanganate is passed through them and this again removed by sucking through a five per cent solution of bisulphite of soda. This last is washed out by sending a considerable quantity of distilled water through the filter, which is then dried and sterilized by heat. The suction necessary for filtration through these filters is usually applied by means of the ordinary suction-pump attached to a run- ning faucet. Slanting of Media. — Solid media which are to be used in slanted form in test tubes should be inclined on a ledge (easily improvised of glass tubing) at the proper slant, after the last sterilization. Agar, the medium most frequently employed in this way, should be left in this position until hard. Indicators. — We cannot in this place go into details concerning the theory of indicators. For this we may refer the reader to the recent treatise by Clark on the "Determination of Hydrogen Ions." 7 According to Ostwald, indicators are acids or bases whose undis- sociated molecules "have a different color from that of their dis- sociation products." This conception has been somewhat modified by more recent work, in that color changes are found to be associated ''Clark, W. Mansfield, Determination of Hydrogen Ions, Williams and Wilkins Co., Baltimore, 1920. THE PREPARATION OF CULTURE MEDIA 147 with tautomeric rearrangements of the original substances, and the state of these substances in a dissociated or undissociated form determines the color. The degree of dissociation as determined by the hydrogen ion concentration brings about a predominance of one or the other tautomeric compound, and the color depends upon which one of these compounds is associated with the color. Chapter three of Clark's book goes into this matter with sufficient thorough- ness for bacteriological work. In cultural work not many indicators are needed. For the actual titration of media, the necessary indicators are given in another section. For actual addition to culture media, we may restrict our- selves to a limited number of useful indicators. Litmus is the indicator most commonly used in former years, is a product obtained from a species of lichen, and is obtained by oxidation of the orcin contained in this plant. Asolitmin is the indicator substance in the litmus, chemically complex and not com- pletely analyzed. The litmus solutions used in the preparation of media are best made up as follows : Litmus in substance — Merck 's purified, or Kaul- baum's — is dissolved in water to the extent of 5 per cent. The solution is made by heating in an Arnold sterilizer for about one to two hours, shaking occasionally. The solution is then filtered through paper and sterilized. It should be kept sterile, as molds will grow in it otherwise. A standard litmus solution, which is marketed for laboratory purposes, known as "Kubel and Tiemann's" solution, may be used. Andrade indicator is made up as follows: 0.5 per cent aqueous acid fuchsin 100 c.c. Normal NaCH 16 c.c. The red color fades out gradually ; the indicator should be yellow after standing two or three hours. If it remains red or reddish, 1 c.c. more of normal NaOH may be added. Acid production turns this indicator red, due to the neutralization of the alkali present and the liberation of the acid color base. The medium in which Andrade indicator is used, must be adjusted to the neutral point of this indicator which is equivalent to P^ 7.2. The reaction of the medium is right for this indicator, when the medium containing 1 per cent of the indicator is red when hot, and colorless when cold. 148 BIOLOGY AND TECHNIQTJE China, Blue Indicator. — Make up 1 per cent solution of China blue, heat almost to boiling in the water bath, add, drop by drop, normal NaOH until completely decolorized. Use 1.5 per cent concentration in medium. This indicator seems to be much less toxic than some of the others, and has been used with success particularly by Teague, to whom we are indebted for these practical details. Neutral red an indicator formerly used extensively by the Ger- mans in the proportions of 1 c.c. of staturated aqueous solution to 100 c.c. of culture medium. It has been particularly used in con- nection with the colon-typhoid group, in that typhoid bacilli do not change the color, whereas B. coli decolorizes it. The list of indicators might be very much increased, but there would be little point in it since most of these indicators are not being used at the present time. ACTUAL STEPS IN THE PREPARATION OP NUTRIENT MEDIA Meat Extract Broth. — To 1000 c.c, of distilled or clear tap water, add 5 grams or 0.5 per cent of Liebig's meat extract, 10 grams or 1 per cent of Witte or any other reliable brand of pepton, and 5 grams or 0.5 per cent of common salt, NaCl. The ingredients are mixed together in a suitable vessel and heated with stirring over a free flame. When the pepton and meat extract are completely dissolved, the vessel is removed from the flame. The medium is titrated by the colorimetric method described above and adjusted to the desired reaction. It is advisable to make the reaction about two points (on the hydrogen ion scale) more alkaline than the final reaction should be, since the heat of the autoclave usually increases the acidity of the medium. Using Liebig's meat extract and Digestive Ferment's pepton, we have found that the addition of 10-12 c.c. of normal NaOH to every liter of extract broth usually brings the reaction, before autoclaving, to 7.6, making it about 7.4 after autoclaving, which is the optimum for most pathogenic bacteria. After the addition of the alkali, the broth is autoclaved for thirty minutes at fifteen pounds pressure. The medium is then filtered, the reaction checked and after tubing and sterilization, is ready for use. It is not necessary in most cases to add eggs to extract broth, since it is easy to obtain clear without this step. Meat Infusion Broth. — Infuse 500 grams8 of lean meat (veal or beef) in 1000 c.c. of distilled or tap water for twelve to twenty -four hours in the ice-box. Strain through wet cheese cloth, squeezing the meat as dry as "Koughly 1 pound (1*4 Ibs.). THE PREPARATION OF CULTURE MEDIA 149 possible. Make up the volume to 1000 e.c. Add 1 per cent pepton and 0.5 per cent salt. Heat over the free flame until the pepton is dissolved, stirring from time to time. Solution usually takes place so rapidly that the loss of water through evaporation is negligible. If desired, however, the medium can be measured after this preliminary heating, and the loss in volume made up by the addition of water. Titrate the medium and bring it to the desired reaction using the colorimetric method. Meat infusion media are usually considerably more acid than meat extract media. The addition of about 20 e.c. normal NaOH per liter in the case of meat infusion broth usually brings the reaction to about 7.4 after autoclaving. The change in reaction after autoclaving is sometimes considerable, and must be carefully checked. After the addition of the alkali, the medium is autoclaved for thirty minutes at fifteen pounds pressure. The broth is now filtered through paper or cotton, and usually comes through clear without any further trouble. After tubing and sterilization, the medium is ready for use. "Hormone Medium." 9 — A curious observation has been made recently which seems to indicate that the filtration of media removes from them certain substances which considerably enhance their nutritive value for bacteria. These substances, for want of a better name, have been spoken of as hormones, and the hormone media, which are used pretty generally, and which we have found to possess unusual advantages over ordinary culture media, and which, consequently, we are using almost altogether in our routine work, are made as follows, the description given being that of Huntoon : 10 The basis for "hormone media" is beef heart instead of the customary beef or veal. It is important that the hearts are fairly fresh. The heart muscle is cut up in the usual way ai d after the removal of fat and large vessels, put through the meat grinder. The chopped meat is weighed and 1 liter of water added for every 500 grams of meat. One per cent pepton and 0.5 per cent salt are added directly, and 1 egg (whole) added for each liter of medium. If the bouillon is to be used for broth, 1 per cent gelatin is added immediately. If the bouillon is to be used as a basis for agar, it is not necessary to add the gelatin. The 3 per cent agar, finely cut up, is added to the other ingredients. When all the ingredients have been placed in the same pot, the mixture is heated over the free flame until it reaches a temperature of about 70° C. and meat begins to turn brown. 25 e.c. of normal NaOH are then added per liter. The pot is placed in the Arnold and allowed to cook for 1^ to 2 hours. At the end of this time, a firm clot has usually formed and the broth or agar can be decanted. 9 Cole and Lloyd, Journal Path, and Baet. vol. 21, 1916. 10 Huntoon, F. M., Jour, of Inf. Dis., 23, 1918, 169. 150 BIOLOGY AND TECHNIQUE The medium is then titrated accurately and brought to the desired reaction. The medium usually gets slightly more acid on autoclaving, so that it is better to adjust to about 2 points more alkaline on the hydrogen ion scale, than the final reaction desired. It is important, in making hormone media, never to filter in any way. Cotton, paper, and cheese cloth filters are equally undesirable. If a firm clot is obtained after the first heating, there is usually no difficulty in obtaining a clear medium. • On the first autoclaving a second precipitate usually forms, from which the clear medium can easily be decanted. It is best to check up the reaction after the final sterilization. The Hormone media can also be cleared with the Sharpless centrifuge. Sugar-free Broth. — Make one liter of infusion broth11 according to the directions given. Inoculate with a young culture of B. coli comnmnis. Incubate for twenty-four to forty-eight hours. The bacteria will ferment and thus destroy any sugar (mono-saccharide) which may be present in the broth, and thus render the medium sugar-free and acid. Arnold for one hour to kill the B. coli. Titrate and adjust. Arnold again for thirty minutes. Filter through paper until clear. This sugar-free medium is used as a basis for fermentation reactions. The different sugars are added in 1 per cent concentrations and the medium is then sterilized for three successive days in the Arnold, since the higher temperatures of the autoclave tend to split the more complex sugars into the simpler ones. Glycerin Broth. — To ordinary, slightly acid or neutral meat infusion broth, add six per cent of C. P. glycerin. Sterilize by fractional method. Calcium Carbonate Broth. — This medium is designed for obtaining mass cultures of pneumococcus or streptococcus for purposes of immunization or agglutination. To 100 c.c. of meat infusion broth in small flasks, add one per cent of powdered calcium carbonate, and one per cent of glucose. It is a wise precaution to sterilize the dried calcium carbonate in the hot-air chamber before using. Small pieces of marble may be used as suggested by Bolduan. METHOD FOR THE PREPARATION OF NUTRIENT BROTH12 (Avery) (for Streptococcus Agglutination). — One pound of lean chopped beef allowed to infuse in a liter of tap water over night in the ice-box. The unfiltered meat infusion boiled for 30 minutes, filtered through paper, and the loss by evapora- tion made up by the addition of water. One per 'cent peptone (Faircliild) and 0.2 per cent sodium phosphate (Na.JIPOJ are now added. The mixture "It is not necessary to filter the infusion if it is to be used for making sugar free broth. 12 We may add that this method of growing streptococcus for agglutination purposes does not always work out successfully — but is the best we know of so far. THE PREPARATION OF CULTURE MEDIA 151 is allowed to boil for 20 minutes and the reaction is adjusted to the desired P7/ (About 0.2 of a PH is allowed for change in the reaction during sterilization. For pneumococcus work the optimus P// is 7.8; in the adjust- ment, therefore, before sterilization, the reaction is set at PH 8.) The broth is sterilized in the Arnold sterilizer for 20 minutes on three successive days. For streptococcus work the final read ion should be P// 7.4. Pepton-Salt Solution (Dunham's solution) : 1. Distilled water 1,000 c.c. Pepton (Witte) 10 gms. NaCl 5 < ' 2. Heat until ingredients are thoroughly dissolved. 3. Filter through filter paper until perfectly clear. 4. Tube or store flasks. Sterilize by discontinuous method. Nitrate Solution: 1. Distilled water 1,000 c.c. Pepton 10 gms. Potassium nitrate 10 gms. 2. Heat until ingredients are thoroughly dissolved. 3. Filter through filter paper until perfectly clear. 4. Tube or store flasks. Sterilize by discontinuous sterilization. Uschinsky's Protein-Free Medium.13 — To one liter of distilled water add: Asparagin 3.4 grams. Ammonium lactate 10 * ' Sodium chlorid 5 ' ' Magnesium sulphate 0.2 ' ' Calcium chlorid 0.1 • ' Potassium phosphate 1.0 ' ' When these substances are thoroughly dissolved, add 40 c.c. of glycerin. Tube and sterilize. Meat Extract Gelatin. — Gelatin is best made in the Arnold and should at no stage be autoclaved. One per cent pepton, 0.5 per cent salt, 0.5 per cent meat extract in one liter of water are dissolved in the Arnold. When these ingredients have been dissolved, 15 to 18 per cent of finest French sheet gelatin14 are added, and the mixture kept in the Arnold until the gelatin is completely dissolved. 13 Uschinsky, Cent. f. Bakt., 1, xiv, 1893. 14 The acidity and consistence of the different commercial gelatins vary con- siderably and care should be taken in selecting a uniform and suitable brand, such as Hesterberg '& gold label gelatin. It is advisable, when working during the summer or in hot climates, to add 18 per cent. 152 BIOLOGY AND TECHNIQUE The medium is then titrated and adjusted. After cooling, the whites of two eggs are added and the media is put in the Arnold for forty-five minutes. The medium is filtered, tubed and sterilized by the fractional method. All unnecessary heating of gelatin is to be avoided. Meat Infusion Gelatin. — Meat infusion gelatin is made in the same way as the above except that fresh meat infusion is substituted for the meat extract. Meat-Extract Agar. — To 1 liter of distilled or tap water add: Thread agar 15 to 30.0 grams15 Pepton 10.0 ' ' . . Meat extract . 5.0 " Common salt 5.0 " Put in autoclave, 15 pounds' pressure, for 15 minutes. The agar can also be dissolved over the free flame, but this takes a long time and is not necessary. However, if so done make up loss by evaporation. Take out of autoclave, and adjust to desired reaction. Cool to 60° C. and add the whites of two eggs. Stir thoroughly. Heat either in the autoclave, fifteen pounds, thirty minutes; or if no autoclave is available, thirty minutes in the Arnold. If the heating is done in the Arnold, take out after half an hour, stir and replace for fifteen minutes more. Titrate, and adjust again if reaction has changed. If a correction in reaction is made, heat again for ten minutes, filter through cotton, tube and sterilize. Meat Infusion Agar.16 — In making meat infusion agar it is best to prepare two solutions, one consisting of the meat infusion to which the pepton and salt are added, the other an aqueous solution of agar which is dissolved in the autoclave. The ingredients are added in double strength to each solution so that when they are finally combined, the desired concentrations are obtained. Solution (1) 500 grams of lean meat in 500 c.c. of distilled or tap water are infused over night in the ice-box. The infusion is then strained through cheese cloth and all the juice squeezed out of the meat. The infusion is measured and the volume made up to 500 c.c. Two per cent pepton or 10 grams, and 1 per cent or 5 grams salt are now added to the 500 c.c. infusion and the mixture is heated over ihe free flame until it reaches a temperature of about 50° C. and the pepton dissolves. This solution is then 15 Amount of agar varies according to stiffness desired. 16 While titrating care should be taken that the medium does not solidify along the sides of the vessel. Glycerin agar is made by adding 6 per cent C.P. glycerin to meat extract or meat infusion agar. THE PREPARATION OF CULTURE MEDIA 153 titrated and sufficient alkali added to make the reaction about two or three points more alkaline than the desired final reaction of the medium. Solution (2) to 500 c.c. of water add the quantity of agar that will give the right concentration for the final volume (1 liter). For instance, if a 3 per cent agar is desired, add 30 grams of agar to 500 c.c. of water and dissolve in the autoclave at fifteen pounds pressure for fifteen minutes. When the agar is dissolved, cool it down to 50° C. and add to it Solution (1). Mix thoroughly and titrate again. Usually the addition of the agar solution does not change the reaction. Add the whites of two eggs well beaten with a little water and autoclave for thirty minutes at fifteen pounds pressure. If no autoclave is available, the medium may be put in the Arnold for forty-five minutes. The autoclave, however, is more satisfactory. It is best to check the titration again after autoclaving. The medium is now filtered, tubed and sterilized. LACTOSE-LITMUS- AGAR (Wurtz). — This is ordinary meat extract agar which is adjusted to P^ 7.5 to 7.8, to which 1 per cent of lactose is added, and enough litmus to give it a bluish purple color when cooled. Instead of litmus, 1 per cent of the Andrade indicator can be used. If the latter indicator is used, the reaction of the medium must be brought down to P^ 7.2. The medium containing 1 per cent of the indicator, if the reaction is correct, will be dark red when hot and colorless when cold. After the addition of the sugar and the indicator, it is best to sterilize by the inter- mittent method on three successive days. The red color fades out gradually, the indicator should be yellow after standing 2 or 3 hours. If it remains red or reddish, 1 or 2 c.c. more of normal NaOH should be added. The above description of the basic media, broth and agar, details chiefly the methods in general use until a few years ago. The recognition that bacteria grow more luxuriantly upon media containing considerable quantities of protein-split products, has resulted in the utilization of trypsinized culture media which has been found to possess considerable advantages for the cultivation of delicate organisms like the meningococci, etc. A simple formula for the production of trypsinized agar is the following, taken from the directions of Gordon : 17 TRYPAGAR. — To one pound of chopped meat, free from fat, add 1 liter tap water and make faintly alkalin to litmus with 20 per cent NaOH solution. Heat in double boiler at 75° to 80° for five minutes. Cool to 37° and add 0.5 gram trypsin. (Fairchild's preparation. For other brands the amount must be determined by experiment.) Incubate for five to six hours. Test for pepton as follows: Take 5 c.c. of the liquid, add 5 c.c. N/l NaOH and 1 c.c. dilute CuSO4. A pink color indicates that trypsinization is 17 Gordon, Br. Med. Journal 2, 1916, 678. 154 BIOLOGY AND TECHNIQUE complete — a bluish purple shade, that it is incomplete. If test is satisfactory, slightly acidify the broth with glacial acetic acid and bring slowly to boiling point and boil gently for ten minutes and filter through paper. Add 2 per cent agar and 0.5 per cent salt, autoclave to dissolve agar, and proceed from this point as usual, clearing with egg and setting to P H 7.5, or any desired reaction. Vedder Starch Agar. — Beef infusion agar is prepared without salt and pepton. This is adjusted to P^ 6.8, 10 grams of corn starch is added to each liter, and the mixture is heated in the autoclave for 20 minutes at 15 pounds. It is tubed and sterilized. This medium has been recommended by Vedder for the cultivation of gooococcus. Welch's Modification of Guarnieri's Medium.™ — This medium is made on a meat infusion basis, according to the directions given for the preparation of meat-infusion agar. It contains 5 grams of agar, 80 grams of gelatin, 5 grams of NaCl, and 10 grams of pepton to one liter. It should be adjusted to a neutral reaction. It is used for stab cultures and is designed chiefly for pneumococcus cultivation and storage. Dorsett Egg Medium. — This medium is chiefly useful for the cultivation of tubercle bacilli. 1. Carefully break eggs and drop the contents into a wide-mouthed flask. Break up the yolk with a sterile platinum wire, and shake up the flask until the whites and yolks are thoroughly mixed. 2. Add 25 c.c. of distilled water to every four eggs; strain through sterile cloth. 3. Pour 10 c.c. each into sterile test tubes and slant in an inspissator and expose to 73° C. for four to five hours on two days. 4. On the third day, raise the temperature to 76° C. 5. The sterilization may be finished by a single exposure to 100° C. in the Arnold sterilizer for fifteen minutes. Before inoculation, add two or three drops of sterile water to each tube. LUBENAU'S GLYCERIN- EGG. — To 1 liter of veal broth containing 2 per cent pepton, 5 per cent of glycerin is added. Neutralize this to litmus, and to every 200 c.c. add 10 fresh eggs. The mixture is thoroughly stirred, and when homogeneous, is tubed, slanted and inspissated, as in the case of other egg media. PETROFIF'S MEDIUM.— I. Meat Juice. 500 grams of beef or veal are infused in 500 c.c. of a 15 per cent solution of glycerin in water, in a cool place. After 24 hours the meat is squeezed in a sterile press and the infusion collected in a sterile beaker. II. Eggs. The shells of the eggs are sterilized by 10 minute immersion in 70 per cent alcohol. They are broken into a sterile beaker, well mixed " Welch, Bull. Johns Hopkins Hosp., 1892, vol. 3, p. 127. THE PREPARATION OF CULTURE MEDIA 155 and filtered through sterile gauze. One part of meat juice is added to two parts of egg by volume. III. Gentian Violet. 1 per cent alcoholic solution of gentian voilet is added to make a final proportion of 1 : 10,000. The three ingredients are well mixed. The medium is tubed and inspis- sated as usual. Petroff recommends for sputum the following technique: Equal parts of sputum and 3 per cent sodium hydroxid are shaken and incubated at 38° C. for 15 to 30 minutes, the time depending on the consistency of the sputum. The mixture is neutralized to litmus with hydrochloric acid and centrifugalized. The sediment is inoculated into the medium described above. Pure cultures are obtained in a large proportion of cases. SYNTHETIC MEDIA FOR THE TUBERCLE BACILLUS. — A considerable number of synthetic media have been made for the growth of the tubercle bacillus. The purpose of these media is to omit complex protein substances as much as possible. The one- we give below is according to the formula used by Petroff with success. 0.35 gram K2HPO4 4.93 grams Mg HPO4. 2H2O 10 c.c normal H2SO4 20 c.c normal (1/3 molar) H3PO4 10 c.c 3 times normal (molar) citric acid 5.29 grams asparagin 20 c.c glycerin 1000 c.c water add 10 c.c. normal NaOH. Potato Media. — Large potatoes are selected, washed in hot water, and scrubbed with a brush. They are peeled, considerably more than the cuticle being removed. The peeled potatoes are washed in running water, following which cylindrical pieces are removed with a large apple corer. The cylinders are cut into wedges. Since the reaction of the potato is normally acid, this should be corrected by washing the pieces in running water over night, or, better, by immersing them in a one per cent solution of sodium carbonate for half an hour. The pieces are then inserted into the large variety of test tubes known as "potato tubes." (See Fig. 21, c.) In the bottom of the tubes a small amount of water (about 1 c.c.) or a small quantity of moist absorbent cotton should be placed in order to retard drying out of the potato. The tubes are sterilized by fractional sterilization, twenty minutes to half an hour in the Arnold sterilizer on three successive days. POTATO BROTH. — Petroff has used extract of potatoes in fluid media for the growth of tubercle bacilli. There are many different ways of preparing the potato extract. The best way is to finely grind thoroughly, or grate the 156 BIOLOGY AND TECHNIQUE potatoes and soak them in tap water for from 12 to 24 hours, using about 500 grams to a liter of water. This mixture can be filtered, or, better, heated before filtration. It may be used as an ingredient with or without glycerin in ordinary broth or agar, or can be used with pepton and salt added, as an independent culture medium. Glycerin Potato. — In preparing glycerin potato the potato wedges are treated as above, and are then soaked in a ten to twenty-five per cent aqueous glycerin solution for one to three hours. A small quantity of a ten per cent glycerin solution should be left in the tubes. In sterilizing these tubes, thirty minutes a day in the Arnold after the sterilizer is hot, will sterilize without altering the glycerin. Milk Media. — Fresh milk is procured and is heated in a flask for fifteen minutes in an Arnold sterilizer. It is then set away in the ice chest for about twelve hours in order to allow the cream to rise. Milk and cream are then separated by siphoning the milk into another flask. It is rarely necessary to adjust the reaction of milk prepared in this way, since, if acid at all, it is usually but slightly so. If, however, it should prove more than 1.5 per cent acid, it should be discarded or neutralized with sodium hydrate. The milk may then be tubed either with or without the addition of an indicator. Litmus gives the most satisfactory results in milk, but at the present time is difficult to obtain. The Andrade indicator can be used also, but usually discolors the milk somewhat. However, if fractional sterilization is carefully carried out, the milk becomes yellowish, but acid production shows up clearly by a distinct reddening of the medium. If the milk is to be used for the differentiation of the anaerobic bacilli isolated from war wounds, it is best not to add any indicator, since the type of coagulum formed is a differential characteristic, and coagulation does not take place readily when Andrade is present. SODIUM OLEATE AGAR (For Influenza Bacilli). — Avery19 has found that sodium oleate will enhance the development of influenza bacilli, and at the same time will inhibit many of the Gram-positive organisms commonly found in sputum. A neutral solution of Kahlbaum's sodium oleate in water is prepared and sterilized in the autoclave. Human or rabbit blood is defibrinated, centri- fuged, the serum removed, and the volume made up to the original with broth. 1 c.c. of the red blood cell suspension and 5 c.c. of the 2 per cent sodium oleate solution are added to 94 c.c. of agar at 80° to 90° C. The agar is preferably a 2 per cent hormone agar with a reatcion of Prt 7.4. Serum Media. — Loeffler's Medium. — Beef blood is collected at the slaughter house in high cylindrical jars holding two quarts or more. It is desirable that attempts should be made to avoid contamination as much as 19 J. A. M. A. 1918, vol. 71, 2050. THE PREPARATION OF CULTURE MEDIA 157 is feasible by previously sterilizing the jars, keeping them covered, and exercising care in the collection of the blood. The blood is allowed to coagulate in the jars, and should not be moved from the slaughter house until coagulated. All unnecessary shaking of jars should be avoided. As soon as the coagulum is fully formed, adhesions between the clot and the sides of the jar should be carefully separated with a sterile glass rod or wire. The jars are then set away in the ice chest for 24 to 36 hours. At the end of this time clear serum will be found over the top of the clot, and between the clot and the jar. This should be pipetted off, preferably with a large pipette of 50 to 100 c.c. capacity, or siphoned off with sterile glass tubing, and transferred to sterile flasks. To three parts of the clear serum is then added one part of a one per cent glucose beef infusion or veal infusion bouillon. The mixture is filled into tubes, preferably the short test tubes commonly used for diagnostic diphtheria cultures. The tubes are then placed in a slanting position in the apparatus known as an inspissator (see p. 71). This is a double- walled copper box covered by a glass lid, eased in asbestos, and •surrounded by a water jacket. It is heated below by a Bunsen flame. Together with the tubes a small open vessel containing water should be placed in the" inspissator to insure sufficient moisture. The temperature of the inspissator is now raised to 70° -75° C., care being taken that the rise of temperature takes place slowly. The temperature is maintained at this point for two hours, and the process is repeated, for the same length of time, at the same temperature, on six successive days, preferably without removing the tubes from the inspissator at any time. It is also possible, though less regularly yielding good results, to sterilize in the inspissator for one day, following this on the second and third days by exposure for thirty minutes to 100° C. in the Arnold steam sterilizer. In doing this, the Arnold should be very gradually heated, at first without outer jacket, this being lowered only after thorough heating has taken place. Serum-Water Media for Fermentation Tests. — For the determination of the fermentative powers of various microorganisms for purposes of differen- tiation, Hiss has devised the following media in which the cleavage of any given carbohydrate is indicated, not only by the production of an acid reaction, but by the coagulation of the serum proteins. Obtain clear beef serum by pipetting from clotted blood in the same way as this is obtained for the preparation of Loeffler's blood-serum medium. Add to this two or three times its bulk of distilled water, making a mixture of serum and water in proportions of one to two or three. Heat the mixture for fifteen minutes in an Arnold sterilizer at 100° C. to destroy any diastatic ferments present in the serum. Add one per cent of a five per cent aqueous litmus solution (the variation in the different litmus preparations as obtained in laboratories necessitates a careful addition of 158 BIOLOGY AND TECHNIQUE an aqueous litmus solution until the proper color, a deep transparent blue, is obtained, rather than rigid adherence to any quantitative directions). With many batches of serum, it will be found that the addition of two or three times its bulk of distilled water is not a sufficient dilution to prevent coagulation. It will often be found necessary to add four or five volumes of distilled water to one volume of serum. One per cent of the Andrade indicator may be substituted for the litmus. Add to the various fractions of the medium thus made one per cent respectively of the sugars which are to be used for the tests. For the preparation of inulin medium, made in this way for pneumococcus- streptococcus differentiation, it is necessary to sterilize the inulin dissolved in the water to be added to the serum in an autoclave at high temperature (15 pounds for 15 minutes) in order to kill spores before mixing- with the • serum. The serum-water media are sterilized by the fractional method at 100° C., at which temperature they remain fluid. Chocolate Media (Park and Williams). — It has recently been observed that for the -cultivation of organisms like the influenza bacillus, meningococcus, and a number of other of the more delicately growing bacteria, an excellent medium can be made up in the following way: Agar or broth are made up as usual, and to them added defibrinated rabbit, beef, horse or human blood in proportions of from 5 per cent to .10 per cent by volume. This mixture is then heated gradually up to about 75°, until the blood begins to coagulate and assume a dark brown chocolate like color. The broth or agar can first be adjusted to the desired reaction, but it is likely that any excess alkali or acidity is corrected by the proteins which are added. The medium can be tubed, or, in the case of agar, plated or slanted, as it is, after distribution of the blood throughout the medium by shaking. In the case of broth the medium can be filtered through paper while hot, and sterilized subsequently by filtration and fractional heating. Such filtrate consists of a clear brownish fluid on which influenza bacilli and other organisms grow with enormous speed. The speed with which influenza bacilli grow on this medium, and its almost complete freedom of hemoglobin, but very much increased cholestrin and other lipoid contents, have lead us to believe that it is not the hemoglobin particularly which is needed for influenza bacillus cultivation. SPECIAL MEDIA FOR COLON TYPHOID DIFFERENTIATION Conradi-Drigalski Medium.-®— Original directions. (a) Three pounds of meat are infused in two liters of water for twelve hours or more. Strain, boil for one hour and add 20 gms. Witte's pepton, 20 gms. of nutrose, 10 gins, of NaCl ; boil one hour and filter. Add 60 gms. (Jonrvdi- VripateM, Zeit, f, Hyg.z xxxix, 1902, THE PREPARATION OF CULTURE MEDIA 159 of agar. Boil for three hours (or one hour in an autoclave) until agar is dissolved. Render weakly alkaline to litmus paper, filter, and boil for half an hour more. (b) Litmus solution: Two hundred and sixty e.c. of litmus solution are boiled for ten minutes. (The litmus solution used by Conradi and Drigalski is the very sensitive aqueous litmus recommended by Kubel and Tiemann, and purchasable under the name.) After boiling, 30 grams of chemically pure lactose are added to the litmus solution. The mixture is then boiled for fifteen minutes, and if, a sediment has formed, is carefully decanted. (c) Add to the hot lactose mixture to the hot agar solution; mix well and, if necessary, again adjust to weak alkaline reaction, litmus paper being used as an indicator. To this mixture add 4 c.c. of a hot, sterile ten per cent solution of sodium carbonate, and 20 c.c. of a freshly made solution of crystal violet (c. p. Hochst), 0.1 gram in 100 c.c, of sterile distilled water. Surface smears are made upon large plates. These are incubated twenty-four hours. Typhoid colonies are small, blue, and trans- parent. Colon colonies are large, red, and opaque. Endo's Medium.-1 — 1. Prepare one liter of meat infusion three per cent agar, containing 10 grams of pepton and 5 grams of NaCl. 2. Neutralize and clear by filtration. 3. Add 10 c.c. of 10% sodium carbonate to render alkaline. 4. Add 10 grams of chemically pure lactose. 5. Add 5 c.c. of alcoholic fuchsin solution, filtered before using. Endo in his original contribution does not mention the strength of this fuchsin solution, which, however, should be saturated. This colors the medium red. 6. Add 25 c.c. of a 10% sodium sulphite solution. This again decolorizes the medium, the color not entirely disappearing, however, until the agar is cooled. 7. Put into test tubes, 15 c.c. each, and sterilize. The medium should be kept in dark. Plates are poured and surface smears made. The typhoid colonies remain colorless, while those of coli become red. The preparation of Endo's medium presents difficulties due to the varying purity of sodium sulphite. Kastle and Elvove22 recom- mend the use of anhydrous sodium sulphite instead of the crystallized variety. Harding and Ostenberg23 add sodium sulphite solution to a measured amount of .5 per cent fuchsin to determine the propor- •lEndo, Cent. f. Bakt., xxxv, 1904. -Kastle and Elvove, Jour. Inf. Dis., xvi, 1909. 23 Harding and Ostenberg, Jour, of Inf. Dis., xi, 1, 1909. 160 BIOLOGY AND TECHNIQUE tions which give the greatest delicacy of reaction as tested with formaldehyd. The proportions so determined are then added to the hot 3 per cent agar. Although Endo described his medium as dependent upon the formation of acid by the bacteria, this is not so. Acids give no coloration of the sulphite-fuchsin mixture. Indeed this mixture is used by chemists under the name of Schiff's reagent as a test for aldehyds. Acids decolorize the red caused by aldehyds, and this accounts for the frequent late discoloration of red colon colonies on prolonged cultivation. The medium is red when hot, and colorless when cold, because the compound between sulphite and fuchsin dis- sociates in the hot solution. Robinson and Rettger's Modification of Endo.24 — This seems to be at present the most useful modification of Endo available. 1. To 1 liter of water add 25 grams of agar, 10 grams of pepton, and 5 grams of meat extract. Dissolve the agar, meat extract and pepton. Bring to P H 6.8, and heat in autoclave for l/2 hour at 15 pounds. Filter through cotton. To this add 10 c.c. of a 10 per cent sodium carbonate solution. Heat for a few minutes, and add 1 per cent lactose. The fuchsin sulphite indicator is then added in the form of 5 c.c. of saturated alcoholic fuchsin, and 10 c.c. of a 10 per cent solution of sodium bisulphite. This is tubed and sterilized. Krumwiede recommends preparing the medium by adjusting the reaction to PH 8.5 in the first place, relying upon sterilization and the addition of the bisulphite to bring the reaction to the desired end point. The best results are obtained by adding the lactose, fuchsin and bisulphite just before use, and this can be done most conveniently if the agar basis is bottled in 100 c.c. amounts. The final reaction of the medium should be P# 8. A more acid reaction favors the diffusion of the indicator. In our own laboratory we have found that the addition of these amounts of fuchsin and sodium bisulphite to the medium inhibit the growth of typhoid in some instances. We have obtained equally good differentiation by using 0.25 per cent fuchsin instead of 0.5 per cent, and 0.5 per cent sodium bisulphite instead of 1 per cent. Kendall's Modification of Endo's Medium.2^ — 1.5 per cent meat extract agar is prepared, and the reaction adjusted faintly alkaline to litmus by the addition of NaOH. This agar is stored in small flasks and it is usually convenient to keep flasks containing 100 c.c. each. Just before use, 1 per cent of lactose is added, and then decolorized fuchsin solution, as in Endo's 2*Kobinson and Eettger, Jour, of Med. Kes., 24, 1916, 363. 28 Kendall, Boston Med. & Surg. Jour. THE PREPARATION OF CULTURE MEDIA 161 medium. Add about 1 c.c. of decolorized fuchsin solution, made up as above by mixing roughly prepared 10 per cent sodium sulphite with saturated alcoholic fuchsin. (The proportions of fuchsin and sulphite are sometimes difficult to adjust, possibly by reason of impurities in the sulphite due to formation of sulphate. The instructions given by most workers at present are to use 10 c.c. of a 10 per cent aqueous solution of sodium sulphite, and to add to this 1 c.c. of a 10 per cent solution of fuchsin in 96 per cent alcohol.) When these flasks containing the various ingredients are hot they are red or pink, but when plates are poured and allowed to harden, the medium should be either colorless or very faintly pinkish. It is best to pour a number of plates rather thickly and then allow them to dry with the covers off. Inoculations from the feces suspension are then made by surface smear, with a bent glass rod. Colon colonies are pinkish and red; typhoid colonies, smaller and grayish. In concluding the description of some of the most important typhoid isolation media, we would like to add that a great deal seems to depend upon the habit-acquired skill which the individual worker attains. None of these stool isolation media are ordinarily successful at once in the hands of anyone, and a certain amount of practice must be attained before one can judge of the usefulness or uselessness of a medium. Brilliant Green Agar for Typhoid Isolation. — Krumwiede has 'recently devised a brilliant green agar with which he has had excellent results.26 The basis is an extract agar like that used for Endo's medium : Beef Extract 0.3% Salt 0.5% Peptone 1.0% Agar 1.5% (Domestic peptones are satisfactory.) Dissolve in autoclave; clear and filter. A clear agar is essential. The final reaction of the medium is to be neutral to27 Andrade's indicator, which in terms of phenolphthalein is 0.6-0.7% acid (normal HC1) or P^ 7.2. It is more convenient to have the reaction set slightly alkaline to litmus at the time of preparation and to acidify each bottle as used. The agar is bottled in 100 c.c. amounts and autoclaved. When needed, the bottles are melted and the volume of each corrected (if necessary) to an approximate 100 c.c.. Add to each bottle: 28 We are indebted to Dr. Krutnwicde for a preliminary account of this method. 21 Andrade's Indicator: 0.5 per cent aqueous acid fuchsin 100 c.c. Normal NaOH 16 c.c. The dye is slowly (2 hours) alkalinized to the color-base; the red tint is restored by acids. 162 BIOLOGY AND TECHNIQUE One per cent Andradc Indicator. Acid to bring to neutral point of the indicator.28 One per cent Lactose.29 0.1 per cent Glucose. Brilliant Green in 0.1 per cent aqueous solution. Two dilutions of dye are • used in routine plating, corresponding to 1-500,000 and 1-330,000 in terms of solid dye (0.2 c.c. and 0.3 c.c. of 0.1 per cent solution per 100 c.c. of agar). The sample of dye which Krumwiede has used is from Bayer, but he has also tested and found equally satisfactory samples from Griibler and Hochst. 0.1 gram of dye is accurately weighed on a foil, washed with boiling H20 into a 100 c.c. volumetric flask and made up to the mark when cool. The flask should be clean and neutral (by test). Fresh solutions vary in activity (see standardization tests) ; they keep about a month. Each bottle is mixed and poured into six plates only (a thick layer of agar gives the most characteristic colonies). Plates are left uncovered until agar has "jellied"; porous tops are used; dry plates are essential to avoid diffusion. Standardization: The agar must have proper "balance." The reaction is important; sediment reduces the activity of the dye and light colored media are better than darker ones. Different lots of agar with the same dye solution act ununif ormly ; a new batch or a new solution must be tested. Any variation in the composition of the media necessitates a readjustment of dye concentration; this statement cannot be over-emphasized. Brilliant green, in appropriate dilutions, not only inhibits all Gram- positive and many Gram-negative bacteria, but exhibits differential action on the colon-typhoid group. Paratyphoid and the B. lactis aerogenes are untouched, typhoid is restrained only at low dilutions, while dysentery and the other colon group are extremely susceptible. The typhoid colony on this medium is characteristic. Looking through the plate against a dark surface, in oblique light the colony has a snowflake appearance; the edge delicately serrate. With artificial light and a hand lens, the texture is that of a coarse woolen fabric. Acid production from the trace of glucose may tinge the colony. The colony is large. Brilliant Green-Eosin Agar.30 — Meat infusion agar is prepared and titrated to +1 to phenolphthalein. The following substances are added: 28 An agar is neutral to Andradc when, hot, the color is a deep red, but fades completely on cooling. This is determined by cooling 3 or 4 c.c. of acidified hot agar in a serum tube under the tap and adjusting accordingly, 29 These are conveniently added from one sterile solution containing 20% lactose and 2% dextrose, 5 c.c. to 100 of agar gives the requisite concentration. 30 Teague and Clurman, Jour, of Infcc. Dis., 18, 1916, 647. THE PREPARATION OF CULTURE MEDIA 163 Eosin 3/50 per cent Brilliant green 1/300 per cent Saccharose 1 per cent Lactose 1 per cent The typhoid colonies on this medium are large and have a grayish pink color. Most strains of B. coli do not grow upon it; the colonies of B. coli that do develop have deep red centers. Meyer and Stickel31 claim that better results are obtained with the brilliant green-eosin medium if peptic digest agar is substituted for the meat infusion agar. They set their reaction at Pff =7 to 6.8. Malachite-Green Bouillon (Peabody and Pratt).32— To 100 c.c. of beef infusion broth add 10 c.c. of one per cent solution of malachite green Hochst 120, made with sterile water. This is tubed. This medium is used as an enriching fluid. One drop of the suspected material (emulsified stool) is added to each tube and after incubation for eighteen to twenty-four hours inoculations may be made upon plates. Peabody and Pratt found a reaction of .5 per cent acidity to phenol- phthalein most favorable. Lead Acetate Agar for the Differentiation of Paratyphoid "A" and "B." 33 — One drop of a ten per cent solution of neutral lead acetate is added to every 4 c.c. of agar. This is the original procedure of Burnet and Weissen- bach. Krumwiede recommends the cooling of the agar to 60°, then adding enough of a 0.25 per cent basic lead acetate solution to bring the concentration to 0.05 per cent. The agar is tubed, and Burnet and Weissenbach recommend inoculating with a fine needle in several places between the agar an_d the walls of the tube. Typhoid and paratyphoid "B" bacilli blacken the medium, while paratyphoid "A" leaves it unchanged. B. Enteritidis and Typhi Murium behave like paratyphoid "B." Bile Medium?* — (Recommended for blood cultures by Buxton and Cole- man.) The medium is prepared as follows: Ox-bile 900 c.c. Glycerin 100 c.c. Pepton 20 grams .t into small flasks containing quantities of about 100 c.c. .each and sterilized ; fractional sterilization. Jackson's Lactose-Bile Medium.™ — This medium is used for isolating B. 31 Meyer, K. F., and Sticlce I, J. K., Jour, of Infec. Dis., 23, 1918, 48. 3- Peabody and Pratt, Boston Med. and Surg. Jour., clviii, 7, 1008. 33 Burnet and Weissenbach, C. R. de la Soc de Biol., vol. 78, 1915, p. 565. 34 Conradi, Dent. med. Woch., 32, 1906. 35JacJcson, "Biol. Studies of Pupils of W. T. Sedgwick," 1906, Univ. Chicago ?ress. 164 BIOLOGY AND TECHNIQUE typhosus and B. coli from water, and serves as a valuable enriching medium in isolating them from other sources. Jackon and Melia36 found that in this medium B. typhosus and B. coli outgrow all other microorganisms and eventually B. typhosus will even outgrow B. coli. It consists of sterilized undiluted ox-bile (or a ten per cent solution of dry, fresh ox-bile) to which is added one per cent pepton and one per cent lactose. It is filled into fermentation tubes and sterilized by the frac- tional method. MacConkey's Bile-Salt Agar: Sodium glyeocholate 0.5 per cent. Pepton 2.0 " " Lactose 1.0 ' ' " Agar '. ... 1.5 " " Tap water q.8. The agar and pepton are dissolved and cleared and the lactose and sodium glyeocholate added before tubing. The B. typhosus produces no change; B. coli, producing acid, causes precipitation of the bile salts. Neutral-Red Medium. — To 100 c,c. of a one or two per cent glucose agar add 1 c.c. of a saturated aqueous solution of a neutral-red. The medium is used in tubes, stab or shake cultures. The typhoid bacillus produces no change, while members of the colon group render the medium colorless by reduction of the neutral-red and produce gas. Bariekow's Medium*1 — To 200 c.c. of cold water, add 10 grams of nutrose and allow to soak for one-half to one hour. Pour this into 800 c.c. of boiling water, and heat for twenty minutes in an Arnold sterilizer at 100° C. Filter through cotton and to the opalescent solution of nutrose add 5 grams of NaCI, 10 grams of lactose, and sufficient aqueous litmus solution to give a pale blue color.38 Russell's Double Sugar Agar.^ — Russell has devised a simple medium for quick identification of typhoid bacilli. A 2% or 3% extract agar is used, about 0.8% acid to phenolphthalein. Enough litmus solution is added to give it the ordinary deep blue. The reaction is then adjusted with sodium hydrate until neutral to litmus. Finally 1% lactose and 0.1% glucose (dissolved in a small amount of hot water) are added, the medium is carefully sterilized in the Arnold sterilizer and slanted. Inoculations are made by surface streak and stab. 36 Jackson and Melia, Jour. Inf. Dis., vi, 1909. 87 BarsieTcow, Wien. klin. Bund., xliv, 1901. 38 Filtration may be done through paper, but takes a long time. *gKussell, Jour. Med. Kesearch, xxv, 1911, 217. THE PREPARATION OF CULTURE MEDIA 165 Sharper results' are obtained with this medium if 1 per cent of the Andrade indicator (described above), is substituted for the litmus. When Andrade is used, the final reaction of agar should be about P^ 7.2. It ib best to standardize the reaction against the particular solution of Andrade used. The reaction of the medium is satisfactory when the mixture containing 1 per cent of the indicator is red when hot, and colorless when cold. Typhoid ,and paratyphoid A and B, and dysentery, on this medium show colorless growths on the slant, the butts, however, where partial anaerobiosis exists, show a deep red color, due to acid fermentation. Typhoid may be dis- tinguished from paratyphoid A and B by the fact that typhoid, since it ferments glucose without the production of gas, forms no gas bubbles in the butt, whereas, there is gas formation with paratyphoid A and B. Typhoid and dysentery give the identical reaction on Russell's medium colorless growth on the slant, and acid formation without gas in the butt, and must be distinguished by the motility test. There is no reliable way of distinguishing between paratyphoid A and B, unless lead acetate is added to the medium (see below). Krumwiede recommends the addition of 1 per cent saccharose to the 1 per cent lactose, and 0.1 per cent glucose as a means of ruling out some .of the "paratyphoid-like" intermediates that ferment saccharose more rapidly than lactose. The non-pathogenic types fermenting lactose, or lactose and saccharose, which are present in 1 per cent concentrations, produce acid on the slant, as well as in the butt, with the formation of gas^ and are thus easily eliminated. Russell Double Sugar Agar with Lead Acetate.40 — The 'best basis for this medium is an infusion agar which has been rendered sugar free and adjusted to P^ 7.4, or neutrality to Andrade indicator. To this medium add 1 per cent Andrade indicator, tube and sterilize in quantities of 5 c.c. to each tube. Make up a solution containing 20 per cent lactose and 2 per cent glucose. Sterilize. Make up a solution of 0.25 basic lead acetate. Sterilize. To each tube of the agar add 0.25 c.c. of the double sugar solution and 1 c.c. of the lead acetate solution. Add both the solutions to the agar at 60° C. under sterile precautions and slant. Typhoid bacilli cause a brown color near the surface of the stab. Para- typhoid "A" and dysentery do not cause any browning. Paratyphoid "B" and other members of the group cause browning. (The volume of agar per tube is not given in the article, but the sugar percentage works out for 5 c.c.) 40 Kligler, Jour, of Experimental Medicine, September, 1918. 166 BIOLOGY AND TECHNIQUE • SPECIAL MEDIA FOR THE ISOLATION OF CHOLERA SPIRILLA Dieudonnc Medium.** — To 70 parts of ordinary 3 per cent agar made neutral to litmus, add 30 parts of a sterile mixture of defibrinated beef blood and normal sodium hydrate. This is sterilized by steam before being added to the agar. This alkali agar is poured into plates and allowed to dry several days at 37°, or 5 minute* at 60°. The material to be examined is smeared on the surface of these plates with a glass rod. Aronson's Medium for Cholera Stool Isolation.42 — This medium is pre- pared as follows: 35 grams of agar are added to 1 liter of tap water and soaked over night. Add 10 grams of meat extract, 10 grams of pepton, 5 grams of sodium chloride and heat in steam sterilizer from 4 to 5 hours. The particles are allowed to settle by letting the hot agar stand, and the clear supernatant agar poured into flasks to hold 100 c.c. each. The following solutions are previously made and sterilized for l/2 hour in the Arnold: 1. 10 per cent solution of sodium carbonate 2. 20 per cent solution of cane-sugar 3. 20 per cent solution of dextrin 4. saturated solution of basic fuchsin 5. 10 per cent solution of sodium sulfite (sterilized by being brought to a boil) To 100 c.c. of agar add 6 c.c. of the 10 per cent solution of sodium carbonate and heat for 15 minutes at 100° C. The agar, because of the alkalinity, becomes brown and cloudy. While hot, add 5 c.c. of the 20 per cent solution of cane-sugar, 5 c.c. of the 20 per cent solution of dextrin, 0.4 c.c. of the saturated solution of basic fuchsin, and 2 c.c. of the 10 per cent sodium sulfite solution. The flask is allowed to stand to let the coarser particles settles, and plates are poured with the clear supernatant fluid. The principle of this medium, like Dieudonne's, depends upon the ability of cholera spirilla to grow on very alkalin media and upon their ability to split polysaccharites with acid and aldehyde formation. Cholera strains, recently from the human body, give large red colonies in from 15 to 20 hours, whereas, the colon colonies are smaller and colorless. Teague and Travis43 found that strains of cholera spirillum that had been out of the human body for some time did not yield red colonies promptly, but they obtained excellent results even with these cultures, if they added 0.25 per cent nutrose to Aronson's medium. "Dieudonne, Cent. f. Bakt., 1., orig., 1909. "Aronson, Deut. med. Woch., 41, 1915, 1027. 43 Teague and Travis, Jour, of Infec. Dis., 18, 1916, 601. THE PREPARATION OF CULTURE MEDIA 167 Teayue and Travis Medium for the Cholera Spirillum.** — They prepare their medium as follows: Two pounds of chopped beef are soaked in 2 liters of distilled water in the ice box over night. The fluid is squeezed out, heated in the Arnold, filtered through filter paper and made neutral to litmus with sodium hydrate, and is heated again. After being allowed to cool, it is inoculated with colon bacillus, and incubated for 2 or 3 days to make it sugar free. Agar is then prepared from it by adding 1 per cent pepton, 0.5 per cent sodium chlorid and clearing with egg. The reaction is adjusted to —0.5 phenolphthalein, and after the agar has been cleared and filtered, 0.25 per cent nutrose is added. A stock aqueous solution of 3 per cent bluish eosin is kept on hand in the dark, also a 1 per cent stock solution of Bismarck-brown. The Bis- marck-brown solution must be made up in water containing 10 per cent of lactose, because it is entirely soluble to 1 per cent in distilled water alone. To 50 c.c. of this nutrose agar add 1 per cent saccharose, 1 c.c. of the 3 per cent eosin solution, and 2 c.c. of the 1 per cent Bismarck-brown solution. After this mixture has been shaken until the stains are uniformly distributed, pour plates. These plates are uncovered and placed on a shelf, face down, in the incubator for 20 minutes to remove excess of moisture before smearing. On this medium the differentiation of the cholera colonies is striking, with large and with red centers, while the colon colonies are uniformly pink. Rabbit's Blood for Ducrey Bacillus Cultivation. — Rabbits are bled from the heart with a sterile syringe and about IVk to 2 c.c. placed into small test tubes. The blood is allowed to clot and inactivated at 56° for y% hour. This makes an excellent medium for the cultivation of the Ducrey bacillus, for streptococci and some other organisms. It is also excellent for the preservation of streptococci and pneumococci in a virulent condition. The preparation of Anaerobic Tissue Tubes for the Cultivation of Spiro- chaetes and other Anaerobes. — This is the Tarrozi and Smith method of using tissue for anaerobic purpose, adapted by Noguchi for the cultivation of various spirochaetes. The proportions of broth, serum and agar are adapted to the particular purpose for which it is to be used. It is necessary, therefore, in this place only to describe the best method of putting up the tissue tubes. High, narrow test tubes are used, about 7 to 8 inches in length, with a diameter not larger than that of a Wassermann tube. One-half inch tubes are convenient. A rabbit is rapidly killed by ether anesthesia. It is best to bleed him from the carotid at the same time, in order not to waste the blood. The animal is immediately opened with sterile precautions. It is best to dissect off the fur and wash the abdominal wall with alcohol before opening the abdomen. The abdomen is then opened carefully and widely, so that tlio organs can T>c easily reached without unnecessary poking about. 44 Teague ami Travis, Jour, of Infec. Dis., 18, 1916, 601. 168 BIOLOGY AND TECHNIQUE The intestines are pulled aside so as to uncover the kidneys. With a fresh set of instruments, the hilum of the kidney is now grasped and the kidney rapidly separated from its capsule and passed through the flame before being placed into a sterile Petri plate. The spleen may be removed in the same way. The kidney and spleen should then be cut up with the utmost precautions of sterility. Work in a dustless place with the windows closed and several Bunsen flames going close to the field of operation. An assistant slightly raises the cover of the Petri plate and the bacteriologist, working with sterile forceps and a sterile old knife which can be constantly flamed, cuts the kidney in pieces of appropriate size against the bottom of the plate. In placing these bits of tissue into tubes, the stopper of the sterilized tube is pulled, and the tube heated around its' lips and upper one-half inch. The tissue is then rapidly passed into the flame, thrust into the mouth of the tube, the cotton stopper flamed and inserted. The tube is then given a rapid flip with the hand, which sends the tissue to the bottom. When these tubes are filled with broth and ascitic fluid or agar, they should be incubated before use and the unsterile ones discarded. In most of Noguchi's work, and some of our own, paraffin oil was used over the tops of these tubes. The sealing properties of this, however, are not what they were formerly supposed to be. Air passes through this paraffin oil, and if sealing is desired it is much better to heat the upper empty part of the tubes thoroughly, and thrust in a paraffin stopper. The top of the fluid also can be covered with melted paraffin which will solidify in the incubator. Cooked Meat Medium, Robertson,45 for the Cultivation of Anaerobes. — 250 grams of beef heart are minced and ground in a mortar. Add 250 c.c. of tap water, heat slowly, cook thoroughly, neutralize to litmus with NaOH, tube and sterilize in autoclave. The simplest method of making cooked meat media which gives satis- factory results with the majority of the anaerobic bacilli, is the following. A few pieces of chopped meat (not necessarily heart) are placed in the bottom of the tube, enough infusion broth, PH 7.8, is added so that there are about 3 c.c. clear broth over the meat. The medium is ready for use after autoclaving. The reaction usually becomes more acid on autoclaving in the presence of the meat fragments. The optimum for the most anaerobic bacilli is PH =7.4. Enriching Substances Used in Media. — For the cultivation of microorganisms which are sensitive to their food environment, it is often necessary or advisable to add to the ordinary media enriching substances, which empirical study has shown to favor the growth ts Robertson, Jour, of Path, and Bacter., January, 1916, 20, No. 3. THE PREPARATION OF CULTURE MEDIA 16§ of the organism in question. The substances most commonly used for such enrichment are glucose, nutrose (sodium casemate), gly- cerin, sodium formate, and uiisolidified animal proteins. As animal and blood serum and whole blood must frequently be used in this way, an understanding of the methods employed in obtaining these substances is necessary. Method of Obtaining Blood and Blood Media. — Blood serum from beef and sheep may be collected in the manner recommended for the collection of such serum in the preparation of Loeffler's medium, pipetted into test tubes, and sterilized in the fluid state by exposure to temperatures ranging from 60° to 65° C., for one hour upon six consecutive days. It is not a simple matter to sterilize serum in this way and requires much time and care. The method most commonly employed, in laboratories which have access to hospitals, for obtaining clear serum depends upon the col- lection of exudate or transudate fluids by sterile methods directly from the pleural cavity, the abdominal cavity, or the hydrocele cavity. Sterile flasks or test tubes are prepared and the fluid is allowed to flow directly out of the cannula into these. It is necessary to avoid carbolic acid or other disinfectants in sterilizing instru- ments and rubber tubing used during the operation. These should be brought into the ward in the water in which they have been boiled and not in strong antiseptic solutions, as is frequently done. The fluid so obtained may be incubated and the contaminated tubes discarded. The serum may then be added, in proportions of one to three, to sterile broth or melted agar. Agar thus used is melted and cooled to 60° C., or below. One- third of its volume of warmed exudate fluid is added, and the plates are poured. Serum may be rendered free of bacteria by filtration through a Berkefeld or Pasteur-Chamberland filter. This is an effectual method, but requires much time and care. Whole blood may be obtained for cultural purposes by bleeding rabbits or dogs or other animals directly from a blood-vessel into tubes of melted agar. In the case of a rabbit, after the administra- tion of an anesthetic (ether), an incision is made directly, over the trachea, and, by careful section, the carotid artery is isolated, lying close to the side of the trachea. Blood may be collected and hemolysed by the gradual addition of the smallest amount of ether which will completely hemolyse the amount treated. This may be 170 BIOLOGY AND TECHNIQUE kept in stoppered sterile bottles and added to agar as desired. This is particularly useful in preserving blood for routine work on menin- gococcus carriers. Methods of bleeding animals are briefly described above. SELECTIVE ACTION OF DYE STUFFS In describing the selective media for typhoid bacilli we have seen that malachite green and crystal violet have been found to exert a certain amount of selective action upon the typhoid and colon groups. The selective influence of various dyes has been recently again studied by Churchman. Churchman46 found that the addition of gentian violet in dilutions of 1 :100,000, to media, in- hibited some bacteria, while others grew luxuriantly in its presence. Extremely interesting, both practically and theoretically, is his ob- servation that upon such gentian violet media bacteria fall into two groups. Those which grow on gentian violet correspond in a general way to the Gram-negative bacteria; those which fail to develop on these media correspond roughly with the Gram-positive species. One strain of the enteritidis group could not be cultivated on gen- tian violet, and this was found to differ from the others also in its agglutination tests. Signorelli47 claims that dahlia is useful in differentiating true cholera strains from similar spirilla. The true cholera strains grew with colored colonies, while others remain colorless, in his experi- ments. Krumwiede and Pratt48 were unable recently to confirm the claims of Signorelli. However they fully confirm the findings of Churchman both as to the selective action of gentian violet and in regard to the classification of bacteria into two groups corresponding to their reaction to the Gram stain. They state that among Gram- negative bacteria a strain is occasionally found which will not grow on the gentian violet media, differing in this respect from other members of the same species. They find also that the reaction is quantitative. The streptococcus-pneumococcus group, according to their in- *• Churchman, Jour. Exp. Med., 16, 1912 ; also Churchman and Michael, ibid. "Signorelli, Centralbl. f. Bakt., Orig. 56, 1912. "Krumwiede and Pratt, Centralbl. f. Bakt., Orig. 68, 1913; and Proc. N. Y. Path. Soc., xiii, 1913. THE PREPARATION OF CULTURE MEDIA 171 vestigations, differs from other bacteria in being able to grow in the presence of quantities of violet which inhibit other Gram-positive species. Dysentery bacilli show variations. They found, however that, in addition to gentian violet, Hoffman violet, crystal violet, dahlia violet, fuchsin, rosanilin and methyl violet will inhibit Gram- positive but not Gram-negative bacteria in dilutions of from 1 to 5000 to 1 to 50,000. CHAPTER VIII METHODS USED IN THE CULTIVATION OF BACTEEIA INOCULATION OF MEDIA THE transference of bacteria from pathological material to media, or from medium to medium, for purposes of cultivation, is usually accomplished by means of a platinum wire or loop. The platinum wire used should be thin and yet possess a certain amount of stiff- ness and be about two to three inches in length. This is fused into the end of a glass rod six to eight inches long. It is an advantage, though not necessary, to use rods of so-called "sealing-in" glass which, having the same coefficient of expansion as platinum, does not crack during sterilization. For work with fluid media, the wire should be bent at its free end so as to form a small loop which will pick up a drop of the liquid. For the inoculation of solid media and the making of stab cultures, a straight "needle" or wire should be used. Other shapes of these wires and spatulae from heavy wire have been devised for various purposes and are easily improvised as occasion demands. When making a transfer from one test tube to another, the tubes should be held between the thumb and first and second fingers of the left hand. (See Fig. 12.) The plugs are then removed by grasping them between the small and ring fingers and ring and middle fingers of the right hand, first loosening any possible adhe- sions between glass and plugs by a slight twisting motion. The platinum wire is held meanwhile by the thumb and index fingers of the right hand in the manner of a pen. The wire is heated red hot in a Bunsen flame, and is then passed into the culture tube without being allowed to touch the glass. It is held suspended within the tube for a few seconds to permit of cooling before touch- ing the bacterial growth. The wire is then allowed to touch lightly the surface of the growth and a small amount is picked up. It is then removed from the tube without allowing it lo 172 METHODS USED IN CULTIVATION OF BACTERIA 173 touch the sides of the glass, and is passed into the tube which is to be inoculated. If the tube contains a slanted medium, such as agar, a light stroking motion from the bottom of the slant to its apex will deposit the bacteria upon the medium evenly along a central line. The needle may also be plunged downward into the substance of the nutritive material so that in the same tube both surface growth and deep growth may be observed. If a stab culture is to be made in unslanted agar or in gelatin, the needle is simply plunged straight downward as nearly as possible along the axis of the medium. If a fluid medium is being inoculated, the wire should be introduced only into the upper part of the liquid and the bacteria gently rubbed into emulsion against the side of the glass. The FIG. 12. — TAKING PLUGS FROM TUBES BEFORE INOCULATION. needle is then removed from the tube, the stopper carefully replaced, and the platinum wire immediately sterilized in the flame. This sterilization of platinum needles after they have been in contact with bacteria should become second nature to those working with bacteria, since an infraction against this rule may give rise to serious and widespread consequences. In burning off platinum needles it is well to remember that a part of the glass rod, as well as the wire itself, is introduced into the tubes and may become contaminated, and for this reason the rod itself, at least in its lower two or three inches, should be passed through the flame as well as the wire. As an extra precaution against contamination, the lips of test tubes and flasks and the protruding edges of cotton plugs may be passed through the flame and singed. 174 BIOLOGY AND TECHNIQUE THE ISOLATION OF BACTERIA IN PURE CULTURE It is obvious that in many cases where bacteria are cultivated Irom water, milk, pathological material, or other sources, many species may be present in the same specimen. It is likewise obvious that scientific bacteriological study of any bacterium can be made only if we obtain this particular species entirely apart from others, in what is known as "pure culture." The earliest methods for accomplishing this were the methods of Pasteur and of Cohn who depended upon the power of one species to outgrow all others, if cultivated for a sufficient length of time in fluid media. This method, of course, was inadequate in that it was often purely a matter of chance which one of the mixture of species was finally obtained by itself. A later method, by Klebs, depends upon serial dilution, in test tubes of fluid media, by which the eventual transference of one germ only, to the last tube was attempted. Such methods, none of them of great practical value, have been entirely displaced by those made possible by Koch's introduction of solid media which may be rendered fluid by heat. The methods now employed for the isolation of bacteria depend upon the inoculation of gelatin or agar, when in the melted state, the thorough distribution of the bacteria in these liquids by mixing, and the subsequent congealing of these media in thin layers. By this means the individual bacteria, distributed in the medium when liquid, are held apart and separate when the medium becomes stiff. The masses of growth or "colonies" which develop from these single isolated microorganisms are discrete and are descendants of a single organism, and can be transferred, by means of a process known as "colony-fishing," to fresh sterile culture media. Plating. — The first method employed by Koch for bacterial isola- tions was one that consisted in the use of simple plates of glass, about 3X4 inches in size, mounted upon a leveling stand. A wooden triangle, supported upon three adjustable screw-feet, formed the base of this apparatus. Upon this was set a covered crystallizing dish which could be filled with ice water. Upon the top of this rested the sterilized plates under a bell jar. By screwing up or down upon the supports of the triangle, leveling of the plate could be achieved and controlled by a spirit-level placed at its side. The inoculated gelatin was poured upon the plate and spread and rapidly METHODS USED IN CULTIVATION OF BACTERIA 175 cooled and hardened by the cold water contained in the crystallizing dish. The original method of Koch has been modified considerably and the method universally employed at present depends upon the use of circular covered dishes, the so-called Petri dishes. These obviate the necessity of a leveling stand and prevent contamination of the plate when once poured. Each Petri dish plate consists of two circular glass dishes; the smaller and bottom dish has an area of 63.6 square centimeters ; the larger is used as a cover for the smaller, and forms a loosely fitting lid. The plates when fitted together are sterilized and thus form a closed cell which, if properly handled, may remain sterile indefinitely. FIG. 13. — INOCULATING. The technique for making a pour plate for the purpose of isolating bacteria from mixed culture is as follows : The actual " pouring" of plates is preceded by the preparation of usually three graded dilutions of the material to be examined. For this purpose three agar or gelatin tubes are melted and, in the case of the agar, are cooled to a temperature of about 42° C. in a water bath. A platinum loopful of the material to be examined is transferred to one of these tubes. The bacteria are then thoroughly distributed throughout the melted gelatin or agar by alternately depressing and raising the plugged end of the tube, giving it a rotary motion at the same time. This thoroughly distributes the bacteria throughout the medium without allowing the formation of air-bubbles, Two loopfuls of this mixture are then transferred to the second tube and. a similar mixing process is repeated. This 176 BIOLOGY AND TECHNIQUE second tube contains the bacteria in much greater dilution than the first and the colonies which will form in the plate poured from this tube, will be farther apart. A third dilution is then made by trans- ferring five loopfuls of the mixture in the second tube to the third. This again is mixed as before. The contents of the tubes are then poured into three sterile Petri dishes. The pouring should be done with great care. The cover of the dish is raised along one margin simply far enough to permit the insertion of the end of the test tube, the plug of which has been removed and the lips passed, with a rotary movement, through the flame. The medium is poured into the dish without the lips of the tube being allowed to touch either the bottom or the cover of the dish. The cover is then replaced and the medium allowed to harden. FIG. 14. — POURING INOCULATING MEDIUM FROM PETRI DISH. When agar has been used, the dishes may be placed in an in- cubator at 37° C. It is well to place the plates upside down in the incubator. This prevents the condensation water, squeezed out of the agar during hardening, from collecting on its surface, and forming channels for the diffuse spreading of bacteria. The same end may be attained by the use of Petri plates provided with porous earthenware lids, as suggested by Hill. Simple inversion of the plates, however, usually suffices. When gelatin has been used, the plates are allowed to remain in a dark place at room temperature or in a special thermostat kept at 22° -25° C. Colonies in agar, kept at 37.5° C., usually develop in eighteen to twenty-four hours ; those in gelatin or agar at room temperature in from twenty-four to forty-eight hours, depending upon the species METHODS USED IN CULTIVATION OF BACTERIA 177 of bacteria which are being studied. Often in the second dilution, more frequently in the third, the colonies will be found well apart and can then be "fished." The process of "colony-fishing" is one which requires practice and should always be done with care, for upon its success depends the purity of the sub-culture obtained. Colonies should never be fished under the naked eye, no matter how far apart and discrete they may appear, since not infrequently close to the edge of or just beneath a larger colony there may be a minute colony of another species which may be too small to be visible to the naked eye, but which, nevertheless, if touched by accident will contaminate the sub-culture. For proper "fishing," the Petri plate with cover removed, should be placed upon the stage of the microscope and examined with a low power objective, such as Leitz No. 2 or Zeiss AA. The sterilized platinum needle, held in the right hand, is then carefully directed into the line of focus of the lens, while the small finger of the hand is steadied upon the edge of the microscope stage. When the point of the needle is clearly visible through the microscope, it is gently depressed until it is seen to touch the colony and to carry away a portion of it, The needle is then withdrawn without again touching the nutrient medium or the edges of the glass or the lens, and transferred to a tube of whatever medium is desired. In this way, individuals of one colony, descendants of a single bacterium of the original mixture — are carried over to the fresh medium. Esmarch Roll Tubes.1 — A simple method of obtaining separate colonies is that devised by von Esmarch and known as "roll-tube ' ' cultivation. Tubes of melted gelatin are inoculated with various dilutions of the bacterial mixture and, while still liquid, are laid in an almost horizontal position upon a block of ice, which has been grooved slightly by means of a test tube filled with hot water. The test tube containing the gelatin, after being placed in this groove, is rapidly revolved by passing the fingers of the right hand across it, while its base is carefully steadied with the left hand. If the revolving is carried out with sufficient speed, the gelatin will harden in a thin layer on the inner surface of the tube. The colonies will develop in this layer and may be "fished" by means of a platinum wire with bent point introduced into the tube. This method is useful for certain purposes, but is too inconvenient for routine work. It is now rarely used. 1 Esmarch, Zeit. f. Hyg., i, 1886. 178 BIOLOGY AND TECHNIQUE Separation of Bacteria by Surface Streaking. — When it is neces- sary to isolate bacteria like the gonococcus Bacillus influenzas, the pneumococcus, and others, which, because of great sensitiveness to environment and possibly a preference for free oxygen, are not readily grown in pour plates, it is often advantageous first to pour plates of suitable media, allow them to harden, and then gently smear over their surfaces dilutions of the infectious material, usually in three or four parallel streaks. Upon such plates, if dilutions have been properly made, and this is only a question of judgment based upon an estimation of the numbers of bacteria in the original material, discrete colonies of the microorganisms sought for may develop, and can be " fished" in -the usual manner. The media most favorable for the cultivation of various micro- organisms will be discussed in the sections dealing with the in- dividual species. BARBER PIPETTE METHOD FOR THE ISOLATION OF SINGLE MICRO- ORGANISMS.— Although we consider this one of the very important methods of bacteriological study, we shall give no extensive descrip- tion of" the apparatus or the manner of using it, because both are too complicated to permit of satisfactory use from text book descrip- tion. The principle of the method depends upon a specially pre- pared mechanical stage adjusted to a compound microscope on which there is a moist chamber closed with a large coverglass, on the bottom of which drops of fluid, containing bacteria can be placed. A very fine glass pipette, manipulated by a specially constructed pipette holder and with a rubber tubing attached, is governed by observation through the microscope and by means of it small drops of the fluid are taken up, an attempt being made to obtain a single microorganism in these drops. With a little practice this can be accomplished, and these separate drops in which the individual bacteria can be seen swimming about, are made to suspend from the bottom of the cover, slip, closing the top of the moist chamber. The apparatus can be understood and worked only by practice and suitable instruction, together with a study of the description given by Barber in his article in the Philippine Journal of Science.2 The apparatus is made by a number of firms under the name of the 2 Barber, Philippine Jour, of Science, Sec. B, Vol. 9, No. 4, August, 1914. METHODS USED IN CULTIVATION OF BACTERIA 179 Barber Single Cell Apparatus, and no description that could be given in a text book would be of sufficient value to be allowed to take up space. ANAEROBIC METHODS We have seen in a preceding chapter (p. 28) that many bacteria, the so-called anaerobes, will develop only in an environment from which free oxygen has been excluded. FIG. 15.— DEEP STAB CULTIVATION OF AN- AEROBIC BACTERIA. FIG. 16. — DEEP STAB CULTIVATION OF AN- AEROBIC BACTERIA. The exclusion of oxygen for purposes of anaerobic cultivation may be accomplished by a variety of methods, depending upon a few simple principles which have been applied, either separately or in combination, by many workers. The earliest methods depended upon the simple exclusion of air by mechanical devices. In other methods, the oxygen of the air is displaced by inert gases (hydrogen), and others again depend 180 BIOLOGY AND TECHNIQUE upon the oxygen-absorbing qualities of alkaline solutions of pyro- gallol. Cultivation by the Mechanical Exclusion of Air. — Koch succeeded in growing anaerobic bacteria upon plates by simply dropping upon the surface of the inoculated agar or gelatin a flat piece of sterile mica. This method, however, rarely succeeds in sufficiently excluding the air. LIBORIUS' METHOD.3 — This method consists in the use of deeply filled tubes of agar or gelatin, from which all oxygen has been removed by boiling for fifteen minutes or more. It is advantageous, as has been pointed out in the section on anaerobiosis, that media used for this purpose should contain carbohydrates in some form, preferably glucose. After boiling, the tubes are rapidly transferred to ice water so that as little oxygen as possible may be absorbed during the hardening of the medium. The tubes are then inoculated by deep stabs. After inoculation, the medium may be covered with a thin layer of agar, gelatin, or oil (albolin), and further sealed with sealing-wax to prevent oxygen-absorption. This method may be utilized for the isolation of anaerobes (as in the original method of Liborius) by inoculating the medium just before it solidifies. The tubes may be gently shaken in order to distribute the bacteria throughout the medium and then rapidly cooled. In this case colonies which develop may be scattered throughout the deeper layers of the agar or gelatin, and may be "fished" after breaking the tube. ESM ARCH'S METHOD.* — Von Esmarch has applied the principles of his roll-tube to the cultivation of anaerobic bacteria. Gelatin tubes are inoculated as above and roll-tubes prepared. The tubes are then set into cold water to prevent melting of the thin gelatin layer and the interior of the tube is filled with melted gelatin. Roux's METHOD.5 — Anaerobic bacteria are cultivated by sucking the inocu- lated gelatin or agar into narrow tubes, which are then closed at both ends by fusing in the flame. After growth has taken place the tubes are broken and the organism recovered by "fishing." FLUID MEDIA COVERED WITH OIL. — Erlenmeyer flasks or other vessels are partially filled with glucose-bouillon over which a thin layer of albolin or other oil is allowed to flow. The oxygen is driven out of the liquid by vigorous boiling for fifteen minutes or more. It should be remembered whenever using this or similar methods that a layer of fluid oil does not form an impermeable seal. By covering an alkaline pyrogallol solution with oil it can easily be shown that oxygen 'Liborius, Zeit. f. Hyg., i, 1886. 4 Von Esmarch, loc. cit. «Koux, Ann. Past., i, 1887. METHODS USED IN CULTIVATION OF BACTERIA 181 slowly diffuses through the oil into the medium below. In using paraffin oil on anaerobic cultures it must be remembered that liquid oil is a very incomplete seal and that solid paraffin or any other solid seal is much more efficient. The simple exclusion of air, also, is the principle underlying the cultiva- tion of anaerobic bacteria in the closed arm of a Smith fermentation tube. WRIGHT'S METHOD.6 — Wright has described a simple and excellent method for the cultivation of anaerobic bacteria in fluid media. The apparatus necessary is easily im- provised with the materials at hand in any laboratory. A short piece of glass tubing, con- stricted at both ends and fitted at each end with a small piece of soft-rubber tubing, is inserted into a test tube containing nutrient broth. The upper end of the inserted glass tubing is con- nected by the rubber with a pipette passed through the cotton plug in the tube. The entire apparatus, plus broth, may be sterilized after being put together. When a transplant is made, the fluid in the test tube is inoculated as usual. The fluid is then sucked up into the glass tubing until this is completely filled. A downflow of the fluid is then prevented by placing the finger over the pipette through which the suction has been made or by constricting a small piece of rubber tubing attached to the upper end of the pipette. The entire system of tubes is then pushed downward in such a way that both pieces of rubber tubing, attached to the ends of the little glass chamber, are kinked. The entire apparatus may then be incubated. Growth of anaerobic bacteria takes place within the air- tight chamber formed by the short glass tubing within the test tube. The fluid in the test tube, outside of this chamber, usually remains clear. When cultivation has been successful, the bacteria may be obtained either for morphological study or for further cultivation, by simply allowing the fluid to flow out of the little air-tight chamber back into the test tube. The method is simple and usually successful. FIG. 17. — WRIGHT'S METHOD OF ANAEROBIC CULTIVATION IN FLUID MEDIA. 6 Wright, J. II. Quoted from Mallory and Wright, "Path. Technique/' Phila., 1904. 182 BIOLOGY AND TECHNIQUE Methods Based upon the Displacement of Air by Hydrogen.— The prin- ciple of air-displacement by hydrogen, first utilized by Hauser,7 has been widely applied to the cultivation of anaerobic bacteria. In substance it consists of the conduction of a stream of hydrogen through an air-tight chamber in which plates or tubes containing inoculated media have been placed. For the production of hydrogen, the most convenient apparatus is the Kipp hydrogen generated from zinc and sulphuric acid and this may be FIG. 18. — NOVY JAR. passed through a series of Woulfe-bottles, containing solutions of lead acetate and of pyrogallic acid, to remove traces of sulphuretted hydrogen and of oxygen, respectively, of Lugol's solution to absorb traces of acid vapor, and of one with a silver-nitrate solution to take up any hydrogen arsenide. For the preparation of anaerobic conditions where very rigid anaerobiosis is necessary, nitrogen may be used, which can be bought in tanks from com- mercial firms. Hauser, ( i Ueber Faulnissbakterien, ' ' 1885. METHODS USED IN CULTIVATION OF BACTERIA 183 For anaerobic cultivation upon solid media, the inoculated tubes or plates are placed in an apparatus such as the Novy jar. This is connected with the hydrogen apparatus and hydrogen allowed to flow through it for five or ten minutes, and the stop-cocks then closed. In applying the hydrogen method to fluid media, flasks containing the broth are fitted with sterile, tightly fitting rubber stoppers perforated by two holes, through which glass tubes are passed. One of these tubes, the inlet, passes below the surface of the liquid. The other one, the outlet, extends only a short distance below the stopper and is always kept above the surface of the medium. The flasks are inoculated and hydrogen is passed through the medium so that it enters the long tube, bubbles up through the fluid, and leaves by the short tube. The broth may be covered with a thin layer of liquid paraffin or albolin. The Use of Pyrogallic Acid Dissolved in Alkaline Solutions for Oxygen Absorption. — Buchner8 has applied the principle of chemical absorption for the removal of oxygen to the cultivation of anaerobic bacteria. This has been made use of in a number of different ways. The method is based upon the fact that alkaline solutions of pyrogallol possess the power of absorbing large quantities of free oxygen. At first such solutions are of a light straw-color, which becomes dark brown as oxygen is absorbed. The absorp- tion of all the oxygen in the environment may be assumed when there is no further deepening of the brown color. Buchner first utilized this principle by placing a small wire or glass holder within a large test tube, dropping dry pyrogallol (pyrogallic acid) into the bottom of this tube, then running thirty per cent sodium hydrate solution into it, and inserting within this large tube a smaller test tube containing the inoculated culture medium. The large tube was then tightly closed with a rubber stopper. In this way, the air space surrounding the smaller tube was rendered oxygen free. An excellent little trick with which to employ the Buchner tube method is to pack lightly over the dry pyrogallic acid in the bottle a small piece of absorbent cotton. This prevents the immediate solution of the pyrogallic acid, and allows one time to pour in the KOH solution, insert the smaller tube inside the Buchner tube, and tightly insert the rubber stopper in place before solution and oxygen absorp- tion has begun. A simple modification of the preceding method of Buchner has been devised by Wright.9 Stab-cultures of gelatin or agar in test tubes are made in the usual way. The cotton stopper closing the tube is then thrust into the tube to such a depth that its upper end lies at least 1 cm. below the mouth of the tube. A small quantity of sodium or potassium hydrate solution * Buchner, Cent. f. Bakt., I, iv, 1888. 9 Wright, Jour, of the Boston Soc. of Med. Sci., Dec., 1900. 184 BIOLOGY AND TECHNIQUE in which some pyrogallic acid has been dissolved, is then allowed to flow on to the cotton of the plug and the mouth of the tube is immediately sealed by a tightly fitting rubber stopper. The cotton stopper in these cases must be made of absorbent cotton; 1.5 to 2.5 c.c. of the pyrogallic acid solution is usually sufficient for test tubes of ordinary size. For cultivation of anaerobic bacteria upon agar slants, a simple technique FIG. 19. — WEIGHT'S METH- OD OF ANAEROBIC CULTIVA- TION BY THE USE OF PYRO- GALLIC ACID SOLUTION. FIG. 20. — JAR FOR ANAEROBIC CUL- TIVATION. may be applied and easily improvised in the laboratory as follows: the tube of slant agar is inoculated with the infectious material in the usual way. It is then, with stopper removed, inverted into a tumbler or beaker containing about a gram of dry pyrogallic acid. A small quantity of a five per cent or three per cent sodium hydrate solution is then run into the tumbler and this is covered with a thin layer of liquid paraffin or albolin before the pyrogallic acid has been completely dissolved. In this way, com- METHODS USED IN CULTIVATION OF BACTERIA 185 pletely anaerobic conditions are obtained in the tube and the growth of anaerobes takes place upon the surface of the slant. For the cultivation of anaerobes in Petri dishes, for purposes of separa- tion, a combination of the pyrogallic acid method and the hydrogen displace- ment methods is often employed. For this purpose the jars devised by Novy and by Bulloch are extremely convenient. In using the Novy jar, the inoculated plates are set upon a wire frame resting about an inch above the bottom of the jar. The cover is then tightly set in place and the air in the jar exhausted by means of a suction pump. The arrangement of the double stop-cock in the top renders it possible now, by simply turning this, to admit hydrogen from a Kipp generator into the jar. The process of alternate exhaustion and admission of hydrogen may be several times repeated. FIG. 21. — APPARATUS FOR COMBINING THE METHODS OF EXHAUSTION, HYDROGEN REPLACEMENT AND OXYGEN ABSORPTION. A combination of air exhaustion, oxygen absorption, and hydrogen replace- ment may be practiced in jars such as that shown in Fig. 21. Dry pyrogallic acid is placed in the bottom of the jar and the cover tightly fitted. An opening in the side of the jar connects its interior with a bottle containing sodium or potassium hydrate solution. Through the stopper of this bottle pass two glass tubes, one of them of such length that it can be pushed down into the alkaline solution, or pulled upward above the level of the fluid. By this means the KOH can be sucked into the jar after a vacuum has been produced with the exhaustion pump. The other glass tube is short, passing just through the stopper and at the top made in the form of a T, one arm of the T being connected with a Kipp hydrogen generator, the other with a suction-pump. A simple method for the separation of anaerobes in plates without the use of hydrogen or of specially constructed jars, may be carried out as follows : 10 The apparatus used consists of two circular glass dishes, fitting ™ Zinsser, Jour. Exp. Med., viii, 1906. 186 BIOLOGY AND TECHNIQUE one into the other as do the halves of a Petri dish, and similar to these in every respect except that they are higher, and that a slightly greater space is left between their sides when they are placed together. The dishes should be about three-fourths to one inch in height, they need be of no particular diameter, although those of about the same size as the usual Petri dish are most convenient. An important requirement necessary for the success of the method is that the trough left between the two plates, when put together, shall not be too broad, a quarter of an inch being the most favorable. Into the smaller of these plates the inoculated agar is poured exactly as into a Petri dish in the ordinary aerobic work. Prolonged boiling of the agar before plating is not essential. When the agar film has become sufficiently hard on the bottom of the smaller dish, the entire ap- paratus is inverted. The smaller dish is now lifted out of the larger, and placed, still inverted, over a moist surface — a towel or the wet surface of the table — to prevent contamination. Into the bottom of the larger dish, which now stands open, there is placed a quantity (about 3 grams) of dry pyrogallic acid. Into this, over the pyrogallic acid, the smaller dish, still inverted, is then placed. A five per cent solution of sodium hydrate is poured into the space left between the sides of the two dishes, in quantity sufficient to fill the receiving dish one-half full. While this is gradually dissolving the pyrogallic acid, albolin, or any other oil (and this is the only step that requires speed), is dropped from a pipette, previously filled and placed in readiness, into the same space, thus completely sealing the chamber formed by the two dishes. If these steps have been performed successfully, the pyrogallic solution will at this time appear of a light brown color, and the smaller plate, with its agar film, will float unsteadily above the other. Very rapidly, as the pyrogallic acid absorbs the free oxygen in the chamber, this plate is drawn down close to the other, and the acid assumes a darker hue, which remains without further deepening even after three or four days' incubation. We have described a considerable number of methods of anae- robic cultivation which have been in use. Following our general purpose, however, of emphasizing the methods that we ourselves have found most useful, we will describe in the following paragraphs the methods which we are using as routine for anaerobic work in our own laboratory, and which we believe are the most practical. For anaerobic cultivation on agar slants, we use the Buchner tube method as described above. For spirochaete cultivation, etc., we use the Noguchi method of narrow deep agar or broth tubes with tissue at the bottom, sterile METHODS USED IN CULTIVATION OF BACTERIA 187 oil at the top, placed into anaerobic jars, or incubated without jars according to purpose. For anaerobic jar methods, we hardly ever use hydrogen, em- ploying one of the two following : Jars may be of any size or shape, provided they are well constructed strong museum jars with well fitting lids, preferably ground glass surfaces on lid and top of jar, and with one perforation in the top. Stoppers and glass tubing are fitted into the top of the jars with Major's cement, and recemented every time the jars are used. Glass tubing which communicates with the exterior through the cemented joints is drawn out in the flame, along its course connecting with the suction pump, and closure is effected by sealing in these narrow places with the flame. The fitting of the lid for closure is accomplished by a thick layer of sculptors' plastocene, a smooth layer of which is placed on the top of the jar, and another on the lips of the lid before the lid is put in place. It is useful to have a vacuum gauge cemented into the lid so that the functioning of the suction pump can be controlled. This, however, is not absolutely necessary. A small tube containing a weak methylene blue solution in 1 per cent glucose broth is placed inside the jar with the cultures. Decolorization of this on the following morning will prove anaerobiosis. I. One method which can be used for Petri plates or tubes consists in placing a suitable amount of dry pyrogallic acid into the bottom of the jar. The cultures are then inserted and next to them is placed an envelope of thick brown paper, such as used by commercial houses, which is torn so as to be open at the top and to form a sort of elongated bag or cornucopia. The glass top of the lid is then connected with the suction pump before it is placed on the jar, the suction pump started while an assistant holds the lid ready for placement on the jar. A suitable amount of 20 per cent KOH is then poured into the envelope and the lid immediately placed in position on the top of the jar, pushed down on all sides so that the plastocene flattens out between the lid and the jar. The suction immediately begins to draw the air out of the jar, before the KOH solution works its way through the envelope and begins to dissolve the pyrogallol. The suction is then continued for a suitable length of time, and while the pump is still going a Bunsen burner is placed under a narrow place in the connection glass tube and this is sealed in the flame. This method if successfully carried out, gives almost complete anaerobiosis. II. The second method is that of Mclntosh and Fildes,11 which is, in our opinion, the best anaerobic method for application to the "Mclntosh and Fildes, Lancet, 190, 1916, 768. Methylene blue added in sufficient quantity to 10 c.c. of a 2 per cent dextrose alkaline broth to give a distinct blue color, is the most convenient anaerobic indicator. It depends on 188 BIOLOGY AND TECHNIQUE growth of tubes and plates in anaerobic jars. It depends upon the removal of oxygen by the oxidation of hydrogen under the influence of palladinized asbestos wool. Pfuhl had previously used the catalytic action of platinum sponge for anaerobic cultivation in broth tubes. Mclntosh and Fildes adopted and improved upon this method. The principle of the method is to suspend in an air tight jar a bit of asbestos wool impregnated with platinum or, better, with palladium black. Hydrogen is then allowed to pass in until no oxygen remains. We have used this method in our own labora- tory with great success where a number of the workers compared it with other methods, and it has been used successfully at the Rockefeller Institute by Olitsky and others. An ordinary museum jar, such as those used for anaerobic work, is the vessel employed. The lid is perforated for the passage of hydrogen, and from the bottom of the lid there is suspended a bit of the impregnated asbestos wool, inclosed in a small cage of copper wire. About 0.25 gram of the asbestos wool is the amount recommended by Mclntosh and Fildes. "The palladium asbestos (about 40 per cent) is made by weighing out 0.25 gram of asbestos wool, placing it in a small evaporating dish, and adding 1.5 c.c. of a 10 per cent solution of palladium chlorid. The wool is then molded into a flat mass about one inch square, and the dish gently heated until the wool is dry. Since the palladium chlorid is difficult to dissolve, the addition of a little concentrated hydrochloric acid may be neces- sary. The palladium chlorid is then reduced by heating the impregnated wool, first in a smoky gas flame until it is coated with a layer of carbon, and then in a blow-pipe. The palladium asbestos should now be able to light a jet of hydrogen which is made to impinge on it." For ordinary purposes the palladium asbestos can be bought. For closure of lids upon the jars, we have used throughout plastocene, the material used by sculptors for molding. In using the jars the culture tubes are placed inside the jars and the lid rim and the jar rim are covered with plastocene. The copper gauze, with the asbestos wool, is detached and held over a flame until red hot, and is then rapidly put in place, closing the jar. Through the perforation with proper connections made (best a glass stopper connection) hydrogen is allowed to flow in very slowly, best from a liquid hydrogen cylinder, with careful regulating check valve. It is very necessary to take careful the fact that in the absence of oxygen the reducing action of an alkaline solution of dextrose changes the methylene blue to the colorless luecomethylene blue. On exposure to the oxygen of the air, the leucomethylene blue is oxidized back to the colored compound. METHODS USED IN CULTIVATION OF BACTERIA 189 precautions that the hydrogen is not allowed to enter too fast, or else explosion may follow, and it is best to make some arrange- ment like that used in our laboratory by Dr. Teague where the hydrogen is first let into a glass chamber over water, and allowed to flow in very gradually by graded pressure. The main thing is to so work the apparatus that a very slow jet of hydrogen is made to flow in close to the palla-dmized asbestos, while this is still hot. A small film of water will begin to form on the sides of the tube, and this is continued until a negative pressure has distinctly developed in the jar. Judgment concerning the amount of hydrogen that should be . let in can be attained with practice. Closure of the jar may be then made more firm with paraffin or any of the other ordinary methods of making such jars air tight. Plastocene in sufficient amounts has usually served our purpose. We do not illustrate this method because illustrations rarely help an experienced bacteriologist to any great degree, and it is best to see the apparatus work in some laboratory where it is in use. Many modifications are possible, but the principle is easy to apply in a great many different ways. By this method it is possible to decolorize methylene blue tubes put into the jars, completely over night, and we believe that this method properly applied gives a practically perfect anaerobiosis. The Use of Fresh Sterile Tissues as an Aid to Anaerobic Cul- tivation.— The addition of small pieces of fresh sterile tissue (rabbit or guinea-pig) to culture tubes, either solid or fluid, greatly favors the growth of anaerobic bacteria. By such a method anaerobes can be made to develop even when other precautions for the establish- ment of anaerobiosis are imperfectly observed. This was noticed first by Theobald Smith and by Tarozzi and has become an extremely useful reenforcement to other methods. It has been utilized most extensively by Noguchi of recent years in his technique for the cultivation of various treponemata. The simplest way to apply this method is to place a piece of freshly excised rabbit kidney, testicle, or spleen into the bottom of a high test tube (20 cm.) and then pouring over it the culture fluid. Kidney or other tissues are more suitable for this purpose than liver tissue since the latter is not easily obtained in a sterile condition, bacteria often getting into it during life through the portal circulation. The action of the tissues depends upon its great reducing power. PARTIAL OXYGEN TENSION FOR THE GROWTH OF BACTERIA. — In 1916 Wherry and Oliver12 found that partial anaerobiosis was favorable 12 Wherry and Oliver, Lancet-Clinic, 115, 1916, 306. 190 BIOLOGY AND TECHNIQUE for the growth of gonococcus. Cohen and Markle13 applied this to meningococcus and found again that partial anaerobiosis favored growth. It was later found by Wherry and Erwin,14 as well as by Cohen and Fleming15 that the growth of a number of different bacteria was favored when the air was partially replaced by carbon dioxide. Gates16 confirmed this, as did Kohman.17 We have tried this out in our own laboratory and, like other observers, have found that, in the case of meningococcus and also with the influenza bacillus, growth is definitely stimulated by replacing about 10 per cent of the air with carbon dioxide'. Both Kohman, Gates and others believe that the principle underlying this is due to the effect of the carbon dioxide on the reaction of the medium. As the organisms grow they produce acid which displaces equivalent amounts of dis- solved carbon dioxide, in consequence of which the acidity is hot materially increased. THE INCUBATION OF CULTURES After bacteria have been transferred to suitable culture media, it is necessary to expose them to a temperature favorable to their development. In the case of many saprophytes, the ordinary room temperature is sufficiently near the optimum to obviate the use of any special apparatus for maintaining a suitable temperature; in the case of most pathogenic bacteria, however, the body temperature of man, 37.5° C., is either a necessary requirement for their growth, or at any rate favors speedy and characteristic development. . For the purpose of obtaining a uniform temperature of any required degree, the apparatus in general use is the so-called incu- bator or thermostat. This may be adjusted for gelatin cultivation at 20 to 22° C., or for agar, broth, or other media at 37.5° C. Incubators, while varying in detail, are all constructed upon the same principles. They consist of double-walled copper chambers, which are fitted with a set of double doors, the outer being made of asbestos-covered metal, the inner of glass. The space between the two walls is filled with water, which, being a poor heat con- 13 Cohen and Markle, Abst. of Bacter., 2, 1918, 10. 14 Wherry and Erwin, Jour, of Infec. Dis., 22, 1918, 194. 15 Cohen and Fleming, Jour, of Infec. Dis., 23, 337, 1918, 19 Gates, Jour, of Exper. Med., 29, 325, 1919. " Kohman, Jour, of Bacter., 4, 1919. METHODS USED IN CULTIVATION OF BACTERIA 191 ductor, tends to prevent rapid changes of temperature within the chamber as the result of changes in the external environment. Both walls are perforated above by openings to admit thermometers into the interior and one wall is perforated so that a thermo-regulator may be inserted into the water jacket. The under surface of the chamber is heated by a gas flame, the size of which is automatically regulated by the thermo-regulator. A number of thermo-regulators are on the market, all of them con- structed upon modifications of the same principle. One of the most efficient of those in common use is that shown in Fig. 22. This consists of a long tube of glass fitted with a metal cap through which an inlet tube (A) projects n i. FIGS. 22, 23. — THERMO REGULATOR. into the interior. Slightly below the middle of the tube there is a glass diaphragm separating its interior into two compartments. In the middle of the diaphragm an aperture leads into a spiral of glass which projects into the lower compartment. The lower compartment is filled with ether and mercury. The lower end of the inlet tube (A) has a wedge-shaped slit. The gas from the supply pipe passing through the tube (A) is conducted through the slit-like opening in its lower end into the inner chamber and passes out to the burner through the elbow (B). When the temperature is raised, the ether and mercury in the lower chamber expand and the mercury rises in the upper chamber, gradually restricting the opening through the V-shaped slit in the inlet tube. Thus the gas supplied to the burner is diminished, the flame reduced, and the temperature again falls. The tem- perature can be arbitrarily adjusted by raising or lowering the inlet tube. A scale at the upper end of the inlet tube allows exact adjustment. Complete 192 BIOLOGY AND TECHNIQUE shutting off of the gas is prevented by a small circular opening placed in the inlet tube just above the slit. Another cheaper and simpler thermo-regulator is shown in Fig. 24. This consists of a long tube open at the top and fitted about 1^ inches from the top with two hollow glass elbows. One of these elbows remains open, the other, situated on a slightly lower level, is closed by a brass screw-cap. The tube is filled with mercury to a point slightly above the level of the elbow containing the screw-cap. The height of the mercury can thus be increased or decreased by screwing in or out upon the cap. Into the upper end of the tube there is fitted another device which consists of a T-shaped system of glass tubes, one arm of the T being open and the other closed, the perpendicular leg of the T tapering to a minute opening at the bottom. FIG. 24. — MOITESSIER GAS PRESSURE REGULATOR. The gas passes into one arm of the T down through the tapering leg and into the space immediately above the mercury. It then passes out through the open elbow of the main tube. As the mercury rises, it gradually diminishes the space between its surface and the small opening in the tapering tube above it, finally completely shutting off the gas from this source. Gas can now pass only through a minute hole perforating the vertical leg of the T an inch above its end. The flame decreases and the temperature again sinks. Since gas pressure in laboratories is apt to vary, it is convenient to interpose between the gas supply and thermo-regulator some one of the various forms of gas-pressure regulators. The use of these is not absolutely necessary but aids considerably in the maintenance of a constant temperature. The one most commonly employed is the so-called Moitessier apparatus. This consists of a cylindrical metal chamber within which there is fitted an inverted metal bell. Glycerin METHODS USED IN CULTIVATION OF BACTERIA 193 is poured into the cylinder to the depth of about two inches. An inlet pipe conducts gas into the open space between the top of the glycerin and the bell. From, the top of the bell is suspended a conical piece of metal which hangs free in the outlet pipe. As the gas pressure under the bell increases, this is raised and the opening of the outlet pipe is gradually diminished by the cone. Thus the relation between the pressure in the inlet pipe and the actual quan- tity of gas passing through is equalized. A cup connected to the top of the bell through the roof of the cylinder by a bar can be filled with birdshot and the pressure against the gas can thus be modified to conform with existing conditions. Colony Study. — Cultures are usually incubated for from twelve FIG. 25. — VARIATIONS IN THE CONFORMATION OP THE BORDERS OP BACTERIAL COLONIES. to forty-eight hours. Considerable aid to the recognition of species is derived from the observation of both the speed of growth and the appearance of the colonies. It is therefore necessary to proceed in the study of developed colonies in a systematic way. The develop- ment of colonies should be observed in all cases both upon gelatin and upon agar. In forming any judgment about colonies, the acidity or alkalinity, and the special constitution of the media should be taken into account. The colonies are carefully examined with a hand lens and with the low power (Leitz No. 2, Zeiss A A, Ocular No. 2) of the microscope. The colonies should be observed as to size, outline, transparency, texture, color, and elevation from the surface of the media. Much information, also, can be obtained by observing whether a colony appears dry, mucoid, or glistening, like a drop of moisture. By a careful observation of these points, definite 194 BIOLOGY AND TECHNIQUE differentiation, of course, can not usually be made, but much cor- roborative evidence can be obtained which may guide us in the methods to be adopted for further identification and for a final summing up of species characteristic as a whole. The Counting of Bacteria. — It is often necessary to determine the number of bacteria per c.c. contained in water, milk, or other sub- stances. For this purpose definite quantities of the material to be analyzed are mixed with gelatin or agar and poured into Petri plates. The exact dilutions of the suspected material must largely depend upon the number of germs which one expects to find in it. The plates, if prepared with gelatin, are allowed to develop at room temperature for twenty-four to forty-eight hours. If agar has been used, they are usually placed in the incubator at 37.5° C. At the end of this time, the colonies which have developed are enumerated. For this purpose, a Petri dish is placed upon a Wolffhiigel plate. This plate consists of a disk or square of glass which is divided into small squares of one square centimeter each. Diagonal lines of these squares running at right angles to each other are subdivided into nine divisions each in order to facilitate counting when the colonies are unusually abundant. The Petri dish is placed upon the plate in such a way that the center of the dish corresponds to the center of the plate. The colonies in a definite number of squares are then counted. The greater the number of squares that are counted the more accurate the estimation will be. When the growth is so abundant that only a limited number of squares can be counted, these should be chosen as much as possible from different parts of the plate, and in practice one counts usually six squares in one direction and six at right angles to these, so as to preclude errors arising from unequal distribution. The final calculation is then made by ascertaining the average number of colonies contained in each square centimeter. If standard Petri dishes have been used, this is multiplied by 63.6, the number of squares in the area of the dish, and then by the dilution originally used. Thus if twelve squares have been counted with a total number of one hundred and forty-four colonies — the average for each square is twelve. Twelve times 63.6 is 763.2, which represents the total number of colonies in the plate. Now if 0.1 c.c. of the original material (water or milk) has been plated, this material may be assumed to have contained 10 X 763.2, or 7,632 bacteria to each cubic centimeter. METHODS USED IN CULTIVATION OF BACTERIA 195 If dishes of an unusual size are employed, the square area must be ascertained by measuring the radius and multiplying its square by TT (w X R2 = area) (IT = 3.141592). The number of bacteria in a given volume of a suspension can be estimated by a variety of methods without cultivation. The one most commonly used is the method developed by Wright, which consists in mixing a small measured amount of the bacterial suspen- sion with an equal volume of a red blood cell suspension in which \ S/ FlG. 26. — WOLFFHUGEL COUNTING PLATE. the number of erythrocytes per cubic millimeter are known. Smears are made and the relative number of bacteria and red blood cells per one or two hundred fields are counted. A simple calculation can then be made. This method has been described in the section dealing with opsonin technique. Another useful technique is the direct counting of dilutions of the bacterial suspension, unstained or stained with methylene blue, in a modification of the Thoma Zeiss counting chamber, known as the Helber chamber. CHAPTER IX METHODS OF DETEEMINING BIOLOGICAL ACTIVITIES OF BACTERIA ANIMAL EXPERIMENTATION Gas Formation. — Bacteria of many varieties produce gas from the protein and the carbohydrate constituents of their environment. Gas formation can be observed in a very simple manner by mak- ing stab cultures in gelatin or agar containing the fermentable nutrient substances. In such cultures bubbles of gas will form along the track of the inoculation, or, in the case of such semisolid media as the tube medium of Hiss, will spread throughout the tube. In the case of some anaerobes gas formation in stab cultures will occur to such an extent that the medium will split and break. It should be borne in mind in carrying out such methods that air is readily carried into the medium with the inoculating needle or loop by splitting of the medium, also that media which have been stored in the cold may absorb air. Expansion of the air in such tubes may simulate small amounts of gas formation and lead to error. It is advisable, therefore, whenever making stab inoculations with the above purpose, to heat the media and rapidly cool them before use. A more accurate method of gas determination is by the use of fermentation tubes, such as those devised by Smith. The gas which is formed collects in the closed arm of the fermentation tube and may be quantitatively estimated. The fermentation, with gas production, of certain substances such as carbohydrates, may be determined by adding these materials in a pure state to the media before inoculation with organisms. In the case of carbohydrates this method has proved of great differential value, since the power of splitting specific carbohydrates with gas production is a species characteristic of great constancy for many forms of bacteria. ANALYSIS OF GAS FORMED BY BACTERIA. — Carbon Dioxide. — Foi the estimation both qualitatively and roughly quantitatively of car- 196 DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 197 bon dioxide produced by bacteria, cultures are grown in fermen- tation tubes containing sugar-free broth, (see page 150) to which one per cent of pure dextrose, lactose, saccharose, or other sugars has been added. The tubes are incubated until the column of gas formed in the closed arm no longer increases (twenty-four to forty- eight hours). The level of the fluid in the closed arm is then accurately marked and the column of gas measured. The bulb of the fermentation tube is then completely filled with ^ NaOH solution, the mouth closed with a clean rubber stopper, and, the bulb inverted several times in order to mix the gas with rr FIG. 27. — TYPES OF FERMENTATION TUBES. the soda solution. The tube is then again placed in the upright position, allowing the gas remaining to collect in the closed arm. The gas lost may be roughly estimated as consisting of C02. Hydrogen. — The gas remaining, after removal of the C02 in the preceding -experiment, at least when working with carbohydrate solutions, may be estimated as hydrogen. When allowed to collect near the mouth, further evidence of its being hydrogen may be gained by exploding it with a lighted match. Hydrogen Sulphid (H2S, Sulphuretted hydrogen). — In alkaline media, sulphuretted hydrogen, if formed, will not collect as. gas, but will form a sulphid with any alkali in the solution. For the estimation of the formation of hydrogne sulphid, bacteria are cul- 198 BIOLOGY AND TECHNIQUE tivated in a strong pepton solution to which 0.1 c.c. of a one per cent solution of ferric tartrate or lead acetate has been added. The addition of these substances gives rise to a yellowish precipitate in the bottom of the tubes. If, on subsequent inoculation, the bacteria produce H2S, this precipitate will turn black. The solution recommended by Pake for this test is prepared as follows : 1. Weigh out 30 grams of pepton and emulsify in 200 c.c. of tap water at 60° C. 2. Wash into a liter flask with 80 c.c. tap water. 3. Add sodium chlorid 5 grams and sodium phosphate 3 grams. 4. Heat at 100° C. for 30 minutes, to dissolve pepton. 5. Filter through paper. 6. Fill into tubes, 10 c.c. each, and to each tube add 0.1 c.c. of a one per cent solution of ferric tartrate or lead acetate. These solutions should be neutral. 7. Sterilize.1 ACCURATE QUANTITAVE GAS ANALYSES of bacterial cultures can be made only by the more complicated methods used in chemical labora- tories for quantitative gas analysis. The gas, in such cases, is collected in a bell jar mounted over mercury, and subjected to analysis by the usual method described in works on analytical chemistry. Acid and Alkali Formation by Bacteria. — Many bacteria produce acid or alkaline reactions in culture media, their activity in this respect depending to a large extent upon the nature of the nutrient material. Many organisms which on carbohydrate media produce acid will give rise to alkali if cultivated upon media containing only proteins. Information as to the production of acid or alkali can be obtained by the addition of one of a variety of indicators to neutral media. The indicators most often employed for this purpose are litmus, neutral red, China blue and the so-called Andrade indicator. An- drade consists of 100 c.c. of a 0.5% aqueous solution of acid fuchsin to which 16 c.c. of accurately normal NaOH has been added. Changes in the color of these indicators show whether acids or alkalis have been produced. Great help in differentiation is obtained by adding chemically pure carbohydrates to media to which litmus has been added, and 1 Quoted from Eyre, ' ' Bact. Technique, ' ' Phila., 1903. DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 199 then determining whether or not acid is formed from these substances by the microorganisms. These tests have been of special importance in the differentiation of the typhoid-colon groups of bacilli. Quantitative estimation of the degree of acidity or alkalinity produced by bacteria may be made by careful titration of definite volumes of the medium before and after bacterial growth has taken place. The variety of acid formed by bacteria depends largely upon the nature of the nutrient medium. The acids most commonly resulting from bacterial growth are: lactic, acetic, oxalic, formic, and hippuric acids. Qualitative and quantitative estimation of these acids may be made by any of the methods employed by analytical chemists. Indol Production by Bacteria. — Many bacteria possess the power of producing indol. Though formerly regarded as a regular accom- paniment of protein decomposition, later researches have shown that indol production is not always coexistent with putrefaction processes and occurs only when pepton is present in the pabulum. Indol formation by bacteria is determined by the so-called nitroso- indol reaction. Organisms are grown in sugar-free pepton broth or in the pepton-salt bouillon of Dunham. (See page 151.) Media containing fermentable substances are not favorable for indol production since acids interfere with its formation. The cultures are usually incubated for three or four days at 37° C. At the end of this time, ten drops of concentrated sulphuric acid are run into each tube. If a pink color appears, indol is present, and we gather the additional information that the microorganism in question has been able to form nitrites by reduction (e.g., cholera spirillum). If the pink color does not appear after the addition of the sulphuric acid alone, nitrites must be supplied. This is done by adding to the fluid about 1 c.c. of a 0.01 per cent aqueous solution of sodium nitrite. The sodium nitrite solution does not keep for any length of time and should be freshly made up at short intervals. VANILLIN TEST.2 — An excellent test for indol is the so-called vanillin test. To 5 c.c. of the culture add 5 drops of 5 per cent vanillin solution in 95 per cent alcohol, and 2 c.c. of concentrated hydrochloric acid or sulphuric acid. If indol is present, an orange color develops within 2 or 3 minutes. Tryptophane gives a reddish - Stcensma, Zeit. £. Physiol, Chem., 47, 1906; Nelson, Jour, of Biol. Chem., 24, 1916. 200 BIOLOGY AND TECHNIQUE violet which grows deeper when the medium, is heated or allowed to stand. Phenol Production by Bacteria. — Phenol is often a by-product in the coarse of protein cleavage by bacteria. To determine its presence in cultures, bacteria are cultivated in flasks containing about 50-100 c.c. of nutrient broth. After three to four days' growth at 37° C., 5 c.c. of concentrated HC1 are added to the culture, the flask is connected with a condenser, and about 10-20 c.c. are distilled over. To the distillate may be added 0.5 c.c. of Milloii's reagent (solu- tion of mercurous nitrate in nitric acid), when a red color will indicate phenol; or 0.5 c.c. of a ferric chloride solution, which will give a violet color if phenol is present. Reducing Powers of Bacteria. — The power of reduction, possessed by many bacteria, is shown by their ability to form nitrites from nitrates. This is easily demonstrated by growing bacteria upon nitrate broth (see page 151). Bacteria are transferred to test tubes containing this solution and allowed to grow in the incubator for four or five days. The presence of nitrites is then chemically determined.3 In bacteriological work, 4 c.c. of the culture fluid are poured into a clean test tube, and to it are gradually added 2 c.c. of the mixed test solutions. A pink color indicates the presence of nitrites, the intensity of the color being proportionate to the amount of nitrite present. The reducing powers of bacteria may also be shown by their ability to decolorize litmus, methylene-blue, and some other anilin dyes, which on abstraction of oxygen form colorless leukobases. 8 We are indebted to Dr. J. P. Mitchell, of Stanford University, for the follow- ing technique for nitrite tests: I. Sulphanilic Acid. — Dissolve 0.5 g. in 150 c.c. of acetic acid of Sp. Gr. 1.04. (Acetic acid of 1.04 prepared by diluting 400 c.c. of cone, of sp. gr. 1.75 with 700. c.c. of water.) II. A-Naphthylamin. — Dissolve 0.1 gr. in 20 c.c. of water, boil, filter (if neces- sary), and to clear filtrate add 180 c.c. of acetic acid, Sp. Gr. 1.04. The solutions are kept separate and mixed in equal parts just before use. In carrying out the test, put 2 c.c. of each reagent in a test tube and add substance to be tested. (In ordinary water analysis use 100 c.c.) Cover tube with watch glass and sot in warm water for 20 minutes. Observe presence or absence of pink color promptly. Always run a blank on the distilled water used for rinsing to avoid errors due to nitrites in the water, or in the air of the laboratory. DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 201 Enzyme Action.4 — The action of the enzymes produced by bac- teria may be demonstrated by bringing the bacteria, or their isolated ferments, into contact with the proper substances and observing both the physical and chemical changes produced. In obtaining enzymes free from living bacteria, it is convenient to kill the cultures by the addition either of toluol or of chloroform. Both of these substances will destroy the bacteria without injuring the enzymes. Enzymes may also be obtained separate from the bodies of the bacteria by nitration. PROTEOLYTIC ENZYMES. — The most common evidences of proteo- T FIG. 28. — TYPES OF GELATIN LIQUEFACTION BY BACTERIA. lytic enzyme action observed in bacteriology are the liquefaction of gelatin, fibrin or coagulated blood-serum, and the peptonization of milk. This may be observed both by allowing the proper bacteria to grow upon these media, or by mixing sterilized cultures with small quantities of these substances.5 The products of such a reac- tion may be separated from the bacteria by filtration and then tested for pepton by the biuret reaction. 4 See also pp. 54 et seq. 8 Bitter, Archiv f. Hyg., v. 1886. 202 BIOLOGY AND TECHNIQUE Proteolytic6 enzymes may also be determined by growing the bacteria upon fluid media containing albumin solutions, blood serum, or milk serum, then precipitating the proteins by the addition of ammonium sulphate (about 30 grams to 20 c.c. of the culture fluid) and warming between 50 to 60° C. for thirty minutes. The pre- cipitate is then filtered off, the filtrate made strongly alkalm with NaOH, and a few drops of copper sulphate solution added. A violet color indicates the presence of pepton — proving proteolysis of the original albumin. DIASTATIC ENZYMES. — The presence of diastatic ferments may be determined by mixing broth cultures of the bacteria with thin starch paste. It is necessary that both the cultures and the starch paste be absolutely free from sugar. After remaining in the incubator for five or six hours, the fluid is filtered and the filtrate tested by methods used for determining the presence of sugars. INVERTING FERMENTS. — Inverting ferments are determined by a procedure similar to the above in principle. Dilute solutions of cane sugar are mixed with old cultures or culture filtrates of the respec- tive bacteria and the mixture allowed to stand. It is then filtered, and the filtrate tested for glucose, preferably by Fehling's solution. ANIMAL EXPERIMENTATION In the study of pathogenic microorganisms, animal experimenta- tion is essential in many instances. The virulence of any given organism for a definite animal species and the nature of the lesions produced are characteristics often of great value in differentiation. Isolation, moreover, of many bacteria is greatly facilitated by the inoculation of susceptible animals and recovery of the pathogenic organism from the heart's blood or from the lesions produced in various organs. That investigations into the phenomena of im- munity would be absolutely impossible without the use of animal inoculation is, of course, self-evident, for by this method only can the action of bacteria in relation to living tissues, cells, and body- fluids be observed. The animals most commonly employed for such observations are guinea-pigs, white mice, white rats, and rabbits. The method of inoculation may be either subcutaneous, intrapleural, intraperitoneal, *Hanlcin and WesfbrooTc, Ann. Past., vi., 1892. DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 203 intravenous, or subdural, etc. It must be borne in mind always that the mode of inoculation may influence the course of an infection no less than does the virulence of the microorganism or the size of the dose. Inoculations are made with some form of hypodermic needle fitted to a syringe. The most convenient syringes are the all-glass Luer or the Debove syringes, which, however, are expensive. Any form of sterilizable syringe may be used. In making inoculations the hair of the animal should be clipped and the skin disinfected with carbolic acid or alcohol. Subcutaneous inoculations are most conveniently made in the abdominal wall, where the skin is thin. After clipping and steriliz- ing, the skin is raised between the fingers of the left hand and the needle plunged in obliquely so as to avoid penetrating the abdominal wall and entering the peritoneum. In making intraperitoneal inoculations, great care must be exer- cised not to puncture the gut. This can be avoided by passing the needle first through the skin in an oblique direction, then turning it into a position more vertical to the abdomen and perforating the muscles and peritoneum by a very short and carefully executed stab. Intravenous inoculations in rabbits are made into the veins run- ning along the outer margins of the ears. The hair over the ear is clipped and the animal held for a short time head downward so that the vessels of the head may fill with blood. An assistant holds the animal firmly in a horizontal position, the operator grasps the tip of the ears with the left hand, and carefully passes his needle into the vein in the direction as nearly as possible parallel to its course. Mice are usually inoculated under the skin near the base of the tail. They may be placed in a jar over which a cover of stiff wire-gauze is held. They are then grasped by the tail, by which they are drawn up between the side of the jar and the edge of the wire cover, so that the lower end of the back shall be easily acces- sible. The skin is then wiped with a piece of cotton dipped in carbolic solution and the needle is inserted. Great care must be exercised to avoid passing the needle too close to the vertebral column. Mice are extremely delicate, and any injury to the spine usually causes immediate death. With proper care mice or rats may be easily injected intra- venously if a sufficiently fine needle is used. There are four super- 204 BIOLOGY AND TECHNIQUE ficially placed veins running along the tail, which stand out prominently when rubbed with cotton moistened with xylol. Into these the injections are made. When inoculating rats or guinea-pigs with Bacillus pestis the Kolle vacination method is used. The skin is merely shaved and a loopful of the culture vigorously rubbed into the shaven area. The various forms of animal holders which have been devised are rarely necessary in bacteriological work unless working un- assisted, immobilization of the animals being easily accomplished by the hands of a skilled assistant. Autopsies upon infected animals must be carefully made. The animals are tied, back down, upon pans fitted in the corners with clamps for the strings. They are then moistened either with hot water or with a weak solution of carbolic acid, so that contamination by hair may be avoided. A median cut is made, the skin is carefully dissected back, and the body cavities are opened with sterile instru- ments. Cultures may then be taken from exudates, blood, or organs under precautions similar to those recommended below for similar procedures at autopsy upon man. Inoculated animals should be, if possible, kept separate from healthy animals. Rabbits and guinea-pigs are best kept in gal- vanized iron-wire cages, which are fitted with floor-pans that can be taken out and cleaned and sterilized. Mice may be kept in battery jars fitted with perforated metal covers. The mice should be supplied with large pieces of cotton upon batting since they are delicately susceptible to cold. The Bleeding of Animals. — Animals are bled for the purpose of obtaining either corpuscles, defibrinated blood or serum. In order to obtain small amounts of blood, that is about 5 or 10 c.c., from rabbits, the ear is shaved and had best be immersed in warm water for a few moments in order to expand the vessels. Gentle rubbing with alcohol is also advantageous. The rabbit is then held with head hanging downward, and a broad needle of the Hagedore needle type is thrust into the vein and withdrawn. The drops can be caught directly in a centrifuge tube, or in the culture media for which it is intended. All blood media should be incubated for 24 hours and the contaminated tubes discarded. A better method is to take blood from rabbits and from guinea pigs directly from the heart. If this is skillfully done the animals can be repeatedly bled without being killed. For taking blood for DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 205 complement in Wassermann reactions, this is among the best methods since large guinea pigs can be alternately bled and rested. Both in rabbits and guinea pigs, bleeding directly from the heart is easily accomplished after a little practice. The anterior thorax of the animal is clipped and painted with tincture of iodin and the operator in feeling for the third interspace close to the sternum had best paint the tips of his fingers with iodin. A twenty-two gauge needle about two inches long is then attached to a syringe and passed downward in the third left interspace close to the sternum, slight suction being exercised at the same time. There is not much purpose in describing this in detail since it can be taught only by practice. Both rabbits and guinea pigs can be bled from the carotid. The animal is anesthetized as above, and the carotid laid bare. It is found vety close to the trachea, in rabbits lying almost in contact with the trachea, and a little behind it. It is carefully separated from the vagus nerve, and tied off in its distal portion. The thread with which it is tied can be used to handle it thereafter. A sterile glass cannula can be thrust into the artery and the blood taken through this, or else, as we prefer to do it, the side of the artery is picked up with a very fine forceps and held with one hand while it is cut across with a sharp scissors. In this way the blood can be directed straight into a wide mouth flask without being allowed to come in contact with anything until it hits the inside of the flask. Larger animals, like sheep, goats, horses, are. easily bled by plunging a sterile needle into the external jugular vein which runs in these animals from a line just behind the angle of the lower jaw to the sterno-clavicular junction. The blood can be run directly into media as for blood agar, blood broth and chocolate medium. If serum is desired, it can be run into containers of various kinds slanted and allowed to clot in the ice-box. If defibrinated blood is desired, the blood can be taken directly into sterile flasks containing pieces of broken glass or beads and attenuated before clot. Such blood can be kept in the ice-chest and added to media subsequently. Blood can be also preserved for culture purposes by the addition of just enough ether to hemolyze it, and added to media in this form. The ether is evaporated off. CHAPTER X THE BACTEEIOLOGICAL EXAMINATION OF MATEEIAL FEOM PA- TIENTS AND AN OUTLINE OF THE BACTERIAL FLORA OF THE NORMAL HUMAN BODY TECHNICAL procedures for the examination of specimens of exudates, stools, sputum, etc., in various conditions are given in appropriate places in the text dealing with the individual diseases. In this chapter we wish to discuss briefly general principles of bacteriological examination which will be useful in properly col- lecting and handling materials which are sent to the laboratory for diagnosis or which the bacteriologist takes from the patient himself. In making bacteriological examinations of material taken from living patients, or at autopsy, the validity of result is as fully dependent upon the technique by which the material is collected, as upon proper manipulation in the later stages of examination. Material taken at autopsy should be, if possible, directly trans- ferred from the cadaver to the proper culture media. If cultures are to be taken from the liver, spleen, or other organs, the surface of the organ should first be seared with a hot scalpel and an incision made through the capsule of the organ in the seared area, with the same instrument. The platinum needle can then be plunged through this incision and material for cultivation be taken with little chance of surface contamination. When blood is to be transferred from the heart, the heart muscle may be incised with a hot knife, or else the needle of a hypodermic syringe may be plunged through the previously seared heart muscle and the blood aspirated. The same end can be accomplished by means of a pointed, freshly prepared Pasteur pipette. In taking specimens of blood at autopsy it is safer to take them from the arm or leg, by allowing the blood to flow into a broad, deep cut made through the sterilized skin, than from the heart, since it has been found that post-mortem contamination of the heart 's blood takes place rapidly, probably through the large veins from the lungs. Exudates from the pleural cavities, the peri- 206 BACTERIOLOGICAL EXAMINATION OF MATERIAL 207 cardium, or the peritoneum may be taken with a sterilized syringe or pipette. Under all circumstances it should be remembered that cultures taken from blood or tissues of the cadaver will be con- taminated, unless cultures are taken within a few hours after death. Bacteria get into the circulation and multiply throughout the body with astonishing speed after death. Materials collected at the bedside or in the operating-room should be transferred directly to the proper media or else into sterile test tubes and so sent to the laboratory. When the material is scanty, it may be collected upon a sterile cotton swab, which should be immediately replaced in the sterilized containing tube and sent to the laboratory. Syringes, when used for the collection of exudates or blood, should be of some variety which is easily sterilizable by dry heat, or boiling. Most convenient of the forms in common use are the all-glass "Luer" syringe, or the cheaper "Sub-Q" model. Instru- ments which can be sterilized only by chemical disinfectants should not be used. When fluids are collected for bacteriological examina- tion, such as spinal fluid, ascitic fluid, or pleural exudates, it is convenient to have them taken directly into sterilized centrifuge tubes, since it is often necessary to concentrate cellular elements by centrifugalization. By immediate collection in these tubes, the danger of contamination is avoided. Examination of Exudates. — Pus. — Pus should first be examined morphologically by some simple stain, such as gentian-violet, and by the Gram stain. It is convenient, also, to stain a specimen by Jenner's stain, in order to show clearly the relation of bacteria to the cells. Such morphological examination not only furnishes a guide to future manipulation, but supplies a control for the results obtained by cultural methods. Specimens of the pus are then trans- ferred to the proper media, and pour-plates made or streaks made upon the surface of previously prepared agar or serum-agar plates. A guide to the choice of media is often found in the result of the morphological examination. In most cases, it is well also to make anaerobic cultures by some simple method. (See page 179 et seq.) The colonies which develop upon the plates should be studied under the microscope, and specimens from the colonies transferred to covor-glasses and slides for morphological examination and to the various media for further growth and identification. Animal inocu- lation and agglutination tests must often also be resorted to. A 208 BIOLOGY AND TECHNIQUE knowledge of the source of the material may furnish considerable aid in making a bacteriological diagnosis, though great caution in depending upon such aid is recommended. If the morphological examination shows Gram-positive micro- cocci, as in staphylococcus boils, any ordinary properly made agar will suffice. If streptococci are present in the Gram stain, it will be useful to employ blood agar plates without glucose. When the pus is gonorrheal, ascitic agar plates with glucose should be used, and the pus transferred directly from the patient to the plate and incubated before it chills. In the case of pus from abrasions of the skin, furuncles or boils that arouse any suspicion of anthrax a careful search for Gram-positive bacilli should be made with the Gram stain, and the characteristic colonies looked for on ordinary agar plates. When plentiful leucocytes are present and the pus shows no organisms in smear, this should not discourage culture since it is not unusual to obtain colonies on culture when nothing can be found by smear. In the examination of peritoneal, pericardial, or pleural exudates it is often advantageous to use the sediment obtained by centrifugaliza- tion. A differential count of the cells present may be of aid in confirm- ing the bacteriological findings. Morphological examination and cul- tural examination are made as in the case of pus. Specimens should also in these cases be stained for tubercle bacilli. Whenever mor- phological examinations of such fluids are negative, no bacteria being found, and especially when among the cellular elements the lymphocytes preponderate, the search for tubercle bacilli should be continued by means of animal inoculation. Guinea-pigs should be inoculated intraperitoneally with specimens of the fluid. The animals will usually die within six to eight weeks, but can be killed and examined at the end of about six weeks if they remain alive. The chances for a positive result are considerably increased if the fluid is set away in the ice-chest until a clot has formed and the animals are inoculated with the material from the broken-up clot. Spinal Fluid. — Normal spinal fluid is a clear, colorless fluid which contains not more than ten to twelve cells per cubic millimeter. Anything above this should be regarded as suspicious. When clear spinal fluid is brought to the laboratory it is always well to shake up the specimen and do a direct count. BACTERIOLOGICAL EXAMINATION OF MATERIAL 200 It is of value also to do a globulin reaction on such clear fluids, which is easily done by Noguchi's butyric acid method as follows: To 0.2 c.c. of spinal fluid add 0.5 c.c. of a 10 per cent butyric acid solution in physiological salt solution. Boil the mixture and add 0.1 c.c. of normal sodium hydrate, and boil again. A flocculent precipitate forms in positive reactions. Clear specimens of fluid of this kind should be examined with an intelligent understanding of the nature of the case. Syphilitic spinal fluids are almost always clear, but the cells are increased to 100 or more per cubic millimeter. The cells consist mainly of lymphocytes. Low counts may be encountered in tabes and general paresis. The determination of these facts will be valuable in con- nection with the subsequent Wassermann reaction or colloidal gold reaction on these fluids, and with bacteriological examination. In infantile paralysis or acute poliomyelitis, the spinal fluid is usually clear. The cells here are increased from the beginning. According to Peabody, Draper and Dochez1 during the early days of the disease, 80 or more per cent of the cells may be polynuclear. After 72 hours, however, the mononuclears preponderate. The cell count may go up even in the prodromal period. The highest cell count is usually found in the first week, gradually coming down until the fourth week of the disease. The globulin reaction is usually highest in the second and third week, but the writers men- tioned above found a percentage of cases in which the cell counts were normal. These facts are given because they should be taken into consideration, together with bacteriological examinations. Tuberculous fluids are entirely clear, or but slightly turbid. When the suspicion of tuberculosis exists, the fluid should be handled as sterilely as possible, and allowed to stand in the ice-chest until a little, white, thread-like clot appears in the center, which sinks to the bottom of the tube. It is in this clot that tubercle bacilli can be found by careful search. It is smeared on the slide and stained by the usual methods. If enough fluid is available, the residue should be injected into one or two guinea pigs in as large quantities as can be obtained. The cells in tuberculous fluid are chiefly lymphocytes. Acute meningitis is most commonly caused by the meningococcus, pneumococcus, streptococcus, less commonly by influenza bacilli and 1 Peabody, Draper and Dochez. 210 BIOLOGY AND TECHNIQUE other organisms. Such fluid may range from slight turbidity to thick purulence. The cells in such fluid consist almost entirely of polynuclear leucocytes during the acute stages. Smears should he made immediately and stained by Gram. If Gram-positive organisms are present, they can immediately be recognized, and cultures taken accordingly. In epidemic meningitis the Gram-negative meningo- cocci will be found mostly intracellular, but some also extracellular. Sometimes a very prolonged search must be made before any meningococci can be found, because these organisms readily undergo autolysis. In the fluid from an acute case of meningitis in which many polynuclear leucocytes are present, and no organisms can be found, it is pretty safe to suspect epidemic meningitis, since we have on a number of occasions encountered fluids of this kind in which no organisms could be seen. Cultures should, in all cases, be taken even when the organisms are found, since this may be of value in determining the meningococcus type, in finding out whether the particular meningococcus is agglutinable in the polyvalent serum used, and the collection of the type of meningococci which are present in an epidemic is a part of the bacteriologist's contribution to the successful production of sera. The cultures are best taken on plates of hormone agar with 0.5 per cent glucose, and hemolyzed blood or ascitic fluid added. Pneumococci, streptococci, influenza bacilli, etc., may be cul- tivated by appropriate methods. The cytological character of the fluid and the relationship of cells to bacteria should always be determined since this may have a certain amount of prognostic significance. Examination of Urine. — Bacteriological examination of the urine is of value only when specimens have been taken with sterile catheters, and care has been exercised in the disinfection of the external genitals. This is particularly important in the female. Many of the numerous finds of bacillus coli in urine are unquestion- ably due to defective methods of collecting material. Urine should be centrifugalized and the sediment examined morphologically and pour-plates and surface smears made upon the proper media. If necessary, animal inoculation may be done. In examining urine for tubercle bacilli, special care should be taken in staining methods so as to differentiate from Bacillus smegmatis. When the question is one of infection of one kidney alone the specimens must of course be obtained by ureteral catheterization. BACTERIOLOGICAL EXAMINATION OF MATERIAL 211 Examination of Feoes. — Human feces contain an enormous num- ber of bacteria of many varieties. Klein,2 by special methods, estimated that there were about 75,000,000 bacteria in one milligram of feces. It has been a noticeable result of all the investigations upon the feces, that although enormous numbers can be counted in morphological specimens, only a disproportionately smaller num- ber can be cultivated from the same specimen. This is explicable upon the ground that special culture media are necessary for many of the species found in intestinal contents and upon the consideration that many of the bacteria which are present in the morphological specimen are dead, showing that there are bactericidal processes going on in some parts of the intestinal tract, possibly through the agency of intestinal secretions, bile, and the action of the products of metabolism of the hardier species present. By far the greater part of the intestinal flora consists of members of the colon group, bacilli of the lactis aerogenes group, Bacillus faecalis alkaligenes, Bacillus mesentericus, and relatively smaller numbers of strepto- cocci, staphylococci, and Gram-positive anaerobes. Many other species, however, may be present without being necessarily con- sidered of pathological significance. Certain writers have recently laid much stress upon a preponderance of Gram-positive bacteria in specimens of feces, claiming that such preponderance signifies some form of intestinal disturbance. Herter3 has recently advanced the opinion that the presence of Bacillus aerogenes capsulatus in the intestinal canal is definitely associated with pernicious anemia. This is discussed in another section. The determination of these bacilli in the stools is made both by morphological examination by means of Gram stain and by isolation of the bacteria. Such isolation is easily done by the method of Welch and Nuttal.4 A suspension of small quantities of the feces in salt solution is made and 1 c.c. of the filtered suspension is injected into the ear vein of a rabbit. After a few minutes the rabbit is killed and placed in the incubator. After five hours of incubation, the rabbit is dissected, and if the Welch bacillus has been present in the feces, small bubbles of gas will have appeared in the liver from which the bacilli may be cul- tivated in anaerobic stab cultures. -Klein, Ref. Cent. f. Bakt., I, xxx, 1901. 3 Herter, <• ' Common Bacterial Infections of the Digestive Tract, ' ' N. Y., 1907. * Welch and Nuttal, Bull. Johns Hopkins Hosp., 1892, 111, 81. 212 BIOLOGY AND TECHNIQUE Bacteriological examination of feces is most often undertaken for the isolation of Bacillus typhosus, dysentery, cholera, etc. These methods are discussed in detail in the chapters dealing with the diseases. See also section on media. The determination of tubercle bacilli in stools is difficult and of questionable significance, in that they may be present in people suffering from pulmonary tuberculosis as a consequence of swallow- ing sputum or infected food, and in that there may be other acid- bacilli, such as the timothy bacillus, present. Perhaps the most reliable method is to treat a suspension of the feces with 5 per cent antiformin over night, centrifugalize thoroughly, wash the sediment, and inject into guinea pigs. Blood Cultures. — The diagnosis of septicemia can be positively made during life only by the isolation of bacteria from the blood. Such examinations are of much value and are usually successful if the technique is properly carried out. A large number of methods are recommended, the writers giving, however, only the one which they have found successful and simple for general use. The blood is taken by preference from the median basilic vein of the arm. If, for some reason (both forearms having been used for saline infusion), these veins are unavailable, blood may be taken from the internal saphenous vein as it turns over the internal malleolus of the ankle joint. The skin over the vein should be prepared before the specimen is taken by painting with iodin, as for a surgical operation. The syringe which is used should be of some sterilizable variety (the most convenient the Luer model), which is easily manipulated and does not draw with a jerky, irregular motion. Its capacity should be at Jeast 10 c.c. It may be sterilized by boiling for half an hour, or preferably, when all-glass syringes are used, they may be inserted into potato-tubes and sterilized at high temperature in the hot-air chamber. Before drawing the blood, a linen bandage is wound tightly about the upper arm of the patient in order to cause the veins to stand out prominently. When the veins are plainly in view, the needle is plunged through the skin into the vein in a direction parallel to the vessel and in the direction of the blood-stream. After perforation of the skin, while the needle is groping for the vein, gentle suction may be exerted with the piston. Great care should be exercised, however, that the piston is not allowed to slip back, and air be, by accident, forced into the vessel. In most cases no suction is necessary, the pressure of the BACTERIOLOGICAL EXAMINATION OF MATERIAL 213 blood being sufficent to push up the piston. After the blood has been drawn, it should be immediately transferred to the proper media. Epstein has recently recommended the mixture of the blood with sterile two per cent ammonium oxalate solution in test tubes, by which means the clotting is prevented, and transfers can be made more leisurely to culture media. While this method is convenient FIG. 29. — BLOOD-CULTURE PLATE SHOWING STREPTOCOCCUS COLONIES. Note halo of hemolysis about each colony. in cases where blood must be taken at some distance from the laboratory, it is preferable, whenever possible, to make cultures from the blood immediately at the bedside. The choice of culture media for blood cultures should, to a certain extent, be adapted to each individual case. For routine work, it is best to employ glucose "hormone" agar and glucose-meat- 214 BIOLOGY AND TECHNIQUE infusion broth. At least six glucose-agar tubes should be melted and immersed in water at 45° C. Before the blood is mixed with the medium, the agar should be cooled to 41° in order that bacteria, if present, may not be injured by the heat. The blood is added to the tubes in varying quantities, ranging from 0.25 to 1 c.c. each, in order that different degrees of concentration may be obtained. Mixing is accomplished by the usual dipping and rotary motion, the formation of air-bubbles being thus avoided. The mouth of each test tube should be passed through the flame before pouring the contents into the plates. Three flasks of glucose broth, contain- ing 100 to 150 c.c. of fluid each, should be inoculated with varying quantities of blood — at least one of the flasks containing the blood in high dilution. The most stringent care in the withdrawal and replacement of the cotton stoppers should be exercised.5 The writers have found it convenient to use, in place of one of these flasks, one containing, in addition to the glucose, 1 gm. of powdered calcium carbonate. This insures neutrality, permitting pneumococci or streptococci, which are sensitive to acid, to develop and retain their vitality. In making blood cultures from typhoid patients, Buxton and Coleman6 have obtained excellent results by the use of pure ox-bile containing ten per cent of glycerin and two per cent of pepton in flasks. The writers have had 110 difficulty in obtaining typhoid cultures by the use of slightly acid meat-extract broth in flasks containing 200 or more c.c. to which comparatively little blood has been transferred. The failure of a proper blood culture service in most hospitals is due, we believe, to the fact that blood cultures are taken by the interne staff, and worked out by the bacteriologist. It is of the utmost importance, in our opinion, that a single individual should be responsible for the entire examination from beginning to end. This is to avoid the great possibility of contamination in blood culture work. ANAEROBIC BLOOD CULTURES. — These cultures may be taken by mixing blood in deep tubes with glucose-ascitic agar, covering with albolene and putting into Novy jars. In estimating the results of a blood culture, the exclusion of 5 Small Florence flasks are preferable to the Erlenmeyer flasks usually employed, ^Buxton and Coleman, Am. Jour, of Med. Sci., 1907. BACTERIOLOGICAL EXAMINATION OF MATERIAL 215 contamination usually offers little difficulty. If the same micro- organism appears in several of the plates and flasks, if colonies upon the plates are well distributed within the center and under the surface of the medium, and if the microorganisms themselves belong to species which commonly cause septicemia, such as streptococcus and pneumococcus, it is usually safe to assume that they emanated from the patient's circulation. When colonies are present in one plate or in one flask only, when they are situated only near the edges of a plate or upon the surface of the medium, and when they belong to varieties which are often found saprophytic upon skin or in air, they must be looked upon with suspicion. It is a good rule to look upon all staphylococcus albus cultures skeptically. Sputum. — In examining sputum, sufficient emphasis cannot be placed upon the necessity of collecting the sputum in a proper way. The sputum collected by patients in the ordinary sputum cup con- sists to a very large extent of material obtained from the mouth and throat. If a successful examination of sputum is to be made, the patient should be taught to rinse out his mouth thoroughly, and the sputum collected directly after a cough. It is very little to ask for this amount of care, if the examination is really worth mak- ing at all. Sputum so collected should not be left in the ward, but should be sent to the laboratory immediately. Smears should be made on such sputum, with an intelligent idea of what is desired. For pneumococcus type examination, the sputum is thoroughly washed and intraperitoneally injected into mice, according to the detailed directions given for typing in the chapter on pneumococcus. For influenza bacillus examinations, thin smears of the sputum should be stained by Gram and dilute carbol fuchsin. The char- acteristic grouping of influenza bacilli is of considerable help. Plates of chocolate agar are then streaked. Sputum for tuberculosis examination is thinly smeared and stained by carbol fuchsin or Hermann's stain. When it is desired to carry the examination beyond this in negative examinations, washed sputum can be injected into guinea pigs, or the sputum can be antiforminized, washed and then injected. Also, the anti- forminized sediment can be examined by stain preparation. Direct culture of tubercle bacillus sputum can be made by treat- ing with sodium hydrate and plating upon Petroff's gentian- violet- egg medium, by the method described in detail in the section on tuberculosis. 216 BIOLOGY AND TECHNIQUE Throat Smears and Throat Examinations. — The bacteriologist, if possible, should take these specimens himself, or the physician tak- ing them should take them only with a clear illumination of the throat, taking his specimen from the exact spot where the lesion is supposed to be located. For diphtheria examination, the specimen is taken with a sterile swab, and plated directly upon Loeffler's medium. This should be incubated without delay, and the swab sent to the labora- tory with the culture. The method has been standardized and is described in the section of diphtheria. For Vincent's angina examination, smears should be taken and stained, best by strong gentian violet, such as used in the Gram stain, and the smear searched for the characteristic spirilla, and fusiform bacilli. If the patient is in the laboratory, it is best to make a dark field examination. Examination of Lesions on the Genitalia. — Lesions suspicious of primary syphilitic nature should be gently washed, the superficial pus removed, and only exudate from the bottom of the lesion taken. If necessary, the lesion can be gently scraped and serum, mixed with as little blood as possible, used. No examination for treponema pallidum is equal to the dark field examination. It is important to use only thick slides. A drop of the exudate is placed on the slide and a cover-slip dropped on it. Then a drop of oil is placed on the bottom of the slide, over the preparation and on the top of the cover-slip, and the preparation is placed on the dark field condenser. In doing this, care should be taken to avoid air bubbles in the oil. When suspicion of chancroid exists, the material should be in- oculated immediately into tubes of coagulated and inactivated sterile rabbit 's blood, and incubated according to the method of Teague. BACTERIA HABITUALLY INHABITING THE NORMAL HUMAN BODY In studying bacteria in disease, it is of considerable importance to have a clear idea of the morphological and cultural characteristics of forms which are frequently encountered in different parts of the human body under normal conditions. Various cavities of the body which communicate with the ex- ternal world, always contain considerable numbers of bacteria repre- senting a large variety of species. Some of these may be habitual saprophytes associated with that particular part of the body, others BACTERIOLOGICAL EXAMINATION OF MATERIAL 217 may be accidental and temporary invaders, members of pathogenic groups which, either because of the reduced virulence of the strains or the increased resistance of the individual are not capable under the circumstances of causing their specific infection. It is such conditions which may lead to many erroneous etiological conclusions and which render the investigation of the causation of diseases in the mouth, intestines and other locations extremely difficult. It is perhaps best to discuss this subject from the point of view of the individual locations studied. Bacteria in the Normal Mouth and Pharynx. — The mouth and pharynx are habitually the habitat of numerous bacteria. Saliva itself is not a good culture medium, and, indeed, may, according to some investigators, show very slight inhibitory or even bacteri- cidal powers. But these, at best, are not very potent, and the saliva thus is a basis for a fluid medium which furnishes water as a solvent and a reaction suitable for a great many different bacteria. Sloughing epithelium, decayed teeth and gums, food particles, etc., furnish suitable nutrition. Catarrhal inflammation which is rarely entirely absent to some degree or in some place in the adult human being, favors the lodgment of bacteria upon the membranes and reduces the resistance of the tissues. In spite of these facts, it is surprising that the frequent accidental injury of the gums and oral and pharyngeal mucous membranes so rarely leads to serious infection, and ends so readily. This is a fact which has not as yet been adequately explained. Staphylococci can almost always be isolated from the mouth. They are usually of the Albus variety, but not infrequently staphylococcus Aureus also can be found. Of the streptococci the Viridans is almost always present. The isolation of a "viridans" from inflammatory processes of the mouth and throat, therefore, has very little true significance, unless it is isolated from a closed process, such as a tooth abscess, or unless other strong corroborative evidence can be adduced. The Hemolyticus variety is less frequently found in the normal mouth, but may be present without causing disease. However, the isolation of a hemoly- ticus from an inflamed tonsil or pharynx is much more likely to mean that there is an etiological relationship, and it is of course well known that many of the severe inflammations in this location are of hemolyticus origin. In examinations made many years ago by the writer, 30 per cent 218 BIOLOGY AND TECHNIQUE of people examined harbored pneumococci in their mouths, at one time or another, in the course of the cold months. Since then, the typing of the pneumococcus has made it possible to show that the pneumococci most frequently present in the mouth belong to Group IV. In the investigations of Dochez and Avery which are described in another place, it was found that this type caused only about 9.8 per cent of pneumonias, but was found with considerable frequency in normal mouths. The other and more virulent types may be found in the normal mouth, as well, but are more apt to represent recent con- tact with pneumonia cases or a transitory carrier state. This, at least, is suggested by the writers mentioned above, though probably a definite, conclusive statement cannot be made concerning it at the present time. Of the non-pathogenic Gram-positive cocci the Micrococcus Candi- cans and occasional pigment forming micrococci are not infrequent. Micrococcus Tetragenus is very often an inhabitant of the mouth and, as a matter of fact, one sees it most frequently in routine work in Loeffler's cultures taken for the purpose of diphtheria diagnosis. Of Gram-negative micrococci there is a considerable variety which, without being pathogenic, may be cultivated from the mouth and throat and add no little confusion to meningococcus carrier examina- tions. Most common among these are the Micrococcus Catarrlialis, which is described in another section, and may be distinguished from the meningococcus by its heavier growth, its growth at room tem- perature and its failure to produce fermentation of dextrose and maltose. The Micrococcus Flavus, which frequently has led to error in similar work, is a pigment forming Gram-negative coccus often found in the throat, which grows at room temperature, and in most cases agglutinates spontaneously in normal horse serum. Another which forms very dry colonies, the Micrococcus Pkaryngis siccus, is often isolated, but easily recognized. In addition to this, Elser and Huntoon have described three different chromogenic groups of similar organisms often found in the mouth and throat. These probably do not exhaust all the possible Gram-negative micrococci that can be isolated from this locality, but it is really only of importance to make sure in human examination whether one is dealing with a true meningococcus, with a micrococcus catarrhalis, or with other sapro- phytes. True meningococci are of course often found in normal or slightly inflamed throats during the carrier state, which is discussed at con- BACTERIOLOGICAL EXAMINATION OF MATERIAL 219 siderable length in another place. As discussed there, these organisms when they are present are usually located high up in the pharynx near its roof, and successful search for carriers depends very largely upon care of reaching the right spot with the swab. Of bacilli, the mouth contains a large variety at different times. Few of these, however, are confusing from the bacteriologist's point of view, except some of the diphtheroids. The pseudo-diphtheria bacillus, or Bacillus Hoffmanni, may be present without having any relationship to disease. It is described in another section. The other larger and more irregular diphtheroids are not uncommon, and are easily distinguished from true diphtheria bacilli by their appearance and cultural characteristics. Chain- forming Gram-positive bacilli and large obviously sapro- phytic varieties may be present in very dirty mouths, but offer no bacteriological difficulties. Of the Gram-negative bacilli, Proteus, Lactis Aerogenes, and special members of the Friedldnder group may be present. We have known one man who habitually had a Friedlander culture in his mouth, with- out ever suffering any harm from its presence. The fusiform bacillus described in another section in connection with Vincent's angina, is almost always present between the gums and the teeth in mouths that are dirty, with carious teeth or where there is some inflammation of the gums themselves. It is an observation that we make almost every year with our students, that, if a platinum loop is passed between the base of the tooth and the gums, and smears taken from a number of students, the bacteria usually associated with Vincent's angina, spirochsetes and fusiform bacilli, can be seen in one or another of the cases examined. Spirilla and spirochaetes are almost habitually present. The Spirillum Milleri, named after Miller, who has made valuable studies upon mouth bacteria, is a small true spirillum, easily cultivated, and not easily confused with other morphologically similar organisms. Miller cultivated three of four varieties of mouth spirilla. True treponema (Noguchi's classification), are almost always present in locations like those described for the fusiform bacilli, and even on the mucous membranes, especially when small spots of necrosis or inflammation occur. Most frequently discussed among these are the large spirochaete, associated with Vincent's angina, the Spironema Vincenti. There are, likewise, present very frequently the treponema macrodentium and microdentium, classified thus by Noguchi. These 220 BIOLOGY AND TECHNIQUE organisms are best observed under the dark field, but can also be stained in smear if strong gentian violet or carbol fuchsin are used. It is important to note that morphologically the macrodentium is very similar to the treponema pallidum, and in the dark field examination of syphilitic lesions of the mouth and throat, this similarity must be carefully taken into account. We have seen cases in which we were unwilling to make a definite diagnosis on these findings alone. It is our belief that whenever extensive necrosis of the tissues of the mouth and pharynx occur in consequence of other infection or or injury, the necrotic tissues are apt to be invaded by fusiform bacilli, and spirochaetes, which in subsequent examination dominate the bac- teriological picture. We believe, however, that in the large majority of these cases, perhaps including the clinical picture spoken of as Vincent's angina, the trepdnemata and fusiform bacilli are secondary to the primary etiological factors, such as those mentioned. These organisms are anaerobic. We believe that the early contention of Tunnicliff that the spirochaetes and fusiform bacilli found in Yin- cent 's Angina are different stages of the same organism, is not generally accepted to-day. The normal mouth is also apt to contain occasional members of the Leptotkrix and Streptothrix groups. One of these, the LeptotJirix innominata of Miller, is supposed to be characteristic of the mouth flora. It may appear as a large Gram-positive bacillus form which is believed by some writers to be a true bacillus, rather than a lepto- thrix, and is spoken of as the Bacillus Maximus Buccalis (Miller). Bacteria in the Nose and Accessory Sinuses. — That the nasal mucosa should be a favorable site for the deposit of numerous microorgan- isms follows from the fact that air is constantly passing in and out during respiration. The varieties of bacteria to be found in the nose, therefore, may belong to any that happen to be present in the inhaled air. The subject of the bacteriology of the nose deserves more atten- tion than has been given to it during recent years, for infections of the nasal sinuses and the conditions which lead to them, are being recognized of the utmost importance upon general health. Earlier investigators claimed that the passage of bacteria in the air to the deeper respiratory organs is very largely arrested by a sort of filtering action in the nose. Thomson and Hewlett7 found that in ''Thomson and Hewlett, Baumgarten's Jahresb., 12, 1896, 767. BACTERIOLOGICAL EXAMINATION OF MATERIAL 221 animals, the tracheal mucus, as well as the mucous membrane of the posterior portions of the healthy nose, are usually sterile, although the vestibulium nasae is usually heavily contaminated. When the nasal cavity and the septum were artificially inoculated with Bacil- lus prodigiosus, the organisms disappeared in about two hours. These observers believed that the healthy nasal mucus is not bac- tericidal, but does not favor growth. They examined air which passed through the nose, and found that in the case of air which con- tained over 20 mould spores and 9 bacteria per cubic centimeter, these organisms almost entirely disappeared in the passage of the air through the nose. Hilderbrandt8 has previously obtained similar results in Baumgarten's laboratory. Wright9 also has made similar investiga- tions, and showed that between 3/4 to 4/5 of the bacterial flora of the inspired air was held back in its passage through the nose. Among the most interesting studies along these lines are those of Neumann10 who studied the nasal secretions of over 200 people, of which about 111 were supposedly normal. Neumann found in normal noses a very large number of different microorganisms. The percentage findings of various bacteria were as follows: Pseudo-diphtheria (probably including diphtheroids) — 98 to 100 per cent Micrococcus albus — 98 per cent Micrococcus aureus — 30 per cent Streptococcus lancelatous (probably pneumococcus) — 4 per cent Friedlander bacilli — 6 per cent Micrococcus citreus — 12 per cent Colon bacilli — 12 per cent Streptococcus — 2 per cent Molds — 20 per cent Sarcinae — 6 per cent Lactis aerogenes — 4 per cent Yeasts — 2 per cent Neumann mentions other microorganisms in addition to these, but the figures given are sufficient to show that the normal nose may contain almost any of the known organisms including a great many of the non-pathogenic forms in air, of which the bacteriologist dealing with disease knows very little as a rule. Calamida and * Hilderbrandt, Baumgarten's Jahresb., 4, 1888, 378. 9 Wright, quoted from Baumgarten 's Jahresb., 5, 1889. "Neumann, Zeit. f. Hyg,, 40, 1902, 33. 222 BIOLOGY AND TECHNIQUE Bertarclli11 also carried out interesting studies on the normal bac- terial flora of the accessory nasal sinuses. Working first with dogs, they found that in 20 dogs of various ages, the frontal sinuses were always sterile. In a single case they isolated an organism which resembled the Colon bacillus. In 8 dogs the ethmoidal sinuses were sterile. In. 16 of 20 dogs the maxillary sinuses, antrum of Highmore, were sterile. In the others they found various cocci. When they inoculated the naso- pharynx of dogs with cultures of B. prodigiosus, pyocyaneus, and subtilis, and killed them 8 to 24 hours later, 3 of them showed entirely sterile accessory sinuses and sterile middle ear. One animal gave a positive culture of prodigiosus in the antrum, frontal sinuses, and the ear. In 3 others, only the antrum was infected. Two of the animals treated with B. pyocyaneus retained sterile sinuses. Of 6 treated with Subtilis, 4 were entirely sterile. It is interesting to note that the animals that were infected, were those which were killed only 8 to 10 hours after inoculation. Of those which were killed between 18 and 24 hours after inoculation, all but one were sterile. They examined 12 fresh human cadavers within a few hours after death, never later than 20 hours. In all but one of these all accessory sinuses of the nose were sterile, and in this one a non- pathogenic Staphylococcus Albus was found. Kuster, who has sum- marized work on the nasal flora in the Kolle and Wassermann, on the basis of a study of the literature as well as his own investiga- tions, comes to the conclusion that we cannot speak of a character- istic nasal flora, that practically all of the organisms with which man can come in contact through the air may settle there for a shorter or longer period. In a healthy nose, however, few organisms can gain a permanent foothold, largely because of unsuitable cultural conditions and of the action of leucocytes and the secretions. Bacteria in the Tissues Themselves. — Recent work has given definite evidence that even the tissues themselves may not be sterile in normal human beings. This has led, • we believe, to a certain amount of error in etiological conclusions when blood cultures and cultures from normal or slightly diseased lymphatic tissues have been taken, and diphtheroid and various coccus forms isolated. According to the experiments done by Adami12 there is a constant "Calamida and Bertarelli, Ziet. f. Bakt., I Orig., 32, 1902, *- Adami, Jour. A. M. A., Dec., 1899, BACTERIOLOGICAL EXAMINATION OF MATERIAL 223 entrance of bacteria into the portal circulation from the intestines. These are very largely disposed of in the liver, but it may well be that the liver does not always eliminate all the bacteria from the portal circulation, and that some of these then lodge in other tissues and become latent there. The latency of bacteria in the healthy body can no longer be questioned. We have long known that treponema pallidum the spirochsetes that infect mice, and many trypanisomes can remain present for a long time in the circulation and in the tissues of animals and man without giving rise to characteristic symptoms or even to any symptoms. We have, ourselves, found pallida in the testes of rabbits four months after inoculation without there having been the slightest tissue reaction, and in human syphilis this latency is well recognized. That tetanus spores may remain latent in the spleen and other organs of guinea pigs under certain experimental conditions, has been shown by the Italian observer, Canfora,13 and recently we have seen a very convincing example of latency of streptococci in the tissues of the hand. A very severe hemolytic streptococcus lesion subsided under surgical treatment, and four months later a purely cosmetic secondary operation was undertaken at a time when there was not the slightest trace of infection, and hemolytic streptococci were again isolated from the tissues at this operation. There was, incidentally, no sign of infection of the wound, which healed uneventfully. The investigations of Torrey and others have shown that from lymph nodes, the seat of various non-bacterial conditions, such as sarcoma, Hodgkin's disease, etc., many varieties of diphtheroids may be isolated, and Roscnau has reported a number of blood culture results in which diphtheroids and cocci were isolated from the blood in the presence of febrile conditions which obviously were not due to the particular organisms isolated. Not much can be said about this problem of latency at the present time because little is known about it, but the possibility should be kept in mind, and should cause great conservatism whenever isola- tions from the tissues are made, and the etiological question is raised. The Bacteriology of the Intestinal Tract. — More than any other part of the body, the intestinal canal has a specific flora of its own. This varies at different ages, with health and disease, and is to "Can-fora, Cent. f. Bakt., 45, 1908. 224 BIOLOGY AND TECHNIQUE a considerable extent dependent upon diet. Also, many of the bac- teria that cause specific diseases of the intestinal canal, such, for instance, as the typhoid bacillus, the paratyphoid bacilli, the dysen- tery group, and some of the doubtfully pathogenic organisms like the Morgan bacilli, are very closely related in morphology and cul- tural reactions to non-pathogenic and saprophytic inhabitants of the bowel. In no type of bacteriological work, therefore, is it more necessary to have an intelligent understanding of the bacterial species that are likely to be found without pathogenic significance. Furthermore, the intestinal canal is a large test tube from which bacterial products can be absorbed in sufficient amounts to cause severe illness. In it, different kinds of food supply nutritive ma- terial which may favor one or another species, and various condi- tions of aerobiosis and anaerobiosis may prevail. It is more than likely, therefore, that many so-called cases of intestinal poisoning, formerly loosely spoken of as ptomain poisoning, may be caused by substances formed within the intestine by bacterial action upon the food, rather than upon the relatively smaller amount of fermen- tative and putrefactive products taken in. with partially decomposed food. The intestinal canal of the child at birth is sterile. The meconium of such children has been found by many investigators to be free from bacteria. But this does not last very long. Within a few hours after birth, infection takes place, and from then until death, the intestinal canal is constantly the seat of a voluminous and varied bacterial life. Kendall,14 who has written much on this subject, and in his book has brought together much of the information, gathered from the researches of Escherich,15 Herter,10 and his own investigations, has classified the different stages of the bacterial flora in man, as follows: 1. Bowel at birth, sterile. 2. First to the third day a period of " adventitious bacterial in- fection.'' After this time there is the period of establishment of the charac- teristic infantile intestinal flora which gradually changes as the diet "Kendall, Bacteriology, General, Pathological and Intestinal, Lea & Febiger, Phila., 1916, p. 580. 15 Escherich, Darmbakterien des Sauglings, Stuttgart, 1886, p. 9. 18 Herter, The Common Bacterial Infections of the Digestive Tract, Harvey Lect., 1906-1907. BACTERIOLOGICAL EXAMINATION OF MATERIAL 225 approaches more and more that of the adult, into the characteristic flora of the adult. In the earliest days during the stage of "adventitious infection/' when the child is getting its first bacteria from the air and subjects with which its mouth comes in contact, the bacterial flora is de- termined largely by accident. When the child begin to take food, it is of great importance for the determination of the bacterial flora, whether it is being breast fed or being fed on artificially modified cows ' milk. In breast fed children, the upper part of the small intestine will usually contain enterococcus, streptococcus lacticus, and a general predominance of the coccoid form. Lower down toward and behind the ileocecal valves, the B. lactis aerogenes and the Colon bacilli appear. In the lower parts of the cecum and the rectum, the anaero- bic Bacillus bifidus of Tissier and similar anaerobes predominate, and many proteolytic bacteria may be present. In contrast to this, in artificially fed infants, the bowel is rela- tively richer in the Colon group, and the B. aerogenes type; B. mesentericus and other anaerobic spore formers will be present in considerable numbers, and in the lower bowel the B. bifidus types are largely replaced by Colon bacilli, B. acidophilus and similar organisms. Very early there may be also present in children a curious little tetanus-like organism spoken of as Bienstock's B. putrificus. Tissier, who has done a great deal of work on this problem, described the flora of a five year-old child in which the gradual transition from the milk to the mixed diet was taking place as follows. We quote from Kuster.17 "Constant fundamental flora, B. bifidus, enterococcus, Colon bacillus, B. acidophilus. Variable adventitious organisms, B. perfringens, cocci, and a number of other Gram-negative bacilli, together with some yeasts.'* As adult life is attained, there is a gradual relative increase of organisms of the Colon type, which eventually constitute about 75 per cent of the intestinal bacteria. In the normal adult the stomach is usually sterile. The duodenum contains a few cocci and Gram-positive and negative bacilli, not of the Colon type. There is a gradual numerical increase of bacteria downward. In the jejunum, or upper ileum, the Colon types begin 17 Kuster, Kolle and Wvssermann, 2nd, Edition, Vol. 6, p. 469. 226 BIOLOGY AND TECHNIQUE to grow numerous, and in the cecum and colon, which are the seat of the greatest bacterial activity, the flora consist chiefly of Colon types, B. mesentericus, a few anaerobic spore formers of the Welch bacillus type, and Gram-positive cocci. All who have studied this subject have found that diet has a definite and important bearing upon the intestinal flora and that definite changes may be brought about in the bacterial contents of the bowel by purposefully adjusting the diet. The studies of Herter16 and of Kendall/4 particularly, have been contributed to our knowledge of this subject. Herter has laid particular stress upon the importance of the Welch bacillus and its subvarieties upon intestinal putrefaction. In this he is not entirely in agreement with Eettger18 and others who believe that the Welch bacillus attacks proteins but slightly, being chiefly concerned with carbohydrate fermentation. Herter has produced indicanuria in dogs by feeding large amounts of meat, and found that with such feeding the colon and ileum contained considerable numbers of anaerobic bacilli. He also believes that this bacillus is particularly concerned with a chronic putrefactive activity which takes place in the large intes- tine, in the course of which anaerobic bacilli produce butyric acid. In consequence of this, there may be a considerable intestinal irrita- tion and carbohydrate intolerance. Considerable anuria may also be a consequence. Other writers like Friedman19 believe that con- stipation favors the increase of these putrefactive organisms. Simonds20 has made an exhaustive study of the relationship of the Welch bacillus group to intestinal conditions, and has reviewed the literature extensively. He summarizes his studies on this problem as follows: "In the case of gas bacillus diarrhea, the presence of an excess of carbohydrates in the intestinal content brings about conditions in the lower ileum and first part of the colon which are particularly conducive to the growth of B. Welchii. The absence of lactic acid producing bacteria, as pointed out by Kendall, renders conditions still more favorable to the multiplication of these or- ganisms. They, therefore, rapidly increase in numbers, produce irritating butyric acid, and are swept on in excessive numbers into the lower bowel. The number of spores produced will be measurably 18 Eettger, Jour, of Biol. Chem., 2, 1906, 71. 10 Friedman, Transac. of Chicago Pathol. Soc., 1901, cited from Simonds. 20 Simonds, Monograph of Rock. Institute, No. 5, 1915. BACTERIOLOGICAL EXAMINATION OF MATERIAL 227 proportional to the number of bacilli which reach the lower part of the bowel; hence, the excessive number of spores "of Bacillus Welchii in the stools in cases of gas bacillus diarrhea." Simonds' results substantiate the work of Kendall and Day21 to the effect that children and adults with diarrhea who showed large numbers of0 gas bacilli in the stools are made worse by feeding sugars, and that prompt improvement results when the diet is changed to one largely composed of protein. An absence of lactic acid by the feed- ing of butter-milk still further aids in eliminating the Welch bacillus. Kendall has shown by prolonged experimentation on monkeys, dogs and cats that feeding with cows' milk, to which sufficient lactose has been added to simulate breast milk, produces a bacterial flora in such animals which approaches that of the normal nursing infant. The stools take on an acid reaction, and organisms like B. bifidus and the Enterococcus begin to predominate. In order to bring this about, he states, it is necessary to continue the feeding for consider- able periods. Kendall divides the pathological cases in which it can be reasonably suspected that abnormal bacterial conditions of the intestinal tract play a causative part, into those which are due to the action of the bacteria upon proteins, and those in which it is chiefly a matter of carbohydrate fermentation. In the case of the abnormal proteolytic processes, there may be a liberation of substances like histamin and other toxic amines, and there may even be the formation of specific toxins such as those which have been recently produced by Bull and others from the Welch bacillus. Abnormal carbohydrate splitting' may result in hyperacidity and in stasis of the bowel, and secondary putrefaction in consequence of this. It is due to studies like those of the writers mentioned above that we may hope to be able to exert considerable therapeutic in- fluence upon abnormal intestinal conditions by altering the flora of the intestine, on the one hand by controlling the diet, and on the other hand by inoculating with, or, in other words, feeding bacteria of a type which may correct the condition that exists. A detailed study of these therapeutic measures cannot be given in tliis place. They are treated of in articles like those of Coleman and Shaffer,22 in Kendall's book from which we have quoted freely, 21 Kendall and- Day, Boston Med. and Surg. Jour., 1911, 741 and 1912, 753. "Coleman and Shaffer, Archiv. Inter. Med., Vol. 4, 1909. 228 BIOLOGY AND TECHNIQUE and in the publications of Herter, Kendall, Rettger, Torrey23 and others. BACTERIA IMPORTANT BECAUSE OF THEIR FREQUENT PRESENCE IN THE INTESTINAL CHANNEL Bacillus Acidophilus.2* — A Gram-positive bacillus which easily undergoes granular degeneration and grows under conditions of acidity, not supported by most other bacteria. It is closely related to the Bacillus bulgaricus, does not form spores, and is closely related to a group of similar organisms which have not been suffi- ciently studied to be conclusively classified. (See Kendall, Jour. Med. Res., 1910, 22, and Rahe, Jour. Infec. Dis., 15, 1914, 41.) Its isolation can, according to Kendall, best be accomplished by in- oculating the original material into dextrose broth containing 0.25 per cent acetic acid. Two or three transfers on a medium like this after intervals of several days will give pure cultures. Does not liquefy gelatin, has been associated by Escherich with acute diarrhea in children. Does not produce gas. Bacillus AcidopMlus Aerogenes. — Described by Torrey and Rahe25 which closely resembles the Bacillus acidophilus, except that it produces gas in mono-, di- and some polysaccharids. Bacillus Bifidus of Tissier. — This is a strictly anaerobic bacillus. It was described by Tissier in 1900. (See also, Noguchi, Jour. Exper. Med., 12, 1910, 182.) It is spoken of as Bifidus because it will often show a bifid branching at the ends, a condition in which it is not shown in smears from the intestinal contents. It is found early in the stools of nursing children. It produces considerable amounts of acid, but no gas from carbohydrates. Morphologically this organism is often described as Gram-positive, but, as a matter of fact, many members are Gram-negative, and often the bacilli themselves may be Gram-negative with Gram-positive granules scat- tered through them. Bacillus Mesentericus. — This is a Gram-positive, aerobic, spore bearing bacillus which is active proteolytic and concerned with putrefaction in the intestinal channel. The organism is more closely 83 Torrey, Jour, of Infec. Dis., 16, 1915, 72. 84 Moro, Wien. klin. Woch., 5, 1900 ; Finkelstein, Deut. med.. Woch., 22, 1900. 25 Torrey and Rahe, Jour, of Infec. Dis., 17, 1915, 437, BACTERIOLOGICAL EXAMINATION OF MATERIAL 229 related to the common hay bacillus or Subtilis. It is actively motile, forms spores and grows with great ease on the simplest media. It differs from the ordinary Subtilis bacillus in that it does not ferment dextrose. Kendall26 has shown that symbiosis of the B. Mesentericus with the Colon bacillus in milk will produce a greatly increased metabolism of both. Bacillus Putrificus of Bienstock.27 — This is a Gram-positive anaerobic bacillus which forms end spores, and, therefore, mor- phologically resembles the tetanus bacillus. It is identical probably with Klein's bacillus cadaveris sporogenes. It is capable of produc- ing powerful proteolytic cleavage in milk, cheese, etc. Bienstock believes that it is present constantly in normal feces. 88 Kendall, Boston Med. and Surg. Jour., 163, 1910. 27 Bienstock, Archiv. f. Hyg., 36, 335, 1899, and 39, 390, 1901. SECTION II INFECTION AND IMMUNITY CHAPTER XI FUNDAMENTAL FACTOKS OF PATHOGENICITY AND INFECTION WHEN microorganisms gain entrance to the animal or human body and give rise to disease, the process is spoken of as infection. Bacteria are ever present in the environment of animals and human beings and some find constant lodgment on various parts of the body. The mouth, the nasal passages, the skin, the upper respira- tory tract, the conjunctivas, the ducts of the genital system, and the intestines are invariably inhabited by numerous species of bac- teria, which, while subject to no absolute constancy, conform to more or less definite characteristics of species distribution for each locality. Thus the colon organisms are invariably present in the normal bowel, Doderlein's bacillus in the vagina, Bacillus xerosis in many normal conjunctivae, and staphylococcus, streptococcus, various spirilla, and pneumococcus in the mouth. In contact, there- fore, with the bodies of animals and man, there is a large flora of microorganisms, some as constant parasites, others as transient in- vaders ; some harmless saprophytes and others capable of becoming pathogenic. It is evident, therefore, that the production of an infec- tion must depend upon other influences than the mere presence of the microorganisms and their contact with the body, and that the occurrence of the reaction — for the phenomena of infection are in truth reactions between the germ and the body defenses — is governed by a number of important secondary factors. In order to cause infection, it is necessary that the bacteria shall gain entrance to the body by a path adapted to their own respective cultural requirements, and shall be permitted to proliferate after gaining a foothold. Some of the bacteria then cause disease by 230 FACTORS OF PATHOGENICITY AND INFECTION 231 rapid multiplication, progressively invading more and more exten- sive areas of the animal tissues, while others may remain localized at the point of invasion and exert their harmful action chiefly by local growth and the elaboration of specific poisons. The inciting or inhibiting factors which permit or prohibit an infection are dependent in part upon the nature of the invading germ and in part upon the conditions of the defensive mechanism of the subject attacked. Bacteria are roughly divided into two classes, saprophytes and parasites. The saprophytes are those bacteria which thrive best on dead organic matter and fulfill the enormously important function in nature of reducing by their physiological activities the excreta and dead bodies of more highly organized forms into those simple chemical substances which may again be utilized by the plants in their constructive processes. The saprophytes, thus, are of extreme importance in maintaining the chemical balance between the animal and plant kingdoms. Parasites, on the other hand, find the most favorable conditions for their development upon the living bodies of higher forms. While a strict separation of the two divisions cannot be made, numerous species forming transitions between the two, it may be said that the latter, class comprises most of the so-called pathogenic or disease-producing bacteria. Strict saprophytes may cause dis- ease, but only in cases where other factors have brought about the death of some part of the tissues, and the bacteria invade the necrotic areas and break down the proteins into poisonous chemical substances such as ptomains, or through their own destruction give rise to the liberation of toxic constituents of their bodies. It Is necessary, therefore, that bacteria, in order to incite disease, should belong strictly or facultatively to the class known as parasitic. It must not be forgotten, however, that the terms are relative, and that bacteria ordinarily saprophytic may develop parasitic and patho- genic powers when the resisting forces of the invaded subject are reduced to a minimum by chronic constitutional disease or other causes. Organisms that are parasitic, however, are not necessarily patho- genic, and there are certain more or loss fundamental requirements which experience has taught us must be met by an organism in order that it may be infectious (or pathogenic) for any given animal; and by infectiousness is meant the ability of an organism 232 INFECTION AND IMMUNITY to live and multiply in the animal fluids and tissues. For instance, an organism which is shown not 'to grow at the body temperature •of warm-blooded animals may safely be assumed not to be infectious for such animals; and experience is gradually teaching us that strictly aerobic organisms, those thriving only in the presence of free oxygen and not able to obtain this gas in available combination from carbohydrates, can also be safely excluded from the infectious class. We have also learned that anaerobic organisms, although infectious when gaining entrance to tissues not abundantly supplied with blood, are practically unable to multiply in the blood stream and give rise to generalized infection. The pathogenic microorganisms differ very much among them- selves in the degree of their disease-inciting power. Such power is known as virulence. Variations in virulence occur, not only among different species of pathogenic bacteria, but may occur within the same species. Pneumococci, for instance, which have been kept upon artificial media or in other unfavorable environment for some time, exhibit less virulence than when freshly isolated from the bodies of man or animals. It is necessary, therefore, in order to produce infec- tion, that the particular bacterium involved shall possess sufficient virulence. Whether or not infection occurs depends also upon the number of bacteria which gain entrance to the animal tissues. A small number of bacteria, even though of proper species and of sufficient virulence, may easily be overcome by the first onslaught of the defensive forces of the body. Bacteria, therefore, must be in sufficient number to overcome local defenses and to gain a definite foothold and carry on their life processes, before they can give rise to an infection. The more virulent the germ, other conditions being equal, the smaller the number necessary for the production of disease. The introduc- tion of a single individual of the anthrax species, it is claimed, is often sufficient to cause fatal infection ; while forms less well adapted to the parasitic mode of life will gain a foothold in the animal body only after the introduction of large numbers. The Path of Infection.— The portal by which bacteria gain en- trance to the human body is of great importance in determining whether or not disease shall occur. Typhoid bacilli rubbed into the abraded skin may give rise to no reaction of importance, while the same microorganism, if swallowed, may cause fatal infection. Conversely, virulent streptococci, when swallowed, may cause no FACTORS OF PATHOGENICITY AND INFECTION 233 harmful effects, while the same bacteria rubbed into the skin may give rise to a severe reaction. Animals and man are protected against invasion by bacteria in various ways. Externally the body is guarded by its coverings of skin and mucous membranes. When these are healthy and undis- turbed, microorganisms are usually held at bay. While this is true in a general way bacteria may in occasional cases pass through uninjured skin and mucosa. Thus the Austrian Plague Commission found that guinea-pigs could be infected when plague bacilli were rubbed into the shaven skin, and there can hardly be much doubt of the fact that tubercle bacilli may occasionally pass through the intestinal mucosa into the lymphatics without causing local lesions. Even after bacteria of a pathogenic species, in large numbers and of adequate virulence, have passed through a locally undefended area in the skin or mucosa of an animal or a human being by a path most favorably adapted to them, it is by no means certain that an infection will take place. The bodies of animals and of man have, as we shall see, at their disposal certain general, systemic weapons of defense, both in the blood serum and the cellular ele- ments of blood and tissues which, if normally vigorous and active, will usually overcome a certain number of the invading bacteria. If these defenses are abnormally depressed, or the invading micro- organisms are disproportionately virulent or plentiful, infection takes place. Bacteria, after gaining an entrance to the body, may give rise merely to local inflammation, necrosis, and abscess formation. They may, on the other hand, from the local lesion, gain entrance into the lymphatics and blood-vessels and be carried freely into the circula- tion, where, if they survive, the resulting condition is known as bacteremia or septicemia. Carried by the blood to other parts of fhe body, they may, under favorable circumstances, gain foothold in various organs and give rise to secondary foci of inflammation, necro- sis, and abscess formation. Such a condition is known as pyemia. The disease processes arising as the result of bacterial invasion may depend wholly or in part upon the mechanical injury produced by the process of inflammation, the disturbance of function caused by the presence of the bacteria in the capillaries and tissue spaces, and the absorption of the necrotic products resulting from the reaction between the body cells and the microorganisms. To a large extent, however, infectious diseases are characterized by the symptoms result- 234 INFECTION AND IMMUNITY ing from the absorption or diffusion of the poisons produced by the bacteria themselves. Bacterial Poisons. — It was plain, even to the earliest students of this subject, that mere mechanical capillary obstruction or the absorption of the products of a local inflammation were insufficient to explain the profound systemic disturbances which accompany many bacterial infections. The very nature . of bacterial disease, therefore, suggested the presence of poisons. It was in his investigations into the nature of these poisons that Brieger1 was led to the discovery of the ptomains. These bodies, first isolated by him from decomposing beef, fish, and human cadavers, have found more extended discussion in another section. Accurately classified, they are not true bacterial poisons in the sense in which the term is now employed. Although it is true that they are produced from protein material by bacterial action, they are cleavage products derived from the culture medium upon the composition of which their nature intimately depends. The bacterial poisons proper, on the other hand, are specific products of the bacteria themselves, dependent upon the nature of the medium only as it favors or retards the full development of the physiological functions of the micro- organisms. The poisons, produced to a greater or lesser extent by all pathogenic microorganisms, may be of several kinds. The true toxins, in the specialized meaning which the term has acquired, are soluble, truly secretory products of the bacterial cells, passing from them into the culture medium during their life. They may be obtained free from the bacteria by filtration and in a purer state from the filtrates by chemical precipitation and a variety of other methods. The most important examples of such poisons are those elaborated by Bacillus diphtherias and Bacillus tetani. If cultures of these bacteria or of others of this class are grown in fluid media for several days and the medium is then filtered through porcelain candles, the filtrate will be found toxic often to a high degree, while the residue will be either inactive or comparatively weak. Moreover, if the residue possesses any toxicity at all, the symptoms evidencing this will be different from those produced by the filtrate. There are other microorganisms, however, notably the cholera spirillum and the typhoid bacillus, in which no such exotoxins are formed. If these bacteria are cultivated and separated from the cul- 1 Brieger, "Die Ptomaine," Berlin, 1885 and 1886. FACTORS OF PATHOGENICITY AND INFECTION 235 ture fluid by filtration, as above, the fluid filtrate will be toxic to only a very slight degree, whereas the residue may prove very poison- ous. In these cases, we are dealing, evidently, with poisons not secreted into the medium by the bacteria, but rather attached more or less firmly to the bacterial body. Such poisons, separable from the bacteria only after death by some method of extraction, or by autolysis, were termed by Pfeiffer endotoxins. The greater number of the pathogenic bacteria seem to act chiefly by means of poisons of this class. The first to call attention to the existence of such intracellular poisons was Buchner, who formulated his conclusions from the results of experiments made with a number of microorganisms, notably the Friedlander bacillus and Staphylococcus pyogenes aureus, with dead cultures of which he induced the formation of sterile abscesses in animals and symptoms of toxemia. The conception of "endotoxins," received its clearest and most definite expression in the work of Pfeiffer2 on cholera poison. Some clarity of conception, based on visual perception, may pos- sibly be gained by comparing some of the products of pathogenic bacteria with bacterial pigments and with insoluble interstitial or intercellular substance, which may be seen accompanying bacteria in cover-glass preparations. Soluble toxic secretions are to be compared to such pigments as the pyocyanin of Bacillus pyocyaneus, which is so readily soluble in culture media ; endotoxins proper, to pigments confined to the bacterial cell, or at least, when secreted, being insoluble in culture media, such for instance as the well-known red pigment of Bacillus prodigiosus, which may often be seen free among the bacteria in irregular red granules like carmine powder. That bodies such as this latter might be extruded from pathogenic bacteria and not be soluble in the usual culture fluids, is not improbable, and the fact that more or less insoluble interstitial substances are not infre- quent among bacteria is well known. In all bacterial bodies, after removal of toxins and endotoxins, a certain protein residue remains which, if injected into animals, may give rise to localized lesions such as abscesses or merely slight temporary inflammations. The nature of this residue has been carefully studied, especially by Buchner, who has named it bacterial protein and he believes the substance to be approximately the same in all bacteria, without specific toxic action, but with a general ability to exert a 2 Pfeiffer, Zeit. f. Hyg., xl, 1892. 236 INFECTION AND IMMUNITY positive chemotactic effect on the white blood cells, thereby causing the formation of pus. The nature of the bacterial proteins is by no means clear, and it is still in doubt whether the separation of these substances from the endotoxins can be upheld. A number of bacteria may give rise to both varieties of poisons. Thus, recently, Kraus has claimed the discovery of a soluble toxin for the cholera spirillum and Doerr for the dysentery bacillus, both of which microorganisms were regarded as being purely of the endotoxin-producing type. It is plain, moreover, that occasionally it may be very difficult to distinguish between a soluble toxin and an endotoxin. In the filtration experiment recorded above, it might well be claimed that the toxicity of the filtrate, when not very strong, may depend upon an extraction of endotoxins from the bodies of the bacteria by the medium. The final test, in such instances, lies in the power of true toxins to stimulate in animals the production of antitoxins; for, as we shall see later, the injection of true soluble toxins into animals gives rise to antitoxins, whereas the formation of such neutralizing bodies in the serum or plasma does not, it is claimed, follow the injection of endotoxins. We could spend much time in analyzing the literature on the exotoxin and endotoxin, and this, of course, would be important were we attempting in this book to cover completely immunological problems. When all is said and done, however, the present status of the question is as follows: Certain bacteria, like the diphtheria bacillus, the tetanus bacillus, B. botulinus, some of the anaerobes of surgical infections, etc., produce secretory products during life which are highly toxic, can be obtained during the life of the culture by simple filtration, and which incite, in carefully treated animals, specific neutralizing substances, or antitoxins, which neu- tralize the action of the toxin, roughly according to the law of mul- tiples. These antitoxins in the serum, therefore, can be shown definitely to prevent the injury of the animal by the toxin. In many bacteria such soluble toxins cannot be demonstrated. Older bacterial culture filtrates and the bodies of the bacteria may be highly toxic, as in the case of typhoid, cholera, and practically all the Gram-negative organisms, but these toxic substances are derived either from direct extraction of the bacterial bodies, or, as claimed by some, may represent split products produced in the course of decomposition or cleavage of the bacterial protein. These FACTORS OF PATHOGENIOITY AND INFECTION 2,37 substances, called by Pfeiffer endotoxins, do not produce antitoxins. It is doubtful, in our minds, whether they may be regarded as strictly specific in all cases. In addition to these poisonous substances, the writer, with Kutt- ner and Parker,3 has recently obtained non-specific toxic substances for a great many different bacteria (streptococci, typhoid bacilli, influenza bacilli, etc.) which appeared in cultures as early as 6 to 18 hours, could be obtained by filtration, and could also be obtained by washing young cultures on solid media with salt solution and filtering. These substances are not unlikely similar to those en- countered by some other writers who have interpreted them as true exotoxins, but, as far as we can determine, they are neither specific nor antigenic. Nevertheless, they are regular in appearance, suffi- ciently potent to make rabbits very sick, though rarely to kill them, and must be taken into account in all work in which the toxic substances of bacteria are studied by the usual methods. We can speak of them, for want of more accurate definition, as bacterial "X" substances. In resistance to chemical action and heat, the various poisons show widely divergent properties. As a general rule, most true soluble toxins are delicately thermolabile, they are destroyed by moderate heating, and deteriorate easily on standing. Their chemical nature is by no means clear, but, on precipitation of toxic solutions with magnesium sulphate, these poisons come down together with the globulins. The nature of the "endotoxins" is still less clearly understood. Most of them are far less liabile than the extracellular poisons. Some powerful intracellular poisons, like those of the Gartner bacillus of meat poisoning and the poison attached to the bodies of typhoid bacilli may undergo exposure to even 100° C. and still retain their toxic properties. The nature of each individual poison will be discussed in connection with its microorganism. It should be remembered, moreover, by those studying bacterial poisons that recent investigations have shown that a number of bacteria (coli, influenza, etc.) may produce substances either identical with or closely related to histamin and tyramin, on pepton media, after five or more days of growth. 97insftcr, Parker and Kuttner, Transact. Soc. for Exp. Biol. and Mod., Jan., 1921. 238 INFECTION AND IMMUNITY The Made of Action of Bacterial Poisons. — Close study of the toxic products of various microorganisms has shown that many of the bacterial poisons possess a more or less definite selective action upon special tissues and organs. Thus, certain soluble toxins of the tetanus bacillus and Bacillus botulinus attack specifically the nervous system. Again, certain poisons elaborated by the staphylococci, the tetanus bacillus, the streptococci, and other germs, the so-called 1 ' hemolysins, ' ' attack primarily the red blood corpuscles. Other poisons again act on the white blood corpuscles ; in short, the char- acteristic affinity of specific bacterial poisons for certain organs is a widely recognized fact. In explanation of this behavior, much aid has been given by the researches of Meyer,4 Overton,5 Ehrlich,6 and others upon the causes for the analogous selective behavior of various narcotics and alka- loids. It seems probable, from the researches of these men, that the selective action of poisons depends upon the ability, chemical or physical or both, of the poisons to enter into combination with the specifically affected cells. From the nature of the combinations formed, it seems not unlikely that the physical factors, such as solubility in the cell plasma, may also play an important part. Observations of a more purely bacteriological nature have tended to bear out these conclusions. Wassermann and Takaki,7 for in- stance, have shown that tetanus toxin, which specifically attacks the nervous system, may be removed from solution by the addition of brain substance. Removal of the brain tissue by centrifugation leaves the solution free from toxin. In the same way it has been shown that hemolytic poisons can be removed from solutions by contact with red blood cells, but only when the red blood cells of susceptible species are employed. Similar observations have been made in the case of leukocidin, a bacterial poison acting upon the white blood cells specifically.8 That bacterial poisons injected into susceptible animals rapidly disappear from the circulation is a fact which bears out the view that a combination between affected tissue and toxin must take * Meyer, Arch. f. exper. Pathol., 1899, 1901. * Overton, "Studien iib. d. Narkose," Jena, 1901. 9 Ehrlich, ' ' Sauerstoffs-Bediirfniss des Organismus," Berlin, 1885. 7 Wassermann und Takaki, Berl. klin. Woch., 1898. 9 Sachs, Hof meister 's Beitrage, 11, 1902. ' FACTORS OF PATHOGENICITY AND INFECTION 239 place. Db'nitz,9 for instance, has shown that within four to eight minutes after the injection of certain toxins, considerable quantities will have disappeared from the circulation. Conversely, Metchni- koff10 has observed that tetanus toxin injected into insusceptible animals (lizards) may be detected in the blood stream for as long as two months after administration. 9 Ddnitz, Deut. mod. Woch., 1897. 10Metchnikoff, "L'immunite dans les malad. infect." CHAPTER XII DEFENSIVE FACTOKS OF THE ANIMAL ORGANISM GENERAL CONSIDERATIONS WE have seen that the mere entrance of a pathogenic microor- ganism into the human or animal body through a breach in the con- tinuity of the mechanical defenses of skin or mucosa does not neces- sarily lead to the development of an infection. The opportunities for such an invasion are so numerous, and the contact of members of the animal kingdom with the germs of disease is so constant, that if this were the case, sooner or later all would succumb. It is plain, therefore, that the animal body must possess more subtle means of defense, by virtue of which pathogenic germs are, even after their entrance into the tissues and fluids, disposed of, or at least prevented from proliferating and elaborating their poisons. The power which enables the body to accomplish this is spoken of as resistance. When this resistance, which in some degree is common to all members of the animal kingdom, is especially marked, it is spoken of as " im- munity. ' ' From this it follows naturally that the terms resistance and im- munity, as well as their converse, susceptibility, are relative and not absolute terms. Degrees of resistance exist, which are determined to a certain extent by individual, racial, or species peculiarities ; and persons or animals are spoken of as immune when they are unaffected by an exposure or an inoculation to which the normal average in- dividual of the same species would ordinarily succumb. The word does not imply, however, that these individuals could not be infected with unusually virulent or large doses, or under particularly unfavor- able circumstances. Thus, birds, while immune against the ordinary dangers of tetanus bacilli, may be killed by experimental inoculations with very large doses of tetanus toxin.1 Similarly, Pasteur rendered 'Quoted from Abel, Kolle und Wassermann, "Handbuch," etc. 240 DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 241 naturally immune hens susceptible to anthrax by cooling them to a subnormal temperature, and Canalis and Morpurgo did the same with doves by subjecting them to starvation. Absolute immunity is exceedingly rare. The entire insusceptibility of cold-blooded animals (frogs and turtles) under normal conditions to inoculation with even the largest doses of many of the bacteria pathogenic for warm-blooded animals, and the immunity of all the lower animals against leprosy, are among the few instances of absolute immunity known.2 Apart from such exceptional cases, however, re- sistance, immunity, and susceptibility must be regarded as purely relative terms. The power of resisting any specific infection may be the natural heritage of a race or species, and is then spoken of as natural im- munity. It may, on the other hand, he acquired either accidentally or artificially by a member of an ordinarily susceptible species, and is then called acquired immunity. Natural Immunity. — SPECIES IMMUNITY. — It is well known that many of the infectious diseases which commonly affect man, do not, so far as we know, occur spontaneously in animals. Thus, infection with B. typhosus, the vibrio of cholera, or the meningococcus occurs in animals only after experimental inoculation. Gonorrheal and syphilitic infection, furthermore, not only does not occur spontane- ously, but is produced experimentally in animals with the greatest difficulty — the consequent diseases being incomparably milder than those occurring in man. Other diseases, like leprosy, influenza, and the exanthemata,3 have never been sucessfully transmitted to animals. Conversely, there are diseases among animals which do not spon- taneously attack man. Thus, human beings enjoy immunity against Rinderpest, and, to a lesser degree, against chicken cholera. Among animal species themselves great differences in susceptibility and resistance toward the various infections exist. Often-quoted examples of this are the remarkable resistance to anthrax of rats and dogs, and the immunity of the common fowl against tetanus. The factors which determine these differences of susceptibility and resistance among the various species are not clearly understood. It has been suggested that diet in some instances may influence these relations, inasmuch as carnivorous animals are often highly resistant 2 Lubarsch, Zeit. f. klin. Medi/., xix. 'With the possible exception of smallpox. 242 INFECTION AND IMMUNITY to glanders, anthrax, and even tuberculous infections, to which herbiv- orous animals are markedly susceptible.4 It is likely, too, that the great differences between animals of various species in their metab- olism, temperature, etc., may call for special cultural adaptation on the part of the bacteria. The fact that the bacillus of avian tuber- culosis— whose natural host has a normal body temperature of 40° C. and above — will grow on culture media at 40 to 50° C., whereas B. tuberculosis of man can not be cultivated at a temperature above 40° C., would seem to lend some support to this view. The difference between warm- and cold-blooded animals has already been noted. The necessity for cultural adaptation, too, would seem to be borne out by the great enhancement observed in the virulence of certain micro- organisms for a given species after repeated passage through in- dividuals of this species. RACIAL IMMUNITY. — Just as differences in susceptibility and im- munity exist among the various animal species, so the separate races or varieties within the same species may display differences in their reactions toward pathogenic germs. Algerian sheep, for instance, show a much higher resistance to anthrax than do our own domestic sheep, and the various races of mice differ in their susceptibility to anthrax and to glanders. Similar racial differences are common among human beings. As a general rule, it may be said that a race among whom a certain disease has been endemic for many ages is less susceptible to this dis- ease than are other races among whom it has been more recently introduced. The appalling ravages of tuberculosis among negroes, American Indians, and Esquimaux, bear striking witness to this fact. Conversely, the comparative immunity of the negro from yellow fever, a disease of the greatest virulence for Caucasians, furnishes further evidence in favor of this opinion. It must not be forgotten, however, in judging of these relations, that the great differences in the customs of personal and social hygiene existing among the various races may considerably affect the transmission of disease and lead to false con- clusions. In so far as the statement made above is true, however, it seems to indicate that the endemic diseases have carried in their train a certain .degree of inherited immunity. ' Hahn, in Kolle und Wassermann, vol. iv. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 243 In other cases5 — as in. the instance of the malaria-immunity of negroes — the resistance seems to be acquired rather than inherited, for, as Hirsch was first to note, death from this disease occurred frequently among the children, while adult negroes were rarely attacked. DIFFERENCES IN INDIVIDUAL RESISTANCE. — In bacteriological ex- perimentation with smaller test animals, a direct ratio may often exist between body weight and dosage in determining the outcome of an infection, provided the mode of inoculation has been the same and the virulence of the germ not excessive. It would seem, therefore, that among these animals the difference in resistance in the face of an artificial infection between individuals of the same race is very slight. In higher animals, however, especially in the case of man, the existence of such apparent individual differences is a well-established fact, although in judging of them we must not forget that the condi- tions of infection are not subject to the uniformity and control which animal experimentation permits. Of a number of persons exposed to any given infection there are always some who are entirely unaffected and there are great variations in the severity of the disease in those who are attacked. In the absence of positive evidence in support of the direct inheritance of this individual immunity, the most reasonable explanation for such differences in resistance seems to lie in attributing them to individual variations in metabolism or body chemistry. De- pressions, for instance, in the acidity of the gastric secretion would predispose to certain infections of gastro-intestinal origin. Anatomical differences, too, may possibly influence resistance. Thus, Birch-Hirsch- feld believed that certain anomalous arrangements of the bronchial tubes predisposed to tuberculosis. Instances of transient susceptibility induced by physical or mental overwork, starvation, etc., should hardly be classified under this head- ing, since the conditions in such cases correspond simply to experi- mental depression of natural species for race resistance. Acquired Immunity. — It is a matter of common experience that many of the infectious diseases occur but once in the same individual. This is notably the case with typhoid fever, yellow fever, and most of the exanthemata, and is too general an observation to require exten- sive illustration. A single attack of any of the diseases of this class 6 Halin, in Kolle und Wassermann, loc. cit. 244 INFECTION AND IMMUNITY alters in some way the resistance of the individual so that further exposure to the infective agent is usually without menace, either for a limited period after the attack, or for life. Resistance acquired in this way is often spoken of as acquired immunity. The protection conferred by certain diseases against further attack was recognized many centuries ago, and there are records which show that attempts were made in ancient China and India to inoculate healthy individuals with pus from small-pox pustules in the hope of producing by this process a mild form of the disease and its consequent immunity. Pasteur, before all others, thought philosophically about the phenomena of acquired immunity, and, with adequate knowledge, realized the possibility of artificially bestowing immunity without inflicting the dangers of the fully potent infective agent. The first observation which, made by him, purely accidentally, inspired the hope of the achievement of such a result, occurred during his experi- ments with chicken cholera. The failure of animals to die after inocula- tion with an old culture of the bacilli of chicken cholera, fully potent but a few weeks previously, pointed to the attenuation of these bacilli by their prolonged cultivation without transplantation. With this observation as a point of departure he carried out a series of investiga- tions with the purpose of discovering a method of so weakening or attenuating various incitants of disease that they could be introduced into susceptible individuals without endangering life and yet without losing their property of conferring protection. The brilliant results achieved by Jenner, many years before, in protecting against smallpox by inoculating with the entirely innocuous products of the pustules of cowpox furnished an analogy which gave much encouraging support to this prospect. The experimental work which Pasteur carried out to solve this problem not only reaped a rich harvest of facts, but gave to science the first and brilliant examples of the application of exact laboratory methods to problems of immunity. ACTIVE IMMUNITY Active Artificial Immunity. — The process of conferring protec- tion by treatment with either an attenuated form or a sublethal quan- tity of the infectious agent of a disease, or its products, is spoken of DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 245 Whatever the method employed, the immunized individuals gain their power of resistance by the unaided reactions of their own tissues. They themselves take an active physiological part in the acquisition of this new property of immunity. For this reason, Ehrlich has aptly termed these processes * ' active immunization. ' ' There are various methods by which this can be accomplished, all of which were, in actual application or in principle, discovered by Pasteur and his associates, and can be best reviewed by a study of their work. ACTIVE IMMUNIZATION WITH ATTENUATED CULTURES. — In the course of his experiments upon chicken cholera, as mentioned above, Pasteur6 accidentally discovered that the virulence of the bacilli of this disease was greatly reduced by prolonged cultivation upon artifi- cial -media. This was especially noticeable in broth cultures which had been stored for long periods without transplantation. By repeated injections of such cultures into fowl, he succeeded in rendering the animals immune against subsequent inoculations with lethal doses of fully virulent strains. During the same year, 1880, in which Pasteur published his ob- servations on chicken cholera, Toussaint7 succeeded in immunizing sheep against anthrax by inoculating them with blood from infected animals, defibrinated and heated to 55° C. for ten minutes. Toussaint wrongly believed, however, that the blood which had been used in his immunizations was free from living bacteria. In repeating this work Pasteur showed that the protection in Toussaint 's cases was conferred by living bacteria, the virulence of which had been reduced by their subjection to heat. In following out the suggestions offered by these experiments, Pasteur8 discovered that he could reduce the virulence of anthrax bacilli much more reliably than by Toussaint 's method, by cultivating the organisms at increased temperatures (42° to 43° C.). By this process of attenuation he was able to produce "vaccines" of roughly measurable strength, with which he succeeded in immunizing sheep and cattle. A successful demonstration of his discovery was made by him at Pouilly-le-Fort, soon after, upon a large number of animals and before a commission of professional men. 6 Pasteur, Compt. rend, do 1'acad. des sci., 1880, t. xc. 7 Toussaint, Compt. rend, de 1'acad. des sci., 1880, t. xei. * Pasteur, Chamberland et Eoux, Compt. rend, de 1'acad. des sci., 1881, t. xcii. 246 INFECTION A^D IMMUNITY It is a fact well known to bacteriologists that certain of the patho- genic microorganisms, when passed through several individuals of the same animal species, become gradually more virulent for this species. In his studies on the bacillus of hog cholera, Pasteur9 ob- served that when this microorganism was passed through the bodies of several rabbits it gained in virulence for rabbits, but became less potent against hogs. He succeeded, subsequently, in protecting hogs against fully virulent cultures by treating them with strains which had been attenuated by their passage through rabbits. A further principle of attenuation for purposes of immunization was, at about this time, contributed by Chamberland and Roux,10 who reduced the virulence of anthrax cultures by growing them in the presence of weak antiseptics (carbolic acid 1:600, potassium bichromate 1:5,000, or sulphuric acid 1:200). Cultivated under such conditions the bacilli lost their ability to form spores and became entirely avirulent for sheep within ten days. A similar result was later obtained by Behring11 when attenuating B. diphtherias cultures by the addition of terchlorid of iodin. ACTIVE IMMUNIZATION WITH SUBLETHAL DOSES OF FULLY VIRULENT BACTERIA. — The use of fully virulent microorganisms in minute quan- tities for purposes of immunization was first suggested by Chauveau,12 and is naturally inapplicable to extremely virulent organisms like B. anthracis. The principle, however, is perfectly valid, and has been experimentally applied by many observers, notably by Ferran13 in the case of cholera. A similar method proved of practical value in the hands of Theobald Smith and Kilborne14 in prophylaxis against the protozoan disease, Texas fever. ACTIVE IMMUNIZATION WITH DEAD BACTERIA. — Suggested by Chau- veau, the method of active immunization with gradually increasing doses of dead microorganisms has been successfully employed by various observers, chief among whom are Pfeiffer, Brieger, Wright, and Wassermann. The method is especially useful against that class of bacteria in which the cell bodies (endotoxins) have been found to 9 Pasteur, Compt. rend, de 1'acad. des sci., 1882, t. xcv. 10 CJiamberland et Eoux, Compt. rend, de 1'acad. dcs sci., 1882, t. xcvi. u Behring, Zcit. f. Hyg., xii, 1892. ™Chauveau, Compt. rend, de Pacad. des sci., 1881, t. xcii. "Ferran, Compt. rend, de 1'acad. des sci., 1895, t. ci. 14 Th. Smith and Kilborne, U. S. Dept. of Agri., Bureau of Ani. Indust., Wash., 1893. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 247 be incomparably more poisonous than their extracellular products (toxins) . From a practical point of view, the method is of the greatest importance in routine laboratory immunization against B. typhosus, Vibrio cholera? asiaticse, B. pestis, and a number of other bacteria. In the therapy of human disease, this method has recently come into great prominence, chiefly through the work of Wright, whose inves- tigations will be more fully discussed in a subsequent section. ACTIVE IMMUNIZATION WITH BACTERIAL PRODUCTS. — Many bacteria when grown in fluid media produce extracellular, soluble poisons which remain in the medium after the microorganisms have been removed by filtration or centrifugalization. Since the diseases caused by such microorganisms are, to a large extent, due to the soluble poisons excreted by them, animals can be actively immunized against this class of bacteria by the inoculation of gradually increasing doses of the specific poison or toxin. This method is naturally most successful against those microorganisms which possess the power of toxin forma- tion to a highly developed degree. Most important among these are B. diphtherias and B. tetani. The first successful application of this principle of active immunization, however, was made by Salmon and Smith15 in the case of hog cholera. PASSIVE IMMUNITY In Pasteur's basic experiments, as in those of the other scientists who followed in his footsteps, the methods of immunization were based upon the development of a high resistance in the treated subject by virtue of its own physiological activities. This process we have spoken of as "active immunization" and it is self-evident that a method of this kind can, in the treatment of disease, be employed prophylactically only against possible infection, or in localized acute infections, or at the beginning of a long period of incubation before actual symptoms have appeared, as in rabies or in chronic conditions in which the infection is not of a severe or acute nature. A new and therapeutically more hopeful direction was given to the study of immunity when, in 1890 and 1892, v. Behring and his collaborators discovered that the sera of animals immunized against 15 Salmon and Smith, Eep. of Com. of Agri., Wash., 1885 and 1886. 248 INFECTION AND IMMUNITY the toxins of tetanus16 and of diphtheria17 bacilli would protect normal animals against the harmful action of these poisons. The animals thus protected obviously had taken no active part in their own defense, but were protected from the action of the poison by the substances transferred to them in the sera of the actively immunized animals. Such immunity or protection, therefore, is a purely passive phenom- enon so far as the treated animal is concerned, and the process is for this reason spoken of as "passive immunization." Passive immunization of this description is practically applicable chiefly against diseases caused by bacteria which produce powerful toxins, and the sera of animals actively immunized against such toxins are called antitoxic sera. In the treatment of the two diseases men- tioned above, diphtheria and tetanus, the respective antitoxic sera have reached broad and beneficial therapeutic application, and in- numerable lives have been saved by their use. Passive immunization against microorganisms not characterized by marked toxin formation was attempted, even before Behring's discovery, by Richet and Hericourt,18 experimenting with cocci, and by Babes,19 in the case of rabies ; and the underlying thought had been the basis of Toussaint's work upon anthrax. Microorganisms, however, which exert their harmful action rather by the contents of the bacterial cells than by secreted, soluble toxins, do not, so far as is known, produce antitoxins in the sera of immunized animals. The substances which they call forth in the process are directed against the invading organisms themselves in that they possess the power of destroying or of causing dissolution of the specific germs used in their production. Such antibacterial sera are extensively used in the laboratory in the passive immunization of animals against a large number of germs, and are fairly effectual when used before, at the same time with, or soon after, infection. Their therapeutic use in human disease, however, has, up to the present time, been disappointing and their prophylactic and curative action has been almost invariably ineffectual or feeble at best, except when the antibacterial sera could be brought in direct contact with the germs, in closed cavities or localized lesions. 16 v. Bearing and Kitasato, Dent. med. Woeh., 49, 1890. 17 v. Bearing and Wernicke, Zeit. f . Hyg., 1892. 18 Richet et Hericourt, Compt. rend, de Paead. dos sei., 1888. 19 Babes et Lepp, Ann. de 1'inst. Pasteur, 1889. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 249 Thus, in epidemic meningitis, such sera have proved extremely useful in the hands of Flexner, when injected directly into the spinal canal. ANTIBODIES AND THE SUBSTANCES GIVING RISE TO THEM In the foregoing sections we have seen that the process of active immunization so changes the animal body that it becomes highly resistant against an infection to which it had formerly in many in- stances been delicately susceptible. In the absence of visible anatomical or histological changes accompanying the acquisition of this new power, investigators, in order to account for it, were led to examine the physiological properties of the body cells and fluids of immunized subjects. While it was reasonable to suppose that all the cells and tissues were affected by, or might have taken part in, a physiological change so profoundly influencing the individual, the blood, because of its unquestionably close relation to inflammatory reactions, and because of the ease with which it could be obtained and studied, claimed the first and closest attention. The bactericidal properties or normal blood serum noted in 1886 by Nuttall,20 v. Fodor,21 and Fliigge, moreover, aided in pointing to this tissue as primarily the seat of the immunizing agents. It is an interesting historical fact, that, long before this time, the English physician Hunter had noted that blood did not decompose so rapidly as other animal tissues. The study of the blood serum of immunized animals as to simple changes in chemical composition or physical properties has shed little light upon the subject. Beljaeff22 in a recent investigation found little or no alteration from the normal in the blood sera of immunized animals as to index of refraction, specific gravity, and alkalinity. Joachim23 and Moll agree in stating that immune blood serum is com- paratively richer in globulin than normal serum. Similar observations had been made by Hiss and Atkinson24 and others. Important and significant as these purely chemical observations are, they have helped little in explaining the nature of the processes going on in immune sera. The first actual light was thrown upon the mysterious phenomena 20 Nuttall, Zeit. f. Hyg., i, 1886. 21 v. Fodor, Deut. med. Woch., 1886. "Beljaeff, Cent. f. Bakt., xxxiii. 23 Joachim, Pfliigers Archiv, xciii. -4Hiss and Atkinson, Jour. Expcr. Med., v, 1900. 250 INFECTION AND IMMUNITY of immunity by the investigations of Nuttall,25 v. Fodor, Buchner, and others, who not only demonstrated the power of normal blood serum to destroy bacteria, but also showed that this property of blood serum became diminished with age and was destroyed completely by heating to 56° C. The thermolabile substance of the blood serum possessing this power was called by Buchner,26 alexin. Soon after this work, Behring, in collaboration with Kitasato27 and Wernicke,28 in 1890 and 1892, made further important advances in the elucidation of the immunizing processes by showing that the blood sera of animals actively immunized against the toxins of diphtheria and tetanus would protect normal animals against the poisons of these diseases. He believed, at the time of discovery, that such sera contained substances which had the power of destroying the specific toxins which had been used in the immunization. He called these bodies antitoxins. While Behring 's first conception of actual toxin destruction soon proved to be erroneous, his discovery of the presence in immune sera of bodies specifically antagonistic to toxins was soon confirmed and extended, and stands to-day as an established fact. Ehrlich,29 soon after Behring 's announcement, showed that specific antitoxins could also be produced against the poisons of some of the higher plants antiricin, antikrotm, antirobin), and Calmette30 pro- duced similar 'antitoxins against snake poison (antivenin). Stimulated by these researches, other observers have, since then, added exten- sively to the list of poisons against which antitoxins can be produced. Kempner31 has produced antitoxin against the poison of Bacillus botulinus, and Wassermann,32 against that of Bacillus pyocyaneus. Antitoxin has been produced by Calmette33 against the poison of the scorpion, and by Sachs34 against that of the spider. Thus a large number of poisons of animal, plant, or bacterial origin have been found capable of causing the production of specific antibodies in the sera of animals into which they are injected. 25 Nuttall, Zeit. f. Hyg., 1886. 26 Buchner, Cent. f. Bakt., i, 1889. 27 Behring und Kitasato, Deut. med. Woch., 1890, No. 49. 28 Behring und Wernicke, Zeit. f. Hyg., 1892. ^Ehrlich, Dent. med. Woch., 1891. ^Calmette, Compt. rend, de la soe. de biol., 1894. 31 Kempner, Zeit. f. Hyg., 1897. *•' Wassermann, Zeit. f. Hyg., xxii. 83 Calmette, Ann. de 1 'inst. Pasteur, 1898. 34 Sachs, Hofm. Beit., 1902. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 251 The formation of antitoxins directed against soluble poisons, how- ever, did not explain the immunity acquired by animals against bacteria like Bacillus anthracis, the cholera vibrio, and others which, unlike diphtheria and tetanus, produced little or no soluble toxin. It was evident that the antitoxic property of immune blood serum was by no means the sole expression of its protective powers. Much light was shed upon this phase of the subject by the discoveries of Pfeiffer in 1894, who worked along the lines suggested by the inves- tigations of Nuttall and Buchner. Pfeiffer35 showed that when cholera spirilla were injected into the peritoneal cavity of cholera- immune guinea-pigs, the microorganisms rapidly swelled up, be- came granular, and often underwent complete solution. The same phenomenon could be observed when the bacteria were injected into a normal animal together with a sufficient quantity of cholera-im- mune36 serum. This process he observed microscopically by abstracting, from time to time, a small quantity of the peritoneal exudate and studying it in hanging-drop preparations. The reaction was specific in that the destructive process took place to any marked extent only in the case of the bacteria employed in the immunization. Metchnikoff,37 Bordet, and others not only confirmed Pfeiffer's observation, but were able to show that the lytic process would take place in vitro, as well as in the animal body. The existence of a specific destructive process in immune serum was thus established for the vibrio of cholera and soon extended to other microorganisms. The constituents of the blood serum which gave rise to this destructive phenomenon were spoken of as b act erioly sins. Following closely upon the heels of Pfeiffer's observation came the discovery of another specific property of immune serum by Gruber and Durham.38 These workers noticed that certain bacteria, when brought into contact with the serum of an animal immunized against them, were clumped together, deprived of motility, and firmly agglutinated. They spoke of the phenomenon as agglutination and of the substances in the serum giving rise to it as agglutinins. The list of antibodies was again enlarged by Kraus,39 who in 1897 35 Pfeiffer, Zeit. f. Hyg., xviii, 1894. 36 Pfeiffer und Isaeff, ibid. 87 Metchnikoff, Ann. de 1 'inst. Pasteur, 1895. 38 Gruber und Durham, Munch, med. Woch., 1896. w Kraus, E., Wien. klin. Woch., 32, 1897. 252 INFECTION AND IMMUNITY showed that precipitates were formed when filtrates of cultures of cholera, typhoid, and plague bacilli were mixed with their specific immune sera. He called the substances which bestowed this property upon the sera precipitins. The treatment of the animal body, therefore, with bacteria or their products gives rise to a variety of reactions which result in the presence of the "antibodies" described above. Extensive investiga- tion has shown, however, that the power of stimulating antibody production is a phenomenon not limited to bacteria and their products alone. Antitoxins, we have already seen, may be produced with a variety of poisons of plant and animal origin. Sensitizing, agglu- tinating and precipitating effects may, likewise, be produced by the use of a large number of different substances. Chief among these, because of the great aid they have given to the theoretical investiga- tion of the phenomena of immunity, are the red blood cells. Bordet40 and, independently of him, Belfanti and Carbone41 showed in 1898 that the serum of animals repeatedly injected with the defibrinated blood of another species exhibited the specific power of dissolving the red blood corpuscles of this species. This was the first demonstra- tion of "hemolysis" — a phenomenon which, because of the ease with which it can be observed in vitro, has much facilitated investigation. The knowledge that specific "cytotoxins" or cell-destroying anti-bodies could be produced by injection of red blood cells naturally suggested the possibility of analogous reactions for other tissue cells. It was not long, therefore, before Metchnikoff42 and, independently of him, Landsteiner43 succeeded, by repeated injections of spermatozoa, in producing a serum which would seriously injure these specialized cells. Von Dungern44 ob- tained similar results with the ciliated epithelium of the trachea. Since then a host of cytotoxins have been produced with the cells of various organs and tissues. Thus, Neisser and Wechsberg45 produced leucotoxin (leucocytes); Delezenne,4G neurotoxin and hepatotoxin; Surmont,47 pan- creas cytotoxin; and Bogart and Bernard,48 suprarenal cytotoxin. 40 Bordet, Ann. de 1'inst. Pasteur, 1898. 41 Belfanti et Carbone, Giornale della E. Acatl. di Torino, July, 1898. 42 Metchnikoff, Ann. de I'inst. Pasteur, 189S. 43 Landsteiner, Cent. f. Bakt., i, 25, 1899. 44 v. Dungern, Munch, med. Woch., 1899. 4*Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901. "Delezenne, Ann. de I'mst. Past. 1900; Compt. rend, de 1'acad. des sci., 1900. " Surmont, Compt. rend, de la soc. de biol., 1901. 48 Bogart et Bernard, ibid., 1891. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 253 One of the most interesting of the cytotoxins, moreover, is nephrotoxin — produced by the treatment of animals with injections of emulsions of kidney tissue. In all cases it was supposed by those first working with these bodies, that the injection of the sera of animals previously treated with any particular tissue substance would produce specific injury upon the organs homologous to the ones used in immunization. It need hardly be pointed out how very important such phenomena would be in throwing light upon the degenerative pathological lesions occurring in disease. As a matter of fact, however, sera so produced have been shown to be specific for certain organs in a limited sense only. The question of specific cytotoxins has been of especial importance in the case of nephritis, where Ascoli and Figari49 and others have suggested an autonephrotoxin as the basis of the pathology of this disease. In the hands of Pearce and others, how- ever, the strict specificity of nephrotoxin could not be upheld and the subject is still in the experimental stage. Recent experiments by Pearce50 suggest that at least a part of the local injury to organs exerted by such "cytotoxic" sera may not be due to a specific action upon the organ cells so much as upon the hema- glutinating action of the sera causing embolism and necrosis. It is a fact also that most cytotoxic sera are usually hemolytic as well. It is not easy to decide, therefore, how much of the action upon the organs is due to their true cytotoxic properties and how much is attributable to the concomitant action upon blood cells. The extravagant hopes at first based upon cytotoxin investigation, especially in regard to the problem of malignant tumors, have been disappointed, and much is still obscure in regard to the cytotoxins which calls for further research. The many points of similarity existing between bacterial toxins and digestive ferments, by animal inoculation, suggested to several observers, the possibility of producing antibodies against the latter. As a result, a number of antiferments have been obtained, chief among which are antilab (Morgenroth51), antipepsin (Sachs52), antisteapsin (Schiitze53), and antilactase (Schiitze). The stimulation of antibody formation in the sera of animals is a consequence, therefore, of the injection of a large variety of sub- stances— some of them poisonous, some of them entirely innocuous. 49 Ascoli and Figari, Borl. klin. Woch., 1902. 60 Pearce, Jour. Exper. Mod., viii, 190(5. 61 Morgenroth, Cent. f. Bakt., 1899. ^Saclis, Fort. d. Med., 1902. ™Schutze, Deut. med. Woch., 1904; Zeit. f. Hyg., 1905. 254 INFECTION AND IMMUNITY The substances possessing this power have been conveniently named antigen or antibody -producers by German writers. The term antigen —though etymologically wrong, nevertheless is convenient and has crept into general usage. It signifies simply a substance which can stimulate the production or formation of an antibody. Such sub- stances, so far as is known, belong to the group of proteins and are derivatives of animal or plant tissues. Being proteins, all antigens are colloids. Recently, however, some crystalloidal substances have been described as possessing antigenic properties. CHAPTER XIII TOXINS AND ANTITOXINS The Toxin-Antitoxin Reaction. — Apart from the therapeutic pos- sibilities disclosed by the discovery of antitoxins, new light of in- estimable value was thrown by these observations upon the biological processes involved in immunization. The most vital problem, of course, which immediately thrust itself upon all workers in this field, was the question as to the nature of the reaction in which toxin was rendered innocuous by antitoxin. The simplest conception of this process would be an actual destruction of the toxin by its specific antitoxin, and it is not un- natural, therefore, that this was the view which, for a short time, found favor with some observers. Roux, and more particularly Buchner,1 however, under the sway of cellular pathology, advanced the opinion that the antitoxins in some way influenced the tissue cells, rendering them more resistant against the toxins. Antitoxin, according to this theory, did not act directly upon toxin, but affected it indirectly through the mediation of tissue cells. Ehrlich,2 on the other hand, conceived that the reaction of toxin and antitoxin was a direct union, analogous to the chemical neutralization of an acid by a base — an opinion in which Behring soon joined him. The conception of toxin destruction received unanswerable refutation by the experiments of Calmette.3 This observer, working with snake poison, found that the poison itself (unlike most other toxins) possessed the property of resisting heat even to 100° C., while its specific antitoxin, like other antitoxins, was delicately thermolabile. He noted, furthermore, that non-toxic mixtures of the two substances, when subjected to heat, regained their toxic properties. The natural inference from these observations could 1 Buchner, ' ' Schutzimpfung, " etc., in Penzoldt u. Stinzing, "Handbuch d. spez. Therap. d. Inf ektkrank., ' ' 1894. 2 Ehrlich, Deut. med. Woch., 1891. 3 Calmette, Ann. de Pinst. Past., 1895. 255 250 INFECTION AND IMMUNITY only be that the toxin in the original mixture had not been destroyed, but had been merely inactivated by the presence of the antitoxin, and again set free after destruction of the antitoxin by heat. A similar observation, made soon after by Wassermann4 and in the case of pyocyaneus toxin and antitoxin, fully supported the results of Calmette. An ingenious proof of the direct action of antitoxin upon toxin was obtained by Martin and Cherry.5 It was found by them that very dense filters, the pores of which had been filled with gelatin, permitted toxin to pass through under high pressure, while the presumably larger antitoxin molecule was held back. Through such filters they forced toxin-antitoxin mixtures, under a pressure of fifty atmospheres, at varying intervals after mixing. They found that, if filtered immediately, all the toxin in the mixtures came through, but that, as the interval elapsing between mixing and filtra- tion was prolonged, less and less toxin appeared in the filtrate, until, finally, two hours after mixing, no toxin whatever passed through the filter. Besides demonstrating the direct action of anti- toxin upon toxin, this work of Martin and Cherry showed that the element of time entered into the toxin-antitoxin reaction, just as it enters into reactions of known chemical nature. The absolute non- participation of the living tissue cells in these reactions was demon- strated by Ehrlich himself. Kobert and Stillmarck6 had shown that ricin possessed the power of causing the red blood cells of de- fibrinated blood to agglutinate in solid clumps, a reaction which could easily be observed in vitro. Ehrlich,7 who had obtained anti- ricin in 1891 by injecting rabbits with increasing doses of ricin, found that this antibody possessed the power of preventing the hemagglutinating action of ricin in the test tube. By a series of quantitatively graded mixtures of ricin and antiricin, with red blood cells as the indicator for the reaction, he succeeded in proving not only that the toxin-antitoxin neutralization was in no way dependent upon the living animal body, but that definite quantitative relations existed between the two substances entirely analogous to those which, according to the law of multiple proportions, govern reac- tions between different substances of known chemical nature. 4 Wassermann, Zeit. f . Hyg., xxii, 1896. 5 Martin and Cherry, Proc. Eoyal Soc., London, Ixiii, 1898. 6 Kobert und Stillmarck, Arb. d. phar. Inst. Dor,pat, 1889. 7 Ehrlich, Fort. d. Med., 1897. TOXINS AND ANTITOXINS 257 Similar quantitative results were subsequently obtained by Stephens and Myers8 for cobra poison and its antitoxin, by Kossel9 for the toxic eel blood serum, and by Ehrlich10 for the hemolytic tetanus poison known as tetanolysin. The introduction of the test-tube experiment into the investiga- tion of these reactions permitted of much more exact observations, and by this means, as well as by careful, quantitatively graded, animal experiments, the further facts were ascertained that toxin and antitoxin combined more speedily in concentrated than in dilute solutions, and that warmth hastened, while cold retarded, the reac- tion— observations11 which in every way seem to bear out Ehrlich 's conception of the chemical nature of the process. Ehrlich 's Analysis of Diphtheria Toxin. — Shortly after the dis- covery and therapeutic application of diphtheria antitoxin, it be- came apparent that no two sera, though similarly produced, could have exactly the same protective value. It was necessary, therefore, to establish some measure or standard by which the approximate strength of a given antitoxin could be estimated. Von Behring12 attempted to do this for both tetanus and diphtheria antitoxins by determining the quantity of immune sera which, in each case, was needed to protect a guinea-pig of known weight against a definite dose of a standard poison. He ascertained the quantity of standard toxin-bouillon which would suffice to kill a guinea-pig of 250 grams, and called this quantity the " toxin unit." This unit was later' more exactly limited by Ehrlich, who, considering the element of time, stated it as the quantity sufficient to kill a guinea-pig of the given weight in from four to five days. Appropriating the terminology of chemical titration, v. Behring spoke of a toxin-bouillon which contained one hundred such toxin units in a cubic centimeter, as a " normal toxin solution" ("DTN1 M250"), and designated as "normal antitoxin" a serum capable of neutralizing, cubic centimeter for cubic centimeter, the normal poison.13 A cubic centimeter of such an antitoxic serum was suffi- 8 Stephens and Myers, Jour, of Path, and Bact., 1898. 9 Kossel, Berl. klin. Woch., 1898. 10 Ehrlich, Berl. klin. Woch., 1898. 11 Knorr, Fort. d. Med., 1897. 12 v. Behring, Deut. med. Woch., 1893. 13 DTN1 M250 signifies : D, Diphtheria ; TN1, Normal Toxin solution ; M2SO, Meer- schweinchen or guinea-pig weighing 250 grams. 258 INFECTION AND IMMUNITY cient, therefore, to neutralize one hundred toxin units, and was spoken of as an "antitoxin unit.5" In the experiments of v. Behring, toxin and antitoxin had been separately injected. Ehrlich14 im- proved upon this method by mixing toxin and antitoxin before in- jection, thereby obviating errors arising from differences which may have existed in the depth of injection or rapidity of absorption. In order, however, that any such method of standardization of antitoxin may be practically applicable, it is necessary to produce either a stable toxin or an unchangeable antitoxin. This Ehrlich achieved for antitoxin by drying antitoxic serum in vacua and pre- serving it in the dark, at a low temperature and in the presence of anhydrous phosphoric acid. By the use of such a stable antitoxin, various toxins may be measured and other antitoxic sera estimated against these. Toxophorex / I Bordet, Ann. de Pinst. Pasteur, 1899. 46 Gengou, Annal. Past., 1904. 47 Landsteiner mid Jagic, Wien. klin. Woch., iii, 1904. 48 Neisser and Friedmann, Munch, med. Woch., 1904, li. 465-827. SENSITIZING ANTIBODIES 295 antibodies, including the so-called amboceptors or sensitizers that take part in the phenomena of lysis and bactericidal action are essentially of one type; that the fundamental phenomenon is the union of the antigen with the specific antibody or its "sensitiza- tion;" that by such sensitizatioii the antigen is now rendered on the one hand more easily agglutinable or precipitable, on the other may be rendered more amenable to the action of the alexin or com- plement or to phagocytosis. The agglutination and precipitation phenomena, moreover, are merely evidences of the fact that these substances are in colloidal suspension and are influenced by agencies which produce precipitations in such suspension. It is interesting to note in this connection, also, that bacteria in neutral suspension carry negative charges which can be weakened by sensitization with serum and weakened or reversed by the addition of acid. These points tend to strengthen such a point of view. The degree of acidity necessary to reverse the normal negative charge of bacteria corresponds roughly to that at which growth is inhibited. This has led us to speculate whether or not vitality of bacteria and the negative charge may be related. Facts Concerning Alexin or Complement. — Muir and Browning claim that, on the filtration of serum, amboceptor or immune body will pass through the filter, whereas alexin or complement is held back. This retention of complement by filters occurs only when new filters are used, and this is probably due to adsorption or comple- ment by the finely divided substances which make up the filter and not due to retention because of the large size of the complement molecule. Alexin can be inactivated by shaking as well as by heat when diluted 1 :10 and shaken for about 20 minutes in salt solution. Ac- cording to Gramenitski it is spontaneously partially reactiviated on standing. Alexin is dependent upon the total volume of the mixture in which it acts, i.e., upon concentration, the same actual quantity of complement acting more strongly in higher than in lower concen- trations, this not being true of amboceptor or sensitizer which acts in direct proportion to its actual quantity independent of the con- centration. Alexin is inhibited by hypertonic salt solution and can be pre- served in 15-25 per cent salt concentration for weeks in the icebox, 296 INFECTION AND IMMUNITY resuming its activity when diluted to isotonicity with distilled water. Removal of salt by dialysis or other means of globulin precipitation divides the complement into two fractions, the globulin fraction and the albumin fraction, neither of which will act alone, but which together possess the properties of undivided complement. The globulin fraction attaches directly to the sensitized cells and is there- fore spoken of by German investigators as "mid-piece." The al- bumin fraction acts upon the sensitized cells only after attachment of the globulin fraction and is therefore spoken of as "end-piece." It is seen, therefore, that a great many of the properties of alexin make it seem rather likely that this substance is quite similar to ferments in its action. The Fixation of Complement by Precipitates. — It has been found by Gengou49 and confirmed by Moreschi, Gay,50 and others, that when the serum of an animal immunized with the serum of another species or with a foreign albumin is mixed with a solution of the substance used in the immunization, the precipitate formed will remove complement from the mixture. In other words, precipitates formed by the reaction of precipitin with its antigen will fix com- plement. This is of great importance in complement-fixation tests ; for because of insufficient washing, the blood cells used in producing the hemolytic amboceptor, may, from the presence of serum, give rise to a precipitin as well as a hemolysin. In the test done subse- quently, a precipitin reaction may take place and by thus removing complement may give a false result. The absorption of complement by such precipitates takes place when the two reacting factors, the precipitin and its antigen, are in dilution — so high a visible precipi- tate can not be observed. This fact, together with others too com- plicated to be discussed in this place, have led us to the belief that the so-called precipitins are true sensitizers, exerting toward unformed proteins the same function that the so-called sensitizer or amboceptor exerts toward cellular formed antigens. (See p. 293.) Quantitative Relationship Between Amboceptor and Complement. — Morgenroth and Sachs51 have succeeded in showing that within certain limits an inverse relationship exists between these two bodies. If for a given quantity of red blood cells a certain quantity of 49 Gengou, Ann. Past., 1902. 5(1 Gay, Cent. f. Bakt., I, xxix, 1905. 51 Morgenroth nnd Sachs, ' ( Gesammel. Arb. f iir Immimitatsf orschung. ' ' Berlin, Hirschwald, 1904. SENSITIZING ANTIBODIES 207 amboceptor and complement suffices to produce complete hemolysis, reduction of either the complement or the amboceptor necessitates an increase of the other factor. As amboceptor is increased, in other words, complement may be reduced and vice versa. This result is of great importance in arguing against the original conception of Ehrlich in supposing these substance's to act together unit for unit. Deviation of the Complement (Complement-Ablenkung).— It was noticed by Neisser and Wechsberg52 that in mixing together bacteria, inactivated bactericidal immune serum (im- mune body), and complement in the test tube, a great excess of immune body hindered rather than helped bactericidal action. As the amount of immune body in the mixture was carried beyond the experimental optimum, bactericidal action became less and less pronounced, and was finally completed suspended. They explain this by assuming that free immune body, un- combined with complement, has a greater affinity for the bacterial receptor than the im- mune body combined with complement. The complement is consequently diverted and pre- vented from activating the amboceptor attached ,11 . n I-, r* -i . -,. FIG. 37. — NEISSER AND to the bacterial cell. Graphically, the condi- WECHSBBRG,S CoN. tions may be illustrated as follows : CEPTION OF COMPLE- The above theory of Neisser and Wechs- MENT DEVIATION. berg is here stated simply because of the wide discussion it has aroused. In the light of our present knowledge concerning the relations between antigen, ambo- ceptor, and complement, their conception is obviously erroneous. The phenomenon of Neisser and Wechsberg is probably a "zone" phenomenon, namely, an occurrence which depends upon the fact that the complete or incomplete union of colloidal substances de- pends to a very great extent upon the relative concentrations of each, and too high a concentration of the anti-serum in experiments, such as those of Neisser and Wechsberg, may result in incomplete union. Thus, it is possible in many colloidal precipitation phenomena to show that too high a concentration of one or the other reacting colloid will result in failure of precipitation, and in some cases, even 52 Neisser und Wechsberg, Munch, med. Woch., xviii, 1901. 298 INFECTION AND IMMUNITY when precipitation has taken place, dispersion will again occur, if one or the other component is added in excess. These phenomena are frequently observed in agglutination and precipitation reactions where the highest concentrations of serum will produce less pre- cipitate, or perhaps none at all when greater dilutions produce heavy precipitation. Fixation of the Complement. — Bordet and Gengou53 in 1901, de- vised an ingenious method of experimentation by which even very small quantities of any given immune body (amboceptor) can be demonstrated in serum. The term "fixation of complement," by which their method of investigation is now generally known, ex- plains itself, as the steps of experimentation are followed. They prepared the following mixtures : (a) (b) Bacteriolytic amboceptor Normal serum, heated (Plague immune serum, heated) + + Plague emulsion Plague emulsion + + Complement Complement (Fresh normal serum) (Fresh normal serum) To both of these after five hours was added Hemolytic amboceptor (Heated hemolytic serum) + Red blood cells Results : (a) showed no hemolysis. (b) showed hemolysis -f-. The conclusion to be drawn from this was that in (a) the presence of immune body had led to absorption of all the complement. In (b), there being no bacteriolytic immune body to sensitize the bacteria and enable them to absorb complement, the latter substance was left free to activate the subsequently added hemolytic ambo- ceptors. The Bordet-Gengou phenomenon has been extensively used by Wassermann and Bruck,54 Neisser and Sachs,55 and others to ™Bordet et Gengou, Ann. de Tinst. Pasteur, 1901. 64 Wassermann und Bruck, Med. Klin., 1905. 55 Neisser und Sachs, Berl. klin. Woch., xliv, 1905, and i, 1906. SENSITIZING ANTIBODIES 299 demonstrate the presence of immune bodies in various sera. (See p. 315.) It should be noted that this method, if valid, must presuppose the identity of the hemolytic and bactericidal complement in the activating serum. Complement fixation will be more - extensively discussed in the section dealing with the Wassermann reaction. WL. .Complement I Together a* 4 I I HaemolyKe StiphiHtic present Aiwl>oceptor _ _ _ Jmmune or ? or 1 37.SO C. antibody not ^ « « f for one Hour aktv I, xxxix, 1905- and ibid., xlvi, 1908; Korschum, Ann. de 1'Inst. Pasteur, xxii, 1908; Zinsser, Jour. Med. lies., xxii, 3, 1910. OPSONINS AND VACCINE THERAPY 347 NON-SPECIFIC PROTEIN THERAPY A very surprising- development of the last ton years has been the observation that profound physiological reactions accompanied by occasional therapeutic benefit in infectious diseases has followed the intravenous injection of bacterial and other proteins which ap- parently had no specific relationship to the nature of the infectious process. The first observations were more or less accidental, in- cident to attempts by various writers to treat diseases like typhoid fever by the intravenous injection of typhoid bacilli. Ichiwaka, Kraus, Gay and others injected sensitized and unsensitized typhoid bacilli intravenously into patients suffering from typhoid fever, ob- serving a sudden drop of temperature with chill, and frequent benficial effects on the course of the disease. It was soon found that similar results could be obtained in these diseases with colon bacilli, paratyphoid bacilli, etc. Holler26 obtained striking results in typhoid fever by injecting deutero-albumose. Other proteins and proteose substances were subsequently used by many observers, and important studies on the theoretical effects of the injection of such substances have been made by Jobling and Petersen.27 It can be regarded as quite definite that these reactions are entirely non- specific. The substances used have been typhoid vaccine, primary and secondary albumoses, gonococcus and other bacterial vaccines, normal serum, leucocyte extracts, etc., etc. The method has been applied to a great many definite infections, such as typhoid fever, general sepsis, pneumonia, gonorrheal infections, and to arthritis and dermatological lesions, etc. Miller28 has used typhoid vaccines in typhoid fever and other conditions, and, in general, concludes that .there can be little doubt that in a limited number of cases rapid and sometimes permanent beneficial results, are obtained after a preliminary slight rise of temperature and subsequent drop, often with a chill. He also concludes that if an amount just sufficient to incite a chill is used, that is, if the dosage is carefully controlled, the treatment is without danger. He has given 2,000 intravenous injections of typhoid vaccine in the Cook County Hospital, without serious consequences, except for the development of delirium tremens ~« Holler, Beit. z. Frank, u. Inkeft., 6, 1917, cited from Miller, Jour. A. M. A., 76, Jan., 1921. "'Jobling and Petersen, Jour. Exper. Med., 20, 1914. ** Miller, loc. cit. 348 INFECTION AND IMMUNITY in some alcoholics. He, however, carefully selected his cases. As Petersen29 concludes, non-specific therapy has produced definite re- sults, though the eventual determination of its definite value cannot yet be made. It is in the experimental stage, and according to Petersen29 concludes, non-specific therapy has produced definite re- sults, though the eventual determination of its definite value cannot yet be made. It is in the experimental stage, and according to Petersen, "its usefulness and ultimate range" cannot yet be fully judged. The effects of the injection, as analyzed by Petersen from his own studies and a study of the literature, are as follows: After injection of the more powerful and active substances, there is at first a chill, sweating, and a definite rise of temperature ; there is a leucopasnia followed by leucocytosis, lowering of the blood pressure, and changes in the blood, such as increase in fibrinogen, a rise of enzyme curve and an increase in blood sugar and antibodies. The less active substances produce some temperature, a slight chill and other symptoms mentioned, to a lesser degree. The beneficial effects may perhaps be to some extent explained by the increase of leu- cocytes and of enzymes, and Petersen makes a point of the fact that if the method is to exert beneficial effects, it is probably neces- sary to use it early in the disease. We are not in any position at present either to recommend or further comment upon the method, but it is an important problem for laboratory experimentation and for careful clinical application in the hands of men trained in ex- perimental studies. The Problem of Virulence. — An extremely obscure chapter in our knowledge of the reaction of animals and man against infection is the one dealing with the questions of varying pathogenicity between different bacterial species and between different races of the same microorganism. We know that certain bacteria may be injected into an animal or human being in considerable quantities, without pro- ducing anything more than the temporary local disturbance follow- ing the subcutaneous administration of any innocuous material. Other bacteria, on the other hand, such as the bacillus of anthrax or the bacillus of chicken cholera, injected in the most minute dosage, may give rise to a rapidly fatal septicemia. Within the same species, furthermore, fluctuations in virulence may take place 29 Petersen, Jour. A. M. A., 76, January, 1921. OPSONINS AND VACCINE THERAPY 349 which may depend upon a variety of influences which have been discussed in another section and need not be recapitulated. Suffice it to say that variations in the susceptibility of inoculated subjects do not, in any way, furnish a sufficient explanation for these phenomena. In an effort to cast light upon this subject, Bail, following in the footsteps of his predecessors, Kruse,30 Deutsch and Feistmantel,31 has formulated his so-called ' i aggressin-theory. " Bail32 was first led to the formulation of his theory by extensive researches which he had made in conjunction with Petterson33 into anthrax immunity. He had noted, as others before him had, that animals, highly susceptible to anthrax, often possessed marked bac- tericidal powers against this bacillus. When such animals, whose serum should surely be capable of bringing about the death of, at least, a few hundred anthrax bacilli, were injected with doses far less than this number they nevertheless succumbed rapidly and the bacilli multiplied enormously in their bodies. He argued from this that the injected microorganisms must possess some weapon whereby they were enabled to counteract the protective forces of the animal organism. In an anthrax-immune animal, as a matter of fact, no proliferation of bacteria took place and the injected germs were rapidly disposed of by the protective forces, foremost of which was phagocytosis. The theory of Bail34 contains the following basic principles:35 Pathogenic bacteria differ fundamentally from non-pathogenic bacteria in their power to overcome the protective mechanism of the animal body, and to proliferate within it. They accomplish this by virtue of definite substances given off by them, probably in the nature of a secretion, which acts primarily by protecting them against phagocytosis. These substances (referred to by Kruse as "Lysins") were named by Bail, * * Aggressins. " The production of aggressins by pathogenic germs is probably absent in test-tube cul- tures, or, at any rate, is greatly depressed under such conditions, 30 Kruse, Ziegler 's Beitrage, xii, 1893. 31 Deutsch und Feistmantel, "Die Impfstoffe in Sera," Leipzig, 1903. 32 Bail, Cent, f . Bakt., I, xxvii, 1900, and xxxiii, 1902. 83 Bail und Petterson, Cent. f. Bakt., I, xxxiv, 1903; xxxv, 1904; xxxvi, 1904. 34 Bail, Arch, f . Hyg., lii, 1905 ; liii, 1905 ; Wien. klin. Woch., xvii, 1905. 85 Bail und Weil, Wien. klin. Woch., ix, 1906; Cent. f. Bakt., I, xl, 1906; xlii. 1906. 350 INFECTION AND IMMUNITY but is called forth in the animal body by the influences encountered after inoculation. These aggressiiis can be found, according to Bail, in the exudates about the site of inoculation in fatal infections. He obtained them, separate from the bacteria, by the centrifugation and subsequent decanting of edema fluid, and pleural and peritoneal exudates. Two experimental observations are brought by Bail in support of the truth of his contentions. In the first place, he was able to show that fatal infection could be produced in animals by the injection of sublethal doses of bacteria, when these were administered with a small quantity of "aggressin." He inferred from this that the injected aggressin had paralyzed the onslaught of phagocytic and other protective agencies, and had thus made it possible for the bacteria to proliferate. The second experimental support of Bail's theory consists in the successful immunization of animals with aggressin. Animals were treated with aggressive exudates, from which all bacteria had been removed by centrifugalization and which had been rendered sterile by three hours' heating to 60° 0. and addition of 0.5 per cent phenol. Animals so treated were not only immune themselves, but contained a substance in their serum which permitted the passive immunization of other untreated animals. Bail explained this by assuming the production of anti-aggressins in the treated subjects. His experi- ments and those of his pupils were conducted with the typhoid and dysentery bacilli, the bacilli of chicken cholera and of plague, the cholera spirillum, and various micrococci. According to whether a microorganism is capable of producing an aggressin and conse- quently of invading the animal body, he divides bacteria into "pure parasites," "half parasites," and "saprophytes." The theory, of Bail has been attacked chiefly, by Wassermann and Citron,36 Wolff,37 and Sauerbeck.38 The criticism which these investigators make of Bail's views has succeeded in placing the "aggressin" theory in doubt. It is claimed by them that much of the "aggressive" character of Bail's exudates is due to their con- taining liberated bacterial poisons (endotoxins). This they have maintained both because the sterile "aggressin" exudates could be shown to possess a considerable degree of toxicity and because the M Wassermann and Citron, Deut. med. Woch., xxviii, 1905. «7 Wolff, Cent. f. Bakt., I, xxxviii, 1906, 38 Sauerbeck, Zeit. f. Hyg., Ivi, 1907, OP8ONINS AND VACCINE THERAPY 351 aggressive action could be duplicated by aqeous extracts of bacteria. Citron,39 was able to show, by the Bordet-Gengou method of com- plement fixation, that the exudates ,of Bail contained quantities of free bacterial receptors, which, in taking up immune body, would neutralize any destructive power on the part of the infected animal. The writer in conjunction with Dwyer40 has done certain experi- ments which seem to indicate that Bail's aggressin may be in the nature of anaphylatoxin. The addition of such anaphylatoxin to bacteria will convert a sublethal into a lethal dose, as will Bail's aggressin, and in principle the manner of production is the same. The nature of the immunity produced in animals by Bail's method of treatment is less easily explained and less exposed to adverse criticism. Whatever may be the truth about the possession of offen- sive weapons on the part of bacteria, it is certainly a fact that microorganisms differ much in their powers of defense against de- struction by the cells in sera of the animal body. Virulent bacteria are not destroyed by serum or agglutinated or taken up by leucocytes as easily as are the non-virulent. In some cases there seems to be no morphological clue to the reason for this. In other cases, like pneumococci, Friedlander bacilli and others, there is a bacterial capsule which seems to insulate these organisms against attack. Many bacteria lose their capsules in" the non- virulent stage on culture media, but form them within the animal body in the process of infection. Again, bacteria rendered non-virulent by cultivation on artificial media may become virulent, inagglutinable, and more resistant to phagocytosis when cultivated on immune sera or passed through the animal body. Thus the power to invade depends possibly upon a combination of offensive properties and defensive qualities on the part of the bacteria. Added to this, some of us believe that the reaction between lytic antibodies and the bacterial protein may produce toxic sub- stances which poison the animal body, prevent positive chemotaxis, and thereby aid the invader. Again, there are microorganisms like the treponema pallidum in syphilis where adaptation between invader and host seems to be of such a nature that an indifferent reaction against the invading organism only is set up. 39 Citron, Cent, f . Bakt., I, xl, 1905 ; xli, 1906 ; and Zeit. f . Hyg. Hi, 1905. 40 Zinsser and Dwyer, Proceedings of the Soc. for Exper. Biol. and Med., 1914, xi, 74-76. CHAPTER XIX HYPERSUSCEPTIBILITY THE phenomena now grouped together under the heading of anaphylaxis and hypersusceptibility have but recently become the subject of systematic experimentation. Nevertheless, manifestations now recognized as belonging to this category had not escaped the attention of a number of the earlier workers in immunity. Although the development of scientific knowledge of hypersus- ceptibility has concerned itself particularly during the last fifteen years with hypersusceptibility phenomena as they apply to antigenic substances, we must deal with the subject a little more compre- hensively than this at the present day, and call attention to the fact that clinicians had for many years noticed so-called idiosyn- crasies against drugs of various kinds, morphin, strychnin, arsenic, etc., etc., and against many things which cannot be regarded at the present time as antigens in the sense in which this term is used in immunological literature. By hypersusceptibility in general, then, we mean that a certain individual, human being or animal, suffers injury from the admin- istration of a substance which, in similar amounts and methods of administration, does not exert any injurious action upon normal members of the same species. In some cases, such as pollen hypersensitiveness and some other food idiosyncrasies, the hypersusceptibility may be congenital, that is, inherited by the individual without traceable previous contact with the substance to which he is sensitive. In most instances, however, some form of previous physiological contact with the particular substance seems to be necessary for the development of the hypersensitive state. Owing to the varied manifestations of specific hypersusceptibility, it has been necessary to classify the phenomena of this nature into two main subgroups. In doing this we follow the classification of Doerr.1 Doerr has grouped together all phenomena of hypersus- 1 Doerr, Kolle and Wassermann Handb., 2, 1913, 947. 352 HYPERSUSCEPTIBILITY 353 ceptibility under the general term of allergy, which means altered reaction. Under this heading he classifies, A, those forms of hyper- susceptibility in which the inciting substance is, as far as we know at the present time, non-antigenic, and, B, that form of allergy in which the inciting substance is a known antigen. We ourselves would rather define these subdivisions as, A, those forms of allergy in which the mechanism of the hypersusceptibility cannot be shown to be due to an antigen-antibody union, and, B, those forms in which an antigen-antibody union within the body can be proved to be responsible. The difference is a slight one, but may, in the future, perhaps become a fundamental one, for our recent studies on the tuberculin reaction make us believe that the conception of the word "antigen" will necessarily change in the course of the next few years of in- vestigation. We do not see any particular purpose in altering the Doerr classification to one suggested by Coca2 in which the general term * * hypersensitiveness ' ' is subdivided into true anaphylaxis and allergy, the term "allergy" here being confined to the reactions in which no true antigen-antibody reaction can be determined. In fact, we believe that this would be harmful, in that Coca thereby implies that there is a general identity of mechanism underlying the mani- festations which he classifies together as "allergy," some of which are certainly open to justified differences of opinion. We will first deal with those forms of hypersusceptibility in which truly antigenic substances are involved, and, for this form, we may reserve the term of True Anaphylaxis. Anaphylaxis. — As early as 1893, Behring3 and his pupils4 had noticed that animals, highly immunized against diphtheria toxin, with high antitoxin content of the blood, would occasionally show marked susceptibility to injec- tions of small doses of the toxin. The phenomena observed by them was interpreted as an increased tissue susceptibility to the toxin, and Wassermann, reasoning on the basis of Ehrlich's side-chain theory, formulated the conception that the increased susceptibility was due to toxin receptors, increased in number by immuniza- 2 Coca, Tice ;s System of Medicine, Vol. 3, 1920. 3Behring, Deut. med. Woch., 1893. 4 Knorr, Dissert., Marburg, 1895 ; Behring und Kitashina, Bert, klin, Woch., 1901, 354 INFECTION AND IMMUNITY tion, but not yet separated from the cells that had produced them; the cells thereby becoming more vulnerable to the poison. In the same category belongs the observation of Kretz, who noticed that normal guinea-pigs did not show any reaction after injections of innocuous toxin-antitoxin mixtures, but that marked symptoms of illness often followed such injections when made into immunized guinea-pigs. Other phenomena which are now re- garded, a posteriori, as probably depending upon the principles involved in anaphylaxis, are the tuberculin and mallein reactions, fully described in another place, and the adverse effects often following the injections of anti- toxins in human beings, conditions spoken of under the heading of "serum sickness." The last-named condition has been made the subject of an exhaus- tive study by v. Pirquet and Schick.5 That the injection of diphtheria antitoxin in human beings is often followed, after an incubation time of from three to ten days, by exanthematous eruptions, urticaria, swelling of the lymph glands, and often albuminuria and mild pulmonary inflammations, has been noticed by many clinicians, who have made extensive therapeutic use of antitoxin. It was recognized early that such symptoms were entirely independent of the antitoxic nature of the serum, but appended upon other constituents or properties peculiar to the antitoxic serum. Moreover, symptoms of this description were by no means regular in patients injected for the first time, but seemed to depend upon an individual predisposition, or idiosyncrasy, v. Pirquet and Schick, however, noticed that in those injected a second time, after intervals of weeks or months, the consequent evil effects were rapid in development, severe, and occurred with greater regularity. The fundamental observations from which our present knowledge of anaphylaxis takes its origin are those made in 1898 by Hericourt and Richet,6 who observed that repeated injections of eel serum into dogs gave rise to an increased susceptibility toward this substance instead of im- munizing the dogs against it. Following up the lines of thought suggested by this phenomenon, Portier and Richet7 later made an interesting observa- tion while working with actino-congestin — a toxic substance which they ex- tracted from the tentacles of Actinia. This substance in doses of 0.042 gram per kilogram produced vomiting, diarrhea, collapse, and death in dogs. If doses considerably smaller than this were given in quantities sufficient to cause only temporary illness, and several days allowed to elapse, a second injection of a quantity less than one-quarter or one-fifth of the ordinary lethal dose would cause rapid and severe symptoms and often death. Similar 5 Pirquet and Schick, "Die Serum Krankheit," monograph, Leipzig and Wien, 1905. 6 Hericourt and Ricliet, Compt. rend, de la soc. de biol., 5,3, 1898. 7 Portier and Eichet, Compt. rend, de la soc. de biol., 1902 j Eiclict, Ann. de Tinst. Pasteur, 1907 and 1908. HYPERSUSCEPTIBILITY 355 observations were made soon after this by Richet with mytilo-congestin, a toxic substance isolated from mussels. In these experiments there remained little doubt as to the fact that the first injection had given rise to a well- marked increased susceptibility of the dogs -for the poison used. It was Richet who first applied to thistphenomenon the term "anaphylaxis" ( avd against, vAo£ts protection), to distinguish it from immunization or prophylaxis. Soon after Richet's earlier experiments, and simultaneously with his later work, Arthus8 made an observation which plainly confirmed Richet's observa- tions, though in a somewhat different field. The observation of Arthus is universally spoken of as the "phenomenon of Arthus." He noticed that the injection of rabbits with horse serum ^a substance in itself without toxic properties for normal rabbits) rendered the rabbits delicately susceptible to a second injection made after an interval of six or seven days. The second injection — even of small doses — regularly produced severe symptoms and often death in these animals. An observation very similar to that of Arthus was made by Theobald Smith9 in 1904. Smith observed that guinea-pigs injected with diphtheria toxin-antitoxin mixtures in the course of antitoxin standardization, would be killed if after a short interval they were given a subcutaneous injection of normal horse serum. The fundamental facts of hypersusceptibility had thus been observed, and Otto,10 working directly upon the basis of Smith's observation, carried on an elaborate inquiry into the phenomenon. Almost simultaneously with Otto's publication there appeared a thorough study of the condition by Rosenau and Anderson.11 The researches of Otto, and Rosenau and Anderson, besides confirming the observations of previous workers, brought out a large number of new facts. They showed conclusively that the action of the horse serum had no relationship to its toxin or to its antitoxin constituents, that the "sensitiza- tion" of the guinea-pigs by the first injection became most marked after a definite incubation time of about ten days. Sensitization was accomplished by extremely small doses (one one-millionth in one case, usual doses 1/250 to 1 c.c.). Rosenau and Anderson, furthermore, excluded hemolysin or precipitin action as explanations of the phenomena, and proved that hyper- susceptibility was transmissible from mother to offspring, and that it was specific — animals sensitized with horse serum not being sensitive to subsequent 8 Arthus, Compt. rend, de la soc. do biol., 55, 1903. ' Th. Smith, Jour. Med. Bes., 1904. 111 Otto, "Leuthold Gedenkschrif t, " 1905. 11 Roscnaik and Anderson, Hyg. Lab. U. S. Pub. Healt'i and Marine Hosp. Serv. Bull., 29, 36, 1906, 1907. 356 INFECTION AND IMMUNITY injections of other proteins. These authors, Vaughan12 and Wheeler, Nicolle,13 and others, furthermore, showed that the reaction was by no means limited to animal sera, but was elicited by proteins in general, pepton, egg albumin, milk, the extract of peas, and bacterial extracts. These observations, together with those of many other workers which we must omit for the sake of conciseness, were the funda- mental ones. In the time immediately following this first work, many theories of anaphylaxis were advanced and many faulty ideas conceived, justifiable in the light of the knowledge available at that time, but no longer tenable as more precise analyses followed. Such, in our opinion, are the earlier ideas of Gay and Southard,14 and that of Besredka,15 both of which depended chiefly upon the premise that the substance which sensitized in the first injection was not the same as that which incited the harmful effect at the second or subse- quent injections. Into the same category at the present time belong the earlier views of Wolf-Eisner,16 and in a less definite way, the theories of Vaughan and Wheeler.17 The latter, however, have had an important influence upon subsequent developments which will be referred to below. Von Pirquet and Schick,18 from the beginning, maintained the analogy of the anaphylactic phenomena to other immune reactions, and believed that the reaction was dependent essentially upon an antigen-antibody union; and similar views were held by Rosenau and Anderson,19 from the beginning. In order to make the subject clear, however, without unnecessarily lengthening its discussion, we must abandon the historic method of treatment, and define the various elements that enter into the reaction more systematically. Wells20 has, in our opinion, most concisely laid down the criteria which must be met in the light of our present knowledge, in order that a condition may be regarded as one of true anaphylaxis. They are, 12 Vaughan, Assn. Am. Phys., May, 1907. 13 Nicolle, Ann. de 1'Inst. Pasteur, 2, 1903. 14 Gay and Southard, Jour. Med. Ees., May, 1907. 15 Besredka and Steinhardt, Ann. de 1'Inst. Past., 1907. 16 Wolf-Eisner, Berl. klin. Woch., 1904. 17 Vaughan and Wheeler, Jour. Inf. Dis., 4, 1907. 18 Von Pirquet and Schick, Die Serum Krankheit, Vienna, 1905. 19 Rosenau and Anderson, Hyg. Lab. U. S. Pub. Health and Marine Hosp. Serv, Bull., 29, 36, 1906 and 1907. ?0 Wells, Physiological Reviews, 1, No. 1, January, 1921, HYPERSUSCEPTIBILITY 357 with a few commentaries and slight modifications, as follows: "The observed toxicity of the injected material must depend upon sen- sitization of the animal, that is, the substance must not produce similar symptoms in the non-sensitized animal" (of the same species). "It should be possible to demonstrate passive sensitization with the serum of a sensitized animal. ' ' This we would modify some- what since, of course, in the early stages of developing hypersus- ceptibility, an animal, though sensitive, may have but slight or even no demonstrable antibodies in his serum. It would perhaps be more accurate to say that it must be possible to produce passive sensitiza- tion to an antigen by the administration of serum which contains antibodies to this antigen. If dealing with guinea pigs, it should be possible by the Dale Method, to demonstrate typical reactions as described below with the uterus of the sensitized guinea pig. After recovery from anaphylactic shock, a condition of desensitization should be apparent if quantitative conditions are taken into account. This is not all that Wells says about it, and we have somewhat modified it for our own purposes, but, in general, these criteria are the chief ones which must be met, and they all boil down to the statement that, in order to be considered true aiiaphylaxis, it must be shown that the mechanism of whatever reaction that may occur is one that is fundamentally based upon the meeting of an antigen with its homologous antibody. Further limitations of this, as to the site and manner of such meeting will be described below. THE ANAPHYLACTIC ANTIGEN. — We may save much discussion for the purposes of this particular book by saying that substances with which true aiiaphylaxis can be produced are all, as far as we know, protein in nature. No conclusive proof has ever been brought that lypoids or carbohydrates can act as antigens, and work with protein- split products has not been sufficiently satisfactory. Racemized proteins have been shown by Ten Broeck21 not to exert antigenic action for anaphylaxis. Wells, himself, states that he has always obtained negative results with protein cleavage products, but says that Fink22 in his own laboratory has occasionally obtained slight anaphylactic reactions with proteose fractions obtained from coagulated egg-white by hydrolysis with steam under pressure, for those parts of the proteose solution which were precipitated by 21 Ten Eroeck, Jour. Biol. Chem., 17, 1914, 369. 22 FinTc, Jour. Inf. Dis., 25, 1919, 97. 358 INFECTION AND IMMUNITY complete and three-fourths saturation with ammonium sulphate. It is interesting to note that these two fractions also incited antibodies, by which precipitin and complement fixations could be obtained, a matter which, in this case, is of considerable importance. For further discussion of other claims of non-protein anaphylactic anti- gens, we refer the reader to the article by Coca and the one by Wells. We may summarize here by saying that, as far as we know at the present time, anaphylactic antigens differ in no way from other antigens, and that no substance at the present time has been proven to be an anaphylactic antigen (in the sense in which we define the term above), with which antibody formation in animals has failed. In other words, any substance that can incite the forma- tion of true antibodies may also be an anaphylactic antigen. THE METHODS OF SENSITIZATION. — Experimental sensitization. may be active or passive, and differs to some extent according to the species of animal under observation. The early observations were chiefly made on guinea pigs. Guinea pigs can be actively sensitized by a single injection of various amount. When dealing with animal sera such as horse serum quantities of anywhere from 0.1 to 1 c.c. are most suitable. Minute amounts, however, will suffice, and Rosenau and Anderson23 succeeded in one case in sensitizing with one one-millionth of c.c. of horse serum. If so sensitized, the animals become hypersusceptible at varying periods, hardly ever in less than six days, the ideal time for reinjection ranging between two and three weeks, somewhat dependent upon the amount given. Various statements have been made as to the relationship of the incubation time and the initial dose given to guinea pigs. The ideal time for injection is that at which the maximum amount of antibody has been formed on the cells Avith the minimum amounts of circulating antigen and antibody in the blood. The statement of Coca is prob- ably right, that, in general, the small amounts injected into guinea pigs require a relatively longer incubation period, but extremely large amounts (5-10 c.c.) may have a similar effect. The method of administration, to some extent, governs the incubation period in the same way that it governs the speed of antibody formation. However, the administration of the antigen to guinea pigs and other animals may be carried out in any way, except by feeding, and even feeding may result in a certain amount of hypersusceptibility 23 Rosenau and Anderson, loc. cit. HYPERSUSCEPTIBIL1TY 359 under unusual or pathological conditions (abnormal permeability of the intestinal mucosa). When sensitizing guinea pigs with bacterial proteins or pollen and some other vegetable proteins, it is necessary to inject anywhere from six to ten times on consecutive days, and testing about three weeks after the last injection. In dogs active sensitization is hard to obtain by one injection. Single doses of 2 to 3 c.c. of normal horse serum are usually followed by sensitization in three weeks, at least such results seem to have been obtained with some regularity by Simonds24 and others. Such sensitization, however, is not as acute and severe as that generally observed in guinea pigs under similar conditions. Weil,25 however, obtained acute shock in dogs by giving two sensitizing injections within a few days, and testing with large quantities of serum after two and three weeks. • Rabbits are difficult to sensitize with a single dose under any circumstances, but may easily be sensitized by repeated injection. The lower monkeys are extremely difficult to sensitize under any circumstances.26 Our knowledge of sensitization in man is of course based entirely upon clinical observation. And it is held, especially by one inves- tigator of anaphylaxis, that man cannot be sensitized. This, how- ever, does not seem to us to be tenable, and anaphylaxis in man is not an uncommon observation as manifested by serum sickness, im- mediate skin reaction and accidents observed especially among asthmatics treated with foreign protein for one or another clinical reason. Fatal acute shock in man, however, is fortunately rare. PASSIVE SENSITIZATION. — It is this phenomenon of passive sensi- tization which has thrown the most important light upon the process. It was first demonstrated by Nicolle,27 by Otto,28 and by Gay and Southard,29 all of whom showed that the hypersusceptible state could be passively transferred to normal animals by injecting them with the serum of anaphylactic animals. In such experiments the serum of the anaphylactic animal is first injected in quantities of 24 Simon ds, Jour. Infcc. Dis., 19, 1916. 25 Weil, Jour. Tmmun., 2, 1917, 429. ^7Ansser, Proc. Soc. Exper. Biol. and Med., 18, 1920, 57. 27 Nicolle, Ann. de 1 'Inst. Past., 2, 1903. 28 Otto, Munch, med. Woch., 1907. 29 Gay and Southard, Jour. Med. Ees., May, 1907. 360 INFECTION AND IMMUNITY 0.5 c.e. or preferably more, and twenty-four hours later an injection of the specific antigen — that is, the protein used for sensitization — is given. The animals so treated show typical symptoms of hyper- susceptibility and often die. Simultaneous inoculation of the two substances, either mixed or injected separately, does not produce the same effect. A fact, observed by Otto, is that the serum of guinea-pigs who have been given the sensitizing or first injection will confer passive anaphylaxis on the eighth or tenth day after injection, before the animals them- selves show evidences of being actively hypersensitized. It is also true that occasionally the serum of antianaphylactic animals will possess the power of conferring passive anaphyiaxis. It is by means of the passive method of sensitization that the relations between anaphylaxis and antibodies have been most suc- cessfully studied. Doerr and Russ30 showed that the power of a serum to convey anaphylaxis passively depended directly upon its contents of specific antibody. It was then' shown by Nicolle,31 Otto,32 and others, that a sharp reaction can be produced by this method only when a distinct interval not less than four to six hours, was allowed to lapse between the injection of the antibodies and the injection of the antigen. This may be taken as an axiom for all cases in which the antigen is an unformed protein in solution. It was also shown by Weil33 that passive sensitization could be conferred by the injection of precipitates formed in the test tube between an antiserum and its antigen, a thing which we can now well understand in view of the knowledge we have of the dissociation of antibody from such precipitates. Anaphylaxis may be transmitted passively by inheritance. Thus the young of anaphylactic guinea-pigs show hypersusceptibility, irrespective of whether the mother became hypersusceptible before or after the beginning of pregnancy. Such anaphylaxis has no reference to the condition of the father, and is not transmitted by the milk. The nature of these anaphylactic antibodies has aroused much discussion. By many observers they are regarded as special 80 Doerr and Russ, Ztschr. f. Immunitatsforsch., 1909, iii. "Nicolle, Bull, de PInst. Past., 1907, v. 32 Otto, Das Theobald Smithsche Phaenomenon, . etc., von Leuthold Gedenk- schrift, 1905, i. 33 Weil, Jour, of Immunol., 1, 1916, 19. HYPERSUSCEPTIBILITY 361 anaphylactic antibodies, separate from precipitins, opsonins, etc., etc. ; Friedberger,34 himself, from the beginning, identified them with precipitins. The direct quantitative relationship between pre- cipitating antibodies and the power to convey passive sensitization. described by Doerr and Russ35 would point in the same direction as would the above mentioned experiments of Weil. Since we have variously expressed in the preceding pages our own opinion that all antibodies developed against a single antigen are one and the same substance, we have no hesitation in stating that we think that the so-called sensitizing antibody is identical with the sensitizing antibody which is formed to the antigen, rendering it amenable to agglutination or precipitation or complement-fixation. Where Does the Reaction Occur. — As we have seen, when the antigen and antibody are injected simultaneously or within a very short period of one another, no anaphylactic symptoms occur. The study of this interval has gradually led to the recognition that the anaphylactic reaction, whatever it may be, takes place upon the body cells and that the interval in passive sensitization is neces- sitated by the time required for the anchoring of the antibodies to the cells of the tissues. Experiments by Pearce and Eisenbrey36 (1910) showed definitely that a hypersusceptible dog remained sen- sitized even when his entire blood volume was substituted with that of a normal dog. The principle has been made especially clear by the introduction of direct methods of observation of the smooth muscle cells of animals, by Schultz37 and by Dale,38 a method which has been particularly developed by Weil.39 It seems fairly clear from this work and a volume of other researches which cannot be reviewed here, that acute protein anaphylaxis as we see it in guinea- pigs and other laboratory animals is due to the direct reaction between antigen and a specific antibody when this reaction takes place upon the body cells and not in the blood stream. Just how much influence the reaction within the blood stream can exert or 34 Friedberger, with Hartoch, Zeit. f . Immunit., 3, 1909. 35 Doerr and Buss, Zeit. f. Immunit., 3, 1909. 36 Pearce and Eisenbrey, Congr. Am. Phys. and Surg., 1910, viii. 37 Schultz, Jour. Pharmacol. and Exper. Therap., 1910, i. 38 Dale, Jour. Pharmacol. and Exper. Therap., 1913, iv. 39 Weil, Jour. Med. Eesearch, 27, 1913; 30, 1914; Proc. Soc. Exper. Biol. and Med., 1914, xi, 86. 362 INFECTION AND IMMUNITY whether it takes any important part in the phenomena is at present a matter of considerable doubt. We can feel safe, therefore, in stating very definitely that the site of the anaphylactic reaction, that is, the place at which the union between antigen and antibody occurs in the production of the various symptoms of anaphylactic injury is upon the cells. That, in other words, the important reaction which determines the train of symptoms which we call anaphylaxis occurs when the antigen goes into relationship with antibodies which are still in some way united to tissue cells, or are, in the jargon of immunology, "sessile" upon the cells. Whether or not any injury may occur when antigen meets anti- body in the circulation is still an open question. There are experi- ments on record by Friedemann40 in wliich he obtained reactions in rabbits by the simultaneous intravenous injection of antigen and antibody, and similar occasional occurrences have been observed by Brion, Scott and ourselves. But these reactions are neither regular in occurrence, nor are they ever very severe. As a matter of fact, as we have shown, the union of antigen and antibody in the circulation is inhibited probably by colloidal protection, and this may be regarded as being very likely a protective mechanism. As a matter of fact, Weil and others have shown that a sufficient amount of antibody in the circulation may even protect the cells to some extent against anaphylactic shock, and it is interesting to note in this connection that very large doses of protective anti- serum are necessary to bring about this result, a circumstance which is again easily explained by the inhibition of union between cir- culating antigen and antibody. Concerning the possibility of the formation of poisonous sub- stances in the circulation, produced by the union of antigen-anti- body, a question which is involved in Friedberger's theory of anaphylaxis, we will have more to say in a subsequent paragraph. SYMPTOMS OF ANAPHYLAXIS. — Anaphylaxis differs in its symp- tomatology and pathology according to the species of animal in which shock is produced. There are certain fundamental systemic reactions which are common to all species, but in each species that has been observed, there are particular localization of the immediate and severe changes which lead to acute death. As general symptoms 40 Friedemann, Zeit. f. Immunit., 2, 1909. HYPERSUSCEPTIBILITY 363 we may enumerate drop in blood pressure, fall of temperature, diminution of leucocytes, increased flow of chyle, and certain meta- bolic disturbances, the identity of which in different animal species have not been worked out. In guinea pigs, as first demonstrated by Auer and Lewis, the typical lung inflation which leads to respiratory death is ' due to spasms of the muscles of the bronchioles. In rabbits, acute death is not respiratory, but is a circulatory one, and has been shown by Coca41 to be due to spasms of the muscular coats of the arterioles of the pulmonary circulation, the rabbit's lung during shock development remarkably increased pres- sure against the passage of perfusion fluid. In dogs, acute anaphylactic symptoms have been localized in the liver by Manwaring42 and others. This peculiar physiological difference in various animals in reaction to the same general mechanism of injury has been difficult to understand, but recent observations of Coca, Simonds, Huber and Koessler43 and others have been correlated by Wells into what seems to us a very rational and likely explanation. Wells calls attention to the fact that acute death in guinea pigs is due to spasm of the bronchial muscles, and that anatomically the guinea pig has a very high development of musculature in the bronchii, the smaller bronchioles being " prac- tically nothing but muscular tubes." Similarly, Coca's findings in relation to the pulmonary circulation of rabbits coincides with the histological demonstration that the pulmonary arteries of the rabbit show a marked muscular development. Simonds has shown that the hepatic veins of dogs differ from those of all other animals in having a highly developed musculature, and it would seem, as Wells points out, as though the localization of acute changes in different organs in the various animals were dependent upon fortuitous differences in the anatomical distribution of the smooth muscle. He also points out, as further evidence, that fatal reactions in man have occurred only or most frequently in persons suffering from chronic pulmonary conditions, chiefly asthma; and Huber and Koessler have shown that asthmatic people develop a hypertrophy of the bronchial mus- culature which in its final histology is closely analogous to that of guinea pigs. 41 Coca, Jour. Immimol., 4, 1919, 219. 42 Manwaring, Jour, of Immimol., 2, 1917, 517. "Huber and Koessler, Arch. Int. Med., 1921. 364 INFECTION AND IMMUNITY This explanation of Wells seems to us eminently logical. We would add to it only the following consideration. Acute death may well be caused directly by the acute spasm of smooth muscle tissue, and the acute pathological manifestations may be dependent upon the distribution of such muscle. This does not, however, exclude the likelihood that severe anaphylactic injury may be caused in other cells in the body as well, but these, not being able to react by acute contraction, or by any other pathological alteration that can cause acute and sudden death, may still be injured severely without there being an immediately noticeable effect. ANTI-ANAPHYLA.XIS. — When sensitized animals recover from anaphylactic shock, they do not react to a subsequent injection of the same substance made within a reasonable interval. This desensitization or ' ' antianaphylaxis ' ' as Besredka and Steinhardt have called it, appears immediately after recovery from the second injection. Antianaphylaxis may also be produced if animals which have received the first or sensitizing dose are injected with comparatively large quantities of the same substance during the preanaphylactic period — or, as it is sometimes spoken of, during the anaphylactic incubation time. This injection should not be done too soon after the first dose, but rather toward the middle or end of the preanaphylactic period. If given within one or two days after the sensitizing injection, anaphylaxis will develop, nevertheless. The desensitized condition is a purely transitory state. Besredka and Steinhardt believe that it lasts a long time, while Otto found guinea-pigs immunized in the above manner to lose their antianaphylaxis within three weeks. It is not at all necessary to actually shock an animal to desen- sitize it. The doses may be given gradually, either in small frac- tions or slowly by means of high dilution, as by the method of Friedberger, and gradual desensitization thereby accomplished without noticeable harm.. This is of great practical importance. Desensitization in the ordinary sense probably means a gradual saturation of the sessile antibodies with antigen. It can be demon- strated not only in the living animal but upon the sensitive uterus in the Dale apparatus. Another form of partial protection against anaphylaxis, by the injection of large amounts of specific antiserum has been mentioned above. The mechanism of this is obvious. Desensitization by injection into the rectum or by feeding has HYPERSUSCEPTIBILITY 365 been accomplished but since the absorption of unchanged antigen by these routes is ordinarily slight, little hope can be expected in this direction for practical purposes. There are certain forms of protection against anaphylactic shock produced by the injection of foreign sera, and other proteins and processes non-specific as far as the particular anaphylactic mechan- ism is concerned, but there is too little positive knowledge about these to permit us to discuss them here. ANAPHYLATOXIN THEORIES, ETC. — Vaughan and Wheeler44 early suggested that anaphylaxis might be a poisoning produced as fol- lows. Antibodies are formed by the first injection, which on subse- quent reaction with the antigen administered in the second injection, lead to poisonous protein-split products. A similar idea was ad- vanced by Wolf-Eisner.45 Proceeding from this general concept, Friedberger, whose extensive experimental work may be found in many articles in the Zeitschrift f. Immunitatsforschung, elaborated a theory of anaphylaxis which may be summarized in the following way: When the antigen and antibody meet in the circulation, the union of the two renders the antigen amenable to complement action, and the action of the alexin or complement upon this complex splits off from it a poison which he calls ' ' anaphylatoxin. ' ' He succeeded in producing poisonous substances in vitro by treating specific pre- cipitates as well as sensitized and unsensitized bacteria with alexin. Injection of these substances into guinea pigs caused acute death, analogous in symptoms to anaphylaxis. Friedberger and Hartoch46 showed that there was a diminution of alexin in the serum of animals suffering from acute shock in the course both of active and of passively transmitted anaphylaxis. He showed that the intravenous injection of substances which inhibited complement action in vitro, such as, for instance concentrated salt solution, would diminish and sometimes prevent shock in sensitized animals, a phenomenon, how- ever, which the writer with Lieb and Dwyer47 showed to be due to diminution of the irritability of smooth muscle caused by hyper- tonic salt. It was subsequently shown, however, that similar poisons could be produced from boiled as well as from normal bacteria, that they could be produced by the treatment of fresh guinea pig serum 44 Vaughan and Wheeler, Jour. Infec. Dis., 4, 1917. 45 Wolf-Eisner, Berl. klin. Woch., 1904. 48 Friedberger and Hartoch, Zeit. f. Immunitat., 1909, 3. 47 Zinsser, Lieb and Dwyer, Jour. Exper. Biol. and Med., 12, No. 8, 1915. INDUCTION AND IMMUNITY with kaolin, barium sulphate. The literature of this subject is exten- sive. The most important contributions recent have hem m;iO,/T. /.-it. f. ImiiMinitiit.. 7, UNO. « Novy and DeKruiff, Jour. A. M. A., (is, 1!>I7, 1524. 80 Jobling and Petersen, Jour. Expci. M,-,!., i«», 11)14, No. 5. HYPER8USCEPTIBILITY 367 period after first injection is less than eight days, while in about 14 per cent it is longer than twelve days. The symptoms of serum sickness usually consist of an eruption at the site of injection, which ordinarily comes on quite early, some time before any general eruption is noticed. It often takes an urticaria! form, and becomes general after several days. There is usually some fever, occasionally albuminuria, and there may bo joint manifestations. The joint manifestations are often of a peculiar nature, with slight tenderness, but considerable stiffness, and very little, if any, objective symptoms of the joints, and we remember distinctly a case in which it was difficult to tell whether or not the patient, who had received tetanus antitoxin ten days before, was developing tetanus or not. The case turned out to be one of serum sickness. Serum sickness may also be accompanied by leucopaenia, aild, according to Weil,51 by drops in blood pressure and decreased coagulability of the blood. The latter manifestations, as Wells points out, bring it still closer to true anaphylaxis. When a human bring is being treated for the second time with horse serum at intervals longer than two or three weeks, the re- semblance to true anaphylaxis is still greater, and as Doerr points out, the procedure is more dangerous and will vary in its mani- festations, according to the length of time elapsing between the two injections. If the injections are not very much more than a month apart, there may be, according to Von Pirquet and Schick, an immediate reaction, which takes the character of severe serum dis- ease. At the point of injection there may be swelling and edema, within twenty-four hours, with general symptoms such as those described above but rather more severe, within one or two days. If the injections are months and years apart, the onset, while usually more rapid than at the first injection, is still likely to occur more rapidly than when the antigen is given the first time. Van Pirquet and Schick, from the beginning, believed that serum sickness was due to the reaction of antigen which had not yet dis- appeared from the circulation of the patient at a time when anti- bodies were already being actively formed. Opinions as to whether' scrum sickness is to be regarded as true anaphylaxis or not, seem to differ at the present time. Coca par- ticularly seems to hesitate about incorporating these phenomena into ri n cil, Jour. Immimol., 2, 1917, 399. 368 INFECTION AND IMMUNITY those of true anaphylaxis. We cannot go into the controversial literature in this place, but may set down our own opinion that we think that the time interval between observation of symptoms and injection, on first administration, the speeding up of symptoms in cases of second and third injections, the nature of the symptoms, and the relationship between serum sickness and antibody formation in the patient, as pointed out by C. W. Wells,52 as well as more recently by MacKensie and Longcope,53 together with the phenomena of desensitization of patients, leave little room for doubt that this peculiar condition is fundamentally of an anaphylactic nature. It is not so easy to include in true anaphylaxis the apparently inherited sensitiveness to foreign proteins most frequently observed as food idiosyncrasies. Whether or not these belong into the cate- gory of anaphylaxis, the future must reveal. SKIN REACTIONS. — There are two kinds of skin reaction, sone which can be obtained, for instance, in horse serum sensitive people by the injection of minute quantities, 0.1 to 0.01 c.c. of horse serum intra- cutaneously injected. In these cases, within a few minutes to one- half hour, a growing urticarial wheal begins to appear which fades again within an hour or longer. This reaction has been variously used to determine whether or not patients possessed a high degree of sensitiveness just before the administration of therapeutic sera. We have recently experimentally studied the relationship of such immediate skin reactions with generalized anaphylaxis in guinea pigs, and have found that the two phenomena correspond with con- siderable accuracy, namely, that guinea pigs give definite immediate skin reactions during the periods at which they are highly sensitive to intravenous injections, and that recovery from severe anaphylactic shock desensitized them not only to general anaphylaxis but to the skin reaction as well. There is another form of skin reaction typified by the tuberculin reaction and the typhoidin reaction in which very little manifest change occurs during the first two hours, but in which after twelve to twenty-four hours, definite inflammatory swellings occur at the point of injection with the occasional development of a little central neucrosis and hemorrhage, and true cell injury. Such reactions fade only after three or four days or longer, and are similar in 62 Wells, C. W., Jour. Infec. Dis., 16, 1915. 53 MacKensie and Longcope, Med. Sec. N. Y. Academy Med. April, 1921. HYPERSUSCEPTIBILI TY 369 many ways to the toxin skin reactions as observed in the Schick test with diphtheria toxin. The relationships of these latter reac- tions to anaphylaxis has been much questioned and with justice. A recent analysis by ourselves in the case of the tuberculin reaction has shown that these reactions are not anaphylactic in the ordinary sense, but are brought about by substances probably proteose in nature which among other things, differ from the true anaphylactic antigens in being more permeable than these, and penetrating into cells. We refer the reader to our article in the J. of Exp. Med., unpublished, probably 1921. METHODS OF TESTING AND DESENSITIZATION IN INDIVIDUALS ABOUT TO BE INJECTED WITH ANTITOXIC OR ANTIBACTERIAL SERA. — A consider- able degree of practical importance attaches to the anaphylactic phenomena in connection with the administration of sera. When small doses of tetanus or diphtheria antitoxin are to be administered for the first time, it is generally unnecessary to precede this with a skin test. Although we should always advise that this is done in asthmatics or in people who have suffered from chronic coughs or the prolonged pulmonary inflammations that are apt to occur in children following measles, influenza or whooping cough. This is necessary for the reasons stated above and pointed out by Huber and Koessler, which demonstrated the hypertrophy of smooth muscles in the bronchioles of such people. When intravenous serum injections are to be made, as in the treatment of pneumonia and meningitis, it is best to precede the injection with a skin test. Such tests have been done more fre- quently than anywhere else, we believe, at the Rockefeller Hospital, where 0.02 c.c. of a 1 :10 dilution of horse serum is injected intra- cutaneously with a similar injetcion of sterile salt solution as control. In unsensitive people the wheal of the injection disappears rapidly. But in sensitive ones it will begin to increase after five or ten minutes, and within four hours may show a large red erythematous, urticaria-like area. In such cases, careful desensitization should be practiced. The precautions to be taken are twofold. One consists of a preliminary attempt at desensitization, the other in slow administration by dilu- tion of the serum during the injection of the therapeutic dose. The desensitization is best accomplished by the method of Besredka54 which consists in the gradual injection of progressively 64 Besredka, Bull, de 1 'Inst. Past., 6, 1908, 826. 370 INFECTION AND IMMUNITY increasing doses of the antigen, in this case horse serum, over an interval of a number of hours. Cole55 states that it is safe to begin with a subcutaneous injection of 0.025 c.c. of scrum, and at one-half hour intervals thereafter, giving further doses, doubling the amount each time. Adverse symptoms should of course lead to still greater care and a lengthening of the interval. If by this careful method finally 1 c.c. has been given at a dose without adverse symptoms, 0.1 c.c. may be given intravenously, and continued every one-half hour with double the dose. Cole recommends continuing this careful procedure until about 25 c.c. total of the serum has been given. He then waits about four days, and gives the remainder of the dose. It stands to reason that no absolute rules for such a procedure can be given and that after the general principle has been under- stood, the individual physician must be guided by close observation of the patient and familiarity with the symptoms to be expected. The adverse symptoms to be expected are immediate respiratory distress, rapidity of the pulse, and there may be coughing. In finally giving the serum, in such cases, in the larger quantities, it should be diluted with sterile salt solution by at least one-half, and may be so given by gravity that the first 10 c.c. should occupy about ten minutes. HAY FEVER, URTICARIA, ETC. — The clinical condition spoken of as hay fever, asthmatic attacks called forth by apparent hypersus- ceptibility to certain kinds of dust, the emanation of animals, etc., etc., cannot at present be classified clearly with true anaphylaxis because of certain apparent differences from ordinary anaphylactic phenomena which cannot be ignored. Dunbar56 at first regarded the susceptibility to pollen or hay fever as due to a toxin. This, however, is not tenable on present experimental evidence. The toxin idea is not tenable because of the fact that normal insusceptible individuals do not react to many times the amounts to which the hay fever patients respond. Numerous studies upon the inheritability of the hay fever tendency seemed to show that a tendency to hyper- sensitiveness of this kind may be hereditary, an observation which in the train of a number of other investigators has recently been corroborated by Cooke and Vander Veer.57 They seem to believe 55 Cole, Monograph of the Eock Inst., 1917, No. 7. 58 Dunbar, cited from Doerr, Kolle and Wassermann Hamlbiich, Edit. 2. 67 Coolce and Vander Veer, J. of Immun. I, 1916, 201. HYPERSUSCEPTIBILITY 371 ' ' that the tendency is inherited as a dominant characteristic. ' ' The question of whether or not hay fever is a phase of protein sensitiza- tion, then, cannot be answered either affirmatively or negatively at the present time. It is accompanied by cutaneous sensitiveness to pollen extracts and, according to Cooke and Vander Veer, subcu-. taneous injections of such extracts into sensitive individuals may produce a general eruption. On the basis of its heredity, Cooke and Vander Veer and Coca58 remove the hay fever complex from the class of protein anaphylaxis. Coca claims in addition that pollen extracts are not anti genie in the ordinary sense , of the word, but in this he is mistaken, since Mrs. Parker in our laboratory recently proved that extracts made by us, as well as the ones used by Cooke and Vander Veer, could be used as anaphylactic antigens if the methods of sensitization and of test were sufficiently delicate. DRUG IDIOSYNCRASIES. — Abnormal sensitiveness to many drugs be- longing to almost all chemical classes of substances, inorganic and organic, has been noted for years by clinicians. Among them are morphin, strychnin, atropin, salicylates, halogens, and their com- pounds, salvarsan, etc. These substances are obviously not antigenic in the ordinary sense. The hypersusceptibility is generally specific at least for the chemical group, and it seems to be a fact that the reaction elicited in the individual does not represent exaggerated symptoms of the physiological effects of the drugs, but are, in a general way, alike, whatever the drug used. The symptoms usually come on rapidly, within a few hours or days, and consist in various kinds of skin rashes, and fever. In such cases, where salvarsan, iodin, etc., preparations have been used, there may be marked and rapidly developing local inflammatory effects at the point of inocula- tion. Drug idiosyncrasies cannot be transmitted passively, and, so far, no conclusively successful experiments on artificial hypersen- sitization of animals with these substances have been made. There, is only one exception to this, namely the experiments of Swift59 with salvarsan in which active sensitization of guinea pigs with salvarsan-guinea pig serum mixtures resulted in apparently success- ful, though inconclusive, results. There is no adequate explanation at the present time for drug idiosyncrasies. The most reasonable suggestion which has been made, however, is the one that drugs enter into some form of com- 58 Coca, Tice's Practice of Medicine, Vol. 2, 1920. 59 Swift, J. Exp. .Med. 24, 1916, 373. 372 INFECTION AND IMMUNITY bination with the serum proteins of the host, which are then so altered in their antigenic properties that they can act as antibody inciting or, in other words, antigenic substances. When horse serum or other animal sera for instance are treated with iodin, an iodin- protein combination is formed, which represents an antigenic altera- tion of the original serum protein. Animals injected with such an iodized horse serum will produce antibodies which are, to some extent, specific for this iodin-protein combination. It is not impos- sible that this is the fundamental basis for drug allergy, but there are very strong arguments against this assumption, particularly the failure up to date to produce such conditions uniformly in animals by active or passive sensitization. It would seem to us futile at the present time to make either positive or negative statements in this respect. ANAPHYLAXIS IN INFECTIOUS DISEASE. — There can be little doubt about the fact that both general and localized hyper sensitiveness play an important role in infectious disease. Whenever an infection becomes subacute or chronic, the body may become sensitized to the coagulable protein in the bacterial body. This we have ourselves shown with typhoid protein, and with tuberculous guinea pigs and tuberculo-protein ; and, in the case of tuberculosis, our experiments done with the Dale method corroborated the previous experiments of Baldwin,60 Krause61 and others who worked by the intravenous method. Thus, in all infectious diseases which last any length of time, we can count upon anaphylactic phenomena to participate in the general symptomatological and pathological picture. This sub- ject is still very much a matter of experimental investigation, and a discussion of it would carry us too far afield in the present connection. Delayed skin reactions, like the typhoidin, tuberculin, etc., reac- tion, are, we believe, phenomena of specific sypersensitiveness to substances of a somewhat less complex molecular structure than the proteins, substances which do not produce antibodies in the usual sense; these substances are more diffusible than are the true pro- teins, can get inside the cells, and their reaction with the cells, then, is an intracellular one in which the intervention of sessile antibodies is not necessary. For a further analysis of this relation- ship and the experimental basis for the statements we make, we refer the reader to our unpublished article mentioned above. "Baldivin, J. Med. Kes., 119, 1910. 61 Krause, Amer. Eev. Tuber., 1, 1917, 65. SECTION III PATHOGENIC MICROORGANISMS CHAPTER XX AN INTRODUCTION TO THE STUDY OF INFECTIOUS DISEASES THE RELATIONSHIP OF BACTERIOLOGY TO THE CLINIC AND TO PUBLIC HEALTH THE problem of infectious diseases is peculiar in that, more than any other branch of medicine, it calls for the intimate cooperation of the laboratory worker, the clinician and the sanitarian. It is a subject in many phases of which problems of engineering are im- portant, in which food production plays a part, and to which the educational and sociological agencies of the community as a whole can contribute very materially. The eventual suppression of in- fectious diseases cannot be accomplished by any one of the agencies mentioned, or by all of them together without the cooperation of the public in general. In many respects the study of infectious diseases is the most logical branch of medicine at the present time. In many of these conditions we are familiar with the causative agents, we know the manner in which they gain entrance to the body, where they lodge and multiply, where and how they form their secondary foci, what manner of poisons they produce, and what reactions they call forth in the living animal tissues. We can often isolate the bacteria from the bodies of the sick and the dead; we can recognize them when we isolate them in locations outside the body; we can study their biological activities, and their poisons both apart from the animal body and in animal experimentation. Moreover, both in animals and man we can study directly the reactions of the body to invasion, and the agencies by which the invaders are destroyed. For these reasons, the first requirement for a thorough clinical 373 374 PATHOGENIC MICROORGANISMS comprehension of infectious disease as it occurs in man, is a funda- mental biological knowledge of the bacteria themselves, their actions upon artificial media, the characteristics by which they can be dif- ferentiated, the conditions under which they grow and produce poisons, and the reactions which they or their poisons elicit in animals. Of course there are many points in such a chain of reason- ing which investigation has not yet cleared up, and there is, especially, much uncertainty and half-knowledge in regard to some of the most important phases of the chemical and immunological reactions which take place between the invaders and the animal body. Moreover, it is quite likely that many of our theories con- cerning the fundamental principles which govern such reactions are defective. But without, at least, a knowledge of the biological facts available, the physician who is confronted by a human infection is working very largely in the dark. It is the task of the specialized laboratory worker to prepare this material and submit it to the clinician so that we may use it in the premises of his reasoning. Considered in this way, an infection in an animal or a human being resolves itself into a balance between the forces of infection, on the one hand, and the injuries and resisting mechanisms of the infected subject, on the other. We may consider the entrance of a foreign living being into the tissues of a higher animal or plant as a process in which a struggle for existence is initiated. The invading microorganism must take its nourishment from the invaded host, abstracting thereby materials needed by the host, and, in the course of its multiplication and digestive processes, it not only injures the host mechanically by local accumulation, but also by the remote action of the substances which it produces in the course of its metabolism, many of which are toxic. The host, if not overwhelmed, responds by reactions which express themselves both in local morphological changes in places where direct contact with the bacteria is established, and by remote systemic reactions incited by absorption not only of the toxic derivatives of the bacteria, but also to some extent of the products of the local struggle in which proteolytic destruction, necrosis, etc., are involved. The symptom complex, therefore, which we recognize as disease results in part from injury, but to a larger extent repre- sents the manifestations of the defense reactions of the host. When a physician makes a diagnosis of a particular infection by "history and physical examination, he does so on the basis of his own observa- INTRODUCTION TO THE STUDY OF INFECTIOUS DISEASES 375 tions and of those of his clinical predecessors, which have taught him that a certain kind of bacterial invader elicits characteristic reactions on the part of the host. In many cases, however, micro- organisms of entirely different biological classes may elicit clinical pictures of great similarity, because of analogous methods of inva- sion, like degrees of virulence and similar selective distribution. Pneumonias caused by pneumococci and Friedlander bacilli may show very little clinical difference for these reasons, and septicemic invasions of the blood by a variety of microorganisms may not be clinically differentiable. On the other hand, one and the same species of microorganism may cause widely divergent clinical pic- tures under conditions of varying balance between the virulence of the invader and the resistance of the host. A streptococcus of low virulence in a vigorous subject may for instance cause only a local- ized abscess, whereas, if the virulence is enhanced and the subject very susceptible, the localized symptoms may be negligible and a septicemia with secondary localizations and fatal in outcome may result. The accurate diagnosis of an infectious disease, therefore, depends first of all upon a clinical understanding of the reactions of the human body with the different microorganisms that can invade it, a knowledge of the manner in which the different forms can enter the body, how they progress and are distributed, where in the body they are apt to accumulate, how great a degree of fluctua- tion in virulence and toxicity can be expected from them, what poisons they produce and what the pharmacological action of these poisons is. While such information may often, and must frequently suffice to make the diagnosis, yet a knowledge of the manner in which blood cultures, urine cultures, stool cultures, throat cultures, etc., can be made, is necessary to affirm the diagnosis in relatively clear cases and to determine it in doubtful cases; and this implies not only a knowledge of the preceding facts, but also both the knowledge and the technical ability supplied by the trained bac- teriologist. Moreover, the dependence of clinical understanding of infectious diseases upon bacteriological knowledge is not a bit less intimate than is that of preventive measures or sanitation. For purposes of prevention, every infected individual or every carrier of a pathogenic organism may be regarded as the potential source for infection of other human beings. 376 PATHOGENIC MICROORGANISMS Some diseases will of necessity remain sporadic because the infec- tion of a new individual can be brought about only by unusually depressed resistance or by accidentally enhanced virulence on the part of the causative agent. And in some diseases transmission requires conditions of contact which are not an ordinary feature of intercourse between the members of communities. Diseases, thus, which under ordinary conditions of civilized life may occur more or less frequently as sporadic scattered cases, may become epidemic only when life in army cantonments, in crowded and unsanitary city quarters, subject to poverty, filth and neglect, produces com- munity susceptibility and facilitates transmission. Such, as we shall see, is the case with many respiratory infections, especially the pneumonias. Other diseases, on the other hand, are characteristically epidemic because the ordinary virulence of the microorganisms is such that practically all normal human beings may be regarded as susceptible and because the most important avenues of invasion are open in the course of normal community association. Such diseases are plague, smallpox, cholera, influenza and, to a less extreme degree, the enteric fevers. For this reason, the ultimate basis of sanitation in infectious diseases depends upon close observation of the possible sources of infection so that they may be circumscribed before broadcast dis- semination has taken place; it involves the routine safeguarding of ordinary community life in such a way that the avenues of trans- mission for the various possible invaders may be controlled, and finally it necessitates attention to the maintenance of the resistance of the community as a whole, both by the hygiene of every day life, the prevention of undue lowering of resistance of large groups by economic or other hardships, or, as in smallpox and typhoid fever, by the artificial reenforcement of community resistance by methods of immunization. The source of infection lies invariably in direct or indirect trans- mission of microorganisms from a human or animal source. Every case of infection represents the possible origin of many others, and necessitates surrounding the patient with all the safeguards appro- priate to the type of infection from which he is suffering, based on knowledge of the manner in which the particular disease is transmitted. If the diseased individual were the only problem, the task would be relatively easy. As we shall see, however, in the INTRODUCTION TO THE STUDY OF INFECTIOUS DISEASES 377 subsequent chapters, a great many infectious agents may live sapro- phytic lives in and upon the bodies of human beings, who themselves are not suffering from the disease. Such individuals are known as carriers, and the carrier problem has infused difficulties into sanitary procedure which, in some cases, it is almost impossible to combat. Thus, every community has in it a definite percentage of typhoid and paratyphoid carriers; many individuals carry meningococci, diphtheria bacilli and virulent pneumococci and streptococci; there are quite surely carriers of poliomyelitis and scarlet fever, and it is not impossible that the carrier state can exist for a number of other diseases in which we have not yet been able to prove the condition by actual experiment. In addition to the case and the carrier, a source of great danger are unrecognized mild cases. Typhoid fever may take a very mild form, especially in vaccinated people; the atypical cases of poliomyelitis which occur in the course of every epidemic and may occur in interepidemic periods may not be recognized until secondary cases occur; and in many adults who possess a relatively high im- munity, diphtheria infection of the throat may be so mild that no suspicion of the disease is aroused. It is in connection with such occurrences that the diagnostic acumen of the practicing physician is especially important, and it is in the recognition of the atypical cases and in the early diagnosis of the ordinary cases, that the practitioner represents the first line of defense against epidemic outbreaks. It is for this part of the protective campaign that we need reporting systems, so that early cases may be centrally collected and charted. Organizations for epidemiological survey to trace the early cases to their sources, and laboratory units to affirm the diagnosis, search out the possible carriers and trace the infection, if possible, to contaminated food or water. Here, too, are necessary arrange- ments for isolation and hospitalization which involve the bac- teriological control by which may be determined when it is safe to release the patient for free association with his fellows. The transmission of infectious disease may be either by direct contact from person to person, by indirect contact through materials that have passed from the sick or the carrier to the new victim by food and water and by conveyance through the agency of insects. These factors will be considered in detail in connection with every individual disease, and it is quite clear that, in order to properly 378 PATHOGENIC MICROORGANISMS safeguard a community, it is necessary to know where in the body of the sick or the carrier the organisms are to be found; how they may leave the body; what the viability of the infectious agent is in nature; how long it can live under different conditions of environ- ment in the interval between its leaving one body and entering the next, and by which channels it most easily infects. Also, the habits of insects that can carry disease must be studied. To interrupt the chain of transmission from source to victim, .also, there must be a routine organization for the safeguarding of water, food and other agencies which in crowded communities are always in danger of contamination. And in special cases it may involve emergency measures of personal hygiene, engineering prob- lems and insect destruction. In thinking of infectious diseases from the point of view of sanitation it is well to carry them in one's mind in the tentative classification based upon means of transmission, since for each par- ticular subdivision of this kind, preventive measures will fall into a definite common plan of procedure. Thought of in this way, all the infectious diseases fall into four groups : (1) The first of these consists of those transmitted by the respira- tory channels. This means that the infectious virus leaves the case, convalescent or carrier, witli the saliva or mucus of the upper respiratory passages and enters the new victim by the same chan- nels. On this basis, the respiratory group would consist in fhe common cold, the pneumonias, influenza, scarlet fever, measles, smallpox, chickeiipox, mumps, whooping cough, tuberculosis, diph- theria, meningitis, poliomyletis and a few others. In dealing with such diseases, the task is to suppress conditions which would make it possible for a wholesale distribution of sputum, for close contact between individuals in sleeping quarters, the avoidance of crowding in homes, schools, institutions, etc., ventila- tion, dust prevention, and, in short, to diminish as much as possible the opportunities for close individual contact in closed spaces. In this group of diseases, prevention is most difficult because it is quite obvious that contacts need no1 be of long duration, and that close association in public vehicles, and in the ordinary course of business and social intercourse may suffice for transmission. In these dis- eases, particularly, when there is universal susceptibility, as in the case of influenza or smallpox, or some of the exanthemata in the INTRODUCTION TO THE STUDY OF INFECTIOUS DISEASES 379 case of children, epidemic spread is almost unavoidable if unsani- tary, close association exists. Fortunately, in many of the diseases so transmitted, namely, pneumonia, meningitis, diphtheria and a number of others, the normal resistance . of the human being is relatively high and epidemic occurrence takes place only when unusual conditions prevail. (2) The intestinal group consisting very largely of typhoid and the paratyphoid fevers, the dysenteries, cholera, the food poisonings, and the simple diarrheas. In this group sanitation, apart from isola- tion of the recognized case, focuses upon a constant vigilance in regard to the distribution of dejecta from human beings, for every infection signifies a sanitary defect in the transmission of the con- tents of the bowels of one human being to the mouth of another by any one of a variety of routes. Sanitation here requires proper sewage disposal, the care of privies and latrines, and careful control of water and food supplies, since naturally large epidemics can be most easily brought about in this group by the contamination of the daily diet of the community. In these diseases, wholesale infec- tion with water and milk is constantly diminishing, as we are im- proving in our sanitary organizations. But contact epidemics from person to person becoming relatively more prominent. Kecent studies are tending, for instance, to show more and more the in- creasing importance of contact infection in typhoid fever. Schule1 studying the reports of the German Laboratory at Trier for 1918, states that of the typhoid cases occurring in the district controlled by this laboratory during that year, 60 per cent were due to contact with cases, 5 per cent were due to contact with carriers and only 1 per cent were due to water and milk each. Of 5,889 cases analyzed at this laboratory, 71 per cent were contact cases. The manner of contact may vary, and Gay2 summarizes in the following way, the manner in which this may take place : 1. Fingers or utensils — mouth. 2. Fingers — food — mouth. 3. Fomites — fingers — food — mouth. 4. Flies — food — mouth. 5. Fingers — flies — food — mouth. In the case of the first route, more or less direct contact with a case is implied. In each of the five routes it is easy to draw 1 Schule, Mil. Surgeon, 45, 1919, 268. 2 Gay, Typhoid Fever, Macmillan Company, N. Y., 1918. 380 PATHOGENIC MICROORGANISMS vertical lines across the dashes and see where and how sanitary interference may interrupt the progress of transmission. Thus, in the prevention of the intestinal infections, the municipal and state authorities must care for the water supplies and sewage disposal plants ; the administrative public health authorities must be supplied with a reporting system for the early recognition and demarkation of foci, and must epidemiologically attempt to trace existing cases to their sources, following this with carrier examinations of sus- pected small groups ; physicians must act as the first line of defense in regard to early diagnosis, intelligent, immediate isolation and the collection of the first significant epidemiological information for the use of the health authorities; city cleaning departments and other agencies must aid in preventing fly breeding, in garbage disposal, etc., and last but not least, general educational campaigns must elicit the intelligent cooperation of the public by supplying the simple information which is necessary for individual protection. As a matter of fact, if the public realized that most cases of typhoid and the paratyphoid fevers could be suppressed at the present time by a regular hand washing after defecation and before meals, the problem would be largely solved. (3) The third group comprises diseases in which intimate contact between infectious material and the external surface of the body of the new victim is necessary. In some of these diseases trans- mission can take place without a visible break in the skin, as perhaps in plague, rabies, syphilis and some others, where the lesion through which the virus can enter may be so microscopical in size that no visible trauma is apparent. In others, such as the pyogenic infec- tions, glanders, anthrax and the anaerobic infections, trauma of some kind is usually necessary. Few of these diseases can ever become epidemic to any great extent in the ordinary sense of the word. Some of them, however, like the venereal diseases may be regarded as so plentifully endemic, owing to the nature of their transmission, that we may look upon the present condition of com- munities as subject to a constant subacute epidemic state. Venereal diseases are a special sanitary group which requires individual treatment which we cannot enlarge upon in this place. (4) In the fourth group are those diseases which are transmitted by insects. Such are malaria, yellow fever and dengue fever, trans- mitted by different species of mosquitoes, African sleeping sickness, pappataci fever, transmitted by flies, Rocky Mountain spotted fever INTRODUCTION TO THE STUDY OF INFECTIOUS DISEASES 381 and African relapsing fever, transmitted by a species of tick, European and Balkan relapsing fever, and kala-azar transmitted by bed bugs, and typhus fever, trench fever and some varieties of relapsing fever transmitted by lice, and plague largely transmitted by fleas. In the sanitation of these diseases it is quite obvious that, in addition to our knowledge of the pathogenic microorganisms which cause the disease, we must study carefully the habits of insects, their seasonal occurrence, their nocturnal or diurnal habits, their methods and places of breeding, the distances which they can travel, the manner in which they acquire the parasites and the manner in which they can be suppressed. Based upon an understanding of the conditions outlined above, moreover, is the science of epidemiology which, in its turn, can in- directly contribute a great deal, both to the solution of the problems of bacteriology and to those of transmission. For, by epidemiological surveys, by a careful study of the manner of spread of disease from group to group, and from place to place, the explosiveness with which it appears and relationship of cases to personal contact, water or milk supplies, etc., many deductions can be made which have important bearing in guiding bacteriological investigation and preventive measures. As to preventive measures, nothing is so im- portant in the suppression of an epidemic disease as the rapid circumscription of the initial focus, a thing which can be accom- plished only by prompt epidemiological survey, backed up by accurate bacteriological diagnosis. Such results can be achieved only when communities have efficient organizations for the prompt reporting of communicable diseases, for the systematic charting of the cases and for intelligent and experienced study of such charts. Indeed, studies of this nature alone may often make possible a causal classification of the epidemic before its bacteriological nature is accurately known. For epidemics will vary in the respects men- tioned above, very largely according to whether they are water borne, distributed by milk or other food, or whether they are spread by contact or by insects. It is probable that small water epidemics from individual rural house supplies may still be quite frequent and simulate contact epidemics. The larger water epidemics of typhoid, cholera, etc., will become, as we have stated, more and more infrequent as water supplies are more carefully supervised, but when they do occur, 382 PATHOGENIC MICROORGANISMS their onsets and courses will be characteristic to a degree which makes it possible for an experienced epidemiologist to suspect the water simply by a study of the incidence of cases in time and place. To some extent, of course, this depends upon whether the water is polluted by a single introduction of large quantities of sewage or whether contamination is continuous over a longer period. In both cases, however, a large number of the people living in the area of the water supply will be infected during a relatively short period of time. The rise of the curve which can be constructed from the cases, therefore, will be steep and rapidly reach a peak. Classical instances of this are the cholera epidemic in Hamburg and an epidemic of typhoid fever in an American city which is described in the section on typhoid fever. The incidence of the cases in places will be sharply limited by the distribution of the water supply and examination of the water will reveal colon bacilli. In milk epidemics, a still more explosive rise of the cases will appear and in such cases as the Stamford epidemic described by Trask, a definite connection between the milk route and the dis- tribution of cases may be traced. On the basis of such suspicion, the bacteriologist can investigate the milk and attempt a determina- tion of recent intestinal disease or the carrier state in milk handlers. Also, it is stated by many who have studied these epidemics that milk epidemics are apt to claim the largest numbers of victims among women and children. Contact epidemics will proceed by a more insidious course. In such epidemics it is extremely important to gather together careful data concerning the earliest cases observed and to attempt to trace the cases to some association at a common meal, a restaurant or at some other common source of food. Sawyer traced a "contact epidemic to the carrier state in a ship's cook by a simple epidemiological study of the individual cases which all led by separate trails to the same ship 's galley. We have, ourselves, traced cases in this way to company kitchens in military units. In large communities contact epidemics may trail along for long periods of time and when association is indiscriminate it may be necessarily impossible to establish accurate relationships. In some contact epidemics, such as those occurring in the Allied Armies in France when intestinal diseases appeared in large numbers, the rise of the curve simulated that of a water epidemic very largely because the indiscriminate distribution of unprotected dejecta over large areas INTRODUCTION TO THE STUDY OF INFECTIOUS DISEASES 383 of recently occupied territory, the prevalence of flies, and the crowd- ing of large masses of men in small areas made frequent contact unavoidable. Such conditions, however, are not likely to happen to any extent at ordinary times, and contact epidemics are usually limited in area and distribution, and careful epidemiological study of three or four cases may lead to the original focus which can then be determined by prompt laboratory investigation. CHAPTER XXI THE STAPHYLOCOCGI (MICROCOCCI) THE power to incite purulent and sero-purulent inflammations and localized abscesses in man and animals is possessed by a large variety of pathogenic bacteria. Most infections, in fact, in which the rela- tive virulence of the incitant and the resistance of the infected subject are so balanced that temporary or permanent localization of the infectious process takes place are apt to be accompanied by the formation of pus. The large majority of acute and subacute purulent processes, however, are caused by the members of a well- defined group of bacteria spoken of as the pyogenic cocci. Among these, pre-eminent in importance, are the " staphylococci" or "micro- cocci." Many of the earlier investigators of surgical infections had seen small round bodies in the pus discharged from abscesses and sinuses and had given them a variety of names. Careful bacteriological studies, however, were not made until 1879 and the years imme- diately following, when Koch, Pasteur, Ogston,1 and others not only described morphologically, but cultivated the cocci from surgical lesions of animals and man. Of fundamental importance are the studies published by Rosenbach2 in 1884, in which the technical methods of modern bacteriology were brought to bear upon this sub- ject for the first time. The group of staphylococci — so named from their growth in irregular, grape-like clusters — is made up of several members, by far the most important of which, pathologically, is the Staphylococcus pyogenes aureus. STAPHYLOCOCCUS PYOGENES AUREUS Morphology and Staining. — This microorganism, the most fre- quent cause of abscesses, boils, and many surgical suppurations, is a spherical coccus having an average diameter of about 0.8 micra, 1 Ogston, Brit. Med. Jour., 1881. 2 Rosenl)acli, f ' Microorganismen bei Wundinf ektion, " 1884. 384 STAPHYLOCOCCUS PYOGENES AUREUS 385 but varying within the extreme limits of 0.4 to 1.2 miera. Any considerable variation from the average size, however, is rare. The perfectly spherical character may not develop, whenever, as is usually the case, two or more are grouped together, unseparated after cell cleavage. In this case, adjacent cocci are slightly flattened along their contiguous surfaces. Examined in smears from cultures or pus, the staphylococci may appear as single individuals, in pairs, or, most frequently, in irregular grape-like clusters, Occasionally, short chains of three or four may FIG. 43. — STAPHYLOCOCCUS PYOGENES AUREUS. (After Gunther.) be seen. In very young cultures in fluid media, the diplococcus form may predominate. The staphylococci stain with all the usual basic aqueous anilin dyes, and, less intensely, with some of the acid dyes. Stained by the method of Gram, they retain the amlin-gentian-violet. Gram's method of staining is excellently adapted for demonstration of these cocci in tissue sections. Although exhibiting marked Brownian movements in the hang- ing drop, staphylococci are non-motile and possess no flagella. They are non-sporogenous and form no capsules. Cultural Characters. — Staphylococci grow readily upon the usual laboratory media. The simpler media, made of meat extract, are quite as efficient for their cultivation as are the freshly made meat- 386 PATHOGENIC MICROORGANISMS infusion products. The optimum temperature for staphylococcus cultivation lies at or about 35° C., though growth readily takes place at temperatures as low as 15° C., and as high as 40° C. Slow but denfiite growth has been observed at a temperature as low as 10° C. While development is most characteristic and luxuriant under aerobic conditions, staphylococci are facultatively anaerobic on suit- able media. They grow readily in an atmosphere of hydrogen. As to the reaction of media, staphylococcus develops most favor- ably upon those having a slightly alkalin titer. Moderately in- creased alkalinity or even moderate acidity of media does not inhibit growth. On gelatin plates, growth occurs readily at room temperature, forming within thirty-six to forty-eight hours, small, shining, pin-head shaped colonies, appearing, at first, grayish-white, and later assuming a yellowish hue, which intensifies into a light brown and often a bronze color as the colony grows older. The intensity of the color differs con- siderably in different races of staphylococci. Liquefaction of the gelatin occurs, and, shallow, saucer-shaped depressions are formed about the colonies after forty-eight hours or more. These zones of fiuidification grow larger as the colonies grow, finally becoming con- fluent. Microscopically, the colonies themselves, before liquefaction has destroyed their outline, are round, rather finely granular, with smooth edges. They are not flat, but rise from the surface of the medium as the segment of a sphere. In gelatin stab cultures in tubes, fluidification leads to the formation of a funnel-shaped depression, with, finally, complete liquefaction of the medium and sedimentation of the bacteria. Liquefaction of gelatin by the staphylococcus is due to a ferment-like body elaborated by it, which is spoken of as "gela- tinasQ. ' ' This substance can • be obtained apart from the cocci by the filtration of cultures.3 It is an extremely thermolabile body. On agar plates the characteristics of the growth, barring liquefac- tion, are much like those on gelatin. Colonies do not show a tendency toward confluence, remaining discrete, and show a rather remarkable difference in the size of the colonies occurring upon the same plate. Upon slanted agar in tubes, rapid growth occurs, at first grayish-white, but soon covering the surface of the slant as a glistening, golden-brown layer. In broth, growth is rapid, leading to a general, even clouding of *Loeb, Cent. f. Bakt., xxxii, 1902. STAPHYLOCOCCUS PYOGENES AUREUS 387 the medium, and giving rise, after forty-eight or more hours, to the formation of a thin surface pellicle. As growth increases, the bacteria sink to the bottom, forming a heavy, mucoid sediment. The odor of old cultures is often peculiarly acrid, not unlike weak butyric acid. FIG. 44. — STAPHYLOCOCCUS COLONIES. In milk, staphylococcus causes coagulation usually within three or four days, with the formation of lactic and butyric acids. On potato, growth is abundant, rather dry and usually deeply pigmented. Upon coagulated animal sera, rapid growth takes place and even- tually slight liquefaction of the medium occurs. In nitrate solutions, reduction of the nitrates to nitrites is caused. In Dunham's broth, indol is not formed. Bayne- Jones and Zin- ninger have studied 115 strains of various staphylococci in all kinds of media suitable for the production of indol, and have not found a single indol producer. In media containing the carbohydrates — dextrose, lactose, or sac- charose— acidification takes place with the formation chiefly of lac- tic, butyric, and formic acids. There is no gas formation, however. 388 PATHOGENIC MICROORGANISMS In protein media free from sugars, the staphylococcus produces alkali. The reducing action of staphylococcus is shown by decolorization in cultures of litmus, methylene-blue, and rosanilin.4 Pigment Formation. — Differentiation between the various mem- bers of the staphylococcus group is based largely upon the formation of pigments. These pigments, so far as we know, seem to be species characteristics. Thus, Staphylococcus pyogenes aureus is recognized primarily by its production of a yellowish-brown pigment, varying in different strains from a pale brown hue to a deep golden yellow. Prolonged cultivation upon artificial media may lead to a diminution in the depth of color produced.5 It appears only when cultivation is carried on under freely aerobic conditions, anaerobic cultivation resulting in unpigmented colonies. The coloring matter is insoluble in water but soluble in alcohol, chloroform, ether, and benzol.6 According to Sch'neider,7 the pigment belongs to the class of "lipo- chromes" of fatty pigments, and is probably composed of carbon, oxygen, and hydrogen, without nitrogen. Treatment with concen- trated sulphuric acid changes it to a green or greenish-blue.8 Neisser9 states that the pigment of staphylococci is excreted into the media by the organisms but does not diffuse because it is not soluble in water. Differences in pigment have been the basis of differentia- tions within the micrococcus group as we shall see below. Resistance. — Although not spore formers, staphylococci are more resistant to heat than many other purely vegetative forms. The thermal death point given for Staphylococcus pyo genes aureus by Sternberg10 lies between 56° and 58° C., the time of exposure being ten minutes. The same author states that, when in a completely dried state, the coccus is still more resistant, a temperature of from 90° to 100° C. being required for its destruction. Against low tem- peratures, staphylococci are extremely resistant, repeated freezing often failing to sterilize cultures. Desiccation is usually well borne, staphylococci remaining alive 4Fr. Muller, Cent. f. Bakt., xxvi, 1899. 5 Fliigge, ' ' Die Microorg., ' ' etc. *Migula, tl System d. Bakt.," Jena, 1897. 7 Sclmeider, Arb. a. d. bakt. Inst,, Karlsruhe, 1, vol. i, 1894. s Fischer, "Vorles. iiber die Bakt,," Jena, 190,°,. 9 Neisser, Kolle and Wassermann, 2nd Ed. vol. 4, p. 369. 10 Sternberg, "Textbook," etc., N. Y., 1901, p. 375. STAPHYLOCOCCUS PYOGENES AUREUS 380 for six to fourteen weeks when dried upon paper or cloth.11 On slant agar, staphylococci may be safely left for three or four months without transplantation, and remain alive.12 The resistance of staphylococci to chemicals, a question of great surgical importance, has been made the subject of extensive re- searches, notably by Liibbert,13 Abbott,14 Franzott,15 and many others. According to Liibbert, inhibition of staphylococcus growth is attained by the use of boric acid 1 in 327, salicylic acid 1 in 650, corrosive sublimate 1 in 80,000, carbolic acid 1 in 800, thymol 1 in 11,000. Staphylococci are killed by corrosive sublimate 1 in 1,000 in ten minutes, by carbolic acid 1 per cent in 35 minutes, 3 per cent in 2 minutes (Franzott). Ethyl alcohol,16 even when absolute, is not very efficient as a disinfectant. Nascent iodin, as split off from iodof orm in wounds, is extremely powerful in . destroying staphy- lococci. Pathogenicity. — Separate strains of Staphylococcus pyo genes aureus show wide variations in relative virulence. The most highly virulent are usually those recently isolated from human suppurative lesions, but no definite rule can be formulated in this respect. The virulence of a given strain, furthermore, may be occasionally en- hanced by repeated passages through the body of a susceptible animal. Prolonged cultivation upon artificial media is liable to decrease the virulence of any given strain, though this is not regularly the case. There are, moreover, unquestionably, many staphylococci constantly present in the air, dust, and water, which although morphologically and culturally not unlike the pathogenic- ally important species, may be regarded as harmless saprophytes. The susceptibility of animals to staphylococcus infection is, like- wise, subject to extreme variations, depending both upon differences between species and upon fortuitous individual differences in sus- ceptibility among animals within the same species. Animals on the whole are less susceptible to staphylococcus than is man. Among the ordinary laboratory animals, rabbits are most susceptible to this microorganism. Mice, and especially the white Japanese mice, show 11 Deslong champs, Paris, 1897. 12Passet, Fort. d. Med., 2 and 3, 1885. "Liibbert, "Bipl. Untersuch.," Wurzburg, 1886. 14 Abbott, Medical News, Phila., 1886. ™ Franzott, Zeit. f. Hyg., 1893. ™Hanel, Beit. z. klin. Chir., xxvi. 390 PATHOGENIC MICROORGANISMS considerable susceptibility. Guinea-pigs possess a relatively higher resistance.17 Subcutaneous or intramuscular inoculation of a susceptible animal usually results in the formation of a localized abscess with much pus formation and eventual recovery. Intraperitoneal inocula- tion is more often fatal. Intravenous inoculation of doses of 0.5 c.c., or more, of fresh broth cultures of virulent staphylococci usually leads to pyemia with the production of secondary abscesses, located chiefly in the kidneys and the heart and voluntary muscles, but not infrequently in other organs as well. In the kidney they occur as small foci, situated most often in the cortex, composed of a central, necrotic pus cavity, surrounded by a zone of acute inflammatory exudation. Staphylococcus lesions form histologically the typical " acute abscess." Not infrequently the pyemic condition is accom- panied by suppurative lesions in the joints. Intravenous injections of virulent staphylococci preceded by injury to a bone is often followed by the development of osteomyelitis. Mechanical or chem- ical injury of the heart valves preceding intravascular staphylo- coccus inoculation may result in localization of the infection on or about the heart valves, leading to "malignant endocarditis." In producing experimental lesions in rabbits all varieties of staphylo- coccus infection may be obtained by suitable methods of injection. If, for instance, a rabbit is given one-half to 1 c.c. of a young broth culture, from which the clumps have been gently centrifuged down, into the ear vein, a rapid fatal septicemia will result with organisms in the heart's blood, but no secondary localization or abscess forma- tion. If, however, Staphylococcus cultures containing clumps are gently centrifuged, the supernatant fluid taken off, and small clumps injected in not too large amounts (and the amounts must be adjusted to the virulence of the culture) the animal will pass through a protracted illness, with secondary abscess formation in kidneys, liver and other organs, in which emboli have been formed, a condi- tion simulating accurately pyemia in human beings. The pyemic conditions following Staphylococcus inoculation usually lead to chronic emaciation and death after an interval dependent upon the relative virulence of the microorganism, the amount injected, and the resistance of the infected subject. 17 Terin, Kef. in Lubarsch und Ostertag, Ergebnisse, 1896 ; Lingelsheim, Aetiol. d. Staph. Inf.," etc., Wien, 1900. {STAPHYLOCOCCUS PYOGENES AUREUS 391 As above stated, the susceptibility of man to spontaneous staphy- lococcus infection is decidedly more marked than is that of animals. The form of infection most frequently observed is the common boil or furuncle. As Garre,18 Biidinger,19 Schimmelbusch,-0 and others have demonstrated by experiments upon their own bodies, energetic rubbing of the skin with virulent staphylococcus cultures may often be followed by the development of a furuncle. Subcutaneous inocu- lation of the human subject invariably gives rise to an abscess. The organisms are apparently present on the skin of human beings with great frequency, and it is not unlikely that in the course of daily life, they may be rubbed into hair follicles and sweat glands, and be present constantly on some part of the body, prepared for immediate invasion of an abrasion or other accident furnishes the opportunity. A simple and frequent disease, furunculosis, is, never- theless, a condition about the pathogenesis of which we are con- siderably in the dark. Reductions of general resistance, especially those accompanying overwork, indoor occupations, and faulty diet, seem to be concerned in furnishing the proper conditions for invasion by the ever present staphylococci. General metabolic diseases, such as nephritis and especially diabetes, render the individual abnormally susceptible. In certain instances it has been suggested, especially by Wright, that reduction in coagulation time of the blood might influence this state of affairs. Staphylococcus lesions of the skin are characteristic in that, after an induration, there occurs a central softening with the forma- tion of liquid pus. It is an important observation, confirmed by much experience, that if incision is practiced in the indurated and inflamed tissue before the process has come to a central head, infection is usually spread, perhaps by the opening of adjacent lymphatics. Therefore, there is much judgment required in treating even these simple lesions. Faulty surgical interference may easily convert a simple furuncle into a dangerous carbuncle. Common among staphylococcus skin infections is paronychia, or infection of the nail bed on the fingers. This may often lead to troublesome extension up the fingers and into the hands. 18 Garre, Beit, z. klin. Chir., x, 1893. w Biidinger, Lubarsch und Ostertag, Ergebnisse, etc,, 1896, 20 Schimmelbusch, Ref . by Biidinger. 392 PATHOGENIC MICROORGANISMS Especially dangerous are boils about the nose and lips, and not infrequently infections in these locations may extend rapidly, and cause fatal septicemia. Impetigo contagiosum, a skin disease consisting of boil-like in- flamed papules and occurring particularly in young children, is caused by staphylococci. In suppurative lesions of the bones, or osteomyelitis, staphylococci are the most frequent causative agents. This may result, after compound fracture, by infection from without, or not infrequently staphylococci will lodge in the site of mechanical injury of bone or fracture, reaching the focus through the circulation. The lesions produced in bone may consist of slow localized abscesses, or may extend along the medullary canal of the entire bone. In addition to these most common lesions, staphylococci may cause abscesses in almost any part of the body. In cases in which resistance if low and the staphylococci particularly virulent, sep- ticemia may follow in any of these. Unlike the rapid, acute sep- ticemia death, however, which is likely to ensue when similar general infection with streptococci takes place, staphylococcus generalization is apt to lead to secondary foci in kidneys, liver and other organs. This leads to the condition of pyemia in which an irregular septic temperature with frequent chills are characteristic. Blood culture in such cases will give a clue to the nature of the infection. Ascending infections of the genito-urinary tract, cystitis and pyelonephritis, may be caused by staphylococci. Staphylococcus empyema and peritonitis are not particularly common, but may occur. Puerperal sepsis, while not as commonly a staphylococcus infec- tion, as it is a streptococcus lesion, may occur. By some writers staphylococci have been held responsible for rheumatism, but there is no convincing evidence of this. Staphylococci may also appear in meningitis. It is a curioi fact that occasionally a very low grade staphylococcus may get int< the meninges, and cause a very slow and apparently mild meningitis. We have seen one such case caused by a staphylococcus albus recover, and another which died after a prolonged illness in which the organisms were repeatedly isolated from the spinal fluid, and, at autopsy, in which the origin was a cerebellar abscess. Prolonged chronic infection with staphylococci may give rise to the so-called amyloid changes in liver, spleen and kidneys. STAPHYLOCOCCUS PYOGENES AUREUS 393 Toxic Products. — Endotoxins. — The dead bodies of staphylococci injected into animals may occasionally give rise to abscess formation, and,21 if in sufficient quantity, may cause death. To obtain the latter result, however, large quantities are necessary, the endotoxic sub- stances within the dead cell body of these microorganisms being prob- ably neither very poisonous nor abundant.22 That dead cultures of Staphylococcus aureus exert a strong posi- tive chemotaxis for leucocytes was shown beyond question by the experiments of Borissow.23 Hemolysins. — In 1900 Kraus24 noticed the hemolytic action of staphylococci growing upon blood-agar plate cultures. Neisser and Wechsberg25 then showed that this hemolytic substance, secreted by the Staphylococcus, could be demonstrated in filtrates of bouillon cul- tures. Such hemolysins are produced by Staphylococcus aureus, and, to a lesser degree, by Staphylococcus albus. The quantity produced varies enormously with different strains and seems to be roughly proportionate to the virulence of the particular microorganism, though exceptions to this rule are not uncommon. Absolutely avirulent races do not, so far as we know, produce hemolysins. Relationship of hemolysin formation to virulence, however, is not by any means as regular as at first supposed. A great many staphylococci may be isolated from human lesions which produce absolutely no hemolysin. The culture medium most favorable to the formation of these sub- stances is, according to Neisser and Wechsberg, a moderately alkalin beef bouillon. Cultivated at 37.5° C., the bouillon contains the maxi- mum amount of hemolytic substance between the eighth and four- teenth day, and this may be separated from the bacteria by filtration through Berkefeld or Chamberland filters. We have not ourselves attempted to confirm these investigations, but in the particular respect of lateness of concentration in the cultures the Staphylococcus hemoly- sin seems to differ distinctly from that produced by streptococci, in which the optimum for a large yield is early, within the first twelve or fourteen hours of cultivation. The hemolytic action may be observed by the general technique for determining hemolysis (given on page 311). It is important to 21 Scltattenfroh, Arch. f. Hyg., xxxi, 1887. 22 v. Lingelslieim, "Aetiol. u. Therap. d. Staph. Krank.," Wien, 1900. 23 Borissow, Zieglers Beitr., xvi, 1894. 24 Kraus, Wien. klin. Woch., iii, 1900. 25 Neisser und Wechsberg, Zeit. f . Hyg., xxxvi, 1901. 394 PATHOGENIC MICROORGANISMS wash the red blood corpuscles used for the experiments, since many animals normally possess small quantities of antihemolysin in their blood-sera (man and horse especially).26 The red blood corpuscles of rabbits, dogs, and guinea-pigs are extremely susceptible to the action of the staphylo-hemolysin. Those of man are less easily injured by it. The hemolytic action takes place, as Todd27 and others28 have shown, not only in vitro, but in the living animal as well. The staphylo-hemolysin is comparatively thermolabile. According to Neisser and Wechsberg, heating it to 56° C. for twenty minutes destroys it. According to some other authors, however, higher tem- peratures (60° to 80° C.) are required. Reactivation of a destroyed staphylo-hemolysin has so far been unsuccessful. The fact that anti- staphylolysin is occasionally present in normal sera has been men- tioned above. This antibody is most abundant in the blood of horses and of man. Artificially antistaphylolysin formation is easily induced by subcutaneous inoculation of staphylolysin into rabbits. Leucocidin. — In 1894, Van de Velde29 discovered that the pleural exudate of rabbits following the injection of virulent staphylococci, showed marked evidences of leucocyte destruction. He was subse- quently able to show that the substance causing the death and partial solution of the leucocytes was a soluble toxin formed by the staphylo- coccus, not only in vivo, but in vitro as well ; for cultures of Staphylo- coccus pyogenes aureus, grown in mixtures of bouillon and blood serum, contained, within forty-eight hours, marked quantities of this ' ' leucocidin. ' ' Other workers since Van de Velde have evolved various methods for obtaining potent leucocidin. Bail30 obtained it by growing virulent staphylococcus in mixtures of one-per-cent glycerin solutions and rab- bit serum. Neisser and Wechsberg31 advise the use of a carefully titrated alkalin bouillon. To obtain the leucocidin free from bacteria, the cultures are passed through Chamberland or Berkefeld filters, after about eight to eleven days' growth at 37° C., at which time the contents in leucocidin are usually at their highest point. The action of leucocidin upon leucocytes may be observed in vivo 26 Neisser, Deut. med. Woch., 1900. 27 Todd, Trans. London Path. Soc., 1902. 2sKraus, Wien. klin. Woch., 1902. 29 Van de Velde, La Cellule, x, 1894. 80 Bail, Arch. f. Hyg., xxxii, 1898. 31 Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901. STAPHYLOCOCCUS PYOGENES AUREUS 395 by the simple method of Van de Velde, of injecting virulent staphylo- cocci intrapleurally into rabbits and examining the exudate. Bail advises the production of leucocytic intrapleural exudates by the use of aleuronat and following this after twenty-four hours by an injection of leucocidin-filtrate. In vitro the phenomenon may be observed by direct examination of mixtures of leucocytes and leucocidin in the hanging drop on a warmed stage, or by the t ' methylene-blue method" of Neisser and Wechsberg. This method is based upon the fact that living leucocytes will reduce methylene-blue solutions and render them colorless, while dead leucocytes have lost this power. Leucocidin and leucocytes are allowed to remain in contact for a given time and to them is then added dilute solution of methylene- blue. If the leucocytes have been actively attacked by leucocidin, no reduction takes place. This method is particularly adapted for quantitative tests. All staphylococcus strains do not produce leucocidin to the same degree. Almost all true Staphylococcus pyogenes aureus cultures produce some of this taxin, but one strain may produce fifty- and a hundred-fold the quantity produced by another. Staphylococcus pyogenes albus gives rise to this substance but rarely, and then in small quantity. Leucocidin seems to be similar to the soluble toxins of other bacteria. It is rapidly destroyed by heat at 58° C., and deteriorates quickly in culture fluids at incubator temperatures. It is distinct from staphylohemolysin as shown by differences in thermostability. Soon after Van de Velde 's discovery of leucocidin, Denys and Van de Velde32 produced an antileucocidin by treating rabbits with pleural exudate containing leucocidin. Neisser and Wechsberg33 later confirmed these results and showed that among staphylococci, leucocidin is not specific, the toxin of all strains of Staphylococcus aureus and albus examined being neutralizable by the same anti- leucocidin. Antileucocidin is often found in the normal sera of horses and man.34 Leucocidin should not be confounded with "leucotoxin," a sub- stance obtained in serum by treatment of animals with leucocytes, a true "cytotoxin," having no connection whatever with the staphy- lococcus. 32 Denys et Van de Velde, La Cellule, xi, 1895. 33Loc. cit. 34 Van de Velde, Presse medicale, i, 1900. 396 PATHOGENIC MICROORGANISMS Staphylococci, besides the toxic substances already mentioned, give rise to gelatinase, spoken of in the section upon cultivation, and to a proteolytic ferment by means of which albuminous media (Loeffler's serum) may be slightly digested. Immunization. — Animals can be rendered actively immuix by repeated inoculations with carefully graded doses of living or d< -ad staphylococcus cultures.35 The production of antistaphylolysin and of antileucocidin in the sera of animals so treated, has b< •< -n allud< •<[ to in the preceding sections. The sera of such actively immuni/' <1 animals possess distinct protective power when adminisi< n-4 to other animals, slightly before or at the same time with an inoculation of staphylococci. They do not, however, exhibit very high bacU-ri- cidal power in vitro, the protective properties depending probably upon their opsonic contents.36 Agglutinins have been demonstrated in staphylococcus immune sera by a number of authors, and have been of some slight valu< in differentiating between the several groups of staphylococc-i. 7 A rather surprising result of these researches has been the recognition that immune sera obtained with pathogenic staphylococci will ag- glutinate other pathogenic staphylococci, whether belonging to tin- group of Staphylococcus pyogenes aureus or that of Staphylocoocus pyogenes albus, but will not agglutinate any of the non-pathogenic members of either group.38 In view of the recent studios on the antigenic classification of streptococci and pneumococci, a re- examination of these relationships within the staphylococcus group should be undertaken. It is more than likely that this group is a heterogeneous one, and that, for purposes of intelligent expM -irm -nta- tion in serum and vaccine therapy, an antigenic classification should be attempted. Active immunization of human beings suffering from staphylo- coccus infections has been extensively practiced by Wright, in con- nection with his work on opsonins. There can be no question about the fact that the opsonic substances in the blood are increased by the injection of dead staphylococci. The procedure is of therapeutic value in subacute and chronic cases. The work of Hiss on the use *Bichet et Hericourt, Compt. rend, de 1'acad. des sci., cvii, 1888. *Kolle und Otto, Zeit. f. Hyg., xli, 1902. " Proscher, Cent. f. Bakt., xxxiv, 1903; v. Lingelsheim, "AetioL u. Therap. d. Staphyl.," etc., Wien, 1900. "Proscher, Deut. med. Woch., xi, 1903. STAPHYLOC :<>(•(' I IS I'YO( !! INKS AURKIIS of leucocyte extracts in animals infected with Staphylococcus pyogenes aureus lias given encouragement for such treatment in human beings. A number of staphylococcus infections in man have, been successfully treated with leucocyte extracts by Hiss and Zinsser. Passive immunization with anti-staphylococcus sera has not been a therapeutic success. Kx tensive work has been done especially by German investigators in animal experimentation, but in most cases it lias been found that serum injections were of little use if administered after the. infection had been established. Injection into the test animals before infection or at the same time with infection sometimes gave favorable results. In man, passive im- munization has not been encouraging, although we believe that hope of some benefit in this direction should not be completely abandoned until the antigcnie classification of the staphylocoeci has been attempted. Hooker4" has recently reported a number of cases in which he lias transfused into patients suffering from staphylococcus scp- ticemia the blood of donors irnmuni/ed with staphylococcus vaccines. This procedure of course would be applicable only to subacute cases, but in the procedure of Hooker there seems to be promise. STAPHYLOCOCCUS PYOOKNKB ALDUS. — This coccus differs from Staphylococeus pyogenes aun-us simply in the absence of the golden yellow coloration of its cultures. Morphologically, culturally, and pathogenically, it is in every way identical with the staphylococcus described in the preceding section, but its toxin- and enzyme-produc- ing powers in general are less developed than those of the aureus variety. Its close biological relationship to aureus is furthermore demonstrated by its agglutination in Staphylocoeeus pyogenes aureus immune sera. ST.M'jjyi.oc'"-' Dfi KPIDKKMIDIS ALBTS. — The Staphylococcus epi- dermidis albus described by Welch is merely one of the rion- mthogemc varieties of Staphylococcus pyogenes albus and possibly loes not deserve separate classification. It may give rise to unim- portant abscesses. ST.\KFJYJ.or<»r CUfi PYDOENHE On BEO& — Staphylococcus pyog<-n«-s '•it IT-US produces a bright yejlow or lemon-colored pigment of dis- tinctly different hue from that of Staphyloeoeeus pyogenes aureus. " Hooker, An. of Surgery, Nov., 1917, p. 51.'i. 398 PATHOGENIC MICROORGANISMS It may be pyogenic and in every way similar to Staphylococcus pyogenes aureus, but is less often found in connection with pathological lesions than either of the preceding staphylococci. A large number of staphylococci, differing from those described above in one or another detail, have bejen observed. They are of common occurrence and are met with chiefly as contaminations in the course of bacteriological work. Few of these have any patholog- ical significance and none of them are toxin-producers, so far as we know. Many of them differ, furthermore, in their inability to liquefy gelatin. All of them belong more strictly to the field of botany than to that of pathological bacteriology. Atypical pathogenic staphylococci have been described by a num- ber of observers. Thus Weichselbaum40 isolated a Staphylococcus from a case of malignant endocarditis which could not be cultivated at room temperature, and grew only in very delicate colonies. Veil- Ion,41 moreover, has described a strictly anaerobic Staphylococcus cultivated from the pus of an intra-abdominal abscess. MICROCOCCUS TETRAGENUS.-^LI 1881 Gaffsky42 discovered a micro- coccus which occurs regularly in groups of four or tetrads. He FIG. 45. — MICROCOCCUS TETRAGENUS. first isolated it from the pus discharged by tuberculous patients with pulmonary lesions. Observed in smear preparations from pus, 40 Weichsellaum, Baumgarten, Jahresb., 1899, Ttef. **Veillon, Compt. rend. soc. de biol., 1893. 42 Gaffsky, Mitteil. a. d. kais. Gesundheitsamt, i, 1881. STAPHYLOCOCCUS PYOGENES AUREUS 399 the tetrads are slightly larger in size than the ordinary staphylo- coccus, flattened along their adjacent surfaces, and surrounded by a thick halo-like capsule. Preparations from cultures often lack these capsules. The micrococcus is easily stained by the usual basic aniliii dyes. Stained by Gram's method, it is not decolorized, retain- ing the gentian-violet. Cultivation. — Micrococcus tetrageiius grows on the ordinary laboratory media, showing a rather more delicate growth than do the staphylococci. On agar, the colonies are at first transparent, later they become grayish-white, but are always more transparent than are staphylo- coccus cultures. On gelatin, growth is rather slow and no liquefaction takes place. Broth is evenly clouded. On potato there is a white, moist growth which shows a tendency to confluence. Milk is coagulated and litmus milk indicates acid formation. Pathogenicity. — Micrococcus tetragenus is especially pathogenic for Japanese mice, which succumb within three or four days to sub- cutaneous inoculation.43 Gray mice, rats, guinea-pigs, and rabbits are less susceptible, showing only a localized reaction at the point of inoculation. The organism has occasionally been isolated from spontaneous abscesses observed in domestic animals. In man, this microorganism is usually found without any par- ticular pathogenic significance, in sputum or saliva. In isolated cases, however, it has been described as the sole incitant of abscesses. Bezanc.on44 has isolated Micrococcus tetragenus from a case of meningitis. A single case of tetragenus septicemia is on record, reported in 1905 by Forneaca.45 In America, this microorganism has not been frequently observed in connection with disease. It is often found, however, in consider- able numbers, in smears of sputum which are being examined for pneumococci or tubercle bacilli. ENTEROCOCCUS. — This is a capsulated streptococcus found fre- quently in the intestines of infants, and first described by Escherich and Tavel. It is a pleomorphic organism which may appear in the "Miiller. Wien. klin. Woch., 17, 1904. 44 Bezan^on, Semaine mod., 1898. 45 Forneaca, Rif. med., 1903. 400 PATHOGENIC MICROORGANISMS stools as a diplococcus or in short chains. Even in the diplococcus form, the individuals may be of different size and shape, some of them oval, rather than round. It may assume diplobacillus-like forms. It is Gram-positive, produces neither gas nor indol, and grows well on ordinary broth and agar. It is aerobic, but may grow under anaerobic conditions. Its relationship to intestinal disease in children has been sus- pected but never conclusively proven. Mice are susceptible to in- oculation, rabbits and guinea-pigs less so. CHAPTER XXII THE STREPTOCOCCI AMONG the pyogenic cocci, there is a large and important group of organisms which multiply by division in one plane of space only, and thus give rise to appearances not unlike chains or strings of beads. The term streptococcus or chain-coccus is, therefore, a purely morphological one .which includes within its limits microorganisms which may differ from each other considerably, both as to cultural and pathogenic properties. Thus, cocci which form chains may be isolated from water, milk, dust, and the feces of animals and man. These may have little but their morphological appearance in common with the pyogenic streptococci which are so important as the incitants of disease. The interrelationship between streptococci from different sources, however, is by no means fully understood, and we are forced at present to content ourselves with the recognition of a large mor- phological group, in no individual case taking the pathogenic or more special cultural characteristics for granted. Of paramount importance among the streptococci are those which possess the power of giving rise to disease processes in animals and in man, and which, because of their frequent association with sup- purative inflammations, are roughly grouped under the heading of Streptococcus pyogenes. The same researches upon surgical infections which led to the discovery of the staphylococci laid the basis for our knowledge of the streptococci. The fundamental studies of Pasteur and Koch1 were followed, in 1881, by the work of Ogston,2 who was the first to differentiate between the irregularly grouped staphylococci and the chain-cocci. Pure cultures of streptococci were first obtained by Fehleisen3 in 1883 and by Rosenbach4 in 1884. The thorough and systematic *Koch, ' ' Untersuch. iiber Wundinfektion, " etc., 1878. 2 Ogston, Brit. Med. Jour., 1881. 'Fehleisen, "Aetiol. d. Erysipelas," Berlin, 1883. *ltosenbach, "Mikroorg. bei Wundinfektion," etc., Wiesbaden, 1884. 401 402 PATHOGENIC MICROORGANISMS researches of the last-named authors, together with those of Passet,5 were of special influence in placing our knowledge of the pathogenic properties of streptococci upon a scientific basis. Morphology and Staining. — The individual streptococcus is a spherical microorganism measuring from 0.5 micron to 1 micron in diameter. Since the line of cleavage of cocci, when in chains, is *• FIG. 46. — STREPTOCOCCUS PYOGE'NES. perpendicular to the long axis of the chain, adjacent cocci often show slight flattening of the contiguous surfaces, forming, as it were, a series of diplococci arranged end to end. As a general rule the streptococci pathogenic for man, when grown upon favorable media, have a tendency to form chains made up of at least eight 6 Posset, "Untersuch. iiber die eitrigen Phlegm./' etc., Berlin, 1885. THE STREPTOCOCCI 403 or more individuals, while the more saprophytic, less pathogenic varieties are apt to he united in shorter groups. Upon this basis a rough morphological distinction has been made by v. Lingelsheim,6 who first employed the terms streptococcus "longus" and "brcvis." A differentiation of this kind can hardly be relied upon, however, since the length of chains is to some degree dependent upon cultural and other environmental conditions. Species which exhibit long and tortuous chains, when grown upon suitably alkalin bouillon, or ascitic broth, may appear in short groups of three or four, or even in the diplo form, when cultivated upon solid media or unfavor- able fluid media. It has often been noticed that some streptococci will form cap- sules.7 We are not referring by this to the so-called "streptococcus mucosus" of Schottmiiller, which is now spoken of as " pneumococcus mucosus" or Type III, but ordinary and probably true hemolytic streptococci may on occasion form capsules which are noticeable in smears from infected animals and in early cultures made upon media rich in animal fluids, but may be lost on subsequent transplantation to simpler media. The capsule here is an attribute of virulence. It has been particularly noticed in connection with the so-called streptococcus epidemicus isolated from milk, to which we will refer in subsequent paragraphs. Streptococci do not form spores, are non-motile, and do not possess flagella. An astonishing change in the size and appearance of streptococci may be noticed under different conditions of cultivation. Strepto- cocci which have grown under anaerobic or partially anaerobic con- ditions will often show chains of the organisms of minute size. Indeed, we have seen, interspersed with chains of the ordinary ap- p'earance, individual chains composed of organisms almost as small as the globoid bodies of Noguchi. It is these small individuals which appear under anaerobic conditions that have been responsible for Rosenau's ideas concerning the etiological role of streptococci in poliomyelitis. Again, in old cultures there may be either at the ends or even in the middle of the chains, large, swollen individuals, almost as big as small yeast cells. When these are first encountered «v. Lingelsheim, "Aetiol. u. Therap. d. Streptok. Infek." Beit. z. Exp. Ther., Hft. 1, 1899 7 Pasquale, Zieglers Beit., xii; Bordet, Ann. de Plnst. Pasteur, 1887; Schott- nyller, Munch, med. Woch., xx, 1903; Hiss, Jour. Exp. Med., vi, 1905. 404 PATHOGENIC MICROORGANISMS by an inexperienced bacteriologist, he may assume for some time that his culture has been contaminated. These swollen individuals may probably be interpreted as involution forms. Streptococci are easily stained by the usual anilin dyes. Stained by the method of Gram, the pyogenic streptococci are not decolorized and invariably retain the gentian-violet. Certain species found in stools and described as Gram-negative, are rare and are non- pathogenic. Others of the "Streptococcus brevis" variety, and purely saprophytic, may stain irregularly by the Gram method. Cultivation. — The pyogenic streptococci are easily cultivated upon all the richer artificial media. While meat extract-pepton media may suffice for certain strains, it is usually better to employ those media which have the beef or veal infusion for a basis. For the cultivation of more delicate strains of streptococci, especially when taken directly from the animal or human body, it is well to add to the media animal albumin in the form of whole blood, blood serum, or ascitic or pleural transudates. Glucose, added in propor- tions of one to two per cent, likewise renders media more favorable for streptococcus cultivation. Prolonged cultivation of all races upon artificial media renders them less fastidious as to cultural require- ments. The most favorable reaction of media for streptococcus cul- tivation is moderate alkalinity (two-tenths to five-tenths per cent alkalinity to phenolphthalein) . Growth may be readily obtained, however, in neutral media or even in those slightly acid. The optimum temperature for growth is at or about 37.5°. Above 43° to 45° C., development ceases. At from 15° to 20° C., growth, while not energetic, still takes place, an important point in the differen- tiation of these microorganisms from pneumococci. While the free access of oxygen furnishes the most suitable environment for most races of streptococci, complete anaerobiosis does not prevent develop- ment in favorable media. Strictly anaerobic streptococci have been cultivated from the human intestinal tract by Perrone8 and others. In alkalin bouillon at 37.5° C., pyogenic streptococci grow rapidly, form long and tortuous chains, and have a tendency to form flakes which rapidly sink to the bottom. Diffuse clouding occurs rarely and is a characteristic rather of the shorter so-called Strepto- coccus brevis. When sugar has been added to the broth the rapid formation of lactic acid soon interferes with extensive development. 8 Perrone, Ann. de Pinst. Pasteur, xix, 1905. % THE STREPTOCOCCI 405 This may be obviated, especially when mass cultures are desired, without sacrifice of the growth-increasing influence of the glucose, by adding to the sugar-broth one per cent of sterile powdered CaCO.9 In milk, Streptococcus pyo genes grows readily with the forma- tion of acid, followed, in most cases, by coagulation of the medium. On agar-plates at 37.5° C., grow.th appears within eighteen to twenty-four hours. The colonies are small, grayish, and delicately opalescent. They are round with smooth or very slightly corrugated or lace-like edges, and rise from the surface of the medium in regular arcs, like small droplets of fluid. Microscopically they appear finely granular and occasionally, under high magnification, may be seen to be composed of long in- tertwining loops of streptococcus chains, which form the lace-like edges. When ascitic fluid or blood serum has been added to agar, growth is more energetic and the colonies correspondingly more rapid in appearance and luxuriant in development. In glucose-ascitic-agar, acid formation from the sugar causes coagulation of albumin with the conse- quent formation of flaky white precipi- tates throughout the medium.10 In gelatin stab-cultures growth takes place slowly, appearing after twenty- four to thirty-six hours as a very thin white line, or as disconnected little spheres along the line of the stab. The colonies on gelatin plates are similar in form to those on agar, but are usually more opaque and more distinctly white. The gelatin is not liquefied by the pyogenic streptococci, though certain of the more saprophytic forms may occasionally bring about slow fluidification. On Loeffler's coagulated blood serum, growth is rapid and luxuriant and may show a slight tendency to confluence if the medium is very moist. Good chain formation takes place on this medium. Upon potatoes, growth is said not to take place.11 On media containing red blood cells, most pathogenic streptococci FIG. 47. — STREPTOCOCCUS COL- ONY ON SERUM AGAR. 9 Hiss, Jour. Exp. Med., vi, 1905. 10 Libman, Medical Record, Ivii, 1900. 11 Frosch und Kolle, in Fliigge, ' ' Die Mikroorganismen, ' ' 1891. 406 PATHOGENIC MICROORGANISMS cause hemolysis and decolorization. It is useful to remember this when examining blood-culture plates, for here the yellow transparent halo of hemolysis and decolorization surrounding the colonies may aid in differentiating these bacteria from pneumococci. This is of especial importance, since many streptococci, when cultivated directly out of the human blood, do not exhibit chain formation, but appear as diplococci. In the inulin-serum media of Hiss,12 streptococci do not produce acid and coagulation. The so-called Streptococcus mucosus, a cap- sule-bearing, inulin-fermenting microorganism, is very probably a sub-species of the pneumococcus (see later section). A very im- portant differential characteristic of the streptococci as a class is the fact that they are not bile soluble. This distinguishes them sharply from the pneumococci. The test is carried out by the method of Neufeld described in the chapter on pneumococci. Ox bile or a ten per cent solution of taurocholate of sodium is added to a young broth culture in proportions of one part of bile or tauro- cholate to nine parts of the culture. Resistance. — Streptococci on the ordinary culture media, without transplantation and kept at room temperature, usually die out within ten days or two weeks. They may be kept alive for much longer periods by the use of the calcium-carbonate-glucose bouillon, if the cultures are thoroughly shaken and the powdered marble thoroughly mixed with the bouillon from time to time.13 Preservation at low temperatures (1° to 2° C.), in the ice chest, considerably prolongs the life of cultures. Virulence is preserved longest by frequent transplantation upon albuminous media. In sputum or animal excreta, streptococci may remain alive for several weeks. Strepto- cocci like pneumococci may be preserved alive and with virulence unchanged by drying in the frozen condition by the method described by Swift, and referred to on page 456. For ordinary purposes preservation in tubes of defibrinated rabbit's blood in the ice-chest is the most practical method. Streptococci are killed by exposure to a temperature of 54° C. for ten minutes.14 Low temperatures, and even freezing, do not destroy some races. *- Hiss, Jour. Exp. MeXc(,l, Arch, of Inter. Mod., 2.°», 1919, 657. 17 Foster and Cookson, Lancet, 2, 1918, 585. "Ktunley, U. S. Pub. Health Serv. Eep., No. 19, May 9th, 1919. 492 PATHOGENIC MICROORGANISMS secondary waves that lasted as long as 1800, and similar observations were made during the epidemics of 1836 and 1837. The most careful studies of such waves were made by Parsons during the 1889 epidemic. He divided this epidemic into the first wave which began in England in the winter of 1889 to 1890, a second wave in the spring of 1891, and a third in the winter of 1891 to 1892. Frost has similarly studied the American epidemics, and has come to analogous conclusions. Brownlee19 has attempted to establish a law of periodicity for the intervals between pandemics, and for the intervals between several waves of each outbreak. In general, his studies seem to show that there is an approximate period of ten years between large epidemics, and that a period of about thirty- three weeks intervenes between individual waves. We cannot go into these purely statistical facts in the present work, but refer the reader to papers by Brownlee, and the more recent paper by Raymond Pearl.20 Secondary and tertiary waves have certain characteristics which it is important to note. In contrast to the relatively mild onset of the primary waves, these later waves are marked by greater severity of the cases, and almost universal secondary infection. The disease takes on a much more dangerous respiratory form. The mortality becomes progressively higher during these waves than during the original outbreak. Leichtenstern, also agrees that, in the secondary and tertiary waves, that the morbidity is lower and the mortality much higher. Similar observations have been made by Wutzdorff21 for the 1889 epidemic. The secondary epidemic waves do not travel with the same speed and to the same extent as do the first waves. Cases are more scat- tered and the period of prevalence is more prolonged. These waves never stop abruptly, but play out, in gradually diminishing ripples, into subsequent years. Also, according to Leichtenstern, these secondary and tertiary waves do not seem to take their origins from a single place, but crop up here and there from many scattered foci. As Netter says, "they have appeared in separate, synchronous or successive explosions, without connection between various reap- pearances in different places, as this was possible during the first appearances in 1889." 19 Krownlce, Lancet, 2, 1919, 856. =° Pearl, E., U. S. Pub. Health Serv. Rep., No. 548, August 8th, 1919, 21 Wutzdorff, Arb. a. d. k. Gesundh., 9, 1894, 414, EPIDEMIOLOGY OF INFLUENZA 433 AVhat is at the bottom of this succession of waves, it is hard to say. The most natural explanation would be that there is a short lived immunity, conferred by the original attack, and that the second and third waves appear at times when the infectious agent is still widely distributed while the immunity has waned. It is very difficult to get at the facts, but serious attempts are being made, particularly by Frost and others. Studies by Jordan and Sharp22 at the Great Lakes Training Station indicate that no marked immunity existed twelve to fifteen months after the first attack. This, too, seems to be the conclusion reached by Frost who states that in Baltimore those persons who were attacked during the 1918 to 1919 epidemic, showed no relative immunity during the epidemic of 1920. It would seem in general that an almost universal infection of a community with the first mild disease conferred a short lived immunity. As a consequence of this, the epidemic would burn itself out and wane. Gradual return of susceptibility in the course of the subsequent period of months, not, however, bringing the community back to the very low universal resistance which it was characterized by before, would now create a soil in which reintroduction of the infectious agent could produce many cases, but in which spread would be less rapid and extensive. Such a view, however, must be regarded as purely tentative, and it is hoped that a final study of the statistics gathered during the great war epidemic will clear this matter up. It is a difficult question to decide where the last war epidemic began. After the 1889 epidemic, it seems that the disease may have remained endemic in a great many different parts of the world. It may have been so universally distributed that we cannot speak, in this case, of a definite focus in China or Turkestan, as this was done in past epidemics. Before 1889, the world was not so con- tinuously traveled over by large numbers of people. There was less travel by railroad communication, steam-ship lines, etc. It may be that this development of civilization has completely altered the epidemic conditions prevailing in regard to influenza. It has been suggested in the case of the last epidemic however, that it came from the East, as did previous outbreaks. McNalty in an article in Nelson's System states that the disease was prevalent in March, 1918, in China, and that in April cases appeared on a Japanese ship in a Chinese port. Frost who has given particular attention to this ^-Jordan and Sharp, Jour, of Infec., Dis., May, 1920, p. 463. 494 PATHOGENIC MICROORGANISMS question, on the other hand, finds that the roots of the epidemic go far deeper than this, since his studies of statistics of respiratory diseases in the United States seem to show that as early as December, 1915, and January, 1916, there occurred, in New York and Cleveland, sudden and considerable rises in mortality from respiratory dis- eases. In January, 1916, influenza was reported in twenty-two cities of the Union. These epidemics were mild and attracted little at- tention. During the winter of 1917, many so-called cases of influenza occurred in Europe among French and British troops. In the winter of 1917, similar cases, supposedly influenza, appeared in many Ameri- can camps. MacNeal states that in November and December, 1917, and January, 1918, there were many cases of so-called influenza in the American Expeditionary Forces. The disease appeared without question at Camp Oglethorpe in March, 1918, a month before it was recognized in any numbers in Europe, almost at the same time at which it seems to have appeared in Spain. It cannot be said with definifeness just where the lasi epidemic began, but if we summarize the evidence available at the present time, it would seem that Frost is probably right in that it did not begin in a single place, as previous epidemics are said to have begun, but started in a great many different places almost at the same time. Like other epidemics it appeared in successive waves, the first wave probably beginning in 1917 in some places, in the spring of 1918 in others. There was an interval, and then in September and October of 1918 the second wave had gathered its full velocity. This was the really fatal wave all over the world. The mortality was enormous. Pearl estimates that in the United States alone, deaths from influenza were not less than 550,000 and this is approximately five times the number (111,179) of American soldiers officially stated to have lost their lives from all causes in the war. In the Surgeon General's report it is stated that influenza, with its complications, is charged with 688.86 admissions of American and native troops for the year 1918, and caused 23,007 deaths, practically 82 per cent of all deaths in the Army being attributed to respiratory diseases. Morphology and Staining.— The bacillus of influenza23 (Pfeiffer bacillus) is an extremely small organism, about 0.5 micron long by 23 Pfeiffer, Deut. med. Woch., ii, 1892; Zeit. f. Hyg., xiii, 1892; Pfeiffer und Beck, Deut. med. Woch., xxi, 1893. EPIDEMIOLOGY OF INFLUENZA 495 0.2 to 0.3 micron in width. They are somewhat irregular in length, but show rounded ends. They rarely form chains. They are non- motile, and do not form spores. Influenza bacilli stain less easily than do most other bacteria with the usual anilin dyes, and are best demonstrated with 10 per cent aqueous fuchsin (5 to 10 minutes), or with Loeffler's methy- lene-blue (5 minutes). They are Gram-negative, giving up the anilin-gentian voilet stain upon decolorization. Occasionally slight polar staining may be noticed. Grouping, especially in thin smears of bronchial secretion, is charac- teristic, in that the bacilli very rarely form threads or chains, usually lying together in thick, irregular clusters without definite parallelism. Isolation and Cultivation. — Iso- lation of the influenza bacillus is not easy. Pfeiffer24 succeeded in growing the bacillus upon serum- agar plates upon which he had smeared pus from the bronchial secretions of patients. Failure of growth in attempted subcultures made upon agar and gelatin, however, soon taught him that the success of his first cultivations depended upon the ingredients of the pus carried over from the sputum. Further experimentation then showed that it was the blood, and more particularly the hemoglobin, in the pus which had made growth possible in the first cultures. Pfeiffer made his further cultivations upon agar, the surface of which had been smeared with a few drops of blood taken sterile from the finger. Hemoglobin separated from the red blood cells was found to be quite as efficient as whole blood. Whole blood taken from the finger may be either smeared over the surface of slants or plates, or mixed with the melted meat-infusion agar. In isolating from sputum, only that secretion should be used which FIG. 49. — BACILLUS INFLUENZA ; Smear from pure culture on blood agar. Pfeiffer, loc. cit. 496 PATHOGENIC MICROORGANISMS is coughed up from the bronchi and is uncontaminated by micro- organisms from the mouth. It may be washed in sterile water or bouillon before transplantation, to remove the mouth flora adherent to the outer surface of the little clumps of pus. The blood of pigeons or that of rabbits may be substituted for human blood. For isolation of the organisms on plates from sputum or other sources, the best medium to use is the Avery sodium oleate blood medium described in the section on technique. The sodium oleate in this medium inhibits a great many of the contaminating organisms and seems to favor the growth of influenza bacilli. FIG. 50. — BACILLUS INFLUENZA; Smear from sputum. (After Heim.) In place of this medium, blood agar plates can be used, rabbit's blood being, in our opinion, somewhat more favorable than other varieties of blood. It is best prepared in small lots by puncturing a rabbit's heart with a sterile needle and transferring the blood directly to tubes of melted agar. For further cultivation, the best medium is a chocolate agar made as described in the section on media, in which a 5 or 10 per cent rabbit's blood agar with a PH of 7.8 is heated to about 90° C. (but not boiled), and plates are prepared by shaking this brownish discolored medium, pouring it into plates or tubes and letting it cool before all the blood has settled to the bottom. For fluid cul- tivation, chocolate broth may be similarly prepared and filtered through cotton or paper while hot. This makes a transparent straw- colored fluid on which influenza bacilli grow with great speed and EPIDEMIOLOGY OF INFLUENZA 497 luxuriance. In the hands of J. T. Parker in this laboratory, this method has given excellent results and it is by means of such a medium that she has produced the toxic substances referred to below. The fact that this fluid medium does not contain much, if any, hemoglobin, but is very rich in lipoidal substances, makes us believe that possibly the nutritive substances in blood necessary for the growth of this bacillus may consist in the lipoidal contents of the blood cells, rather than the hemoglobin. This, too, is confirmed by recent observation of Avery.25 'The general view, however, is the one whic)? attributes to the hemoglobin in the blood the nutritive function. FIG. 51. — COLONIES OF INFLUENZA BACILLUS ON BLOOD AGAR. (After Heim.) A more luxurious growth of influenza bacilli may be obtained by cultivation in jars in which about 1/10 the volume of air has been replaced by C02. For preservation of laboratory cultures of influenza bacilli, the best medium is fresh, defibrinated rabbit's blood, kept at room temperature in the dark. In this way, laboratory strains after several generations of cultivation outside the body can be kept alive for weeks and even months. They will not keep well either in the ice-box or in the incubator. Avery, Proc. Soe. Exper. Biol. and Med., 18, 1921, 6. 498 PATHOGENIC MICROORGANISMS Other .substances which, added to neutral or slightly alkalin agar, have been used for the cultivation of influenza bacilli are the yolk of eggs'"0 (not confirmed) and spermatic fluid.27 None1 of these, however, are as useful as the blood media. Symbiosis with staphy- lococci,-8 too, has been found to create an environment favorable for their development. Influenza bacilli do not grow at room temperature. Upon suit- able, media at 37.5° C. colonies appear at the end of eighteen to twenty-four hours, as minute, colorless, transparent droplets, not unlike spots of moisture. These never become confluent. The limits of growth are reached in two or three days. To keep the cultures alive, tubes should be stored at room temperature and transplanta- tions done at intervals not longer than four or five days. Biology. — The bacillus is aerobic, growing in broth-blood mix- tures only upon the surface, hardly at all in agar stab cultures, and not at all under completely anaerobic conditions. As it does not form spores, it is exceedingly sensitive to neat, desiccation, and disinfectants. Heating to 60° C. kills the bacilli in a few minutes. In dried sputum they die within one or two hours. They are easily killed even by the weaker antiseptics. Upon culture media the bacilli, if not transplanted, die within a week or less, the time depending to some extent upon the medium used. Toxin Formation. — The opinion in former years has been that the poisonous substances produced by the influenza bacillus were in the nature of endotoxins and a great many observers noted toxic symptoms on the injection of whole cultures into rabbits and guinea pigs. There is no question about the fact that such cultures in quantities of a cubic centimeter or more can exert powerful poison- ous action. In our laboratory, Julia T. Parker29 recently showed that culture filtrates of young influenza bacilli would kill rabbits in doses of from 1.5 c.c. upward. The best poisons are produced by cultivating the organisms on broth of a PH of 7.8, with 5 to 10 per cent defibrinated rabbit 's blood. They were also produced actively in the chocolate broth produced by the filtration of such rabbit's blood broth as described above. The nature of these poisons is un- certain. The symptoms in the rabbits consist in marked prostration, "'Nastjulcoff, Cent. f. Bakt., Ref., xix, 1896. 27 Cantani, Cent. f. Bakt., xxii, 1897. 28 Grassberger, Zeit. f. Hyg., xxv, 1897. 29 Parker, Jour. A. M. A., 72, 1919, 476. EPIDEMIOLOGY OF INFLUENZA 499 flattening out on the bottoms of the cages, muscular weakness and death in a considerable percentage of the cases within two to six hours. A characteristic feature is the incubation time which is regularly between forty-five minutes and one hour and one-half. These poisons have been studied in parallel series with similarly produced streptococcus and typhoid filtrates by Zinsser, Parker and Kuttner30 and belong to a class of substances probably non-specific and non-antigenic, described by us as "X" substances, the exact nature of which is at present uncertain. INFLUENZA BACILLI NOT ASSOCIATED WITH EPIDEMIC INFLUENZA. — The question of the etiological relationship of the influenza bacillus to the epidemic disease has been sufficiently discussed above. It is an important fact that the influenza bacillus is a common respiratory invader of man in the interepidemic periods, and without any apparent relationship to the typical epidemic disease. Subsequent to the epidemic of 1889, its wide distribution was established by a multitude of investigations. Pfeiffer31 noted its frequent presence in tuberculous processes in his early studies, and this observation was confirmed by many others. It is especially frequent in bron- chiectatic cavities. Boggs32 reported a number of such cases in which influenza bacilli were present symbiotically in the cavity fluids in cases that showed negligible symptoms. A number of observers have isolated influenza bacilli from the blood in cases that had died of other conditions. Jaehle33 obtained the bacilli from the heart's blood at autopsy from two scarlet fever cases. He also found the organisms in blood culture at autopsy in fifteen cases of measles. In one of these cases the influenza bacillus was present when the only other influenza bacillus lesion present was a tonsillar infection. He also found the organisms in the blood five times in nine cases of chickenpox, and twice in twenty-four cases of whooping cough. He found them in the respiratory passages in fifteen cases of diph- theria. Wynekoop34 has made similar studies, especially in con- nection with lesions of the larynx, and in chronic laryngitis he found the organisms in pure culture. He described a special form of severe tonsillitis due to the influenza bacillus. Some of these were clinically 30 Zinsser, Parker and Kuttner, Jour. Exper. Biol. and Med., 18, 1920, 49. 81 Pfeiffer, Deut. med. Woch., 18, 1892, 28. 32 Boggs, 130, 1905, 902. 83 Jaehle, Zeit. f . Heilkd., 22, 1901, 190. 34 Wynelcoop, Jour. A. M. A., 40, 1903, 574. 500 PATHOGENIC MICROORGANISMS mistaken for diphtheria. Madison35 collected from the literature thirty cases in which influenza bacilli were present in the blood during life. Influenza bacilli in the meninges have been described by Woll- stein36 and others and we have seen them in a number of cases in they were associated in this location with streptococci. Influenza bacillus meningitis is not uncommon in children. In diseases of the cavities of the skull, the antrum of Highmore, the frontal sinuses etc., influenza bacilli may be chronically present and have been held responsible for intermittent attacks of asthma. Wollstein has made an extensive study of the presence of in- fluenza bacilli in children at the Babies Hospital in New York. In the interepidemic periods, Wollstein has found them present fre- quently in bronchopneumonia, less frequently in cases of lobar pneu- monia. In thirteen cases of bronchopneumonia at autopsy she found the organism three times. Six times she found them in connection with tuberculosis. Other workers, as well as Wollstein, have fre- quently found the organisms in whooping cough where they were reported as being almost regularly present after the disease was well established. In the lungs in measles, scarlet fever and diph- theria their presence is very frequent. Wollstein states that she has found the organisms rarely in the throats of healthy infants, and that whenever it was present, it seemed to have a definitely aggravating influence upon the existing disease. The carrier state may persist after infection with influenza bacilli for long periods. During epidemics this seems to be particularly important as found by the investigations of Pritchett and Stillman37 and of Opie38 and his collaborators. Varieties of the Influenza Bacillus. — A great many investigators have reported organisms almost identical with the influenza bacillus which, however, they have regarded as sufficiently different to be considered as distinct types. It is the opinion of the workers at the New York Department of health, moreover, that the Gram-negative, hemophilic organisms found in trachoma must be regarded also as belonging to the general influenza bacillus type, and cannot be sharply separated. 35 Madison, Amer. Jour. Med. Sc., 139, 1910, 527. 36 Wollstein, Jour. Exper. Med., 1905, 7, 335. 87 Pritchett and Stillman, Jour. Exper. Med., 29, 1919, 259. ^Opic, et al, Surgeon General's Rep., Jour. A. M. A., 72, 1919, 168. EPIDEMIOLOGY OF INFLUENZA 501 The recent epidemic has given much opportunity for bac- teriological and serological study upon influenza bacilli from many different types of lesion, from many different parts of the world. The result of these studies has been confusing. Studies of Valentine and Cooper39 have shown that agglutination reactions do not in- dicate hemogenicity of the influenza group. In a large number of isolations and agglutinin reactions, they found that very few of the strains fell into antigenically identical groups. Of ten autopsy strains isolated by them, all strains seemed to be distinct. Of seventy-three miscellaneous strains, no two strains were identical. Two strains from different individuals were found to be identical, but in a family in which there were six cases of influenza, all the different races were distinct. It would seem that either influenza bacilli were composed of an infinite number of antigenic varieties or that the agglutinin reaction in these particular organisms, be- cause of their small size, is not a suitable test for biological relation- ship. For the present, the biological subdivision of the influenza bacilli into well defined groups cannot be regarded as settled. Dr. Anna Williams40 has recently studied hemoglobinophilic bacilli isolated from the eye in cases of trachoma. She believes that trachoma is probably caused by bacteria of this group. At first an acute infection or acute conjunctivitis occurs. Later when chronic productive inflammation supervenes the clinical picture is that of trachoma. Experimental infection of animals reveals susceptibility only in monkeys. Pfeiffer and Beck41 produced influenza-like symptoms in monkeys by rubbing a pure culture of the bacillus upon the un- broken nasal mucosa. Intravenous inoculation in rabbits produced severe symptoms, but the bacilli do not seem to proliferate in these animals, the reaction probably being purely toxic. Cultures killed with chloroform may produce severe transient toxic symptoms in rabbits.42 Immunity produced by an attack of influenza, if present at all, is of very short duration. PSEUDO-INFLUENZA BACILLUS. — In the broncho-pneumonic proc- 39 Valentine and Cooper, Rep. 84, Dept. Health, City of N. Y., 1919, December. ""/>/•. Anna IVUHdnix, Ir.lcr. Congress of Hygiene and Demography, Washing- ton, 1912. 41 Pfeiffer mid Beck, Deut. mod. Woeh., xxi, 1893. 42 Pfeiffer, loc cit. 502 PATHOGENIC MICROORGANISMS esses of children, Pfeiffer43 found small, non-motile, Gram-negative bacilli, which he was forced to separate from true influenza bacilli because of their slightly greater size, and their tendency to form threads and involution forms. These microorganisms are strictly aerobic and grow, like true influenza bacilli, only upon blood media. They are differentiated entirely by their morphology upon twenty- four-hour-old blood-agar cultures. Wollstein/44 who has made a careful study of influenza-like bacilli, both culturally and by agglu- tination tests, has come to the conclusion that these bacilli are so similar to the true influenza organisms that the term pseudo-influenza should be discarded. Strains of similar bacilli isolated from cases of FIG. 52. — KOCH-WEEKS BACILLUS. pertussis, while differing from the others in some of their character- istics, could not properly be maintained as distinct species. KOCH-WEEKS BACILLUS. — Koch,45 in 1883, Weeks46 and Kartulis, in 1887, described a small Gram-negative bacillus found in connec- tion with a form of acute conjunctivitis which occurs epidemically. The bacillus is morphologically similar to B. influenzae, but is generally longer than this and more slender. The bacilli grow only at incubator temperature. Kecent studies by Anna Williams at the 43 Pfeiffer, Zeit. f. Hyg., xiii, 1892. 14 Wollstein, Jour. Exp. Med., viii, 1906. 45 Koch, Arb. a. d. kais. Gesundheitsamt, iii ; Cent. f. Bakt., 1, 1887. 46 Weeks, N. Y. Eye and Ear Infirmary Kep., 1895; Arch. f. Augenheilk., 1887. EPIDEMIOLOGY OF INFLUENZA 503 New York Department of Health seem to indicate that the Koch- Weeks bacillus may be merely a variety of the true influenza bacillus. BACILLUS OF PLEURO-PNEUMONIA OF RABBITS. — This is a small Gram- negative bacillus, described by Beck, not unlike that of influenza. These microorganisms are slightly larger than the Pfeiffer bacilli and grow upon ordinary media even without animal sera or hemoglobin. BACILLUS MURISEPTICUS AND BACILLUS RHUSIOPATHI^E. — While mor- phologically similar to the microorganisms of this group, these bacilli are culturally easily separated because of their luxuriant growth on simple media. The last two microorganisms are more closely related to the groups of the bacilli of hemcrrhagic septicemia. CHAPTER XXV BOEDET-GENGOU BACILLUS, WHOOPING COUGH AND MORAX-AXEN- FELD BACILLUS, ZUE NEDDEN'S BACILLUS, DUCREY BACILLUS BORDET-GENGOU BACILLUS ("Microbe de la Coqueluche," Pertussis bacillus, Bacillus of whooping- cough.) WHOOPING cough is endemic in most cold countries and, on occasion, in schools, infant asylums and other places where children are crowded, may assume epidemic proportions. Occasional cases may occur in adults, though they are rare. The disease seems to occur sporadically all through the year in large centers, but is more common during the winter. According to Rosenau1 suscep- tibility is pretty general and he states that the disease causes at least 10,000 deaths a year in the United States. Indeed, Eosenau who has analyzed the statistics for the United States for the year 1910, states that whooping cough caused almost as many deaths as scarlet fever. It is the pulmonary complications that follow on the initial infection, however, which, in this disease, are responsible for the deaths and it is the subacute and chronic inflammations of the lung which lead to prolonged illness and .pave the way for tuberculosis and other secondary infections. For sanitary purposes, an arbitrary incubation time of about two weeks has been regarded as probably nearest the facts. During the incubation time, the infectiousness does not seem to be great, the most contagious period being the first few days of the actual disease when the Bordet-Gengou bacilli are brought up in large numbers. It is at this time only that pure cultures of the organisms can be obtained. Very soon after the onset, influenza bacilli and other secondary invaders are found. Transmission is probably by direct and indirect contact as in other 1 Rosevau, Preventive Medicine and Hygiene, D. Appieton and Co., New York, 1921. 504 BORDETrGENGOU BACILLUS 505 respiratory infections, and the general infectiousness is very high, since susceptible children definitely exposed do not often escape. After the disease has set in, the patient may remain infectious for others all through the disease and long into convalescence. It is best to keep children isolated for several weeks after the cough has subsided. It has been possible to reproduce conditions simu- lating the disease in monkeys and in dogs, and there is a suspicion that the disease may be transmitted by domestic animals, especially dogs. Though this is not absolutely certain, Rosenau and other sanitarians advise care in this regard. Prevention consists in early diagnosis and isolation, exclusion from school and absolute avoidance of close contact with other children. Quarantine should continue, as stated above, for several weeks after the cough has completely subsided. In 1900 Bordet and Gengou2 observed in the sputum of a child suffering from pertussis a small ovoid bacillus which, though similar to the influenza bacillus, showed a number of morphological char- acteristics which led them to regard it as a distinct species. As they were at first unable to cultivate this organism, their discovery remained questionable until 1906, when cultivation succeeded and the biology of the microorganism was more fully elucidated. Morphology. — The morphology of this organism is described by them as follows: The organism in the sputum, early in the disease, is scattered in enormous numbers indiscriminately among the pus ** — FIG. 53. — BORDET-GENGOU BACILLUS. cells, and at times within the cells. It is extremely small and ovoid, and frequently is so short that it resembles a micrococcus. Often its polos stain more deeply than the center. In general, the form - Bordet et Gengou, Ann. de 1 'inst. Pasteur, 1906. 506 PATHOGENIC MICROORGANISMS of the organisms is constant, though occasionally slightly larger individuals are encountered. They are usually grouped separately, though occasionally in pairs, end to end. Compared with the influenza bacillus in morphology, the bacillus of pertussis is more regularly ovoid and somewhat larger. It has, furthermore, less tendency to pleomorphism and involution. Staining. — The Bordet-Gengou bacillus may be stained with al- kaline methylene-blue, dilute carbol-fuchsin, or aqueous fuchsin solu- tions. Bordet and Gengou recommended as a staining-solution carbolated toluidin-blue made up as follows: Toluidin-blue 5 gms. Alcohol 100 c.c. Water 500 c.c. Allow to dissolve and add 500 c.c. of 5 per cent carbolic acid in water. Let this stand one or two days and filter. Stained by the method of Gram, the bacillus of Bordet and Gengou is decolorized. Cultivation. — Early attempts at cultivation made by the discov- erers upon ordinary ascitic agar or blood agar were unsuccessful. They finally obtained successful cultures from sputum by the use of the following medium : One hundred grams of sliced potato are put into 200 e.c. of 4 per cent glycerin in water. This is steamed in an autoclave and a glycerin extract of potato obtained. To 50 c.c. of this extract, 150 c.c. of 6-per-cent salt solution and 5 grams of agar are added. The mixture is melted in the autoclave and the fluid filled into test tubes, 2 to 3 c.c. each, and sterilized. To each tube, after sterilization, is added an equal volume of sterile defibrinated rabbit blood or prefer- ably human blood, the substances are mixed, and the tubes slanted. On such a medium, inoculated with sputum, taken preferably during the paroxysms of the first day of the disease, colonies appear, which are barely visible after twenty-four hours,3 plainly visible after forty-eight hours. They are small, grayish, and rather thick. After the first generation the organisms grow with markedly greater luxuriance and speed. On the potato-blood medium after several generations of artificial cultivation, they form a grayish glistening layer which, after a few days, becomes heavy and thick, almost 3 Wollstein, Jour. Exp. Med., xi, 1909. BORDET-GENGOU BACILLUS 507 resembling the growth of typhoid bacilli. In these later generations, also, they develop readily upon plain blood agar or ascitic agar and in ascitic broth or broth to which blood has been added. In the fluid media they form a viscid sediment, but no pellicle. Culturally, the bacillus varies from B. influenzae in growing less readily on hemoglobin media than the latter, on first cultivation from the sputum. Later it grows much more heavily on such media and shows less dependence upon the presence of hemoglobin than does B. influenzae. It also grows rather more slowly than the influ- enza bacillus. It is strictly aerobic and in fluid cultures is best grown in wide flasks with shallow layers of the medium. The Bordet-Gengou bacillus grows moderately at temperatures about 37.5° C., but does not cease to grow at temperatures as low as 5° to 10° C. On blood agar and in ascitic broth it may remain alive for as long as two months (Wollstein). Pathogenicity. — As regards the pathogenicity and etiological specificity of this organism for whooping-cough, no positive state- ment can as yet be made. The fact that it has been found in many cases in almost pure cultures during the early paroxysms, renders it likely that the organism is the specific cause of the disease. Mallory and Homer 4 have found bacilli appearing to be Bordet and Gengou 's organisms lying between the cilia in the tracheal epithelium of whooping cough cases that have come to autopsy. However, in early cases true influenza bacilli have been often found, and these latter seem to remain in the sputum of such patients for a longer period and in larger numbers than the bacillus of Bordet and Gengou. Endotoxins have been obtained from the cultures of the bacilli by Bordet and Gengou by the method of Besredka.5 The growth from slant cultures is washed up in a little salt solution, dried in vacuo, and ground in a mortar with a small, measured quantity of salt. Finally, enough distilled water is added to bring the salt into a solution of 0.75 per cent and the mixture is centrifugalized and de- canted. One to two c.c. of such an extract will usually kill a rabbit within twenty-four hours after intravenous inoculation. Subcu- taneous inoculation produces non-suppurating necrosis and ulcera- tion without marked constitutional symptoms. Inoculation of monkeys with the bacilli themselves by the respira- tory path has failed to produce the disease. 4 Mallory and Homer, Journ. Med. Ees. xxvii, 1912, p. 115. fi Bordet, Bull, de la Soc. Eoy. de Brux., 1907. 508 PATHOGENIC MICROORGANISMS Specific agglutinins may be obtained in immunized animals which prove absolutely the distinctness of this organism from Bacillus influ- enzas.6 In the serum of afflicted children the agglutination is too irregular to be of value. Specific complement fixation with the serum of patients is reported by Bordet and Gengou, but failed in the hands of Wollstein. MORAX-AXENFELD BACILLUS In 1896 Morax 7 described a diplo-bacillus, which he associated etiologically with a type of chronic conjunctivitis to which he applied the name " conjonctivite subaigue." Soon after this, a similar micro- organism was found in cases corresponding to those of Morax by Axenfeld.8 The condition which these microorganisms characteris- tically produce is a catarrhal conjunctivitis which usually attacks both eyes. The inflammation is especially noticeable in the angles of the eye, most severe at or about the caruncle. There is rarely mujch swelling of the conjunctiva and hardly ever ulceration. The condition runs a subacute or chronic course. Its diagnosis is easily made by smear preparations of the pus which is formed with especial abun- dance during the night. Morphology. — In smear preparations from the pus, the micro- organisms appear as short, thick bacilli, usually in the form of two placed end to end, but not infrequently singly or in short chains. Their ends are distinctly rounded, their centers slightly bulging, giving the bacillus an ovoid form. They are usually about two micra in length. They are easily stained by the usual anilin dyes, and, stained by the method of Gram, are completely decolorized. Cultivation. — The Morax-Axenfeld bacillus can be cultivated only upon alkalin media containing blood or blood serum. It grows poorly, or not at all, at room temperature. Upon Loeffler's Hood serum, colonies appear after twenty-four to thirty-six hours as small indentations which indicate a liquefaction of the medium. Axenfeld states that eventually the entire medium 6 Wollstein, loc. cit. 7 Morax, Ann. de 1'inst. Pasteur, 1896. 8 Axenfeld, Cent. f. Bakt,, xxi, 1897. ZUR NEDDEN'S BACILLUS 509 may become liquefied. Upon serum agar delicate grayish drop-like colonies are formed which are not unlike those of the gonococcus. In ascitic bouillon general clouding occurs within twenty-four hours. FIG. 54.— MORAX-AXENFELD DIPLO-BACILLUS. Pathogenicity. — Attempts to produce lesions in the lower animals with this bacillus have been universally unsuccessful. Subacute con- junctivitis, however, has been produced in human beings by inocula- tions with pure cultures. ZUR NEDDEN'S BACILLUS In ulcerative conditions of the cornea, Zur Nedden has frequently found a bacillus to which he attributes etiological importance. The bacillus which he has described is small, usually less than one micron in length, often slightly curved, and generally found singly. It may be found in the diplo form but does not form chains. It is stained by the usual dyes, often staining poorly at the ends. Stained by Gram's method it is decolorized. The bacillus is non-motile. Cultivation. — It is easily cultivated upon the ordinary laboratory media. Tpon agar it forms, within twenty-four hours, transparent, slightly fluorescent colonies which are round, raised, rather coarsely granular, and show, a tendency to confluence. 510 PATHOGENIC MICROORGANISMS Gelatin is not liquefied. Milk is coagulated. Upon potato, there is a thick yellowish growth. Upon dextrose media, there is acid formation, but no gas. The bacillus forms no indol in pepton solutions. Pathogenicity. — Corneal ulcers have been produced by inocula- tion of guinea-pigs. / BACILLUS OF DUCREY The soft chancre,- or chancroid, is an acute inflammatory, de- structive lesion which occurs usually upon the genitals or the skin surrounding the genitals. The infection is conveyed from one individual to another by direct contact. It may, however, under conditions of surgical manipulations, be transmitted indirectly by means of dressings, towels, or instruments. The lesion begins usually as a small pustule which rapidly ruptures, leaving an irregular ulcer with undermined edges and a necrotic floor which spreads rapidly. It differs clinically from the true or syphilitic chancre in the lack of induration and in its violent inflammatory nature. Usually it leads to lymphatic swellings in the groin which, later, give rise to abscesses, commonly spoken of as "buboes." In the discharges from such lesions, Ducrey,9 in 1889, was able to demonstrate minute bacilli to which he attributed an etiological relationship to the disease, both because of the regularity of their presence in the lesions and the successful transference of the disease by means of pus containing the microorganisms. Morphology and Staining. — The Ducrey bacillus is an extremely small bacillus, measuring from one to two micra in length and about half a micron in thickness. It has a tendency to appear in short chains and in parallel rows, but many of the microorganisms may be seen irregularly grouped. It is not motile, possesses no flagella, and does not form spores. Stained by the ordinary anilin dyes, it has a tendency to take the color irregularly and to appear more deeply stained at the poles. By the Gram method, it is decolorized. In tissue sections, it may be demonstrated by Loeffler's methylene-blue method, and in such preparations has been found within the granulation tissues forming * Ducrey, Monatschr. f. prakt. Dermal., 9, 1889. BACILLUS OF DUCREY 511 the floor of the ulcers. In pus, the bacilli are often found within leucocytes. Cultivation and Isolation. — Early attempts at cultivation of this bacillus were universally unsuccessful in spite of painstaking experi- ments with media prepared of human skin and blood serum. In 1900, Besanc,on, Griffon, and Le Sourd 10 finally succeeded in obtaining growths upon a medium containing agar to which human blood had been added. They were equally successful when dog's or rabbit's blood was substituted for that of man. Since the work by these authors, the cultivation by similar methods has been carried out by a number of investigators. Coagulated blood, which has been kept for several days in sterile tubes, has been found to constitute a favor- able medium. Freshly clotted blood cannot be employed, probably because of the bactericidal action of the serum. Serum-agar has occasionally been used with success, but does not give results as satisfactory as those obtained by the use of the whole blood. The best method of obtaining pure cultures upon such media consists in puncturing an unruptured bubo with a sterile hypodermic needle and transferring the pus in considerable quantity directly to the agar. If possible, the inoculation of the media should be made immediately before the pus has had a chance to cool off or to be exposed to light. When buboes are not available, the primary lesion may be thoroughly cleansed with sterile water or salt solution, and material scraped from the bottom of the ulcer or from beneath its overhanging edges with a stiff platinum loop. This material is then smeared over the surface of a number of blood-agar plates. Upon such plates, isolated colonies appear, usually after forty- eight hours. They are small, transparent, and gray, and have a rather firm, finely granular consistency. The colonies rarely grow larger than pinhead size, and have no tendency to coalesce. At room temperature, the cultures die out rapidly. Kept in the incubator, however, they may remain alive and virulent for a week or more. On the simpler media, glucose-agar, broth, or gelatin, cultivation is never successful. On moist blood-agar and in the condensation water of such tubes, the bacilli have a tendency to grow out in long chains. Upon media which are very dry, they appear singly or in short chains. During recent years interest has again been aroused in the 10 Besangon, Griffon, et Le Sourd, Presse med., 1900. 512 PATHOGENIC MICROORGANISMS chancroidal lesions because of the apparent relative frequency of such lesions among venereally infected soldiers in Europe. We are informed by Walker that during the post-armistice periods of the existence of American troops in France, the proportion of chancroids to other venereal infections rose quite beyond the ordinary relative proportion of this variety of infection, apparently for the reason that prophylaxis as practiced had less effect upon chancroidal infec- tion than it did upon the syphilitic and gonorrheal infections. Since there had apparently developed in the minds of genito-urinary specialists, a certain amount of skepticism regarding the role played by the Ducrey bacillus at this time, the matter was reinvestigated in our laboratory by Teague and Deibert.11 They developed a method for direct diagnostic cultivation of Ducrey bacilli from chan- croidal lesions which has so much practical value that it will be well to quote it in considerable detail. The method as described by them is as follows: A rabbit is bled from the heart with a sterile 20 c.c. syringe and the blood is distributed in amounts of 1 c.c. in small test tubes, a little larger than the ordinary Wassermann tube. The blood is allowed to clot at room temperature and is then heated for five minutes at 55° C. It can thus be preserved in the ice-box or can be used immediately. Equally good results can be obtained when the tubes are kept in the ice-box for three to four days before use with- out heating. Pieces of stiff iron wire, gauge 18, about 5y2 inches long are bent upon themselves at one end for about % inch. Ten or twelve of these wires are placed in a 6-inch test tube and are heated in the dry sterilizer. The patient removes the dressing and a bit of the pus is picked up with the bent end of the wire, the latter having been first rubbed gently over the base of the ulcer or under its undermined edge. The pus is then transferred to a tube of clotted blood and distributed in the serum by passing the wire around the clot. A second tube is prepared in the same way. After twenty-four hours incubation at 37° C. the serum around the clot is thoroughly stirred with a platinum loop and a smear is made. Examination with the oil-immersion lens shows characteristic chains of small Gram- negative bacilli, sometimes in pure culture, sometimes in mixed cul- ture. The organism is usually so characteristic that such an exam- ination is sufficient basis for a positive diagnosis. Even when anti- 11 league and Deibert, Jour, of Urology, 4, 1920, 543. BACILLUS OF DUCREY 513 septic power or ointments have been applied, repeated positive cul- tures have been obtained by finding a bit of pus free from drug. It is not even necessary to wash the ulcer before taking cultures. At the time of the publication of their first paper, Teague and Deibert had cultured by the above method, 274 sores. In most cases these were indiscriminately cultured, even in many cases when no clinically characteristic picture was apparent. Of these 274 sores, 140 yielded positive Ducrey cultures. Of the 134 negative cases, satisfactory notes were obtained of only 69, and from these notes it is apparent that 42 of these 69 negative cases at least were not chancroidal but primary syphilitic lesions. It seems to Teague fair to assume that by this method probably over 90 per cent of true chancroids can be diagnosed, and it is so simple that the physician in the clinic can take the cultures as directed and send them to the laboratory. Isolations can subsequently be made by inoculating blood agar plates from the clotted blood tubes after 24 hours. The nutrient agar should have a PH of 7.2 or 7.3, and the agar must be neither too stiff nor its surface too dry. Teague 's results not only furnish a simple method for the determination of mixed infection, but also reaffirm the etiological importance of the Ducrey bacillus in chancroids. As to prophylactic treatment, the recent experience seems to indicate that warm water and soap very thoroughly applied is prob- ably more effective in the prophylaxis of this type of infection, than are the specific methods used for venereal prophylaxis. Pathogenicity. — Besanc,ori, Griffon, and Le Sourd, and others, have succeeded in producing lesions in man by inoculation with pure cultures. Inoculation of the lower animals has, so far, been entirely without result. CHAPTER XXVI MIOROCOCCUS INTRACELLULARIS MENINGITIDIS (MENINGOCOCCUS) AND EPIDEMIC CEEEBBOSPINAL MENINGITIS INFECTIOUS processes in the meninges may be caused by many different microorganisms. Meningitis may be primary or secondary. Secondary meningitis may often occur during the course of pneumonia, when pneumococci, carried to the meninges by the blood stream, give rise to a usually fatal form of the disease. More rarely a similar process may occur as a secondary manifestation of typhoid fever or influenza. Menin- gitis may also result secondarily by direct extension from sup- purative lesions about the skull, such as those occurring in diseases of the middle ear or frontal sinuses or after compound fractures. In such cases the invading organisms are usually staphylococci, strepto- cocci, or pneumococci. Isolated cases of meningeal infection with B. coli, B. para- typhosus, Bacillus pestis, and Bacillus mallei have been reported- A frequent more chronic form of the disease is caused by Bacillus tuberculosis. Primary acute meningeal infection, however, is due chiefly to two microorganisms, Micrococcus intracellularis meningitidis, and the pneumococcus. A tabulation of the comparative frequency with which the vari- ous microorganisms are found in the meninges has been attempted by Marschal.1 This author estimates that about 69.2 per cent of all acute cases are due to the meningococcus, 20,8 per cent to Diplo- coccus pneumonias, and the remaining 10 per cent to the other bac- teria mentioned. The cases caused by the pneumococcus and the other less frequent incitants usually occur sporadically. When the disease occurs in epidemic form, it is almost always due to the meningococcus. 1 Marschal, Diss. Strassburg, 1901, Quoted from Weichselbaum, in Kollc und Wassermann, ' ' Handbuch. ' ' 514 MICROCOCCUS INTRACELLULARIS MENINGITIDIS 515 Diplococcus intracellularis meningitidis was first seen in menin- geal exudates by Marchiaf ava and Celli 2 in 1884. These authors not only described accurately the morphological characteristics now recognized, but also called attention to the intracellular position of the microorganism and to its gonococcus-like appearance. They failed, however, to cultivate it. Observations confirmatory of the Italian authors were, soon after, made by Leichtenstern.3 Cultivation and positive identification as a separate species was not accomplished, however, until Weichsel- baum,4 in 1887, reported his observations upon six cases of epidemic cerebrospinal meningitis in which he not only found the cocci mor- phologically, but was able to study their biological characteristics in pure culture. The researches of Weichselbaum were soon confirmed and extended by elaborate studies 5 which left no doubt as to the specific relationship between the microorganism cultivated by him and the clinical condition. Morphology and Staining. — Stained in the spinal fluid from an infected patient, the meningococcus bears a striking similarity to the gonococcus. The microorganisms appear intra- and extracellu- larly, usually in diplococcus groups, sometimes as tetrads, or even in larger agglomerations. The individual diplo-forms are flattened on the sides facing each other, presenting somewhat the biscuit-form of the gonococcus. The variation in size of the cocci in the same smear is a noticeable feature and of some diagnostic importance. This dissimilarity in size is noticeable also in cultures, which, especially when older than twenty-four hours, contain forms double or even triple the size of the average coccus. These may possibly be involution forms. The meningococcus is non-motile and non-spore forming. It stains easily with all the usual aqueous anilin dyes. Its behavior toward Gram's stain was long a subject of controversy, owing to the error of Jaeger,6 who claimed to have found it Gram-positive. There is no question now, however, that the cocci decolorize by Gram's method when this is carefully carried out. 2 Marchiaf ava and Celli, Gaz. degli ospedali, 8, 1884. 3 Leichlenstern, Dent. med. Woch., 1885. 4 Weichselbaum, Fort. d. Med., 1887. 5 Councilman, Mallory, and Wright, Special Eep. Mass. Board of Health, 1898, Albrecht und Ghon, Wien. klin. Woch., 1901. 6 Jaeger, Zeit. f. Hyg., xix, 1895. 516 PATHOGENIC MICROORGANISMS In spinal fluid satisfactory preparations may be obtained by staining in Jenner 's blood stain. Councilman, Mallory, and Wright 7 were the first to notice that, when stained with Loeffler 's methylene- blue, meningococcus stains irregularly, showing metachromatic gran- ules in the center of the cell bodies. These granules can be demon- strated more clearly with the Neisser stain employed for similar demonstration in the case of B. dipththeriae and have some value in differentiating meningococcus from gonococcus. RT 'J *** * .*»>* *•* V ** V * ^ :v^ ;* , ¥ FIG. 55. — MENINGOCOCCUS PURE CULTURE. It is important to remember that meningococci in spinal fluid undergo solution very readily, a solution which is probably an autolysis, with the result that spinal fluid which may be full of polymorphonuclear leucocytes, contains very few recognizable or- ganisms. This readiness of meningococci to go into solution will be spoken of below in connection with problems of cultivation. Cultivation of the Meningococcus. — The meningococcus is peculiar in that there is considerable difference in the ease with which separate strains can be made to grow upon artificial media. Some meningo- cocci grow readily upon all meat infusion culture media. They may even grow upon some meat extract media, but growth upon these is never profuse. It is never well to rely upon media to which no enriching substance has been added, or that have not been especially made for meningococcus cultivation when attempts are made at first 7 Councilman, Mallory , and Wright, Eep. Mass. State Bel. of Health, 1898. MICROCOCCUS INTRACELLITLARIS MENINGITIDIS 517 isolation from human material. After the bacteriologist is familiar with the individual strains, he may at times carry his strains on the simpler media, meat infusion agar and broth. Growth is more luxuriant and rapid upon media to which animal protein in the form of blood serum or ascitic fluid has been added. Coagulated serum is not liquefied. For cultivation of the meningo- coccus directly from the human body it is wise to use the richer serum or blood media. Agar to which whole rabbit's blood has been added FIG. 56. — MENINGOCOCCUS IN SPINAL FLUID. forms an excellent medium, both for cultivation and for keeping the organism alive. Loeffler's blood serum is less favorable. It is advisable, too, when cultivating directly from spinal fluid, to plant rather large quantities (1 to 2 c.c.), since many of the cocci in the exudate will fail to develop colonies, possibly because of their prolonged exposure either to the body fluids or to their own products in a closed space. Upon broth, growth is slow and takes place chiefly upon the sur- face, the sediment consisting mainly of dead bacteria. Glucose added to agar or to broth renders the medium more favorable for rapid 518 PATHOGENIC MICROORGANISMS growth, but, owing to acid formation, tends to cause a more rapid death of the culture. In flasks of broth containing glucose one per cent, and CaC03 one per cent, however, cultures have been kept alive for as long as fourteen months (Hiss). On milk, growth takes place without coagulation of the casein. Potatoes are not a favorable medium, though growth occasionally takes place. While slight alkalinity or acidity does not inhibit, the most favor- able reaction of media is a Pw of 7.4 to 7.6. H Oxygen is necessary for development. Complete anaerobiosis, while not absolutely inhibitory, is extremely unfavorable, unless proper carbohydrates be present. Recently, work by Wherry and Erwin,8 as well as by Gates,9 has shown that the growth of the meningococcus is definitely stimulated by replacing about ten per cent of the air by carbon dioxide. The plates are placed into a closed jar, into which freshly produced carbon dioxide is allowed to pass, and the jars are then sealed and incubated. This matter has been discussed in a previous section on partial oxygen tensiort in bacterial cultures. We have confirmed this in our labora- tory, and find that the colonies grow larger and growth is more rapid under the partial C02 atmosphere. While growth may take place at temperatures ranging from 25° to 42° C., the optimum is 37.5° C. It is an important aid to the recognition of true meningococci that they never grow at ordinary room temperature. Apart from the remarkable viability displayed upon calcium-carbonate broth, the average length of time during whiclTthe meningococcus will remain alive without transplantation is rather short. Recently isolated cultures grown on agar or serum-agar may die within two or three days. Accustomed to artificial cultivation through a number of generations, however, the cultures become more hardy and transplantation may safely be delayed for a week or even longer. Albrecht and Ghon10 have kept a culture alive on agar for one hundred and eighty-five days. It is a strange fact that after pro- longed artificial cultivation some strains of meningococcus may grad- ually lose their growth energy and finally be lost because of their refusal to develop in fresh transplants. It is our belief that this phenomenon which hitherto we have been at a loss to explain, may have some connection with the so-called 8 Wherry and Erwin, Jour, of Infec. Dis., 22, 1918, 194. 8 Gates, Jour. Exper. Med., 29, 1919, 325. 10 Albrecht und Ghon, Wien. klin. Woch., 1901. MICROCOCCUS INTRACELLULARIS MENINGITIDIS 519 "bacteriophage" phenomena discussed in a separate section. Since the extensive investigation of the autolytic properties acquired by bacteria under certain conditions, there has been no investigation of the meningococcus problem from this point of view. We ourselves have often found meningococcus cultures to lose their viability under conditions which, retrospectively, we suspect now may have been due to this kind of development. It is also not unlikely that the extensive autolysis of the organism in the spinal fluid may have a similar sig- nificance. Storage is best carried out at incubator temperatures. At room temperatures or in the ice chest, the diplococcus dies rapidly.11 Special Meningococcus Media. — For the routine cultivation of meningococcus, there are certain media which are better than others and which are, therefore, described in this section. As a basis for meningococcus media, we like to use hormone agar or hormone broth, or trypagar or trypsinized broth as described in the section on media. To these basic media enriching substances are added. The necessity for these enriching substances may have a more complex cause than the simple addition of nutrition, since, as Lloyd has sug- gested, the occasional first growth on simple media of meningococci directly from the human body, may depend upon the presence of a certain amount of "vitamine" furnished by the animal fluids present in the exudate. The most convenient substances to add to these media are blood in one form or another. Many different varieties of blood are favorable, and human, horse, or rabbit blood can be most con- veniently used. The blood may be defibrinated and added directly in quantities of about five per cent, and if agar for plating is used, melted agar is mixed with the blood and thoroughly shaken just as the plates are poured. Laked blood is very convenient, and may be pre- pared by mixing whole blood with about four parts of sterile distilled water. This laked blood may be kept and mixed with the agar just before pouring the plates, after the agar has been cooled below 50°. The blood may also be laked in ether and in this way can be kept sterile for a long time before being added to the basic medium. Blood serum and ascitic fluid can be used, but do not seem to give as good results as does laked or whole blood. The addition of one-half to one per cent of glucose is always favorable. The special pea-powder-blood-agar 11 A very thorough biological study of meningococcus and related organisms lias recently been made by Elser and IFuntoon (Jour. Med. Res., N. S. vol. xv, 1909), which may be consulted for a more detailed description of cultural characteristics. 520 PATHOGENIC MICROORGANISMS used by the British during the war is not described in detail because we believe that for ordinary laboratory work its production is too com- plicated, without offering sufficient advantages over other media. To summarize, we, therefore, recommend for plating media, carrier work, and isolation from spinal fluid, hormone or trypagar with a PH of 7.4 to 7.5, containing one-half per cent glucose, to which about 5 or 10 per cent of defibrinated or laked blood is added just before the plates are poured. For the storage of stock cultures, Vedder's starch agar described in the section on media has been used with satisfaction. Gordon 12 and others also have used coagulated egg media in slants for storage of stock cultures with good results. Egg -yolk Medium for the Storage of Meningococcus Cultures.13 — The egg-yolk used may be the yolks of eggs, the whites of which have been used for clearing media. One volume of the egg-yolk is mixed with one-half volume of physiological salt solution. The yolk and salt are thoroughly mixed, tubed and slanted. The slants are then inspissated in the usual way. This can be done in an autoclave by bringing the temperature up gradually without letting out the air, until 14 pounds pressure has been reached, and then maintaining this for twenty minutes. Great care should be taken to prevent bubbles in the medium. The tubes should be plugged with paraffin, since water of condensation is necessary to make the medium useful for storage. For fermentation reactions, solid or fluid media with various sugars and litmus indicator, may be used. Gordon, whose experience in this kind of work has been extensive, used for his fermentations a liquid medium of simple pepton water with one per cent blood serum and the sugar to be investigated, added. Elser and Huntoon 14 em- ployed among other things, for their extensive fermentation work, sheep serum water, ascitic broth, and broth made with nutrose. Resistance. — The meningococcus is killed by exposure to sunlight or to drying within twenty-four hours.15 It is extremely sensitive to heat and cold and by the common disinfectants is killed in high dilutions and by short exposures, At 0° C. it usually dies within two or three days. ^-Gordon, FlacTc and Hinex, Med. Kes. Com., Special Report Series, No. 3, London, 1917. "Directions of Major Foster from Gordon's Laboratory. 14 Elser and Huntoon, Jour. Med. Eos., 20, 1909, 371. 15 Councilman, Mallory, and Wright, Boston, 1898; Albrecht and Ghon, loc. cit. MICROCOCCUS INTRACELLULARIS MENINGITIDIS 521 A special study of the resistance of meningococci to various dye stuffs has been made by Binger.16 An interesting result of these investigations was that Binger found that methylene-blue had a specific inhibiting action upon meningococcus and gonococcus at dilutions too IOAV to inhibit other pathogenic microorganisms, and that this inhibitory action was not interfered with by the presence of the protein in spinal fluid or other exudates. Toxic Products of the Meningococcus. — No soluble exotoxin has ever been conclusively isolated from meningococcus cultures. A substance which causes acute symptoms in rabbits within an hour can be recovered from young broth cultures of meningococci, and from filtered wash- ings from meningococcus cultures on agar. These substances are analogous to the so-called "X" substances which one of the writers with Kuttner and Parker17 has described, which are non-specific, can be obtained from many different microorganisms and are non- antigenic. That they are a very real and important substance in connection with meningococci we are persuaded to believe by the fact that those who immunize horses for serum production with meningo- cocci find that it is necessary to wash the agar cultures once before injecting into horses, otherwise severe symptoms occasionally result. A number of investigators have found that cultures that have been kept in broth long enough for a certain amount of extraction or autolysis to occur, yield toxic products which are in general identical in their action to that of whole meningococci injected in analogous quantities. This has been the experience of Flexner,18 Kraus and Doerr,19 and others. Extracts of meningococci made with salt solution, weak sodium hydrate, etc., kill guinea pigs within 24 hours, with symptoms of general intoxication, peritoneal exudates, and often pleural exudates. Intravenous injection of sufficient quantities of such extracts or of dead meningococci may kill rabbits. No reliable or con- stant results with such substances have been obtained, but it is quite definite that the bodies of meningococci, like the bodies of typhoid and colon bacilli, are toxic for animals by what is generally spoken of as an endotoxin action. 16 Binger, Jour. Infec. Dis., 25, 1919, 277. 17 Zinsser, Kuttner and Parker, Proc. Soc. Exper. Biol. and Med., November, 1920. 18 Flexner, Cent, f . Bakt., 43, 1907. 19 Kraus and Doerr, Wien. klin. Woch., 1908. 522 PATHOGENIC MICROORGANISMS Types of Meningococci. — Until 1909 it was believed that the men- ingococcus group, was homogeneous, and that no essential difference between individual members of the group existed. In this year, Dopter 20 found that some of the meningococci isolated from cases which occurred in Paris and environment, could be distinguished "by specific agglutination reactions from the ordinary or normal type. This para-meniiigococcus, as Dopter called it, opened the way for investigations aimed at the serological classification of the group, and, as was to be expected, it was found that there were a considerable number of different meiiingococcus sub-types. Wollstein 21 confirmed Dopter 's work and found, among other things, that the various para- meningococeus strains were not wholly homologous, and suggested their possible further subdivision. Gordon 22 examined a large num- ber of meningococci from cases occurring among British and Canadian troops, and found that all the organisms studied by him could be divided into four definite types. He used not only the agglutination reaction, but controlled them with absorption tests. Tulloch,23 follow- ing up Gordon's work on a considerable material, found that, out of 356 cocci investigated, 234 gave specific results with the four type sera used by Gordon's laboratory. He found that, with remarkably few exceptions, the organisms responsible for the outbreaks among British troops were comprised in the four Gordon types. He did, however, find some organisms in the nasopharyngeal cultures of carriers which, though closely resembling meningococci, did not react with any of the type sera. There was some question, however, in his mind as to whether these represented true virulent meningococci. An important result of Gordon's investigations was to show that very many of the organisms obtained from carriers belong to one of the four types known to exist in actual cases of meningeal infection. In America, Flexner 24 and his associates have investigated the group relationships of the meningococci very carefully, and their results indicate that there are probably two main types, the normal and the para-meningococcus of Dopter; and, in addition to this, a considerable number of heterogeneous intermediate types which are related to each other and to the fixed types more or less in the same 20 Dopter, Cdmpt. Eend. de la Soc. de Biol., 67, 1909, 74. 21 Wollstein, Jour. Exper. Med., 20, 1914. 22 Gordon, Brit. Med. Res. Commit. Eeports, London, 1915 and 1917. 28 Tulloch, Jour. Royal Medical College, February, 1918, p. 9. 24 Flexner, Bulletin, Rock. Inst. for Med. Res., 1917. MICROCOCCUS INTRACELLULARIS MEN1NGITIDIS 523 way, but somewhat more closely than are the different members of the viridans group of streptococci, a point which makes it plain that a diagnostic or curative serum, to be truly polyvalent, must be produced with many different representatives of organisms isolated from cases. The correspondence of the different types, as named in various coun- tries, is as follows: Gordon's type I = para-meningococcus Gordon's type II = normal meningococcus Gordon's types III and IV = intermediate or irregular strains, of which there are a considerable number of different ones, shading into each other, serologically. As far as the prevalence of type is concerned, no definite rule can be established at present. In the extensive investigations of Gordon and his co-workers, it was found that the earliest cases were mostly his type I, later came his type II, especially in the London district, and after March type IV cases began to appear, but no type III cases were noticed until July. Agglutination. — Immunization of animals by repeated inocula- tions of meningococcus 25 results in the formation in the blood serum of agglutinins. Kolle and Wassermann 26 obtained from horses a serum which had an agglutinating value of 1 :3,000 for the homol- ogous strain, and of as much as 1 :500 for other true meningococcus strains. Similar experiments by Dunham27 and others have proved the unquestionable value of agglutination for species identification of this group. Great differences may, however, exist between indi- vidual races in their agglutinability in the same immune serum. Kutscher has recently called attention to the fact that strains which cannot be 'agglutinated in specific sera at 37° C. will often yield positive results when subjected to 55° C., a fact of some prac- tical importance if confirmed. Elser and Huntoon 28 have shown that in the serum of infected human subjects agglutination of some strains takes place in dilutions as high as 1 :400. The Production of Agglutinating Sera for Meningococcus Deter- mination in • Laboratories. — For this purpose, rabbits are best K Albreclit and Ghon, Wien. klin. Woch., 1901. 26 Kolle und Wassermann, Deut. med. Woch., 15, 1906. 27 Dunham, Jour. Inf. Dis., 11, 1907. 28 Elser and Huntoon, loc. cit. 524 PATHOGENIC MICROORGANISMS .employed. Amoss has found that young rabbits are more satisfac- tory than older ones for this purpose, and he uses rabbits weighing between 1500 and 1800 grams. He grows his meningococci on glu- cose agar slants, and washes up the growth in salt solution. 0.001 of a culture is inoculated as the first dose. For the rapid production of agglutinating sera, he injects his rabbits for three succeeding days, giving a rest of five days, and then another course of three days injection. He bleeds the animal two or three days after the second course of inoculation. For ordinary purposes, the slow method of three or four day intervals, about five or six injections, with bleeding eight or nine days after the last injection, may be used. Among English workers, Hine 29 injects culture suspensions grown on 25 per cent hemoglobin serum agar, killed at 65° and brought to a standard opacity. 0.5 per cent carbolic acid is added for preservation. He standardizes all his suspensions by opacity comparisons against suspensions of freshly precipitated barium sulphate. He compares by diluting his suspension in a test tube of similar dimensions as the standard tube, until the image of a small flame is just visible in the same distance from the flame as in the case of the standard tube. With such suspensions he immunizes rab- bits, beginning with an injection of two doses of five hundred million cocci at an interval of one hour. Six days later he gives three million cocci, and, if the serum is satisfactory on the eighth day later, he bleeds. This method was satisfactory in the hands of Hine, with types I and III. With the other types he has had to give larger and more frequently repeated doses. In all such immunization, experience and judgment, with frequent titration of samples of the rabbit serum taken from the ear, are necessary. Nicolle 30 at the Pasteur Institute uses for immunization, pow- dered meningococcus antigen prepared from growth on agar slants by suspension in salt solution, centrifugation and drying. Hine also recommends the use of rabbits ranging from 800 to 1500 grams. Agglutination Technique ivith Meningococci. — Agglutination of meningococci present considerable difficulties because of the relative inagglut inability of many meningococcus cultures. This is a peculiar- ity of these organisms which has necessitated much investigation and many technical modifications. Hine has found that allowing the 29 Hinc, Med. lies. Committee, Spec. Eep., Series 3, No. 3, p. 99, 30 Nicolle, MICROCOCCUS INTRACELLULARLS MENINGITIDIS 525 diluted carbolic saline suspension to stand for twenty-four hours, increases agglutinability, and recommends this technique if time per- mits. Tulloch31 has called attention to a number of precautions both in handling of the cultures and the serum for agglutination which seem to us sufficiently important to note. He recommends the use of standardized suspensions of the meningococci as described by Hine, and recommends great caution in the nature of the medium on which the cultures are grown. He advises getting rid of the condensation water in the slants before washing off the growth, owing to the possi- bility of alkaliii or acid reactions in this condensation water. The strength of the phenol in standard suspensions should never be more than 0.5 per cent. Also he warns against getting any of the agar into the suspension because he believes that it may act under certain cir- cumstances as a protective colloid. On the basis that moderate heat increases the agglutinability of organisms like the typhoid bacillus and meningococci, the workers in the British Central Laboratory used the routine method described under carrier determination, that is, incubating the serum culture agglutination mixtures in a water bath at 55°, for varying periods, usually twelve hours before the final readings are made. Hot air ovens at 55° are not good substitutes because of the great evaporation which, according to Hine, occasionally leads to spontaneous agglutination. (See also section on Carriers.) A'gglutinin Absorption Experiments for Meningococcus Typing.32 — A thick suspension of the meningococci to be examined, quantity 0.5 c.c., is mixed with the various monovalent type sera, 0.5 e.c., dilu- tions 1 to 25, in saline. Similar serum dilutions were set up without suspensions. The tubes are thoroughly shaken, set in the water bath at 37° for one hour, and then at room temperature overnight. The tubes are then centrifugalized at high speed until the supernatant fluid is clear. The supernatant fluids of the tubes containing the suspensions, as well as the fluids similarly treated without suspension, now repre- sent serum dilutions of 1 to 50. Each suspension-absorption tube now has a serum control which has been exposed to the same temperature in the same dilutions with- out meningococci. From each set of two tubes now other tubes are made, into which 0.4, 0.2, 0.1, and 0.05 c.c. are taken, and with salt 31 Tulloch, Royal Army Med. Jour., February, 1918. 32 The description given is that given by the British Medical Research Com- mittee, loc. cit. 526 PATHOGENIC MICROORGANISMS solution the volume of all of these tubes is brought up 0.4 c.c. To each of the tubes now 0.4 c.c. of the homologous meningococcus sus- pension is added, giving serum dilutions ranging from 1 to 100, to 1 to 800. These are now incubated at 55° for twenty-four hours, as in the first agglutination. The agglutinating titers of the absorbed sera are now compared with those of the un-absorbed, and diminutions of titer are noted. Animal Pathogenicity. — Animals are not very susceptible to infection with Diplococcus meningitidis. Subcutaneous inoculation is rarely followed by more than a local reaction unless large quan- tities are used. White mice are rather more susceptible than other species. Intraperitoneal and intravenous inoculation of sufficient quantities usually results in the death of mice, rabbits, guinea-pigs, and dogs. Occasional strains have been found to possess a not inconsiderable degree of toxicity for rabbits, grave symptoms or even death following intravenous injection of but moderate quanti- ties without any traceable development of the microorganisms in the organs of the animals. Similar observations have been made by Albrecht and Ghon,33 who succeeded in killing white mice with dead cultures. It would seem, therefore, that the effect of this coccus upon animals depends chiefly upon the poisonous substances contained in the bacterial bodies (endotoxins). Lepierre 34 has obtained the meningococcus toxin by alcohol precipitation of broth cultures. Weichselbaum himself succeeded in producing meningeal sup- puration and, in one case, brain abscess, by subdural inoculation of dogs. Councilman, Mallory, and Wright produced a disease in many respects similar to the human disease by intraspinous inoculation of a goat. More recently, Flexner 35 has succeeded in producing in monkeys a condition entirely analogous to that occurring in human beings. THE DISEASE IN MAN The disease produced in man consists anatomically in a suppurative lesion of the meninges, involving the base and cortex of the brain and the surface of the spinal cord. The nature of the exudate may vary 33 Albrecht nnd Ghon, loc. cit. "Lepierre, Jour, de phys. et de path, gen., v, No. 3. 85 Flexner, Journ. of Exp. Med., 1906. MICROCOCCUS INTRACELLULARIS MENINGITIDIS 527 from a slightly turbid serous fluid to that of a thick fibrinous exudate. In chronic cases encephalitis and dilatation of the ventricles may take place. Apart from their presence in the meninges and in the naso- pharynx, meningococci have not been satisfactorily demonstrated in any of the complicating lesions of the disease. Reports of their pres- ence in the conjunctivas, in the bronchial secretions from broncho- or lobar pneumonia, and in otitis media, have been reported but are not very common. The occurrence of this microorganism in the circulating blood of meningitis cases has been definitely proved by Elser,36 who found it in ten cases. In the discussions on epidemiology, below, we will see that Her- rick and others claim that the meningococcus is probably, in the majority of cases, in the blood before it reaches the meninges, making its way to the central nervous system by way of the blood stream rather than directly along the lymphatics at the base of the skull. It seems fair to assume from blood culture evidence that this cer- tainly happens in many cases even though it may not be the rule. During epidemics, also, there are occasional cases in which a general septicemia due to meningococci occurs, without ever giving rise to symptoms pointing to meningeal involvement. These cases are always violent in course, usually fatal and accompanied by a profuse petechial rash. BACTERIOLOGICAL MANAGEMENT OF THE MENINGITIS CASE AND SERUM TREATMENT In the light of our present knowledge of the bacteriology and serum treatment of epidemic meningitis, a considerable responsibil- ity rests with the bacteriologist. The difference between recovery and death may depend directly upon the speed with which a bac- teriological diagnosis is made and a proper management of the serum treatment. When a case of suspicious fever in which slight stiffness of the neck, and a developing Kernig sign is associated with the other indications of an acute infection, the first step must consist of lumbar puncture. A sterile lumbar puncture needle is thrust into the spinal canal, a little to one side of the third or fourth lumbar space, and the fluid 36 E Iscr, Jour. Med. Ees., xiv, 1906. 528 PATHOGENIC MICROORGANISMS which is always under some pressure, is taken directly into a centri- fuge tube. This fluid must then be examined as above indicated in the technical section on spinal fluid, and the diagnosis made. If pos- sible, a smear should be made at the bed side, and an immediate Gram stain done with the first drop of fluid that flows. In this way, it may be possible to inject the first dose of serum immediately after the withdrawal of the diagnostic fluid. Examination of Spinal Fluid. — The spinal fluid of meningococcus cases is slightly turbid in the very early periods, becoming increas- ingly purulent, with large numbers of polymorphonuclear leucocytes. In some cases the fluid which has been very purulent may clear up considerably, and then become purulent again, a matter probably dependent upon sacculation in parts of the subarachnoid space. The fluctuations in the nature of the spinal fluid under intraspinous serum treatment will be spoken of in another place. A certain amount of prognostic information can be obtained from the spinal fluid in that in severe cases that are not doing well, there will be a considerable number of organisms, extracellular. Ordinarily, the majority of the meningococci are intracellular. Such spinal fluid should be taken into sterile centrifuge tubes, brought to the labora- tory without delay, slides smeared from the sediment, and stained by Jenner and by Gram. It is important to remember that because of the extensive autolysis of meningococci in the fluid, it may under circumstances be very difficult to find meningococci. In such cases, if prolonged search has failed to reveal organisms, our ex- perience has taught us to assume that purulent fluid from a case of an acute meningitis in which there are a preponderance of poly- morphonuclear leucocytes without organisms is probably " meningo- coccus " in origin. Streptococcus and pneumococcus fluids invariably show Gram-positive cocci. The fluid should be cultured upon blood agar plates, the medium prepared as described above. It is well to inoculate the plates heavily since the viable organisms present, even in acute fluids, may be relatively few in numbers. To be on the safe side it is sometimes well too, to place a portion of the fluid in the original centrifuge tube in the incubator for three or four hours before inoculating media from it. The typing of meningococci derived from spinal fluid is desirable since the preliminary injection of polyvalent serum upon first diag- nosis may be advantageously followed by the injection of type sera, MICROCOCCUS INTRACELLULARIS MENINGITIDIS 529 homologous to the organisms found in the patient. This method is impracticable on a large scale since so many types, shading into each other, are possible in this disease. However, the method is used to a considerable extent in France. As stated in the section on the manner of entrance of the menin- gococci into the subarachnoid space, it is a question now under discussion whether the organisms travel along the lymphatics to the base of the skull directly, or whether bacteriemia precedes menin- geal infection. It is well, always, in cases of early meningitis, to take blood cultures. In taking blood cultures it is best to inoculate hormone glucose broth flasks containing not less than 100 c.c. of culture fluid and to make a number of glucose hormone agar plates with varying amounts of blood. The presence of menmgococci in the blood is, of course, an indication for intravenous as well as intraspinous injection of serum, a procedure which is in our opinion advisable in all cases, since it is quite likely that meningococcus septicemia, constant or intermittent is a regular feature of the pathology of the disease. Serum Therapy of Meningitis. — During recent years, attempts have been made to treat epidemic meningitis by injections, subcu- taneous and intraspinous, of meningococcus-immune serum. Wasser- mann,37 in 1907, reported results of such treatment in one hundred and two patients, with a recovery of 32.7 per cent. The serum, manufactured by Wassermann and his associates, was obtained from horses immunized with cultures of meningococcus and with toxic meningococcus extracts. More recently Flexner and Jobling38 have used a similar serum in the United States with apparently excellent results. The serum, in Flexner 's cases, as in the technique first used by Jochmann, is injected intraspinously after a quantity of spinal fluid had been withdrawn. The cases treated by Flexner and Jobling 's method have now reached large numbers, both in this and foreign countries and the value of the serum as a therapeutic agent seems firmly established. The Serum. — In America polyvalent serum is used almost universally. Horses, as for other serum production, are the animals employed. The cultures with which the horses are im- munized must be many containing representatives of the normal 37 Wassermann, Dent, med. Woch., 39, 1907. 38 Flexner and Jobling, Jour. Exper. Med., x, 1908. 530 PATHOGENIC MICROORGANISMS and para-types, and a considerable number of intermediates, if pos- sible, to represent individuals from various parts of what we may call the spectrum of intermediate agglutination types. The choice of cultures is perhaps the most important single feature in serum production, and those who undertake to produce serum should be constantly receiving cultures from various parts of the country, isolated from cases, checking them up with their serum product, and adding them to their immunizing collection, if they are not represented by antibodies in the polyvalent serum. It is still a question of demanding some research, whether or not a definite limitation of the number of strains used for immunization would be of advantage, since the use of too many different strains may keep down the agglutination value of the serum of the immunized horse. The cultures injected into the horse are grown on agar, and once washed in salt solution before injection. Various routines for the injection of horses have been devised, the most useful method at the present time consisting of injecting on two or three consecu- tive days, giving rests of seven or eight days, between courses of injection. By this method, antibody production may be speeded up. It is unnecessary here, however, to go into the details of the actual technical procedures and measurements used in the production of serum. These methods are constantly changed and can be learned only by taking part in the process in a well equipped producing laboratory. Serum must be standardized before it can be marketed. This has been a very difficult matter and a number of suggestions have been made. Flexner and Jobling39 first attempted standardization by opsonin contents. Complement fixation has been recommended by some writers, but the usual method at the present time is that of agglutination. As a general rule Flexner states that the poly- valent sera are ready for use when they agglutinate the normal and para-types in dilutions of 1 :1500 or 1 :2000. Such sera should also agglutinate intermediate strains in dilutions of 1 :200 and upward. Administration of Serum. — The most important single considera- tion in serum treatment of meningitis is the early recognition of the case and avoidance of delay in starting the specific treatment. Failure of serum treatment can probably in most cases be referred 89 Flexner and Jobling, Jour. Exper. Med., 10, 1908. MICROCOCCUS INTRACELLULARLS MENINGITIDIS 531 to delay. Lumbar puncture, therefore, should be done as early as the first suspicion is aroused, and, if meningococci are found, the injection of serum should follow as rapidly as possible. It is probably best, in the long run, to inject serum immediately upon obtaining a turbid fluid in a case in which the clinical suspicion points strongly to epidemic cerebrospinal meningitis. The technique of serum injection consists in first withdrawing spinal fluid by tapping the canal with a sterile needle and allowing the fluid to flow out, of course without suction, holding a centrifuge tube directly over the butt of the needle. The flow is allowed to continue until the drops begin to come quite slowly, that is, a drop every ten or twenty seconds, and then the serum is injected, either by gravity or with a syringe through the same needle. It is im- portant that the serum at body temperature shall enter the canal very slowly, and, for this reason, the gravity method is advised. A gravity arrangement can easily be constructed by attaching about eighteen inches of catheter tubing, sterilized, to the end of the needle with a small sterile funnel at the other end. The withdrawal of large amounts of fluid suddenly sometimes causes trouble, the patient breathing rapidly, and showing symptoms of threatened collapse, but this is rare, and a ' little judgment in withdrawing fluid which has been under considerable pressure too rapidly will usually guard against accident. During the injection of the serum, the patient should be carefully watched, since occasionally alarming symptoms may arise from too rapid increase of internal pressure. The physician must be on the alert for such symptoms and imme- diately discontinue the injection for the time being. Flexner40 advises 10 minutes for the injection of the entire amount of serum used. The dosage of serum should, to some extent, depend upon the amount of fluid withdrawn, and the amount injected should usually be less by several centimeters than the amount withdrawn. The average dose for an adult should be about 30 c.c., though more may be given when large quantities of fluid have been withdrawn, and when the case is very carefully watched by an experienced man. Sophian41 has recommended controlling the withdrawal of spinal fluid and the injection 'of the serum by blood pressure measurements. 40 Flexner, Bulletin, Eock. Inst. for Med. Ees., 1917. n, Epidemic Ccrebrospinal Meningitis, St. Louis, 1913, p. 54. 532 PATHOGENIC MICROORGANISMS Sudden drops of blood pressure in either case, should lead to caution, and perhaps interruption of the procedure. Repetition of the injections is as important as the initial injec- tion, as far as cure is concerned. The action of the serum may be compared somewhat to the action of anti-serum in a Pfeiffer reaction in a guinea pig's peritoneum. Thus, probably some bac- teriolysis and considerable stimulation to phagocytosis by opsonic action may result. The spinal fluid shows changes in that the num- bers of organisms are diminished and the extracellular ones dis- appear. Purulent spinal fluid may become clearer and may even become entirely free from organisms or leucocytes. There is prob- ably a certain amount of poison neutralization by the serum. Repeti- tions of the doses, therefore, must be governed to some extent by the progress of the case, clinical conditions pointing to changes in the meningeal inflammation, and observation of the spinal fluid. One injection a day for three to six days usually controls a case that is treated with sufficient promptness. In addition to the intraspinous administration, it is wise to inject from 30 to 50 c.c. intravenously, preceding this by withdrawal of blood for blood culture, and being governed as to repetition by subsequent blood culture control.' Everyone dealing with meningitis during an epidemic must re- member that occasionally meningococcus septicemia cases occur which never show meningeal infection. We have mentioned these in another place, but believe that more attention should be given them, since they are very apt to be fatal, either without meningitis, or subsequently followed by a violent meningeal involvement. Such cases displayed the clinical picture of a general severe septic in- fection with usually a profuse eruption in which petechial spots not unlike those of typhus fever may cover the entire body. There is an irregular septic temperature with a high leucocytosis and sometimes delirium. Blood culture will diagnose these cases and vigorous intravenoiis serum treatment would be indicated. Effects of Serum Treatment. — The mortality of meningitis in the days before serum was used varied between 60 and 80 per cent. Higher mortalities have been noted in individual epidemics. The average for many different parts of the world fluctuates about 70 per cent. Since serum treatment was begun just before the year 1906, a great many statistical studies have been made which are of course subject to great error, owing to the fact that the treated MICROCOCCUS INTRACELLULARIS MENINGITIDIS 533 eases must have included a great many treated too late to permit any kind of treatment to be effective. Flexner 's statistics of cases under serum treatment show that, of 1211 cases, analyzed, those treated between the first and third day (199) showed a mortality of 18.1 per cent, those treated between the fourth and seventh day (346) showed a mortality of 27.2 per cent, and those treated later than the seventh day (666) showed a mortality of 36.5 per cent. The following table taken from a paper by Flexner, published by the Rockefeller Institute as a Bulletin in 1917, gives similar com- parative mortality statistics reported by different observers. COMPARATIVE MORTALITY REPORTED BY VARIOUS OBSERVERS 42 Treatment Begun. Flexner, Per Cent. Netter, Per Cent. Dopter, Per Cent. Christo- manos, Per Cent. Levy, Per Cent. Flack, Per Cent. Before third dav 18 1 7 1 8 2 13 0 13 2 9 09 From fourth to seventh day 27.2 11.1 14.4 25.9 20.4 After seventh day 36.5 23.5 24.1 47.0 28.6 50.00 Altogether, then, it seems quite 'clear that serum treatment has made a tremendous difference in the mortality from this otherwise so fatal disease. OTHER GRAM-NEGATIVE MICROCOCCI WHICH MUST BE DIFFERENTIATED FROM MENINGOCOCCI MICROCOCCI CATARRHALIS. — This organism is more particularly described in a separate section below. It is one of the common organisms which may confuse carrier examinations because of its frequent presence in the nose and throat of normal human beings. Its fermentation reactions are described in the table from Elser and Huntoon43 also given below. The organisms are slightly larger than meningococci, grow readily on the simplest media, the colonies are larger, thicker, opaque and white, and have a tendency to dryness quite distinct from the dew-drop like appearance of meningococcus colonies, and do not agglutinate in specific serum. They show a 42 Flexner, Bulletin of the Rock. Inst. for Med. Ees., 1917, 43 Elser and Huntoon, Jour. Med. Res., 20, 1909, 371, 534 PAT H(Xi UN 1C M ICUOOKU AN ISMS tendency to spontaneous agglutination in salt solution and in horse serum. MICROCOCCUS FLAVUS. — A common inhabitant of the normal throat which grows easily on simple media and may be grown at room temperature at or below 25°, temperatures at which the meningo- coccus ceases to grow. It is always important to expose suspected cultures at room temperature in the dark. A yellowish pigment is formed by the cultures, but often does not come out for several days. The very young colonies may very closely resemble meningo- coccus colonies, but are easily distinguished in sub-cultures, es- pecially when the growth is forty-eight or more hours old. There are a considerable number of chromogenic organisms closely related to the Flavus. Elser and Huntoon describe three chief chromogenic groups, one of which has a greenish gray or greenish yellow ap- pearance by reflected light, with an opacity that approximates the meningococcus colony. The second group is the one most closely resembling Lingelsheim 's M. Flavus. Their third chromogenic group also makes a greenish yellow pigment, and, except for this, is very similar to the M. catarrhalis. A curious fact has been noted by Elser and Huntoon, namely, that some of their chromogenic organisms were easily distinguishable from meningococcus colonies at first isolation, but in the course of artificial cultivation they lost some of their original characters and their power to produce pigment, and gradually approximate the appearance of meningococcus, at least as it appears in strains long isolated. The Flavus group gives pernaps most difficulty in meningococcus carrier examinations, since the young colonies of these organisms may look very much like the young meningococcus cultures. The chief points of differentiation, apart from sugar fermentation, which confirm them, are: The fact that Flavus colonies will grow out at room temperature on slants of simple media; that they begin to form pigment after forty-eight hours or so, and that they will agglutinate in normal horse serum in dilutions often as high as 1 to 50, and in the meningococcus sera, indiscriminately, often as high as 1 to 100. Meningococci do not agglutinate in salt solution spon- taneously, unless under the conditions mentioned above as noted by Hine, and under the influence of abnormal acid or alkalin reactions. In all series in which the specific a i>^luti nation test is used for 1he determination of a meningococcus, therefore, normal hoi-sc scrum MICROCOCCUS INTRACELLUDARIS MENINGITIDIS 535 tubes should be set up in dilutions ranging up to 1 to 50 at least, in order to exclude organisms of the Mavus type. MICROCOCCUS PHARYNQIS Siccus. — This organism described by Lingelsheim44 is a Gram-negative diplococcus often found in the normal pharynx, and is recognized by its dry, creiiated colonies on simple media. According to Elser and lluntoon, it sediments spontaneously in salt solution and this, together with the fact that the colonies are formed in a way almost impossible to break up, makes it easy, according to these observers, to distinguish it from the meningococcus. It is a little more difficult to distinguish from M. Catarrhalis; but can be easily separated from this organism by means of the fermentation test. DIPLOCOCCUS CRASSUS. — This is the organism that Kutscher45 described as probably identical with the so-called "Jaeger" variety of meningococcus. According to Kutscher and von Lingelsheim, this organism has a tendency to wander from the normal pharynx into the central nervous system in cases of meningitis of other origin. Lingelsheim claims to have found it in the fluids of traumatic meningitis and tuberculous meningitis. It has the peculiarity that the cultures are said to be composed of Gram-negative and Gram- positive organisms some of the cocci retaining the Gram-stain. Ac- cording to Von Lingelsheim, the colonies are smaller and more compact than meningococcus colonies, and it will grow at room temperature. FERMENTATION REACTIONS OF GRAM NEGATIVE DIPLOCOCCI Strains Tested. Strains Dex- trose. Mal- tose. Levu- lose. Sacch- arose. Lac- tose. Gal- actose. Meningococcus 200 -f- + 0 0 0 0 Pseudomeningococcus Gonococcus 6 15 + + + 0 0 0 0 0 0 0 0 0 Micrococcus catarrhalis Micrococcus pharyngis siccus.. . Chromogenic group I 64 2 28 0 + + 0 + + 0 + + 0 + + 0 0 0 0 0 0 Chromogenic group II 11 4. + -f 0 0 0 Chromogenic group III. . . Jaeger meningococcus, Krai .... Diplococcas crassus Krai 9 1 1 + + + + + + 0 + + 0 + + 0 + + 0 + + Table taken from Elser and Huntoon, loc. cit. 44 Lingelsheim, Klin. Jahrb., 15, 1906. 45 Kutscher, Kolle and Wassermann, Vol. 4, Second Edition, p. 603. 536 PATHOGENIC MICROORGANISMS DIPLOCOCCUS Mucosus. — A form of Gram-negative diplococcus, the description of which we take from Elser and Huntoon. Its colonies may resemble meningococcus colonies on ascitic agar. They are said to differ from the meningococcus colonies by being more mucoid, resembling the colonies of the B. capsnlatus mucosus. The colonies have a tendency to confluence and the above writers say that the luxuriance of its growth on serum free media helps to tell it from the meningococcus. It also grows at room temperature, and shows capsules with capsule stains. EPIDEMIOLOGICAL PROBLEMS IN MENINGITIS Although sporadic cases of meningitis may occur in a community for a considerable number of years after an epidemic is over, and the disease may, therefore, be regarded as, to some extent, endemic in all crowded cities, it is chiefly important for its epidemic occur- rence. In 1905 a recognizably described epidemic occurred in Switzerland. Since that time epidemics have been reasonably fre- quent, especially at times of war, when they appeared among armies in barracks and mobilization camps. During the many continental campaigns in the time of Napoleon, outbreaks occurred in the various armies, and secondary epidemics among the civilian popula- tion in many cities followed in the train of these. In America, a number of limited epidemics occurred in the States along the Eastern sea board during the early half of the 19th century, and in these civilian epidemics the disease particularly selected children and young adults. Extensive civilian epidemics occurred in different parts of the world in the early years of the 20th century. In 1903 the disease appeared in East Prussia and spread to other parts of Germany from there. In 1904 and 1905 it appeared in New York City, and the adjacent country, on an extensive scale causing the death of 3,000 people and altogether about 7,000 cases in New York City alone. In the summer following its appearance in New York, it extended to Canada, and in the years since then, small outbreaks and sporadic cases have appeared with gradually decreasing fre- quency all through the more thickly populated parts of North America. During the late war, there was little meningitis among the European armies until an extensive outbreak occurred among the MICROCOCCUS INTRACELLULARIS MENINGIT1DIS ( 537 Canadian troops on Salisbury Plains. The increase of cases among these troops took place in February, 1915, and after this time the disease began to appear in the overseas expeditionary troops, al- though among these no extensive epidemic occurred at any time. Among American troops the disease was most prevalent in 1917 and 1918 among the troops gathered in the cantonments in the United States. According to the epidemiological studies of Vaughan ( and Palmer4'5 in the camps in 1918, meningitis showed "of all dis- eases, the greatest excess over the disease in civilian communities.'* Vaughan estimates that meningitis was forty-times as frequent in the Army as in civilian life. The highest morbidity occurred at Camp Jackson where it reached a rate of 25.1 per thousand, and a death rate of 7.05. Next to pneumonia, it was the most serious disease occurring in the camps. In the Surgeon General's report for 1918, the disease stood fifth as a cause of death for enlisted men in the United States and Europe, with a case mortality of 34.8 per cent. During the Army epidemics there was a definite racial difference in that, according to Surgeon General Ireland's report, the admission rate for colored troops in the United States was 2.44, whereas, it was only 1.2 for whites, and the death rate for colored troops was 0.98 against 0.41 for whites. As to seasonal prevalence, meningitis usually develops in the late autumn and winter months, the largest case rates being coin- cident with the cold and wet weather, when a basic catarrhal inflam- mation of the upper respiratory tract, creates favorable conditions for the lodgment of organisms and for the general distribution of saliva by coughing, sneezing and spitting. During the war, the highest admission rates in the United States usually fell into the months of November, December and January, which is the time of the highest case rate for most respiratory epidemics. Yet cases will usually trail along through the hot weather. Meningitis epidemics, therefore, will occur chiefly in the tem- porate zones during the winter months at times when, during the prevalence of generalized respiratory disease, large numbers of people are crowded in close quarters, under conditions which render attention to hygiene and sanitation difficult. The reasons for this will become apparent as we study the manner of transmission. The meningocoecus does not survive easily outside the body, and 46 Vauglian and Palmer, Jour. Lab. and Clin. Med., 4, 1919, 647. 538 PATHOGENIC MICROORGANISMS rapidly dies out in dust, or even in sputum, under conditions of low temperature, deficient moisture and competition with other microorganisms. As far as we know, it is not carried by any of the domestic animals and, therefore, the origin of infection lies in the secretions of cases and of carriers. The microorganisms are found in the noses and throats of the sick, sometimes in the secre- tions of the eye where a meningococcus conjunctivitis may exist. With the secretions of these mucous membranes it reaches the outer world. The meningococci may be present in cases for a long time after convalescence, and, as we know, they are present in a consider- able percentage of people who have never had meningitis, with whose secretions the organisms may be constantly transferred to contacts. Transmission probably occurs by close contact between carrier or case and new host, through the nasopharynx, where the organisms lodge and multiply. From this lodgment they pass into the meninges, either directly along the lymphatic channels to the base of the skull, or perhaps indirectly by way of the general circulation. The former route is the one favored by most observers. However, during the early periods of the war, a few cases were reported by British clinicians, in which blood culture was positive before symptoms of meningitis had developed, and during these army epidemics, we, as well as others, saw occasional cases of general meningococcus septicemia which died without ever developing meningitis. In- cidentally, it may be stated that these cases develop a generalized rash which, in some of its stages, is not unlike that of typhus fever. The writer recalls a case in which he made a probable diagnosis of typhus fever which in the light of subsequent experience seems to him to have possibly been a case of meningococcus septicemia. Positive blood culture in such cases will differentiate between the two diseases. Herrick47 studied this phase of the problem at Camp Jackson in 1918 with great care, and came to the conclusion that in 50 per cent of the cases early blood culture will reveal general infection before clinical evidences of meningeal invasion are ap- parent. This observation is of the greatest importance, indicating the desirability of early blood culture work for doubtful diagnosis, and also throwing light upon the wisdom of intravenous serum therapy combined with the intraspinous injections. Infection of a healthy individual from a case is of very rare 41 llerrick, Arch. Inter. Med., 21, 1918, 541. MICROCOCCUS INTRACELLULARIS MENINGITIDIS 539 occurrence, and since there are in every epidemic a very much larger number of carriers than of cases, the carrier is the chief epi- demiological problem. As far as infection of new individuals from patients is concerned, experience during the New York epidemic showed only two or three cases of infection of doctors and nurses, although the hospitals in the city were constantly handling consider- able numbers of the sick. In our epidemiological experience with the army, the actual tracing of one case to a preceding one was also relatively very rare. This does not mean that the greatest precautions should not be taken to prevent such transmission in hospitals and sick room. But the epidemiological emphasis lies with the carrier. This rareness of transmission from cases to the healthy is in our opinion due to the peculiar conditions of susceptibility that pre- vail in relation to meningitis, and the fact that the number of people with whom the sick come in contact is relatively small. The susceptibility of man to meningitis is a curious one, different in some aspects from susceptibility relations to almost all other infections, except perhaps poliomyelitis. In the general population there seems to be a great variability in individual susceptibility to infection with the meningococcus, a variation which can be traced to no determinable cause. Unlike pneumonia, temporary fluctua- tions in well being, produced by respiratory disease, malnutrition, exposure to cold, etc., do not seem to play a determining role. The disease indiscriminately picks out individuals here and there, some of them in the most robust health, strong and hardy, while sparing associates who may be feeble and run down. It is obvious that some individuals are normally resistant and will not come down, in spite of considerable exposure, while others are delicately susceptible. The difference may possibly have been produced fortuitously by the fact that some individuals may have been carriers at one time or another, and have become, thereby, spontaneously immunized. It is difficult to get at this question experimentally, and there are no serum or other reactions which we can apply at the present time, by which we can discriminate between the susceptible and the non- susceptible of a community. There is no available method, more- over, by which we can distinguish between virulent and non- virulent strains of meningococci. The Carrier Problem. — As stated above, the meningococcus car- rier probably is the source of infection in most of the cases that 540 PATHOGENIC MICROORGANISMS develop during an epidemic. Earlier carrier work has lost value to a considerable extent, owing to the fact that the criteria of meningococcus identification of which we are now more thoroughly informed were neglected in these early studies. The flora of the nose and throat contains many Gram-negative diplococci, mentioned above under the heading of identification, some of which were mis- taken in this earlier work for true meningococci. Micrococcus Catarrhalis, Micrococcus Flavus, and a number of other similar microorganisms probably represent a definite percentage of the earlier statistics. The criteria of meningococcus determination have been discussed in a special section above, and these, in general, were applied in the extensive meningococcus carrier work which was done during the years of the war, especially by British and American bacteriologists. The studies of Bassett-Smith,48 Gordon,49 Mathers and Herrold,50 show that in the American camps under conditions of ordinary life and weather, there may be anywhere from two to five per cent of meningococcus carriers. Mathers and Herrold at the Great Lakes Naval Training Station examined over 15,000 men, finding over 4 per cent to be carriers and between 1 and 2 per cent to be chronic carriers. Their work also showed that the carrier rate is higher among those taking care of cases, and that over 38 per cent of those recovering from the disease may remain carriers during convalescence for variable periods. Contacts showed a carrier rate of 36.7 per cent during a period in which the general carrier rate in the camps (15,000 men examined) was slightly over 4 per cent. The hospital Corps showed a carrier rate of 13.5 per cent. It is natural that there have been many endeavors to establish relationship between new cases and contact with carriers. This line of investigation has not been conclusive, owing to the great difficul- ties incident to such investigation. The transmission of respiratory organisms may take place during a very brief contact, in conversa- tion, close association in barracks, moving picture shows, public conveyances, sleeping quarters, etc., and the innumerable associa- tions of this kind established by each man in the course of a day, makes it almost impossible to trace them with accuracy. Among the most interesting studies made in this connection are those of 48 Bassett-Smith, Lancet, 194, 1918, 290. "Gordon, Med. Res. Com., Spec. Rep. Ser., No. 3, London, 1917. 50 Mathers and Herrold, Jour. Infect. Dis., 22, 1918, 523. MICROCOCCUS INTRACELLULARIS MENINGITIDIS 541 Glover,51 who swabbed the throats of a considerable number of men in overcrowded barrack rooms, in the course of sanitary supervision during which the spacing between beds was among the many pre- cautionary measures taken. It will be seen here that sanitary measures, including the spacing out in sleeping quarters, brought about a very considerable drop in the carrier rate, 'with coincident diminu- tion of cases of meningitis. Meleney and Ray52 traced fourteen out EFFECTS OF "SPACING OUT" ON "SEVERELY OVER CROWDED" BARRACK-ROOMS * Unit. Date of First Swabbing. Percentage Carrier Rate before Spacing. Out. Period Spaced Out Approxi- mately. Date of Second Swabbing. Percentage Carrier Rate after Spacing Out. No 1 Sept 29 22.0 8 weeks Dec. 6 2.0 No. 2 Oct. 2 28.0 6 weeks Nov. 23 7.0 One room of No. 2 No 4 Oct. 2 Oct 26 38.5 28 0 6 weeks 5 weeks Nov. 23 Nov. 30 4.5 4.5 *Glover's Table of twenty-four cases which occurred in an American camp to contact with carriers, and found parallelism between the incidence of cases and the rise of the carrier rate. These examples, however, are excep- tional and it is relatively rare that a definite relationship of this kind can be established. It is a fact that carrier rates are high in such camps during the cold months, and in connection with the general spread of respiratory disease, and that, at such times, the incidence of the disease increases, and it is absolutely logical to assume that the new cases arise by contact with the carriers. It is of importance, however, to recognize that the tracing of the case to the individual from whom he has been infected, at times when high carrier rates exist, is not often possible, and comprehension of this must considerably influence the measures instituted for the control of the epidemic. It is our belief that the extensive carrier examinations made during epidemics and the wholesale isolation of carriers, were rela- tively ineffective during the war, and that it is far better to bend all one's energies upon a general improvement of the respiratory 51 Glover, Jour. Hyg., 17, 1918, 367. 52 Meleney and Kay, Jour. Inf. Dis., 23, 1918, 317. 542 PATHOGENIC MICROORGANISMS sickrate with the reduction of carriers, focusing the carrier ex- aminations upon the small epidemiologically determined intimate group from which the case has come, rather than making wholesale carrier examinations of carriers of meningococcus through whole regiments and divisions. Carrier Determination. — The bacteriological analysis of a carrier is not a simple procedure, and implies the proper control of a great many conditions which necessitate special description. To obtain the material properly the swab must be taken from high up in the pharynx, behind the soft palate. A general swabbing of the pharynx and throat is not sufficient. The best swabs for this purpose are made by the West tube method, as follows: A cotton swab is fixed on the end of a copper wire, about eighteen centimeters long, and this is inserted in a glass tube, bent upward at the swab end, in such a way as to permit passage upward behind the soft palate. The swab is placed into the tube, both ends plugged with cotton, and is so sterilized. For large scale work it is sufficient to take copper wire swabs, sterilize them in a box, and bend them up carefully with the finger, being careful not to touch the cotton, just before use. Swabbing through the nose has also been practiced, but we do not believe that it is as efficient as the method described above. The swab must be taken with the patient facing the light. A tongue depressor is used, and the swab inserted so as to pass behind the soft palate. The copper wire is then thrust forward so that the swab emerges from the tube and touches the posterior and upper pharyngeal wall. Slow motion to and fro brings the cotton in contact with the sides of the upper pharynx. The swab is then immediately passed over the surface of the plate medium. It is best not to carry the inoculated swab back to the laboratory, but to plate it directly upon removing the material from the patient. The media employed are various, but for ordinary use a glucose- hormone-agar, PH of 7.4 with addition of 1-10 defibrinated or hemolyzed human or rabbit's blood, is best. The plates thus inoculated should be kept warm and immediately taken to the laboratory where they are incubated. The British prefer trypagar to the hormone agar as the basic medium for such work. After eighteen to twenty-four hours incubation, the plates are examined and the colonies suspected of being meningbcocci are fished. This is not a matter which can be taught by book. The colonies are of small, rounded appearance, the recognition of which MICROCOCCUS INTRACELLULARIS MENINGITIDIS 543 is a matter of judgment. Every bacteriologist confronted with the problem should immediately plant plates of the medium which he is going to use, with spinal fluid or with cultures recently isolated, and familiarize bimself with the colonies on this medium, at various stages of growth. In spite of our not inconsiderable experience, we would do this ourselves. Plates that are too thickly covered with 'colonies are of no use. On blood medium, the true meningococcus colonies do not produce any change in the blood. They are slightly translucent and look somewhat stringy. They are homologous and slightly glistening. They are practically indistinguishable from young colonies of M. Flavus which is also a Gram-negative diplococcus, but which is easily distinguished subsequently by the fact that it will grow at and below 25° C., produces a yellow pigment on further cultivation, and has a tendency to agglutinate spontaneously in normal horse serum. Having ringed the suspicious colonies, some of them are now picked and stained by Gram. Our habit is to take up part of a colony which we strongly suspect of being meningococcus, plant part of it immediately, and from the rest make the Gram stain, since plating after taking material for Gram stain may increase the chances for contamination. This point, however, is not of very great importance. The suspicious colonies are now planted upon blood agar slants, the medium being made up as for the plates. These slants should not be used directly from the ice-box, but should be warmed. Dried media must not be used. If two tubes can be inoculated, one should be kept at room temperature. Growth in the incubator for about twelve hours gives sufficient growth for further identification. Gram stains are now made from the tubes. If the morphological and staining properties are proper, agglu- tination is carried out. Diagnostic Agglutination. — The organisms are emulsified in isotonic salt solution. Agglutination may be done against type sera, or against polyvalent serum. When large numbers of cases are examined, as in times of epidemic, it is best first to agglutinate in polyvalent serum, preferably one in which the agglutinin titre for a great many different meningococcus strains has been con- trolled. As a general rule, the polyvalent sera used in this country will show specific agglutinations for practically all meningococcus 544 PATHOGENIC MICROORGANISMS strains in dilutions of 1 to 100. For this reason, 1 to 100 was the dilution adopted for such work in the American Army laboratories. One-half c.c. of the bacterial emulsion is mixed with 0.5 c.c. of the polyvalent serum. A control of a similar amount of the culture suspension in 1 to 50 normal horse serum must always be made to guard against spontaneous agglutination. It is always well also, to run a tube with a known meningococcus. Since meningococci show a certain amount of resistance to ag- glutination, Gordon has recommended the method in general use during the war, that is, placing the tubes in a water bath at 50° for twelve to eighteen hours. Evaporation must be guarded against. Olitsky has recommended saving time by growing the organisms in normal horse serum broth directly from the colonies on the plate, discarding all those that grow in a granular form. We, ourselves, have used a method that we have not published because we have not had a chance to use it on material on a large scale, which depends upon the thread reaction. Dilutions of poly- valent sera are made in broth tubes, so that the final concentration is 1 to 100. The colony is directly inoculated into this, and in the case of true meningococci grow in granular form. A similar inoculation is made in control tubes of 1 to 50 normal horse serum. These procedures sometimes save time. Nicolle in France makes his diagnosis by another method, in that he uses, instead of a dilution of serum, the serum in concentrated form, checking it up with the bacterial suspension, and noting the speed of agglutination. In such concentrations he often gets rapid agglutination of true meningococci in the concentrated serum. After the preliminary identification has been made, typing of the meningocococus may be desirable by agglutination against type serum. Carriers occasionally will develop meningitis some time after they have been recognized as carriers, showing that the organisms may remain in the nasopharynx for some time, without penetrating, insusceptible individuals. We know of a number of cases in which this seems to have occurred. Gordon mentions a number of cases in which "meningismus" developed among carriers, namely, carriers complained of severe headache, pains in the back of the neck, slight fever up to 102°, and slight Kernig. One case he mentions had been in contact for a few hours with a case of cerebro-spinal meningitis, which died within twenty-four hours. It was swabbed M1CROCOCCUS INTRACELLULARIS MENINGITIDIS 545 and found to be negative. A month later, he went to a military hospital with the symptoms above enumerated, and the swab from his nasopharynx revealed meningococcus, but curiously not of the same type as that of the case with which he had been in contact. He mentions other similar cases. An interesting point comes up in regard to whether or not the presence of meningococcus in the nasopharynx leads, in itself, to a catarrhal inflammation. Fliigge in the early days of meningococcus carrier investigation believed that the carrier state was usually associated with local inflammations. Gordon, however, finds that, in general, there was no nasopharyngeal catarrh associated with the carrier state. But he also finds that_ cases with tonsillar or pharyn- geal inflammations were much more difficult to free from meningo- coccus than others. The same he says is true of convalescents, a point which indicates the importance of bringing the mucous mem- branes to normal in connection with the cure of carriers. The question of how we are to deal with meningococcus carriers in times of epidemic is a difficult one. Local treatment .of the nose and throat has been tried with antimeningococcus serum, with astringent solutions, and various disinfectants, without encouraging result. Sprays of Dichloramine T and other chlorin preparations have been tried, also, in our opinion, without marked success. Dur- ing the war the British constructed rooms of about one thousand cubic feet capacity, along the sides of which steam pipes were placed at about the height of a man's waist, and jets were fitted to them in such a way that a spray of steam could be ejected. These sprays were connected with bottles containing 1 to 2 per cent chloramine, or 0.5 per cent zinc sulphate. The carriers were put into these inhaling rooms for from fifteen to twenty minutes a day, during which they inhaled the medicated spray through their nos- trils. By this method, they claimed to clear up all but the most resistant cases of so-called pure meningococcus carriers. In general, it may be said that cases in which only a few meningococcus colonies form on the plates, clear up rather readily, and that the others in which the cultures are almost pure are extremely resistant to any kind of treatment. Our own impression from some experience with the various methods would lead us to conclude that the best treat- ment for a carrier would be careful attention to the nasopharynx, with an attempt to bring it back to normal as far as the condition of the mucous membrane is concerned, correction of tonsillar, adenoid, 546 PATHOGENIC MICROORGANISMS or septum defects, cessation from smoking or other habits that irritate the mucous membrane, and outdoor life, especially in the sunlight, with sea baths if available. Specific antiseptic treatment in general seems to us to have been a failure as far as the handling of large numbers of men is concerned. The virulence of meningococci is a matter that is very difficult to determine because of our inability to produce invasive infections with regularity in any known laboratory animal. So far, extensive attempts to determine the virulence of standardized injections into mice have not succeeded. Death in most laboratory animals is due to the toxic effects and not by invasion. This is a very unfortunate circumstance, inasmuch as our failure to be able to distinguish between virulent and non-virulent strains makes it impossible for us to tell a dangerous carrier from one who is relatively harmless, as we can in the case of diphtheria carriers. All we can do at the present time is to regard as dangerous any. carrier whose meningo- coccus agglutinates in a polyvalent serum. Those with strains which neither agglutinate nor absorb with the polyvalent serum at our disposal, if culturally they seem to be true meningococci, must be regarded as suspicious. CHAPTER XXVII DIPLOCOCCUS GONORRHCE^E (GONOCOCCUS), MICROCOCCUS CATARRHALIS, AND OTHER GRAM-NEGATIVE COCCI DIPLOCOCCUS GONORRHOEA NEissER,1 in 1879, described diplococci which he had found regularly in the purulent secretions of acute cases of urethritis and vaginitis and in the acute conjunctivitis of the new-born. His researches were purely morphological, as were the numerous confirm- atory investigations which rapidly followed his announcement. Cultivation of this diplococcus, now usually spoken of as gonococcus, was not definitely suc- cessful until 1885, when Bumm 2 obtained growth upon tubes of coagulated human blood serum. Bumm was not only able to keep the organisms alive by transplantation in pure culture, but produced the disease by inoculation of his cul- tures upon the healthy urethra. Morphology and Staining. — The gonococcus is usually seen in the diplococcus form, the pairs being characteristically flattened along the sur- faces facing each other. This gives the cocci a peculiar coffee-bean or biscuit shape. The size of the diploforms is about 1.6 micra in the long diameter, about 0.8 micron in width. Stained directly in gonorrheal pus from acute cases, the microorganisms are found both intra- and extra- cellularly, a large number of them crowded characteristically within the leucocytes. They are never found within the nucleus. The phagocytosis which produces this picture has beeji shown by Scholtz 3 ? FIG. 57. — GONORRHEAL Pus FROM URETHRA, SHOWING THE Cocci WITHIN A LEUCOCYTE. 1Neisscr, Cent. f. d. med. Wiss., 1879. 2 Bumm, ''Boitr. z. Kermtniss des Gonococcus, " Wiesbaden, 1885. 3 Sclioltz, Arch. f. Dermat., 1899. 547 548 PATHOGENIC MICROORGANISMS and others to take place in the free secretions, not in the depth of the tissues. The intracellular position, which is of considerable diagnostic importance, is lost to a great extent in secretions from chronic cases. In smears made from pure cultures the arrangement in groups of two may often be less marked than in pus, clusters of eight or more being common. The gonococcus is non-motile and does not form spores. It is easily stained with aqueous anilin dyes. Methylene-blue alone, or eosin followed by methylene-blue, give good results. An excellent picture is obtained with the Pappenheim-Saathof stain consisting of Methyl green 0.15 Pyronin 0. 5 96 per cent alcohol 5. 0 Glycerin 20. 0 2 per cent carbolic acid water ad 100. 0 Fix; stain 1-2 min. Gram's method of staining, however, is the only one of differential value, gonococcus being Gram negative. The Gram stain applied to pus from the male urethra, while not absolutely reliable, is, for practical purposes, sufficiently so to make a diagnosis. In exudates from the vagina or from the eye the morphological picture is not so reliable, owing to the frequent presence in these regions of other Gram-negative cocci. The great scarcity of gono- cocci in very chronic discharges necessitates thorough cultural investi- gation ; negative morphological exami- nation in such cases can not be regarded as conclusive.4 Cultivation. — The gonococcus is delicate and difficult to cultivate. Bumm5 obtained his first growths 58.-GoNococcus. Smear uPon human blood serum which from pure culture. had been heated to partial coagu- lation. The medium most commonly used at the present day was intro- duced by Wertheim,6 and consists of a mixture of two or three parts of 4 Heiman, Medical Record, 1896. 5 Bumm, Dent. med. Woch., 1885. 6 Wertheim, Arch. f. Gynakol., 1892. t. • *.• DIPLOCOCCUS GONORRHOEA 549 meat infusion-agar with one part of uncoagulated human ascitic fluid, hydrocele fluid, or blood serum. The agar is melted and cooled to 45° before the serum is added. The mixture may then be slanted in the test tube or poured into a Petri plate. One per cent of glucose may be added. Cultures in fluid media may be obtained by similar additions of serum to meat-infusion-pep ton-broth. Whole rabbit's blood added to agar, or the swine-serum-nutrose medium of Wassermann 7 may occa- sionally be used with success. Plates may be made by smearing for enrichment a drop of blood from the finger over the surface of agar in the manner of Pfeiffer's method for influenza-bacillus cultivation. Inoculations from gonorrheal material are best made by surface smearing upon plates, since the gono- coccus grows best in the presence of free oxygen. Growth becomes more luxuriant after prolonged cultivation upon artificial media. The most favorable reaction of media is neutrality or slight acidity. When the gonococcus has been successfully cultivated from pus upon media without serum additions, the success has probably been due to the substances carried over in the pus. The ease of cultivation differs considerably with different strains of gonococci. Some grow very heavily after first isolation, but the majority show a very delicate growth even on rich ascitic glucose agar. After several generations of growth on artificial media, however, the organism develops with increasing ease and on simpler media. It may eventually be cultivated on plain agar, especially when this is made of veal infusion. Recently a medium upon which gonococci after first cultivation can be grown with ease has been recommended by Edward B. Vedder.8 The medium consists of a 1.5 to 1.75 per cent agar made with beef infusion neutral to phenol- phthalein, and after clearing, 1 per cent of corn starch added. The corn starch is best added after grinding with a little agar to avoid clumps, this then being poured into the bulk of the agar and thoroughly mixed. The medium should be sterilized at not over 15 Ibs. pressure to avoid changes in the starch. Recently we have isolated several strains of gonococci which grew very heavily on simple media without ascitic fluid in the second culture generation. The gonococcus will develop sparsely under anaerobic conditions 7 Wasserma-nn, Berl. klin. Woch., 1897. (Fifteen c.c. swine-serum, 35 c.e. of water, 3 c.c. glycerin, with two per cent nutrose. The nutrose is dissolved by boiling and the solution sterilized. This is then added to agar, in equal parts, and used in plates.) 8 Vedder, Jour. Infec. Dis., May 15, 1915, xvi, 385. 550 PATHOGENIC MICROORGANISMS but has marked preference for aerobiosis. The optimum temperature is 37.5° C. Growth ceases above 38.5° and below 30°. Upon suitable media colonies appear as extremely delicate, grayish, opalescent spots, at the end of twenty-four hours. The separate colo- nies do not tend to confluence and have slightly undulated margins. Touched with a platinum loop their consistency is found to be slimy. In fluid media, growth takes place chiefly at the surface. Types of the Gonococcus. — As in the case of so many other organisms, it has been found that the Gram-negative diplococci which cause gon- orrheal infections are not a single type, but must be regarded as rep- resenting a group, including many closely related,' but antigenically differentiable subgroups. Such subgrouping of species formerly regarded as homogeneous has been a very natural development of the more intensive study of serum reactions, incident to diagnostic agglutination and complement fixation, and to the control of specific therapy. Torrey9 and Teague and Torrey 1 ° in 1907 showed that the gonococcus group is not homologous, but that agglutination and agglutinin absorption divided this group into at least three separate subtypes. Agglutinin absorption seemed to show more types than complement fixation,- a matter which we ourselves would rather expect at the present day because of the almost universal experience that complement fixation reactions are not as strictly specific as agglutination. Torrey's claims have been to some extent misquoted in the past ten years, in that he himself never supposed that his ten strains represented the entire gon- ococcus group. In 1910 Watabiki n made a similar study of the gon- ococcus group, and studying a limited number of strains, confirmed the heterogeneous nature of the group by referring to them as " compara- tive but not distinctive differences between individual strains." In 1915 Louise Pearce 12 made a comparison between gonococci isolated from adult males and from the vulvovaginitis of children. She came to the conclusion that strains from these two sources constituted fairly definite serologically distinct groups, that at least there was a relative distinc- tion between the two types. This, in view of the important sanitary problem involved in these infections in children, would be of great importance if confirmed. We will refer to it again below. More 9 Torrey, Jour. Med. Res., 16, 1907, 329. 10 Teague and Torrey, Jour. Med. Res., 17, 1907, 223. » Watabiki, Jour. Infec. Dis., 1910, 7, 159. 12 Pearce, Jour. Med. Res., 21, 1915, 289. D1PLOCOCCUS GONORRHCE^ 551 recently, Hermanies 13 studied 85 gonococcus strains from various sources, using for cultivation the partial tension method of Wade W. Oliver. He concluded that gonococci fall into distinct types, with little relationships to each other. Agglutinins produced by one type cannot be absorbed by strains of other types, and his 85 strains fell into six distinct groups. At the time of the present writing, Torrey is again working on the same subject, since the conflicting evidence of these researches and earlier work shows clearly that the subject cannot be regarded as closed. The work Torrey is doing with Buckell is not yet completed, and it would, therefore, be rash to make a final statement concerning his opinion, but, since he has devoted a considerable amount of time and energy to this subject, and since his bacteriological judgment is unusually sound, we quote as follows from advanced information he has courteously furnished us in a personal communication. Tor- rey's impressions at the present time are that it is not possible to demon- strate definite groups, such as the three fixed pneumococcus types, or even groupings, comparable to that established for the meningococcus and discussed in another section. A study of 50 strains from many sources and from different parts of the world by agglutination and agglutinin absorption, has shown that there are certain generalized strains which possess antigenic properties common to a considerable part of the " whole" species. On the other hand, there are a large number of variants among recently isolated strains which show no serological relationship to one another as far as specific agglutination is concerned, and which quite often show some specific relationship to one or more of the specific strains. These - variants overlap and show such intergradations that it is not possible to subdivide them into definite groups. It has also been noticeable in his work that some of the original strains which he studied as long as fourteen years ago, and which at that time seemed to be unrelated to each other, are now beginning to show some specific relationships. All the older strains show greater relationships than do similar groups of recently isolated ones. Torrey has not been able to confirm the contentions of Louise Pearpe that there is a recognizable distinction between vulvovaginitis strains from chil- dren and those from adult males. Recognition of the Gonococcus. — Speaking of sugar fermentations, Torrey has come to the conclusion that, taken together with typical colony formation, morphology, staining reactions and absence* of growth on ordinary laboratory media, sugar fermentations form Ihe most , Jour. In foe. Dis., 28, 192], 1 :'.:'.. 552 PATHOGENIC MICROORGANISMS reliable methods of recognition of the gonococcus. Of 60 strains exam- ined by him, all except one fermented glucose, and none of them fer- mented maltose, thus -differentiating from meningococcus. The one exception was an old strain, all the recent ones fermenting true to type. A gonococcus can, therefore, be recognized bacteriologically by the Gram-negative diplococcus form, the typical colony formation on ascitic agar, its failure to grow on simpler media or at room temperature, its ability to split dextrose and failure to split maltose. Resistance. — Recent cultures of gonococcus, if not transplanted, usually die out within five of six days at incubator temperature. At room temperature they die more rapidly. The resistance of the gonococcus to light and heat is slight. A tem- perature of 41° to 42° kills it after a brief exposure. Complete drying destroys it in a short time. Incompletely dried, however, and pro- tected from light (gonorrheal pus) it may live, on sheets and clothing, for as long as eighteen to twenty-four hours.14 It is easily killed by most disinfectant solutions 15 in high dilution and seems to be almost specifically sensitive to the various silver salts, a fact of therapeutic importance. Pathogenicity. — Gonorrheal infection occurs spontaneously only in man. True gonorrheal urethritis has never been experimentally pro- duced in animals. In human beings, apart from the infection in the male and female genital tracts, and in the conjunctive, the gonococcus may produce cystitis, proctitis, and stomatitis. It may enter the cir- culation, giving rise to septicemia,16 to endocarditis and arthritis. Iso- lated cases of gonorrheal periostitis and osteomyelitis have been reported.17 The acute infections of the genito-urinary passages are often fol- lowed by prolonged chronic infection, which, though quiescent, may for many years be a source of social danger. In children, especially females, the infection is not rare, and may assume epidemic characters, traveling from bed to bed in institutions. Such hospital epidemics can be stopped only by the. most rigid isolation. This is more specifically dealt with in the paragraphs on Sanitary Considerations, below. While inoculation of animals has never resulted in active prolifera- tion of the gonococcus upon the new host, local necrosis, suppuration, 14 Heiman, Medical Eecord, 1896. 15 Schaeffcr und Steinschn eider, Kong. Deut. Dermat. Geselis., Breslau, 1894. 16Eeview of cases of Gon. Septicemia, Faure-Beaulieu, Thesis, Paris, 1906. >7 Ullmann, Wien. med. Presse, 1900. DIPLOCOCCUS GONORRHCEjE 553 and temporary systemic reactions have been produced by subcutaneous and intraperitoneal inoculation. A toxin has been isolated by Niko- laysen 18 by extraction from the bacterial bodies with distilled water or sodium hydrate solutions. It was found to be resistant to a tempera- ture of 120° and to remain potent after complete drying. The same author found that the isolated toxin and dead cultures were fully as toxic for animals as living cultures, 0.01 gram killing a white mouse. Specific injury to the nervous system by injections of gonococcus toxin has been reported by Moltschanoff.19 The secretion of a true soluble toxin by the gonococcus, asserted by Christmas,20 is denied by Wassermann,21 Nikolaysen,22 and others. Christmas,23 and, more recently, Torrey,24 have reported successful active immunization of animals by repeated injections of whole bacteria. Torrey and others apparently have successfully treated human cases by injections of the serum of immunized animals. Antibodies to Gonoooccus. — Patients infected with gonococci seem to produce antibodies against the organisms. Although in the ordinary gonorrheal urethritis, or vaginitis, it is relatively simple to make the diagnosis by finding gonococci in the discharges, diagnosis may be difficult in cases of gonorrheal rheumatism, or endocarditis, when isolation of the bacteria fails or when the connection between the local venereal disease and the general condition is obscure. Various sero- logical diagnostic methods have been attempted, and of recent years the complement-fixation test has' been found to be very useful. The method has been especially developed by Archibald McNeil, at the New York Department of Health. It consists in making a polyvalent antigen, using the 10 Torrey strains which are kept in stock transplants on glucose ascitic agar. It has been found that the best medium for antigen production is an agar made of "bob veal." For the production of antigen, stock cultures are transplanted on "bob veal" agar, without salt, glucose or ascitic fluid, the reaction carefully adjusted to an acidity of 0.1 per cent to 0.2 per cent. Twenty-four hour growths on this medium are scraped off and emulsified in neutral sterile distilled water. The emulsion is autolyzed one hour in a water bath at 56° and heated ™NiTcolaysen, Cent. f. Bakt., 1897. "Moltschanoff, Munch, med. Woch., 1899. 20 Christmas, Ann. de 1 'Inst. Pasteur, 1897. 21 Wassermann, Zeit. f. Hyg., xxvii, 1897. 22 Nikolaysen, Fort. d. Med.,.xxi, 1897. 23 Christmas, loc cit. 24 '±orrey^ Jour. Amer. Med. Assn., xlvi, 1906. 554 PATHOGENIC MIRCOORGANISMS one hour at 80° C. It is then filtered through a sterile Bcrkefcld filter. The filtrate is aseptically bottled and sterilized three days at 56°, half an hour each day. It is then made isotonic and is ready for titration. Vaccine therapy in systemic gonorrheal infection has been tried and is promising/ though as yet unconvincing. The vaccine, if possible, should be made with the organism isolated from the patient, for reasons described above. Passive immunization with the serum of gonococcus- immune animals has also been attempted, but records on it at present are not sufficiently complete to permit definite judgment. SANITARY CONSIDERATIONS IN CONNECTION WITH GONOCOCCUS INFECTION Of the three prevalent venereal infections, those caused by the gonococcus are probably the most common. For more exhaustive statistical studies of the prevalence of these diseases the reader is referred to such books as those of Pusey, Morrow, and the larger text-books of hygiene, such as that of Rosenau. Although it has been well known that gonorrhea was extremely common, the astonishing prevalence among young men of draft age was revealed during the late war when the figures of the Surgeon General25 show that about 5.6 per cent of the men who came into the military service were infected with a veneral disease. Considering that these diseases, in their early detectable acute stages, do not last very long, that many cases still apparent to a slight degree must surely be missed in physical examinations of large numbers of men, it seems to indicate that the estimate by many authorities of a prevalence of venereal disease in civilian life as high as 10 per cent may be very near the truth. The percentage of gonorrhea to other venereal diseases is probably pretty well exemplified by the percentage of these diseases for the entire army during 1918, during which, accord- ing to the Surgeon General's report, there were 44,213 cases of syphilis, 16,173 cases of chancroid, and 167,475 cases of gonococcus infection, a total of 227,861 cases of venereal disease. The rate for syphilis was 17.56 per 1000, for chancroid 6.42, for gonorrhea 66.50. One of the great dangers in connection with gonorrheal infection has been the relative indifference of the public to these diseases. In the past, there has been a remarkable lack of appreciation of the seriousness of the infection, which actually, in its economic and sociological impor- 23 Rep. of Surgeon General, U. S. A., 1919, Vol. 1, p. 956. GONOOOCCUS INFECTION 555 tance, is equal to, if not more serious, than syphilis. The gonococcus is primarily infectious for the genital organs, but may also infect the eye, and in its secondary manifestations cause disease of the prostate, epidydimis and bladder of the male, of the fallopian tubes and ovaries of the female. In both it may and often does cause sterility. Invad- ing the blood stream, it may cause endocarditis, and not infrequently an acute and .subacute arthritis which is characterized by its frequent localization in single joints or the bursse about joints, and cause peri- articular inflammation. It may, but rarely does attack other organs. A most important consideration is the difficulty of complete cure. A male who has contracted gonorrhea may seem to be completely cured, but if a posterior urethritis has occurred, the organisms may •remain viable and capable of infecting others for a great many years. Individuals who, therefore, seem to have been cured for years, may still cause infection upon marriage, a fact which is the most frequent cause of gynecological lesions in women. It goes without saying that even the most careful bacteriological examination of such individuals may often fail to reveal the gonococci, even though they may be present. Infection with gonococcus is almost invariably by sexual contact, though the organism may remain viable on wearing apparel, bed cloth- ing, towels, hands, etc., for brief periods, especially if protected from light and drying, and others may be infected in this way. The danger of self-infection of eyes by people who are suffering from an acute dis- charge is a frequent one, and physicians and nurses, especially, are liable to such infection. Gonorrheal infection of the eye is one of the most serious infections that can occur in this organ. Ophthalmia of the new born may be due to other organisms, but is almost invariably caused by the gonococcus. It is acquired by the child in the course of delivery, from the secretions of the mother, and if not attended to, may lead to blindness. The importance of this infection may be estimated from the following figures quoted by Rosenau from Kerr, who states that in the United States and Canada 23.9 per cent of 351 admissions to schools for the blind in 1910 the blindness was the result of gonorrheal infection. Fortunately, the method introduced by Crede has, to a very large extent, done away with this accident. Crede, many years ago, intro- duced the method of instilling a 2 per cent silver nitrate solution into the conjunctiva] sa.cs of every child at birth. Since his time oilier silver salts have been in use, the most popular ones at the present time being protargol, 5 per cent solution, and argyrol, 20 per cent solution, which 556 PATHOGENIC MICROORGANISMS are dropped into the eye at birth. It is extremely important that this should be done properly and the entire conjunctival sac bathed in the fluid. The method is so important that it is regarded as a matter of very serious and inexcusable omission, if, under any circumstances, in dealing with any class of the population, the physician managing a childbirth fails to carry out this measure as soon as feasible after birth. Another very important gonorrheal problem is the vulvovangitis which occurs in children. In our own experience, this infection has oc- curred most often in connection with the children's wards in hospitals. The condition has, however, been observed in schools and in small family groups where children were infected by sleeping in the same beds with adults. In hospitals the disease may spread in epidemics, and from bed to bed, with an ease that is astonishing when one considers the delicate life of the gonococcus outside the body. It has often been extre nely difficult to stop such bed to bed infection, in spite of the most rigid precautions. Epidemics are so difficult to arrest, and the conse- quences for the child so grave from many points of view, that it has becone the custom in all well-managed hospitals to delay the admission of female children to the general children's ward until vaginal smears have been made and examined for gonococci. It is in our opinion extremely important that when such smears are made, they should be taken not only from the visible secretion, but should be taken from high up in the vagina through a small Kelly speculum, with good illumina- tion. When there is danger of spread and a case has been inadver- tently admitted, only the greatest care in avoiding indirect contact from bed to bed can stop it. As a matter of routine in children's wards, there should be separate thermometers, unless all thermometers are very carefully sterilized, thermometers should be kept in weak carbolic solutions and washed with alcohol before use; the sterilization of diapers and towels should be attended to, nurses handling cases with discharge should wear gloves, and there should be no common use of towels and washing utensils. Great attention should be given to the scouring of bath tubs, and bed linen, night clothes, etc., should be sterilized by boiling. Public Health Management of Venereal Diseases. — During the past ten years there has been a very wholesome increase of interest in venereal disease prevention. There are certain general fundamental principles which apply to all venereal diseases equally. In the first place, it is necessary to look upon venereal infections as preventable GONOCOCCUS INFECTION 557 diseases. It has been unfortunate that the sanitary and moral issues have been so closely interwoven in these diseases, that it has been impossible to create the free discussion and spread the information necessary to obtain the cooperation of the public in these matters. Without public education and cooperation, large scale public health results cannot be achieved. In our opinion, one of the most important factors that have prevented earlier progress in the prevention of venereal disease has been the ignorance of the public in regard to these matters. It has been especially wrong that women of the marriageable age have been kept in ignorance about facts concerning, these infections, an ignorance which has often left them absolutely at the mercy of chance. Accurate and clear information, free of sensationalism, will do more eventually to reduce the venereal rate than any other single factor. It is not the function of a book of this kind to go into the very com- plex problems of general sex education, and the moral issues involved. We will restrict ourselves entirely to the purely sanitary phases of the problem. Chief among these are : 1. Diagnosis. — Education and knowledge of the seriousness of these infections should lead to a gradual attraction of patients, away from quacks, to reliable clinics and physicians. The development of diag- nostic clinics by departments of health, the improvement of clinical facilities in large cities, and the better understanding by physicians, as a whole, of the sanitary importance of these relatively simple infections, must lead to more accurate diagnosis and proper instruction of the patient. 2. Reporting of Venereal Diseases. — In a great many communities at the present time gonorrheal infections, as well as other venereal diseases, are regarded, like other communicable diseases, as subject to report. There are many reasons why such reporting systems will meet with objections, and will for many years be unsatisfactory. This, too, we believe is a matter of education, and the fact that it will fail for the present is no reason why the principle should not be upheld. Event- ually we believe it will be accepted as a sensible and necessary step. These diseases are communicable to others during certain stages, and when they are regarded primarily as possibilities for the spread of dis- ease and the public stress is not laid purely on the moral issue, objections to reporting will cease. In our opinion the chief objection that has been raised against the reporting of these diseases is the permanent record, apparent disgrace and perhaps opportunity for blackmail which is opened by the public registration of an individual in this way. 558 PATHOGENIC MICROORGANISMS When we consider, on the other hand, these dangers as balanced against the danger of the uncontrolled circulation among their fellows of indi- viduals capable of infecting others, there seems very little choice in our minds between the two evils. Moreover, we believe it would be possible to develop a system of reporting whereby the reported individual could have the record destroyed when he could bring a certificate of cure from a responsible clinic or physician. This, we believe, would add a further inducement to proper care and cure. At any rate, we believe that the prompt report of cases, following them up from municipal health bureaus, and prompt destruction of the record when the individual has been cured, will greatly aid in this matter. 3. H capitalization. — It will probably be impossible to hospitalize all infectious cases of venereal diseases because, unfortunately from the public-health point of view, these patients are not incapacitated during their most infectious stages. We are able to confine a case of smallpox with or without consent, but diseases that in their remote possibilities are responsible for far greater injury and unhappiness, are permitted to walk about and follow their own devices, through the course of their illnesses. The eventual ideal would consist in making physicians responsible for the isolation of cases which came under their care and to hospitalize those who could not be taken care of in their own quarters. Hospitalization in separate hospitals would confer so great a stigma that it would probably be impossible. It might also be impossible to admit these cases into general hospitals in spite of special arrangements. We do not ourselves believe that compulsory hospitalization could be enforced at the present time. It should be looked upon, however, as an attempt worth making, as soon as educa- tion and general public cooperation has reached a point at which success would seem at least not totally out of question. It is our opinion that the sooner the attitude toward these diseases is made one purely of sanitary principles, and the more purely moral factors are allowed to take care of themselves under the influence of increased civilization and sense of community responsibility, the sooner these ends may be accomplished. While it is of course quite impossible to do justice to as funda- mentally important a problem as the sanitary control of venereal dis- eases in a section of this kind, it has seemed to us of great importance to at least point out to physicians and bacteriologists who may read this book, the enormous responsibility that falls upon them whenever they are in a position to deal with cases of this kind. (joNororrrs IN FICTION 559 Prophylaxis of Gonorrhea. — As practiced in the United States army stations during the war26 this consisted in injecting about 10 c.c. of a 2 per cent protargol into the urethra — enough to thoroughly dis- tend it, with a glass hand syringe, holding it there with the syringe in place, for one-half minute. The procedure is twice repeated. Its success depends very largely upon early application after intercourse. As to general efficiency in regard to gonorrhea we are not in a position as vet to submit reliable statistics. MICROCOCCUS CATARRHALIS Micrococcus catarrhalis is a diplococcus described first by R. Pfeiffer,27 who found it in the sputum of patients suffering from catarrhal inflammations of the upper respiratory tract. It was subsequently carefully studied by Ghon and H. Pfeiffer.28 According to these authors the pathogenic significance of the micrococcus is slight, though occasion- ally it may be regarded as the causative factor in catarrhal inflammations. Its chief claim to attention, however, lies in its similarity to the meningo- coccus and the gonococcus, from neither of which it can be morpholog- ically distinguished. It is decolorized by Gram's stain, appears often in the diplococcus form, and has a tendency, in exudates, to be located intracellularly. Not unlike the two microorganisms mentioned, too, it shows but slight pathogenicity for animals. Differentiation from gonococcus is extremely simple in that Micro- coccus catarrhalis grows easily on simple culture media and shows none of the fastidious cultural requirements of the gonococcus. From meningococcus the differentiation is less simple and, because of the presence of both microorganisms in the nose, is of great impor- tance. Distinction between the two is made entirely upon cultural charac- teristics and agglutination reactions. Culturally, Micrococcus catar- rhalis grows more heavily than meningococcus upon the ordinary culture media. The colonies of Micrococcus catarrhalis are coarsely granular and distinctly white in contradistinction to the finely granu- lar, grayish meningococcus colonies.29 Micrococcus catarrhalis will 26 Bayard Clark, Medical Times, April, 191!). -' Vliifif/<; "J)ie Mikroorg.," 'M <''/r;/.v/or/,, 1'WI. (1. M ; ll'cif/crl, I>out. uio.l. \Vnch., .Iss.l. 9 Xichl, Deut, nied. Woch., 1883; Ncelscn, "Lehrb. <1. allff. Path.," 1894, 588 PATHOGENIC MICROORGANISMS either with 5 per cent nitric acid, 5 to 20 per cent sulphuric acid, or 1 per cent hydrochloric acid, until most of the red color has disappeared (a few seconds), and the preparation appears pale pink. This results in decolorization of all microorganisms with the exception of members of the acid-fast group. Thorough washing in 80 to 95 per cent alcohol is now employed to complete the decolorization. The preparation is then rinsed in water and counterstained with 1 per cent aqueous methy- lene-blue. Tubercle-bacillus staining has been further simplified by Gabbett,10 who combines decolorization and counterstaining. In this method preparations are stained with Ziehl's carbol-fuchsin as in the preceding; they are then rinsed in water and covered with a solution containing methylene-blue 1 gram, concentrated sulphuric acid 25 grams, and distilled water 100 c.c. This is allowed to act for from two to four min- utes, at the end of which time all elements in the preparation except the acid-fast bacilli will be decolorized and counterstained. Another excellent stain for tubercle bacilli, which has the advantage of greater clearness of contrast over the carbol-fuchsin stain is that of Hermann in which Crystal Violet is used. It is described in the section on staining. Tubercle bacilli in very young culture are often not acid-fast and it is not always possible to demonstrate acid-fast bacilli in pus from cold abscesses in sputum, in serous exudates, and in granulomatous lesions of the lymph nodes which can be shown by animal inoculation to be tuberculous. Much n demonstrated in such material Gram-positive granules which lay singly, in short chains or in irregular clumps, and which he believed to be non-acid-fast tubercle bacilli. He found similar granules in cultures of tubercle bacilli which showed on further incuba- tion numerous acid-fast bacillary forms. His work has been repeatedly confirmed, and there seems little doubt but that these granules are really tubercle bacilli. Their demonstration is not, however, of great diag- nostic value, as other bacilli form granules of the same appearance. Small rods and splinters are also found which stain by Gram's method, but not by carbol-fuchsin.12 To find "Much's granules," smears or sections are steamed in a solution of methyl violet B.N. (10 c.c. of saturated alcoholic solution of the dye in 100 c.o. of distilled water containing 2 per cent phenol). 10 Gabbett, Lancet, 1887. 11 Much, Ben. klin. Woch., 1908, xlv, 700. 12 Liebermeister, Deutsche med. Woch., 1909, xxxv, 1324, THE TUBERCLE BACILLUS 589 They are then treated with Gram's iodine solution one to five minutes; 5 per cent nitric acid one minute; 3 per cent hydrochloric acid ten sec- onds; absolute alcohol and acetone equal parts, until decolorized. The granules may be stained by other modifications of Gram's method. Weiss 13 has devised a combination stain. One part of Much's methyl violet is mixed with three parts of ZiehFs carbol-fuchsin and filtered; slides are stained for twenty-four to forty-eight hours in the mixture. They are then decolorized as in Much's method and counterstained with Bismarck brown or safranin 1 per cent. Both acid-fast and Gram- positive forms are stained by this method and in the red may be seen blue-black granules. While the acid-fast group of bacteria is composed of a number of organisms to be mentioned later, a few only of these offer difficulties of differentiation from the tubercle bacillus. Those to be considered practically are the bacillus of leprosy and that of smegma. The latter bacillus, because of its distribution, is not infrequently found to con- taminate feces, urine, or even sputum, and it is sometimes desirable to apply to suspected specimens one or the other of the stains devised for the differentiation of the smegma bacillus from Bacillus tuber- culosis. The one most frequently employed is that of Pappenheim.14 The preparations are stained in hot carbol-fuchsin as before; the carbol-fuchsin is then poured off without washing and the preparation immersed in solution made by saturating a 1 per cent alcoholic solu- tion of rosolic acid with methylene-blue and adding 20 per cent of glycerin. In such preparations tubercle bacilli remain red, smegma bacilli appear blue. Stained by Gram, tubercle bacilli retain the gentian- violet. When tubercle bacilli are very sparsely present in sputum and other material it may be impossible to find them by direct examination, and often the only method of finding them will be animal inoculation. However, a number of methods have been devised by which the bacilli may be concentrated in such a way that they may be found even when a few only are present. One of these is to add peroxide of hydrogen to the sputum. By this the mucus is dissolved out and the solid particles sstth or may be centrifugalized. A method very commonly employed to-day is that which depends on the use of "antiformin." This is a preparation used extensively for the cleansing of vats in breweries. It 13 Weiss, Bcrl. klin. Woch., 1909, xlvi, 1797. 14 Pappenheim, Berl. klin. Woch., 1898. 590 PATHOGENIC MICROORGANISMS is described by Rosenau 15 as consisting of equal parts of liquor sodse chlorinatse and a 15 per cent solution of caustic soda. The formula for liquor sodse chlorinatse he gives as : Sodium carbonate 000 Chlorinated lime 400 Distilled water 4,000 If sputum is poured into a 10 to 15 per cent solution of antiformin and allowed to stand for several hours, most of the other elements of the sputum, cells, and bacteria, will dissolve out, and acid-fast bacilli be left in the residue. 'Strangely enough they are not killed by this process and if sufficiently washed may be cultivated or can produce lesions in guinea-pigs. Isolation and Cultivation. — Tubercle bacilli are not easily cul- tivated. Their slowness of growth precludes isolation by plating. The first isolations by Koch 16 were made upon coagulated blood serum from tuberculous tissue. Isolation from tuberculous material may be aided by inoculation into guinea-pigs. These animals will withstand the acute infection produced by the contaminating organisms and succumb later (four to six weeks) to tuberculosis. The bacilli may then be obtained by cultivations from lymph nodes or other foci which contain only tubercle bacilli. When isolation from sputum is attempted whether directly or by means of animal inoculation, the sputum may be rendered comparatively free from contaminating bacteria by washing. The sputum is rinsed in running water to free it from pharyngeal mucus. It is then washed in eight or ten changes of sterile water. The material selected is taken from the center of the washed mass, if possible from the flakes of caseous material visible in such sputum. For the isolation of tubercle bacilli from sputum and other materials in which contaminating bacteria of other species are present, Petroff17 has devised an excellent method which has been tried out and used with success in our laboratory. The principles on which PetrofPs method rests are, first of all, the bactericidal power of 3 per cent sodium hydroxid on non-acid-fast bacteria, and the selective action of dyes like gentian violet on bacterial growth, as first practically utilized by Churchman. 15 Rosenau, ' ' Preventive Medicine and Hyigene, ' ' D. Appleton, X. Y., 1913 ; Uhlenhuth, Berl. klin. Woch., Xo. 29, 1908. 18 Koch, loc cit. 17 Petroff, Johns Hopkins Hosp. Bull., vol. xxvi, Xo. 294, August, 1915, p. 276. THE TUBERCLE BACILLUS 591 The medium used by Petroff is made as follows: I. Meat Juice. Five hundred grams of beef or veal are infused in 500 c.c. of a 15 per cent solution of glycerin in water, in a cool place. After twenty-four hours the meat is squeezed in a sterile press and the infusion collected in a sterile beaker. II. Eggs. The shells of the eggs are sterilized by ten minute immersion in 70. per cent alcohol. They are broken into a sterile beaker, well mixed and filtered through sterile gauze. One part of meat juice is added to two parts of egg by volume. III. Gentian Violet. One per cent alcoholic solution of gentian violet is added to make a final proportion of 1 :10,000. The three ingredients are well mixed. The medium is tubed and inspissated as usual. Petroff recommends for sputum the following technique: Equal parts of sputum and 3 per cent sodium hydroxid are shaken and incubated at 38° C. for fifteen to thirty minutes, the time depending on the consistency of the sputum. The mixture is neutralized to litmus with hydrochloric acid and centrifugalized. The sediment is inoculated into the medium described above. Pure cultures are obtained in a large proportion of cases. PetrofFs method has been applied by him to feces, in which the problem is made more difficult by the presence of many spore-formers which resist sodium hydroxid. Feces is collected and diluted with three volumes of water, and then filtered through several thicknesses of gauze. The filtrate is saturated with sodium chlorid and left for half an hour. The floating film of bacteria is collected in a wide-mouthed bottle and an equal volume of normal sodium hydroxid is added. This is shaken and left in the incubator for three hours, shaking every half hour. It is then neutralized to litmus with normal hydrochloric, centrifugalized, and the sediment planted. Once isolated, the bacilli are best grown on glycerin egg medium which is described in the section on Media. On this medium colonies of the human bacilli begin to appear after six or eight days as yellowish white moist crust-like flakes. On blood serum at 37.5° C., colonies become visible at the end of eight to fourteen days. They appear as small, dry, scaly spots with corru- gated surfaces. After three or four weeks, these join, covering the sur- face as a dry, whitish, wrinkled membrane. Coagulated dog serum is regarded by Theobald Smith 18 as a favorable media for the growth of tubercle bacilli. ls Tli. NM////J, .lour. Hxp. Mod., iii, 1898. 592 PATHOGENIC MICROORGANISMS Slants of agar, to which whole rabbit's blood has been added in quan- tities of from 1 to 2 c.c. to each tube, make an excellent medium. Cultivation methods were simplified by the discovery by Roux and Nocard that glycerin facilitates cultivation. Upon glycerin-cigar (glycerin 3 to 6 per cent), at 37.5° C., colonies become visible at the end of from ten days to two weeks. Glycerin bouillon (made of beef or veal with pepton 1 per cent, glycerin 6 per cent, slightly alkaline) is a favorable medium. It should be filled, in shallow layers, into wide- mouthed flasks, since oxygen is essen- tial. Transplants to this medium should be made by carefully floating flakes of the culture upon the surface. In this medium the bacilli will spread out upon the surface, at first as a thin, opaque, floating membrane. This rapidly thickens into a white, wrinkled, or granular layer, spreading over the entire surface of the fluid in from four to six weeks. Later, portions of the membrane sink. In old cultures, the membrane becomes yellowish. These cultures emit a peculiar aromatic odor. Cultures when first grown on solid media are a little difficult to start on glycerin broth. To accomplish this it is best to grew them for a few weeks on egg or glycerin egg slants containing considerable amounts of condensation water. At the end of this time the growth will have begun to grow over the surface of the condensation water, and from this pellicle a bit can be picked up with a bent loop, carefully removed with- out allowing it to immerse in the fluid and this carefully floated on the surface of the glycerin broth in the flask. Glycerin potato forms a favorable culture medium for the bacillus. Hesse 19 has devised a medium containing a proprietary preparation FIG. 64. — CULTURE OF BACILLUS TUBERCULOSIS IN FLASK OF GLYCERIN BOUILLON. 18 Hesse, Zeit. f. Hyg.. xxxi. THE TUBERCLE BACILLUS 593 known as "Nahrstoff Heyden/' upon which tubercle bacilli are said to proliferate more rapidly than other bacteria. His method has yielded excellent results. It is prepared as follows : ' ' Nahrstoff Heyden " -° 10 grams Sodium chlorid 5 'll Glycerin 30 " Ag-ar 10 l ' Normal sodium solution 5 c.c. Aq. dcst 1,000 < ' Biological Considerations. — The tubercle bacillus is dependent upon the access of oxygen. Its optimum temperature is 37.5° C. Temperatures below 30° and above 42° C. inhibit its growth. In fluid media, the bacilli are killed by 60° in fifteen to twenty minutes, by 80° in five minutes, by 90° in one to two minutes. They will withstand dry heat at 100° C. for one hour. They are resistant to cold. The com- paratively high powers of resistance of the bacillus are attributed to the protective qualities of the waxy cell membrane.21 The life of cultures, kept in favorable environment, is from two to eight months, varying to some extent with the nature of the culture medium. The viability of the bacilli in sputum is of great hygienic importance. In most sputum they may remain alive and virulent for as long as six weeks, in dried sputum for more than two months.22 Five per cent carbolic acid kills the bacilli in a few minutes.23 Used for sputum disinfection, where the bacilli are protected, complete disin- fection requires five to six hours. Bichloride of mercury is not very efficient for sputum because of the formation of albuminate of mercury. Direct sunlight kills in a few hours. Pathogenicity. — The tubercle bacillus gives rise in men and sus- ceptible animals to specific inflammations which are so characteristic that a diagnosis of tuberculosis may be made by histological examina- tion, even though tubercle bacilli themselves are not found. These foci, known as tubercles, were first studied in detail by Baumgarten,24 and, since then, have been the object of many pathological investiga- 20 " Nahrstoff Heyden '; is prepared in Germany. It is a white powder similar to nutrose. 21 Tli. Smith, Jour. Exper. Med., 1899 ; Grancher et Ledoux-Lebard, Arch, de med. exper., 1892; Galtier, Compt. rend, de 1'acad. des sci., 1887. 22 Schell und Fischer, Mitt. a. d. kais. Gesundheitsamt, 1884. 23 De Toma, Ann. di med., 1886. *4 Baumgarten, Bcrl. klin. Woch., 1901. 594 PATHOGENIC MICROORGANISMS tions. The lesions are fundamentally alike wherever they occur, though in their detailed histological structure they may vary somewhat accorck ing to the tissue in which they appear. They begin as microscopic agglomerations of concentrically arranged epithelium-like, or epitheloid cells, around which eventually lymphocytes accumulate. These micro- scopic tubercles may gradually enlarge individually, or they may grow by coalescence with neighboring tubercles. Characteristic giant cells, with marginal centers of nuclei, appear near the centers of the tubercles and in these giant cells the tubercle bacilli are usually found. As the tubercles grow in size, the central mass becomes necrotic. Fluid pus does not form, and the centers assume a grumous and friable condition which is generally described as caseous or cheesy. Such tubercles may result from the injection of dead bacilli, as well as living ones as Prudden and Hodenpyl have shown. The cheesy degeneration may be in part due to the toxic action of the substances of the tubercle bacilli and in part by pressure and lack of vascularization. It is astonishing how difficult it is to find tubercle bacilli histologically in such lesions. This may be due perhaps to the fact that, owing to degeneration, most of the tubercle bacilli have lost their acid-fastness. If tubercles heal, as they often do, they undergo a fibrinous change, are surrounded by con- nective tissue and, if central necrosis has begun at the time that healing sets in, calcification results. For more detailed descriptions we refer the reader the Text books of Pathology of Adami's, MacCallum's or Delafield and Prudden. There has been a great deal of discussion concerning the manner in which tubercle bacilli enter the human and animal body in the course of spontaneous infection. A thorough discussion of this will be found in the recent book by Calmette, L' Infection Bacillaire de la Tuberculose (Masson, Paris, 1920), Chapter 8, p. 110. Calmette believes that when a tubercle bacillus "is deposited on the surface of the skin or a mucous membrane or is introduced into the healthy body by another route" it becomes the prey of leucocytes which carry it into the lymphatic circu- lation and into the blood. The leucocytic enzymes are not capable of digesting the organism and eventually the organism is deposited in the lymphatics when the leucocyte degenerates. Tubercle bacilli may remain latent in the body, in lymph nodes, especially, for long periods. It appears that the point of entrance of the tubercle bacilli into the body may be through the tonsils, and secondarily thence through the lymphatics, then to other organs. Pulmonary in- fection may be either by direct inhalation or indirectly through the THE TUBERCLE BACILLUS 595 lymphatics. Calmette believes that actual direct infection of the lung by inhaled bacilli is relatively rare and bases this upon experimental evidence. This, however, is not in agreement with the bulk of evi- dence, and direct inhalation is probably the most common manner of invasion. According to the researches of Bartel and many others, it appears that direct infection through the apparently uninjured mucous mem- brane of the intestinal tract may take place, and after such entrance the bacilli may be carried by the lymphatics and blood to the lungs and other parts of the body. Calmette states that, in all susceptible ani- mals, man included, and in all its varieties of localization, tuberculosis in the large majority of cases originates in a primary infection of the lymphatics which takes its origin by entrance of the tubercle bacilli through the mucous membranes of the digestive tract, chiefly the mucous membranes of the mouth, pharynx and intestine. This is the extreme view, but one that is favored in addition to Calmette by Von Behring, Ravenel and others. Opie 25 as a result of recent studies, states that first infection with tuberculosis may occur either by way of the lungs or the gastro-intestinal tract, and the occurrence of one lesion tends to prevent the other. In man, tuberculosis is most common in the lungs where it usually starts in the apices. The apical situation of early tubercles is not entirely explained. A number of theories have been advanced, most of them based on anatomical reasoning. In the lungs there may be a miliary distribution of tubercles or, by coalescence of these, large areas of consolidation may occur, which are then spoken of as phthisis. Extension to the pleura is common. Although pulmonary infections constitute the very large majority of cases of tuberculosis in adult life, this is not strictly true of childhood. In statistics quoted by Calmette for Europe, it was found by Ham- berger and Sluka 2G that of 160 cases in children there were only 50 per cent pulmonary lesions. According to Dr. Holt's statistics for New York, however, of 119 autopsies of tuberculous children, pulmonary lesions were found in 99 per cent. In 1515 autopsies studied by Comby during fifteen years, involvement of the tracheo-bronchial lymph nodes was found in all. Aside from the pulmonary and lymphatic infections, tuberculosis may occur in practically all other parts of the body. 23 Opie, Am. Kev. of Tuberculosis, vol. 4, 1920, p. 629. 26 Hamburger and Sluka, cited from Calmette, loc. cit. 5% PATHOGENIC MICROORGANISMS Tuberculosis of the skin or lupus is a common disease. Involve- ment of the bones and joints may occur, and according to Fraser27 may in many cases occur without any previous tuberculosis. Tuber- culous meningitis is not infrequent in children, and is always fatal. Calmette quotes Grunberg's studies of the comparative frequency of mortality of children from tuberculosis in 568 families. He analyzed 209 deaths by tuberculosis in children from birth to fifteen years, from birth to one year, 82 per cent were meningitic, 6 per cent were pulmonary and 3 per cent other forms of tuberculosis. At fifteen years, only 6 cases were meningitis,? pulmonary and 5 of other forms. The liver may be the site of tubercles, and tuberculosis of the spleen has been observed though it is not particularly common. The kidneys and the geni to-urinary system are frequently involved, and the supre- renal gland may be tuberculous, and in this case may lead to a condition spoken of as Addison's disease. In the intestines themselves, various forms of tuberculosis have been described. It appears that the only parts of the body in which tuber- culosis is not common are the muscle tissues themselves, and the wall of the stomach. Rosenberger 28 has reported finding tubercle bacilli in the circu- lating blood of all cases of human tuberculosis which he examined. This announcement aroused much interest and has led to many investi- gations by other workers. Rosenberger 's results were obtained by morphological examination of smears of citrated blood taken from the patients, dried upon slides and laked with distilled water. Many other observers have failed to confirm Rosenberger's results. Anderson 29 examined 47 cases in which tubercle bacilli were found in the sputum and one case of joint tuberculosis. In none of these 48 cases was he able to obtain tubercle bacilli, either by morphological examination nor by guinea-pig inoculation. Brem30 subsequently found that laboratory distilled water may frequently contain acid-fast saprophytes — a fact which may account in many cases for errors when morphologi- cal examination alone is relied upon and blood examined by the tech- nique of Rosenberger. This, too, is suggested by the finding of acid- fast bacilli in the blood of perfectly healthy individuals. Therefore, although the bacilli may be present in the blood in a certain number of 27 Fraser, Jour. Exper. Med., No. 4, 16, 1912. 28 Rosenberger, Am. Jour, of Med. Sc., cxxxvii, 1909. 29 Anderson, U. S. P. H. Service, Hygienic Lab., Bull. 57, 1909. 30 Brem, Jour. A. M. A., liii, 1909. THE TUBERCLE BACILLUS 597 cases it does not seem likely that they are so distributed in anything like the high percentages found by Rosenberger.31 Although, therefore, in patients suffering from tuberculosis, the presence of the tubercle bacilli in the blood is generally slight or intermit- tent, there may be times when large numbers of tubercle bacilli are thrown into the blood stream, and, according to the manner and quan- tity thus distributed, secondary foci or general miliary tuberculosis may occur. Bacillus tuberculosis (typus humanus) is pathogenic for guinea- pigs, less markedly for rabbits, and still less so for dogs. It is slightly pathogenic for cattle, a question spoken of more extensively below. Secondary Infection in Tuberculosis. — An important consideration in the symptomatology and prognosis of pulmonary tuberculosis is the fact that on the basis of the chronic inflammatory condition of bronchii and alveoli in the neighborhood of tuberculous processes in the bron- chiectatic cavities and perhaps in cavities communicated with bron- chioles, masses of bacteria of various species may accumulate and habit- ually lodge. Staphylococci, streptococci, Gram-negative cocci, and frequently influenza bacilli may be present in such cases and materially contribute to the illness of the patient by superimposing acute and subacute inflammatory processes upon the tuberculous one. FREQUENCY AND TRANSMISSION In man, tuberculosis is the most common of diseases. Naegeli's statistics, based on a large series of autopsies, show not only the fre- quency of the disease, but its relation to age. Before one year of age he finds it very rare. From the first to the fifth year it is rare, but usually fatal. From the fifth to the fourteenth year, one-third of his cases showed tuberculosis; from the fourteenth to the eighteenth year, one-half of the cases. Between eighteen and thirty, almost all the cases examined showed some trace of tuberculous infection. Three-quarters of these were active, one-quarter healed. Two-fifths of all deaths occur- ring at these ages were due to tuberculosis. After the age of thirty, active lesions gradually diminished, healed lesions increased. In 1900 it was stated that the average yearly mortality from tuber- culosis in New York amounted to 6000, and that in Manhattan alone there were .constantly 20,000 tuberculous persons. Cornet estimates 31 Suzuki and TaJcaki, Centralbl. f. Bakt., Ixi, 1911. 598 PATHOGENIC MICROORGANISMS that in 1894 the deaths in Germany from all other infectious diseases amounted to 116,705, those from tuberculosis alone to 123,904. Similar statistics might be chosen from the health reports of any large city. While the disease is less common in rural districts than in large towns, the difference is not so striking as is generally supposed. Kober 32 states that, owing to active measures of prevention, the death rate from tuberculosis has been reduced from 326 per 100,000 in 1888 to 147.6 in 1913, which he says means that, if the former rate of mortality had been continued "the number of deaths from this disease last year (1914) would have been 322,027, instead of 143,000," meaning the saving of over 179,000 lives. Although there has been much discussion concerning the different methods of infection, there seems to be very little doubt at the present time that inhalation is the most common means of human infection. In coughing, expectoration and sneezing, small droplets of fluid in which all kinds of microorganisms are found, are sprayed into the air, and these may be deposited upon the mucous membranes of people in close contact with the disease. The striking distance of such droplet infection is not very large, but is, as Kober points out, particularly dangerous because the bacilli thus enter the respiratory passages directly from body to body. In addition to this, the tubercle bacilli may remain alive in dust sufficiently dry to be blown about by draughts and winds. Although the bacilli are not spore bearers, their acid-fast nature renders them somewhat more resistant to desiccation and sun- light than are most other germs. Next to direct inhalation, the most frequent method of transmission is probably through the digestive tract. Such infection may take place by direct contamination of food from the expectoration and saliva of consumptives or by indirect infection of food, and milk, through the agency of fingers, flies, etc. Milk Infection. — The question of intestinal infection, however, is particularly important in connection with transmission of bovine tuber- culosis to man through the agency of infected milk. The general public has probably very little idea of the frequency of tuberculosis in cattle. In a community supervised more closely than usual, the work of Public Health Service bacteriologists in Washington, revealed 6.72 per cent of samples of market milk infected with tubercle bacilli. This percentage is probably very much lower than that which would naturally be found in districts with a less well-developed dairy supervision, and in some 82 Kober, Kep. No. 309, U. S. P. H. S. Keports, October, 1915. THE TUBERCLE BACILLUS 599 of the poorer farm districts of the country the cattle tuberculosis situa- tion is actually appalling. The question of how frequently infection with bovine tubercle bacilli through the intestinal tract .may occur is still a matter of some controversy. V. Behring expressed the belief that a large percentage of all cases of tuberculosis originated in childhood from infection through the in- testinal tract. He determined that tubercle bacilli may penetrate the intestinal mucosa without causing lesions. Behring's contention caused a great deal of discussion, and the question he raised is intimately bound up with the problem of the virulence of bovine tubercle bacilli for human beings, as he assumes that the infection is due to the use of infected milk, COMBINED TABULATION, CASES REPORTED AND OWN SERIES OF CASES (From Park and Krumwiede, loc. cit.) Diagnosis. Adults, 16 Years and Over. Children, 5 to 16 Years. Children Under 5 Years. Human. Bovine Human Bovine. Human. Bovine. Pulmonarv tuberculosis 568 2 22 15 6 28 4 18 11 1 2 1? 1 3 ' 1 1 1 1 11 4 33 7 2 4 1 7 2 26 1 1 20 7 3 1 1 12 2 15 6 13 28 3 45 14 21 1 1 20 13 10 5 8 ' 1 2 Tuberculous adenitis, axillary or inguinal Tuberculous adenitis, cervical Abdominal tuberculosis . . . Generalized tuberculosis, alimentary ori- gin Generalized tuberculosis Generalized tuberculosis, including men- inges, alimentary origin Generalized tuberculosis, including men- inges Tuberculous meningitis Tuberculosis of bones and joints Genito-urinary tuberculosis Tuberculosis of skin t Miscellaneous Cases Tuberculosis of tonsils Tuberculosis of mouth and cervical nodes Tuberculous sinus or abscesses Sepsis, latent bacilli Totals 677 9 99 33 161 59 Mixed or double infections, 4 cases. 600 PATHOGENIC MICROORGANISMS The problem is plainly of the greatest importance, and for this reason has been diligently investigated during the last few years. The only reliable method of approaching it has been to isolate the tubercle bacilli from diseased human beings and determine for each case whether the organism obtained belonged to the human or bovine type. These types can be differentiated definitely by cultural characteristics and pathogenicity, and it is not likely that the type changes during the sojourn in the human body. Granted this permanence of tyre, it is naturally of much value in revealing the source of an infection, to deter- mine whether or not a human being is harboring a bacillus of the human type or one of the bovine type. One of the most valuable contributions made to this problem during the last three years is that of Park and Krumwiede.33 The above tabulation is taken from their paper and represents a summary of their own cases and those reported by others. From this table it is evident that out of a total of 1042 cases, 101 only were bovine in origin and over 50 per cent of these occurred in children under five years of age. Fifty-one out of the 59 cases occurring in the 161 infants were directly or indirectly traced to alimentary infec- tion. It seems reasonably accurate, therefore, to state the case as follows: Human adults are relatively insusceptible to bovine infection. Such infection can take place, but is unusual. Below sixteen years of age the human race is relatively more susceptible and up to this age the danger of milk infection is unquestionably great, this source accounting for about one-third of the cases. Below five years the danger is greatest. This table alone should form sufficient evidence to silence absolutely any doubts as to the dangers of milk infection and remove any objections to the most rigid sanitary control of milk supplies. On the other hand, it also shows that Behring's original claims were far too sweeping and can not be upheld. Kober 34 also calls attention to the fact that one must not be deceived into believing that childhood is the only really dangerous age for infec- tion with tuberculosis, and quotes the results of a French committee which, in a small group carefully investigated, found 64 cases in which the disease was transmitted from husband to wife, 43 cases in which it was transmitted from wife to husband, 38 cases transmitted from brother R3 Park and Krumwiede, Jour, of Med. Ees., Oct., 1910. 31 Kober, Repr. No. 309, TJ. S. P. H. S. Reports, October, 1915. THE TUBERCLE BACILLUS 601 to sister, 19 from mother to child, 16 from other relatives, and in 33 cases it was traced to people who were not relations, but with whom the patient had been in communication. He also quotes Zasetsky who reports the case of a tuberculous woman who, in the course of eleven years married three husbands who had been previously healthy. The first one died of tuberculosis seven years after marriage, the second three years later, and the third at the time of the report had the disease, the wife in the meantime having died of tuberculosis. The fact that tubercle bacilli may be conveyed in dust has been indicated above, but there are other means by which habitual inhalation of dust may favor the spread of tuberculosis, namely, by virtue of the irritant properties of inhaled dust in predisposing the lung to infection. Attention to the dangers of trades in which dust is an habitual environmental factor has been particularly empha- sized by Winslow and Greenberg. Sommerfeld, whom we quote from Kober, made a statistical study in which he showed that in the population of Berlin, the average tuberculosis death rate was 4.93 per 1000. The rate in the non-dusty trades, was 2.39, and in the dusty trades 5.2. He also states that the analysis of tuberculosis in the towns in Vermont where granite and marble cutting is carried out showed a tuberculosis rate of 2.2 per 1000 against a rate of 1.3 for the whole state, and Ropke is stated by the same writer to have shown that the mortality from tuberculosis of the population in the large cutlery center, Solingen, in Germany was reduced from 5.4 per 1000 in 1885, to 1.8 in 1910, by measures aimed at the control of the dust in work rooms. A recent study by Drury 35 shows that polishers and grinders in axe factories are subject to a death rate from pulmonary tuberculosis considerably above that of others in the same mill. In the two decades from 1900 to 1919, the polishers and grinders showed a death rate of 19 per 1000 as against 6.5 of the entire mill population, and between 1 and 2.4 of the general population of the district. In regard to the predisposing factors to tuberculous infection, many phases enter into the problem in this disease which exert a much less direct or perhaps negligible influence in connection with other infections. There can be no question about the fact that poverty, with its coincident crowding in living quarters, close personal contact at night, insufficient warmth, and particularly undernutrition and low fat diet, play a role 35 Drury, Pub. Health Reeports, U. S. P. H. S., Feb., 1921, Vol. 36, No. 5. 602 PATHOGENIC MICROORGANISMS of immense importance in tuberculosis. In no disease is prevention so intimately influenced by general sociological and economic improve- ment as in tuberculosis. Wernicke 36 in a study of the relationship of diseases to social conditions shows an almost direct relationship between the provision of air space and parks in cities to tuberculosis. The statistics of the influence of the war upon tuberculosis have not yet become avail- able for study, but it is important to note that at the present writing we are informed by sanitarians who have returned from Europe that the sanitary problem in the European States is very largely one of tuber- culosis, and that the effects of prolonged undernutrition, especially upon children during the war years has resulted in an enormously increased tuberculosis rate. The question of the inheritance of tuberculosis has frequently been raised, and a large literature on this subject has accumulated, but an analysis of this literature seems to show that inheritance must be regarded as predisposition rather than as a method of direct infection. Children of tuberculous parents are likely to be more susceptible to tuberculosis and, of course, are expos3d to tuberculosis more intimately during the early years of life than are children of normal parents. There is no direct proof that tuberculosis is transmitted from mother to the foatus. Prevention. — As to preventive measures, we must refer the reader to special books on the subject since the problem is too large to be dealt with briefly with anything like completeness. The following summary of preventive measures is based largely upon the conclusions reached in Kober's discussion of this subject: We may assume, as premises for prevention, that tuberculosis can be transmitted at all periods of life and that foci acquired in youth may be arrested but light up under conditions of general undernutrition, malnutrition, etc., etc., in later years. Infection may be direct from person to person, indirect through contaminated food, fomites, flies; through dust, and in childhood through milk from infected cattle. The most common manner of acquiring tuberculosis is by inhalation and, next to that, probably through the digestive tract. The most important factor in the prevention of tuberculosis is education. This must elucidate the method of infection and the im- portance of the economic and sociological factors as they effect habits of food, sleep and fresh air. 36 Wernicke, quoted from Kober. THE TUBERCLE BACILLUS 603 • Direct infection must be prevented by compulsory notification, care and disinfection of expectorations, isolation, at least as far as the possibilities of sputum infection are concerned, in the home, in hospitals, etc., with introduction of pocket sputum flasks and the other simple measures by which a well-controlled tuberculosis patient can avoid infecting others. The actual prevention in deed, as well as word, of expectoration in public places, the protection of public drinking places, introduction of individual cups and public cleanliness in general; espe- cial supervision of these conditions in places of public lodgment and public amusement in schools and public conveyances; introduction of vacuum cleaning, etc. Attention in regard to the marriage of tuberculous people. Supervision of dairies and the marketing of milk by governmental grading, and control, with pasteurization of suspicious milk or milk not produced under the required conditions for grade "A" milk. Public provision for the proper and humane care of tuberculous people in state and municipal sanatoria so arranged that the poor will regard them as havens of hope, rather than as penalties imposed for disease. The public must be educated in knowing that tuberculosis is a cu- rable disease, provided that the diagnosis is made early and clinical facilities must be so arranged in cities so that accurate diagnosis in the early stages may be made and proper fresh air and nutritional care instituted if necessary, at public expense. In our cities, roofs, play- grounds, parks, etc., should be provided for school children. Summer care of children living in the crowded districts must be developed on a more generous and more important scale. The nutrition of school children in the public schools must be supervised and subsidized so that no child in a civilized community should suffer at any time from under nutrition. The prevention of tuberculosis is only in small part a medical prob- lem and must rest in its last analysis on the prevention of the means of direct infection and the predisposing considerations in which the sociol- ogist and the educator must play as important a part as the physician. Chemical Analysis of Tubercle Bacilli.37 — Diligent efforts by many investigators to isolate the specific toxins which lend tubercle bacilli their pathogenic properties have led to careful chemical analysis of the organisms. About 85.9 per cent of the bacillus consists of water; 20 37 Hammerschlag, Cent. f. klin. Med., 1891; Weyl, Deut. med. Woch., 1891; De'Schweinits and Dorset, Jour. Amer. Chem. Soc., 1895; Hammerschlag, loc. cit. 604 PATHOGENIC MICROORGANISMS to 26 per cent of the residue can be extracted with ether and alcohol. This material consists of fatty acids and waxy substances (fatty acids in combination with the higher alcohols). The residue after alcohol- ether extraction is composed chiefly of nitrogenous constituents. These can be extracted with dilute alkaline solutions, and consist chiefly of nucleoproteins. After removal of the so-called nucleoproteins — that is the material which comes down on treatment with dilute acetic acid in the cold, there remains a small amount of coagulable protein and, we have recently observed, small amounts of a substance that reacts like Bence-Jones protein.38 The final residue contains alcohol precip- itable substances — proteoses and polypeptids that constitute the active substances of the tuberculin reactions. Cellulose is also found and is supposed to represent the framework of the cell membrane, and there is an ash rich in calcium and magnesium. Toxins of the Tubercle Bacillus. — THE TUBERCULINS. — Filtrates of bouillon cultures of Bacillus tuberculosis39 will occasionally produce slight emaciation when injected into guinea-pigs, and when adminis- tered to tuberculous subjects in sufficient quantity will give rise to marked increase of temperature. It is likely, therefore, that the tubercle bacillus actually secretes a soluble toxin.40 The chief toxic principles, however, of Bacillus tuberculosis are probably endotoxins or bacterial proteins, bound during cell life to the body of the bacillus. Dead bacilli will produce sterile abscesses when inj ected into animals. Prudden and Hodenpyl,41 Straus and Gamaleia,42 and others,43 moreover, have shown that the injection of dead and care- fully washed cultures of this bacillus will produce lesions histologi- cally similar to those occurring after infection with the living germs, and will often lead to marasmus and other systemic symptoms of poisoning. The hope of actively immunizing with substances obtained from dead bacilli led Koch to employ various methods of extraction of cul- tures for the manufacture of tuberculin. "Old Tuberculin"** (Koch) ("T.A.K.").— The first tuberculin made 88 Zinsser, Journ. exp. med., Nov., 1921. 39 Straus and Gamaleia, Arch. med. exp., 1891. 40 Denys, ' < Le Bouillon Filtre, ' ' Louvain, 1905. 41 Prudden and Hodenpyl, N. Y. Med. Jour., June, 1891; Prudden, ibid., Dec. 5. 42 Straus and Gamaleia, loc. cit. 43Mafucci, Cent. f. allg. Path., 1890. 44 Koch, Cent. f. Bakt., 1890; Deut. med. Woch., 1891. THE TUBERCLE BACILLUS 605 by Koch is produced in the following manner: Tubercle bacilli are grown in slightly alkaline 5 per cent glycerin-pepton bouillon for six to eight weeks. At the end of this time, growth ceases and the corrugated pellicle of tubercle bacilli, which during growth has floated on the surface, begins, here and there, to sink to the bottom. The entire culture is then heated on a water-bath at about 80° C., until reduced to one-tenth of its original volume. It is then filtered either through sterile filter paper or through porcelain filters. The resulting filtrate is a rich brown, syrupy fluid, containing the elements of the original cul- ture medium and a 50 per cent glycerin extract of the tubercle bacilli. While the glycerin is of sufficient concentration to preserve it indef- initely, 0.5 per cent phenol may be added as an additional precaution. Dilutions of this fluid are used for diagnostic and therapeutic purposes. "New Tuberculin"*5 (Koch) (TA, TO, TR).— Koch believed that the immunity resulting from treatment with the old tuberculin was purely an antitoxic immunity, devoid of all antibacterial action. The use of whole dead tubercle bacilli for immunization purposes, however, was impracticable; because, injected subcutaneously, they were not absorbed, and introduced intravenously they were deposited in the lungs and gave rise to lesions. Koch was led, therefore, to resort to more energetic extraction of the bacilli in the hope of procuring a substance which could be easily absorbed and would at the same time give rise, when injected, to antibodies more definitely bactericidal. By extract- ing tubercle bacilli with decinormal NaOH, for three days, filtering through paper and neutralizing, he obtained his TA (alkaline tubercu- lin). This preparation seemed to fulfill some of the hopes of its dis- coverer, but had the disadvantage of often producing abscesses at the points of injection. Koch then resorted to mechanical trituration of the bacilli. The method he subsequently followed for tuberculin pro- duction is now extensively used, and is carried out as follows:46 Virulent cultures of tubercle bacilli are dried in vacuo and thor- oughly ground in a mortar. Grinding is continued until stained prep- arations reveal no intact bacilli. (This is done by machinery in all large manufactories.) One gram of the dry mass is shaken up in 100 c.c. of sterile distilled water. This mixture is then centrifugalized at high speed. The supernatant fluid, known as TO (Tuberculin-Oberschicht), contains the water-soluble constituents of the bacillus, gives no precip- 45 Koch, Deut. med. Woch., 14, 1897. "Ruppel, Lancet, March 28, 1908. 606 PATHOGENIC MICROORGANISMS itate on the addition of 50 per cent glycerin, and has the same physiolog- ical action as the old tuberculin. The residue TR (Tuberculin-Ruck- stand), after pouring off TO, is again dried and ground up, and again shaken in water and centrifugalized. This process is repeated several times, and eventually, after three or four repetitions, all the TR goes into emulsion. The total volume of water used for these TR extractions should not exceed 100 c.c. All of the TR emulsions are then mixed together. This gives TR a precipitate with 50 per cent of glycerin, and is supposed by Koch to contain substances important in producing an antibacterial immunity. For purposes of standardization, the amount of solid substance in 5 c.c. of the TR is determined by evaporation in vacuo and drying. To the rest are added a little glycerin and formalde- hyd and enough water to allow each cubic centimeter of the solution to contain 0.002 gram of solid material. Thus the culture and the medium remaining the same, fairly accurate standardization is possible. "New Tuberculin-bacillary Emulsion."47 — In 1901, Koch combined "TO" and "TR" by putting forth a preparation referred to as "Bazil- lenemulsion." This consists of an emulsion of pulverized bacilli 1 : 100 in distilled water. After several days of sedimentation to remove the coarser particles, the supernatant fluid is poured off and fifty per cent volume of glycerin is added to it for purposes of preserva- tion. This preparation contains 5 milligrams of solid substance in each cubic centimeter. Bouillon F litre (Denys)48 — This preparation consists of the filtrate (through Chamberland filters) of 5 per cent glycerin-pepton-bouillon cultures of Bacillus tuberculosis. Phenol 0.25 per cent is added to insure sterility. The filtered bouillon corresponds to the unconcen- trated old tuberculin of Koch, but, not having been heated, is supposed by Denys to contain important soluble and possibly thermolabile secretory products of the bacillus. Tuber culoplasmin (Buchner and Hahn)49 — Buchner and Hahn, by crushing tubercle bacilli by subjecting them to a pressure of 400 atmos- pheres, obtained a cell-juice in the form of an amber fluid, to which they attributed qualities closely analogous to those of TR. Other tuberculins are those of Beraneck,50 highly recommended "7 Koch, Dent. mod. Woc.h., 1901. 48 Dei\ys, ' 'Le Bouillon Filtre, " Lonvain, 1905. 49 Buchner mid llahn, Miinch. med. Woch., 1897; Halm, ibid. 50 Beraneck, Compt. rend, de 1'acad. des. sci., 1903. THE TUBERCLE BACILLUS 607 clinically by Sahli,51 that of Klebs,5- and the tuberculin produced from bovine tubercle bacilli by Speiigleiv™ Diagnostic Use of Tuberculin. — Subculancous*Usc. — The prepara- tion usually employed for diagnostic purposes is Koch's "Old Tubercu- lin" ( Alttuberculin) . This preparation is administered by hypodermic injection of small quantities obtained by means of dilutions. The dilutions are best made with a 0.5 per cent aqueous carbolic acid solution. In practice a 1 per cent solution is made by pipetting 0.1 c.c. of tuber- culin into 9.9 c.c. of the 0.5 per cent carbolic solution. A cubic centi- meter of this then contains 0.01 c.c. of tuberculin. One cubic centi- meter of this solution added to 9 c.c. of 0.5 per cent carbolic acid gives a solution in which each cubic centimeter contains 0.001 c.c., or 1 mm. of tuberculin. The initial dosage in adults in Koch's 54 early work, and as used by Beck55 on a large number of patients, was 1 mgm. This, according to present opinions, is too high, and most clinicians to-day prefer 0.1 to 0.2 of a milligram. If after three or four days no reaction has occurred, a second dose of 1 mm. is given. The reaction itself is recognized chiefly by the changes in tem- perature. In a positive reaction the patient's temperature will begin to increase within six to eight hours after injection, rising sharply within a few hours to 0.5 or 1.5° higher than the temperature before injection. It then sinks more gradually than it rose, the reaction usually being complete within thirty to thirty-six hours. With the tempera- ture there maybe nausea, a chill, rapid pulse, and general malaise. Locally visible tuberculous processes, such as lupus, lymph nodes, etc., may become tender or swollen, and if the tuberculosis is pulmonary, there may be coughing and increased expectoration. The temper- atures of persons subjected to the test should be taken regularly for three or four days before tuberculin is used. Ophthalmo-tuberculin Reaction. — Wolff-Eisner 56 and, soon after him, Calmette,57 proposed a method of using tuberculin for diagnostic purposes by instillation into the conjunctival sac. In tuberculous 51 Sahli, Corrbl. d. Schw. Aerzte, 1906. tiKlebs, Cent. f. Bakt% 1896; Dent. med. Woch., 1907. 53 Spengler, Deut. med. Woch., xxxi, 1904 ; xxxi and xxxiv, 1905. M Koch, Deut. med. Woch., 1890. 55J5ecfr, Deut. med. Woch., 1899. 66 Wolff-Eisner, Berl. med. Gesell., May 15, 1907. 57 Calmette, Acad. des sci., June 17, 1907. 008 PATHOGENIC MICROORGANISMS patients this process is followed by a sharp conjunctival congestion lasting from one to several days. The preparation used for this purpose is produced in the following way: "Old Tuberculin" is treated with double its volume of 95 percent alcohol, the precipitate allowed to settle and the alcohol then filtered off through paper. The sediment is washed with 70 per cent alcohol until the filtrate runs clear, then pressed between layers of filter paper to remove excess of moisture, scraped into a dish, dried in vacuo over H^SCU, and broken up in a mortar under a hood. Solutions of the powder are made in sterile normal salt solution, 1 per cent by weight, boiled and filtered. The solutions are used in strengths of 0.5 to 1 per cent, a drop of which is instilled into the con- junctival sac.58 Cutaneous Tuberculin Reaction. — Von Pirquet 59 has suggested the cutaneous use of tuberculin for diagnostic purposes. A 25 per cent solution of "Old Tuberculin" was first used. At present the undi- luted substance is employed. After sterilization of the patient's forearm, two drops of this solu- tion are placed upon the skin about 6 cm. apart. Within each of these drops scarification is done, and the skin between them scarificed as a control. Within twenty-four to forty-eight hours, in tuberculous patients, erythema, small papules, and herpetiform vesicles will appear. According to recent investigations, about 70 per cent of adults show a positive reaction. This diminishes its diagnostic value for adults. Moro 60 has modified this by making a 50 per cent ointment of tuberculin in lanolin and rubbing it into the skin without scarification. Complement Fixation in Tuberculosis.61 — The problem of comple- ment fixation for diagnostic purposes in tuberculosis has been very actively investigated of recent years. The most promising results have been reported with an antigen made by Besredka of a filtrate of an egg-meat-broth, upon which the tubercle bacilli had been grown for several weeks with a similar filtrate of cultures on a watery extract of potato with glycerin, used by Petroff, and with an antigen made by Miller and Zinsser by triturating dead tubercle bacilli with dry crystals 58 The conjunctival test is not in general use at the present time owing to pos- sible dangers to the eyes. m-v. Pirquet, Berl. Win. Woch., xx, 1907; Med. Klinik, xl, 1907. 69 Moro, Munch, med. Woch., 1906, p. 216. 61 H. E. Miller, Jour. Lab. & Clin. Med., 1916, i, 816. THE TUBERCLE BACILLUS 609 of NaCl and adding distilled water to isotonicity. Craig, Bronfenbren- ner and the above-named writers have reported good results with these various antigens, and, although it is too early to say which will prove most useful, it is clear that complement fixation methods can aid in the diagnosis of active tuberculosis. We can, of course, judge con- cisely only of the method used in our laboratory, where Miller has followed carefully a considerable number of cases on which the method has been used. It would appear at present that about 70 per cent of the fixation results correspond with clinical findings. The Tuberculin Test as Applied to Cattle. — In cattle, the symptoms of tuberculosis are not easily detected by methods of physical diag- nosis until the disease has reached an advanced stage. In consequence, cows may be elements of danger without appearing in any way diseased. In consequence, routine examination of herds by the tuberculin test has become one of the necessary measures of sanitation. According to Mohler,62 an accurate diagnosis may be established in at least 97 per cent of the cases. It is natural that a good deal of objection to the test is encountered on the part of dairy farmers and cattle raisers, and it has been claimed that the cattle are injured by the test. There is, how- ever, no scientific basis for this belief, if the test is carried out carefully and intelligently. As a matter of fact, the systematic use of the test would eventually be distinctly advantageous to the owners of the cattle them- selves, since it has been shown that cows, even in the early stages of the disease, may expel tubercle bacilli, either during respiration or with the feces, and thus become a menace to healthy members of the herd. The tuberculin test on cattle should be made as follows: (The directions given below are taken directly from the circular sent out. from the Bureau of Animal Industry at Washington.) 1. Begin to take the rectal temperature at 6 A.M., and take it very two hours thereafter until midnight. 2. Make the injection at midnight. 3. Begin to take the temperature next morning at 6 A.M., and con- tinue as on preceding day. To those who have large herds to examine, or are unable to give the time required by the above directions, the following shortened course is recommended: 1. Begin to take the temperature at 8 A.M., and continue every 2 hours until 10 P.M, (omitting at 8 P.M., if more convenient) ; or take the temperature at 8 A.M., 12 M., and 10 P.M. « Mohler, Pub. H. and Mar. Hosp. Serv. Bull., 41, 1908. 610 PATHOGENIC MICROORGANISMS 2. Make the injection at 10 P.M. 3. Take the temperature next morning at 6 or 8 A.M., and every two hours thereafter until 6 or 8 P.M. Each adult animal should receive 2 c.c. of the tuberculin as it is sent from the laboratory. (The tuberculin sent out from the central labora- tory at Washington is already diluted; 2. c.c. represents 0.25 c.c. of the concentrated " Old Tuberculin " of Koch.) Yearlings and two-year-olds, according to size, should receive from 1 to 1.5 c.c. Bulls and very large animals may receive three c.c. The injection should be made beneath the skin of the neck or shoulders behind the scapula, after washing the area with a weak carbolic acid solution. There is usually no marked local swelling at the seat of the injection. There are now and then uneasiness, trembling, and frequent passage of softened dung. There may also be slight acceleration of the pulse and of breathing. The febrile reaction in tuberculous cattle following the subcutaneous injection of tuberculin begins from six to ten hours after the injection, reaches the maximum nine to fifteen hours after the injection, and returns to normal eighteen to twenty-six hours after the injection. A rise of two or more degrees Fahrenheit above the maximum tem- perature observed on the previous day should be regarded as an indica- tion of tuberculosis. For any rise less than this a repetition of the injec- tion after four or six weeks is highly desirable. It is hardly necessary to suggest that for the convenience of the one making the test the animals should not be turned out, but fed and watered in the stable. It is desirable to make note of the time of feed- ing and watering and of any temperature fall after watering. The tuberculin should, not be used later than six weeks after the date on the bottle, nor if there is a decided clouding of the solution. Therapeutic Uses of Tuberculin.— Tuberculin was first used therapeutically, by Koch,63 shortly after its discovery. Hailed with the most optimistic enthusiasm, its possibilities were overestimated and hopeless cases were treated unskillfully, with unsuitable dosage. The consequence was that harm was done, the method was attacked by Virchow and others and the new therapy fell into almost complete neglect. At present, the use of tuberculin has again been revived, but with greater caution and with a thorough understanding of its limitations. The tendency has been toward smaller dosage and the limitation of the 63 Koch, Deut. med. Woch., iii, 1891. THE TUBERCLE BACILLUS 611 agent to early cases. No two institutions use tuberculin in exactly the same manner, and it is, therefore, impossible to do more than outline the general scheme of treatment. It must never be forgotten, however, that all forms of tuberculin treatment consist in an " active immuniza- tion " in which, for the time being, the toxemia of the patient is increased rather than neutralized. It is obvious, therefore, that only cases in which the process is not a very acute one, are at all suitable for treat- ment. The general principle of modern tuberculin therapy seems to lie in choosing doses so small that no marked general reaction shall follow. The preparations most frequently employed are Koch's "Alttuber- culin," his "TR," his "Neu Tuberkulin-Bazillen Emulsion/' and the Bouillon filtre of Denys. Initial doses of Alttuberculin range from 0.1 to 0.01 of a milligram. In case of complete absence of a reaction, the injection may be repeated, gradually increasing, about twice a week. The occurrence of a reaction should be the signal for a longer interval and a slower advance in the size of the dose. The initial dose of "TR" is, as advised by Koch,64 about 0.002 mgm. This usually causes no reaction. The dose is doubled, at reasonable intervals, up to 1 mgm. After this, further increase is carefully gauged by the clinical indications. The maximum dose is about 20 mgm. "Neu Tuberkulin-Bazillen Emulsion,"65 is begun with a dose of 0.001 mgm. Gradual increase as with the other preparations is then practiced. The maximum dose is about 10 mgm. Bouillon filtre has been used chiefly by Denys 66 who claims ex- cellent results. Denys is very emphatic in advising the absolute avoidance of any reaction. He begins with a millionth or even the tenth of a millionth of a cubic centimeter of the bouillon and increases with extreme caution. His dilutions are made with glycerin broth. Active immunization with tubercle bacilli of reduced virulence has been suggested and attempted at various times but, so far, without definite success. No strikingly favorable results can be justly claimed for any of these preparations. Passive Immunization in Tuberculosis. — Numerous attempts have been made to immunize tuberculous subjects with the sera of actively immune 64 Koch, Deut. med. Woch., xiv, 1897. 83 Bandelier and Eoeplce, "Lehrb. d. specifisch, Tub. Ther,/7 Wurzburg, 1908; Koch, Deut. med. Woch., 1901. 88 Denys, ' ' Le Bouillon filtre, ' ' Louvain, 1905. 612 PATHOGENIC MICROORGANISMS animals. The most widely used method of producing such serum is that of Maragliano. Maragliano's Serum.Q7 — Maragliano believes that a toxalbumin is present in tubercle-bacillus cultures which is destroyed by the heating employed in the usual tuberculin production. He procures this substance by nitration of unheated cultures and precipitation with alcohol (tossina prageipitata). He furthermore makes an aqueous extract of the bacillary bodies. With these two substances he immunizes horses. He draws blood from these after four to six months of treatment. The serum is extensively used in Italy. Its value is, at present, very doubtful. Marmorek's Serum.68 — Marmorek claims that the poisons produced by Bacillus tuberculosis depend largely upon the medium on which it is grown. He advanced the view in 1903 that the substances obtained in tuberculin were not the true toxins of the tubercle bacillus, that there was a marked difference between these and the poisons elaborated by a younger (primitive) phase of the bacillus as it occurs only within the animal body or on media composed of animal tissue. He consequently grows his cultures on a medium composed of a leucotoxic serum (produced by inoculating calves with guinea- pig leucocytes) and liver tissue. Such cultures, he claims, contain no tuber- culin. To the sera produced by immunization with these cultures he attributes high curative powers. We may say with considerable confidence at the present time that no method of passive immunization in tuberculosis has, up to the pres- ent, had any degree of success. Bacilli Closely Related to the Tuberculin Bacillus. — The Bacillus of Bovine Tuberculosis. — Tuberculosis of cattle (Perlsucht) was studied by Koch 69 in connection with his early work on human tuberculosis. Koch 'did not fail to recognize differences between the reactions to infection in the bovine type of the disease and that of man. He attrib- uted these, however, to the nature of the infected subject rather than to any differences in the infecting agents. This point of view met with little authoritative contradiction, until Theobald Smith,70 in 1898, made a systematic comparative study of bacilli isolated from man and 6T Maragliano, Eerl. klin. Woch., 1899; Soc. de biol., 1897. 68 MarmoreJc, Berl. klin. Woch., 1903; p. 1108; Med. Klinik, 1906. 69 Koch, Arb. a. d. kais. Gesimdheitsamt, 11, 1882. 70 Th. Smith, Jour. Exp. Med., Ill, 1898. THE TUBERCLE BACILLUS 613 from cattle and pointed out differences between the two types. The opinion of Smith was fully accepted by Koch71 in 1901. Since that time, the question, because of its great importance to prophylaxis, has been the subject of many investigations, most of them confirming Smith's original work. Morphologically, Smith 72 found that the bovine bacilli were usually shorter than those of the human type and grew less luxuriantly than these upon artificial media. He deter- mined, furthermore, that, grown upon slightly acid glycerin bouillon, the bovine bacillus gradually reduces the acidity of the culture medium until the reaction reaches neutrality or even slight alkalinity. Fluc- tuations after this do not exceed 0.1 to 0.2 per cent on either side of neutrality. In the case of the human bacillus, on the other hand, there is but slight reduction of the acidity during the first weeks of growth; after this acidity increases and, though subject to fluctuations, never reaches neutrality. This behavior is probably due to action exerted upon the glycerin, since on ordinary bouillon no such differences between the two varieties can be noticed. These observations of Smith were confirmed by Ravenel,73 Vagedes,74 and others. The cultural differences between the two types have been studied with especial care by Wolbach and Ernst,75 and Kossel, Weber, and Heuss.76 All of these observers bear out Smith's contention that luxuriance and speed of growth are much more marked in the human than in the bovine variety. Marked differences, furthermore, have been shown to exist in the pathogenic properties of these bacilli toward various animal species. Guinea-pigs inoculated with the bovine type77 die more quickly and show more extensive lesions than those infected with human bacilli. The difference in the pathogenicity of the two organisms for rabbits is sufficiently striking to be of diagnostic value. The bovine bacilli usually kill a rabbit within two to five weeks; the human bacilli produce a mild and slow disease, lasting often for six months, and occasionally fail to kill the rabbits at all. The practical importance of distinguishing between the two types, iiKoch, Dent. med. Woch., 1901. 72T/i. Smith, Jour. Exp. Med., 1905. 73 Ravenel, Lancet, 1901; Univ. Penn. Med. Bull., 192. ™Vagedes, Zeit. f. Hyg., 1898. 75 Wolbach and Ernst, ' ' Studies from the Eockef eller Inst., ' ' 11, 1904. 76 Kossel, Weber, und Heuss, Arb. a. d. kais. Gesundheitsamt, 1904 and 1905. n Smith, loc. cit., and Medical News, 1902. 614 PATHOGENIC MICROORGANISMS of course, attaches to the question as to whether the bovine and the human disease are mutually intercommunicable. This has been dis- cussed in the preceding section dealing with the human type. Summary of the Differentiation between Bovine and Human Tubercle Bacilli. — Morphologically the bovine bacillus is a little plumper and thicker than the human type, but this cannot be regarded as suf- ficiently constant to be reliable for differentiation. On glycerin broth, the final reaction in the case of human bacilli is considerably acid, the final reaction with the bovine is very slightly above the neutral point. The bovine bacillus does not grow as readily as the human and is not aided by the addition of glycerin to the media to the same extent as the human. The growth of the human bacillus is apt to be more luxuriant than that of the bovine, especially in earlier generations. As to virulence, the bovine is much more virulent for all the ordinary laboratory animals than is the human. The difference is particularly marked in rabbits. Small doses of human bacilli inoculated into rab- bits will kill them very late, and if quantities of less than 0.1 of a milli- gram are used intravenously, the rabbits may live for longer than two months, or may survive. Similar injection of the bovine type into rabbits kills with greater regularity and more extensive lesions, usually within two months. The Bacillus rf Avian Tuberculosis. — A disease resembling in many features the tuberculosis of man is not uncommon among chickens, pigeons, and some other birds. Koch was the first to discover in the lesions of diseased fowl, bacilli much resembling Bacillus tuberculosis. It was soon shown, however, by the studies of Nocard and Roux,78 Mafucci,79 and others, that the bacillus of the avian disease represented a definitely differentiate species. Morphologically, and in staining characteristics, the bacillus is almost identical with that of the human disease. In culture, however, growth is more rapid and takes place at a temperature of 41° to 45° C.80 (the normal temperature of birds), while the human type is unable to thrive at a temperature above 40°. The organisms grow more easily than do either the human or bovine bacilli. Colonies appear, on glycerin agar within a week and cultiva- tion may also be successful on media without glycerin. It is char- ts Nocard et Roux, Ann. 2 and hydrogen. The bacillus grows well on media containing urine and on those containing bile. Upon the latter fact methods for the isolation of the colon bacillus from water and feces have been based. Isolation of the colon bacillus from mixed cultures is most easily accomplished by plating upon lactose-litmus-agar, the Conradi-Drigal- ski medium, or the Endo medium after preliminary enrichment if necessary in bile or malachite-green broth. (In the case of feces such enrichment is superfluous.) Distribution. — The colon bacillus is a constant inhabitant of the intestinal canal of human beings and animals. It is also found occasion- ally in soil, in air, in water, and in milk and is practically ubiquitous in all neighborhoods which are thickly inhabited. When found in nature its presence is generally taken to be an indication of contamination from human or animal sources. Thus, when found in water or milk, much hygienic importance is attached to it. Recently, Papasotiriu3 and independently of him, Prescott,4 have reported finding bacilli apparently identical with Bacillus coli upon rye, barley, and other grains. They believe, upon the basis of this discovery, that Bacillus coli is widely distributed in nature and that its presence, unless it appears in large numbers, does not necessarily indicate recent fecal contamination. These reports, however, have not found confirmation by the work of others. In man, Bacillus coli appears in the intestine normally soon after birth, at about the time of taking the first nourishment.5 From this time on, throughout life, the bacillus is a constant intestinal inhabitant apparently without dependence upon the diet. Its distribution within the intestine, according to Gushing and Livingood,6 is not uniform, it being found in the greatest numbers at or about the ileocecal valve, diminishing from this point upward to the duodenum and downward as far as the rectum. Adami7 and others claim that, under normal con- ditions, the bacillus may invade the portal circulation, possibly by the 3 Papasotiriu, Arch. f. Hyg., xli. 4 Prescott, Cent. f. Bakt., Eef., xxxiii, 1903. * Schild, Zeit. f. Hyg., xix, 1895; Lemblce, Arch. f. Hyg., xxiv, 1896. *Cushing and Livingood, "Contributions to Med. Sci. by Pupils of Wm. Welch," Johns Hopk. Press, 1900. 7 Adami, Jour, of Amer. Med. Assn., Dec., 1899. 632 PATHOGENIC MICROORGANISMS intermediation of leucocytic emigration during digestion. After death, at autopsy, Bacillus coli is often found in the tissues and the blood without there being visible lesions of the intestinal mucous membrane.8 It is probable, also, that it may enter and live in the circulation a few hours before death during the agonal stages. The distribution of the colon bacilli in the human intestine at dif- ferent periods of life, and under varying dietetic conditions has been considered in the section on the " normal flora of the intestinal canal," p. 223. Extensive investigations have been carried out to determine whether or not the constant presence of this microorganism in the intestinal tract is an indication of its possessing a definite physiological function of advantage to its host. It has been argued that it may aid in the fer- mentation of carbohydrates. The question has been approached experi- mentally by a number of investigators. Nuttall and Thierfelder 9 delivered guinea-pigs from the mother by Cesarean section and suc- ceeded in preserving them from infection of the intestinal canal for thirteen days. Although no microorganisms of any kind were found in the feces of these animals, no harm seemed to accrue to them, and some of them even gained in weight. Schottelius,10 on the other hand, obtained contradictory results with chicks. Allowing eggs to hatch in an especially constructed glass compartment, he succeeded in keeping the chicks and their entire environment sterile for seventeen days. During this time they lost weight, did not thrive, and some of them were moribund at the end of the second week, in marked contrast to the healthy, well-nourished controls, fed in the same way, but under ordinary environmental conditions. Although insuffi- cient work has been done upon this important question, and no definite statement can be made, it is more than likely that the function of the Bacillus coli in the intestine is not inconsiderable if only because of its possible antagonism to certain putrefactive bacteria, a fact which has been demonstrated in interesting studies by Bienstock n and others.12 Pathogenicity. — The pathogenicity of the colon bacillus for animals is slight and varies greatly with different strains. Intraperi- toneal injections of 1 c.c. or more of a broth culture will often cause death in guinea-pigs. Intravenously administered to rabbits it may 8 Birch-Hirschfeld, Ziegler 's Beitr., 24, 1898. 9 Nuttall und Thierf 'elder, Zeit. f . Physiol. Chemie, xxi and xxii. 10 Scltottelvus, Arch. f. Hyg., xxiv, 1889. 11 Bienstock, Arch. f. Hyg., xxix, 1901. 12 Tisser and Martelly, Ann. de Pinst. Pasteur, 1902. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 633 frequently cause a rapid sinking of the temperature and death with symptoms of violent intoxication within twenty-four to forty-eight hours. Subcutaneous inoculation of moderate doses usually results in nothing more than a localized abscess from which the animals recover. In man, a large variety of lesions produced by Bacillus coli have been described. It is a surprising fact that disease should be caused at all, in man, by a microorganism which is so constantly present in large numbers in the intestine and against which, therefore, it is to be expected that a certain amount of immunity should be developed. A number of explanations for this state of affairs have been advanced, none of them entirely satisfactory. It is probable that none of the poi- sonous products of the colon bacillus are absorbed unchanged by the healthy unbroken mucosa and that, therefore, the microorganisms are> strictly speaking, at all times, outside of the body proper. Under these circumstances, no considerable process of immunization would be antici- pated. It is also possible that, whenever an infection with Bacillus coli does occur,the infecting organism is one which has been recently acquired from another host, having no specific adaptation to the infected body. Virulence may possibly be enhanced by inflammatoryfprocesses caused by other organisms. Considering the subject from another point of view, colon-bacillus infection may possibly take place simply because of unusual temporary reduction of the resistance of the host. Whether or not altered cultural conditions in the intestine may lead to marked enhancement in the virulence of the colon bacilli cannot at present be decided. The opinion has been frequently advanced, however, without adequate experimental support. Septicemia, due to the colon bacillus, has been described by a large number of observers. It is doubtful, however, whether many of these cases represent an actual primary invasion of the circulation by the bacilli, or whether their entrance was not simply a secondary phe- nomenon occurring during the agonal stages of another condition. A few unquestionable cases, however, have been reported, and there can be no doubt about the occurrence of the condition, although it is prob- ably less frequent than formerly supposed. The writer has observed it on two occasions in cases during the lethal stages of severe systemic disease due to other causes. An extremely interesting group of such cases are those occurring in new-born infants, in which generalized colon-bacillus infection may lead to a fatal condition known as WinckeFs disease or hemorrhagic septicemia.13 Prominent among disease processes 13 Kamen, Ziegler '9 Beitr., U, 1896, 634 PATHOGENIC MICROORGANISMS attributed to these microorganisms are various diarrheal conditions, such as cholera nostras and cholera infantum. The relation of these maladies to the colon bacillus has been particularly studied by Escherich,14 but satisfactory evidence that these bacilli may specifically cause such conditions has not been brought. While it is not unlikely that under conditions of an excessive carbohydrate diet, colon bacilli, may aggravate morbid processes by a voluminous formation of gas, they do not, of themselves, take part in actual putrefactive processes. It is likely, therefore, that in most of the intestinal diseases formerly attributed purely to bacilli of the colon group, these microorganisms actually play but a secondary part.15 It is equally difficult to decide whether or not these bacilli may be regarded as the primary cause of peritonitis following perforation of the gut. Although regularly found in such conditions, they are hardly ever found in pure culture, being accompanied usually by staphylococci, streptococci, or other microorganisms, whose relationship to disease is far more definitely established. Isolated cases have been reported, however, one of them by Welch, in which Bacillus coli was present in the peritoneum in pure culture without there having been any intestinal perforation.16 Granting that the bacillus is able to proliferate within the peritoneum, there is no reason for doubting its ability of giving rise to a mild suppurative process. Inflammatory conditions in the liver and gall-bladder have fre- quently been attributed to the colon bacillus. It has been isolated from liver abscesses, from the bile, and from the centers of gall-stones. Welch has reported a case of acute hemorrhagic pancreatitis in which the bacillus was isolated from the gall-bladder and from the pancreas. In the bladder, Bacillus coli frequently gives rise to cystitis and occasionally to ascending pyonephrosis. No other microorganism, in fact, is found so frequently in the urine as this one. It may be present, often, in individuals in whom all morbid processes are absent. The condition is frequently observed during the convalescence from typhoid fever. It may disappear spontaneously, or cystitis, usually of a mild, chronic variety, may supervene. Localized suppurations due to this bacillus may take place in all parts of the body. They are most frequently localized about the anus and the genitals, and are usually mild and amenable to the simplest surgical treatment. "Escherich, loe. cit. 15 Herter, "Bact. Infec. of Digest. Tract," N. Y., 1907. 16 Welch, Med. News, 59, 1891. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 635 For a consideration of the distribution of colon bacilli in the intestines of human beings at various ages and under modified dietary conditions the reader is referred to the section on the normal flora of the intestinal canal. Poisonous Products of the Colon Bacillus. — The colon bacillus belongs essentially to that group of bacteria whose toxic action is sup- posed to be due to the poisonous substances contained within the bacillary body. Culture filtrates of the colon bacillus show very little toxicity when injected into animals; whereas the injection of dead bacilli produces symptoms almost equal in severity to these induced by injection of the live microorganisms. Corroborative of the assumption of this endotoxic nature of the colon-bacillus poison is the fact that, so far, no antitoxic bodies have been demonstrated in serum as resulting from immunization. Dead colon bacilli have a very high toxicity for rabbits and some what less for guinea pigs. Immunization with the Colon Bacillus. — The injection into animals of gradually increasing doses of living or dead colon bacilli gives rise to specific bacteriolytic, agglutinating, and precipitating substances. The bacteriolytic substances may be easily demonstrated by the technique of the Pfeiffer reaction. In vitro bacteriolysis is less marked than in the case of some other microorganisms such as the cholera spiril- lum or the typhoid bacillus. Owing probably to the habitual presence of colon bacilli in the intestinal tracts of animals and man, considerable bacteriolysis may occasionally be demonstrated in the serum of normal individuals. Agglutinins for the colon bacillus have often been produced in the sera of immunized animals in concentration sufficient to be active in dilutions of 1 : 5000 and over. The agglutinins are produced equally well by the injection of live cultures and of those killed by heat, if the temperature used for sterilization does not exceed 100° C. It is 17 a noticeable fact that the injection of any specific race of colon bacilli produces, in the immunized animal, high agglutination values only for the individual culture used for immunization, while other strains of colon bacilli, although agglutinated by the serum in higher dilution than are paratyphoid or typhoid bacilli, require much higher concen- tration than does the original strain. The subject has been extensively studied by a number of observers and illustrates the extreme individual specificity of the agglutination reaction. Thus a serum which will " Wolff, Cent. f. Bakt., xxv, 1899. 636 PATHOGENIC MICROORGANISMS agglutinate its homologous strains in dilutions of one to 1000 will often fail to agglutinate other races of Bacillus coli in dilutions of 1 : 500 or 1 : 600. The normal serum of adult animals and man will often agglutinate this bacillus in dilutions as high as 1 : 10 or 1 : 20 — a phenomenon pos- sibly referable to its habitual presence within the body. Corroborating this assumption is the observation of Kraus and Low,18 that the serurn of new-born animals possesses no such agglutinating powers. The fact that agglutinins for the colon bacillus are increased in the serum of patients convalescing from typhoid fever or dysentery is probably to be explained, partly by the increase of the group agglutinins pro- duced by the specific infecting agent, and partly by the invasion of colon bacilli, or the absorption of its products induced by the diseased state of the intestinal mucous membrane. Varieties of the Colon Bacillus. — During the earlier days of bac- teriological investigations, a large number of distinct varieties of colon bacilli were described, many of which may now be dismissed as based simply upon a temporary depression of one or another cultural charac- teristic of Bacillus coli communis, while others can be definitely included within other closely related, but distinct groups. That secondary features, such as dimensions, motility, and luxuri- ance of growth upon various media, may be markedly altered by arti- ficial cultivation is a common observation. It has not, however, been satisfactorily shown that cardinal characteristics, such as the forma- tion of indol from pepton, or the power to produce gas from dextrose and lactose, can be permanently suppressed without actual injury or inhibition of the normal vitality of the microorganism. Such alter- ation is, in fact, contrary to experience, which demonstrates that whenever such changes do occur, they are purely temporary and a few generations of cultivation under favorable environmental conditions will regularly restore the organism to its normal activity. Distinct and constant varieties of the Colon Bacillus or, at least, close biological relatives do occur. It is necessary to consider the organ^ isms as a group for this reason, since, in sanitary work, it is of the utmost importance to recognize forms which should properly be classified under this category. It may be well, therefore, to reiterate the criteria for identification of the group established by the American Public Health Association Committee on Standard Methods of Water Analysis.19 This report defines the general characteristics of the group, as follows: 15 Kraus und Low, Wien. klin. Woch., 1899. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 637 short bacillus form, failure of spore formation, facultative anaerobiosis, growth on gelatin without liquefaction in fourteen days, Gram-negative stain and fermentation of dextrose and lactose with gas formation. They add that there is a positive reaction with esculin. The organisms which are placed under the Colon group in this report are the B. Coli communis described above, the B. Coli communior of Durham, the B. aerogenes of Escherich, and B. acidi-lactici of Htieppe. The chief differential characteristics are as follows : Dextrose. Lactose. Saccharose. Dulcit. B coli communis + + + B coli communior 4- , , , B. lact. aerogenes B. lact. acidi t t - Individual descriptions of these organisms follow: B. COLI COMMUNIOR. — This organism first described by Durham was called Communior by him because of his belief that it was more abundant in the human and animal intestine than the Communis type. It possesses all the characteristics of the Colon group. It is a Gram- negative bacillus, motile, non-sporulating, and morphologically indis- tinguishable from the Communis variety. It does not liquefy gelatin, it produces indol from pepton, coagulates and acidifies milk, and grows characteristically upon agar and potato. It differs from B. coli com- munis in that it produces acid and gas from saccharose as well as from dextrose and lactose, whereas the former does not form acid or gas from saccharose. Several varieties have been described by Melia, and by A very. The Melia type differs from the ordinary variety in not pro- ducing indol. The Avery type did not coagulate milk. B. LACTIS AEROGENES. — Bacillus lactis aerogenes is the type of a group which is closely similar to the colon group and often distin- guished from it with difficulty. It was first described in 1885 by Escherich 19 who isolated it from the feces of infants. Since then it has been learned that this bacillus is almost constantly present in milk, and, together with one or two other microorganisms, is the chief cause of the ordinary souring of milk. Apart from its occurrence in milk, moreover, the bacillus is widely distributed in nature, being found in feces, in water, and in sewage. 19 A. P. H. A. Standard Methods of Water Analysis, 1915. 638 PATHOGENIC MICROORGANISMS It is distinguished from the Colon bacillus chiefly by the fact that it is less motile, hardly ever forms chains, and, when cultivated upon suitable media, especially milk, it possesses a distinct capsule. It differs from other forms of the Colon group in not fermenting dulcite, and differs from B. acidi-lactici in fermenting saccharose. It ferments with gas production, dextrose, lactose, saccharose, mannite and raffinose. It produces indol, reduces nitrate, possesses either no motility, or is very slightly motile. It coagulates milk, and when grown on milk or lactose bile it often makes a stringy viscous cult- ure, On agar and gelatin it makes heavy white colonies of a some- 1 2 3 FIG. 67. — BACILLUS COLI COMMUNIOR. Grown in: 1. Dextrose, 2. Lactose, 3, Saccharose broth. what mucoid appearance, certainly more mucoid than most colon colonies. It does not liquefy gelatin. In broth it causes general cloud- ing with later a pellicle, and a sour odor. It grows heavily on potato. It is a facultative anaerobe and grows at room temperature. Varieties have been described depending upon minor cultural char- acteristics which have no particular importance in this connection. The pathogenicity of Bacillus lactis aerogenes for man is slight. Its chief claims to importance lie in its milk-coagulating properties and its almost constant presence in the human intestine. In infants, it may give rise to flatulence and it has been occasionally observed as the sole incitant of cystitis. Among such cases rare instances have been BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 639 observed in which it has formed gas in the bladder (pneumaturia). When this occurs the urine is not ammoniacal but remains acid. Different strains of this bacillus vary much in their pathogenicity for animals. Wilde claims that it is more pathogenic for white mice and guinea-pigs than is the bacillus of Friedlander. He speaks of it as the most virulent member of this group. Kraus, writing in Fluegge's "Mikroorganismen," rates its pathogenicity less high. Closely related to this bacillus, as well as those of the Friedlander group, is an encapsulated bacillus isolated from a case of broncho- pneumonia by Mallory and Wright,20 which is strongly pathogenic for mice, guinea-pigs, and rabbits. B. ACIDI-LACTICI. — This organism, like the others, is a Gram- negative, non-liquefying, non-sporulating bacillus. Just like the B. aerogenes, it has no motility. It differs from the Colon bacilli proper in not fermenting dulcite. It differs from the Lactis aerogenes in failing to ferment saccharose. Like the Lactis aerogenes, it is non- motile. It forms indol, reduces nitrate, and coagulates milk. It is commonly present in milk and may be present in water. It is often found in the intestinal canal but as far as we know has no patho- genic significance. BACILLUS FECALIS ALKALIGENES. — In 1896 Petruschky21 described a bacillus which is a not infrequent inhabitant of the human intestine, being found chiefly in the lower part of the small intestine and in the large intestine. This organism, which he called Bacillus fecalis alkaligenes, is of little pathogenic importance, although Neufeld states that he has seen a case of severe gastroenteritis in which the watery defecations contained this bacillus in almost pure culture. As a rule, however, this organism cannot be regarded as pathogenic, and is important chiefly because of the ease with which it may be mistaken for Bacillus typhosus. Bacillus fecalis alkaligenes is an actively motile, Gram-negative bacillus, possessing, like the typhoid bacillus, numerous peritrichal flagella. On the ordinary culture media it grows like the typhoid bacillus. It does not coagulate milk. It produces no indol, and on sugar media in fermentation tubes produces no acid or gas. On potato, its growth, while somewhat heavier than that of the typhoid bacillus, is not sufficiently so to permit easy differentiation. It differs from Bacillus typhosus in that it produces no acid on any of the sugar media, and is therefore easily differentiated by cultivation upon Hiss serum- 20 Mallory and Wright, Zeit. f. Hyg., 20, 1895. 21 Petruschky, Cent. f. Bakt., I, ixix, 1896. 640 PATHOGENIC MICROORGANISMS water media or on pep ton waters containing sugars. On the Hiss semi-solid tube-medium Bacillus fecalis alkaligenes, while clouding the medium throughout, grows most heavily on the surface, where, eventually, it forms a pellicle, BACILLI OF THE PROTEUS GROUP There are a great many other organisms which are similar to the Colon Bacillus in general appearance and superficial morphological and cultural characteristics, and which are found frequently associated with it in feces, water and sewage. It will be important for this reason to speak of them briefly. The most important of these is the Proteus Group, which is sharply separable from the Colon and allied bacteria by its gelatin liquefaction. The bacilli of this group have little pathological interest, but are important because of the frequency with which they are encountered in routine bacteriological work. They may confuse the inexperienced because of a superficial similarity to bacilli of the colon-typhoid group. In form they may be short and plump or long and slender, staining easily with anilin dyes and decolorizing with Gram's method. They are actively motile and possess many flagella. Individuals stain irregularly, often showing unstained areas near the center. The so-called Bacillus proteus vulgaris described by Hauser 22 in 1885 is the type of the group. Bacilli of this group are widely distributed, being found in water, soil, air, and wherever putrefaction takes place. In fact, proteus is one of the true putrefactive bacteria possessing the power to cause the cleav- age of proteins into their simplest radicles. Bacillus proteus vulgaris grows best at temperatures at or about 25° C. and develops upon the simplest media. It is a facultative anaerobe and forms no spores. In broth, it produces rapid clouding with a pellicle and the formation of a mucoid sediment. In gelatin, the colonies are characteristically irregular, giving the name to this group. Gelatin is Rapidly Liquefied. — Liquefaction, however, is diminished or even inhibited under anaerobic conditions. On agar and other solid media, as well as upon gelatin before lique- faction has taken place, characteristic colonies are produced. From the central flat, grayish-white colony nucleus, numerous irregular streamers grow out over the surrounding media, giving the colony a stellate appearance. On potato, it forms a dirty, yellowish growth. 22 Hauser, "Ueber Faulniss-Bakt., ' ' Leipzig, 1885. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 041 In milk, there is coagulation and an acid reaction at first; later the casein is redissolved by proteolysis. Blood serum is often liquefied, but not by all races. A great many really dissimilar bacteria have been described under the name of Proteus. The type of the group is the so-called Proteus vulgaris (Hauser, 1885). Other organisms spoken of as proteus are the Proteus mirabilis, which differs in slower gelatin liquefaction from vulgaris, the Proteus Zenkeri, which does not liquefy gelatin, the Proteus septicus, and the Bacillus Zopfi, a Gram-positive organism. A good many of these were formerly classified as of Bacterium termo. Closely related is the slow liquefying organism known as Bacillus cloacce, com- mon in sewage. There is no group which so urgently requires study as this, since organisms belonging here are so often found in the human body and human excreta. In urine we have encountered a non-gelatin liquefying Gram-negative bacillus belonging to this group which has given us much trouble in identification. As far as we can establish any general characteristics for the group at all, we may say that they are Gram- negative, non-spore-bearing, motile bacilli, which on the surface of gelatin plates show colonies characterized by spreading streamers, most of which liquefy gelatin, a few of which, however, do not. All of them ferment dextrose and saccharose with gas, but few of them attack lactose. The pathogenic powers of proteus are slight. Large doses injected into animals may give rise to localized abscesses. In man proteus infections have been described in the bladder, in most cases, however, together with some other microorganism. The Urobacillus lique- faciens septicus described by Krogius was a variety of this group. Epidemics23 of meat poisoning have been attributed to the proteus family by some observers. Thus Wesenberg24 cultivated a proteus from25 putrid meat which had caused acute gastroenteritis in sixty- three individuals. Similar epidemics have been reported by Silber- schmidt,25 Pfuhl,26 and others. B. CLOACAE. — This organism was first described by Jordan and is one of the commonest of the sewage bacteria. It is closely related to the Proteus organisms, but is less motile than they. It coagulates milk, and liquefies gelatin, but its gelatin liquefaction is not as active as that X8chniteler, Cent. f. Bakt., viii, 1890. 24 Wesenberg, Zeit. f . Hyg., xxviii, 1898. 25 Silberschmidt, Zeit. f. Hyg., xxx, 1899. 29 Pfuhl, Zeit. f. Hyg., xxxv, 1900. 642 PATHOGENIC MICROORGANISMS of the Proteus group. It forms indol and produces acid and gas on dextrose and saccharose, but one of its chief characteristics is its slight action on lactose. Jordan states as one of its chief characteristics the relatively large proportion of C02 formed as compared with hydrogen, the ratio being in some cases as high as 5 to 1. Kendall, Day and Walker27 have observed the same thing. The same investigators state that after three days' growth, even sugar broths become alkalin, owing to protein decomposition. Recently certain stains of Proteus have become important because of their apparently specific agglutination in Typhus Serum (Weil- Felix Reaction). See chapter on Typhus. Were we following a purely biological order of presentation, we should now proceed to a description of the organisms belonging to the so-called Mucosus Capsulatus or Friedlander group. These bacilli are closely related to the Colon type, more particularly to the B. aero- genes variety, and have been regarded by some observers, notably Fitzgerald, as perhaps representing members of the Colon group which have acquired capsulation and virulence. In practice, however, these bacteria are rarely encountered under conditions where differentiation from Colon bacilli is necessary, and their heavy mucoid colonies and capsulated morphology renders their recognition relatively easy. It will be better, therefore, from the point of view of practical discussion, to proceed directly to the study of organisms of the typhoid and dysen- tery groups, since these are the ones which in medical and sanitary bacteriology, are associated in the human body, in water and sewage with members of the Colon group and which, therefore, present the most frequent differential problems. 27 Kendall, Day and Walker, Jour. Amer. Chem. Soc., 1913, 35. CHAPTER XXXII BACILLI OF THE COLON-TYPHOID-DYSENTtfRY GROUP (Continued) THE BACILLUS OF TYPHOID FEVER (Bacillus typhosus, Bacillus typhi abdominalis) TYPHOID FEVER, because of its wide distribution and almost con- stant presence in most communities, has from the earliest days been the subject of much etiological inquiry. A definite conception as to its infectiousness and transmission from case to case was formed as early as 1856 by Budd.1 But it was not until 1880 that Eberth 2 discovered in the spleen and mesenteric glands of typhoid-fever patients who had come to autopsy, a bacillus which we now know to be the cause of the disease. Final proof of such an etiological connection was then brought by Gaffky,3 who not only saw the bacteria referred to by Eberth, but succeeded in obtaining them in pure culture and studying their growth characteristics. Morphology and Staining1. — The typhoid bacillus is a short rod from 1-3. 5ju in length with a varying width of from .5 to .8/x. In appear- ance it has nothing absolutely distinctive which could serve to differen- tiate it from other bacilli of the typhoid-colon group, except that it has a general tendency to greater slenderness. Its ends are rounded without ever being club-shaped. Contrary to the descriptions of the earlier observers, typhoid bacilli do not form spores. They are actively motile and have twelve or more flagella peripherally arranged. The bacilli stain readily with the usual anilin dyes. Stained by Gram's method, they are decolorized. Cultivation. — Bacillus typhosus is easily cultivated upon the usual laboratory media. It is not delicately susceptible to reaction, but will grow well upon media moderately alkaline or acid. It is an aerobic and facultative anaerobic organism, when the proper nutriment is present. Upon agar plates growth appears within eighteen to twenty- lBudd, "Intestinal Fever," Lancet, 1856. 2 Eberth, Virch. Archiv., 81, 1880, and 83, 1881. ? Gaffky, Mitt. a. d. kais. Gesundheitsamt, 2, 1884. 643 644 PATHOGENIC MICROORGANISMS FIG. 68. — BACILLUS TYPHOSDS, from twenty-four-hour culture on agarl FIG. 69.— BACILLUS TYPHOSUS, showing flagella. (After Frankel and Pfeiffer. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 645 four hours as small grayish colonies at first transparent, later opaque. Upon agar slants growth takes place in a uniform layer. There is nothing characteristic about this growth to aid in differentiation. In broth, the typhoid bacillus grows rapidly, giving rise to an even clouding, rarely to a pellicle. Upon gelatin, the typhoid bacillus grows readily and does not liquefy the medium. In stabs, growth takes place along the entire extent of the stab and over the surface of the gelatin in a thin layer. In gelatin plates the growth may show some differences from that of other mem- bers of this group, and this medium was formerly much used for isolation of the bacillus from mixed cultures, Growth appears within twenty- FIG. 70. — SURFACE COLONY OF BACILLUS TYPHOSUS ON GELATIN. (After Heim.) four hours as small transparent, oval, round, or occasionally leaf-shaped colonies which are smaller, more delicate, and more transparent than contemporary colonies of the colon bacillus. They do not, however, show any reliable differential features from bacilli of the dysentery group. As the colonies grow older they grow heavier, more opaque, and lose much of their early differential value. On potato the growth of typhoid bacilli is distinctive, and this medium was recommended by Gaffky4 in his early researches for purposes of identification. On it typhoid bacilli, after twenty-four to forty-eight hours, produce a hardly visible growth, evident to the naked eye only 4 Gaffky, loc. cit. 646 PATHOGENIC MICROORGANISMS by a slight moist glistening, an appearance which is in marked contrast to the grayish-yellow or even brown and abundant growth of colon bacilli. If the potato medium is rendered neutral or alkaline, this distinction disappears, the typhoid bacillus growing more abundantly. In milk, typhoid bacilli do not produce coagulation. In litmus-milk, during the first twenty-four hours, the color is changed to a reddish or violet tinge by the formation of acid from the small quantities of mono- saccharid present. Later the color becomes deep blue owing to the formation of alkali. In Dunham's pepton solution no indol is produced. According to Peckham, however, continuous cultivation in rich pepton media may lead to eventual indol formation by typhoid bacilli. This fact has no bearing on the value of the indol test, as indol is never have formed under the usual cultural conditions. Tested for its power to form acid from sugars commonly used in differential tests, typhoid bacilli form add, but no gas, on the mono- saccharides, on mannit, maltose and dextrin, and neither add nor gas on lactose and saccharose. (See Table, p. 718.) In the Hiss tube medium (formerly employed extensively) the typhoid bacillus within eighteen to twenty-four hours produces an even clouding by virtue of its motility, but does not form gas. In contradis- tinction to this, dysentery bacilli grow only along the line of inocula- tion, while bacilli of the colon group move in irregular sky-rocket-like figures away from the stab, at the same time breaking up the medium by the formation of gas-bubbles. Some actively motile colon bacilli cloud the medium, but the ruptures caused by the gas are always evident. The differentiation of the typhoid bacillus in pure culture from similar microorganisms by means of its growth upon media has been the sub- ject of many investigations. It is neither practicable nor desirable to enumerate all the various media which have been devised and reported. The aim has been chiefly the differentiation of typhoid bacilli from the bacilli of the colon group, and most of the media have been devised with this end in view. (See section on Media.) Rothberger 5 devised a mixture of glucose agar to which is added 1 per cent of a saturated aqueous solution of neutral-red. Shake- cultures or stab-cultures are made in tubes of this medium. The typhoid bacillus causes no changes in it, while members of the colon group, by 5 Rothberger, Cent, f . Bakt., xxiv, 1898. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 647 reduction of the neutral-red, decolorize the medium and produce gas by fermentation of the sugar. Utilizing the fact that bile-salts are precipitated in the presence of acids, MacConkey devised a medium composed of sodium glycocholate, pepton, lactose, and agar (the composition of this medium is given on page 164), in which Bacillus typhosus grows without causing such change, but distinct clouding results from the growth of the colon bacillus which, producing acid from the lactose, causes precipitation of the bile-salts. On Wurtz's lactose-litmus-agar (see page 153) the typhoid bacillus produces no acid, but eventually deepens the purple color to blue; the colon bacillus produces acid and in stab-cultures gas bubbles and the color changes to red. In Barsiekow's (see page 164) lactose-nutrose-litmus mixture the typhoid bacillus causes no change, while the colon bacillus produces coagulation and an acid reaction. The differentiation of the typhoid bacillus from other similar organ- isms of the typhoid, dysentery, colon group, is based chiefly on growth, upon differential media in which the inability of the typhoid bacillus to form acid or gas from lactose has been the most commonly used basis for differentiation. Various indicators to show whether acid has been formed, added to such media will sharply separate this organism from the colon bacilli and their close relatives. Failure to produce gas with dextrose, differentiates it from the paratyphoid group. The reader is referred to the differential tables given on page 687 and 718, for the basic reactions upon which cultural differentiation is made. The media most convenient for this purpose are, in plates, the Conradi- Drigalski medium, the Endo medium, the Krumwiede brilliant green medium, or the Teague eosin-methylene-blue medium, all of which are described in the section on media; and, in tubes, some of the most convenient media are the Hiss semi-solid mentioned above, Barsiekow's medium, or the Russell double sugar agar. The Russell double sugar agar is particularly useful to give a quick index of differentiation, since it contains both lactose and glucose, and, whereas, the colon group give redness throughout, and a few gas bubbles, the typhoid gives no gas, a red butt due to its action in the depths of the stab on the glucose and an uncolored surface growth. Final differentiation is best based upon specific agglutination. Winsloir, Klu/lcr and Hothenberg, Jour, of Bacter., 4, 1919, 426. 648 PATHOGENIC MICROORGANISMS Winslow, Kligler and Rothberg,6 on the basis of recent careful invests gations, describe the typhoid bacillus as a Gram-negative, non-spore forming, actively motile rod which forms translucent irregular colonies on gelatin, and a colorless growth on potato. It produced strong and prompt acid, but no gas, on media containing the hexoses, maltose, mannit, sorbit, xylose (rapid or slow), and dextrin; it does not attack arabinose, rhamnose, or lactose; produces a slight initial reddening of litmus milk, which, after two weeks, reverts to neutrality or slight alka- linity. It does not form indol, nor liquefy gelatin, does not grow in asparagin-mannitol medium, does not reduce neutral red, and causes browning of lead acetate medium (irregular) . It has low tolerance for acid, but high tolerance for malachite and brilliant green dyes. It has characteristic serum agglutination. Differences Within the Typhoid Group. — Recent work has shown that not all typhoid bacilli are culturally alike, there being two distinct groups, one which ferments xylose rapidly, the other slowly. Since there are also antigenic differences it may be necessary in the future to speak rather of a typhoid group than of the typhoid bacillus. Xylose fermentations of typhoid bacilli have recently been studied in more detail by Krumwiede, Kohn and Valentine,7 and by Morishima.8 The first-named authors inoculated 37 strains of typhoid bacilli into xylose broth and found that 29 of them produced acid within twenty- four hours, while 8 of the strains required from five to thirteen days for this result. Morishima, of this "laboratory, obtained rapid and slow xylose fermenters from a single strain by repeatedly fishing different colonies on plates. An atypical strain has recently been described by Bull and Pritchett9 whose bacillus agglutinated in typhoid serum typ- ically up to 1 to 20,000, but which gave positive indol reactions. Biological Considerations. — The typhoid bacillus is an aerobic and facultatively anaerobic organism growing well both in the presence and in the absence of oxygen when certain sugars are present, showing a slight preference, however, for well aerated conditions. It grows most luxuriantly at temperatures about 37.5° C., but continues to grow within a range of temperature lying between 15° and 41° C. Its thermal death point, according to Sternberg, is 56° C. in ten minutes. It remains alive in artificial cultures for several months or even years if moisture is sup- plied. In carefully sealed agar tubes Hiss found the organisms alive after thirteen years. In natural waters it may remain alive as long 7 Krumwiede, Kohn and Valentine, Jour, of Med. Res., 38, 1918, 89. 8 Morishima, Jour, of Bacter., March, 1921. 9 Bull and Pritchett, Jour, of Exper. Med., 24, 1916, 55. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP G49 as thirty-six days, according to Klein.10 In ice, according toJPruddeia,11 it may remain alive for three months or over. Against the ordinary disinfectants, the typhoid bacillus is comparatively more resistant than some other vegetative forms. It is killed, however, by 1 : 500 bichlorid or 5 per cent carbolic acid within five minutes. Pathogenicity. — In animals, some early investigators to the con- trary, typhoidal infection does not occur spontaneously and artificial inoculation with the typhoid bacillus does not produce a disease anal- ogous to typhoid fever in the human being. Frankel 12 was able to produce intestinal lesions in guinea-pigs by injection of the bacilli into the duodenum, and recovered the bacteria from the spleens of the animals after death, but the disease produced was in no other respect analogous to typhoid fever in the human being. It is probable that typhoid bacilli injected into animals do not multiply extensively and that most of the symptoms produced are due to the poisons liberated from the dead bacteria. In corroboration of this view is the observation that inocu- lation with dead cultures is followed by essentially the same train of symptoms as inoculation with live cultures.13 The injection of large doses into rabbits or guinea-pigs intravenously or intraperitoneally is usually followed by a rapid drop in temperature, often by respiratory embarassment and diarrhea. Occasionally blood may be present in the stools. According to the size of the dose or the weight of the animal, death may ensue within a few hours, or, with progressive emaciation, after a number of days, or the animal may gradually recover. Welch and Blachstein 14 have shown that typhoid bacilli injected into the ear vein of a rabbit appear in the bile and may persist in the gall-bladder for weeks. Doerr,15 Koch,16 Morgan,17 and more recently Johnston 18 have all confirmed this, the last named showing that the typhoid bacillus could not only remain latent for a long time in the gall-bladder of rabbits, but would appear in the blood stream with considerable regularity after the seventh or ninth day, and persist in the gall-bladder for as long as one hundred and 10 Klein, Med. Officers' Report, Local Govern. Bd., London, 1894. 11 Prudden, Med. Rec., 1887. 12 Frankel, Cent, f . klin. Med., 10, 1886. 13 Petruschky, Zeit. f . Hyg., xii, 1892. 14 Welch and Blachstein, Bull. Johns Hop. Hosp., ii, 1891. 15 Doerr, Centralbl. f. Bakt., 1905. 16 Koch, Zeitschr. f. Hyg., 1909. 17 Morgan, Jour, of Hyg., 1911. 18 Johnston, Jour, of Med. Res., xxvii, 1912. • 650 PATHOGENIC MICROORGANISMS twenty-five days. Gay and Claypole19 have been able to produce the carrier state in rabbits with great regularity by growing the typhoid cultures used for inoculation upon agar containing 10 per cent defib- rinated rabbit's blood. Such cultures are not as readily agglutinated by immune serum as are those grown on plain agar, and it may well be that they have acquired a certain degree of resistance to the serum antibodies which renders them more competent to survive in the body of the rabbit. Gay has used rabbits inoculated with such cultures for the determination of the efficacy of his sensitized vaccines. Typical typhoid fever simulating the disease in man has not been produced in any animals except in chimpanzees, by Metchnikoff and Besredka,20 who produced it in connection with their experiments on protective vaccination. They produced a disease almost identical with human typhoid by feeding cultures to chimpanzees. TYPHOID FEVER IN MAN. — It is not within the province of a book of this kind to give an accurate clinical description of the disease as it occurs in man in all its details. The disease is one in which a wide range of variation may occur, and in which complications are various and manifold. We will, therefore, give only a brief account of the infection as it is relevant to bacteriological work. The organisms enter by mouth, with food, water or contact with fingers, direct or indirect, as described in the epidemiological section. Subsequently, the organ- isms, which pass through the stomach uninjured, multiply in the intes- tine, but cause no symptoms for anywhere from seven to fourteen days. During this time they probably begin to proliferate partly within the mucous membrane of the bowel, although there is little definite knowledge concerning this. The symptoms of the disease begin insidiously by gradual malaise, headache, loss of appetite, sleep- lessness, and during the first week of the actual signs of infection, the organisms have probably penetrated or are penetrating into the lym- phatics. At this time there is a swelling of the lymphoid nodules of the intestine and Peyer's patches, and there is a moderate catarrhal inflam- mation of the mucous membrane. At this time too the bacilli enter the blood stream and can be found in blood culture. Though formerly regarded as primarily an intestinal disease, the disease is in truth at this time a bacteriemia, and it is not impossible that the intestinal lesions are as much due to the action of toxic products which are excreted in part through the intestinal wall, as they are due 19 Gay and Claypole, Arch, of Inf. Med., Dec., 1913. '20 Metchnikoff and Besredka, Ann. de Flnst. Past., 1911, 25, 193. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 651 to the direct reaction caused in the intestine by local growth of the bacilli. Secondarily, the bacilli appear and can be cultivated from the spleen, the liver, and can be demonstrated in the sinuses and tissues of the lymphatic and retroperitoneal lymph nodes. Typhoid Bacilli in the Blood during the Disease. — The investigations of many workers have shown that typhoid bacilli are present in the circulating blood of practically all patients during the early weeks of the disease. Series of cases have been studied by Castellani,21 Schottmul- ler22 and many others. More recently Coleman and Buxton23 have reported their researches upon 123 cases, and have at the same time analyzed all cases previously reported. Their analysis of blood cultures taken at different stages in the disease is as follows Of 224 cases during first week, 89 per cent were positive. Of 484 cases during second week, 73 per cent were positive. Of 268 cases during third week, 60 per cent were positive. Of 103 cases during fourth week, 38 per cent were positive. Of 58 cases after fourth week, 26 per cent were positive. The technique recommended by Coleman and Buxton for obtaining blood cultures is that recommended by Conradi,24 slightly modified. The blood is taken into flasks each containing about 20 c.c. of the following mixture : Ox-bile 900 c.c. Glycerin 100 c.c. Pepton 20 grams About 3 c.c. of blood are put into each flask. The ox-bile, besides pre- venting coagulation, may possibly neutralize the bactericidal sub- stances present in the drawn blood. The flasks are incubated for eigh- teen to twenty-four hours, at the end of which time streaks are made upon plates of lactose-litmus-agar and the organisms identified by agglutination or by cultural tests. European workers have generally preferred to make high dilution of the blood in flasks of bouillon, small quantities of blood, 1 to 2 c.c., being mixed with 100 to 150 c.c. of nutrient broth. Epstein 25 has reported excellent results from mixing the blood in 21 Castellani, Riforma medica, 1900. 22 Schottmuller, Deut. med. Woch., xxxii, 1900, and Zeit. f. Hyg., xxxvi, 1901. 2! Coleman and Kuxton., Am. Jour, of Med. Sci., 133, 1907. 24 Conradi, Deut. med. Woch., xxxii, 1906. 25 Epstein, Proc. N. Y. Path. Soc., N. S., vi, 1906. 652 PATHOGENIC MICROORGANISMS considerable concentration with 2-per-cent glucose agar and pouring plates. The writers in hospital work have had equally good results with the bile medium and with broth in flasks, rather less uniform but still satis- factory results with the plating method. In general it may be said that any one of these methods carried out with reasonable accuracy may be satisfactorily employed. Typhoid Bacilli in the Stools. — The examination of the stools for typhoid bacillus is performed for diagnostic purposes chiefly in obscure cases. It may, furthermore, furnish information of great hygienic importance. Thus Drigalski and Conradi 26 have succeeded in isolating typhoid bacilli from the stools of ambulant cases so mild that they were not clinically suspected. It is by means of such examinations that the so-called typhoid-carriers are detected, a problem which is considered at length in the section dealing with epidemiology. Such cases have been known to harbor the bacilli for periods as long as several years. The examination itself is fraught with difficulties, owing to the pre- ponderating numbers of colon bacilli found in all feces and the diffi- culty of isolating the typhoid bacilli from such mixtures. Reviewing the data collected by a number of investigators, it seems probable that the bacilli do not appear in the stools, at least in numbers sufficient for recognition, much before the middle of the second week, or, in other words, as pointed out by Hiss, about the time that the intestinal lesions are well advanced and ulceration is occurring. Thus Wiltschour27 could not determine their presence before the tenth day; Redtenbacher, 28 in reviewing the statistics, states that in a majority of cases the bacilli first appear toward the end of the second week, and Horton-Smith 29 could not find the bacilli before the eleventh day. Hiss,30 in an investigation of the same subject, obtained the following results: First to tenth day, inclusive, twenty-eight cases examined; typhoid bacilli isolated from three; percentage of positive cases 10.7 per cent. Eleventh to twentieth day, inclusive, forty-four cases examined; typhoid bacilli from twenty-two; percentage of positive cases 50 per cent. 26 Drigalski and Conradi, Zeit. f . Hyg., xxxix, 1902. 27 Wiltschour, Cent. f. Bakt., 1890. 28 Redtenbacher, Zeit. f . klin. Med., xix, 1891. 29 Horton-Smith, Lancet, May, 1899. 30 Hiss, Med. News, May, 1901. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 653 Twenty-first day to convalescence, sixteen cases examined; typhoid bacilli isolated from thirteen; percentage of positive cases 81.2 per cent. Stool Examination and Method of Typhoid Carrier Detection. — Fecal carriers of typhoid bacilli may be detected by cultural methods applied either to specimens of feces or to duodenal contents, obtained by a tube passed through the stomach into the duodenum. The simplest method, of course, is direct examination of the feces. The duodenal tube, however, will probably be used considerably in the future, since in some cases it may be positive when stool cultures are negative. As a matter of fact, in the hands of Garbat31 and Nichols,32 the duodenal method seems to have given more regular results than the stool method. Stool Examinations. — Stool material for typhoid examination should be fresh. Preserving stools for as long as twelve hours will diminish positive findings by 50 per cent. If large numbers are to be examined, it is a good plan to give mild, saline cathartics in the morning, so that all specimens can be collected at about the same time. It is best to collect specimens by cotton swabs, on swab sticks thrust into tubes in which there are a few drops of salt solution to prevent drying. We have found that rectal swabbing, if properly carried out, may be a valuable method of collect- ing material. If it is absolutely necessary to ship stools some distance, the addition of 20 per cent glycerin is of advantage. A suspension of about one part of feces to twenty-five parts of salt solution is made, thoroughly emulsified, and allowed to stand to allow the large particles to settle. With this material, surface smears are made with a glass rod upon plates of either Robinson and Rettger's modification of Endo's medium, or Krumwiede's brilliant green medium, as described in the section on media. It is of advantage to use the large plates. A bent glass rod is dipped into the emulsion and rubbed over the surface of a plate, beginning in the center, by passing in concentric circles so that the entire plate is gently smeared. A second plate is inoculated in the same way, without redipping. It is sometimes well to make similar plates with a 1 : 5 dilution of the original suspension. Plates for this purpose should be poured and allowed to dry on a laboratory desk for a few hours before use, and should be kept in the dark if Endo's medium is used. Great care in the accurate production and testing out of the media, should be taken as indicated in the section describing these media. The plates should either be inverted in the incubator, or else earthen-ware covers should be used. Large pieces of blotting paper inserted under the lid serve the same purpose. 31 Garbat, The Typhoid Carrier Problem, Monographs of the Rock. Inst., in press. 32 Nichols H. J., Jour. Exp. Med., xxiv, 1916, 497; Jour, of A. M. A., Ixviii, 1917, 958. 654 PATHOGENIC MICROORGANISMS After eighteen hours growth, the plates should be examined for typical colonies. Suspicious colonies should be immediately inoculated upon tubes of Russell double-sugar medium. Slide agglutinations against 1 :100 dilution of a high titer stock typhoid antiserum should be made for preliminary identification from suspicious colonies, of course together with morphological determination by smear and stain. Much information can be obtained after twelve more hours, by observa- tions of the growth in the Russell double-sugar medium. From this tube, then, the growth can be emulsified in salt solution, and macroscopic agglu- tinations set up. This usually is sufficient to identify the organism, but it is always well to set up a few sugar fermentation tubes. Duodenal examinations are made by means of the Einhorn duodenal tube, which is sterilized by boiling, and given to the patient the evening before the examination is to be made, about three hours after the last meal. We take our description chiefly from Garbat who has had considerable experience with this method. The patient, properly instructed, swallows the tube without gagging, with ease, and retains it throughout the night. On the following morning it has usually passed into the duodenum, and bile can be aspirated with a sterile 20 c.c. Luer syringe. Suction must usually be exerted, and Garbat recommends a well fitting syringe because such suction must often be strong. In about 5 per cent of Garbat's cases the tube remained in the stomach and more difficulty was experienced with the test. When there is difficulty in obtaining sufficient bile, the patient is made to sit up in bed with His head bent forward, pressing upward on the abdomen with the palms of his hands. Sometimes the flow of bile can be stimulated by a cold drink. The bile is handled bacteriologically on Endo plates. Typhoid Bacilli in the Urine. — Careful investigation has revealed typhoid bacilli in the urine in about 25 per cent of all patients. Neu- mann 33 discovered the bacilli in eleven out of forty-six and Karlinsky 34 in twenty-one out of forty-four cases. Investigations by Petruschy,35 Richardson,36 Horton-Smith,37 Hiss,38 and others have confirmed these results. In general the bacilli have not been found before the fifteenth day of the disease, and examination of the urine, therefore, can be of little early diagnostic value. A series of seventy-five cases examined by Hiss before the fourteenth day of the disease did not once reveal typhoid bacilli in the urine. On the other hand, they have been found 33 Neumann, Berl. klin. Woch., xxvii, 1890. 34 Karlinsky, Prag. med. Woch., xv, 1890. 35 Petruschy, Cent, f . Hyg., xxiii, 1898. 36 Richardson, Jour. Exp. Med., 3, 1898. 37 Horton-Smith, Lancet, May, 1899. *Hi88, Med. News, May, 1901. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 655 to be present for weeks, months, and, in isolated cases, for years after convalescence, the examination thus having much hygienic importance. They are probably present in about 12 per cent of cases during the early days of convalescence. In most of these, albumin is present in the urine in considerable quantities. The bacilli usually appear and dis- appear with the albuminuria. An obstinate cystitis caused by typhoid bacilli may follow in the path of typhoid fever. Such cases have been reported by Blumer,39 Richardson,40 and others. Suppurative processes in the kidneys are less frequent. It is noteworthy, also, that in the course of, and fol- lowing, typhoid fever Bacillus coli is often present in the urine. This may obstinately persist for considerable periods after convalescence. For examination of the urine for typhoid bacilli, specimens should be taken with aseptic precautions and planted directly into equal volumes of broth. Direct plates on Endo can also be made, or had better be made at the same time. It is relatively easy under such circumstances to obtain the organism if present. Typhoid Bacilli in the Rose Spots. — Neufeld 41 obtained positive results in thirteen out of fourteen cases. According to his researches and those of Frankel,42 the bacilli are localized not in the blood, which is taken when the rose spots are incised, but are crowded in large numbers within the lymph spaces. Typhoid Bacilli in the Sputum. — In rare cases typhoid bacilli have been found in the sputum of cases complicated by bronchitis, broncho- pneumonia, and pleurisy. Such cases have been reported by Chante- messe and Widal,43 Frankel,44 and a number of others. Empyemia, when it occurs in connection with such cases, is usually accompanied by a mixed infection. From a hygienic point of view the spread of typhoid fever by means of the sputum is probably of rare occurrence. Suppurative Lesions Due to Typhoid Bacillus. — In the course of typhoid convalescence or during the latter weeks of the disease, sup- purative lesions may occur in various parts of the body. The most frequent localization of these is in the periosteum, especially on the long bones, and in the joints. A considerable number of such lesions have 39 Blumer, Johns Hopk. Hosp. Rep., 5, 1895. 40 Richardson, loc. oit. 41 Neufeld, Zeit. f . Hyg., xxx, 1899. ri Frankel. Zeit. f. Hy^., xxxiv, 1909. 4:i ('hantemesse and Widiil, Arch. d(> physiol. norm, ct path., 1887. 44 Frankel, Dent. med. Woch., xv and xvi, 1899. 656 PATHOGENIC MICROORGANISMS been described by Welch, Richardson, * and others. They usually take the form of periosteal abscesses, often located upon the tibia, occurring either late in the disease or months after convalescence, and are char- acterized by very severe pain. Osteomyelitis rnay also occur, but is comparatively rare. Subcutaneous abscesses and deep abscesses in the muscles, due to this bacillus, have been described by Pratt.45 Synovitis may also occur. Meningitis, due to the typhoid bacillus, occurs occasionally, usually during convalescence from typhoid fever. A case of primary typhoid meningitis has been reported by Farnet.46 Peritoneal abscesses, due to the typhoid bacillus, have been reported. The writer 47 has reported a case in which typhoid bacilli were found free in the peritoneal cavity during typhoid fever without perforation of the gut. Isolated instances of typhoid bacilli in abscesses of the thyroid and parotid glands and in brain abscesses have been observed. Typhoid Fever without Intestinal Lesions, — A number of cases have been reported in which typhoid bacilli have been isolated from the organs after death or from the secretions during life of patients in whom the characteristic lesions of typhoid fever have been lacking. Most of these cases must be regarded as true typhoid septicemias. In some cases the bacilli were isolated from the spleen, liver, or kidneys; in others, from the urine or the gall-bladder. In a case observed by Zinsser the bacilli were isolated from an infarct of the kidney removed by operation. In this case the clinical course of the disease had pointed only toward the existence of an indefinite fever accompanied by symp- toms referable to the kidneys. The Widal test, however, was positive. A summary of such cases, together with several personally observed, has been given by Flexner.48 Poisons of the Typhoid Bacillus. — Investigation of the toxic products of the typhoid bacillus has occupied the attention of a large number of workers. The first to do experimental work upon the sub- ject was Brieger49 soon after the discovery and cultivation of the bacillus. That toxic substances can be obtained from typhoid cultures is beyond question. There is, however, a definite difference of opinion * Richardson, Jour. Boston Soc. Med. Sci., 5, 1900. « Pratt, Jour. Boston Soc. Med. Sci., 3, 1899. 46 Farnet, Bull, de la soc. med. des hop. dc P., 3, 1891. 47 Zinsser, Proc. N. Y. Path. Soc., 1907. 48 Flexner, Johns Hopk. Rep., 5, 1896. 49 Brieger, Deut. med. Woch., xxvii, 1902. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 657 as to whether these poisons are so-called endotoxins only, or whether they are in part composed of soluble toxins comparable to those of diphtheria and tetanus, following the injection of which antitoxic sub- stances may be formed. The evidence so far seems to bear out the original contention of Pfeiffer,50 who first advanced the opinion that the poisonous substances are products of the bacterial body set free by destruction of the bacteria by the lytic substances of the invaded animal or human being. These poisons, when injected into animals for purposes of immunization, in Pfeiffer's experiments, did not incite the production of neutralizing or antitoxic bodies, but of bactericidal and lytic substances. That these endotoxins constitute by far the greater part of the toxic products of the typhoid bacillus can be easily demonstrated in the laboratory, by the simple experiment of filtering a young typhoid culture (eight or nine days old) and injecting into separate animals the residue of bacilli and the clear filtrate respectively. In such an experiment there will be little question as to the overwhelmingly greater toxicity of the bacillary bodies as compared with that of the culture filtrate. On the other hand, if such cultures, especially in alkaline media, are allowed to stand for several months and the bacilli thus thoroughly extracted by the broth, the toxicity of the filtrate is found to be greatly increased. Nevertheless, more recent experiments by Besredka,51 Macfadyen,52 Kraus and Stenitzer,53 and others have tended to show that, together with such endotoxic substances, typhoid bacilli may produce a true toxin which is not only obtainable by proper methods from comparatively young typhoid cultures, but which fulfills the necessary requirement of this class of poisons by producing in treated animals a true antitoxic neutralizing body. The typhoid endotoxins may be obtained by a variety of methods. Hahn 54 has obtained what he calls " typhoplasmin " by subjecting them to a pressure of about four hundred atmospheres in a Buchner press. The cell juices so obtained are cleared by filtration. Macfadyen has obtained typhoid endotoxins by triturating the bacilli after freezing 50 Pfeiffer, Deut. med. Woch.. xlviii, 1894; Pfeiffer und Kolle, Zeit. f. Hyg., xxi, 1896. 51 Besredka, Ann. de 1'inst. Pasteur, 1895, 1896. 52 Macfadyen and Rowland, Cent. f. Bakt., I, xxx, 1901; Macfadyen, Cent. f. Bakt., I, 1906. 53 Kraus und Stenitzer, Quoted from "Handb. d. Tech.," etc., 1, Fischer, Jena, 1907. i4 Hahn, Munch, med. Woch , xxiii, 190G. 658 PATHOGENIC MICROORGANISMS them with liquid air and extracting in 1 : 1000 potassium hydrate. Besredka obtained toxic substances by emulsifying agar cultures of bacilli in salt solution, sterilizing them by heating to 60° C. for about one hour, and drying in vacuo. The dried bacillary mass was then ground in a mortar and washed in sterile salt solution which was again heated to 60° C. for two hours. The remnants of the bacterial bodies settle out and the slightly turbid supernatant fluid contains the toxic substances. Vaughan 55 has obtained poisons from typhoid bacilli by extracting at 78° C. with a 2-per-cent solution of sodium hydrate in absolute alcohol. In this way he claims to separate by hydrolysis a poisonous and a non-poisonous fraction. He claims, moreover, that this poison- ous fraction is similar to the poisons obtained in the same way from Bacillus coli and the tubercle bacillus, and other protein substances, believing that the specific nature of such proteids depends upon the non-toxic fraction. A simple method of obtaining toxins from typhoid bacilli is carried out by cultivating the microorganisms in meat-infusion broth, rendered alkaline with sodium hydrate to the extent of about 1 per cent. The cultures are allowed to grow for two or three weeks and then sterilized by heating to 60° C. for one hour, and allowed to stand for three or four weeks at room temperature. At the end of this time the cultures may be filtered through a Berkefeld or Pasteur-Chamberland filter and will be found to contain strong toxic substances. The accounts concerning the thermostability of the various toxins obtained are considerably at variance. In general, corresponding with other endotoxins, observers agree in considering them moderately resistant to heat, rarely being destroyed at temperatures below 70° C. We have ourselves often boiled typhoid suspensions without destroying their toxicity for guinea pigs. Intravenous inoculation of rabbits with typhoid endotoxins, if in sufficient quantity, produces, usually within a few hours, a very marked drop in temperature, diarrhea, respiratory embarrassment, and death. If given in smaller doses or by other methods of inoculation — sub- cutaneous or intraperitoneal — rabbits are rendered extremely ill, with a primary drop in temperature, but may live for a week or ten days and die with marked progressive emaciation, or may survive. Guinea- pigs and mice are susceptible to the endotoxins, though somewhat less so than rabbits. In unpublished experiments we have perfused the isolated guinea pig heart with typhoid extracts for prolonged periods without killing it, BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP G59 showing that the poison does not act upon the normal heart muscle directly. Zinsser, Parker and Kuttner 55 have recently shown that in broth cultures of the typhoid bacillus as young as five to six hours, a mildly toxic substance is formed which can be recovered in filtrates, and which, injected into rabbits intravenously, gives rise to definite symptoms, after an incubation time of an hour or more. This substance is not specific in that it is formed by many other different bacteria similarly grown, and is not antigenic in all probability. It can also be obtained by washing young agar growths repeatedly in salt solution, and filter- ing. Whether or not this substance plays a part in the disease cannot be stated at the present time. IMMUNITY AND ANTIBODIES Animals, may be actively immunized by the injection of typhoid bacilli in gradually increasing doses. In actual practice, this is best accomplished by beginning with an injection of about 1 c.c. of broth culture heated for ten minutes at 60° in order to kill the bacilli. After five or six days, a second injection of a larger dose of dead bacilli is administered; at similar intervals, gradually increasing doses of dead bacilli are given and finally considerable quantities of a living and fully virulent culture may be injected without serious consequences to the animal. While this method is convenient and usually successful, it is also possible to obtain satisfactory immunization by beginning with very small doses of living microorganisms, according to the early method of Chantemesse and Widal,56 and others. Such active immunization, successfully carried out upon rabbits and guinea-pigs, within a short time after the discovery of the typhoid bacil- lus, was believed to depend upon the development of antitoxic sub- stances in immunized animals. This point of view, however, was not long tenable, and was definitely disproven by the investigations of Pfeiffer and Kolle 57 in 1896. These investigators, as well as a large number of others working subsequently, have shown that there are present in the blood serum of typhoid-immune animals and human beings, bacteriolytic, bactericidal, and agglutinating substances, and to a lesser extent, precipitating and opsonic bodies but no true anti- toxins. 55 Zinsser, Parker and Kuttner, Proc. of the Soc. Exper. Med. and Biol., Meeting, Nov., 1920. 56 Chantemesse and Widal, Ann. de Finst. Pasteur, 1892. 57 Pfeiffer und Kolle, Zeit. f. Hyg., xxi, 1896. 660 PATHOGENIC MICROORGANISMS One attack of typhoid fever protects against subsequent infection. Accurate statistics upon the -matter have been difficult to obtain, however, because histories of the disease are apt to be indefinite, and until recently, no proper differentiation was made between true typhoid fever and the paratyphoid group. However, taking into consideration these possibilities of error, the estimations made by various clinicians who have studied the subject, indicate that a second attack of typhoid fever occurs in not more than from 0.7 to 4 per cent of all cases. Two to 3 per cent represents a fair average of all estimates made. When typhoid fever does occur for the second time, it is usually of a milder type than the first attack, though, according to V. Vaughan, Jr., this is not always the case. Circulating antibodies disappear from the typhoid convalescent usually within the first seven months after recovery. Permanent immunity cannot, therefore, be explained upon the basis of serum anti- bodies. The ultimate cause for permanent immunity, in all diseases in which it occurs, must be regarded as depending upon the physiological unit, namely, the tissue cell. It is likely that individuals who have passed through an infection of this nature, thereafter retain a capacity to react more rapidly and effectively to small quantities of introduced antigen. A case in point is the well-known experiment of Wassermann, who immunized a number of rabbits to typhoid bacilli until a highly agglutinin titer was produced. He kept these rabbits until their blood had returned to normal and no agglutinins could be found. Subse- quently he reinoculated them with typhoid bacilli, at the same time giving a number of normal control rabbits similar injections. The previously treated rabbits responded with a rapid and powerful anti- body production in contrast to the slower antibody curve of those that had received the typhoid antigen for the first time. Recent observa- tions by Moon on revaccination of previously vaccinated people, have given analogous results; and it appears from this that a person, once immunized, is capable of reacting with much greater promptness than a normal individual. Our own idea would be somewhat as follows: When the typhoid bacillus enters the bowel of the infected subject it begins to proliferate and gradually enters into the lymphatic system. As a consequence, a small amount of antigen is gradually introduced into the circulation, reaches the cells and stimulates antibody produc- tion. In the normal individual this reaction is a slow one, and the typhoid bacilli multiply with a speed disproportionate to the appearance of antibodies. In the previously immunized individual or in the person who has had the disease, the first absorption of small amounts of antigen BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 661 is followed by a tissue reaction (a part of which is evident as antibody production) so rapid that the protective processes are developed with sufficient potency to check the infection before it has reached a phase at which symptoms become apparent. Bactericidal and Bacteriolytic Substances. — The bacteriolytic sub- stances in typhoid-immune serum may be demonstrated either by the intraperitoneal technique of Pfeiffer or in vitro. In the former experi- ment a small quantity of a fresh culture of typhoid bacilli is mixed with the diluted immune serum and the emulsion injected into the peritoneal cavity of a guinea-pig. Removal of peritoneal exudate with a capillary pipette and examination in the hanging drop will reveal, within a short time, a swelling and granulation of the bacteria — the so-called Pfeiffer phenomenon. The test in vitro, as recommended by Stern and Korte,58 may be carried out by adding definite quantities of a fresh agar culture of typhoid bacilli to progressively increasing dilu- tions of inactivated immune serum together with definite quantities of complement in the form of fresh normal rabbit or guinea-pig serum. At the end of several hours' incubation at 37.5° C. definite quantities of the fluid from the various tubes are inoculated into melted agar and plates are poured to determine the bactericidal action. Careful colony counting in these plates and comparison with proper controls will not only definitely demonstrate the presence of bactericidal sub- stances in the immune serum, but will furnish a reasonably accurate quantitative estimation. (For technic of these tests see page 307.) Although normal human serum contains in small quantity sub- stances bactericidal to typhoid bacilli, moderate dilution, 1 : 10 or 1 : 20, of such serum will usually suffice to eliminate any appreciable bactericidal action. The bactericidal powers of immune serum, on the other hand, are often active, according to Stern and Korte, in dilutions of over 1 : 4000. The specificity of such reactions gives them a con- siderable degree of practical value, both in the biological identification of a suspected typhoid bacillus in known serum and in the diagnosis of typhoid fever in the human patient by the action of the patient's serum on known typhoid bacilli. In the publication of Stern and Korte, quoted above, it was found that typhoid patients during the second week often possess a bactericidal power exceeding 1 : 1000, whereas the blood of normal human beings was rarely active in dilutions exceeding 1 : 50 or 1: 100. While scientifically accurate the practical application of bactericidal determinations for diagnosis 58 Stem und Korte, Berl. klin. Woch., x., 1904. 662 PATHOGENIC MICROORGANISMS presents considerable technical difficulties and gives way to the no less accurate and much simpler method of agglutination. Agglutinins. — Agglutinins are formed in animals and man inoculated with typhoid bacilli, and in the course of typhoid fever. It was, in fact, while studying the typhoid bacillus that the agglutinins were first dis- covered by Gruber and Durham. In animals, by careful immunization, specific typhoid agglutinins may easily be produced in sufficient quantity to be active in dilution of 1 : 10,000, and occasionally even 1 : 50,000 or over. In the blood of typhoid patients, the agglutinins may often be found in dilutions. of 1 : 100 and over. It is interesting to note that irrespective of the agglutinin contents of any given serum, there may occasionally be noted differences in the agglutinability of various typhoid cultures, a point which is practically important in the choice of a typhoid culture for routine diagnosis work. Weeny 59 has called attention to the fact that bacilli which do not readily agglutinate when directly cultivated from the body, may often be rendered more sensitive to this reaction by sev- eral generations of cultivation upon artificial media. Walker has noted 60 a loss of agglutinability if the bacilli are cultivated in immune serum. A similar alteration in the agglutinability of typhoid bacilli was noted by Eisenberg and Volk61 when they subjected the micro- organism to moderate heat or to weak acids such as ^ HC1. Morishima 62 has recently studied the same phenomenon, and has confirmed the observations of Eisenberg and Volk,63 and others. He has also shown, however, that if organisms are cultivated in anti-serum for a sufficiently long time, their preliminary inagglutinability will eventually, after twenty to seventy-five days, revert to almost normal agglutinability . Practical application of agglutination to bacteriological work is found in the identification of suspected typhoid bacilli, and in the diagnosis of typhoid fever. When it is desired to determine whether or not a given bacillus is a typhoid bacillus, mixtures may be made of young broth cultures, or preferably of emulsions of young agar cultures in salt solution, with dilutions of immune serum. The tests are made microscopically in the hanging-drop preparation or, preferably, macroscopically in 69 Weeny, Brit. Med. Jour., 1889. 60 Walker, Jour, of Path, and Bad., 1892; Totxtikn, Holt. f. HyR., xlv, 1903. 01 Eixenberg und Volk, Zeit., f. Hyg., xlv, 190:5. ^Morishima, Jour, of Baeter., March, 1921. 63 Eisenberg and Volk, Erbeg. der Immunit. Exper. Ther. Bakt. u. Hyg., 1913, 73. BACILLI OF THE COLON-TYPHOlB-DYSENTERY GROUP 663 small test tubes. In all cases it is desirable first to determine the agglutinating power of the scrum when tested against a known typhoid culture. (For detailed technique, see chapter on Technique of Serum Reactions, page 302, 282.) In scientific investigations, specific agglutinations in high dilutions of immune serum constitute very strong proof of the species of the micro- organism and may often furnish much information as to the biological relationships between similar species. It is found in immunizing ani- mals with any given strain of typhoid bacilli, that there are formed the " chief" or " major" agglutinins which are specific and active against the species used in immunization, and the "-group" or "minor" agglutinins, active also against closely related microorganisms. The following extract from a table will serve to illustrate this point in the case of typhoid and allied bacilli. Highly Immune Typhoid Scrum. 1 : 100 1 : 250 1 : 500 1 : 1000 1 : 2500 B. typh B. paratyph. (Schottmiiller) ...... B. enteritidis + + + + + + + + + + B. ooli communis The sera of most adult normal animals and human beings usually contain a small amount of agglutinin for these bacilli. Immunization with the typhoid bacillus, while increasing chiefly the agglutinin for this bacillus itself, also to a slighter extent increases the group agglutinins for other closely allied species. That these group agglu- tinins are separate substances and not merely a weaker manifestation of the action of the typhoid agglutinin itself upon these other micro- organisms, may be demonstrated by the experiments of agglutinin absorption. (See section on Agglutinins, page 288.) In the clinical diagnosis of typhoid fever, the phenomenon of agglu- tination was first utilized by Widal.64 This observer called attention to the fact that during the last part of the first or the earlier days of the second week of typhoid fever, as well as later in the disease and in con- valescence, the blood serum of patients would cause agglutination of typhoid bacilli in dilutions of 1 : 10, or over, whereas the serum, of normal individuals usually exerted no such influence. Upon this basis 64 Widal, Bull, de la soc. mcd. des hopit., vi, 1896; Widal et Sicard, Ann. de 1'inst. Pasteur, xi, 1897. 664 PATHOGENIC MICROORGANISMS he recommended, for the diagnosis of the disease, the employment of a microscopic agglutination test carried out by the usual hanging-drop technique. The reaction of Widal is, at present, widely depended upon for diagnostic purposes and although not universally successful, owing to irregularities in agglutinin formation in some patients and because of differences in agglutinability of the cultures employed, it is nevertheless of much* value. The fact that the recent work of Hooker and of Weiss has shown that typhoid bacilli differ in antigenic properties, and may on the basis of agglutination and agglutinin absorption be divided into a number of groups, is not of sufficient practical importance to necessitate the use of a variety of strains since the atypical antigenic ones are rela- tively rare. Original conclusions as to the dilutions of the serum which must be employed, have, however, necessarily been modified. Owing to the fact that Gruber,65 Stern,66 Frankel,67 and a number of others have found that occasionally normal serum will give rise to agglutina- tion of typhoid bacilli in dilutions exceeding 1 : 10, it has been found necessary, whenever making a diagnostic test, to make several dilutions, the ones most commonly employed being 1 : 20, 1 : 40, 1 : 60, and 1 : 80. The wide application of the method has given rise to the development of a number of technical procedures, all of them devised with a view toward simplification. In ordinary hospital work, it is most convenient to keep on hand upon slant agar, a stock typhoid culture, the agglutinability of which is well known. From this stock culture, fresh inoculations upon neutral bouillon should be made each day, so that a young broth culture may always be on hand to furnish actively motile, evenly distributed bacteria. These bouillon cultures may be grown for from six to eight hours at incubator temperature or for from twelve to eighteen hours at room temperature. The tempera- tures at which the broth cultures are kept .must depend, to a certain extent, upon the peculiarities of the typhoid bacillus employed, since some strains are more actively motile and furnish a more suitable emulsion if kept at a temperature lower than 37.5° C. A false clumping in the broth cultures due to a too-high acidity of the bouillon or a too- prolonged incubation must be carefully guarded against. It is also possible to use for this test an emulsion of typhoid bacilli prepared by rubbing up a small quantity of a young agar culture in salt solution. Uniformity in the preparation of broth cultures or of emulsions should be observed, since the quantitative relationship between typhoid bacilli 65 Gruber, Verhand. Congr. f. inn. Med., Wiesbaden, 1896. 66 Stern, Cent. f. inn. Med., xlix, 1896. 67 Frankel, Deut. med. Woch., ii, 1897. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 665 and agglutinins will markedly affect the completeness or incompleteness of the reaction. In high dilutions an excess of typhoid bacilli may bring about complete absorption of all the agglutinins present, without agglu- tinating all the microorganisms. The blood of the patient to be used for a Widal test may be obtained in a number of ways. The most convenient method is to bleed the patient from the ear or ringer into a small glass capsule, in the form of that used in obtaining blood for the opsonin test, or into a small centri- fuge tube. About 0.5 to 1 c.c. is ample. These capsules or tubes, after clotting of the blood, may be placed in the centrifuge which in a few revolutions will separate clear serum from clot. The dilutions of the serum are then made. It is best to use sterile physiological salt solution as a diluent, but neutral broth may be used. The dilutions may be made either by means of an ordinary blood-counting pipette or by means of a capillary pipette upon which a mark with a grease pencil, made about an inch from the tip, furnishes a unit of measure, and upon which suction is made by means of a rubber nipple. It is convenient to have at hand a small porcelain palette such as that used by painters, in which the various cup-like impressions may be utilized to contain the various dilutions. Dilutions of the serum are made, ranging from 1 : 10 to 1 : 50. A drop of each of these dilutions is mixed with a drop of the typhoid culture or emulsion upon the center of a cover-slip and the cover- slip inverted over a hollow slide. A control with normal serum and with the same culture should always be made and also one with the culture alone to exclude the possibility of spontaneous clumping. Mixture with the typhoid culture, of course, each time doubles the dilutions so that, for instance, a drop of serum dilution 1 : 10, plus a drop of the typhoid culture, gives the final dilution of 1 : 20. The preparation may be examined with a high power dry lens or an oil immersion lens. In a positive reaction, the bacilli, which at first swim about actively, singly or in short chains, soon begin to gather in small groups and lose much of their activity. Within one-half to one hour, they will be gathered in dense clumps between which the fluid is clear and free from bacteria, and only upon the edges of the agglutinated masses may slight motility be observed. The degree of dilution and the time of exposure at which such a reaction may be regarded as of specific diagnostic value have been largely a matter of empirical determination. It is generally /iccepted at present that complete agglutination within one hour in dilutions from 1 : 40 to 1 : 60 is definite proof of the existence of typhoid infection. Exceptions, however, to this rule may occur. Agglutina- tions of typhoid bacilli in dilutions of 1 : 40, and over, have occasionally 666 PATHOGENIC MICROORGANISMS been observed in cases of jaundice and of tuberculosis, and these condi- tions must occasionally be considered, though their importance was formerly exaggerated. The method of making the Widal test from a drop of whole blood dried upon a slide, is not to be recommended' since accuracy in dilution by this method is practically impossible. As stated above, the agglutinin reaction rarely appears in typhoid fever before the beginning of the second week. It may continue during convalescence for as long as six to eight weeks and occasionally, in cases where there is a chronic infection of the gall-bladder, a Widal reaction may be present for years after an attack. For very exact work, even in clinical cases, the microscopic agglu- tination method may be replaced by macroscopic agglutination, accord- ing to the technique described in another section (page 303.) In order to avoid both the necessity of keeping alive typhoid cul- tures for routine agglutination tests and also to preclude the danger of infection by the use of living culture, Ficker68 has recommended typhoid bacilli killed by formalin. This method has no advantages for practical purposes and in scientific bacteriological work it is, of course, not to be considered in comparison with the more exact methods. The more recently introduced general practice of vaccination in typhoid fever has added a complicating factor to diagnostic agglutina- tion. Individuals so vaccinated develop agglutinins in consequence of the inoculations, which may persist for six months or more, and even after they have disappeared from the blood stream, various non-typhoid febrile conditions may induce their appearance in the circulation for reasons not well understood. In consequence of this, it is necessary, before drawing conclusions concerning the meaning of a Widal reac- tion, to be thoroughly informed concerning the vaccination history of the patient and the time which has elapsed since the vaccination was done. A certain amount of reliable information may be obtained even in such cases by the study of the quantitative changes in the agglutinins in the patient's blood by the comparative method of Dreyer as in use in the United States Army. This method, however, is, in our opinion, not sufficiently useful or simple to be recommended for ordinary clinical use. In the Dreyer method, standardized suspensions of Bacillus typhosus or the para- typhoid types are used. To obtain these, cultures of the bacilli are grown for about two weeks by daily transplant in broth, a procedure 68 Picker, Berl. klin. Woch., xlvii. 1903. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 667 which is supposed to increase agglutinability. After this, it is planted in flasks in broth, allowed to grow overnight, and 0.1 per cent formalin is added.09 This formalinized culture is placed in the refrigerator and shaken frequently during four or five days. It is then standardized for opacity against an arbitrary standard kept on hand, and prepared in the Army Medical School. Necessary dilutions are made with physiological salt solution to which 0.1 per cent formalin has been added. The suspension is then standardized for agglutinability against a known agglutinating immune serum. For this purpose incubation at 55° for two hours is a method used for the final reading. It is easy, from this test, then, to determine the agglutinability factor of the new suspension. The example cited in the Army Medical School War Manual, No. 6, is as follows: If the dilution of the solution in which the standardized suspension is agglutinated is 1 to 6400, while that of the new suspension is 1 to 3200, then the factor of the new bacterial suspension is one-half. By the use of such suspension it is clear that comparative titrations of the rise and fall of agglutinins in the patient's serum can be made, and information obtained which may have considerable importance in deciding whether the appearance of agglutinins in the patient is due to previous vaccination, or is present in response to a fresh infection. Precipitins. — The investigations of Kraus,70 by which the pre- cipitins were discovered, revealed specific precipitating substances, among others, also in typhoid immune sera. Since Kraus' original investigation, these substances have been studied by Norris 71 and others.72 Opsonins. — A number of observers have shown that opsonins specific for the typhoid bacillus are formed in animals immunized with these organisms. Opsonins are formed also in patients suffering from typhoid fever, but exact opsonic estimations in all these cases are extremely difficult because of the rapid lysis which these bacteria may undergo both in the serum, and intracellularly, after ingestion by the leucocytes. Klein 73 has attempted to overcome this difficulty by working with dilutions of serum, at the same time using comparatively thick bacterial emulsions and exposures to the phagocytic action not exceed- 69 U. S. Medical War Manual No. 6. Lea & Febiger, 1919. 70 Kraus, Wien. klin. Woch., xxxii, 1897. 71 Norris, Jour, of Inf. Dis., I, 3, 1904. 72 Barker and Cole, 22d Ann. Session, Assn. of Amer. Phys., Wash., 1897. 73 Klein, Bull. Johns Hopkins Hosp., 1907. 668 PATHOGENIC MICROORGANISMS ing ten minutes. Chantemesse 74 has claimed that the opsonic index of typhoid patients was increased after treatment with a serum obtained by him from immunized horses, and Harrison 75 has reported similar results in patients treated by a modification of Wright's method of active immunization. Klein claims to have demonstrated that in typhoid-immune rabbits, after five injections, the opsonic contents of the blood were increased to an equal extent as the bactericidal sub- stances. He concludes from this interesting observation that it may well be that the opsonins are quite as important in typhoid immunity as are the latter substances. For diagnostic purposes in typhoid fever the estimation of the opsonic index, so far, has not been proven to be of great value. SANITARY CONSIDERATIONS IN TYPHOID FEVER Typhoid fever is a disease which has been constantly diminishing in frequency in civilized countries during the last one hundred years, but is still a very formidable cause of death rate and disability. The morbidity rates and death rates for typhoid fever vary considerably in different communities according to the extent to which sanitary supervision of water supplies, garbage and sewage disposal, etc., have been developed. In general, the United States has been considerably behind most European communities. In a table given by Gay 76 a comparison of mortality averages per 100,000 population, comparing a group of over 31,000,000 people compiled from the statistics of the 33 largest European cities, with 21,000,000 people representing the pop- ulations of 57 of the largest American cities, the European mortality average was 6.5, and the American 19.59. In similar compilations taken by Gay largely from the report of the New York State Depart- ment of Health for 1914, it is shown that there has been a progressive decrease since 1910, running parallel to increased attention to water supplies and general sanitation. For more extensive figures on the prevalence of typhoid fever the reader is referred to the above-mentioned compilation of Gay. He states that in 1900 there were about 350,000 cases of typhoid fever in the United States as estimated by Whipple, who at the same time calculates that the cost to the community of these cases must have been approximately $212,000,000. Since, as we shall see, it has been variously shown during the last ten years that typhoid 74 Chantemesse, 14th Internal!. Cong, for Hyg., Berlin, 1907. 75 Harrison, Jour. Royal Army Med. Corps, 8, 1907. V6 Gay, F. P., Typhoid Fever, Macmillan Company, New York, 1918. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 669 fever is a distinctly preventable disease, amenable perhaps not to com- plete eradication on account of the difficulties of the carrier problem, but certainly readily subject to material diminution, much of this suffering and economic loss would seem unnecessary. Infection with typhoid fever always means that intestinal contents of a case or a carrier have come into direct or indirect contact with something ingested by the patient. Since this is true, we may best begin the description of the circle of infection from man to man by first considering the manner in which the organism leaves the body of the patient and the carrier. In the patient the typhoid bacillus begins to accumulate in the intes- tines during the later stages of the incubation time, and at this time will begin to appear in the feces. The organisms increase in the intestines from this time on, being distributed in very considerable numbers after the second week, and decreasing only towards the end of the disease, remaining present, however, throughout convalescence and sometimes, as we shall see, for months or years thereafter. During the second and third or later weeks, the organisms appear in the urine. It is generally stated that about 30 per cent of typhoid cases will show the organisms in the urine, but it seems likely that this is too low an estimate. Rau- bitschek, by precipitating considerable quantities of urine with ferric chlorate succeeded in finding the bacilli in 100 per cent of his cases in the^ earlier stages of the disease, and it is not at all Unlikely that in slight numbers, and perhaps intermittently, they may appear in the urine of all typhoid cases. Other routes of distribution from the patient, such as suppurations, sputum, etc., are occasionally mentioned, but may be dismissed as of no practical sanitary importance. Since the recognized typhoid case is usually well guarded from a sanitary point of view, the greater danger of typhoid infection lies in the mild, atypical, unrecognized case and in the carrier. Atypical, mild cases will probably become more and more frequent as typhoid vaccination becomes a more generalized habit. Such a case may show nothing more than a very slight febrile movement, with intestinal dis- turbances and diarrhea. Unless typhoid fever is particularly looked for and suspected, many of these cases may never be put upon typhoid precautions and the resulting possibilities of spread are obvious. More important from the sanitary point of view, under the con- ditions of modern community life, however, is the typhoid carrier. TYPHOID CARRIERS. — The great importance of the typhoid carrier in the spread of the disease has led to extensive studies of the problem in many countries during the last ten years. We may mention par- G70 PATHOGENIC MICROORGANISMS ticularly the studies of Conradi and Drigalski77 the paper of Sacquepee,78 the summary given by Kutscher 79 in the Second Edition of the Kolle and Wassermann Handbook, the summary of Gay in the book men- tioned above, and the article by Garbat, not yet published, but about to appear as one of the Monographs of the Rockefeller Institute. The first definite suggestion of the danger of typhoid infection emanating from convalescents long after the disease itself had been cured, came from Koch.80 He based this opinion at first upon purely epidemiological evidence, but in 1904 Drigalski 81 began to isolate bacilli from individuals who were apparently in complete health. Sacquepee 78 classifies typhoid carriers chiefly into convalescent carriers who become free of the bacilli within three months after the termination of their disease, and chronic carriers who continue to harbor the bacilli for many years, and perhaps permanently. In addition to this, there are a certain number of so-called healthy carriers in whom no history of their ever having had the disease can be adduced. The distinction between a temporary carrier and a chronic carrier must, of course, be to a certain degree arbitrary, but in general it may be said that in most typhoid cases the organisms disappear from the urine and feces within from six weeks to three months after recovery. Sacquepee classifies as chronic carriers only those in which the organisms are still present three months after complete recovery. After this period, the length of time to which the carrier may persist is variable, depending upon whether or not chronic lesions are established. These will be discussed below. The frequence with which chronic carriers following typhoid fever occur may be gathered from the table compiled by Gay and published in the book mentioned above. If we consider that the figures presented in this table must neces- sarily represent underestimates because of the technical difficulties attending the discovery of small numbers of typhoid bacilli, it becomes apparent that the number of potential foci for infection in a community is enormous. Gay estimates, on a basis of a 5 per cent minimum, that we have 7500 added annually to the carriers present in the United States. On this basis about 0.2 per cent to 0.3 per cent of the general population may be assumed to be carriers. According to the foci upon which the carrier state depends, typhoid 77 Conradi and Drigalski, Zeit. f. Hyg., 34, 1902, 283. 78 Sacquepee, Bull, do I'lnst. Past,, 8, 1910, 521 and 689. 79 Kutscher, Kolle and VVassonnann Ilandbuch, 2d Edition, Fischer, Jena, 1913. ™Koch, Ver. a. d. Militarsanitatswesen, H. 21, 1902. 81 Drigalski, Cent. f. Bakt,, 35, 1904, 776. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 671 carriers have been subdivided by a number of writers into intestinal carriers, gall-bladder carriers, and liver or bile duct carriers. We will see how the newer methods of duodenal tube examination have made these distinctions between carriers possible. PERCENTAGES OF CHRONIC TYPHOID CARRIERS FOUND BY VARIOUS INVESTIGATORS IN A STUDY OF CONVALESCENT CASES * Author. Date. Number of Cases. Percentage Carriers for 3 Months and More. Lentz 1905 400 3 0 Conradi 1907 400 0.5 Klinger . . 1907 482 1 7 Kayser 1907 101 3 5 Semple and Greig 1908 86 11.6 Park 1908 68 5.9 Tsuzuki 1910 51 5 8 Bruckner 1910 316 3.8 Stokes and Clarke 1916 810 1 85 * Table taken from Gay, F. P., Typhoid Fever, MacMillan & Co., New York, 1918. By far the most common localization of typhoid bacilli in the body of the carrier is the gall bladder. In speaking of the sequela? of typhoid fever we have seen that cholecystitis is almost always related to a pre- ceding attack of typhoid fever. As a matter of fact in the course of typhoid fever the organisms are always present in the gall bladder. This was noted by Chiari 82 as early as 1894, by Pratt,83 and by many others. Longcope is quoted by Gay to have taken bile cultures as a routine in suspected typhoid deaths at the Pennsylvania Hospital, and found typhoid bacilli in all positive cases. In the gall bladder appar- ently the organisms find a protected nidus where they can persist for years. If gall stones are formed later, typhoid bacilli can often be isolated from them. We ourselves have reported a case in which we isolated the organisms from gall stones seventeen years after the attack of typhoid. That liver-duct carriers, however, may exist independently of gall- bladder infection has been shown by such cases as the one cited by Garbat in the essay mentioned above. He speaks of two patients 82 Chiari, Cent, f . Bakt., Orig., 15, 1894. 83 Pratt, Amer. Jour. Med. Sciences, 1901. 672 PATHOGENIC MICROORGANISMS who, during typhoid convalescence, manifested gall-bladder symptoms. Direct culture of the bile by means of the duodenal tube method showed typhoid bacilli in " A," but not in "B." In both, the gall bladder was removed and a pure culture of typhoid bacilli obtained from both gall bladders. At the time of operation, the negative culture in "B" was explained by the fact that a large stone was fixed in the cystic duct which completely occluded the passage. The bile from "B," before operation had come directly from the liver, and had not entered the gall bladder which was in this case, the only site of infection. After operation however, typhoid bacilli completely disappeared from "B," where the bile that had come from the liver had been found sterile by the original duodenal culture, but in "A," in spite of the complete removal of the gall bladder and cystic duct, repeated duodenal cultures remained posi- tive. Similar cases have been reported in the literature, but none which seem quite as convincing as a proof for the existence of the true liver carrier as these instances reported by Garbat. The manner in which typhoid bacilli get into the gall bladder has occupied the attention of a number of investigators. According to Kiister 84 and a more recent report by Garbat ascending infection of the gall bladder from the duodenal is a possibility, though it is probably not the most common method of infection. Nichols 85 too has admitted the possibility of such a process, although no one believes that this is very common. The fact also that, according to Blumenthal 86 Lauben- heimer 87 and others, colon bacilli are very commonly found in the gall bladder, gives support to the possibility of ascending infection. The opinion, however, that the bile is hematogeneously infected by way of the hepatic circulation in most cases is generally accepted. The existence of pure intestinal carriers has been suggested by Kraus and others, and in addition to the cases cited by Kraus, there is one by Garbat in which duodenal cultures were repeatedly negative, whereas the feces remained positive. Cholecystectomy on this case did not relieve the carrier condition. The intestinal carrier type, according to Kraus 88 may be associated with chronic intestinal ulcerations, chronic appendicitis, etc., but is unquestionably extremely rare, a large majority of carriers being due to actual gall-bladder infection. Chronic urinary carriers are less common than chronic feces carriers. MKu ter, Beitr. f. Klin. d. Infkrankh, etc., 7, 1918, 98. 85 Nichols, Jour, of the A. M. A., 68, 1917. 86 Blumenthal, Arch, f . klin. Med., 88, 1907, 509. 87 Laubenheimer, Zeit. f . Hyg., 58, 1909. 88 Kraus, Wien. klin. Woch., 27, 1914, 1443. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 673 Yet, when they occur, they are of much greater danger to others because of the more indiscriminate distribution of urine. According to Gar- bat 89 about 6.8 per cent of all typhoid cases show typhoid bacilluria for one or two months after the fever has disappeared. In such cases often the organisms are discharged intermittently, and for this reason repeated examination is necessary. Chronic urinary carriers have been reported by Prigge,90 Houston,91 and others, and are usually associated with some pathological lesion of the genito-urinary tract. In a case reported by Mayer and Ahreiner 92 there was a pyonephrosis, and in other cases, cystitis, or other inflammations of the bladder, ureter, and kidney have been found. By so-called healthy carriers are meant individuals who harbor typhoid bacilli in the stools and in .whom no history of typhoid infection at any time in their lives can be obtained. We know more about typhoid fever than we did formerly, and we think that it is quite clear to most students of the disease that a negative history of this kind, especially if obtained from people who have lived vigorous, active, physical lives, must be very unreliable. Extremely mild cases of typhoid fever, while not common, do occur, and it is not impossible that an individual with an unusual resistance may have been ill for a few weeks, perhaps with a little fever at sometime without going to a doctor. On the other hand, Scheller93 in examining a group of people, in connection with the investigations made of a mild epidemic, found a considerable number of temporary carriers who did not develop the disease. These people had taken milk infected from a carrier, 18 of a total of 44 acquiring the organisms without getting sick, while 32 of the same group actually got sick. It is not impossible, therefore, that individuals associated with typhoid cases and during epidemics may become temporary carriers. Carriers may increase enormously in the course of epidemics espe- cially if these epidemics take place among the large groups of vaccinated people. Such conditions prevailed among the Allied and probably among the German Armies during the war, when the enormously in- creased opportunities for fecal transmission produced incident to active warfare, with open latrines, unprotected kitchens, unlimited fly breeding, and defective scattered small water supplies, made sanitary control impossible. Hundreds of thousands of men suffered from diarrheas and 89 Garbat, Jour. A. M. A., Nov., 1916, 1493. 90 Prigge, Klin. Jahr., 22, 1909-1910, 245. 91 Houston, with Irwin, Lancet, 1, 1909, 311. 92 Mayer and Ahreiner, cited from Gay, loc. cit. 93 Scheller, Cent, f . Bakt., Erste Abt., Orig., 45, 1908, 385. 674 PATHOGENIC MICROORGANISMS mild intestinal disease, without, or with very slight febrile manifesta- tions, and the investigations of many bacteriologists, as well as our own, show that a considerable percentage of these people were actually infected with organisms of the typhoid, paratyphoid, and dysentery groups. As to the relative importance of the typhoid carrier in the morbidity of typhoid fever, it is very difficult to adduce accurate data. It is pretty safe to say that the carrier is growing relatively more important, will in the future probably be the chief source of typhoid morbidity in well sanitated communities, and is the only stumbling block which will probably prevent the complete eventual eradication of the disease. Of recent years, as water, milk, and food supplies are coming more and more directly under the vigilant eyes of health authorities, the estimates of the percentage of cases due to carriers, as contrasted with other sources of infection, is growing larger and larger. PATHOLOGICAL CONSEQUENCES OF THE CARRIER STATE. — Perhaps the most common sequelum of the chronic carrier state is cholelith- iasis. According to Exner and Heyworski 94 typhoid bacilli have a particular property of decomposing the bile salts, giving rise to a pre- cipitation of cholesterin, and Dorr 95 experimentally produced small concretions in the gall bladder of infected animals. Typhoid bacilli have often been found in gall stones. Furthermore, obstruction of the bile and stagnation due to inflammatory processes may be indirectly responsible for stone formation. It is probable that typhoid carriers possess an especially high resistance to second attacks, higher even than that of the ordinary individual who recovers without developing the carrier state. Kuster reports that of 800 chronic carriers observed in the military hospital at Cologne during two and one-half years, not a single clinical disturbance attributable to the typhoid bacillus could be determined. The occur- rence of cystitis, pyelitis and renal stones in typhoid carriers is not par- ticularly common. Occasionally, typhoid carriers may possess -agglutinins and other antibodies in the blood higher than normal. Lentz examined a number of chronic carriers and found positive Widals in 10 out of 11; however, only in dilutions of 1 : 20. Gaethgens 96 found both agglutinins and opsonins higher in chronic carriers than in normal people, but Schone 97 94 Exner and Heyworski, Wien. klin. Woch., 1908, 7. 95 Don', cited from Klistcr, loc. cit. 96 Gaethgens, Deut. med. Woch., 1907, 1337. 97 Schone, Munch, med. Woch., 1908, 1063. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 075 found no increase in complement fixation. In general, we would not hope for very much light from serological investigations upon the ques- tion of whether or not an individual was a carrier, but would imme- diately proceed to bacteriological examination, either by feces, urine, or duodenal examination. , THE TREATMENT AND CURE OF TYPHOID CARRIERS. — The great importance of the typhoid carrier from the epidemiological point of view has led to innumerable medical and surgical attempts at cure. That there can be no doubt about the possibility of cure in most cases, by surgical gall-bladder extirpation, is, of course, certain. It will be neces- sary in the future, however, especially on the basis of the recent work of Nichols, Garbat and some German observers, to precede such operations by fecal and duodenal examinations, and it must be remembered that there will always be a certain percentage of cases which are liver-duct carriers, in contrast to gall-bladder foci, which will not clear up. Also, the operation is not without danger, with a certain amount of mor- tality, and, of course, cannot be applied generally upon the enormous numbers of carriers that exist. According to Garbat and others, attempts to cure by other means necessarily depend to a veiy large extent upon early diagnosis of the carriers before the condition has become stubbornly chronic. But in all cases, cure by other than surgical means has been discouraging. Many different methods have been attempted. Vaccination with the ordi- nary typhoid vaccines has given discouraging results in the hands of Park and many others, although much was hoped from it at first. Irwin and Houston 98 claimed to have cured a urinary carrier by vacci- nation, but Houston and Thomas " failed in other cases. Petruski 10° in 1902 claimed that vaccination during the course of the disease might prevent the development of the carrier state. But, on the whole, vaccination has not brought the results that have been hoped for it, and we are rather reluctant to believe that on a theoretical basis it is encouraging, since the organisms in the chronic carrier are physiologically outside the body, and not in contact with antibodies or leucocytes. Medicinal treatment has been tried with many agents, but without much success. Discouraging results have been obtained with urotropin, methylene blue, saliciyates, iodin and arsenic preparations. Conradi 101 in 1910 tried chloroform with apparently successful results in rabbits 98 Inrit/ and Ifonxton, Lancet, 1, 1909, 154. 99 HouKlon and Tlunmi*, Cent. f. Bakt. Rcf., 45, 1910, 390. 100 Petmski, cited rrom Kiister, loc. cit. 101 Conradi, Centralb. f. Immimit., 7, 1910, 158. 676 PATHOGENIC MICROORGANISMS experimentally converted into typhoid carriers. Bully 102 tried this treatment upon human carriers, giving 0.5 c.c. of chloroform in capsules four times a day for twenty days, without any results. Neosalvarsan has been tried without effect. The only encouraging reports we can find in an extensive review of the literature to date are the recent ones of Kalberlah 103 who administered tincture of iodine together with animal charcoal, and those of Geronne 104 who similarly combined charcoal with thymol. None of these methods have, however, been sufficiently confirmed to encourage great hope. THE TYPHOID BACILLUS IN TRANSIT FROM SOURCE TO VICTIM. — The typhoid bacillus which reaches the outer world in the feces and urine of carriers and cases is fortunately not a very resistant organism. It requires moisture and a favorable temperature approaching 37.5° C. for multiplication, and suitable nutritive material. These conditions being unfavorable, it is -subjected to a rapid diminution in concentration by dilution, and dies out with relative speed. In sewage and feces, moreover, it is subject to rapid destruction in the competition with the more hardy plebeians with which it comes in contact. In feces the organisms will live for very variable periods according to temperature and conditions governing decomposition. They may be destroyed within a day or two, and in cesspools, etc., where they are immediately mixed with large numbers of decomposing feces, they live for probably not longer than a few days under any conditions. If feces are frozen, that is, deposited in the open in the winter, the organisms may live throughout the winter and enter water sheds, etc., with the thaw. In water, as a rule, they do not live more than a few days or perhaps a week, and according to Rosenau they live longer in clean than in con- taminated water. In sewage their life is short. Freezing does not kill them. According to the investigations of Gartner105 the typhoid bacilli could be found in the flowing water of the Paris water supply after a day and one-half. In all statements of this kind it must be remembered by the sanitarian that no absolute rules can be set up, since we know that the inability of all microorganisms like the typhoid bacillus depend very delicately upon temperature, nutrition, the presence of other bacteria, moisture, heat, light reaction, etc. We have ourselves seen typhoid bacilli alive and viable after many years of sealing in agar cultures preserved in the dark and in a cool place, and, while in nature 102 Bully, Zeit. f. Hyg., 61, 1911, 29. ^Kalberlah, Med. Klinik., 1915. 104 Geronne, Berl. klin. Woch., 1915. 105 Gartner, Klin. Jahrb., 9, 1902. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 677 the organisms disappear with relative speed, no rule can be set up. Klein 106 claims to have found typhoid bacilli alive in natural waters for as long as thirty-six days. Since the organisms can remain in the soil for limited periods of time, unwashed vegetables, salads, etc., are dangerous, and, as we shall see, oysters grown near sewage outlets, may also be sources of infection. CHANNELS OF TRANSMISSION. — Suspicion of typhoid infection by means of water supplies dates back to the early writing of the English physician, Budd, in 1856, who not only believed that sewage contam- inated water conveyed the disease, but suggested that the origin of this pollution lay in human feces. Since that time, of course, bacteriological investigation and sanitary water purification on a large scale has indis- putably proven the danger of water supplies in this respect. In a few cases, direct proof of typhoid bacilli in the water supply has been brought, but, as a rule, indirect proof has had to be adduced, since the rapid dilution, the usual lateness of water investigation after the occurrence of cases, and the many agencies which lead to the destruc- tion of typhoid bacilli in water supplies, has made it extremely difficult to find the organisms in the water. Indirect evidence, however, has been sufficiently convincing in that colon bacillus tests have revealed massive human feces contamination of water to which typhoid infection could be epidemiologically traced. Also, in many localities the direct diminution of typhoid fever in a community after purification of the water supply has left little room for doubt. Thus, in Schuder's107 inves- tigations of 640 epidemics, 72 per cent were directly traceable to water. Water was unquestionably in former years the most important means of the conveyance of typhoid fever when it occurred in definite epi- demics, and whenever typhoid cases occur in any considerable number in cities and towns, the water supply must first be excluded as a source of infection. It must also always be taken into consideration when typhoid fever occurs in country districts where small well supplies are the chief sources of drinking water. In Schiider's statistics, 110 of his 640 epidemics could be indirectly traced to milk. Gay76 states that in the rural communities typhoid fever has remained more or less stationary during the last ten years, while, in the cities, owing probably to water-supply supervision, it has been diminishing progressively. The methods of examining water under such circumstances will be detailed in the section on water, where emphasis will also be placed lofi Klein, Medical Officers Report, Local Govern. Board. 107 Schiider, Zeit. f. Hyg. u. Infec., 38, 1901, 343. 678 PATHOGENIC MICROORGANISMS upon tnc fact that bacteriological water examinations of this kind must always be associated with careful sanitary survey of the water shed and engineering examination of the purification plant, if there is one avail- able. It is important for the sanitarian to remember that, while water epidemics are constantly diminishing as large scale water purification becomes more and more universal, there are still occasional epidemics in which accidents have occurred to ordinarily properly functioning purification plants. ' Such an epidemic was recently reported from Salem, Ohio,108 where in September and October of 1920, following a rainy period, enteritis of unknown origin afflicted about one-half the population. Subsequent investigation showed that 3 cases of typhoid fever had occurred in early September; and typhoid fever reports began in late October and early November. Investigation of the water supply revealed pollution probably due to the contamination of one of the gravity lines connecting a group of wells with the reservoir. Aside from the earliest cases mentioned above, the first cases appeared about October 1st, and reached a peak of 54 new cases on November 1st, which was the highest daily number of the epidemic. Up to November 20th, a total number of 785 cases, with 12 deaths occurred. Recently, we have heard of another epidemic which occurred in a California town, where a small explosive outbreak of typhoid fever occurred owing to accident to the water supply followed by direct pumping from the river, for one day, necessitated by repairs. The considerable and unexceptional diminution of typhoid fever in all cities where water supply purification plants have been installed, may be found tabulated in such books as Rosenau's Hygiene, Mason's book on water supply, and others. Milk may act as a distributor of typhoid fever either by direct infec- tion of the milk from milk handlers who are carriers, or from bottles that are returned from houses where typhoid fever or typhoid carriers exist. A considerable number of milk epidemics have been traced beyond doubt, and have usually been characterized by an explosive onset and by the fact that the majority of the patients were women and children. Milk is an excellent culture media for the typhoid bacillus, and an enormous increase of the organisms in the milk between contam- ination and consumption may occur without visible changes in the milk. Uncooked vegetables, salad, radishes, etc., may be responsible for typhoid infection, and of recent years it has also been shown that oysters may 108 Jour, of the A. M. A., 75, 1920, 1498. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 679 be a source of danger. Conn 109 was the first to suggest this, tracing an epidemic, which broke out, to this cause. Experiments by Foote no showed that typhoid bacilli may be found alive in oysters, within three weeks after they had disappeared from the surrounding water. While there is very little question as to the possibility of this form of infection, it probably does not occur very often. In the investigations of Rose- nau, Lurnsden and Kastle m it was found to be a negligible factor in the cases of typhoid fever occurring in the District of Columbia. That flies play a very important role in the carrying of typhoid bacilli from feces to food, was suggested by Vaughan and by Veeder 112 in 1898. Vaughan studied this particularly, and showed that in the Army camps in 1898, flies flew directly from the latrines to the kitchen; in fact, he found hypochlorite of lime on the food, picked up by the flies in the latrines. During the late war, there can be very little question about the fact that the enormous morbidity of intestinal diseases which occurred in the Allied Armies at various times, and especially during the July offensive at Chateau Thierry, was caused by open latrines and flies, typhoid epidemics being avoided only by the universal vaccination of the Armies. As water supplies, milk supplies, etc., are being supervised and, therefore, excluded as sources of typhoid infection, contact infection is becoming more and more important. As a matter of fact, the recent studies of typhoid morbidity seem to indicate that contact infection is growing to be the chief problem in the prevention of typhoid fever. Frosch who analyzed 978 cases, concluded that 65.6 per cent were con- tact infections, and Drigalski makes similar estimates. Such infections may be from individual to individual by close contact. They may be from cook and kitchen personnel, to raw food to consumer. Instances of such infection are frequent, the most famous one being that of ''Typhoid Mary" who was recently made the subject of a special pub- lication by Soper.113 This woman, a cook, worked for eight families in the course of ten years, during which time 7 outbreaks directly trace- able to her occurred. Since that time her movements from place to place 'have usually been followed by circumscribed epidemics. Again, 109 Conn, Medical Record, December, 1894. 110 Foote, Medical News, 1895. 111 Rosenau, Lumsden and Kastle, Pub. No. 52, Hyg. Lab. U. S. Pub. Health Serv., 190S. 112 Veeder, Medical Record, 45, 1898. 113 Soper, Military Surgeon, 45, 1919, 1. 680 PATHOGENIC MICROORGANISMS contact infection may take place from fomites — fingers — food to mouth, that is, towels, bed clothing, underclothing, etc., and emphasizes the importance of sanitary paper towels, etc., in toilets. Since in contact infection the case is of relatively little danger, largely because of the fact that danger of a case is so well recognized and precautions against transmission from such a source are easily taken, and have become matters of routine in well-regulated sick-rooms and hospitals, the interest in these infections centers upon the typhoid carrier. The Prevention of Typhoid Fever.114 — The measures which are necessary for the prevention of typhoid fever can be easily deduced from a consideration of the material in the foregoing paragraphs. Of great importance is recognition of cases, hospitalization and isola- tion. During such hospitalization there should be careful attention to disinfection of discharges, sterilization of bedding, bed-pans, eating utensils, etc., etc. Patients should never be discharged from hospitals until the urine and feces have been found free from typhoid bacilli, and several examinations at intervals of two or three days should be negative before this is considered to be the case. . . Attention to sewage disposal, water supplies, filtration and chlorina- tion of water with constant supervision of such plants from both a bac- teriological, chemical and engineering point of view. Similar supervision of milk supply, with especial attention to the carrier state of the personnel of dairies and milk handlers. Public health arrangements for the immediate epidemiological study of cases which occur and laboratory facilities for the tracing of carriers indicated by such epidemiological studies. Eventual examination for the carrier state of food handlers, pro- fessional cooks, and exclusion from such professions of people found to be carriers. Community measures for the suppression of fly-breeding places and flies, screening of kitchens, and the absolute elimination of open latrines of any kind. The prevention of oyster culture near sewage outlets. Finally, more and more attention must be given to generalized vaccination. Vaccination and Specific Therapy in Typhoid Fever. — The failure to produce a soluble toxin from typhoid cultures has naturally so far 114 See Rosenau's Hygiene — Also Zinsser — Prevention of Com. Dis. in Nelson's System, 1921. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 681 precluded the possibility of an antitoxin therapy, such as that which has been successful in diphtheria. In the light of our present knowledge of the poisonous products of the typhoid bacillus it seems but natural that attempts by earlier investigators to apply the -principles of Beh- ring's work to typhoid fever were doomed to fail. Attempts to employ specific bactericidal and bacteriolytic sera for therapeutic purposes in this disease have also been without favorable result. Active Prophylactic Immunization. — We have seen that work by Pfeiffer and Kolle and later by many others has shown that it is com- paratively easy to immunize animals actively against typhoid infection by the sytematic injection of graded doses, at first of dead bacilli, later of fully virulent live cultures. Attempts to apply these principles pro- phylactically have been made recently on a large scale by Wright and his associates upon English soldiers in South Africa, and by German observers in East Africa. The first recorded experiment of this sort which was done upon human beings was that of Pfeiffer and Kolle,115 who in 1896 treated two individuals with subcutaneous injections of an agar culture of typhoid bacilli which had been sterilized at 56° C. The first injection was made with 2 cmm. of this culture. Three or four hours after the injection the patient suffered from a chill, his temperature gradually rose to 105° F., and there was great prostration and headache, but within twenty-four hours the temperature had returned to normal. This experiment showed that such injections could be practiced upon human beings without great danger. Simultaneously with the work of Pfeiffer and Kolle, Wright116 conducted similar experiments on officers and privates in the English army. The actual number of persons treated directly or indirectly under Wright's 117 supervision in an investigation covering a period of over four years comprised almost one hundred thousand cases. The methods employed by Wright have been modified several times in minor details; the principles, however, have remained consistently the same. In the first experiments Wright employed an agar culture three weeks old, grown at 37° C., then sterilized at a temperature below 60° C., and pro- tected from contamination by the addition of five-tenths per cent of 115 Pfeiffer und Kolle, Deut. med. Woch., xxii, 1896, xxiv, 1898. 116 Wright, Lancet, Sept., 1896. 117 Wright and Semple, Brit. Med. Jour., 1897; Wright and Leishmann, Brit. Med. Jour., Jan., 1900. 682 PATHOGENIC MICROORGANISMS carbolic acid. Later, Wright ll8 employed bacilli grown in a neutral 1-per-cent pepton bouillon in shallow layers of flasks. Great importance is attached both to the virulence of the typhoid strain, which may to a moderate extent be standardized by passage through guinea-pigs, and to care in using low temperatures for final sterilization. The temperature recommended by Harrison, is 52° C. after which the cultures are carbolized. It is nevertheless extremely difficult to tabulate satisfactory statis- tics from a mass of experiments observed by a large number of indi- viduals. On the whole, however, it seems fair to state that advan- tageous results followed the active immunization practiced by Wright. Wright's own estimation, in a careful attempt to present the subject fairly, gives a reduction of the morbidity from typhoid fever in the British army of 50 per cent, and a reduction of the mortality of those who became infected in spite of inoculations of 50 per cent also. It is not at all impossible that a number of different strains will have to be used eventually for the ideal vaccine, inasmuch as the antigenic differ- ences which have been recently discovered would make it seem that no single strain can be expected to produce antibodies which would protect against all other strains. It is not impossible that some individual strain may combine the antigenic properties of the entire group. This, however, has still to be worked out. The method of Pfeiffer and Kolle consists in the injection of salt- solution emulsions of fresh agar cultures sterilized at 60° C. The results reported were in general favorable. Extensive tests in the United States Army, observed by Russell,119 have removed any doubt which may have existed as to the efficacy of prophylactic typhoid vaccination. Russell's statistics show a steady decline of typhoid in the U. S. Army beginning with the introduction of compulsory vaccination in 1910. In 1913 there was but one case among over 80,000 men. The method at present employed is as follows: The "Rawlings" strain of typhoid, obtained from Wright, is used. Eighteen-hour agar cultures in Kolle flasks are washed off with sterile saline to an approxi- mate concentration of one billion to the cubic centimeter. The sus- pension is killed at 53° C. for one hour and 0.25 per cent tricresol is added. Aerobic and anaerobic culture controls are made and a rabbit 118 Wright, Brit. Med. Jour., 1901; Lancet, Sept., 1902; Brit. Med. Jour., Oct., 1903. 119 Russell, Am. Jour, of Med. Sc., cxlvi, 1913. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 683 and mouse inoculated to insure sterility. For immunization 3 to 4 doses are given ranging in quantity from 500 million to one billion at seven- to ten-day intervals. The protection probably lasts about two years, though this is not certain. Another point of importance in this connection has recently been raised by Metchnikoff and Besredka.120 They vaccinated chimpanzees with typhoid bacilli and found that when emulsions of the clear bac- teria were used, protection was only slight. Better results were obtained —that is, apparently complete protection within eight to ten days — when living sensitized bacteria were injected (bacteria which had been exposed to the action of inactivated immune serum.) Broughton has applied this method to human beings. Gay76 has also prepared a sen- sitized dead typhoid vaccine which he has used in a considerable number of cases. It will take some time, however, before a statistical estimation of the superiority of this method over the older vaccination with dead bacteria will be possible. Gay76 sensitizes his bacilli with strong immune serum, precipitates with alcohol, dries and grinds them into powder, and uses weighed amounts of the powder. During recent years in France another form of vaccine has been used which has advantages which lead to its general adoption, if its im- munizing value can be successfully proven. This is the so-called "lipo- vaccine." It consists of typhoid cultures grown on agar, taken up in salt solution, the sediment partially dried, and then shaken up with olive oil, the formula for which is not available. It can also be produced by grind- ing up the typhoid sediment with lanolin and shaking up this well-ground paste with sterile olive oil. Great difficulty has been encountered in the sterilization of this vaccine, a matter which still needs much investiga- tion. The vaccine has the advantage of giving much diminished reac- tions, and it is claimed that three times the amounts given in the ordinary saline suspensions can be given without serious discomfort to the patient. There is probably slow absorption of this vaccine and it is claimed that a single dose, because of the slow absorption of the organ- isms, may be sufficient to vaccinate. During the recent war, typhoid vaccination has thoroughly justified itself. We are not in possession at the present writing, of consolidated reports of all the European Armies, but the Surgeon General's report for the United States Army, published in 1919, shows the magnificent results obtained by vaccination in American troops. During the pre-vaccination 120 Metchnikoff and Besredka, Am. dc Tinst. Past., 1911. 684 PATHOGENIC MICROORGANISMS days in the Civil War and the Spanish American War, the admission rates for typhoid fever were enormous. During the first year of the Civil War the annual admssion rate was 70.69, with a death rate of 19.61, and it is likely that, in addition to this, a large number of unrecognized cases occurred. During the Spanish American War and the Philippine Insurrection in the years 1898 to 1899, the annual admission was 91.22, and the death rate 9.67. During the last World War the method of vaccination used consisted in three inoculations at seven-day intervals, of the salt solution suspension triple vaccine, containing typhoid "Rawlings," paratyphoid A and B, the first dose containing one-half million bacilli, and the second and third containing a billion each. The typhoid rate was so low in the camps in the United States that a young man in the camp had 45 times less opportunity of getting typhoid fever than did the same age group in civilian life during the same period. Although approximately three million men passed through the camps during the course of 1918, the actual admission rate for the United States was 0.17. In Europe, in spite of the most terrific sanitary condi- tions in some of the battlefields during the summer, and with perhaps two million troops in France, there were only 488 cases with 88 deaths, and this, in spite of the fact, as we, ourselves, observed, that the oppor- tunities for transmission were enormous in battle areas in which sani- tation was practically impossible, and water supplies were bad and could not be corrected. The question still remains as to how long typhoid vaccination can be regarded as efficient. There is no absolute information upon which opinions can be based. Vaccination is not a complete protection at any time, and a recently vaccinated individual may still occasionally con- tract the disease if he is injected with a large dose of virulent organisms. The protection, however, is very powerful and will prevent contraction of the disease from the ordinary chance infection. We should state on general information that repetition every two years ought to be suf- ficient for civilian purposes. For the armies in the field, we ourselves would favor a first vaccination with three doses as stated above, and single or double doses repeated every six months. Specific Treatment of Typhoid Fever.7® — Anti-sera against typhoid fever have been produced by a large number of workers, notably Chante- messe 121 and Besredka 122 both of whom used the serum of horses immunized with typhoid bacilli or "endo-toxin," so-called. Garbat 121 Chantemesse, Prog, med., 7, 1989, 245. 122 Besredka, Ann. Inst. Past., 16, 1902, 918. BACILLI OF THE COLON -TYPHOID-DYSENTERY GROUP 685 and Meyer 123 believed that an improvement of results could be obtained by mixing the sera of animals that had been immunized with sensitized bacteria and those treated with normal typhoid bacilli. At the present time, however, practice has not sustained the hopes of a specific passive immunization in the treatment of typhoid fever. Since 1893, various workers have tried to treat typhoid fever by injecting killed cultures or vaccines of typhoid bacilli. No results of importance were obtained until Ichikawa 124 in 1914 began to inject dead typhoid bacilli, intravenously. Gay and Claypole 125 and others have since taken up this method. The intravenous injection of vac- cines in this way has given most astonishing results in that the injection has usually resulted in a violent reaction, with often a chill and sudden drop of temperature, and, subsequently, very often definite improve- ment of the cases. Although this method was at first regarded as spe- cific by the writers mentioned above, Kraus 126 in 1915 showed that he could produce similar reactions in typhoid patients with colon bacilli, as well as with typhoid bacilli, and similar observations were made by Liidke 127 and others. It is now quite clear that, whatever results are obtained by such treatment of typhoid patients, they cannot be regarded as specific reactions in the ordinary sense of the word. 123 Garbat and Meyer, Zeit. f. exper. Path., 8, 1910, 1. 124 Ichikawa, Mitteil. d. medic. Gessellsch. zu. Tokio, 1914, 28, H. 21. 125 Gay and Claypole, Arch. Inter. Med., 12, 1913, 613. 126 Kraus, Wien. klin. Woch., 1915, 29. 127 Liidke, Munch, med. Woch., 1915, 321. * For a thorough and clear treatment of the problem connected with typhoid fever, see Gay, Typhoid Fever, MacMillan and Company, New York, 1918. CHAPTER XXXIII BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP (Continued) BACILLI OF THE PARATYPHOID— ENTERITIDIS GROUP AND THE PARATYPHOID INFECTIONS (Bacilli of Meat Poisoning and Paratyphoid Fever) THERE is an extensive group of Gram-negative bacilli which because of their morphology, cultural behavior, and pathogenic properties, are classified as intermediate between the colon and the typhoid types. The microorganisms belonging to this group have been described, most of them, within the last fifteen years, but few of them have been fully identified with one another. They have been variously designated as the " hog-cholera group," "the enteritidis group," the "paracolon group" or "paratyphoid group," because of the pathological conditions with which the chief members under investigation have been found associated. Attempts to systematize the group by the comparative study of a large number of its members have been made, notably by Buxton l and by Durham,2 and the work of these writers, based on cultural and agglutinative studies, has added materially to our knowledge of these organisms. The microorganisms of this group are morphologically indistinguish- able from the colon and typhoid bacilli. They are Gram-negative and possess flagella. Their motility is variable, but usually approaches that of the typhoid bacilli in activity. They correspond, furthermore, to the two other groups in their cultural characteristics upon broth, agar, and gelatin. On potato they vary, some of them approaching in delicacy the typhoid growth upon this medium, others more closely approximating the heavy brownish growth of B. coli. Indol is rarely formed by them, though this has not been absolutely constant in all descriptions. As a group, they are easily distinguished from Bacillus 1 Buxton, Jour. Med. Res. N. S., iii, 1900. 2 Durham, Jour. Exper. Med., v, 1901. 686 BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 087 typhosus on the one hand, and from Bacillus coli on the o.ther> by the following simple reactions tabulated by Buxton.3 B. Coli. Paratyph. etc. B. Typhosus. Coagulation of milk -f- Production of indol Fermentation of lactose with gas -f Fermentation of dextrose Wtth gas Agglutination in typhoid-immune serum - (ac.) The simplest differentiation between the large groups can be made by fermentation tests as follows : . Gas upon Dextrose. Gas upon Lactose. Gas upon Saccharose. Gas on Dulcit. B typhosus Intermediates B. coli communis B. coli communior + + + + + + + + B. acidi lactici B. lactis aerogenes 4 + -}- + + + 4 Jackson. Pathogenically, the bacilli of this " intermediate group" have attracted attention chiefly in connection with meat poisoning, and with protracted fevers indistinguishable from mild typhoidal infections. General Survey of the Group. — In 1888, Gartner5 described a bacillus which he isolated from the meat of a cow, the ingestion of which had produced the symptoms of acute gastrointestinal catarrh in 57 people. One of these died of the disease and the bacilli could be demonstrated in the spleen and blood of the patient. This bacillus, called Bacillus enteritidis by Gartner, was actively motile, formed no indol, but produced gas in dextrose media. Acute gastrointestinal symptoms could be induced by feeding the organisms to mice, guinea-pigs, rabbits, and sheep, and the bacilli could be recovered from the infected animals. The bacterial bodies themselves were found by Gartner to be toxic, containing a poison which was extremely resistant to heat. Sterilized cultures showed the same pathogenic effects as the living bacilli. Epidemics of meat poisoning sim- 3 Buxton, loc. cit. 5 Gartner, Corresp. Bl. d. Aerzt. Vereins, Turingen, 1888. 688 PATHOGENIC MICROORGANISMS ilar to the one described by Gartner, in which similar bacteria were isolated, were those described by Van Ermengem,6 occurring at Morseele in 1891, the one described by Hoist,7 the Rotterdam epidemic described by Poels and Dhont,8 the one described by Basenau, and many others. Bacillus Morseele of Van Ermengem, Bacillus bovis morbificans of Basenau,9 and bacilli isolated in similar epidemics by others, are, except for slight differ- ences in minor characteristics, almost identical with Gartner's microorganism. In 1893, Theobald Smith and Moore 10 noted a great similarity between the so-called hog-cholera bacillus, the bacilli of the Gartner group, and Bacillus lyphi murium isolated by Loeffler. These observers first used the term " hog- cholera" group for the organisms under discussion. In 1899 Reed and Carroll n noted that Bacillus icteroides, associated by Sanarelli with yellow fever, was culturally similar to the bacillus of hog cholera. Meanwhile, other observers had been isolating bacilli, similar to those spoken of above, from cases of protracted fevers in human beings, often closely simulating typhoid infections. The first cases of this kind on record were those of Achard and Bensaude.12 In 1897, Widal and Nobecourt 13 described a bacillus which they had isolated from an esophageal abscess following typhoid fever, which closely resembled Bacillus psittacosis of Nocard,14 and which, following a nomenclature previously suggested by Gilbert,15 they designated the paracolon bacillus. This micro- organism, isolated from a parrot by Nocard, showed a close resemblance to bacilli of the Gartner group. There are a large number of apparently nonpathogenic organisms sometimes referred to as paratyphoid C, but better perhaps as "heterogeneous types," which are culturally identical with the paratyphoid but do not agglutinate in either paratyphoid A or B sera. An anti-serum produced with these types usually reacts with the homologous strain only. Such organisms have been studied by Krumwiede and many others, including ourselves. Strains recently isolated at this laboratory came from casss of nephritis, German measles, jaundice, and the stools of healthy soldiers, plated as a matter of routine. In 1898, Gwyn16 reported a case at the Johns Hopkins Hospital, which pre- 6 Van Ermengem, Bull, Acad. d. mcd. de Belgique, 1892; "Trav. de lab. de Puniv. de Gand," 1892. 7 Hoist, Ref. Cent. f. Bakt., xvii. 1805. 8 Poels und Dhont, Holland Zeit. f. Tierheilkunde, xxiii, 1894. 9 Basenau, Arch. f. Hyg., xx, 1894. 10 Th. Smith and Moore, U. S. Bureau of Animal Industry Bull, vi, 1894. 11 Reed and Carroll, Medical News, Ixxiv, 1899. 12 Achard and Bensaude, Bull, de la soc. d. hopitaux de Paris, Nov., 1906. 13 Widal et Nobecourt, Semaine mod., Aug., 1897. 14 Nocard, Ref. Baumgartcn's Jahresb., 1896. 15 Gilbert, Semaine mcd., 1895. 16 Gwyn, Johns Hopkins Hosp. Bull., 1898. BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP 689 sented all the symptoms of typhoid fever, but lacked serum agglutinating power for Bacillus typhosus. From the blood of the patient, Gwyn isolated an organ- ism, with cultural characteristics similar to those of the Gartner bacillus, which he called a "paracolon bacillus." This bacillus was agglutinated specifically by the serum of the patient. Gushing,17 in 1900, isolated a similar microorganism from a costochondral abscess, appearing during convalescence from supposed typhoid fever. In the same year, Schottmuller 18 reported five cases from which similar bacilli were isolated. Careful cultural studies of the microorganisms here obtained showed that they could be divided into two similar, yet distinctly different types, one of them, the "Miiller" organism (later "A" type), approach- ing closely to the typhoid type, especially in its growth upon potato; the other, the "Seeman" type (later "B" type) corresponding more closely to the Gart- ner bacilli. Similar cases were reported by Kurth,19 Buxton and Coleman,20 Libman,21 and others. The two types of organisms, paratyphoid A and B described by Schott- muller and studied by many other observers, can be culturally differentiated, though not without difficulty. Differential Considerations. — The differentiation of the various organisms within the paratyphoid, enteritidis group is a very difficult matter. The paratyphoid "A" organisms split off rather easily from the rest of them, and seem to represent a fairly homologous group. The paratyphoid " A" organisms, as shown by Ford,22 and more particularly by Krumwiede and Kohn,23 differ from the rest of the organisms of this general group in not fermenting xylose. Also, they do not change lead acetate agar. Serologically, it may be said in a general way, though it is not absolutely true, that paratyphoid "A" represents an antigenically homologous group and that a serum produced with one member of the "A" group will, in a general way, possess antibodies against other "A" organisms. The remaining organisms of this paratyphoid group differ materially from each other and cannot be subdivided into final groups, as yet. However, a tentative grouping, based partly upon the sources from which they were obtained and partly upon fermentations and serological reactions, can be attempted, and a great deal of valuable work in this direction has been done by Theobald Smith and his co- 17 Gushing, Johns Hopkins Hosp. Bull., 1900. 18 Schottmuller, Deut. med. Woch., 1900; Zeit. f. Hyg., xxvi. 19 Kurth, Deut. med. Woch., 1901. 20 Buxton, and Coleman. Proc. N. Y. Pathol. Soc., Feb., 1902. 21 Libman, Jour. Med. Res., N. S., iii, 1902. 22 Ford, Med. News, June 17, 1905. 23 Krumwiede and Kohn, Jour. Med. Res., 36, 1917, 509. 690 PATHOGENIC MICROORGANISMS workers, by Smith and Ten Broeck,24, by Krumwiedc, Pratt and Kohn,25 and many others. The following fermentation chart indicates briefly a summary of the reactions of the more common members of this group, chiefly constructed according to the work of Krumwiede and his co- workers. Lead Dex- trose. Man- nit. Lac- tose. Xylose Rham- nose. Sac- cha- Dulcit Ace- tate Indol. Motil- ity. rose. Agar. B. parat. "A". . . © © _ _ + _ slow _ C *» £3-2 + B. parat. "B". . . © © — + + — + + ^^ y -*-» + B. enteritidis © © — -f- 4. — -j- 4. c"wi"c c 4- B. abortus equi . . © © - + + - + - s ° v ° + B. hog cholera. . . © © — -f -f- — irreg. — g^ ^"c+i 4. B. typhi murium . © © - + + - + S-SjS-S'g -j- ^^ v ' FIG. 71. — BACILLUS MUCOSUS CAPSULATUS. dyes, but is decolorized when stained by the Gram-method. Capsules may often be seen when the more intense anilin dyes are employed. They are brought out with much regularity by any of the usual capsule stains. Cultivation. — B. mucosus capsulatus is easily cultivated. It grows readily on all the usual culture media, both on those having a meat- infusion basis and on those made with meat extract. Growth takes place at room temperature (18° to 20°) and more rapidly at 37.5° C. A temperature of 60° C. and over kills the bacilli in a short time. The thermal death-point- according to Sternberg is 56° C. Growth 722 PATHOGENIC MICROORGANISMS ceases below 10° to 12° C. Kept at room temperature and protected from drying, the bacillus may remain alive, in cultures, for several months. The bacillus is not very fastidious as to reaction of media, grow- ing equally well on moderately alkaline or acid media. It is aerobic and facultatively anaerobic; growth under anaerobic conditions, however, is not luxuriant. On agar, growth appears in the form of grayish-white mucus-like colonies, having a characteristically slimy and semi-fluid appearance. Colonies have a tendency to confluence, so that on plates, after three or four days, a large part of the surface appears as if covered with a film of glistening, sticky exudate, which, if fished, comes off in a tenacious, stringy manner. It is often possible to make a tentative diagnosis of the bacillus from the appearance of this growth. In broth, there is rapid and abundant growth, with the formation of a pellicle, general clouding, and later the development of a pro- fuse, stringy sediment. Stab cultures in gelatin show, at first, a white, thin line of growth along the course of the puncture. Soon, however, rapid growth at the top results in the formation of a grayish mucoid droplet on the surface, which, enlarging, gives the growth a nail-like appearance. This nail-shape was originally described by Friedlander and regarded as diagnostic for the bacillus. The gelatin is not fluidified. As the culture grows older the entire surface of the gelatin tube may be covered with growth, flowing out from the edges of the nail-head. The gelatin acquires a darker color and there may be a few gas bubbles below the surface. Microscopically, colonies on gelatin plates have a smooth outline and a finely granular or even homogeneous consistency. On blood serum, a confluent mucus-like growth appears. On potato, abundant growth appears, slightly more brownish in color than that on other media. In pepton solutions, there is no indol formation. In milk, there is abundant growth and marked capsule develop- ment. Coagulation occurs irregularly. In considering the general cultural characteristics of the Fried- lander bacillus, it must not be forgotten that we are dealing with a rather heterogeneous group, the individuals of which are subject to many minor variations. Capsule development, lack of motility, in- ability to fluidify gelatin, failure to form indol, and absence of BACILLUS MUCOSUS CAPSULATUS 723 spores, are characteristics common to all. In size, general appear- ance, gas formation, and pathogenicity, individual strains may vary much, one from the other. Strong4 has studied various races as to gas formation and concludes that most strains form gas from dextrose and levulose, but that lactose is fermented by some only. About two-thirds of the gas formed is hydrogen, the rest C02. Acid formation, according to Strong, is also subject to much variation among different races. Similar studies by Perkins5 show that most of the ordinary cultural characteristics of bacilli of this group are extremely variable and can not serve as a basis for differentiation. Reactions on sugars, however, are more constant. Perkins suggests the following tentative division classes on this basis: I. All carbohydrates fermented with the formation of gas. II. All carbohydrates, except lactose, fermented with the forma- tion of gas. III. All carbohydrates, except saccharose, fermented with the formation of gas. Type I. corresponds to B. aerogenes (Migula), type II. to B. Friedlander or Bacterium pneumoniae (Migula), and type III. to Bacillus lactis aerogenes. Differentiation by means of serum reactions has not proved satis- factory.6 Pathogenicity. — When Friedlander first described this micro- organism, he assumed it to be the incitant of lobar pneumonia. Sub- sequent researches by Weichselbaum7 and others have shown it to be etiologically associated with pneumonia in about seven or eight per cent of all cases. The percentage in this country is probably lower. Such cases can often be diagnosed by the presence of the bacilli in the sputum, which is peculiarly sticky and stringy. Cases 4 Strong, Cent, f . Bakt., xxv, 1899. 5 Perkins, Jour. of. Infect. Dis., I, No. 2, 1904. 6 J. G. Fitzgerald, who has recently made a careful study of the mucosus cap- sulatus group has concluded that present methods do not permit a subdivision of these organisms into separate species. He offers the following "tentative sug- gestion": "It is conceivable that mutations based on the necessity of maintaining a parasitic existence have caused Gram-negative bacilli found normally in the body elsewhere than in the intestinal tract to develop capsules for protection and a new group has arisen which we designate B. mucosus capsulatus; and the varieties B. aerogenes and B. acidi lactici connect the group with the non-encapsulated colon group." 7 Weichselbaum, loc. cit. 724 PATHOGENIC MICROORGANISMS of Friedlander pneumonia are extremely severe and usually fatal. The bacillus has been found in cases of ulcerative stomatitis and nasal catarrh; in two cases of severe tonsillitis in children; in the pus from suppurations in the antrum of Highmore and the nasal sinuses (Frankel and others), and in cases of fetid coryza (ozena), of which disease it is supposed by Abel8 and others to be the specific cause. Whether the ozena bacillus represents a separate species or not, can not at present be decided. The bacillus of Friedlander has been found in empyema fluid, in pericardial exudate (after pneumonia), and in spinal fluid.9 Isolated cases of Friedlander bacillus septicemia have been described.10 Being occasionally a saprophytic inhabitant of the normal intestine, it has been believed to be etiologically associated with some forms of diarrheal enteritis. B. mucosus capsulatus is pathogenic for mice and guinea-pigs, less so for rabbits. Inoculation of susceptible animals is followed by local inflammation and death by septicemia. If inoculation is intraperitoneal, there is formed a characteristically mucoid, stringy exudate. The question of immunization against bacilli of the Friedlander group is still in the stage of experimentation. Immunization with carefully graded doses of dead bacilli has been successful in isolated cases. Specific agglutinins in immune serum have been found by Clairmont,11 but irregularly and potent only against the particular strain used for the immunization. OTHER BACILLI OF THE FRIEDLANDER GROUP Bacillus of Rhinoscleroma.— This bacillus, described by v. Frisch12 in 1882, is a plump, short rod, with rounded ends, mor- phologically almost identical with Friedlander 's bacillus; it is non- motile and possesses a distinct capsule. Although at first described as Gram-positive, it has been shown to be decolorized with this method of staining. It forms slimy colonies, has a nail-like appearance in gelatin stab cultures, and in pepton solutions produces no indol. It differs from B. mucosus capsulatus (Wilde13) in forming no gas in M6eZ, Zeit. f. Hyg., xxi. 9 Jager, Zeit. f . Hyg., xix. 10 Howard, Johns Hopkins Hosp. Bull., 1899. 11 Clairmont, Zeit. f. Hyg., xxxix. 12 Frisch, Wien. med. Woch., 1882. 13 Wilde, Cent, f . Bakt., xx, 1896. OTHER BACILLI OF THE FRIEDLANDER GROUP 725 dextrose bouillon, in producing no acid in lactose bouillon, and in never coagulating milk. Pathogenicity. — The bacillus of rhinoscleroma is but moderately pathogenic for animals delicately susceptible to the bacillus of Fried- lander. Rhinoscleroma, the disease produced by this bacillus in man, consists of a slowly growing granulomatous inflammation, located usually at the external nares or upon the mucosa of the nose, mouth, pharynx, or larynx. It is composed of a number of chronic, hard, FIG. 72. — BACILLUS OF RHINOSCLEROMA. Section of tissue showing the micro- organisms within Mikulicz cells. (After Frankel and Pfeiffer.) nodular swellings, which, on histological examination, show granu- lation tissue and productive inflammation. In the meshes of the abundant connective tissue lie many large swollen cells, the so-called ' ' Mikulicz cells. ' ' 14 The rhinoscleroma bacilli lie within these cells and in the intercellular spaces. They can be demonstrated in his- tological sections and can be cultivated from the lesions, usually in pure culture. Rhinoscleroma is rare in America. It is most prevalent in Southeastern Europe. The disease is slowly progressive and comparatively intractable to surgical treatment, but hardly ever affects the general health unless by mechanical obstruction of the air passages. 14 Mikulicz, Arch. f. Chir., xx, 1876. 726 PATHOGENIC MICROORGANISMS B. Ozaense. — The work of Abel15 and others has shown that ozena, or fetid nasal catarrh, is almost always associated witli a bacillus morphologically and culturally almost identical with B. mucosus capsulatus. The bacillus can not be definitely separated from the latter. According to Wilde it forms no gas in dextrose bouillon and is less pathogenic for mice than B. Friedlander. Whether it is a separate species, or merely an atypical form changed by environ- ment, can not be stated at present. Perez Bacillus of Ozsena. — Perez16 in 1899 described another microorganism which he connects etiologically with ozaena. The Perez bacillus is Gram-negative, pleomorphic, non-motile and non- capsulated. It grows easily on ordinary media, does not liquefy gelatin, and makes indol. Its cultures have a characteristic fetid odor. Intravenously injected into rabbits it seems to produce a localized lesion in the nasal cavity on the turbinated bones. Hofer17 has also isolated it, but recent work leaves its importance as the causative agent in doubt. 16 Abel, Zeit. f. Hyg., xxi. 18 Perez, Animal de PInst. Past. 1899. 17 Hofer, Wien. klin. Woch., vol. 26, pp. 1011 and 1628. CHAPTER XXXVI THE ANAEROBIC BACILLI. TETANUS AND BACILLUS TETANI. BOTULISMUS AND THE BACILLUS BOTULINUS LOCKJAW or tetanus, 'though a comparatively infrequent disease, has been recognized as a distinct clinical entity for many centuries. The infectious nature of the disease, however, was not demonstrated until 1884, when Carlo1 and Rattone succeeded in producing tetanus in rabbits by the inoculation of pus from the cutaneous lesion of a human case. Nicolaier,2 not long after, succeeded in producing tetanic symptoms in mice and rabbits by inoculating them with soil. In connection with the lesions produced at the point of inoculation, Nicolaier described a bacillus which may have been Bacillus tetani, but which he was unable to cultivate in pure culture. Kitasato,3 in 1889, definitely solved the etiological problem by obtaining from cases of tetanus pure cultures of bacilli with which he was able again to produce the disease in animals. Kitasato succeeded where others had failed because of his use of anaerobic methods and his elimination of non-spore-bearing con- taminating organisms by means of heat. His method of isolation was as follows : The material containing tetanus bacilli was smeared upon the surface of agar slants. These were permitted to develop at incubator temperature for twenty-four to forty-eight hours. At the end of this time the cultures were subjected to a temperature of 80° C. for one hour. The purpose of this was to destroy all non-sporulating bacteria, as well as aerobic spore-bearers which had developed into the vegetative form. Agar plates were then in- oculated from the slants and exposed to an atmosphere from which oxygen had been completely eliminated and hydrogen substituted. On these plates colonies of tetanus bacilli developed. Morphology and Staining. — The bacillus of tetanus is a slender bacillus, 2 to 5 micra in length, and 0.3 to 0.8 in breadth. The 1 Carlo e Rattone, Giornale d. R. Acad. d. Torino, 1884. 2 Nicolaier, Inaug. Diss., Gottingen, 1885. 8 Kitasato, Deut. med. Woch., No. xxxi, 1889. 727 728 PATHOGENIC MICROORGANISMS vegetative forms which, occur chiefly in young cultures are slightly motile and are seen to possess4 numerous peritrichal flagella, when stained by special methods. After twenty-four to forty-eight hours of incubation, the length of time depending somewhat on the nature of the medium and the degree of anaerobiosis, the bacilli develop spores which are characteristically located at one end, giving the bacterium the diagnostic drumstick appearance. FIG. 73. — BACILLUS TETANI. Spore stain. As the cultures grow older the spore-bearing forms completely supersede the vegetative ones. Very old cultures contain spore- bearing bacilli and spores only. The tetanus bacillus is easily stained by the usual amlin dyes, and reacts positively to Gram's stain. Flagella staining is successful only when very young cultures are employed. Distribution. — In nature, the tetanus bacillus has been found by Nicolaier and others to occur in the superficial layers of the soil. The earth of cultivated and manured fields seems to harbor this 4 Vottaler, Zeit. f . Hyg., xxvii. THE ANAEROBIC BACILLI 729 organism with especial frequency, probably because of its presence in the dejecta of some of the domestic animals. Biological Characteristics. — The bacillus of tetanus is generally described as an obligatory anaerobe. While it is unquestionably true that growth is ordinarily obtained only in the complete absence of oxygen, various observers, notably Ferran5 and Belfanti,6 have successfully habituated the bacillus to aerobic conditions by the gradual increase of oxygen in cultures. Habituation to aerobic con- ditions has usually been accompanied by diminution or loss of pathogenicity and toxin-formation. Anaerobic conditions may like- wise be dispensed with if tetanus bacilli be grown in symbiosis with some of the aerobic bacteria. The addition to culture media of suit- able carbohydrates, and of fresh sterile tissue, has also been found to render it less exacting as to mechanical anaerobiosis.7 Anaerobically cultivated, Bacillus tetani grows readily upon meat- infusion broth, which it clouds within twenty-four to thirty-six hours. Upon meat-infusion gelatin at 20° to 22° C. the tetanus bacillus grows readily, growth becoming visible during the second or third day. There is slow liquefaction of the gelatin. On agar, at 37.5° C., growth appears within forty-eight hours. Colonies on agar. plates present a rather characteristic appearance, consisting of a compact] center surrounded by a loose mesh work of fine filaments, not unlike the medusa-head appearance of subtilis colonies. In agar stabs, fine radiating processes growing out in all directions from the -central stab tend to give the culture the appear- ance of a fluff of cotton. Milk is a favorable culture medium and is not coagulated. On potato, growth is delicate and hardly visible. The most favorable temperature for the growth of this bacillus is 37.5° C. Slight alkalinity or neutrality of the culture media is most advantageous, though moderate acidity does not altogether inhibit growth. All the media named may be rendered more favor- able still by the addition of one or two per cent of glucose, maltose, or sodium formate.8 In media containing certain carbohydrates, tetanus bacilli produce acid. In gelatin and agar, moderate amounts of gas are produced, consisting chiefly of C02,9 but with the admix- 5 Ferran, Cent. f. Bakt., xxiv, No. 1. 6 Belfanti, Arch, per le sci. med., xvi. 7 Th. Smith, Brown, and Walker, Jour. Med. Res., N. S., ix, 1906. 8 Kitasato, Ztschr. f. Hyg. 1891. 9 v. Eisler and Pribram in Kraus and Levaditi, Handbuch, etc., Jena, 1907, 730 PATHOGENIC MICROORGANISMS tures of other volatile substances which give rise to a characteristic- ally unpleasant odor, not unlike that of putrefying organic matter. This odor is due largely to H2S and methylmercaptan. The vegetative forms of the tetanus bacillus are not more re- sistant against heat or chemical agents than the vegetative forms of other microorganisms, Tetanus spores, however, will resist dry FIG. 74. — YOUNG TET- ANUS CULTURE IN GLU- COSE AGAR, FIG. 75. — OLDER TET- ANUS CULTURE IN GLU- COSE AGAR. heat at 80° C. for about one hour, live steam for about five minutes; five per cent carbolic acid kills them in twelve to fifteen hours; one per cent of bichlorid of mercury in two or three hours. Direct sunlight diminishes their virulence and eventually destroys them. Protected from sunlight and other deleterious influences, tetanus spores may remain viable and virulent for many years. Henri jean10 has reported success in producing tetanus with bacilli from a splinter of wood infected eleven years before. 10 Henrijean, Ann. de la soc. med. Chir. de Liege, 1891. THE ANAEROBIC BACILLI 731 Tetanus Toxin. — The pathogenicity of the tetanus bacillus depends entirely upon the soluble toxin which it produces. This toxin is produced in suitable media by all strains of virulent tetanus bacilli, individual strains showing less variation in this respect than do the separate strains of diphtheria bacilli. While partial aerobiosis does not completely eliminate toxin formation, anaerobic conditions are by far more favorable for its development. The medium most frequently employed for the production of tetanus toxin is neutral or slightly alkaline beef-infusion bouillon containing five-tenths per cent NaCl and one per cent pepton. Glu- cose, sodium formate, or tincture of litmus may be added, but while these substances increase the speed of growth of the bacilli they do not seem to enhance the degree of toxicity of the cultures. Glu- cose is said even to be unfavorable for strong toxin development. It is important, too, that the bouillon shall be freshly prepared.11 There does not seem to be any direct relationship between the amount of growth and the degree of toxicity of the cultures. Under anaerobic conditions in suitable bouillon and grown at 37.5° C., the maximum toxin content of the cultures is reached in from ten days to two weeks. After this time the toxin deteriorates rapidly. Tetanus toxin has been produced without resort to mechanical anaerobic methods by several observers, notably by Debrand,12 by cultivating the bacilli in bouillon in symbiosis with Bacillus subtilis. By this method, Debrand claims to have produced toxin which was fully as potent as that produced by anaerobic cultivation. The tetanus toxin, in solution in the bouillon cultures, may be separated from the bacteria by filtration through Berkefeld or Cham- berland filters. Since the poison in such filtrates deteriorates very rapidly, much more rapidly even than diphtheria toxin, various methods have been devised to obtain the toxin in the solid state. The most useful of these is precipitation of the poison out of solu- tion by saturation with ammonium sulphate.13 Very little of the toxin is lost by this method and, thoroughly dried and stocked in vacuum tubes, together with anhydrous phosphoric acid, it may be preserved indefinitely without deterioration. The precipitate thus formed is easily soluble in water or salt solution, and therefore 11 Vaillard et Vincent, Ann. de Pinst. Pasteur, 1891. 12 Debrand, Ann. de 1'inst. Pasteur, 1890, 1902. 13 Brieger und Cohn, Zeit. f. Hyg., xv. 732 PATHOGENIC MICROORGANISMS permits of the preparation of uniform solutions for purposes of standardization. Brieger and Boer14 have also succeeded in precipitating the toxin out of broth solution with zinc chloride. Vaillard and Vincent15 have procured it in the dry state by evaporation in vacuo. Brieger and Cohn,16 Brieger and Boer,17 and others have at- tempted to isolate tetanus poison, removing the proteins from the ammonium sulphate precipitate by various chemical methods. The purest preparations obtained have been in the form of fine yellowish flakes, soluble in water, insoluble in alcohol and ether. Solutions of this substance have failed to give the usual protein reactions. The toxin when in solution is extremely sensitive to heat. Kita- sato18 states that exposure to 68° C. for five minutes destroys it completely. Dry toxin is more resistant,19 often withstanding tem- peratures of 120° C. for more than fifteen minutes. Exposure to direct sunlight destroys the poison in fifteen to eighteen hours.20 Interesting experiments as to the action of eosin upon tetanus toxin have been carried out by various observers. Flexner and Noguchi21 found that five per cent eosin added to the toxin would destroy it within one hour. This action is ascribed to the photo- dynamic power of the eosin. Tetanus toxin is one of the most powerful poisons known to us. Filtrates of broth cultures, in quantities of 0.000,005 c.c., will often prove fatal to mice of ten grams weight. Dry toxin obtained by ammonium sulphate precipitation22 is quantitatively even stronger, values of 0.000,001 gram as a lethal dose for a mouse of the given weight not being uncommon. Brieger and Cohn23 succeeded in pro- ducing a dry toxin capable of killing mice in doses of 0.000,000,05 gram. Different species of animals show great variation in their sus- ceptibility to tetanus toxin. Human beings and horses are probably 14 Brieger und Boer, Zeit. f. Hyg. xxi. 15 Vaillard et Vincent, Ann. de 1'inst. Pasteur, 1891. 16 Brieger und Cohn, loc. cit. 17 Brieger und Boer, Zeit. f. Hyg., xxi. 18 Kitasato, Zeit. f . Hyg., x. 19 Morax et Marie, Ann. de 1'inst. Pasteur, 1902. 20 Fermi und Pernossi, Cent, f . Bakt., xv. 21 Flexner and Noguchi, "Studies from Rockefeller Inst.," v., 1905. 22 Brieger und Cohn., loc. cit. * Brieger und Cohn, Zeit. f . Hyg., xv. THE ANAEROBIC BACILLI 733 the most susceptible species in proportion to their body weight. The common domestic fowls are extremely resistant. Calculated for grams of body weight, the horse is twelve times as susceptible as the mouse, the guinea-pig six times as susceptible as the mouse. The hen, on the other hand, is 200,000 times more resistant than the mouse. After the inoculation of an animal with tetanus toxin there is always a definite period of incubation before the toxic spasms set in. This period may be shortened by increase of the dose, but never entirely eliminated.24 When the toxin is injected subcutaneously, spasms begin first in the muscles nearest the point of inoculation. Intravenous inoculation,25 on the other hand, usually results in general tetanus of all the muscles. . The feeding of toxin does not produce disease, the poison being passed through the bowel un- altered. The harmful action of tetanus toxin is generally attributed to its affinity for the central nervous system. Wassermann and Takaki26 show that tetanus toxin was fully neutralized when mixed with brain substance. Other organs — liver and spleen, for instance — showed no such neutralizing power. The central origin of the tetanic contractions was made very evident by the work of Gumprecht,27 who succeeded in stopping the spasms in a given region by division of the supplying motor nerves. The manner in which the toxin reaches the central nervous system has been extensively investigated, chiefly by Meyer and Ransom, and Marie and Morax. Meyer and Ransom28 from a series of careful experiments reached the conclusion that the toxin is conducted to the nerve centers along the paths of the motor nerves. Injected into the circulation,29 the toxin reaches simultaneously all the motor nerve endings, producing general tetanus. In this case too, there- fore, the poison from the blood can not pass directly into the central nervous system, but must follow the route of nerve tracts. These observations have been of great practical value in that they pointed to the desirability of the injection of tetanus antitoxin 24 Courmont et Doyen, Arch, de phys., 1893. 25 Ransom, Deut. med. Woch., 1893. 26 Wassermann und Takaki, Berl. kiln. Woch., 1898. 27 Gumprecht, Pfluger's Arch., 1895. 28 Meyer und Ransom, Arch., f . exp. Pharm. u. Path., xlix. 29 Marie et Morax, Ann. de 1'inst. Pasteur, 1902. 734 PATHOGENIC MICROORGANISMS directly into the nerves and the central nervous system in active cases. Tetanolysin. — Tetanus bouillon contains, besides the "tetano- spasmin ' ' described xabove which produces the familiar symptoms of the disease, another substance discovered by Ehrlich30 and named by him "tetanolysin. " Tetanolysin has the power of causing liemol- ysis of the red blood corpuscles of various animals, and is an entire ly separate substance from tetanospasmin. It may be removed from toxic broth by admixture of red blood cells, is more thermolabile than the tetanospasmin, and gives rise to an antihemolysin when injected into animals. Pathogenicity. — The comparative infrequency of tetanus infection is in marked contrast to the wide distribution of the bacilli in nature. Introduced into the animal body as spores, and free- from toxin, they may often fail to incite disease, easily falling prey to phagocy- tosis and other protective agencies before the vegetative forms develop and toxin is formed. The protective importance of phagocy- tosis was demonstrated by Vaillard and Rouget,31 who introduced tetanus spores inclosed in paper sacs into the animal body. By the paper capsules the spores were protected from the leucocytes, not from the body fluids. Nevertheless, tetanus developed in the animals. The nature of the wound and the simultaneous presence of other microorganisms seem to be important factors in determining whether or not the tetanus bacilli shall be enabled to proliferate. Deep, lacerated wounds, in which there has been considerable tissue de- struction, and in which chips of glass, wood splinters, or grains of dirt have become embedded, are particularly favorable for the development of these germs. The injuries of compound fractures and of gunshot wounds are especially liable to supply these condi- tions, and the presence in such wounds of the common pus cocci, or of other more harmless parasites, may aid materially in furnishing an environment suitable for the growth of the tetanus bacilli. Apart from its occurrence following trauma, tetanus has been not infre- quently observed after childbirth,32 and isolated cases have been reported in which it has followed diphtheria and ulcerative lesions of the throat.33 30 Ehrlich, Berlin, kl. Woch., 1898. 31 Vaillard et Rouget, Ann. de 1'inst. Pasteur, 1892. 32 Baginsky, Deut. med. Woch., 1893, , Wien. med. Woch., 1895. THE ANAEROBIC BACILLI 735 A definite period of incubation elapses between the time of in- fection with tetanus bacilli and the development of the first symp- toms. In man this may last from five to seven days in acute cases, to from four to five weeks in the more chronic ones. Experimental inoculation of guinea-pigs is followed usually in from one to three days by rigidity of the muscles nearest the point of infection. This spastic condition rapidly extends to other parts and finally leads to death, which occurs within four or five days after infection. Autopsies upon human beings or animals dead of tetanus reveal few and insignificant lesions. The initial point of infection, if at all evident, is apt to be small and innocent in appearance. Further than a general and moderate congestion, the organs show no pathological changes. Bacilli are found sparsely even at the point of infection, and have been but rarely demonstrated in the blood or viscera. Nicolaier succeeded in producing tetanus with the organs of infected animals in but eleven out of fifty-two cases. More re- cently, Tizzoni34 and Creite35 have succeeded in cultivating tetanus bacilli out of the spleen and heart 's blood of infected human beings. The researches of Tarozzi86 and of Canfora37 have shown also that spores may be transported from the site of inoculation to the liver, spleen, and other organs, and there lie dormant for as long as fifty-one days. If injury of the organ is experimentally practiced and dead tissue or blood clot produced, the spores may develop and tetanus ensue. These experiments may explain cases of so-called crypto genie tetanus. In man tetanus may take either an acute or chronic form, the word "chronic" here meaning simply that the onset is less abrupt, the incubation time longer, the symptoms slower in development and the prognosis more favorable. In the acute form, the incubation time ranges from three or four days to ten or fourteen days, the common very rapid cases taking about seven. In the so-called chronic form the incubation time may occasionally exceed a month. The first symptoms usually consist in headache and general depres- sion; followed rather rapidly by difficulties in opening the mouth, due to spasms or trismus of the masseters. There is slight stiffness of the neck which makes it difficult for the patient to bring the 34 Tizzoni, Ziegler's Beit., vii. 35 Creite, Cent. f. Bakt., xxxvii. 36 Tarozzi, Cent. f. Bakt. Orig. xxxviii. 37 Canfora, Cent. f. Bakt. Orig. xlv. 736 PATHOGENIC MICROORGANISMS chin forward on the chest. Gradually there develops a spasm of the muscles of the cheeks which results in a drawing up of the tissues about the mouth, giving a curious and characteristic expres- sion. Gradually the spasms extend to the trunk and back, with the development of opisthotonos after several days. Difficulty in swallowing may ensue, and there may be involuntary movements of urine and feces. The localization of the symptoms to some extent follows the location of the injury. Tetanus may occur in the new born, occasionally, developing soon after birth. For differ- ential diagnosis, it is best to refer to books on general medicine and surgery. Many types of atypical tetanus in untreated and in prophy- lactically treated cases have been reported, a description of which can be found in extenso in the volume on "The Abnormal Forms of Tetanus" by Courtois-Suffit and Giroux in the British Medical War Manuals, published in 1918. They speak of splanchnic tetanus char- acterized especially by the involvement of the muscles of deglutition and respiration, with great dysphagia. Simple cephalic tetanus in which the infection may be confined to the head, is a type in which dysphagic and paralytic symptoms are never present, and which result most frequently from wounds of the head. It may be char- acterized only by unilateral and bilateral trismus, or by contraction of muscles of the face. There is, however, a dysphagic form of this in which pharyngeal spasms precede trismus. Rarely they have noticed a so-called hydrophobic form in which convulsions accom- pany the spasms. PROPHYLACTIC USE OF TETANUS ANTITOXIN The most important use for tetanus antitoxin which has been found hitherto, is its prophylactic administration. The methods of applying this have varied in different parts of the world and in different armies. That it is of great value was demonstrated by the almost immediate reduction of tetanus in wounded soldiers after the universal introduction of prophylactic tetanus antitoxin in all the armies in the field. The wounds which are particularly danger- ous as far as tetanus is concerned are those in which there is con- siderable laceration, especially injury to bone, and in which dirt, and especially manured soil or soil from cultivated fields, and feces, are likely to be present. The growth of tetanus bacilli is favored by the presence of dead tissue and other infected organisms. Studies THE ANAEROBIC BACILLI 737 by members of the United States Public Health Service have shown that tetanus can be produced with regularity if staphylococcus in- fection is added to the infection with tetanus spores, and injury of tissue by the injection of small quantities of such substances as quinine, may start the growth of latent tetanus spores with subse- quent development of the disease. Tetanus spores pass through the intestinal canals of animals and man without injury, and are dis- tributed in the soil where they can live for almost unlimited periods. Wounds inflicted upon men in the field, especially -by the blunt and ragged projectiles of high explosives, and by any injury passing through' soil and filth covered clothing, through unwashed skin, furnish an ideal nidus for infection. In consequence, in prac- tically all the allied armies every wounded man was given an injection of about 1,000 to 1,500 units of tetanus antitoxin as soon after the injury as he came under medical observation. In civilian life, the wounds that require similar prophylactic treatment are those inflected with much traumatism and under dirty conditions, especially those in which compound fractures are involved. We refrain from giving any set rules for prophylactic treatment. The principles involved are that the injection of from 500 to 1,500 or even up to 5,000 units should be made subcutaneously as soon as possible after the injury. It should be remembered that the first injection may not be sufficient. The antitoxin gradually dis- appears in the course of about twelve days, and wounds that are slow in cleaning up or cases in which secondary interference, such as removal of sequestrum, resetting of bones, etc. becomes necessary, may call for a second injection after six to eight days, with due precautions to prevent anaphylaxis. In such cases, according to the judgment of the surgeon, second injections should become almost the rule since experience in the war has shown that after two injections tetanus is very rare in appearance. Recently, very important advances in our knowledge of tetanus have been made by W. J. Tulloch38 who in 1917 showed that tetanus bacilli could be classified into at least three and perhaps four types by agglutination with anti-bacterial serum prepared by injection of the bodies of tetanus bacilli. The important point which arises in this connection is, of course, whether the toxins produced by these various agglutinative types differ either qualitatively or quantita- 38 Tulloch, Journal of the R. A. M. C., Dec. 1917. 738 PATHOGENIC MICROORGANISMS lively. Subsequent investigations by Tulloch39 showed that of a large number of isolations from infections on the Western Front, his type I organisms were the most frequent, type II and type III next, and type IV the least common. It seemed, in these investiga- tions, that the agglutinin titer of an antitoxic serum is no index to its antitoxic value; the agglutinating sera, even when of very high titer, does not bring out group reactions, but maintains a sharp separation of the types and that the types remain true even after prolonged cultivation. The types could be demonstrated by opsonic as well as by agglutination experiments. Most important is the observation that the spasm-producing toxin is not specific to the types, although there may be quantitative differences in toxin production. Tulloch 's investigations suggest that anti-bacterial properties in tetanus sera, if polyvalent, would aid considerably in the phagocytosis of the organisms, and, therefore, have a prophy- lactic and therapeutic value. His work, also, seems to indicate that small amounts of the tetanospasmin do not locally aid the growth of the tetanus bacillus to any considerable extent, an observation in distinct contrast with the earlier work on this subject. However, B. Welchii toxin and, to a smaller extent, that of Vibrion Septique, considerably aid the growth of tetanus, by, as he expresses it, "devitalizing" the tissues. This leads Tulloch to favor a procedure, advised by other observers on purely bacteriological grounds, namely the combining of antitoxins against the poisons of B. Welchii and of Vibrion Septique with Tetanus antitoxin, in the prophylactic treatment of war wounds. Even when this is done, however, he cautions against any feeling of false security which might lead to the neglect of surgical prophy- laxis. He emphasizes the great importance of early excision of the wound area. No particular dressings seem to make a great deal of difference, but thorough excision seems to have considerable influ- ence on the development of tetanus and on the mortality. The Treatment of Developed Tetanus.— To speak of the specific treatment of tetanus without saying a few words about the surgical treatment would be taking the risk of conveying a false impression. Therefore, though our business here is concerned largely with specific treatment, we wish to emphasize that surgical treatment must al- 89 Tulloch, Journal of Hygiene, vol. 18, 1919, p. 103. THE ANAEROBIC BACILLI 739 ways be carried out whatever method of serum therapy be employed. This must consist in thoroughly cleansing the wound, removal of foreign bodies, fragments of projectiles, clothing, gross dirt, etc., and, as the late war has shown, it is perhaps best whenever possible to carry out debridement or excision of the wound. From Tulloch 's40 studies it would appear that no dressing is particularly superior to any other, and we doubt very much whether oxidizing agents, like the insufflation of oxygen, peroxides, etc., are of much use, because of the reducing powers of tissues. As to specific serum treatment, it must be admitted that earlier results were very disappointing, and the mere subcutaneous injec- tion even of large doses of tetanus antitoxin has usually been dis- appointing in the acute forms of the disease. This has perhaps been largely due to the fact that the injected antitoxin could have no possible influence on the toxin which had already become united with the substances of the nerve tissues. A great many modifications in the method of injection have been employed, such as injection directly into the central nervous system and into the nerve trunks, themselves. It may be stated that the relative acuteness of the tetanus infection very definitely influences the results of serum therapy. The following table taken from Etienne and copied from Courtois-Suffit and Giroux in the series of British War Manuals mentioned above, gives a general idea of the usefulness of serum therapy in this disease: Mortality before the Incubation. Recoveries. Deaths. Mortality. Introduction of Serotherapy, according to Brunner. 1 to 5 days 3 7 70% 90% 5 to 10 days 20 7 29% 70% 10 to 12 days 7 1 13 3% Over 12 days . 15 1 6 6% This table cited from Courtois-Suffit and Giroux, Military Medical Manuals, Univ. of London Press, London, 1918, page 193. It would be useless to go into the various methods of adminis- tering tetanus antitoxin that have been tried, and we will confine 40 Tulloch, Jour. R. A. M. C., December, 1917, Jour. Hygiene, 18, 1919, 103. 740 PATHOGENIC MICROORGANISMS ourselves to the intraspinal method developed in this country by Park and Nicoll,41 and in France by Doyen, and gradually coming into general use. As advised by Park and Nicoll, a spinal puncture is made and a moderate amount of spinal fluid taken out. Then, slowly by gravity from 3,000 to 5,000 units of tetanus antitoxin are injected until a total amount of 3 c.c. has been reached, the amount injected approximately replacing the amount of fluid withdrawn. At the same time, 10,000 units are given intravenously or intramus- cularly. This procedure must be repeated according to indications. For other forms of treatment such as carbolic acid injections, magnesium sulphate, etc., we must refer the reader to books on surgical therapy. BACILLUS BOTULINUS Meat poisoning was formerly regarded as entirely dependent upon putrefactive changes in infected meat, resulting in the production of ptomains or other harmful products of bacterial putrefaction. It was not until 1888 that certain of these cases were definitely recognized as true bacterial infections, in which the pre- formed poison probably aided only in establishing the infection. Gartner, in that year, discovered the Bacillus enteritidis, a micro- organism belonging to the group of the paratyphoid bacilli, and demon- strated its presence both in the infecting meat and in the intestinal tracts of patients. The characteristics of this type of meat poisoning have been discussed more particularly in the section describing the bacillus of Gartner and its allied forms. There is another type of meat poisoning, however, which is not only much more severe, but is characterized by a clinical picture more significant of a profound systemic toxemia than of a mere gastroenteric irritation. The etiological factor underlying this type of infection was first demonstrated by Van Ermengem,42 in 1896, and named Bacillus botulinus. Van Ermengem isolated the bacillus from a pickled ham, the ingestion of which had caused disease in a large number of persons. Of the thirty-four individuals who had eaten of it, all were attacked, about ten of them very severely. Van Ermengem found the bacilli in large numbers lying between the muscle fibers in the ham, and was able to cultivate the same micro- 41 Park and Nicoll, Jour. A. M. A., 63, 1914, 245. 42 Van Ermengem, Cent, f, Bakt., xix, 1896; Zeit. f, Hyg., xxvi, 1897. THE ANAEROBIC BACILLI 741 organism from the stomach and spleen of one of those who died of the infection. The results of Van Ermengem have been confirmed by Romer,43 and others. Morphology and Staining. — Bacillus botulinus is a large, straight rod with rounded ends, 4 to 6 micra in length by 0.9 to 1.2 micra in thickness. The bacilli are either single or grouped in very short chains. Involution forms are numerous on artificial media. The bacillus is slightly motile and possesses from four to eight undulated flagella, peripherally arranged. Spores are formed in suitable media, most regularly in glucose-gelatin of a distinctly alkaline titer. The spores are oval and usually situated near the end of the bacillus, rarely in its center. Spores are formed most abundantly when cultivation is carried out at 20° to 25° C., and are usually absent when higher temperatures are employed. The bacillus is easily stained by the usual aqueous anilin dyes, and retains the anilm-gentian-violet when stained by Gram. It is necessary, however, in carrying out Gram's stain to decolorize care- fully with alcohol since overdecolorization is easily accomplished, leaving the result doubtful. Cultivation. — The bacillus is a strict anaerobe. In anaerobic en- vironment it is easily cultivated on the usual meat-infusion media. It grows most readily at temperatures about 25° C., less luxuriantly at temperatures of 35° C. and over. The bacillus is delicately susceptible to the reaction of media, growing only in those which are neutral or moderately alkaline. In deep stab cultures in one per cent glucose agar, growth is at first noticed as a thin, white column, not reaching to the surface of the medium. Soon the medium is cracked and split by the abundant formation of gas. On agar plates, the colonies are yellowish, opalescent, and round, and show a finely fringed periphery. On gelatin, at 20° to 25° C., growth is rapid and abundant, and differs little from that on agar, except that, besides the formation of gas, there is energetic fluidification of the medium. On glucose- gelatin plates, Van Ermengem describes the colonies as round, yel- lowish, transparent, and composed of coarse granules which, along the periphery in the zone of fluidification, show constant motion. The appearance of the surface colonies on glucose-gelatin plates is regarded by the discoverer as diagnostically characteristic. 43 Romer, Cent, f . Bakt. xxvii, 1900. 742 PATHOGENIC MICROORGANISMS In glucose Irotli there is general clouding and large quantities of gas are formed. At 35° C. and over, the gas formation ceases after four or five days, the broth becoming clear with a yellowish-white flocculent sediment. At lower temperatures this does not occur. Milk is not coagulated and disaccharids and polysaccharids are not fermented. The gas formed in cultures consists chiefly of hydrogen and methane. All cultures have a sour odor, like butyric acid, but this is not so offensive as that of some of the other anaerobic organisms. The bacillus lives longest in gelatin cultures, but even upon these, transplantations should be done every four to six weeks, since the spores of this bacillus show less viability and resistance than do those of most spore-formers. Isolation. — The isolation of B. botulinus from infected material is often quite easy, since as Burke states, few other organisms may be present in the canned or pickled food products. Often anaerobic shake cultures in agar made directly, serve to isolate the organisms. In most contaminated material, however, she recommends inoculation from the original material into Von Ermengem's broth, inoculating quite richly, duplicate cultures being made and heated at 60° for one hour, to destroy non-spore bearers. These cultures are then incubated at 28° after which parts of them are filtered, and the filtrate in quantities of 1 c.c. injected into 250-gram guinea-pigs. If the guinea-pigs die within four days, the pigs are again tested with specific botulismus antitoxin. The presence of the organisms having been thus proven in the broth, isolation is now carried out by careful anaerobic plating or shake cultures. We do not know whether the platinized asbestos method has been used in botulismus work, but would recommend its use in cases, such as those men- tioned. Botulismus Toxin. — Botulismus toxin is produced under condi- tions of strict anaerobiosis on any media on which the organism will grow readily. According to Dickson44 the toxin is much more potent if the organisms are grown on an alkalin medium and in the dark. Von Ermengem45 obtained his best toxin by growing the organisms on a beef infusion broth to which he added 1 per cent 44 Dickson, Monograph of the Rock. Inst., No. 8, July 31, 1918, (la). Jour, of the A. M. A., 65, 1915, 492. 45 Von Ermengem, quoted from Kolle and Wassermann, Second Edition. THE ANAEROBIC BACILLI 743 sodium chloride, 1 per cent pepton and 2 per cent glucose. Leuchs46 used a pork infusion with 0.5 per cent sodium chlorid, 1 per cent glucose and 1 per cent pepton. Landmann47 claimed that animal protein was necessary for good toxin production. According to Dickson this is not essential. He has produced toxin in media made from string beans and from peas, and found that, although an alkalin reaction is favorable, an acid reaction does not prevent toxin formation. According to Burke48 toxin is produced as readily at 37.5° as it is at 28° C. The toxin is destroyed at temperatures of about 80°. Thorn, Edmonson and Giltner49 claim that their toxin was destroyed by ten minutes' heating at 75° C. Von Ermen- gem's original report was that heating at 56° for three hours killed it, as does heating at 80° for one-half hour. According to Dickson, it is rapidly destroyed by exposure to sunlight and to air, but will maintain its virulence for six months if kept in the dark as it would be in preserved foods. It is not affected by drying and is insoluble in alcohol, ether and chloroform. Normal soda, 20 per cent by volume, is stated by Dickson to destroy it, though similar amounts of acid do not reduce its virulence in twenty-four hours. Its potency is considerable. Dickson produced his strongest toxin in pork and beef infusions, but also obtained potent prepara- tions in media of string beans, peas, green corn, and less virulent toxin in media prepared from asparagus, artichokes, peaches, and apricots. Brieger and Kempner50 obtained a toxin of which 0.000,001 of a c.c. would kill a 250-gram guinea-pig in four days, and (we quote from Dickson), Von Ermeiigem found in one of his outbreaks that 200 grams of the poisonous ham caused the death of one patient. He quotes another case in which a piece of preserved duck the size of a walnut was sufficient to cause a disease lasting six weeks, and in his own series, a patient died after tasting a small spoonful of spoiled corn, another died after " nibbling a portion of a pod of the spoiled string beans." A third was quite ill after tasting, but not swallowing a pod of beans. An important point is the claim that has been made by Von Ermengem and others that the organism will not produce toxin in the tissues. Injection of the bacilli alone 46 Leuchs, Zeit. f. Hyg., 1910, 65, 55. 47 Landmann, cited from Dickson, loc. cit. 48 Burke, Jour. Bacter., 4, 1919, 555. 49 Thorn, Edmonson and Giltner, Jour. A. M. A.. Vol. 73, 1919, page 901. 50 Brieger and Kempner, Deut. med. Woch., 23, 1897, 521. 744 PATHOGENIC MICROORGANISMS into suitable animals produces no botulismus, and intravenous injec- tion or feeding of the bacilli alone, may not produce symptoms. The conclusion drawn by Von Ermengem was that the toxin could not be produced in the bodies of mammals. The toxin is potent for monkeys, rabbits, guinea-pigs, cats, and various birds. Dickson found chickens highly susceptible, and also found that dogs were not as resistant as formerly thought to be. The most susceptible animals seem to be mice, guinea-pigs and monkeys ; rabbits, cats, dogs and rats are relatively resistant. In studying various isolations of the Bacillus; Dickson found that the strains isolated by him were not entirely homologous, and that the toxins produced by some of them were not neutralized by the antitoxin produced with others. Burke also found two types that produced heterologous toxin and claims that the strains could be easily identified by a toxin-antitoxin test. Pathology. — In animals, according to Von Ermengem there is a general hyperemia of the organs and especially of the nervous system. Dickson has made a thorough review of the pathological work done by Von Ermengem, Vander Stricht,* Marinesco51 and others,52 summarizing the pathology as follows: In the central nervous system the meninges at the base of the brain, especially around the pons and the medulla, are usually more markedly con- gested than at the cortex, and there may be hemorrhage in the upper part of the cord and at the base of the brain. The lungs may be hyperemic, heart muscles flabby, but nothing characteristic. An important and regular lesion found by Ophuls53 was multiple throm- bosis in both the arteries and veins of the central nervous system. Ophuls believes this is due to a certain vasodilation with slowing of the blood stream due to a powerful paralyzing effect of the poison on the unstriped muscles. The thromboses are particularly common at the base of the brain. Ophuls, too, differs from others in believing that the specific action of the toxin on the nerve cells themselves has been very much exaggerated in that his histological examina- tions of the brains of fatal cases did not bear out this earlier opinion. Transmission and Occurrence of Botulismus, — Kempner and Pol- * Stricht, Quoted from Van Ermengem, in Kolle and Wassermann. 51 Marinesco, Compt. rend, de 1'acad. des sci., Nov., 1896. 62 Kempner und Pollack, Deut. med. Woch., xxxii, 1897. 63 Ophuls, with Wilbur, Arch. Int. Med., 14, 1914, 589. THE ANAEROBIC BACILLI 745 lack54 in 1897 isolated B. botulismus from the intestines of a normal hog, but Dickson was unable to find the organism in the intestinal canals of 250 grain-fed hogs in the slaughter houses of San Fran- cisco. He also collected soil from gardens, but in a considerable series of specimens did not find organisms. Burke55 examined ma- terials from many different localities in California where a num- ber of epidemics had occurred, cultivating from large varieties of substances, such as water, hay, vegetables, fruits, manure from horses, hogs, chickens, etc. She obtained seven cultures of the B. botulinus from the following sources: Moldy cherries, leaves touched with droppings of insects, sprouts from bush-bean plants, manured bush beans, manure from a hog, and moldy hay. These, and other investigations seem to indicate that the B. of botulismus is quite common in nature, and may be present in the intestines of domestic animals, such as hogs. It may possibly be disseminated by insects, and may often be present on vegetables and fruits at the time that they are picked for canning. The outbreaks first reported were largely due to meat and it is interesting to note that Van Ermengem's first isolations were from pickled meat, thus showing that ordinary salting or brine preserva- tion does not kill the botulinus spores. The epidemics that have occurred are summarized in a monograph of Mayer,56 and more recently in an admirable study of the disease by Dickson, published in a Monograph of the Rockefeller Institute,57 and carried out at the Stanford University Pathological Laboratory. At the time of Mayer's publication (in 1913 ), 800 European cases had been observed since 1882, 200 of which had been fatal. Meat in general, or animal food had been the source of the disease ; and the generally prevalent idea at that time was that meat was the chief source of danger. During recent years, however, the studies of Dickson and others have shown that vegetable foods, especially canned vegetables were equally as dangerous. Of sixty-four cases collected by Dickson for the United States during the past twenty-five years, fifty-four occurred in California. Wilbur and Ophuls58 reported an outbreak in 1914 due to the eating of canned beans. Since that time, a 54 Kempner and Pollack, Deut. med. Woch., 23, 1897, 505. 55 Burke, Jour. Bacter., 4, 1919, 541. 56 Mayer, Deut. Vrtjschr. off. Gsndhtspflg., 35, 1913, 8. 57 Dickson, Monograph of the Rock. Inst., No. 8, July 31, 1918. 68 Wilbur and Ophuls, Arch. Int. Med., 14, 1914, 589. 746 PATHOGENIC MICROORGANISMS number of other cases have occurred in California and the rest of the United States, many of which have been studied by Dickson and his associates, most of them originating from canned corn and string beans. Thorn, Edmonson and Giltner, reported an outbreak occurred in 1919, traceable to canned asparagus. Armstrong59 carefully studied an outbreak in 1919 which was traced to ripe olives. It is impossible in this space to do justice to the large and valuable botulismus literature which has developed during the last few years, since the studies of Dickson and others have renewed the interest of laboratory workers in the disease. The mortality of the disease has been high, and for the United States generally, as stated by Dickson, it has been over 64 per cent. Attention has also been recently called to the relationship of botulismus in man to the disease called limber neck in chickens. In Hillsboro, Oregon, Dickson states60 that fifty chickens came down after eating home-canned corn which had caused the death of a woman who tasted it. In Hornbrook, California, between fifty and one hundred chickens became paralyzed and died at the same time as the woman who cared for them died of "bulbar paralysis." In the San Jose district, eight chickens died after eating home-canned string beans which had caused the death of a woman; and seven chickens died in Fallbrook, California, after eating home-canned apricots which had killed five people. Dickson obtained the carcasses of some of the chickens of the San Jose and Hillsboro outbreaks, and from the gizzard of one of those which had eaten the canned corn and from the crop of three which had eaten canned beans, he obtained B. Botulinus. He also succeeded in producing symptoms by feeding chickens with the infected material obtained in this way. He described the symptoms as follows: The chickens refuse to eat, remaining quiet, and gradually develop weakness of the neck, wings and legs, finally drooping completely. In the experimental cases death occurred within twenty-four hours after feeding. This, apart from its diagnostic value and experimental importance, may have considerable bearing on epidemiological studies; and limber neck in chickens should always be inquired into when human cases are observed or suspected. Clinical Manifestations of Botulismus. — Botulismus is character- istic in its clinical manifestations and should not present great 59 Armstrong, Pub. Health Reports, 1919, 54, December. 60 Dickson, Jour. Amer. Vet. Assoc., January, 1917. THE ANAEROBIC BACILLI 747 diagnostic difficulties, once the disease is suspected. Since the toxin is preformed before ingestion, the symptoms are not long in follow- ing the eating of infected food, coming on usually within twenty-four hours or less. Delays of two or three days, however, may occur, and should not throw out possible positive diagnosis. The earliest symptoms usually consist in a general weakness and lassitude, with fatigue and some headache. Characteristic is the frequent lack of any symptoms pointing to the gastrointestinal canal. Constipation is the rule. Very early in the disease, disturbances of vision may occur which are due to impairment of the muscles of the eye-ball. There is, particularly, involvement of the third cranial nerve, with blepharoptosis, mydriasis, impaired light reflex and diplopia. There may be photophobia. For a detailed discussion of the symptoma- tology, the reader is referred to the Monograph of Dickson.61 Im- pairment of the pharyngeal muscles may produce difficulties in swallowing with inability to chew, and sluggishness of the tongue with thickness of speech. Absence of temperature is an important feature, and in the early stages there is usually no fever and no change in the heart rate. Fatal cases usually end in death within three to seven days, due either to cardiac failure or terminal asphyxia. In discussing the differential diagnosis, Dickson mentions particularly, poliomyelitis, cerebrospinal syphilis, early stages of bulbar paralysis, belladonna poisoning and methyl alcohol poisoning. The last must be particularly thought of during the present period of drought. Specific Therapy. — Potent antitoxins may be produced by the treatment of susceptible animals with toxin. Kempner62 in 1897 was the first to experiment on this extensively, using the Von Ermengem strain, and producing antitoxin in goats. The immuniza- tion of small laboratory animals is comparatively difficult, unless minute doses and attenuated toxin is used. The chief studies on these phases of the problem have been made by Forssman and Lunstrom63 and by Leuchs.64 More recently, Dickson and Howitt65 have produced potent antitoxins in goats, though their products, they state, were not as powerful as those reported by Kempner and 61 Dickxon, Monograph of the Rock. Inst., No. 8, July 31, 1918. 82 Kempner, Zeit. f. Hyg., 26, 1897, 481. 83 Foreman and Limstrom, Ann. do I' Inst. Past., 16, 1902, 294. 64 Leiichs, Kollc and Wasserrnann Handb., Second Edition, 4, 939. 85 Dickson and Howitt, Jour. A. M. A., 74, 1919, 718. 748 . PATHOGENIC MICROORGANISMS some other observers. It is very important to note that in these experiments antitoxin produced against one series of strains had no appreciable effects upon the toxins of three other strains. This brings out the great importance of producing curative sera by the use of toxins from a number of different strains. According to Dickson, there are at least two types which must be used. As to the therapeutic value, reports have been confusing. Dickson advises intravenous injection and states that his procedure would be as follows: The usually precautions for the administration of horse serum should be observed, and the patient tested for skin sensitive- ness. If no such sensitiveness is found, the serum should be injected immediately into a vein at the rate of not more than 1 c.c. a minute. Comparatively large doses should be given, since the amount of toxin ingested may be quite large. Prevention of Botulismus. — Deducing preventive measures from the facts cited in the above paragraphs, it would seem that, in the first place, all people in the habit of preparing canned food should be thoroughly alive to the possibilities of contamination and know that B. botulinus spores may be present on fruit, vegetables, etc., before they are preserved. It should be well understood that food may be contaminated with botulinus, without being changed in any way in its gross appearance, and that not even the slightest rancid odor which sometimes indicates its presence, need be apparent. The sterilization of canned food, sausages, preserved meat, etc., should be thoroughly attended to and no home-canned preparations be eaten under any circumstances unless cooked before eating. CHAPTER XXXVII THE ANAEROBIC BACILLI (Continued) THE ANAEROBIC ORGANISMS ASSOCIATED WITH TRAU- MATIC INJURIES, WITH A CONSIDERATION OF THEIR IMPORTANCE IN WAR SURGERY,1 ALSO BACILLUS OF SYMPTOMATIC ANTHRAX THE anaerobic bacilli which infect wounds have been studied extensively during the last war, and the literature which has ap- peared on this subject since 1914, is voluminous'. Unfortunately much that has been written is inaccurate, due to the fact that in many instances the work was carried on in poorly equipped labora- tories and under difficulties. The two most important sources of confusion in this field are: the nomenclature and the impurity of cultures. During the war many previously described bacilli were rediscovered and given new names, and the literature is full of papers claiming or disclaiming that certain organisms isolated from wounds are identical with organisms described by earlier workers. Even such a well-known species as B. Welchii appears in the litera- ture under four names. It is now generally considered that in all probability most of the early descriptions of anaerobic bacilli cannot be relied upon, because of the extreme difficulty of isolating the anaerobes in pure culture. Many investigators even during the first year of the war were describing a mixture of two or more anaerobes when they thought they were dealing with a pure culture. It was not until the development of the newer anaerobic methods2 which made sur- face growths feasible that the purity of anaerobic cultures could be relied upon. It is only by repeated plating of these anaerobic spore-bearing bacilli, as emphasized repeatedly by English workers, that pure cultures can be obtained. The French workers have adhered for the most part to the older method of Veillon which consists of making varying dilutions of the material to be examined 1 For the thorough revision of this group of organisms we are indebted to Miss Ann Kuttner, Instructor in this Laboratory, Zinsser. 2 Macintosh and Fildes, Lancet, 1, 1916, 768, April 8th. 749 750 PATHOGENIC MICROORGANISMS in deep agar shake tubes until isolated colonies are obtained. The tube is then filed through at the level of 1lie colony and the isolated colony can be fished. This is a laborious method and is unreliable. Barber3 has developed a technique whereby he can isolate a single bacillus and has applied this method to the problem of the purifica- tion of the anaerobic bacilli. The source of anaerobic bacilli in war wounds can always be traced to the contamination of the wound either directly or in- directly with fecally contaminated soil. Most of the anaerobic bacilli have been shown to be normal inhabitants of the intestinal tract of man or animals, and are present in great numbers in cultivated ground. During the war the incidence of anaerobic bacilli in wounds and their relation to the development of gas gangrene was studied in detail. In civilian practice gas gangrene is generally attributed to the presence of B. Welchii. In war wounds, however, it was found that although B. Welchii was isolated from the majority of the cases of gas gangrene, it practically never occurred in pure culture, and was usually associated, besides aerobes, with other anaerobic bacilli which in many instances proved to be more pathogenic than B. Welchii. The separation and identification of these anaerobic bacilli, and the development of methods for the control of such infections were among the most important problems of bacteriologists during the war. In this book only the most important and frequently occurring anaerobic bacilli will be discussed. The reader is referred for more detailed description to the book of Weinberg and Seguin "La Gangrene Gazeuse," and to the Report of the British Medical Research Committee. The anaerobic bacilli found in war wounds can be divided into two general groups, as first suggested by Von Hibler4 in his book on Anaerobes, published in 1908 : the sacchrolytic and the proteolytic. The saccharolytic group includes as its most important members : B. Welchii Vibrion Septique B. Oedematiens B. Fallax 3 Barber, Jour. Exper. Med., 32, 1920, 295. 4 Von Hibler, " Untersuchungen iiber die pathog. anserob.," Jena, 1908. THE ANAEROBIC BACILLI 751 The proteolytic group includes: B. sporogcnes B. histolyticus B. putrificus This classification, like most others, is not a rigid one. It merely means that members of the saccharolytic group have a much greater avidity for carbohydrates than have members of the proteolytic group. It may be possible to demonstrate proteolytic activity of the organisms included in the saccharolytic group on special media from which all carbohydrate has been removed, but on the ordinary culture media the difference between the two groups is striking. Members of the proteolytic group can be distinguished from the saccharolytic group by the fact that they liquefy coagulated horse serum. Organisms of the saccharolytic group fail to liquefy this medium even after prolonged incubation. In milk the saccharo- lytic bacilli produce acid and varying amounts of gas. The pro- teolytic bacilli digest milk with the production of alkali. The organisms of the proteolytic group are not in themselves pathogenic, but complicate wounds by their intense proteolytic action. They are saprophytes, they have no power of invading the tissues and if present without members of the saccharolytic group usually do not interfere with the healing of the wound. Whether the organisms of the saccharolytic group are sapro- phytes or not, is an open question. De Kruif5 concludes that B. Welchii cannot be classified as a pure saprophyte, because a twice washed bacillary emulsion in doses of 0.1 c.c. to 1 c.c. will kill a guinea pig. But in this case even the most careful intramuscular injection will kill some of the tissue at the point of inoculation, and with even a very small amount of dead tissue, B. Welchii can produce a toxin which has tremendous aggressive action and has the property of killing the tissue with which it comes in contact. This makes possible the further invasion of B. Welchii. B. Welchii. — B. Welchii is the organism most frequently found in gas gangrene. It was present in about 72 to 80 per cent of the cases of gas gangrene studied during the war, and has been con- sistently associated with civilian cases of gas gangrene. It is gener- ally considered the most important etiological factor in this disease. * De Kruif, Jour, Infec. Dis., 21, 1917, No. 6. 752 PATHOGENIC MICROORGANISMS On the other hand, it must also be stated that B. Welchii was fre- quently present in wounds which never developed gangrene. Taylor reports that B. Welchii was found in 80 per cent of all wounds examined and that only 10 per cent of these developed gas gangrene. The development of gas gangrene depends on the virulence of the strain of B. Welchii, the amount of dead tissue present, and the anaerobic conditions in the wound. In war wounds B. Welchii was practically never present in pure culture. It was usually associated with aerobes and with other anaerobes of the saccharolytic and the proteolytic type. B. Welchii was discovered independently in three countries. It was first discovered by Welch and Nuttall6 in 1892, and called by them Bacillus aero genes capsulatuSj a name still used by the majority of English writers. In this country this organism is usually called B. Welchii. Welch isolated it from the blood and organs of a cadaver dead eight hours. In 1893 Fraenkel7 isolated a similar organism in Germany from several cases of gaseous phlegmons, calling it B. Phleg- monis enephysematosae, but soon recognized that he was working with the same bacillus previously described by Welch. However, this organism is still referred to as the Fraenkel bacillus in German litera- ture. In 1897, without having heard either of Welch's or FraenkePs work, this organism was again described by Veilon and Zuber8 in France and called by them B. perfringens, B. Welchii, Bacillus aerogenes capsulatus, Fraenkel bacillus, B. perfringens are all names for the same organism. B. Welchii is a short, square Gram-positive bacillus, occurring singly or in pairs. Chains are not formed as a rule. It is non-motile and has a capsule. It grows best under strictly anaerobic condi- tions, but its requirements for anaerobiosis are less rigid than those of tetanus. It grows well in media containing tissue such as cooked meat medium after simple boiling. With milk boiling is not always sufficient to obtain good growth, and it is best to put milk tubes in anaerobic jars. The majority of strains do not form spores readily. Alkaline sugar-free media rich in protein, such as alkaline egg, are necessary to demonstrate spore formation with the majority of strains. The spore of B. Welchii is large, oval, and central or 6 Welch and Nuttatt, Bull. Johns Hopkins Hosp., 3, 1892, 81. 7 Fraenkel, Cent. f. Bakt., Bd. 13, 1893, 13. 8 Veillon and Zuber, Arch, de Med. Exper., 10, 1898, 517. THE ANAEROBIC BACILLI 753 subterminal. B. Welchii is the most active fermenter in the sac- charolytic group. It ferments all the common sugars with the pro- duction of large amounts of gas. Lactic and butyric are the two acids most frequently formed, the latter often giving cultures of B. Welchii a characteristic odor. Glucose agar is sometimes frag- mented to such an extent that the plug is blown off the tube. Simonds9 has been able to divide the B. Welchii group into sub- divisions depending on the ability to ferment either glycerine or inulin, or both, or neither, and this classification has been confirmed by Henry.10 In wounds, B. Welchii ferments the muscle sugar producing gas in the tissues and for this reason is commonly called the "gas" bacillus. The crepitation thus produced is characteristic of gas gangrene and indicates the extent of the infection. The rapid fermentation of the lactose in milk gives a characteristic reaction in this medium which is diagnostic for B. Welchii. The acid clot torn by gas bubbles and the separation of the milk into coagulum and whey is easily recognized and is not given by other anaerobes. The inoculation of a mixed culture from a wound into milk makes possible the diagnosis of B. Welchii within twelve to eighteen hours. Opinions as to the ability of B. Welchii to liquefy gelatin vary greatly. B. Welchii does not grow well on sugar-free gelatin and it is, therefore, difficult to draw conclusions as to its action on gelatin. B. Welchii as pointed out by Rettger in 1906,11 never attacks proteins if carbohydrates are present and even in the absence of carbohydrate shows only a very slight proteolytic activity. No indol is formed from broth, and coagulated serum is not liquefied or blackened. The hemolytic power and pathogenicity of different strains of B. Welchii vary greatly. B. Welchii is particularly pathogenic for guinea-pigs and pigeons, the latter being used in the standardization of B. Welchii toxin. B. Welchii in fatal cases usually invades the blood stream shortly before death, and can usually be isolated from the blood after death. Spores are never formed in the animal body. Rabbits and mice are much less susceptible. Agglutinin production in response to injections of B. Welchii in rabbits and horses is extremely poor. Simonds obtained a serum in rabbits which agglu- tinated the homologous strain in a dilution of 1—80. Ten strains 9 Simonds, Mon. Rock. Inst., No. 5, 1915. 10 Henry, Jour. Pathol. and Bacter., 21, 1917, -344. 11 Rettger, Jour. Biol. Chem., 11, 1906, 71. 754 PATHOGENIC MICROORGANISMS of B. Wclchii failed to agglutinate with this scrum, and ten others agglutinated only in a dilution of 1-20. The agglutination reaction for the identification of anaerobes has so far proved unsatisfactory. TOXIN PRODUCTION. — Klose in 191612 reported the isolation of a toxin from B. Welchii prepared by growing B. Welchii for fourteen days in a 5 per cent glucose broth. The antitoxin produced by injections of this toxin only protected guinea-pigs against three lethal doses of B. Welchii cultures. The antigenic properties of this toxin were too feeble to consider it a true toxin. The most important contribution to the bacteriology of B. Welchii was made in 1917 by Bull and Pritchett13 who were able to prepare a soluble toxin which, when injected into a suitable animal, produced a potent antitoxin possessing protective and curative properties against B. Welchii infections, in animals. One c.c. of antitoxin per 100 grams body weight injected subcutaneously protected guinea-pigs against 300 lethal doses of culture.14 The production of a powerful toxin (0.3 c.c. to 3 c.c. being the M. L. D. for a pigeon of 300 gr. injected intramuscularly) depended on the" virulence of strain, a short in- cubation period, twenty-one to twenty-four hours, and the presence of fresh muscle and glucose in the broth. Bull and Pritchett found no variations in the ability of different strains of B. Welchii, irre- spective of the source of the culture, to produce toxin. The toxin production of less active strains could be increased by raising the virulence of the culture by animal passage. Caulfield in a recent paper15 finds that he does not get good toxin production unless the virulence of his strain is such that 0.02 c.c. of supernatant fluid of a young broth culture will kill a 300 gr. pigeon. Caulfield also emphasizes the importance of fresh muscle in the culture medium, although DeKruif and the Hygienic Laboratory in Washington obtained good results by substituting chopped veal which can be autoclaved, for the fresh muscle tissue. The most potent toxins, however, seem to be obtained by inoculating the infected muscle of a pigeon dying of a B. Welchii infection directly into the medium to be used for toxin production, or, at most, allowing one short culture generation (ten hours) to intervene between the last animal passage and the inoculation of the broth for toxin. By preparing 12 Klose, Munch, med. Woch., Bd. 63, 1916, 723. 13 Bull and Pritchett, Jour. Exper. Med., 26, 1917, 867. 14 Bull and Pritchett, Jour. Exper. Med., 26, 1917, 867. « Caulfield, Jour. Infec. Dis., 27, 1920, No. 2. THE ANAEROBIC BACILLI 755 a toxin in this way, Bengston of Hygienic Laboratory has been able to prepare a B. Welchii antitoxin in which 1 c.c. of serum contains one unit, one unit neutralizing 1000 M. L. D. of B. Welchii toxin.16 In laboratory animals infected with pure cultures of B. Welchii, the antitoxin gives complete protection. Protection in laboratory animals is also afforded by injections of antimicrobial sera prepared by injections of whole broth culture of B. Welchii by Weinberg and Seguin, but the antitoxin content of these sera has not been determined. Bull and Pritchett in their original paper, state that the toxin produced by B. Welchii is comparable to the toxins produced by Tetanus and Diphtheria, and judged by its antigenic properties it must certainly be classified as a true exotoxin. It differs from the classical toxins in that toxin production varies directly with the virulence of the strain and that it has no definite incubation period. B. Welchii antitoxin cannot protect against mixed infections where B. Welchii is associated with either Vibrion Septique or B. Oedema- tiens. In this case the animal always dies of the Vibrion Septique or B. oedematiens infection. However, since both these organisms occur in a smaller percentage of cases and have rarely been isolated from civilian cases of gas gangrene, B. Welchii antitoxin will prob- ably prove of great value. Isolation. — B. Welchii is a normal inhabitant of the intestinal tract of adults and may be found in the stools of infants. Simonds found B. Welchii present in eight out of nineteen stools of babies under one year of age. It can be easily isolated from stools by the following procedure: 5 c.c. of a fecal suspension in saline are inoculated into a tube of milk which has been freshly boiled and cooled. The tube is heated at 80° for one hour to kill off the vegetative forms of the fecal flora, and is then incubated. The development of the ''stormy fermentation" described above indi- cates the presence of B. Welchii. Purification is best completed by plating anaerobically from the milk culture. Animal inoculation is also useful in the isolation of B. Welchii. The material suspected of containing B. Welchii is injected intra- venously into a rabbit. After five minutes the rabbit is killed and placed in the incubator for five to eight hours. At the end of this time, the animal is usually distended with gas. At autopsy gas 16 Bengston, Hygienic Laboratory Bulletin, No. 122, 1920. 756 PATHOGENIC MICROORGANISMS bubbles will be found distributed throughout the organs, especially in the liver. B. Welchii if present, can usually be isolated from the liver and the heart's blood. Cultures can also be identified by injecting them intramuscularly into two guinea-pigs, one normal, the other protected by a dose of B. Welchii antitoxin. If both pigs die, and an anaerobic organism is isolated from the heart's blood, it indicates the presence of some other pathogenic anaerobe, not B. Welchii. If the normal pig dies with an anaerobic, capsulated, non-motile Gram-positive bacillus in the heart's blood, and the anti- toxin pig survives, it is a fairly sure indication that the organism in question is B. Welchii. VIBRION SEPTIQUE (Synonyms, Bacillus of Oh on and Sachs, and Bacillus III of Von Hibler). — Vibrion septique according to Wein- berg and Seguin, occurred in 12 per cent of the wounds examined by them. Henry isolated it in 16 per cent of his cases. Before the war, cases of human gas gangrene due to vibrion septique alone were very few in number. Such cases were described by Ghon and Sachs,17 by Von Hibler, Gould,18 and by Muir and Ritchie.19 During the war, vibrion septique was usually associated with other anaerobes, notably B. Welchii. Weinberg and Seguin cite only one case of gas gangrene in which vibrion septique was the only anaerobe present. Vibrion septique was first described by Pasteur20 in 1877 who isolated it from the blood of a cow dead three days, and from the blood of a horse dead one day, both animals having supposedly died of anthrax. Pasteur called this organism a vibrion, although it is in reality a bacillus, because it is extremely motile in animal exudates and may look slightly curved when in motion. In 1881, Koch21 in studying the etiology of anthrax, isolated an organism which he called the bacillus of malignant edema. Koch considered his organism identical with Pasteur's vibrion septique, although the bacillus of malignant edema had marked proteolytic properties, which Pasteur did not mention in the description of vibrion septique. A great amount of confusion has arisen out of this controversy and the literature is full of papers discussing whether or not Pasteur 17 Ghon and Sachs, Cent, f . Bakt., 1 Abt. Orig., 48, 1909, 396. 18 Gould., Annals of Surgery, 38, 1903, 481. 19 Muir and Ritchie, Manual of Bacteriology, 2d Edit., Edinburgh, 1899. 20 Pasteur et Jourbet, Bull, de 1'Acad. Med., Scien., Vol. 6, 793. 21 Koch, Mitt. a. d. k. Gesundheitsamt,, 1, 1881, 53*. THE ANAEROBIC BACILLI 757 and Koch were working with the same organisms and with attempts to identify organisms isolated from wounds with one or the other of these bacilli. The majority of workers now consider that Pasteur was working with a strictly saccharolytic organism which is identical with what we call vibrion septique at the present time. The bacillus of malignant edema of Koch is thought by most investigators to belong to the proteolytic group and is fairly definitely identified with B. sporo genes. Vibrion septique is a motile, slender Gram-positive bacillus with slightly rounded ends. It is a strict anaerobe. It forms spores readily in most media. The spore is oval, occurring either centrally or subterminally, and appears at the end of twenty-four to forty- eight hours. It has no capsule. It ferments the common sugars with the exception of saccharose. It produces a loose clot in milk in one to four days. Gelatin is liquefied, but coagulated serum is not attacked. Vibrion septique is hemolytic. It is always pathogenic for laboratory animals and guinea-pigs, mice, pigeons and rabbits are all susceptible. It invades the blood stream producing a sep- ticemia. The occurrence of long filamentous forms in the livers of guinea-pigs dying of a vibrion septique infection is characteristic and is used in the identification of vibrion septique. Robertson22 has recently divided the vibrion septique group into four serological types, based upon the agglutination reaction. Miss Robertson again stresses the necessity of minute care in purifying cultures and points out that impure cultures fail to agglutinate. It is difficult, however, to attach much importance to variations in agglutinin production of different strains, since there is no differ- ence in toxin production, and since the antitoxin produced by the injection of the toxin of strains belonging to one serological type neutralizes the toxins produced by members of the other types. The agglutination reaction in the case of vibrion septique subdivides strains that agree in every other respect, and may in this instance be regarded as ultraspecific, as Miss Robertson suggests. She was able to obtain agglutinating sera with a titer of 1-25,000. TOXIN PRODUCTION. — A powerful soluble toxin is produced by all strains of vibrion septique and does not depend on the virulence of the culture. According to Robertson as potent toxins are produced by old laboratory strains of vibrion septique as by recently 22 Robertson, Jour. Bacter. and Pathol., 1920, 758 PATHOGENIC MICROORGANISMS isolated cultures. The toxin, like that of B. Welehii, has no incuba- tion period. The toxin of vibrion septique often fails to produce death in guinea pigs when injected subcutaneously or intramus- cularly, merely producing local necrosis. Toxin production is tested both in rabbits and guinea pigs by intravenous injection. 0.5 c.c. of toxin injected intravenously kills a guinea pig in five minutes. 0.1 to 1 c.c. injected into rabbits intravenously kills them without a latent period, with symptoms of respiratory disturbance, paralysis and convulsions. It is difficult to establish an M. L. D. for rabbits of the same v/eight owing to individual variation. In some instances, death is produced immediately in one rabbit, whereas another rabbit of the same weight will show severe symptoms followed by recovery. METHOD OF PRODUCING TOXIN. — The Hygienic Laboratory obtains a powerful toxin using a 0.2 per cent glucose broth containing 10 per cent horse serum. Robertson recommends using liver of a pig dying of a vibrion septique infection with which to inoculate the broth, but the difficulties of obtaining a liver without gross contaminations are such that the former method is preferable. The broth is in- cubated twenty-four to forty-eight hours. Care should be taken in the selection of the filter since certain filters seem to hold back a large percentage of the toxin. This point is emphasized both by Weinberg and Seguin and by Robertson. Antitoxins are prepared by injecting the toxin into horses or sheep. The French standard requires that 1/1000 c.c. of the antitoxin should neutralize two fatal doses of the toxin after thirty minutes incubation of the mixture at room temperature. The vibrion septique antitoxin is specific; it does not protect against B. oedematiens. OCCURRENCE. — Vibrion septique has been isolated from milk. Heller23 in an excellent summary of anaerobic infections in animals has shown that spontaneous infections by vibrion septique occur in sheep, horses, and hogs. Meyer24 in 1915 reported the isolation of typical vibrion septique from two cases of symptomatic anthrax in hogs. Cattle, according to Heller, are less susceptible to vibrion septique infections than the other animals mentioned. Herbivorous animals are subject to infections with vibrion septique, both follow- lowing and not following demonstrable wounds, whereas infections in man seem to occur only as the result of wounds. " Heller, Jour. Infec. Dis., 27, 1920, 385. 24 Meyer, Jour. Infec. Dis., 12, 1915, 458. THE ANAEROBIC BACILLI 759 Differentiation of Vibrion Septique and B. Chauvaei (Bacillus of symptomatic anthrax, Blackleg). — B. chauvaei was in no instance isolated from wound cultures, and has never been known to cause an infection in man. Vibrion septique, on the other hand, frequently infects animals and a bacteriological differentiation between vibrion septique and B. chauvaei must be made. These two organisms are closely related and very similar, and a reliable differentiation is difficult even for a bacteriologist familiar with anaerobic bacilli. Robertson distinguishes between B. chauvaei and vibrion septique by the fact that the former ferment saccharose and not salicin, whereas vibrion septique ferments salicin and not saccharose. Long snake-like filaments are demonstrable in smears from the liver of guinea-pigs dead of vibrion septique infection; these are entirely lacking in B. chauvaei infections. Vibrion septique is more patho- genic for laboratory animals and produces more gas in the tissues than B. chauvaei. B. chauvaei grows more slowly than vibrion septique. Vibrion septique is Gram-positive, whereas most inves- tigators consider B. chauvaei Gram-negative. Protection tests with a known vibrion 'septique antitoxin ought to prove the most reliable way of identifying vibrion septique. B. oedematiens. — Weinberg and Seguin claim to have isolated this organism in 34 per cent of the wounds examined by them. This is a higher proportion than that obtained by other workers. Henry found B. oedematiens in five out of fifty cases examined. B. oedema- tiens was isolated in 1915 by Weinberg and Seguin. In 1916 a similar organism was isolated by Sacquepee25 under the name "bacille de Toedeme gazeuse malm." Later Sacquepee called this organism B. Bellonensis. B. Bellonensis and B. oedematiens are now considered to be the same organism by the majority of workers. According to Heller, B. oedematiens is closely related to but not identical with a bacillus discovered in 1894 by Novy and called by him B. oedematiens maligin II.26 B. oedematiens is a strict anaerobe. It is a large Gram-positive bacillus, resembling anthrax in appearance. It is practically non-motile. It forms chains in culture and often shows curved forms after two or three days. Filaments are not formed in the animal body. It forms oval sub- terminal spores readily in all media. It ferments most of the 26 Sacquepee, Ann. de 1'inst. Past., 30, 1916, 76. , Zeit. f. Hyg., 17, 1894, 209. 760 PATHOGENIC MICROORGANISMS common sugars,27 and forms a loose clot in milk in three or four days. It liquefies gelatin, but does not attack coagulated serum. PATHOGENICITY. — B. oedematiens is usually pathogenic, although Weinberg and Seguin report the isolation of two non-virulent strains, rabbits are less susceptible than guinea-pigs. Mice are also sus- ceptible. It may or may not enter the blood stream. The lesion in the animal is characterized by a whitish gelatinous exudate and the absence of gas. The* production of agglutinating sera with B. oedematiens has not been satisfactory, because B. oedematiens tends to agglutinate spontaneously. It is feebly hemolytic much less so than vibrion septique and B. perfringens. TOXIN. — B. oedematiens forms a soluble toxin. Different strains vary in their toxin production. With a potent strain 1/100 c.c. of toxin injected intravenously kills a 300-400-gram guinea-pig in forty-eight hours. This toxin differs from those of vibrion septique and B. Welchii in that it never kills acutely on intravenous injection. The toxin is prepared according to Weinberg and Seguin, by grow- ing B. oedematiens in broth containing chopped veal for six to ten days. ANTITOXIN. — Rabbits, sheep and horses have been used to produce antitoxic sera. Immunization is difficult and small doses must be used at first. Weinberg and Seguin prepared an antitoxin in a horse of such a titer that 1/10,000 dilution neutralized two lethal doses (guinea-pig). B. Fallax. — B. fallax was discovered during the war by Wein- berg and Seguin. It is a much less important factor in gas gangrene than the members of the saccharolytic group already described. It is. usually associated with other pathogenic anaerobes. Weinberg and Seguin cite one case in which it invaded the blood stream and caused death. It was isolated by Henry in three cases out of a series of fifty. It is an anaerobic Gram-positive bacillus, resembling vibrion septique in appearance. It has a capsule and is slightly motile. It does not form spores readily in most culture media. Spores are formed on coagulated serum and are subterminal. B. fallax coagulates milk very slowly. It does not liquefy gelatin or coagulated serum. B. fallax is not hemolytic. It is only slightly pathogenic and soon becomes avirulent on artificial cultivation. 27 Wolf, Jour. Pathol. and Bacter., 23, 1920, 254. THE ANAREOBIC BACILLI 761 Proteolytic Group. — The organisms of this group can never produce gas gangrene without the presence of one or more bacilli of the saccharolytic group. The members of the proteolytic group digest milk without the formation of a clot and liquefy and often blacken coagulated serum. These two characteristics together with the fact that cultures of the proteolytic organisms usually have a very offensive odor, make it comparatively easy to distinguish them from the saccharolytic group. Sugars are fermented by the pro- teolytic type, but much less rapidly and with the production of less acid and gas than in the case of the saccharolytic group. The members of the proteolytic group produce spores readily in all media. None of these organisms are very pathogenic and produce no general picture of toxemia in spite of the tremendous liquefaction of tissue caused by them. The ferments28 of several of the pro- teolytic anaerobes have been isolated and have been found to split proteins to animo acids so rapidly that there is no time for the intermediate products to intoxicate the animals. The separation of the proteolytic anaerobes from the saccharolytic is extremely difficult. The members of the two groups are usually present to- gether, arid what will seern to be a pure culture of a saccharolytic organism if held for any length of time, will often show a con- tamination with a proteolytic organism. The best methods for separation of the two groups are : by rapid transplantation in sugar media, where the saccharolytic organisms outgrow the proteolytic, combined with frequent plating, or by animal inoculation. The latter is the most satisfactory. In the animal body after intramus- cular injection, the more pathogenic organisms belonging to the saccharolytic group frequently invade the blood stream and may be isolated from the heart's blood. B. sporogenes. — This organism was next to B. Welchii most frequently found in wound cultures. Weinberg and Seguin isolated it in 27 per cent of their cases. B. sporogenes was the anaerobe usually responsible for the foul odor of wounds. According to most authors, the pathogenicity of this organism is negligible. Weinberg and Seguin claim to have isolated a few toxic strains, but these may possibly have been mixed with members of the saccharolytic group. Heller has not found any proteolytic anaerobes that are pathogenic for animals. 23 Blanc and Pozerski, Compt. Rend. Soc. Biol., 87, 1920, 29. 762 PATHOGENIC MICROORGANISMS B. sporogencs was definitely described by Metchnikoft'29 in 1908. Whether B. sporogenes is identical or not with Koch's bacillus of malignant edema, will probably never be definitely settled. It is considered identical by many workers, although this is emphatically denied by others. B. sporogenes is a Gram-positive, anaerobic bacillus, actively motile, forming oval subterminal spores readily in all media and in the animal body. It is intensely proteolytic, liquefying gelatin and coagulated serum, and digesting and blacken- ing meat. Most strains of B. sporogenes are not hemolytic. Occa- sionally a feebly hemolytic strain has been isolated. It does not produce a soluble toxin and is not pathogenic for laboratory animals unless injected in large quantities. B. Histolyticus. — This organism was discovered by Weinberg and Seguin and isolated by them from eight wound cultures. Like B. sporogenes, it is intensely proteolytic and is of interest chiefly because of the striking lesion it produces in the animal body. It is a Gram-positive anaerobic, motile bacillus with rounded ends. Sporulation takes place in all media, different strains varying in the time required for spore formation. The spores are large and oval, and occupy a terminal position. No gas is formed in cultures of B. histolyticus and no putrid odor develops. Gelatin and coagu- lated serum are liquefied. It does not produce a soluble toxin. It is not hemolytic. The injection of large doses, 2 to 3 c.c. intra- muscularly into guinea pigs, of the whole culture digests the tissues so rapidly that at the end of twelve to twenty-four hours, the bone may be exposed. The picture is striking, one of the characteristics of B. histolytic infection being that in spite of a tremendous local lesion, the animal appears well. B. putrificus. — B. putrificus was occasionally found in putrid wounds. It was first discovered in 1884 by Bienstock and is char- acterized by a terminal oval spore. Bienstock isolated B. putrificus from the intestine of a cadaver. It is a Gram-positive anaerobe, motile, forming spores in all media. It is actively proteolytic, producing a foul odor. No pathogenic strains have been isolated. It has been studied by Tissier and Martally who found it in putrid meat, Klein worked with a similar organism which he called B. sporogenes cadaveris. Hibler considers B. putrificus and B. sporogenes cada- veris the same. 29 Metchnikoff, Ann. de 1'Inst. Past,, 22, 1908, 419. THE ANAEROBIC BACILLI 763 Identification of Anaerobes Present in Wound Cultures. — From the point of serum treatment of infected wounds, the prompt iden- tification of the members of saccharolytic group, B. Welchii, B. oedematiens and Vibrion Septique, is most important. The process of purification and identification by cultural methods is at best slow, and Henry30 has, therefore, suggested the inoculation of the unknown material into immunized guinea-pigs as the quickest and most reliable method. The procedure he outlines is as follows : inoculate the unknown mixed culture into cooked meat medium, and incubate. The next day inoculate the supernatant fluid into milk, and inject in- tramuscularly into two immunized guinea-pigs, one pig having received a mixture of B. Welchii and Vibrion Septique antitoxin, the other a mixture of B. Welchii and B. oedematiens. The stormy fermenta- tion of milk is diagnostic for B. Welchii and this reaction takes place within twenty-four hours. If the pig that was protected against Vibrion Septique (the B. Welchii factor having been eliminated in both pigs) dies, it indicates the presence of some other pathogenic anaerobe, probably B. oedematiens. The diagnosis of B. oedematiens is further indicated if the guinea-pig that received the B. oedematiens combination of sera survives. If the animal inoculations come out in the opposite way, the presence of Vibrion Septique is indicated. The pathogenic organism can usually be isolated from the heart's blood of the animal that succumbs. By using a "filter" of protected guinea-pigs in this way, the pathogenic organisms can be separated out and the specific sferum injected into the patient within forty-eight hours. THE COOPERATION OF SURGERY AND BACTERIOLOGY IN THE MANAGEMENT OF TRAUMATIC WOUNDS (WAR WOUNDS) The extensive experience gained by surgeons, during the war, in connection with infected wounds has developed a number of important bacteriological methods which are likely to remain as parts of the routine work of civil hospitals, especially those in which traumatic cases are handled. It is not our intention to go into the various problems and con- troversies that have arisen among surgeons concerning the value of irrigation with Dakin's solution or with other antiseptics. This 30 Henry and Lacy, Jour. Pathol. and Bacter., 32, 1920, No. 3. 764 PATHOGENIC MICROORGANISMS is not directly concerned with the question of bacteriological control, a matter which is desirable and seems eminently logical whatever method the surgeon chooses to use for the disinfection of the wound. It is, however, chiefly in connection with Dakin's solution irrigation that this method was developed by Carrel. The most complete treatise on the entire matter may be found in the book lay Carrel and De Helly, "The Treatment of Infected Wounds," (Hocber, New York, 1919). The usual type of war wound or, for that matter, any kind of traumatic wound, presents conditions in regard to the possibilities of infection which are quite different from those ordinarily en- countered in aseptic surgery. From the skin and clothing bacteria, both aerobic and anaerobic, are carried by the projectiles or other foreign bodies into the tissues. Tissues are destroyed to a variable degree, and such devitalized tissues furnish an excellent medium for bacterial growth. There is always an interval or latent period between contamination of the wound and proliferation and penetra- tion of the organisms. The duration of this latent period varies, but usually approximates six hours. The immediate aim of treatment is the prevention or limitation of infection, and, for this reason, the rational method of determining whether this purpose is being accomplished and what the next procedure should be is bacteriological control. The first step in limiting infection in such wounds is accom- plished by debridement, that is, excision of the tract with removal of all the devitalized and contaminated tissues, together with foreign bodies, bits of projectile, clothing, etc. Bacteria are greatly diminished though not eradicated by this procedure. Bacteriological control of the original infection of the wound and its progress under treatment is carried out by a method of systematic smear examination of the wound, supplemented by cultures, first practically developed by Carrel. The smear method, introduced by Carrel and employed since that time by many surgeons on a large material, is simple, can be carried out by any well trained assistant or technician without the aid of a highly trained bacteriologist, and has apparently yielded results of value. Our description is taken almost entirely from Carrel 's own writ- ings. "Wounds should be examined every two or three days, and when the time for secondary closure appears, perhaps every day. The principle consists in the examination of the secretions of the wounds THE ANAEROBIC BACILLI 765 by means of smears in such a way that an approximate estimate of the number of bacteria contained in the wounds can be made. Although the method is very inaccurate, its value does not depend upon its revealing slight differences, the significant variations being so widely apart that the necessary error in the comparative enumera- tions does not render the method useless. The examination need not begin earlier than twelve hours after the infliction of the wound, since up to that time few bacteria will be found. At the end of this time, when hemorrhage has completely stopped, smears are taken with a platinum loop from different parts of the wound. The points from which cultures are taken should always be those in which bacteria are most likely to be present in large numbers. Thus, Carrel chooses points in contact with foreign bodies, necrotic bits of bone, and from deep in the sinuses and crevices of the wound. Specimens should never be taken from bleeding points. Specimens should always be taken from a consider- able number of different places in the same wound. Care should be exercised to avoid taking smears from the skin adjacent to the wound. With the end of a small platinum loop small amounts of secretion are picked up, and smeared upon slides in such a way that approximately the same area is covered by the different loopfuls of secretion. With loops of uniform size and a little prac- tice, a surprising uniformity of technique can be developed. These smears are allowed to dry, and may be stained in a variety of ways. Carbol thionin has been extensively used, but we believe that a Gram stain which is almost as simple, will give a little more useful information. The stained slides can now be examined under the microscope and the number of bacteria per field, counted. If the number exceeds fifty or more to the field, more accurate counting will yield no valuable information because the wound still contains too many bacteria to warrant closure or relaxation of the local therapy that is being applied. Gradually, as the wound improves, less and less bacteria will appear in the daily series of slides, and when it has dropped below fifty per field, careful counting may give an index of daily variations. Eventually, they will drop to only one microorganism per five, ten or twenty fields, in which case the daily report can be expressed in fractions, as 1/5, 1/10, or 1/20, etc. The daily counts can, in this way, be numerically charted, and constructed into a curve which 766 PATHOGENIC MICROORGANISMS will show the surgeon by a giance* the numerical progress of the baeterial infection. Carrel states that it is useless to take any smears as long as hemorrhage exists. If the wound is being irrigated with Dakin's solution or other antiseptic fluids, the treatment must be omitted for at least two hours before the smears are taken. Smears taken from the surface of smooth muscles are practically useless, since smooth muscle becomes sterile early in the healing process. There- fore, the choosing of the point of smear is of the utmost importance. The depth of the wound may begin to become sterile at times when individual little foci around necrotic bone, small pockets, etc., may still contain numerous bacteria. This must be borne in mind and an intelligent survey of the wound made by the bacteriologist who takes the smear. To overlook such dangerous points would seriously imperil the life of the patient, were the wound closed. When absolutely no bacteria are found in such smears, it does not mean that the wound is completely sterile. It is still possible that cultures might reveal organisms, and when the period of secondary closure approaches, especially when streptococci have been present at a previous time, we would regard it of the greatest importance to take a culture aimed particularly at the demonstration of hemolytic streptococci, before the actual suture is carried out. Cultural examinations should be made at the beginning by taking specimens from parts of the wound selected as indicated above, and smearing them upon fresh blood-agar plates (without glucose). This is primarily aimed at determining whether cocci, and especially hemolytic streptococci or staphylococci, are present. If the smears show a great many bacilli resembling the ordinary anaerobes, it may be well, too, to make anaerobic cultures, but anaerobic analysis is not of great immediate value to the surgeon as far as further procedure is concerned because of the long time consumed by such examinations. Suture is not carried out if hemolytic cocci of any kind are present, and for this season, with a smear as a preliminary indication, frequent culture upon blood plates should be undertaken during the progress of the treatment. In discussing the subject, it is not possible to give an intelligent survey of the bacteriological methods, without, to some extent, entering into the surgical considerations involved. For this reason .we quote from Pool,31 whose experience with this type of wound has been extensive. « Pool, E. H., Jour, A. M, A., 73, 1919. THE ANAEROBIC BACILLI 767 Debridement should be carried out as soon as possible after infliction of the wound. Primary suture may be employed only during quiet periods in case of war, and in hospitals where the patient may be retained for careful observation. Otherwise suture of the wound may lead to enclosure, within an imperfectly debrided wound, of various microorganisms, including those which produce gas gangrene. In regard to delayed primary and secondary suture, the following observations of Dr. E. H. Pool are not without interest. "The determination as to when a wound may be sutured depends on bac- teriologic findings and clinical observation. It must be emphasized that the co-operation of a bacteriologist is indispensable in making a decision aS to the indications for delayed primary and secondary sutures. The practical func- tion and indisputable importance of the bacteriologist in war surgery lies in this. In the consideration as to whether a wound is suturable or not, reliance must be placed chiefly on cultures, the important feature being the determina- tion of the presence or absence of hemolytic cocci. For this, a routine blood- agar examination is essential. Bacterial counts are far from exact, yet they give an in.dication as to the degree of bacterial contamination of a wound, especially the progress from day to day, and are of value especially for one untrained in estimating clinically the indications and contraindications for suture. From eighteen to forty-eight hours after the original operation of de- bridement or excision of tissues, the wound is dressed and a culture and a smear are made. A report is returned as soon as possible. This contains the approximate number of organisms per field and the varieties of organisms. If no organisms are found, suture is indicated. If hemolytic cocci are present, suture is not considered. In the absence of hemolytic cocci, if the wound is clinically suturable, the presence of a few anaerobes or other organisms (approximately one in two fields) does not contraindicate suture. A con- siderable number of organisms of any kind indicates delay of suture, until the bacterial growth declines. A culture and a smear should be repeated at the following dressing ; the results of this examination will determine suturing or further delay. If the wound is left open for a considerable period, e.g., over a week, or is definitely infected, a smear is made every two days. It is also advisable to make a culture occasionally. Care must be taken not to touch the skin surface in making the smear, since skin contamination vitiates the value of the report. From the smear a bacterial curve is plotted accord- ing to Carrel's plan. When the organisms in two successive counts are few, that is, approximately one per two fields, and a culture shows an absence of hemolytic cocci, the wound is considered susceptible of secondary suture except when the wound has contained hemolytic cocci at any time. In that case careful cultures are made from granulation tissue and from the discharge 708 PATHOGENIC MICROORGANISMS from all parts of the wound, and absence of hemolytie cocci should be estab- lished by two successive negative cultures before suture is made. It has been observed that streptococci are prone to lie dormant in small numbers, but to flare up and cause virulent infection after closure of the wound." In compound fractures the same principles apply, except that, as stated by Pool, expedition, thoroughness and early closure is particularly important because it means the conversion of open into closed fracture. In such fractures of the long bones, delayed primary suture, that is, suture not later than six days after the infliction of the wound, should be aimed at. He states that it lias been demonstrated repeatedly that severe' fractures of long bones, except the femur, may be closed in from three to six days after debridement. If this cannot be done, secondary suture may often be made successfully under proper bacteriological control. In the case of joints, the principle of treatment consists in com- plete debridement of the tract of the wound into the soft parts and bone, with the removal of foreign bodies and irrigation of the joint, followed by absolute closure of the joint by suture, with or without closure of the superficial parts. If a joint becomes distended after the operation and infection is suspected, the effusion should be aspirated and examined by smear and culture. If such examination indicates infection, the joint should be reopened and treatment for suppurative arthritis begun. In civil surgery the principles worked out with war wounds can be applied with still greater hope of success, since here the nature of the trauma and infection is apt to be less extensive. As to serological treatment in civilian surgery, this will be applicable chiefly in cases in which there has been a considerable delay in the proper surgical treatment of the wound after its inflic- tion. It seems most probable at the present time that the most hopeful prospect for future therapy will lie in the combination of antitoxic sera against B. Welchii, B. oedematiens and Vibrion Sep- tique, with Tetanus antitoxin, prophylactically injected in the same way in which Tetanus antitoxin alone has been used, hitherto. Whether it will be possible to produce this in polyvalent sera pro- duced in the same horse or whether they will have to be separately injected, will depend upon future investigation in large scale serum production. Bacillus of Symptomatic Anthrax (Bacillus anthracis symp- tomatici, Rauschbrand, Ckarbon symptomatique, Sarcophysematos THE ANAEROBIC BACILLI 769 bovis). — Symptomatic anthrax is an infectious disease occurring chiefly among sheep, cattle, and goats. It is spoken of as "quarter-evil" or "blackleg." The disease has never been ob- served in man. 'It was formerly confused with true anthrax, because of a superficial similarity between the clinical symptoms of the two maladies. Bacteriologically, the two microorganisms are in entirely different classes. Symptomatic anthrax is of wide distribution and infection is usually through the agency of the soil in which the bacillus is present, in the form of spores which may retain viability .for several years. \ . ! ••* FIG. 76. — BACILLUS OP SYMPTOMATIC ANTHRAX. After Zettnow. Morphology and Staining. — The bacillus of symptomatic anthrax is a bacillus with rounded ends, being about four to six micra long, and five-tenths to six-tenths inicra wide. It is usually seen singly and never forms long chains. The bacillus in its vegetative form is actively motile and possesses numerous flagella placed about its periphery. In artificial media it forms spores which are oval, broader than the rod itself, and placed near, though never actually at, the end of the bacillary body. This gives the bacillus a racket- shaped appearance. It is readily stained with the usual anilin dyes, but is easily decolorized by Gram's method of staining. However, von Hibler claims that when, very carefully stained the bacillus can be shown 770 PATHOGENIC MICROORGANISMS to be Grain-positive — at least when taken from the animal body.32 Cultivation. — The bacillus is a strict anaerobe. It was obtained in pure culture first by Kitasato.33 Under anaerobic conditions it is easily cultivated upon the usual laboratory media, all of which are more, favorable after the addition of glucose, glycerin, or nutrose. In all media there is active gas formation, which, owing to an admixture of butyric acid, is of a foul, sour odor. The bacillus is not very delicate in its requirements of a special reaction of media, grow- ing equally well on those slightly acid or slightly alkaline. On gelatin plates, at 20° C., colonies appear in about twenty-four hours, usually round or oval, with a compact center about which fine radiating filaments form an opaque halo. The gelatin is liquefied. Surface colonies upon agar plates are circular and made up of a slightly granular compact center, from which a thinner peripheral zone emanates, containing microscopically a tangle of fine threads. In agar stabs, at 37.5° C., growth appears within eighteen hours, rapidly spreading from the line of stab as a diffuse, fine cloud. Gas formation, especially near the bottom of the tube, rapidly leads to the formation of bubbles and later to extensive splitting of the medium. In gelatin stab cultures growth is similar to that in agar stabs, though less FIG. 77. — BA- rapid. CILLUS OF SYMP- Pathogenicity.— Symptomatic anthrax bacilli TOMATIC ANTHRAX. Culture in glucose are Path°genic for cattle, sheep, and goats. By far agar. the largest number of cases, possibly the only spon- taneous ones, appear among cattle. Guinea-pigs are very susceptible to experimental inoculation. Horses are very little susceptible. Dogs, cats, rabbits, and birds are immune. Man also appears to be absolutely immune. Spontaneous infection occurs by the entrance of infected soil into abrasions or wounds, usually of the lower extremities.- Infection depends to some extent upon the relative degree of virulence of the bacillus — a variable factor in this 32 ,'on Hibler, Kolle, Wassermann, etc,., p. 792, vol. iv. **Kitasaio, Woch. f. Hyg, 1889. THE ANAEROBIC BACILLI 771 species. Twelve to twenty-four hours after inoculation there appears at the point of entrance a soft, puffy swelling, which on palpation is found to emit an emphysematous crackling. The emphysema spread rapidly, often reaching the abdomen and chest within a day. The course of the disease is extremely acute, the fever high, the general prostration extreme. Death may result within three or four days after inoculation. At autopsy the swollen area is found to be infiltrated with a thick exudate, blood-tinged and foamy. Subcutaneous tissue and muscles are edematous and crackle with gas. The internal organs show parenchymatous degeneration and hemorrhagic areas. The bacilli, immediately after death, are found but sparsely distributed in the blood and internal organs, but are demonstrable in enormous numbers in the edema surrounding the central focus. If carcasses are allowed to lie unburied for some time, the bacilli will attain a general distribution, and the entire body will be found bloated with gas, the organs filled with bubbles. Practically identical conditions are found after experimental inoculation. Toxins. — According to the investigations of Leclainche and Vallee,34 the bacillus of symptomatic anthrax produces a soluble toxin. It is not formed to any extent in ordinary broth, but is formed in considerable quantities in broth containing blood or albuminous animal fluids. The best medium for obtaining toxin, according to the same authors, is the bouillon of Martin,35 made up of equal parts of veal infusion and a pepton solution obtained from the macerated tissues of the stomachs of pigs. The toxin contained in filtrates of such cultures is quite resistant to heat, but rapidly deteriorates if free access of air is allowed. Immunity. — Active immunization against the bacillus of symp- tomatic anthrax was first accomplished by Arloing36 and his col- laborators by the subcutaneous inoculation of cattle with tissue- extracts of infected animals. The work of these authors resulted in a practical method. of immunization which is carried out as follows : 34 Leclainche et Vallee, Ann. de 1'inst. Pasteur, 1909. 135 Martin, Ann. de 1'inst. Pasteur, 1898. 36 Arloing, Cornevin, et Thomas, "Le Charbon Sympt.," ete., Paris, 1887. Ref. from Grassberger und Schattenfroh, Kraus und Levaditi, "Handbuch " etc vol i, pt. 2, 772 PATHOGENIC MICROORGANISMS Two vaccines are prepared. Vaccine I consists of the juice of infected meat, dried and heated to 100° C. for six hours. Vaccine II is a similar meat- juice heated to 90° C., for the same length of time. By the heating, the spores contained in the vaccines are attenuated to relatively different degrees. Vaccine I in quantities of 0.01 to 0.02 c.c. is emulsified in sterile salt solutions and injected near the end of the tail of the animal to be protected. A similar quantity of Vaccine II is injected in the same way fourteen days later. This method has been retained in principle, but largely modified in detail by various workers. Kitt37 introduced the use of the dried and powdered whole meat instead of the meat juice, and made only one vaccine, heated to 94° C., for six hours. This method has been largely used in this country.38 Passive immunization with the serum39 of actively immunized sheep and goats has been used in combination with the methods of active immunization. 37 Kitt, Ref . from Grassberger und Schattenfroh, loc. cit. 38 Report of Bureau of Animal Ind., Wash., 1902. 39 Arloing, Leclainche et Vallee, loc. cit. CHAPTER XXXVIII BACILLUS ANTHRACIS AND ANTHRAX (Milzbrand, Charbon) ANTHRAX is primarily a disease of the hcrbivora, attacking especially cattle and sheep. Infection not infrequently occurs in horses, hogs, and goats. In other domestic animals it is exceptional. Man is susceptible to the disease and contracts it either directly from the living animals or from the hides, wool, or other parts of the cadaver used in the industries. The history of the disease dates back to the most ancient periods and anthrax has, at all times, been a severe scourge upon cattle- and sheep-raising communities. Of all infections attacking the domestic animals' no other has claimed so many victims as anthrax. In Russia, where the disease is most 'common, 72,000 horses are said to have succumbed in one year (1864). * In Austria-Hungary, Germany, France, and the Eastern countries, each year thousands of animals and numerous human beings perish of anthrax. In England and America the disease is relatively infre- quent. No quarter of the globe, however, is entirely free from it. Especial historical interest attaches to the anthrax bacillus in that it was the first microorganism proved definitely to bear a specific etiological relationship to an infectious disease. The discovery of the anthrax bacillus, therefore, laid, as it were, the cornerstone of modern bacteriology. The bacillus was first observed in the blood of infected animals by Pollender in 1849, and, independently, by Brauell in 1857. Davaine,2 however, in 1863, was the first one to produce experimental infection in animals with blood containing the bacilli and to suggest a direct etiological relationship between the two. Final and absolute proof of the justice of Davaine 's con- tentions, however, was not brought until the further development of bacteriological technique, by Koch,3 had made it possible for 1 Quoted from Sobernheim, Kolle und Wassermann, vol. ii. 2 Davaine, Comptes rend, de 1'acad. des. sci., Ivii, 1863. *Koch, Cohn's "Beitr. z. Biol. d. Pflanzen," ii, 1876. 773 774 PATHOGENIC MICROORGANISMS this last observer to isolate the bacillus upon artificial media and to reproduce the disease experimentally by inoculation with pure cultures. Morphology and Staining. — The anthrax bacillus is a straight rod, 5 to 10 micra in length, 1 to 3 micra in width. It is non-motile. In preparations made from the blood of an infected animal, the bacilli are usually single or in pairs. Grown on artificial media they form tangles of long threads. Their ends are cut off squarely, in sharp contrast to the rounded ends of many other bacilli, The FIG. 78. — BACILLUS ANTHRACIS. From pure culture on agar. corners are often sharp and the ends of bacilli in contact in a chain often touch each other only at these points, leaving in consequence an oval chink between the ends of the organisms. The appearance of a chain of anthrax bacilli therefore, has been not inaptly com- pared to a rod of bamboo. On artificial media the anthrax bacillus forms spores. Oxygen is necessary for the formation of these spores and they are consequently not found in the blood of infected sub- jects. The spores are located in the middle of the bacilli and are distinctly oval. They are difficult to stain, but may be demonstrated by any of the usual spore-staining procedures, such as Holler's or Novy's methods. The bacilli themselves are easily stained by the usual anilin dyes, and gentian- violet or fuchsin in aqueous solution BACILLUS ANTHRACIS AND ANTHRAX 775 may be conveniently employed. They are not decolorized by Gram's method. In preparations from animal tissues or blood, stained by special procedures, the anthrax bacillus may occasionally be seen to possess a capsule. The capsule is never seen in preparations from the ordinary artificial media. Some observers have demonstrated them in cultures grown in fluid blood serum. In chains of anthrax bacilli, the capsule when present seems to envelop the entire chain and not the individual bacteria separately. Isolation. — Isolation of the anthrax bacillus from infected ma- terial is comparatively simple, both because of the ease of its cultiva- tion and because of the sharply characteristic features of its mor- phological and cultural appearance. Cultivation. — The anthrax bacillus is an aerobic, facultatively anaerobic bacillus. While it may develop slowly and sparsely under anaerobic conditions, free oxygen is required to permit its luxuriant and characteristic growth. The optimum temperature for its cultivation ranges about 37.5° C. It is. not, however, delicately susceptible to moderate variations of temperature and growth does not cease until temperatures as low as 12° C. or as high as 45° C. are reached. By continuous cultivation at some of the temperatures near either the higher or the lower of these limits, the bacillus may become well adapted to the new environment and attain luxuriant growth.4 The anthrax bacillus may be cultivated on all of the usual artificial media, growing upon the meat-extract as well as upon the meat-infusion media. It may be cultivated also upon hay infusion, various other vegetable media, sugar solutions, and urine. While moderate acidity of the medium does not prevent the growth of this bacillus, the most favorable reaction for media is neutrality or slight alkalinity. On gelatin plates, colonies develop within twenty-four to forty- eight hours as opaque, white disks, pin-head in size, irregularly round and rather flat. As- the colonies increase in size their outlines become less regular and under the microscope they are seen to be made up of a hair-like tangle of threads spreading in thin wavy layers from a more compact central knot. The microscopic appear- ance of these colonies has been aptly described as resembling a 4 Dieudonne, Arb. a. d. kais, Gesundheitsamt, 1894. 776 PATHOGENIC MICROORGANISMS Medusa head. Fragments of a colony examined on a slide with a higher power show the individual threads to be made up of parallel chains of bacilli. After a day or two of further growth, the gelatin about the colonies becomes liquefied. In gelatin stab cultures, growth appears at first as a thin white line along the course of the puncture. From this, growth proceeds in < FIG. 79. — BACILLUS ANTHRACIS. In section of kidney of animal dead of anthrax. thin spicules or filaments diverging from the stab, more abundantly near the top than near the bottom of the stab, owing to more active growth in well oxygenated environment. The resulting picture is that of a small inverted "Christmas tree." Fluidification begins at the top, at first a shallow depression filled with an opaque mixture of bacilli and fluid. Later the bacilli sink to tlic bottom of the flat depression, leaving a clear supernatant fluid of poptonizod gelatin. In broth, growth takes place rapidly, but does not lead to an even, general clouding. There is usually an initial pellicle formation at BACILLUS ANTHRACIS AND ANTHRAX 777 the top where the oxygen supply is greatest. Simultaneously with this a slimy mass appears at the bottom of the tube, owing to the sinking of bacilli to the bottom. Apart from isolated flakes and threads the intervening broth is clear. Shaken up, the tube shows a tough, stringy mass, not unlike a small cotton fluff, and general clouding is produced only by vigorous mixing. Upon agar plates, growth at 37.5° C. is vigorous and colonies appear within twelve to twenty-four hours. They are irregular in FIG. 80. — BACILLUS ANTHBACIS. In smear of spleen of animal dead of anthrax. outline, slightly wrinkled, and show under the microscope the char- acteristic tangled-thread appearance seen on gelatin, except that they are more compact than upon the former medium. The colonies are slightly glistening and tough in consistency. On agar slants, the colonies usually become confluent, the entire surface soon being covered by a grayish, tough pellicle which, if fished, has a tendency to come away in thin strips or strands. On potato, growth is rapid, white, and rather dry. Sporulation upon potato is rapid and marked, and the medium is favorable for the study of this phase of development. Milk is slowly acidified and slowly coagulated. This action is chiefly upon the casein; very few, if any, changes being produced 778 PATHOGENIC MICROORGANISMS either in the sugars or in the fats of the milk. The acids formed are, according to Iwanow,5 chiefly formic, acetic, and caproic acids. Biological Considerations. — The anthrax bacillus is aerobic and facultatively anaerobic. It is non-motile and possesses no flagella. In the animal body it occasionally forms capsules. In artificial cultures in the presence of oxygen, it sooner or later invariably forms spores. The spores appear after the culture has reached its maximum of development. Sporulation never occurs in the animal body, probably because of the absence of sufficient free oxygen. FIG. 81. — ANTHRAX COLONY ON GELATIN. (After Giinther.) Spores are formed most extensively6 at temperatures ranging from 20° C. to 30° C. Spore formation ceases below 18° C. and above 42° C. For different strains these figures may vary slightly, as has been shown from the results of various observers. Spores appear most rapidly and regularly upon agar and potato media. The spore — one in each bacillus — appears as a small, highly refractile spot in the center of the individual bacterium. As this enlarges, the body of the bacillus around it gradually undergoes granular degeneration and loses its staining capacity.7 5 Iwanow, Ann. de 1'inst. Pasteur, 1892. 6 Koch, loc. cit. 7 Behring, Zeit. f. Hyg., vi and vii, 1889; Dent. med. Woch., 1889. BACILLUS ANTHRACIS AND ANTHRAX 779 If anthrax bacilli are cultivated for prolonged periods upon media containing hydrochloric or rosolic acid or weak solutions of carbolic acid,8 cultures may be obtained which do not sporulate and which seem permanently to have lost this power, without losing their virulence to the same degree. Similar results may be obtained by continuous cultivation at temperatures above 42° C. By this procedure, however, virulence, too, is considerably di- minished. Resistance. — Because of its property of spore formation, the anthrax bacillus is extremely resistant toward chemical and physical environment. The vegetative forms themselves are not more resist- ant than most other non-sporulating bacteria, being destroyed by a temperature of 54° C. in ten minutes. Anthrax spores may be kept in a dry state for many years without losing their viability.9 While different strains of anthrax spores show some variation in their powers of resistance, all races show an extremely high resist- ance to heat. Dry heat at 140° C. kills them only after three hours.10 Live steam at 100° kills them in five to ten minutes. Boil- ing in water destroys them in about ten minutes. Low temperatures have but little effect upon them. Ravenel11 found that, frozen by liquid air, they were still viable after three hours. The variability shown by different strains of spores in their resistance to heat is even more marked in their behavior toward chemicals.12 Some strains will retain their viability after exposure to 5 per cent carbolic acid for forty days,13 while others are destroyed by the same solution in two days. Corrosive sublimate, 1 :2,000, kills most strains of anthrax in forty minutes. Direct sunlight destroys anthrax spores within six to twelve hours.14 Pathogenicity. — The anthrax bacillus is pathogenic for cattle, sheep, guinea-pigs, rabbits, rats, and mice. The degrees of sus- ceptibility of these animals differ greatly, variations in this respect existing even among different members of the same species. Thus, 8 Chamberland et Roux, Comptes rend, de 1'acad. des sci., xcvi, 1882. 9 Surmnnt et Arnould, Ann. de 1'inst. Pasteur, 1894. 10 Koc.h und Wolff hiigel, Mitt. a. d. kais. Gesundheitsamt, 1881. 11 Raw'n-rl, Medical News, vii, 1899. 12 FranM, Zeit. f. Hyg., vi, 1889. 13 Koch, loc. oit. 14 Momont, Ann. de Finst. Pasteur, 1892. 780 PATHOGENIC MICROORGANISMS the long-haired Algerian sheep show a high resistance, while the European variety are highly susceptible; and, similarly, the gray rat is much more resistant than the white rat. Dogs, hogs, cats, birds, and the cold-blooded animals are relatively insusceptible. For man the bacillus is definitely pathogenic, though less so than for some of the animals mentioned above. While separate races of anthrax bacilli may vary much in their degree of virulence, a single individual strain remains fairly constant in this respect if preserved, dried upon threads or kept in sealed tubes, in a cold, dark place. Virulence may be reduced15 by various attenuating laboratory procedures which are of importance in that they have made possible prophylactic immunization. Heating the bacilli to 55° C. for ten minutes considerably reduces their virulence. Similar results are obtained by prolonged cultivation at tempera- tures of 42° to 43° C., or by the addition of weak disinfectants to the culture fluids.16 Once reduced, the new grade of virulence remains fairly constant. Increase of virulence may be artificially produced by passage through animals. Experimental infections in susceptible animals are most easily accomplished by subcutaneous inoculations. The inoculation is fol- lowed, at first, by no morbid symptoms, and some animals may appear perfectly well and comfortable until within a few hours or even moments before death, when they suddenly become visibly very ill, rapidly go into collapse, and die. The length of the disease depends to some extent, of course, upon the resistance of the infected subject, being in guinea-pigs and mice from twenty-four to forty- eight hours. The quantity of infectious material introduced, on the other hand, has little bearing upon the final outcome, a few bacilli, or even a single bacillus, often sufficing to bring about a fatal infection. Although the bacilli are not demonstrable in the blood until just before death, they nevertheless invade the blood and lymph streams immediately after inoculation, and are conveyed by these to all the organs. This has been demonstrated clearly by experiments where inoculations into the tail or ear were immediately followed by amputation of the inoculated parts without prevention of the fatal general infection. The bacilli are probably not at first 15 Toussaint, Comptes rend, de 1'acad. des sci., xci, 1880; Pasteur, Chamberlan et Roux, Comptes rend, de 1'acad. des sci, xcii, 1881. 16 Chamberkmd et Rowx, bid., XCVI, 1882. BACILLUS ANTHRACIS AND ANTHRAX 781 able to multiply in the blood. At the place of inoculation and probably in the organs they proliferate, until the resistance of the infected subject is entirely overcome. At this stage of the disease, no longer held at bay by any antagonistic qualities of the blood, they enter the circulation and multiply within it. Autopsy upon such animals reveals an edematous hemorrhagic infiltration at the point of inoculation. The spleen is enlarged and congested. The kidneys are congested, and there may be hemorrhagic spots upon the serous membranes. The bacilli are found in large numbers in the blood and in the capillaries of all the organs. The mode of action of Bacillus anthracis is as yet an unsettled point. It is probable that death is brought about to a large extent by purely mechanical means, such as capillary obstruction. Neither a true secretory toxin nor an endotoxin has been demonstrated for the anthrax bacillus. The decidedly toxemic clinical picture of the disease, however, in some animals and in man, precludes our definitely concluding that such poisons do not exist. It is a matter of fact, however, that neither culture filtrates nor dead bacilli have any noticeable toxic effect upon test animals, and exert no appre- ciable immunizing action. Spontaneous infection of animals takes place largely by way of the alimentary canal, the bacilli being taken in with the food. The bacteria are swallowed as spores, and therefore resist the acid gastric juice. In the intestines they develop into the vegetative forms, increase, and gradually invade the system. The large majority of cattle infections are of this type. Direct subcutaneous infection may also occur spontaneously when small punctures and abrasions about the mouth are made by the sharp spicules of the hay, straw, or other varieties of fodder. When infection upon a visible part occurs, there is formed a diffuse, tense local swelling, not unlike a large carbuncle. The center of this may be marked by a black, necrotic slough, or may contain a pustular depression. Infection by inhalation is probably rare among animals. Trans- mission among animals is usually by the agency of the excreta or unburned carcasses of infected animals. The bacilli escaping from the body are deposited upon the earth together with animal and vegetable matter, which forms a suitable medium for sporulation. The spores may then remain in the immediate vicinity, or may be scattered by rain and wind over considerable areas. The danger 782 PATHOGENIC MICROORGANISMS from buried carcasses, at first suspected by Pasteur, is probably Very slight, owing1 to the fact that the bacilli can not sporulate in the anaerobic environment to which the burying-process subjects them. The disease, in infected cattle and sheep, is usually acute, killing within one or two days. The mortality is extremely high, fluctuating about eighty per cent. In man the disease is usually acquired by cutaneous inoculation. It may also occur by inhalation and through the alimentary tract. Cutaneous" inoculation occurs usually through small abrasions or scratches upon the skin in men who habitually handle live-stock, and in butchers, or tanners of hides. Infection occurs most fre- quently upon the hands and forearms. The primary lesion, often spoken of as "malignant pustule," appears within twelve to twenty- four hours after inoculation, and resembles, at first, an ordinary small furuncle. Soon, however, its center will show a vesicle filled with sero-sanguineous, later sero-purulent fluid. This may change into a black central necrosis surrounded by an angry red edematous areola. Occasionally local gangrene and general systemic infection may lead to death within five or six days. More frequently, how- ever, especially if prompt excision is practiced, the patient recovers. The early diagnosis of the condition is best made bacteriologically by finding the bacilli in the local discharge. The pulmonary infection, known as "wool-sorter's disease," occurs in persons who handle raw wool, hides, or horse hair, by the inhalation or by the swallowing of spores. The disease is fortunately rare in this country. The spores, once inhaled, develop into the vegetative forms17 and these travel along the lymphatics into the lungs and pleura. The disease manifests itself as a violent, irregular pneumonia, which, in the majority of cases, leads to death. The bacilli in these cases can often be found in the sputum before death. Infection through the alimentary canal may occasionally, though rarely, occur in man, the source of infection being usually ingestion of the uncooked meat of infected animals. This form of infection is rare, because in many cases the bacilli have not sporulated in the animal and the ingested vegetative forms are injured or de- stroyed by the acid gastric juice. When viable spores enter the gut, however, infection may take place, the initial lesion being localized usually in the small intestine. The clinical picture that follows is one of violent enteritis with bloody stools and great 17 Eppinger, Wien. med. Woch., 1888. BACILLUS ANTHRAC1S AND ANTHRAX 783 prostration. Death is the rule. The diagnosis is made by the dis- covery of the bacilli in the feces. General hygienic prophylaxis against anthrax consists chiefly in the destruction of infected animals, in the burying of cadavers, and in the disinfection of stables, etc. The practical impossibility of destroying the anthrax spores in infected pastures, etc., makes it necessary to resort to prophylactic immunization of cattle and sheep. Immunity Against Anthrax. — Minute quantities of virulent an- thrax cultures usually suffice to produce death in susceptible animals. Dead cultures are inefficient in calling forth any immunity in treated subjects. It is necessary, therefore, for the production of active immunity to resort to attenuated cultures. The safest way to, .accom- plish such attenuation is the one originated by Pasteur,18 consisting in prolonged cultivation of the bacillus at 42° to 43° C. in broth. Non-spore-forming races are thus evolved. The longer the bacilli are grown at the above temperature the greater is the reduction in their virulence. Koch, Gaffky, and Loeffler,19 utilizing the variations in susceptibilities of different species of animals, devised a method by means of which the relative .attenuation of a given culture may be estimated and standardized. Rabbits are less susceptible than guinea-pigs, and virulent anthrax cultures, grown for two or three days after the stated conditions, lose their power to kill rabbits, but are less virulent for guinea-pigs. After ten to twenty days of further cultivation at 42° C. the virulence for the guinea-pig disappears, but the culture is potent against the still more susceptible mouse. Even the virulence for mice may be entirely eliminated by further cultivation at this temperature. The method of active immunization first practiced by Pasteur, and still used extensively, is carried out as follows: Two anthrax cultures of varying degrees of attenuation are used as vaccins. The premier vaccin is a culture which has lost its virulence for guinea-pigs and rabbits, and is potent only against mice. The deuxieme vaccin is a culture which is still definitely virulent for mice and guinea-pigs, but not potent for rabbits. Forty-eight-hour broth cultures of these strains, grown at 37.5° C., form the vaccin actually employed. Vaccin I is subcutaneously injected into cattle in doses of 0.25 c.c., sheep receiving about half this quantity. After twelve days have elapsed similar quantities of Vaccin II are injected. 18 Pasteur, loc. cit. 19 Koch, Goffky, und Loeffler, Mitt. a. d. kais. Gesundheitsamt, 1884. 784 PATHOGENIC MICROORGANISMS Pasteur's method has given excellent results and confers an immunity which lasts about a year. Chauveau20 has modified Pasteur 's method by growing the bacilli in bouillon at 38° to 39° C., at a pressure of eight atmospheres. Cultures are then made of races attenuated in this way, upon chicken bouillon and allowed to develop for thirty days. Single injections of 0.1 c.c. each of such cultures are said to protect cattle. Active immunization of small laboratory animals is very difficult, but can be accomplished by careful treatment with extremely at- tenuated cultures. Passive immunization by means of the serum of actively immune animals was first successfully accomplished by Sclavo.21 The subject of passive immunization has been especially inves- tigated and practically applied by Sobernheim.22 The serum used is produced by actively immunizing sheep. It is necessary to carry immunization to an extremely high degree in order to obtain any appreciable protective power in the serum. This is accomplished by preliminary treatment with Pasteur's or other attenuated vac- cines, followed by gradually increasing doses of fully virulent cul- tures. Treatment continued at intervals of two weeks, for two or three months, usually produces an effective serum. Horses and cattle may also be used for the process, but they are believed by Sobernheim to give less active sera than sheep. Bleeding is done about three1 weeks after the last injection. The sera are stable and easily preserved. Injections of 20 to 25 c.c. of such a serum have been found to protect animals effectually from anthrax and to confer an immunity lasting often as long as two months. Animals already infected are said to be saved by treatment with 25 to 100 c.c. of the serum. Neither specific bactericidal nor bacteriolytic properties have, so far, been demonstrated in these immune sera. In fact, these properties are distinctly more pronounced against Bacillus anthracis in the normal sera of rats and dogs. Agglutinins have not been satisfactorily demonstrated in sera, partly because of the great technical difficulties encountered in the active chain-formation of the bacillus in fluid media. An increase of opsonic power of such 20 Chauveau, Comptos rend, de 1'acad. des sci., 1884. 21 Sclavo, Cent, f . Bakt., xviii, 1895. 22 Sobernheim, Zeit. f. Hyg., xxv, 1897; xxxi, 1899. BACILLUS ANTHRACIS AND ANTHRAX 785 serum over normal serum has not been satisfactorily demonstrated. Bacteria Closely Resembling Bacillus Anthracis. — In most labora- tory collections there are strains of true anthrax bacilli so attenuated that they are practically non-pathogenic. These do not differ from the virulent strains in any morphological or cultural characteristics. Besides such strains there are numerous non-virulent bacteria cul- FIG. 82. — BACILLUS SUBTILIS. (Hay Bacillus.) turally not identical with Bacillus anthracis, but resembling it very closely. B. ANTHRACOIDES (Hueppe and Wood23). — A Gram-positive bacil- lus, morphologically different from B. anthracis in that the ends arc more rounded. Culturally, somewhat more rapid in growth and more rapid in gelatin fluidification. Non-pathogenic. Otherwise indistinguishable from B. anthracis. B. RADICOSUS (Wurzel Bacillus). — Cultivated from water — city water supplies. Morphologically somewhat larger than Bacillus an- 23 Hueppe und Wood, Berl. klin. Woch., xvi, 1889. 786 PATHOGENIC MICROORGANISMS thracis, and the individual bacilli more irregular in size. Very rapid fluidification of gelatin and growth most active at room temperature. Non-pathogenic. B. SUBTILIS (Hay Bacillus}. — Although not very closely related to the anthrax group, this bacillus is somewhat similar and conveniently described in this connection. It is of importance to workers with pathogenic bacteria, because of the frequency with which it is found as a saprophyte or secondary invader in chronic suppurative lesions. Morphology and Cultivation. — Straight rod, 2 to 8 micra long, 0.7 micron wide. Spores formed usually slightly nearer one pole than the other. Grows in long chains and only in such chains are spores found. It does not decolorize by Gram's method. Is actively motile in young cultures in which the bacilli are single or in pairs. In older cultures chains are formed and the bacilli become motionless. Gelatin is liquefied. On gelatin and agar the bacilli grow as a dry corrugated pellicle. Microscopically, the colonies are made up of interlacing threads, being ^irregularly round with fringed edges. There is a tendency to confluence. ! The bacillus is found in brackish water, infusions of vegetable matter, etc., and is practically non-pathogenic, occurring only occasionally as a saprophyte in old sinuses and infected wounds. * CHAPTER XXXIX BACILLUS MALLEI AND GLANDERS (Glanders Bacillus) GLANDERS is an infectious disease prevalent chiefly among horses, but transmitted occasionally to other domestic animals and to man. The microorganism causing the disease, though seen and described by several earlier authors, was first obtained in pure culture and accurately studied by Loeffler and Schiitz1 in 1882. Morphology and Staining. — The glanders bacillus or B. mallei is a rather small rod with rounded ends.2 Its length varies from 3 to 4 micra, its breadth from 0.5 to 0.75 micron. Variation in size between separate individuals in the same culture is characteristic. The rods are usually straight, but may show a slight curvature. The bacillus is non-motile. There are no flagella and no spores are formed. The grouping of the bacilli in smears shows nothing very characteristic. Usually they appear as single bacilli lying irregularly parallel, often in chains of two or more. In old cultures, involution forms appear which are short, vacuolated, and almost coccoid. While the glanders bacillus stains rather easily with the usual anilin dyes, it is so easily decolorized that especial care in preparing specimens must be observed. Stained in the usual manner with methyleiie-blue, it shows marked irregularity in its staining quali- ties; granular, deeply staining areas alternating with very faintly stained or entirely unstained portions. This diagnostically helpful characteristic has been variously interpreted as a mark of degenera- tion or a preparatory stage for sporulation. It is probably neither of the two, but an inherent irregularity in the normal protoplasmic composition of the bacillus, not unlike that of B. diphtherias. The bacillus is decolorized by Gram's method of staining. Cultivation. — The glanders bacillus is easily grown on all of the usual meat-infusion media. It is practically indifferent to moderate 1 Loeffler und Schutz, Deut. med. Woch., 1882. 2 Loeffler, Arb. a. d. kais. Gesundheitsamt, 1886. 787 788 PATHOGENIC MICROORGANISMS variations in reaction, growing equally well upon neutral, slightly acid, or slightly alkaline culture media. Glycerin or small quantities of glucose added to media seem to render them more favorable for the cultivation of this bacillus. Upon agar the colonies show little that is characteristic. They appear after twenty-four hours at 37.5° C. as yellowish-white spots, at first transparent, later more opaque. They are round, with an even border, and microscopically appear finely granular. The older the cultures are, the more yellow do they appear. On gelatin at room temperature, growth is slow, grayish-white, and FIG. 83. — GLANDERS BACILLUS. From potato culture. (After Zettnow.) no liquefaction of the gelatin occurs. Growth upon this medium is never abundant. In broth, there is, at first, diffuse clouding, later a heavy, tough, slimy sediment is formed. At the same time the surface is covered with a similarly slimy pellicle. The broth gradually assumes a dark brown color. In milk, coagulation takes place slowly. In litmus milk, acidifica- tion appears. The growth upon potato presents certain features which are diag- nostically valuable. On potatoes which are not too acid growth is abundant and within forty-eight hours covers the surface as a yellowish, transparent, slimy layer. This gradually grows darker BACILLUS MALLEI AND GLANDERS 789 until it has assumed a deep reddish-brown hue. In using this feature of the growth diagnostlcally, it must not be forgotten that a very similar appearance upon potato occurs in the case of B. pyocyaneus. Biological Considerations. — Bacillus mallei is aerobic.3 Growth under anaerobic conditions may take place, but it is slow and impoverished. The most favorable temperature for its cultivation is 37.5° C. It fails to develop at temperatures below 22° C. or above 43° C. On artificial media, if kept cool and in the dark, and in sealed tubes, the glanders bacillus will retain its viability for months and years. On gelatin and in bouillon, it lives for a longer time than on the other media. Exposed to strong sunlight it is killed within twenty-four hours. Heating to 60° C. kills it in two hours, to 75° C. within one hour. Thorough drying kills the glanders bacillus in a short time. In water, under the protected conditions that are apt to prevail in watering-troughs, the bacillus may remain alive for over seventy days. The resistance to chemical disinfectants is not very high.4 Carbolic acid, one per cent, kills it in thirty minutes, bichlorid of mercury, 0.1 per cent, in fifteen minutes. Pathogenicity. — Spontaneous infection with the glanders bacillus occurs most frequently in horses. It occurs also in asses, in cats, and, more rarely, in dogs. In man the disease is not infrequent and is usually contracted by those in habitual contact with horses. Experimental inoculation is successful in guinea-pigs and rabbits. Cattle, hogs, rats, and birds are immune to experimental and spon- taneous infections alike. Spontaneous infection takes place by entrance through the broken skin, through the mucosa of the mouth or nasal passages. Infection in horses not infrequently takes place through the digestive tract.5 In all cases, so far as we know, previous injury to either the skin or to the mucosa is necessary for penetration of the bacilli and the development of the disease. Glanders in horses may occur in an acute or chronic form, depending upon the relative virulence of the infecting culture and the susceptibility of the subject. The more acute form of the disease is usually limited to the nasal mucosa and upper respiratory tract. The more chronic type of the disease is often accompanied by mul- 3 Loeffler, loc. cit. 4 Finger, Ziegler's Beitr., vi, 1889. 5 Nocard, Bull, de la soc. centr. de med. vet., 1894. 790 PATHOGENIC MICROORGANISMS tiple swellings of the skin and general lymphatic enlargement. This form is often spoken of as * 'farcy." Acute glanders in the horse begins violently with fever and prostration. After two or three days there is a nasal discharge, at first serous, later seropurulent. At the same time there is ulcera- tion of the nasal mucosa and acute swelling of the neighboring lymph nodes. These may break down and form deep pus-discharging V- V i v FIG. 84. — GLANDERS BACILLI IN TISSUE. (From a drawing furnished by Dr. James Ewing.) sinuses and ulcers. Finally, there is involvement of the lungs and death within four to six weeks. When the disease takes the chronic form the onset is more gradual. Concomitant with the nasal inflammation there is a forma- tion of subcutaneous swellings all over the body, some of which show a tendency to break down and ulcerate. Together with this the lymphatics all over the body become enlarged. The disease may last for several years, and occasionally may end in complete cure. BACILLUS MALLEI AND GLANDERS 791 In horses the chronic form of the disease is by far the more frequent. In man the disease is similar to that of the horse except that the point of origin is more frequently in some part of the skin rather than in the nasal mucosa, and the clinical symptoms differ accord- ingly. The onset is usually violent, with fever and systemic symp- toms. At the point of infection a nodule appears, surrounded by lymphangitis and swelling. A general papular eruption may occur. The papules may become pustular, and the clinical features may thus simulate variola. This type of the disease usually ends fatally in eight to ten days. The chronic form of the disease in man is much like that in the horse, but is more frequently fatal. The histological appearance of the glanders nodules is usually one of diffuse leucocytic infiltration and the formation of young connective tissue which preponderates more and more as the disease becomes chronic. Virchow has classed these lesions with the granu- lomata. From the center of such nodules B. mallei may often be obtained in pure culture. The nodules may be generally distributed throughout the internal organs. The bacilli themselves are found, apart from the nodules, in the nasal secretions, and occasionally in the circulating blood.6 The bacteriological diagnosis of glanders may be made by isolating and identifying the bacilli from any of the above-mentioned sources. When superficial nodules can be opened for the purpose of diagnosis this may prove an easy task. The most diagnostically helpful medium in such cases is potato. In a majority of cases, however, isolation is extremely difficult and resort must be had to animal inoculation. The most suitable animal for this purpose is the male guinea-pig. Intraperitoneal inoculation of such animals with ma- terial containing glanders bacilli leads within two or three days to tumefaction and purulent inflammation of the testicles. Such an experiment, spoken of as the * ' Strauss test, ' ' 7 should always be reinforced by cultural examination of the testicular pus, the spleen, and the peritoneal exudate of the animals employed. Toxin of Bacillus mallei. — The toxin of B. mallei, or mallein, belongs to the class of endotoxins. The toxic products have been invariably obtained by extraction of dead bacilli.8 Mallein differs 6 Wassilieff, Deut. med. Woch., 1883. ''Strauss, Arch, de mcd. exp., 1889. *Kresling, Arch. d. sci. biol., 1892; Preuser, Berl. thierarzt. Woch., 1894. 792 PATHOGENIC MICROORGANISMS from many other bacterial poisons in being extremely resistant. It withstands temperatures of 120° C. and prolonged storage without noticeable loss of strength.9 In its physiological action upon healthy animals, mallein is not a powerful poison. It can be given in considerable doses without causing death. Mallein may be obtained by a variety of methods. Helman and Kalning, the discoverers of this toxin, used filtered aque- ous and glycerin extracts of potato cultures. Houx10 cultivates virulent glanders bacilli in flasks containing 250 c.c. each of 5 per cent glycerin bouillon. Growth is allowed to continue at 35° C. for one month. At the end of this time, the cultures are sterilized at 100° for thirty minutes, and evaporated on a water bath to one- tenth their original volume. They are then filtered through paper. This concentrated poison is diluted ten times with 0.5 per cent carbolic acid before use. Concentration is done merely for purposes of conservation. The diagnostic dose of such mallein for a horse is 0.25 c.c. of the undiluted fluid. At the Washington Bureau of Animal Industry, mallein is pre- pared by growing the bacilli for five months at 37.5° C. in glycerin- bouillon. This is then boiled for one hour and allowed to stand in a cool place for one week. The supernatant fluid is then decanted and filtered through clay filters by means of a vacuum pump. The filtrate is evaporated to one-third its original volume on a water bath, and the evaporated volume resupplied by a 1 per cent carbolic acid solution containing about 10 per cent of glycerin. Diagnostic Use of Mallein. — The injection of a proper dose of mallein into a horse suffering from glanders is followed within six to eight hours by a sharp rise of temperature, often reaching 104° to 106° F. (40° C. +). The high temperature continues for several hours and then begins gradually to fall. The normal is not usually regained for several days. Locally, at the point of injection, there appears Within a few hours a firm, hot, diffuse swelling, which gradually extends until it may cover areas of 20 to 30 centimeters in diameter. The swelling is intensely tender during the first twenty- four hours, and lasts for three to nine days. Together with this there are marked symptoms of general intoxication. In normal animals the rise of temperature following an injection is trifling, 9 Wladimiroff, in Kraus und Levaditi, "Handbuch," etc., 1908. lnRoux et Nocard, Bull. d. 1. soc. centr. v6t., 1892. BACILLUS MALLEI AND GLANDERS 793 and the local reaction is much smaller and more transient. Injec- tions are best made into the breast or the side of the neck. The directions given by the United States Government for using mallein for the diagnosis of glanders in horses are as follows : ' * Make the test, if possible, with a healthy horse, as well as with one or more affected or supposed to be affected with glanders. Take the temperature of all these animals at least three times a day for one or more days before making the injections. "The injection is most conveniently made at 6 or 7 o'clock in the morning, and the maximum temperature will then usually be reached by or before 10 P.M. of the same day. "Use for each horse one cubic centimeter of the mallein solution as sent out, and make the injection beneath the skin of the middle of one side of the neck, where the local swelling can be readily detected. "Carefully sterilize the syringe after injecting each horse by naming the needle over an alcohol lamp or, better, use separate syringes for healthy and suspected animals. If the same syringe is used, inject the healthy animals first, and flame the needle of the syringe after each injection. "Take the temperature every two hours for at least eighteen hours after the injection. Sterilize the thermometer in a 5 per cent solution of carbolic acid, or a 0.2 per cent solution of corrosive sublimate, after taking the temperature of each animal. "The temperature, as a rule, will begin to rise from four to eight hours after the injection, and reach its maximum from ten to sixteen hours after injection. On the day succeeding the injection tako the temperature at least three times. "In addition to the febrile reaction, note the size, appearance, and duration of any local swelling at the point of injection. Note the general condition and symptoms of the animal, both before, during, and after the test. "Keep the solution in the sealed bottle and in a cool place, and do not use it when it is clouded or if it is more than six weeks old ; when it leaves the laboratory of the Bureau it is sterile. ' ' If the result of first injection is doubtful, the horse should be isolated and retested in from one to three months, when the slight immunity conferred by the first injection will have disappeared. The second injection into healthy horses usually shows no reaction whatever. 794 PATHOGENIC MICROORGANISMS Mallein may cause reactions in the presence of other diseases than glanders, such as bronchitis, periostitis, and other inflammatory lesions and is not so specifically valuable as tuberculin for diagnosis. Complement Fixation in Glanders. — Diagnostic complement fixa- tion for the diagnosis of glanders has been developed by McNeil and Olmstead at the New York Department of Health. The antigen is made by growing the glanders bacilli on a 1.6 per cent glycerin potato agar. From this stock cultures transplanted are made upon a neutral meat-free-veal-peptone agar. Twenty-four-hour growths are washed off with distilled water sterilized at 80° C. for four hours and filtered through a Berkefeld. After filtration the antigen must again be sterilized at 80° for one hour. Immunity. — Recovery from a glanders infection does not confer immunity against a second inoculation.11 Artificial active immuniza- tion has been variously attempted by treatment with attenuated cultures, with dead bacilli, and with mallein, but without convincing results. The serum of subjects suffering from glanders contains specific agglutinins.12 These are of great importance diagnostically if the tests are made with dilutions of, at least, 1 in 500, since normal horse serum may agglutinate B. mallei in dilutions lower than this. 11 Finger, Ziegler's Beitrage, vi, 1899. 12 Galtier, Jour, de med. vet., 1901. CHAPTER XL THE BACILLUS MELITENSIS (MICROCOCCUS MELITENSIS), BACILLUS BRONCHISEPTICUS, BACILLUS OF CATTLE ABORTION, BACILLUS OF GUINEA-PIG PNEUMONIA, AND THE BACILLUS PYOCYANEUS. MALTA FEVER AND THE BACILLUS MELITENSIS MALTA fever probably has its endemic focus in the Mediterranean islands and along the Mediterranean coast. From here it appears to have spread into continental Europe, France, Italy and Spain and into the Balkans. Naturally enough it has been found to occur in Northern Africa and cases have been reported along the East African coast. Castellani and Chalmers1 state that it has also been found in parts of Russia, in South Africa, in Uganda, China and the Philippines and in North and South America and in the West Indies. Castellani also has reported cases from Ceylon. It appears, thus, that the disease is very widely distributed, but centralizes chiefly about the Mediterranean, being most common in the warmer temperate and subtropical climates. Studies on its dis- tribution have been made particularly by Bassett-Smith,2 who has mapped its distribution throughout the world. A reproduction of his map may be found in Castellani and Chalmer's book. Morphology. — The micrococcus melitensis is an extremely small bacterium which has been described both as a bacillus and a coccus. It has been the custom of bacteriologists who have studied it more recently to regard it as a bacillus and speak of it as the Bacillus Melitensis. Eyre3 describes it as an extremely small coccus which has bacillary forms on various media which he regards as involution forms. It is Gram-negative and non-motile and does not form spores. In cultures it appears both singly and in short chains of two or more. Chains of any considerable length are uncommon. 1 Castellani and Chalmers, Textbook of Tropical Medicine, William Wood & Co., N. Y., 1919. 2 Bassett-Smith, British Med. Jour., 2, 1904. 3 Eyre, Kolle and Wassermann Handb., Second Edition, Vol. 4, 421. 795 796 PATHOGENIC MICROORGANISMS Cultivation. — The organism can usually be cultivated from the spleens of those who have died of the disease, or by spleen puncture, a method by which Bruce4 obtained it in his early studies. It can also be obtained from the blood stream in active cases, from the urine and from the milk of infected goats. Eyre states that the optimum is at 37° C. and that it will grow but slightly and slowly on media at room temperature at 20° or thereabout. It will grow both aerobically and under conditions of limited anaerobiosis. Growth is relatively slow and does not become luxuriant for three days or more. It does not seem to be very delicate in its nutritive requirements and has been cultivated on most of the ordinary media. It will grow on gelatin without liquefying the gelatin. Its growth on potato is hardly visible. Animal Pathogenicity. — According to Eyre, the B. melitensis is pathogenic for almost all laboratory animals, although it may take a very long time to kill. Eyre states that guinea pigs will live for as long as 100 or more days after an injection of B. melitensis, but that the virulence of the organism can be greatly enhanced by animal passage. It spontaneously infects goats which, as we shall see, is an important point in its epidemiology, and apparently it may similarly infect horses, cattle and sheep. The Disease. — In a number of ways, Malta fever is similar to typhoid fever. It probably gets into man in most cases by way of mouth, passing from the mucous membrane of the intestinal canal into the' blood without causing any considerable lesions. A bac- teriemia follows during which there is a typhoid fever-like tempera- ture and an enlarged spleen. The incubation time seems to be about two weeks, and the onset of the disease in its generalized symptoms again has the indefinite characters, malaise, headache, etc., that are associated with typhoid. There is no leucocytosis and a relative lymphocytosis. Usually cases are protracted, the disease passing through a prolonged febrile period, lasting two, three, or four weeks. There may be acute cases in which the onset is sudden and the course of the disease violent. Secondary symptoms may consist in neuritis, parenchymatous nephritis and pulmonary congestion, arthritis and orchitis. 4 Bruce, Practitioner, 1887. THE BACILLUS MELITENSIS 797 Diagnosis can be made by agglutination and isolation of the organisms. According to Wright and Semple,5 Bassett-Smith, Eyre6 and others, the agglutinins in the blood are very high and may be detected in dilutions of 1 :1,000 or over, even as early as the end of the first week. Blood cultures may be positive very early in the disease. Later, the organisms appear in the urine. Epidemiology. — It seems to be unquestionable at the present time that the disease is transmitted chiefly by the ingestion of the milk of infected goats, and according to Eyre the goat is the natural host of the infection, keeping the disease going in endemic regions. Apparently a very large percentage of the goats in infected regions are infected. According to Castellani and Chalmers in Malta 50 per cent of the goats are infected, and in parts of Northern Africa the percentage of infection in goats as indicated by various re- searches ranges from less than 4 to 34 per cent. Since goats' milk is. a common human food in these regions, the spread of the disease by this means is natural. There are probably carriers among goats and human beings that recover. Among other forms of transmission, direct infection from individual to individual probably takes place, and indirect infection through food and flies is not out of question. It has been suggested that mosquitoes, too, may transmit the disease and Eyre states that it has been possible to demonstrate the organisms in the stomachs of a number of different mosquito species. However, it seems quite clear that the most important method of transmission is by means of infected milk. Immunity. — It is the general belief that one attack of Malta fever protects against a second attack. However, there are a great many cases in which the fever recurs several times in the same individual in the form of relapses and recrudescences. The inter- missions between such attacks often last for months. Prophylactic vaccination has not yet been carried out on a sufficiently large scale to permit the formulation of conclusions. 5 Wright and Semple, Lancet, 656, 1897. 6 Eyre, etc., Report of the Mediterranean Fever Commission, 1907. 798 PATHOGENIC MICROORGANISMS B. Bronchiseptisus. — Prior to 1910 a great deal of inconclusive work was done on canine distemper, but in this year Ferry7 published a preliminary paper in which he reported the isolation of a bacillus which was often present in pure culture in the smaller bronchii and the tracheae of dogs killed early in the disease. At almost the same time, and independently of Ferry, McGowan8 reported similar observations. Soon after this the subject was very thoroughly studied by Torrey and Rahe.9 The B. bronchisepticus was described as a short, Gram-negative organism, occasionally coccoid in appear- ance, slowly motile, which grows very slowly at first isolation, the colonies being hardly visible on agar in twenty-four hours but definitely visible in forty-eight hours. It grows well on glycerin agar, will grow at 20° with an optimum at about 37.5°. It is not an obligate aerobe, but grows poorly without oxygen. It does not liquefy gelatin. It renders broth uniformly turbid with a slight deposit and no pellicle. No acid or gas is formed on carbohydrate media, and the media are rendered slightly alkalin in the course of four or five days. Torrey could determine no indol formation. The typical media, according to McGowan, Ferry and Torrey are litmus milk and potato. Torrey states that the use of these two media alone is sufficient for identification in the hands of a practiced observer. On litmus milk it grows like the B. fecalis alkaligenes. There is a progressive alkalinization, and after about twenty-four hours at 37° a ring of deep blue appears, extending about 3/8 inch from the surface. In from five to ten days the whole medium has assumed a blue-black color. On potato in twenty-four hours a marked yel- lowish-brown growth appears with sometimes a greenish darkening of the potato. In this respect again it is very similar to B. fecalis alkaligenes. Torrey also reports that the organism produces a hemolysin for rabbit and guinea pig erythrocytes. He states that typical distemper can be induced in susceptible dogs by injec- tion with pure cultures, and dogs which have recovered from attacks induced by the bacillus are protected by exposure to the disease in other dogs. 7 Ferry, Amer. Veterinary Review, 499, 1910 (quoted from Torrey) Jour. Infec Dis., 8, 1911, 399. 8 McGowan, Jour. Path, and Bacter., 15, 1911, 372 and 1916, 257, 9 Torrey and Rahe, Jour. Med. Res., 27, 1912, 291. THE BACILLUS MELITENSIS 799 Bacillus of Cattle Abortion (B. Abortus of Bang).— We have already mentioned, in speaking of the organism which causes abor- tion in mares, that there was another bacillus described by Bang10 in 1897 which was found by him to be the etio logical factor in abortion of cattle, but which was distinctly different from that subsequently found by Smith and others in the analogous disease of horses. This organism has been observed by a great many writers since Bang, and it was Smith who first pointed out its similarity to the B. bronchisepticus of canine distemper and to a bacillus which causes epidemic pneumonias in guinea pigs. Alice Evans11 subsequently pointed out the similarity of B. Abortus and of B. bronchisepticus, to the B. melitensis which causes Malta fever. Evans' description of Bacterium Abortus as studied from strains obtained by the Dairy Division of the Bureau of Animal Industry, United States Department of Agriculture, is as follows : It is a short, slender pleomorphic rod with rounded ends, some- times so short as to appear coccoid. Obtained from the condensa- tion water of a twenty-four hour culture on agar, it is non-motile and does not form spores. It is Gram-negative. It is difficult to grow on artificial media on first isolation, and in these early cultures its growth is favored by partial anaerobiosis, which Evans obtained by incubation in a closed jar in the presence of cultures of B. subtilis. Glycerin or serum agar are favorable media for isolation, but after prolonged cultivation it grows well on ordinary media. Colonies on agar plates develop after about two days in very small dewdrop form. It never heavily clouds broth. Milk is rendered slightly alkaline. On potato there is a slight grayish brown growth after several days, and subsequently, the brownish tinge discolors the potato itself. It forms no acid or gas on any of the sugars, but slightly reduces the hydrogen ion concentration of broth cultures. It produces ammonia from amino acids such as asparagin. It does not liquefy gelatin. It is easily distinguished from B. bronchisepticus by its lack of motility, and less rapid and abundant growth on artificial media, as well as by agglutination reactions. Evans states from her studies that B. melitensis is closely related to B. abortus and can be dis- tinguished from it only by means of agglutination tests. Fleischner, 10 Bang. Zeit. f. Tier Med., 1, 1897, 241. 11 Evans, Jour. Infec. Dis., 22, 1918, 580. 800 PATHOGENIC MICROORGANISMS Meyer and Shaw12 have recently examined skin reactions in connec- tion with B. abortus bovis and B. melitensis, and claim that this method establishes a very close association between the two. Of great importance to the sanitarian is the fact that the B. abortus may appear in the milk of cattle that have aborted and may also be present in the milk of cattle that are carriers and have suffered no abortion themselves. Cotton13 has shown that the Bacil- lus may persist in the genital organs for forty-six days after aborti9n has taken place. From the studies of Schroeder and Cotton,14 Stafseth15 and others, it appears that the B. abortus does not es- tablish itself permanently in the uterine cavity, and Stafseth 's recent investigations seem to show that it does not penetrate into the deeper layers of the mucous membrane and remain there as a latent infection. • Theobald Smith16 in 1912 called attention to the tuberculosis- like lesions in guinea pigs caused by B. abortus, a subject which later was studied in more detail by Smith and Fabyan.17 Fabyan, as well as Schroeder demonstrated the presence of the B. abortus in milk by guinea pig injection.. Characteristic lesions not unlike tubercles developed in such animals in about eight to ten weeks, and Evans found the Bacillus in the milk of six out of twenty-four cows that had not aborted. Huddleson18 has recently studied methods of isolating B. abortus from milk, other than by guinea- pig injection. He uses a liver or spleen infusion agar, developed by Stafseth, a medium in the preparation of which overheating and filtration through paper is avoided. The optimum hydrogen ion concentration is PH 6.6. He adds to this medium gentian violet to a final concentration of 1 :10,000. Ten c.c. of milk is centrifuged at 2,000 revolutions for about two hours. 0.1 c.c. of the sediment is taken out with a capillary pipette and distributed over the surface of a gentian violet agar plate. These plates are incubated in jars in which about 10 per cent of the air has been displaced "by C02. 12 Flcischner, Meyer and Shaw, Rep. Hooper Foundation, Univ. of Calif., 4, 1918. 13 Cotton, Amer. Veter. Rev., 44, 1913, 307. 14 Schroeder and Cotton, Jour. Amer. Vet. Assoc., 3, 1916, Quoted from Stafseth (3). 15 Stafseth, Stud, of Infectious Abortion, Mich. Agric. College, 49, 1920. 16 Smith, Theobald, Footnote in article by Cotton, quot. from Fabyn, Jour. Med. Res., 28, 85, 1913. 17 Smith and Fabyan, Cent. f. Bakt. Orig., 61, 1912, 549. 18 Huddleson, Mich. Agric, College Exper. Station, 49, Nov. 1920, 25. THE BACILLUS OF GUINEA-PIG PNEUMONIA 801 By this method he was able to obtain results comparable in regularity to guinea-pig inoculation, and obtained cultures in about four days as against eight weeks by the guinea-pig inoculation. It is important in making routine guinea-pig injections of milk for the determination of tuberculosis to remember the warning of Theobald Smith19 and not to jump at conclusions from mere gross appearance at autopsy of the animals. Whether or not the presence of B. abortus in milk is a danger to man is not certain, but it seems possible that the organism may cause diarrheal and perhaps other diseases, and the milk of aborted cattle should not be used for some time after abortion, and perhaps subjected to bacteriological test. The striking similarity described by Evans and others of the B. abortus of cattle and the B. melitensis, taken together with the fact recently observed that other animals, especially the goat, may be infected by the B. melitensis, suggests the possibility of human pathogenicity for B. abortus, though at the present time this can be mentioned merely as a conjecture and a subject for inquiry. Bacillus of Guinea-Pig Pneumonia. — In 1914 Theobald Smith20 called attention to a minute motile bacillus originally described by Tartakowsky which he found to be the cause of epidemic pneumonia in guinea-pigs. He found it to be similar to the organism described by Strade and Traina as the B. pneumoniae caviarum and Selter's B. caviae septicus. This organism he identified with the B. bron- chisepticus of McGowan.21 He describes the bacillus of guinea-pig pneumonia as follows : It is a minute rod with rounded ends. From agar slants it measures about 0.7 micron in length and about 0.5 micron broad. Longer rods are occasionally seen. The organisms stain solidly and are Gram-negative. They are rapidly motile. They grow moderately well on gelatin at room temperature, but do not liquefy the gelatin. On potato the bacillus makes a rich yellowish-brown color appearing within a week. It clouds broth within twenty-four tours, making a delicate iridescent membrane on the surface after prolonged growth. Later, a ropy deposit appears. It makes no visible change on milk. It does not produce indol. It is strictly aerobic and on sugar media 19 Smith, Jour. Exper. Med., 30, 4, 325, 1919. -° Smith, Theobald, Jour. Med. Res., 29, 1914, 291. 21 McGowan, Jour. Path, and Baet., 15, 1910, 372. 802 PATHOGENIC MICROORGANISMS does not produce gas or acid, but renders broth slowly alkalin. He states that in its bio-chemical aspects it approaches the pyo- cyaneous group, especially in regard to its strict aerobiosis and its lack of activity on sugars. In this respect, Smith calls attention to the fact that it is similar to the bacillus of cattle abortion. Smith compared the organism with a culture of the B. bronchisepticus sent him by Torrey which was supposed to be the cause of distemper in dogs, and found that his bacillus agreed with Torrey 's strain in every particular, in regard to morphology and cultural char- acteristics. He also identified his guinea-pig bacillus with an organism observed by Mallory in the ciliated epithelium of the air tubes in fatal cases of whooping cough. In his description of the growth of his guinea-pig organism on potato, Smith calls attention to the similarity of this bacillus to the growth on potato of the bacillus of cattle abortion of Bang. Bacillus Pyocyaneus. — It is a matter of common surgical ex- perience that many suppurating wounds, especially sinuses of long standing, discharge pus which is of a bright green color. The fact that this peculiar type of purulent inflammation is due to a specific chromo genie microorganism was first demonstrated by Gessard22 in 1882. The bacillus which was described by Gessard has since become the subject of much careful research and has been shown to hold a not unimportant place among pathogenic bacteria.23 Morphology and Staining. — Bacillus pyocyaneus is a short rod, usually straight, occasionally slightly curved, measuring, according to Fliigge, about 1 to 2 micra in length by about 0.3 of a micron in thickness. The bacilli are thus small and slender, but are subject to considerable variation from the measurements given, even in one and the same culture. "While ordinarily single, the bacilli may be arranged end to end in short chains of two and three. Longer chains may exceptionally be formed upon media which are especially unfavorable J!or its growth, such as very acid media or those con- taining antiseptics. Spores are not found. The bacilli are actively motile and possess each a single flagellum placed at one end. Bacillus pyocyaneus is stained easily with all the usual dyes, but is decolorized by Gram's method. Irregular staining of the bacillary 22 Gessard, Th&se de Paris, 1882. in, "La maladie pyocyanique, " Paris, 1889. THE BACILLUS PYOCYANEUS 803 body is common, but is always an indication of degeneration, and not a normal characteristic, as, for instance, in the diphtheria group. Cultivation. — The pyocyaneus bacillus is aerobic and faculta- tively anaerobic. It can be adapted to absolutely anaerobic environ- ments, but does not produce its characteristic pigment without the free access of oxygen. The bacillus grows readily upon the usual laboratory media and is not very sensitive to reaction, growing equally well upon moderately alkaline or acid media. Development takes place at temperatures as low as 18° to 20° C., more rapidly and luxuriantly at 37.5° C. On agar slants, growth is abundant and confluent, the surface of the agar being covered by a moist, grayish or yellowish, glistening, even layer. The pigment which begins to become visible after about eighteen hours soon penetrates the agar itself and becomes diffused throughout it, giving the medium a bright green fluorescent appear- ance, which grows darker as the age of the culture increases. In gelatin stabs, growth takes place much more rapidly upon the surface than in the depths. A rapid liquefaction of the gelatin takes place, causing a saucer-shaped depression. As this deepens, pigment begins to form in the upper layers, often visible as a greenish pellicle. In gelatin plates, the colonies have a characteristic appearance. They are round and are composed of a central dense zone, and a peripheral, loosely granular zone, which extends outward into the peripheral fluidified area in a fringe of fine filaments. When first appearing, they are grayish yellow, later assuming the characteristic greenish hue. In broth, growth is rapid and chiefly at the surface, forming a thick pellicle. Below this, there is moderate clouding. The pigment is formed chiefly at the top. In old cultures there is a heavy floc- culent precipitate. In fluid media containing albuminous material, strong alkalinity is produced. On potato, growth develops readily and a deep brownish pigment appears, which is not unlike that produced by B. mallei upon the same medium. Milk is coagulated by precipitation of casein and assumes a yellow- ish-green hue. In older cultures the casein may again be digested and liquefied. The pigment of Bacillus pyocyaneus has been the subject of much investigation. It was shown by Charrin24 and others that 24 Charrin, loc. cit. 804 PATHOGENIC MICROORGANISMS this pigment had no relation to the pathogenic properties of the bacillus. It is found in cultures as a colorless leukobase which assumes a green color on the addition of oxygen. Conversely, the typical green "pyocyanin/' as the pigment is called, may be de- colorized by reducing substances. This explains the fact that it is not found in cultures sealed from the air. Pyocyanin may be extracted from cultures with chloroform and crystallized out of such solution in the form of blue stellate crystals. These, on chemical analysis, have been found to belong to the group of aromatic com- pounds, with a formula, according to Ledderhose,25 of C14H14N20. Besides pyocyanin, Bacillus pyocyaneus produces another pig- ment which is fluorescent and insoluble in chloroform, but soluble in water.26 This pigment is common to other fluorescent bacteria, and not peculiar to Bacillus pyocyaneus. The reddish-brown color seen in old cultures27 and supposed by some writers to be a third pigment, is probably a derivative from pyocyanin by chemical change. Chloroform extraction of pyocyanin from cultures may serve occasionally to distinguish the pyocyaneus bacilli from other similar fluorescent bacteria. Ernst has claimed that there are two types of B. pyocyaneus, an a-type which produces only the fluorescent, water- soluble pigment, and a /?-type which produces both this and pyo- cyanin.28 Pathogenicity. — Bacillus pyocyaneus is one of the less virulent pathogenic bacteria. It is widely distributed in nature and may be found frequently as a harmless parasite upon the skin or in the upper respiratory tracts of animals and men. It has, however, occa- sionally been found in connection with suppurative lesions of various parts of the body, often as a mere secondary invader in the wake of another incitant, or even as the primary cause of the inflam- mation. In most cases where true pyocyaneus infection has taken place, the subject is usually one whose general condition and resist- ance are abnormally low.29 Thus pyocyaneus may be the cause of chronic otitis media in ill-nourished children. It has been cultivated 25 Ledderhose, quoted from Boland, Cent. f. Bakt., xxv, 1889. 26 Boland, loc. cit. 27 Gessard, Ann. de 1'inst. Pasteur, 1890, 1891, and 1892. 28 Ernst, Zeit. f. Hyg., ii, 1887. "Rohner, Cent. f. Bakt., xi, 1892, THE BACILLUS PYOCYANEUS 805 out of the stools of children suffering from diarrhea, and has been found at autopsy generally distributed throughout the organs of children dead of gastro-enteritis.30 It has been cultivated from the spleen at autopsy from a case of general sepsis following mastoid operation. The bacillus has been found, furthermore, during life in pericardial exudate and in pus from liver abscesses.31 Brill and Libman,32 as well as Finkelstein,33 have cultivated B. pyocyaneus from the blood of patients suffering from general sepsis. Wassermann34 showed the bacillus to have been the etiological factor in an epidemic of umbilical infections in new-born children. Similar examples of B. pyocyaneus infection in human beings might be enumerated in large numbers, and there is no good reason to doubt that, under given conditions, fatal infections may occur. Such cases, however, are still to be regarded as depending more upon the low resistance of the individual attacked than upon the great pathogenicity of B. pyocyaneus. Many domestic animals are susceptible to experimental pyo- cyaneus infection, chief among these being rabbits, goats, mice, and guinea-pigs. Guinea-pigs are killed by this bacillus with especial ease. Intraperitoneal inoculation with a loopful of a culture of average virulence usually leads to the death of a young guinea-pig within three or four days. Toxins and Immunization. — Emmerich and Low have shown that filtrates of old broth cultures of B. pyocyaneus contain a ferment-like substance which possesses the power to destroy some other bacteria, apparently by lysis. They have called this substance ' l pyocyanase ' ' and claim that, with it, they have succeeded in protecting animals from anthrax infection. During recent years' pyocyanase has been employed locally for the removal of diphtheria bacilli from the throats of convalescent cases. Broth-culture filtrates evaporated to one-tenth their volume in vacuo are used for this purpose. Pyocyanase is exceedingly thermostable, resisting boiling for several hours, and is probably not identical with any of the other toxins or peptonizing ferments produced by B. pyocyaneus. 80 Neumann, Jahrb. f. Kinderheilk., 1890. "Kraunhals, Zeit. f. Chir., xxxvii, 1893. 32 Brill and Libman, Amer. Jour. Med. Sci., 1899. "Finkelstein, Cent. f. Bakt., 1899. 84 Wassermann, Virchow 's Arch., clxv, 1901. 806 PATHOGENIC MICROORGANISMS The toxins proper of B. pyocyaneus have been the subject of much investigation, chiefly by Wasserrnann.35 Wassermann found that filtrates of old cultures were far more poisonous for guinea-pigs than extracts made of dead bacteria. He concludes from this and other observations that B. pyocyaneus produces both an eudotoxin and a soluble secreted toxin. The toxin is comparatively ther- mostable, resisting 100° C. for a short time. Animals actively im- munized with living cultures of B. pyocyaneus give rise in their blood serum to bacteriolytic antibodies only. Immunized with filtrates from old cultures, on the other hand, their serum will contain both bacteriolytic and antitoxic substances. The true toxin of B. pyocyaneus never approaches in strength that of diphtheria or of tetanus. Active immunization of animals must be done care- fully if it is desired to produce an immune serum, since repeated injections cause great emaciation and general loss of strength. Specific agglutinins have be'en found in immune sera by Wasser- mann36 and others. Eisenberg37 claims that such agglutinins are active also against some of the fluorescent intestinal bacteria. Bulloch and Hunter38 have recently been able to show that old broth cultures of B. pyocyaneus contain a substance capable of hemolyzing the red blood corpuscles of dogs, rabbits, and sheep. This "pyocyanolysin" seems intimately attached to the bacterial body. Prolonged heating of cultures does not destroy it. Heating of hemolytic filtrates, however, destroys it in a short time. The filtration of young cultures yields very little pyocyanolysin in the filtrate. In old cultures, however, a considerable amount passes into the filtrate. Whether or not the hemolytic power is due to a specific bacterial product or is dependent upon changes in the culture fluid, such as alkalinization, etc., can not yet be regarded as certain. Gheorghiewski39 claims to have found a leucocyte-destroying ferment in pyocyaneus cultures. 35 Wassermann, Zeit. f. Hyg., xxii, 1896. 36 Wassermann, Zeit. f. Hyg., 1902. 37 Eisenberg, Cent. f. Bakt., 1903. 38 Bulloch imd Hunter, Cent. f. Bakt., xxviii, 1900. 39 GheorghiewsJci, Ann. de 1'inst. Pasteur, xiii, 1899- CHAPTER XLI PLAGUE AND BACILLUS PESTIS (The So-called HaemorrJiagic Septicaemia Group] Plague. — The history of epidemic diseases has no more terrifying chapter than that of plague.1 Sweeping, time and again, over large areas of the civilized world, its scope and mortality were often so great that all forms of human activity were temporarily paralyzed. In the reign of Justinian almost fifty per cent of the entire popula- tion of the Roman Empire perished from the disease. The " Black Death" which swept over Europe during the fourteenth century killed about twenty-five million people. Smaller epidemics, appear- ing in numerous parts of the world during the sixteenth, seventeenth, and eighteenth centuries, have claimed innumerable victims. In 1893 plague appeared in Hong Kong. During the epidemic which followed, Bacillus pestis, now recognized as the etiological factor of the disease, was discovered by Kitasato2 and by Yersin,8 inde- pendently of each other. By both observers the bacillus could invariably be found in the pus from the bubos of afflicted persons. It could be demonstrated in enormous numbers in the cadavers of victims. The constancy of the occurrence of the bacillus in patients, shown in the innumerable researches of many bacteriologists, would alone be sufficient evidence of its etiological relationship to the disease. This evidence was strengthened, moreover, by accidental infections which occurred in Vienna in 1898, with laboratory cultures. Since that time the investigations of plague cases and plague outbreaks by individual bacteriologists and by commissions of many governments have established the relationship between the disease and the bacillus to such a degree that there is not the shadow of a doubt as to its etiological significance. TTaiulb. <1. liistor.-£ooj?r. Path.," 1881. *Kitaxato, Lancet, 1894. 9 Yersin, Ann. dc 1'inst. Pasteur, 1894. 807 808 PATHOGENIC MICROORGANISMS Morphology and Staining. — Bacillus pestis is a short, thick bacillus with well-rounded ends. Its length is barely two or two and a half times its breadth (1.5 to 1.75 micra by 0.5 to 0.7 micron). The bacilli appear singly, in pairs, or, more rarely, in short chains of three or more. They show distinct polar staining. In size and shape these bacilli are subject to a greater degree of variation than are most other microorganisms. In old lesions or in old cultures the bacilli show involution forms which may appear either as swollen coccoid forms or as longer, club-shaped, diphtheroid bacilli. De- generating individuals appear often as swollen, oval vacuoles. All these involution forms, by their very irregularity, are of diagnostic importance. They appear more numerous in artificial cultures than FIG. 85. — BACILLUS PESTIS. (After Mallory and Wright.) in human lesions. A very important property of the plague bacillus in this connection is the formation within twenty-four to forty-eight hours of vacuolated and swollen involution forms upon salt agar, that is, agar to which 3 to 5 per cent of salt is added. Such a medium is of great value in diagnostic work. According to Albrecht and Ghon,4 the plague bacillus may, by special methods, be shown to possess a gelatinous capsule. It does not possess flagella and does not form spores. The plague bacillus is easily stained with all the usual anilin dyes. Diluted aqueous fuchsin and methylene-blue are most fre- quently employed. With these stains the characteristically deeper * Albrecht und Ghon, Wien, 1898. PLAGUE AND BACILLUS PESTIS 800 staining of the polar portions of the bacillus is usually easy to demonstrate. Special polar stains have been devised by various observers. Most of these depend upon avoidance of the usual heat fixation of the preparations, which, in some way, seems to interfere with good polar staining. Fixation of the dried smears with absolute alcohol is, therefore, preferable. The bacillus is decolorized by Gram's method. Isolation and Cultivation. — The bacillus is easily isolated in pure culture from the specific lesions of plague patients, during life or at autopsy. It is worth noting that smears from bubos and other plague lesions will often show the typical bacilli in very small \ ««> FIG. 86. — BACILLUS PESTIS, 'INVOLUTION FORMS. (After Zettnow.) numbers only, possibly because of the ease with which they undergo degeneration. The bacillus grows readily and luxuriantly upon the meat-infusion media. The optimum temperature for its cultivation is about 30° C. Below 20° C. and above 38° C., growth is sparse and delayed, though it is not entirely inhibited until exposed to temperatures below 12° C., or above 40° C. The most favorable reaction of culture media is neutrality or moderate al- kalinity, though slight acidity does not prevent development. On agar, growth appears within twenty-four hours as minute colonies with a compact small center surrounded by a broad, irregularly indented, granular margin. 810 PATHOGENIC MICROORGANISMS On gcMin, similar colonies appear after two or three days at 20° to 22° C. The gelatin is not liquefied. In bouillon, the plague bacilli grow slowly. They usually sink to the bottom or adhere to the walls of the tube as a granular deposit and may occasionally form a delicate pellicle. Chain-formation is not uncommon. In broth cultures, moreover, a peculiar stalactite- like growth is often seen, when the culture fluid is covered with a layer of oil and the flasks are incubated in a place where shaking or vibration can be prevented. Delicate threads of growth hang down from the surface of the medium into its depths like stalactites. Characteristic involution forms are brought out best when the .bacilli are grown upon agar containing 3 to 5 per cent NaCl. Milk is not coagulated. In litmus-milk there is slight acid forma- tion. On potato and on blood serum the growth shows nothing char- acteristic or of differential value. On pepton media no indol is formed. Biological Considerations. — Bacillus pestis is aerobic. Absence of free oxygen is said to prevent its growth, at least under certain conditions of artificial cultivation. It is non-motile. Outside of the animal body the bacilli may retain viability for months and even years if preserved in the dark and in a moist environment. In cadavers they may live for weeks and months if protected from dryness. In pus or sputum from patients they may live eight to fourteen days. These facts are of great hygienic importance. Complete drying in the air kills the bacilli within two or three days.5 Thoroughly dried by artificial means they die within four or five hours. Dry heat at 100° C. kills the bacillus in one hour.6 Live steam or boiling water is effectual in a few minutes. The bacilli possess great resistance against cold, surviving a temperature of 0° C. for as many as forty days. Direct sunlight destroys them within four or five hours. The common disinfectants are effectual in the following strengths: car- bolic acid, one per cent kills them in two hours, five per cent in ten minutes ; bichloride of mercury 1 :1,000 is effectual in ten minutes. In a recent communication to the New York Pathological Society, Dr. Wilson reported that plague cultures which he had kept sealed °Kitasato, Lancet, 1894. • Abel, Cent. f. Bakt.. xxi. 1897. PLAGUE AND BACILLUS PESTIS 811 for as long as ten years in the ice chest were found living and virulent at the end of this time. In regard to the viability of plague bacilli in air at different atmospheric temperatures and conditions of humidity, there are many important sanitary problems involved which are of particular significance in connection with the spread of pneumonic plague. Teague and Barber7 worked on this subject in connection with the Manchurian epidemic of pneumonic plague, and found that plague bacilli contained in fine droplets of pneumonic plague sputum would suffer death from drying in a few minutes unless they were sus- pended in an atmosphere with a very small water deficit; in other words, the humidity or the degree of saturation of the atmosphere with water is a very important factor in determining the length of time for which plague bacilli will remain alive in such droplet- spray. Such atmospheres under ordinary circumstances are common in cold climates and droplets of sputum will, therefore, remain infectious longer in cold, wet climates than in warm ones. Animal Pathog"enicity. — Bacillus pestis is extremely pathogenic for rats, mice, guinea-pigs, rabbits, and monkeys. The most sus- ceptible of these animals are rats and guinea-pigs, in whom mere rubbing of plague bacilli into the unbroken skin will often produce the disease. This method of experimental infection of guinea-pigs is of great service in isolating the plague bacillus from material contaminated with other microorganisms. For the same purpose, infection of rats subcutaneously at the root of the tail may be employed. Such inoculation in rats is invariably fatal. The studies of McCoy8 of the United States and Public Health Service upon guinea-pigs and white rats show that individual plague cultures may vary considerably in virulence. The size of the dose, always excepting enormous quantities such as a whole agar culture, seems to make little difference in the speed with which the animals die. There may be considerable variation in the susceptibility of individual animals. Prolonged cultivation on artificial media may gradually reduce the virulence of plague bacilli, though, as stated above, this has not been the experience of all observers. T Teague and Barber, Philippine Jour, of Science, B, 7, 1912. 8 McCoy noted the surprising fact that, in San Francisco a considerable per- centage of wild rats — especially old ones, showed a high natural immunity to plague. 812 PATHOGENIC MICROORGANISMS In rats, spontaneous infection with plague is common and plays an important role in the spread of the disease. The pneumonic type of the disease is common in these animals and has been pro- duced in them by inhalation experiments. During every well- observed plague epidemic, marked mortality among the domestic rats has been noticed. Although it was formerly supposed that rat infection took place because of the gnawing of dead cadavers by other rats, the work of the British Indian Plague Commission has shown that rats, like man, are spontaneously infected by means of fleas which pass from the infected to the uninfected animal. In his work in California McCoy showed that the weasel and chipmunk are susceptible to plague infection, and therefore, poten- tial means of spread if once infected. Toxin, Formation. — The systemic symptoms of plague are largely due to the absorption of poisonous products of the bacteria. Al- brecht and Ghon,9 Wernicke,10 and others were unable to obtain any toxic action with broth-culture filtrates and concluded that the poisons of B. pestis were chiefly endotoxins, firmly attached to the bacterial body. Kossel and Overbeck,11 however, on the basis, of a careful investigation, came to the conclusion that, in addition to the endotoxin, there is formed in older broth cultures a definite and important true, soluble toxin. This, however, is unlikely in the light of a general survey of experimental work and conditions as they exist in the disease itself. It is most likely that the toxic symptoms here are those generally spoken of as endotoxin and also, we believe, perhaps some of the proteose substances suggested by us in connection with other bacteria. Immunization. — A single attack of plague usually protects human beings from reinfection. A second attack in the same individual is extremely rare. Immunization in animals produces specific agglu- tinating and bacteriolytic substances which are of great importance in the bacteriological diagnosis of the bacillus. The agglutinating action of the serum of patients is clinically important in the diagnosis of the disease, even in dilutions of one in ten, since undiluted normal human serum has no agglutinating effect upon plague bacilli. 8 Albrecht und Ghon, loc. cit. 10 Wernicke, Cent. f. Bakt., Ref ., xxiv, 1898. 11 Kossel und Overbeck, Arb. a. d. Gesundh., xviii, 1901. PLAGUE AND BACILLUS PESTIS 813 The curative plague serum prepared by Yersin and others by the immunization of horses with plague cultures has been extensively used in practice and though often disappointing, a definitely benefi- cial influence on the milder cases has been noted. The sera are standardized by their protective power as measured in white rats. The question of prophylactic vaccination and active immuniza- tion will be taken up in connection with plague prevention below. Plague in Man. — There are two chief methods by which the disease is acquired by man. The first is by entrance of the bacilli through the skin as a consequence of the bite of an infected flea. During the act of biting, the flea may either regurgitate blood, or, as is usually the case, deposit feces on the skin. The possibilities of entrance of the plague bacillus through minor injuries in the skin are so great that perhaps the infection may take place through the lesion caused by the fleabite, but more likely is rubbed in by the clothing or by scratching as the fleabite becomes inflamed and irritated. The other method by which plague is transmitted to man is by direct inhalation of sputum spray, a mode of infection which causes pneumonic plague. According to Castellani and Chalmers12 and others, about 2.5 per cent of the cases occurring during epidemics of bubonic plague are of the pneumonic variety, and there may be special epidemics of pneumonic plague like the one described in another section and studied by Strong, Teague and others13 in Manchuria and a more recent one which occurred in Northern China. The incubation time of the disease is usually less than ten days, and may be no longer than two or three. The organisms entering through the skin may cause a localized lesion at the point of entrance. This may be of negligible size or may show a considerable inflammatory reaction. The organisms enter the lymphatics and cause the so-called bubo. The primary bubos are situated in the glands into which the infected area drains and, for this reason, the most common seat for these lesions is in the glands of the groin, but they also may be first seen in the axillary, cervical or other glands. Secondary bubos may arise in other parts of the body, along the distribution of lymphatics, and the organisms rapidly enter the blood stream, causing septicemia. 12 Castellani and Chalmers, Manual of Tropical Medicine, W. Wood & Co., N. Y., 1919. 13 Teague and Strong, Philippine Jour, of Science, Sec. B, No. 7, 1912. 814 PATHOGENIC MICROORGANISMS The onset is usually sudden, with high fever and the general symptoms of a severe toxemia. Castellani states the bacilli can be found in blood cultures in about 30 per cent of the cases. The disease may take a considerable number of forms which depend very largely upon the virulence with which the organisms overwhelm the body. It may be relatively mild, or may take an acute septicemic form which is rapidly fatal. The pneumonic type is very severe and apt to kill rapidly. The onset of the pneumonic type according to Strong and Teague is abrupt, without prodromal symptoms. There is often a chill, head- ache, and fever which reaches 103° or 104° within a day of the onset, accompanied by a very rapid pulse. Cough appears within twenty-four hours. The expectoration soon becomes abundant and consists of blood-tinged mucus. When later it becomes thick and bright red, it contains enormous numbers of plague bacilli. There are marked signs of cardiac involvement, and delirium and coma, frequently appear. The same observers state that plague bacilli may frequently be found in the blood in such numbers that simple microscopical examination suffices for their detection. They state that in the Manchurian epidemic not a single case in which bac- teriological diagnosis was complete, was known to have recovered. The pathology of the lungs in this condition consists of general engorgement and edema. There are hemorrhages under the pleura, often fresh fibrinous pleurisy, and if a case lasts long enough there may be pneumonic infiltration. The distribution of the pneumonic areas may be cither lobar or lobular. Bacteria are found in enormous numbers in the peribronchial lymph spaces and in the adjoining alveoli. They may also be present in large numbers in the inter- lobular septa and under the pleura. Epidemiology. — Owing to the frequency and wide-spread nature of plague epidemics in the history of the world from most ancient times, it is quite impossible to more than very briefly outline the epidemiology of the disease. For fuller treatment of the epi- demiological aspects the reader is referred to such books as Rosenau's14 Preventive Medicine and Castellani and ChalmerV5 work on tropical medicine. The prevalence of the disease in ancient times ™Rosenau, Preventive Medicine and Hygiene, D. Appleton & Co., N. Y. & London, 1921. 15 Castellani and Chalmers, Manual of Tropical Medicine, William Wood & Co., N. Y., 1919. PLAGUE AND BACILLUS PESTIS 815 has been mentioned in the introduction. Through the Middle Ages a number of plague epidemics swept through Europe and frequently reached the commercial ports of Italy, Asia Minor and other parts of Eastern Europe from the Orient. In India it has long been known as a fatal form of epidemic disease and since the early part of the nineteenth century, has probably been endemic there. Cas- tellani and Chalmers15 state that it was introduced into China prob- ably in the first half of the eighteenth century by Mohammedans returning from Mecca via Burma to the Province of Yunnan. Here it has been epidemic ever since. In 1894 the study of the Hongkong epidemic revealed the causative agent of the disease. There was a very serious epidemic in 1894 which started in China, spread throug«h Bombay to other parts of India, thence to Madagascar, into the Malay States, the Philippine Islands, other islands of the Pacific, reaching North and South America and Europe; finally in 1900, it appeared in Cape Town and on the British Isles. Clemow, whom we quote from Castellani and Chalmers, stated that in 1900 plague was 'endemic in Mongolia, Southern China, the Himalayas, Mesopotamia, Persia, Uganda, parts of Russia and Northern Africa. In Africa the same author states that there are two endemic areas, one in Tripoli and the other in Uganda from which occasional African epidemics take origin. The disease is, thus, a constant menace in many different parts of the world and must remain an important source of concern to national public health organizations. In the United States the problem is perhaps more important than is appreciated by the people at large. In 1903 the disease appeared in California and for several years after that human cases occurred, though the disease never took on the menace of an epidemic. This was prevented probably by the energetic work of the United States Public Health Service under Rupert Blue, McCoy,16 Curry, and Wherry17 who instituted energetic methods of rat extermination, rat proofing and other neces- sary sanitary measures. More recently, foci have appeared in Texas and Now Orleans and it must never be forgotten that the conditions of climate and in other respects are not by any means unfavor- able to the development and spread of plague in some parts of America. To convey an idea of the prevalence of plague in the world to-day 16 McCoy, Pub. Health Report., July, 1913, No. 37. 17 Wherry, Jour. Infec. Dis., 5, 1908. 816 PATHOGENIC MICROORGANISMS we insert a record of plague cases reported from various places in 1911, which we take from Jackson's Book on Plague.18 This record appeared in the British Medical Journal for September 16th, 1911 : India. — Deaths from plague in India during the first six months (of 1011), 604,634. Most prevalent (1) United Provinces, 281,317 (2) Punjab, 171,084; (3) Bengal, 58,515; (4) Bombay Presidency, 28,109. Deaths in July, not included above, 8,990. Hong Kong. — April 24 to August 21 (1911), 255 cases, 194 deaths. China. — Since January 1, 1911, plague was reported in varying intensity in (provinces and towns) Manchuria, Peking, Tientsin, Chefoo, Shantung, Shanghai, Amoy, Foochow, Swatow, Canton, Pakhoi and Laichow. Indo-China. — At Saigon, in March and April (1911), many cases reported. April 17 to May 7, 56 cases; 17 deaths. May 22 to May 28, 37 cases; 12 deaths. Siam. — In Bangkok plague was more severe during 1911 than in any previous year. March 15 to April 15, 33 cases and 29 deaths. Java and Sumatra. — In Java, May 25 to June 3 (1911), 105 cases and 62 deaths (one province). In Sumatra plague was present, no statistics. Straits Settlements. — A few cases mostly imported, reported in 1911. Japan. — A few cases at Kobe in 1911. In Formosa, from April 2 to April 15, 31 cases ; 24 deaths. Egypt. — Plague reported from Port Said, Suakin (on board ship), Cairo and Alexandria ; also from 11 provinces. The province of Kena had a severe outbreak, May 5 to May 31 (1911), 51 cases and 49 deaths. Persia. — Several cases reported from ports on the Persian Gulf. Turkey in Asia. — A few cases at Muscat, Basra and at Port of Jeddah. British East Africa. — Kismayu and Port Florence reported a few cases in April (1911). Mauritius. — January 1 to April 11 (1911), 110 cases and 70 deaths. Portuguese East Africa. — Plague was reported present at Nahoria in May (1911). Eussia. — In the Kirgis Steppe in the Astrakan Government in January (1911), 50 cases; 30 deaths. South America. — Plague prevailed during 1911 in Peru, Ecuador, Brazil, Chile and Venezuela. No severe outbreak except in Peru, where from February to May many cases occurred and died. At Libertad, in March, 60 cases and 23 deaths were reported. Plague is primarily a disease of rodents. The bacillus is pathogenic for rats, mice, guinea-pigs, rabbits, for the California 18 Jackson, Plague, Lippincott & Co., 1916. PLAUGE AND BACILLUS PESTIS 817 ground squirrel,19 and for various species of ground moles such as the Manchurian tarbagan (Arctomys bobac).20 The spread of plague by rats has long been recognized and even in ancient times mortality in rats has been associated with large epidemic outbreaks. The most important recent experimental work on this matter was done by the British Indian Plague Commission at Bombay. This commission demonstrated the relationship between rats and plague infection in carefully conducted experiments in which considerable numbers of rats were used. According to the Commission, the most important species of rats are the Epimys nor- vegicus and Epimys rattus. Over thirteen hundred of some seventeen hundred rats found infected, belonged to these two species. Other rats can also be infected and the danger of plague exists wherever rats are found. The rat problem is a very important one, not only in connection with plague, but in connection with economic loss as well. Creel of the United States Public Health Service21 has called attention to the necessity of rat extermination for economic reasons alone. The dis- tribution and number of rats in the world is much greater than anyone ordinarily supposes. Creel states that in the cane producing tropical and semi-tropical countries, Porto Rico, the West Indies, the Hawaiian Islands and the Philippines, there is an enormous rat population. He states that on one cane plantation in Porto Rico where there were less than 500 people, 25,000 rats were killed in six months. He estimates that in the United States the rat population is probably as great as the human population, and the annual economic cost per rodent is higher than $1.00 a piece. Computing the upkeep of rats as one-half cent per day and estimating their number as above, Creel says that a sum of $167,000,000 is lost annually to the country by rat depredations. According to the British Plague Commission, the usual way by which rats are infected from others is by means of fleas, and this, as first suggested in 1898 by Simond, is the method by which the disease is carried to man. In the British Indian Plague Commission experiments, when healthy and infected rats, entirely free from fleas were placed together, no plague developed, even when these rats were in contact with the urine and feces of the infected ones and with polluted food. But when fleas were introduced, infection occurred. The most 19 McCoy, Jour. Infec. Dis., 5, 1909. 20 Wu Lien Teh., Jour. Hygiene, 13, 1913. n Creel, Rep. No. 135, TT. S. Pub. Health Serv., Vol. 28, No. 27, 1913. 818 PATHOGENIC MICROORGANISMS common flea found on rats is the Xenopsylla cheopis. The disease can also be transmitted by Ceratophyllu-s fasciatus and by Puiex irritant. Fleas habitually infesting dogs and cats may also infest rats which means that flea extermination must be general. It also indicates that the climatic and geographical distribution of fleas, as well as that of rats, must be taken into account in dealing with the disease. In rats the first development is a generalized blood infection during which enormous numbers of bacilli may be present in the blood. These are then taken into the intestine of the flea where they can live for a long time, and may be deposted upon the skin of the victim during feeding, since the flea is apt to regurgitate blood and to deposit feces at this time. It may also be that some of the bacilli are directly introduced with the bite, but it is probably more common that the organisms thus distributed will be rubbed in either by the clothing or iri scratching the fleabite. It is thus established with considerable certainty that while contact infection and other means of direct and indirect transmis- sion may, of course, occur, the usual manner of spread of plague is from rat to rat, rat to man, or man to man, by the agency of fleas. It is the Epimys rattus which lives in closest relationship to man, and is perhaps the most dangerous for this reason. The ordinary rat flea leaves the body of the rat within about three days of its death and is capable of remaining alive about three or four weeks. The plague bacilli may multiply tremendously in the intestine of the flea during the period between feedings. In the California outbreak infection from ground squirrels to man was definitely shown in a number of cases, and in Manchuria the tarbagan men- tioned above has also been suspected of being the direct source. McCoy22 in 1921, summarizing the results of recent plague studies, states that in the United States natural infection has taken place among ground squirrels of California, the black rats of Hawaii, and a species of wood rat and field rodent in Louisiana. Human cases have been unquestionably traced to ground squirrels, and almost always, he says, have the peculiarity of showing the primary bubos in the axillae, because the fleas in the course of the infection, attack the upper extremities, whereas when the disease is contracted from rats, the fleas are more apt to bite on the legs. Squirrel 22 McCoy, Amer. Jour. Hyg., March, 1921. PLAGUE AND BACILLUS PESTIS 819 infection, however, according to McCoy, form very few cases, not more than about seventeen in all having been found since the squirrel origin was first studied. The squirrel flea can carry plague from squirrel to squirrel and from squirrel to other rodents. Such transmission does not hold good, however, for the pneumonic form of the disease. Careful studies have been made on the pneumonic form by Strong, Teague, Crowell and Barber23 who observed the Manchurian epidemic which occurred during the winter of 1910 to 1911, and during which, within three months, 50,000 people died of the disease. According to these writers the infection here is not as formerly supposed primarily a septicemic condition, during which the lungs become secondarily involved, but occurs by direct inhalation into the bronchi. The organisms either pass along the bronchioles into the alveoli, or through the walls of the bronchioles into the lungs, giving rise first to peribronchial inflammations and later to more diffuse processes, followed by pneumonic changes of the lobar or lobular type. After this, the blood becomes quickly infected and bacteriemia is, therefore, secondary to pneumonia. As mentioned above, the organisms are coughed out with the drop- lets of sputum, and thus sprayed into the atmosphere. If the atmosphere is dry, they will rapidly die out. If, however, the weather is cold and the atmosphere charged with moisture the organisms may remain alive for considerable periods and inhalation of virulent organisms may take place easily. Acording to the same writers, the organisms are not usually exhaled by the expired air during ordinary respiration or even during the labored respirations of the pneumonic case, but only during coughs when they may be sprayed out in enormous numbers even when the naked eye can detect no visible spray. In this form of plague, then, the transmission is very largely direct. McCoy states that pneumonic plague rarely occurs from rat infection, and states that it is an interesting and perhaps "significant fact" that in plague squirrels there seems to be a definite tendency to localize in the lungs, a thing which rarely happens in rats. From a study of the plague cases in the United States, he states that except for one single focus of thirteen cases, this form of the disease has not occurred. This pneumonic outbreak originated from a bubonic case of squirrel origin which developed secondary pneu- 23 Strong, Teague, Crowell and Barber, Philippine Jour. Science, Sec. B, 7, 1912. 820 PATHOGENIC MICROORGANISMS monia and spread through four transmission generations in man in the autumn of 1919. Plague Prevention. — From what has been said in regard to the transmission of the disease it is apparent that the prevention of plague becomes very largely a question of rat extermination and protection against fleas. Vigilance in observation of the mortality among rats in endemic centers, for the discovery of early rodent foci is important. International precautions depend upon quarantine against rats which may easily be carried, and have been carried from country to country, by ships and by rail. The disinfestation of ships by S02 by means of the Clayton apparatus, and by hydrocyanic acid gas as described by Creel and Faget of the United States Public Health Service, are among the important methods in use for the disinfesta- tion of ships, sleeping cars, etc. Quarantine regulations and the supervision of incoming ships is important. In the United States a quarantine of seven days is imposed on ships arriving from plague ports, a period which is probably not long enough. Precautions must be taken to prevent the travel of rats along hawsers when ships are docked at a wharf, and this is usually accomplished by the application of large circular shields along the course of the hawsers in such a way that rats cannot cross. When foci of plague are discovered in any community, wholesale rat destruction and isolation of the focus, by destruction of build- ings, ratp roofing of cellars, etc., must be resorted to. Blake has introduced a system of which Castellani and Chalmers speak very highly, the principle of which is that the rat extermination and other precautionary measures are started in a wide circle about the focus, working in toward the center, since work beginning at the focus itself in an outward circle may easily serve to scatter rats, rather than circumscribe them. In the Philippines and in villages in which natives live in primitive huts, actual burning of the houses has been resorted to, but this, too, may easily result in merely scattering the rat population into the neighboring districts. On a large scale, rat extermination is usually carried out by poisons in which phosphorous paste is perhaps the most important method. Of especial importance is the protection of food stores, and particular attention to all depositories of food, grain, etc., about which rats are apt to accumulate. BACTERIOLOGICAL DIAGNOSIS OF SUSPECTED PLAGUE CASES. — Since the bacteriological diagnosis of the earliest cases that occur is one of PLAGUE AND BACILLUS PESTIS 821 the most important problems of prevention, various governments have laid down methods of collection and shipment of material that should be followed in the case of suspected cases in man and rats. Public Health Reports, Volume 35, Number 37, lays down the method in which material is to be collected for the United States. This we quote in toto from this Bulletin as follows: To the Officers of the Public Health Service and State and Local Health Officers : Owing to the appearance of plague in several American ports it is important that all cases of suspected plague, both in man and animals, be subjected to a bacteriological examination. 1. The following material from persons or rodents suffering from plague may be sent to laboratories: Human Cases (Living) (a) Pus or gland fluid from buboes aspirated by syringe or collected after incision, on agar slants. (b) Portions of tissues affected, removed at operation, in sterilized bottles, securely stoppered. (c) Blood specimens, in sterilized sealed glass ampules or test tubes. (d) Cultures of suspected organisms, on agar slants. Human Cases (Necropsy) (a) Portions of the affected tissues — preferably bubo, lung and spleen — in sterilized glass bottles, securely stoppered. Rodents (a) The whole rodent carcass, in fruit preserving jar. 2. Do not place tissues or rodents in a preservative. The bacteriological diagnosis of plague rests upon the production of the disease in laboratory animals, and the isolation and growth of the causative organism, Bacillus pestis. Any preservative that kills this organism will defeat the purpose of the examination. If decomposition of the specimen is feared, it may be placed in a tight container and this in turn surrounded by ice in a larger container, preferably of wood. Every specimen should be plainly marked preferably by ordinary pencil, showing the date and the exact location from which it was taken. 3. The shipper must make certain that the specimen is packed in such manner as to prevent possible danger to those handling the same, provided the package is properly handled. In this connection it is necessary that specimens be wrapped in sufficient cotton or other absorbent material, to prevent leakage of fluid from the container should the glass be broken. The Following Instructions should be explicitly observed. 1. Ship by express — Federal laws prohibit the shipping of plague-infected material, or cultures, by mail. 822 PATHOGENIC MICROORGANISMS 2. Do not make packages too small, as small packages are more likely to be lost in transit, or overlooked. 3. Each package should be marked as follows: Notice This package contains perishable specimens for bacteriological examination Please Expedite Careful autopsy must of course be made on all cases, animal or man, and the lesions studied. The lesions in rats have been fully described in another section. Cultures are taken on agar and smears taken from buboes or sputum, stained by Loeffler's methylene-blue, the bipolar appearance and degeneration forms of the organisms looked for. Cultural diagnosis is then made by the appearance of the growing organisms, and their colonies, the staining properties, appearance on salt agar, agglutination in immune sera, and, above all, inoculation of rats and guinea-pigs with observation of the characteristic lesions in these animals. Since the examination of rats for plague is an important phase of the study of epidemics, it may be well to review the typical lesions in these animals as described by an experienced American student of plague, George W. McCoy.24 McCoy, agreeing with the Indian Plague Commission, states that the naked eye is superior to the microscopical examination. There is engorgement of the subcutane- ous vessels and a pink coloration of the muscles. The bubo when present is sufficient for diagnosis. Marked injection surrounds it and sometimes there is hemorrhagic infiltration. The gland itself is firm but usually caseous or occasionally hemorrhagic. In the liver there is apparent fatty change, but this is due to necrosis. Pin-point spots give it a stippled appearance as though it had been dusted with pepper. Pleural effusion is an important sign. The spleen is large, friable, and often presents pin-point granules on the surface. One or two per cent of rats may present no gross lesions. Cultures should of course be made. The method of examination consists in immersing the rat in any convenient antiseptic to kill fleas and other ectoparasites. The rats ar*e nailed by their feet to a shingle and the skin is reflected from the whole front of the body and neck, so as to expose the cervical, axillary, and inguinal regions. The thoracic and abdominal cavities are then opened and examined. 24 McCoy, Jour, of Inf. Dis., vi, 1909 ; George W. McCoy, Public Health Beports, July, 1912. PLAGUE AND BACILLUS PESTTS 823 An excellent example of the circumscription of a plague focus at its first discovery is one which we take from a note by Rucker in the United States Public Health Service Report, No. 28, 1915, based largely on the work of Passed Assist. Surg. R. A. Kearny. In September, 1914, a dead Mus norvegicus was found on a street corner in New Orleans. Laboratory examination proved this plague infected. The district was searched for other rats and on the 16th of September a similar plague rat was found in the neighborhood in a Chinese restaurant located in a ramshackle frame building, situated between a rat-proof brick building and an open lot. Behind the restaurant was a frame shed which was not rat proof. A survey of the district followed, in which thirty-eight infected rats were discovered, all of them of the same species as the preceding. One hundred and thirteen dead rats were found, and two infected rats were found on a neighboring street corner. Twenty-one were found in the Chinese restaurant, and one in the open lot and the other in the neighborhood. Rucker believes that the focus was eliminated largely because there was plenty of food in this particular neighborhood, and rats could not easily leave there without entering the street, a thing which they would have done only under the pressure of shortage of food supply. He calls attention to the fact that if this had been a focus of Mus ratus or Mus alexandrinus which are climbing rats, the original focus would rapidly have been spread. But since the Mus norvegicus is a ground rat, it was closed in by the neighboring brick walls. In the operations following these discoveries, the building chiefly infested was torn down and the frame sheds behind it were rendered not inhabitable for rats. Many rats were found dead and a considerable number were killed. Fumigation was carried out on the premises, and these and other premises washed down with tank oil for the purpose of killing fleas. Very few rats escaped. An interesting control was carried out which has been introduced into plague campaigns, namely, that guinea-pigs were placed into the fumigated premises after fumigation. One of these contracted plague and died, and the place was, therefore, refumigated. Guinea-pigs which had been used as controls in other places remained alive. Only one human case was attributable to this focus. A Bulletin published by the United States Public Health Service in November, 1920, (35, No. 45) has laid down ordinances for rat- proofing. These we quote in toto directly from this Bulletin. "The rat-proofing of buildings is generally secured either by elevation of the structure, with the underpinning open and free, or by marginal rat-proof walls of concrete or of stone or brick laid in cement mortar, sunk two feet into the ground, and fitting flush to the floor above. The wall must fit tightly to the flooring and not merely extend to the joists or 824 PATHOGENIC MICROORGANISMS supporting timbers, as this would result in open spaces, permitting the entrance of rodents. Groceries, stables, warehouses, markets, and food depots in general are best rat-proofed by having a concrete floor in addition to concrete walls. In these structures, untenanted as they are at night, rats might well enter by a doorway or window carelessly left open, or be intro- duced concealed in merchandise, and, gnawing through plank flooring, obtain a well-protected hiding place. "In addition to concrete floor and walls, these food depots must have tight-fitting doors, and all windows and other openings should be properly screened. A 12-guage wire is preferable on account of its strength and durability, and the mesh should not be larger than one-half inch. "Rat-proofing by elevation of the building is chiefly applicable to small and medium sized frame dwellings. The purpose is to have a sufficient elevation, about two feet, so that the ground area beneath will be as exposed and free from covert as land unbuilt upon. Marginal rat-proofing will suffice in more pretentious dwellings where sufficient care can be exercised to prevent rats from gnawing through the plank floors. "Chicken pens can be protected by marginal concrete walls, sunk into the ground two feet or more, and by covering the sides and top with ^-inch mesh wire netting. Garbage cans should be made of serviceable metal and should have properly fitting tops. "Plank sidewalks and plank coverings for yards should be avoided. Cinders and concrete should be used instead. The latter should have marginal protection to prevent rats from burrowing beneath it. "Double walls, with a dead space between, should be avoided, or, if used, they should be rat-proofed at the top and bottom with heavy wooden timbers, 4 by 4-inch fillers, or by a concrete fill. Attics should be well opened and kept free of rubbish or other refuge for rats. "These precautions against rat harborage and for the protection of food supplies, in connection with careful trapping and poisoning, will be attended with considerable success in the destruction of rats. "The appended model ordinance is applicable, with perhaps slight modifi- cations, to any urban community. It should be examined by competent local counsel for changes in form, or in substance if necessary, as dictated by special constitutional, legislative, or charter considerations." Plague Vaccination. — The immunization of animals with suspen- sions of plague bacilli, killed by moderate heating, 50° for one hour, was first attempted by Yersin, Calmette and Borrel in 1897. Kolle,25 Haffkine26 and others studied plague vaccination particularly in the subsequent years. A great many different vaccines have been x Kolle, loc. cit. "Haffkine, loc. cit. PLAGUE AND BACILLUS PESTIS 825 introduced since then. The one most extensively used is that of Haffkine, which consists of cultures grown in broth in shallow bottles for six weeks at room temperature and shaken once a day. At the end of this time they are sterilized at 65° for several hours. The material then consists of degenerated organisms and extracts of the organisms. Glycerinated broth cultures have been introduced, but so far have had little practical application. The German Plague Commission27 in 1899 introduced the use of heated cultures to which 0.5 per cent carbolic acid had been added. Strong28 believing that attenuated living bacilli might be more efficient than dead cultures, produced vaccines from a three years' old laboratory culture, sub- sequently cultivated at temperatures above 41°. These living cul- tures after such treatment had lost their virulence for guinea-pigs and monkeys almost completely. He vaccinated forty-two individ- uals with these cultures without harm, and with resulting develop- ment of specific antibodies. The method is probably quite efficient but because of the possible danger involved in it, has not been extensively employed. The so-called nucleo-protein vaccins of Lus- tig and Galeotti,29 that is, plague bacilli extracted with weak alkalin solutions and precipitated with acid in the cold, have been studied extensively by Rowland30 and others, and Rowland made several vaccines of his own in one of which he extracted the bacterial mass with sodium sulphate. He also used cultures killed with chloroform. None of these vaccines have had any extensive application except that of Haffkine which has been used by the British Government Sanitary organization in India on a very large scale. Bannermann, Bitter and more recently, Major Glen Listen31 have analyzed the results obtained with Haffkine 's virus of which over eight million doses were distributed in India between 1886 and 1899. According to these studies the Haffkine virus seems to be of definite prophy- lactic value, though not completely protective as one would expect from the nature and virulence of the disease. Major Listen states in his report of the Bombay Bacteriological Laboratory for the years 1913 to 1916, that it is quite impossible to give any positive state- ment for India, but that in isolated epidemics in which careful figures "German Plague Commission Report, 1899. ** Strong, Philip. Jour. Science., Sec. B, 1907 and 1912. *Lustig and Galeotti, Deut. med. Woch., 1897-1912. 80 Rowland, Journal of Hygiene, 1910-1914. "Liston, Maj. Glen, Bombay Bacter. Lab. Rep., 1913-1916. 826 PATHOGENIC MICROORGANISMS could be secured, the indications are that the vaccine has been of great value. In. one town in India, 794 individuals were inoculated, and 286 uninoculated, and among the inoculated there were only twelve cases and three deaths, while among the smaller number of the untreated, thirty cases developed and twenty-five died. In one house there were four vaccinated and three unvaccinated. All of the unvaccinated died, and only one of the vaccinated contracted the disease and he recovered. A number of similar studies are cited by Major Liston. There seems, therefore, to be very little doubt as to the protective value of some form of plague vaccination. Whether or not the Haffkine virus is the most useful and final method, cannot of course be stated at the present time. THE PLAGUE-LIKE DISEASE OF RODENTS (McCOY)32 Bacterium Tularense (McCoy and Chapin)33 McCoy has described a disease occurring in Californian ground squirrels (Citellus beechyi) which presents lesions very similar to those of plague in these animals. In fact the disease was noticed in the course of the systematic examination of rodents by McCoy at the Federal Laboratory in San Francisco. Although McCoy was able to transmit the disease to guinea-pigs, mice, rabbits, monkeys, and gophers, and plague-like lesions could be produced in most of the animals, he was at first entirely unable to cultivate any organism from these lesions. In 1912 McCoy and Chapin finally succeeded in growing the specific bacterium on an egg medium made entirely of the yolk. Morphologically it is a very small rod, 0.3 to 0.7 micron in length and often capsulated. The rods stain poorly with methylene blue, better with carbol fuchsin or gentian violet. They are found in large numbers in the spleen of animals dead of the disease. THE BACILLI OF THE HEMORRHAGIC SEPTICEMIA GROUP In many of the lower animals there occur violently acute bacterial infections characterized by general septicemia, usually with petechial hemorrhages throughout the organs and serous membranes and severe intestinal inflammations. These diseases, spoken of as the "hemorrhagic septicemias, " are caused by a group of closely allied 82 McCoy, U. S. Public Health, Bull. 43, 1911. 83 McCoy and Chapin, Jour, of Inf. Dis., x, 1912. PLAGUE AND BACILLUS PESTIS 827 bacilli, first classified together by Hueppe34 in 1886. Some confusion has existed as to the forms which should be considered within Hueppe 's group of "hemorrhagic septicemia," a number of bac- teriologists including in this class bacilli such as Loeffler's Bacillus typhi murium, and Salmon and Smith's hog-cholera bacillus, micro- organisms which, because of their motility and cultural character- istics, belong more properly to the "Gartner," "enteritidis," or "paratyphoid" group, intermediate between colon and typhoid. The organisms properly belonging to this group are short bacilli, more plump than are those of the colon type, showing a marked tendency to stain more deeply at the poles than at the center. They are non-motile, possess no flagella, and do not form spores. They grow readily upon simple media, but show a very marked preference for oxygen, growing but slightly below the surface of media. By some observers they are characterized as "obligatory aerobes, " but this is undoubtedly a mistake. While showing considerable variations in form and differences in minor cultural characteristics, the species characteristics of polar staining, decolorization by Gram, immobility, lack of gelatin lique- faction, and great pathogenicity for animals, stamp alike all mem- bers of the group. Its chief recognized representatives are the bacillus of chicken cholera, the bacillus of swine-plague (Deutsche Schweineseuche), and the Bacillus pleurosepticus which causes an acute disease in cattle and often in wild game. Because of certain cultural and pathogenic characteristics, it seems best to consider the bacillus of bubonic plague with this group. BACILLUS OP CHICKEN CHOLERA (Bacillus avisepticus) The bacillus of chicken cholera was first carefully studied by Pasteur35 in 1880. It is a short, non-motile bacillus, measuring from 0.5 to 1 micron in length. Stained with the ordinary anilin dyes, it displays marked polar staining qualities, which often give it the appearance of being a diplococcus. It is decolorized by Gram's method. It does not form spores, but may occasionally form vaciiolated degeneration forms, not unlike those described for Bacil- lus pestis. 34 Hueppe, Berl. klin. Woch., 1886. 35 Pasteur, Comptes rend, cle Pacad. des sci., 1880. 828 PATHOGENIC MICROORGANISMS The bacillus is easily cultivated from the blood and organs of infected animals, it grows well upon the simplest media at tempera- tures varying from 25° to 40° C. In broth, it produces uniform clouding with later a formation of a pellicle. Upon a gar it forms, within twenty-four to forty-eight hours, minute colonies, white or yellowish in color, which are at first transparent, later opaque. Upon gelatin, it grows without liquefaction. Upon milk, the growth is slow and does not produce coagulation. According to Kruse,36 indol is formed from pepton bouillon. Acid, but no gas, is formed in sugar broth. Among barnyard fowl, this disease is widely prevalent, attacking chickens, ducks, geese, and a large variety of smaller birds. The infection is extremely acute, ending fatally within a few days. It is accompanied by diarrhea, often with bloody stools, great exhaus- tion, and, toward the end, a drowsiness bordering on coma. Autopsy upon the animals reveals hemorrhagic inflammation of the intestinal mucosa, enlargement of the liver and spleen, and often broncho- pneumonia. The specific bacilli may be found in the blood, in the organs, in exudates, if these are present, and in large numbers in the dejecta. Infection takes place probably through the food and water con- taminated by the discharges of diseased birds.37 Subcutaneous inoculation or feeding of such animals with pure cultures, even in minute doses, gives rise to a quickly developing septicemia which is uniformly fatal. The bacillus is extremely pathogenic for rabbits, less so for hogs, sheep, and horses, if infection is practiced by subcutaneous inoculation. Infection by ingestion does not seem to cause disease in these animals. Historically, the bacillus of chicken cholera is extremely in- teresting, since it was with this microorganism that Pasteur35 carried out some of his fundamental researches upon immunity, and suc- ceeded in immunizing chickens with attenuated cultures. The first attenuation experiment made by Pasteur consisted in allowing the bacilli to remain in a broth culture for a prolonged period without transplantation. With minute doses of such a culture (vaccin I) he inoculated chickens, following this, after ten days, with a small dose of a fully virulent culture. Although enormously important in principle, the practical results from this method, as applied to 88 Kruse, in Fliigge 's ' ' Die Mikroorganismen. ' ' 87 Salmon, Rep. of the Com. of Agriculture, 1880, 1881, and 1882. PLAGUE AND BACILLUS PESTIS 829 chicken cholera, have not been satisfactory. It was with this bacil- lus, furthermore, that Pasteur was first able to demonstrate the existence of a free toxin which could be separated from the bacteria by filtration. BACILLUS OF SWINE PLAGUE (Bacillus suisepticus, Schweineseuche) This microorganism is almost identical in form and cultural characteristics with the bacillus of chicken cholera. It is non-motile, forms no spores, is Gram-negative, and does not liquefy gelatin. The bacillus causes an epidemic disease among hogs, which is char- acterized almost regularly by a bronchopneumonia followed by general septicemia. There is often a sero-sanguineous pleura! exudate, a swelling of bronchial lymph glands and of liver and spleen. The gastrointestinal tract is rarely affected. The bacilli at autopsy may be found in the lungs, in the exudates, in the liver and spleen, and in the blood. The disease is rarely acute, but, in young pigs, almost uniformly fatal. It is probable that spontaneous infection usually occurs by in- halation. Experimental inoculation is successful in pigs, both when given subcutaneously and when administered by the inhalation method. Mice, guinea-pigs, and rabbits are also susceptible, dying within three or four days after subcutaneous inoculation of small doses. Active and passive immunization of animals against Bacillus suisepticus has been attempted by various observers. Active im- munization, if carried out with care, may be successfully done in the laboratory. Passive immunization of animals with the serum of actively immunized horses has been practiced by Kitt and Mayr,38 Schrieber,39 and Wassermann and Ostertag. The last-named ob- servers, working with a polyvalent serum produced with a number of different strains of the bacillus, have obtained results of consider- able practical value. The researches of Kitt and Mayr have revealed a fact pointing to the interrelationship of the bacilli of the "hemor- rhagic septicemia" group. They were able to show that the serum of horses immunized with chicken cholera bacilli was able to protect, somewhat, against Bacillus suisepticus. *Kitt and Mayr, Monatsschr. f. Thier Leilk., vol. 8, 1897. 39 Schrieber, Berl. Thierarztl. Wochenschr., vol. 10, 1899. 830 PATHOGENIC MICROORGANISMS Infection with the bacillus of swine plague, in hogs, is often accompanied by an infection with the hog-cholera bacillus (Schweine- pest). The latter, as we have seen, is a microorganism belonging to the enteritidis group, intermediate between Bacillus coli and Bacillus typhosus, and differing from suisepticus in being actively motile, possessing flagella, not showing the polar staining, having a more slender morphology, and producing gas upon dextrose broth. A confusion between the two bacilli frequently occurs because of their nomenclature. Bacteriologically and pathogenically, they are quite distinct. Bacillus suisepticus produces an acute septicemia, accompanied by bronchopneumonia and usually not affecting the gastro-intestinal canal. The bacillus of hog cholera produces an infection localized in the intestinal canal. CHAPTER XLII ASIATIC CHOLERA AND THE CHOLEEA ORGANISM (Spirillum choleroz asiaticce, Comma Bacillus) THE organism, of Asiatic cholera was unknown until 1883. In this year, Koch/ at the head of a commission established by the German government to study the disease in Egypt and India, dis- covered the " comma bacillus" in the defecations of patients, and satisfactorily determined its etiological significance. Koch's investigations were carried out on a large number of cases and many investigations have since then corroborated his results. Apart from the evidence of the constant association of the cholera spirillum with the disease, the etiological relationship has been clearly demonstrated by several accurately recorded accidental in- fections occurring in bacteriological workers, and by the famous experiment of Pettenkofer and Emmerich, who purposely drank water containing cholera spirilla. Both observers became seriously ill with typical clinical symptoms of cholera, and one of them nar- rowly escaped death. Morphology and Staining. — The vibrio or spirillum of cholera is a small curved rod, varying from one to two micra in length. The degree of curvature may vary from the slightly bent, comma-like form to a more or less distinct spiral with one or two turns. The spirals do not lie in the same plane, being arranged in corkscrew fashion in three dimensions. The spirillum is actively motile and owes its motility to a single polar flagellum, best demonstrated by Van Ermengem's flagella stain. Spores are not found. In young cultures the comma shapes predominate, in older growths the longer forms are more numerous. Strains which have been cultivated artificially for prolonged periods without passage through the animal body have a tendency to lose the curve, assuming a more bacillus- like appearance. The spirilla are stained with all the usual aqueous anilin dyes. They are decolorized by Gram's method. In histological , Deut. med. Woch., 1883 and 1884. 831 832 PATHOGENIC MICROORGANISMS section they are less easily stained, but may be demonstrated by staining with alkaline methylene blue. Cultivation. — The cholera spirillum grows easily upon all the usual culture media, thriving upon meat-extract as well as upon meat-infusion media. Moderate alkalinity of the media is prefer- able, though slight acidity does not prevent growth. In gelatin plates growth appears at room temperature within twenty-four hours as small, strongly refracting yellowish-gray, pin- head colonies. As growth increases the gelatin is fluidified. Under & \ ' V '«? " "*/V>**-: ^-' /. >^f «?> -gf^-Wr'- ^V/Pflt CB*^! ^ / 'A FIG. 87. — CHOLERA SPIRILLUM. (After Frankel and Pfeiffer.) magnification these colonies appear coarsely granular with margins irregular because of the liquefaction. Liquefaction, too, causes a rapid development in such colonies of separate concentric zones of varying refractive power. Old strains, artificially cultivated for long periods, lose much of their liquefying power. In gelatin stab cultures fluidification begins at the surface, rapidly giving rise to the familiar funnel-shaped excavation. Upon agar plates, within eighteen to twenty-four hours, grayish, opalescent colonies appear, which are as a rule easily differentiated by their transparency from the other bacteria apt to appear in feces. Agar plates, therefore, are important in the isolation of these organisms. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 833 Coagulated blood serum is liquefied by the cholera vibrio. On potato, growth is profuse and appears as a brownish coarse layer. In milk, growth is rapid and without coagulation. In broth, general clouding and the formation of a pellicle result. The rapidity and luxuriance of growth of the cholera spirillum upon alkaline pepton solutions render such solutions peculiarly useful as enriching media in isolating this microorganism from the stools of patients. In pepton solution, too, the cholera spirillum gives rise to abundant indol, demonstrated in the so-called " cholera-red" reaction. This reaction has a distinct diagnostic value, but is by no means specific. In the case of the cholera spirillum the mere addition of strong sulphuric acid suffices to bring out the color reaction. This is due FIQ. 88. FIG. 89. FIG. 88.— CHOLERA SPIRILLUM. Stab Culture in Gelatin, three days old. FIG. 89. — CHOLERA SPIRILLUM. Stab Culture in Gelatin, six days old. (After Frankel and Pfeiffer.) to the fact that, unlike some other indol-producing bacteria, the cholera organism is able to reduce the nitrates present in the medium to nitrites, thus itself furnishing the nitrite necessary for the color reaction. The medium which is most suitable for this test is that proposed by Dunham,2 consisting of a solution of 1 per cent of pure pepton and .5 per cent NaCl in water. Dieudonne has recommended a selective medium upon which cholera spirilla will grow well, but upon which the colon bacillus will grow either very sparsely or not at all. Cocci will produce minute pin-point colonies only and other common bacilli like those of the proteus group will grow hardly more easily than bacillus coli. Its preparation is very simple. 2 Dunham, Zeit. f. Hyg., ii, 1887. 'Dieudonne A., Cent. Bakt., 1., orig., 1909, 834 PATHOGENIC MICROORGANISMS To seventy parts of ordinary 3 per cent agar, neutralized to litmus, there are added thirty parts of a sterile mixture of equal parts of defibrinated beef blood and normal sodium hydrate. The latter is sterilized by steam before being added to the agar. This pure alkali agar is poured out in plates and allowed to dry several days at 37° or five minutes at 60°. The material to be examined is smeared upon the surface of these plates with a glass rod. If the blood-alkali mixture is prepared beforehand and allowed to stand for four or five weeks, the plates may be used immediately after pouring (Teague). The principle of this medium is that cholera will grow in the presence of an amount of alkali which inhibits other fecal bacteria. For other cholera media see the section on media in the first part of this book. The rational basis for the isolation of cholera spirilla from fecal or other material is found in two chief properties of the spirilla. These have been described to us by Teague as follows: (1) It grows on media of an alkalinity that retards or completely inhibits the growth of most of the fecal bacteria. (2) It comes to the surface of fluid media, rich in oxygen, to a greater extent than do the fecal bacteria. The best results in the practical isolation of the cholera spirilla from stools are obtained by making use of both of these properties from the beginning. A portion of the stool is seeded directly into alkalin-pepton water. The broth used should be distinctly alkalin, titrated to —0.5, or —1.0, with phenolphthalein. After six to twelve hours, a loopful from the surface of these pepton water tubes is plated upon plates of Dieudonne's medium, and is also transferred to a second series of alkalin-pepton water tubes. Once isolated, the spirilla are identified by their morphology and motility, by the appearance of their colonies, by their manner of growth upon gelatin stabs, by the cholera-red reaction, and, finally, by agglutinative tests in immune sera. Owing to the existence of other spirilla morphologically and culturally similar, the serum reac- tions are the only absolutely positive differential criteria. For isolation of the bacteria from water, it is, of course, necessary to use comparatively large quantities. Fliigge and Bitter advise the distribution of about a liter of water in ten or twelve Erlen- meyer flasks. To each of these they add 10 c.c. of sterile pepton-salt solution (pepton ten per cent, NaCl five per cent). After eighteen ASIATIC CHOLERA AND THE CHOLERA ORGANISM 835 hours at 37.5° C. the surface growths in these flasks are examined both microscopically and culturally as before. Biological Considerations. — The cholera spirillum is aerobic and facultatively anaerobic. It does not form spores. The optimum temperature for its growth is about 37.5° C. It grows easily, how- ever, at a temperature of 22° C. and does not cease to grow at temperatures as high as 40°. Frozen in ice, these bacteria may live for about three or four days. Boiling destroys them imme- diately. A temperature of 60° C. kills them in an hour. In impure water, in moist linen, and in food stuffs, they may live for many days. Associated with saprophytes in feces and other putrefying material, and wherever active acid formation is taking place, they are destroyed within several days. Complete drying kills them in a short time. The common disinfectants destroy them in weak solutions and after short exposures (carbolic acid, five-tenths per cent in one-half hour ; bichlorid of mercury, 1 :100,000 in ten minutes ; mineral acids, 1:5,000 or 10,000 in a few minutes). Cholera in Man. — In man the disease is contracted by ingestion of cholera organisms with water, food, or any contaminated material. The disease is essentially an intestinal one. The bacteria, very sensitive to an acid reaction, may often, if in small numbers, be checked by the normal gastric secretions. Having once passed into the intestine, however, they proliferate rapidly, often completely outgrowing the normal intestinal flora. Fatal cases, at autopsy, show extreme con- gestion of the intestinal walls. Occasionally ecchymosis and localized necrosis of the mucosa may be present and swelling of the solitary lymph-follicles and Peyer's patches. Microscopically the cholera spirilla may be seen to have penetrated the mucosa and to lie within its deepest layers close to the submucosa. The most marked changes usually take place in the lower half of the small intestine. The intestines are filled with the characteristically fluid, slightly bloody, or "rice-water" stools, from which often pure cultures of the cholera vibrio can be grown. The microorganisms can be cultivated only from the intestines and their contents, and the parenchymatous degenerations taking place in other organs must be interpreted as being purely of toxic origin. Tli ere is at the same time a profound toxemia due, in part, at least to the absorbed cholera substances. The incubation time of the disease is usually short, lasting from a few hours to several days. The disease usually begins, with diar- 836 PATHOGENIC MICROORGANISMS rhea which gradually becomes more violent until the colorless typical, rice water stools appear. Castellani and Chalmers describe the further course as follows: "Vomiting generally appears early, food being first expelled, fol- lowed later by watery fluid with which bile and occasionally blood may be mixed. As the purging and vomiting persist the urine diminishes and may stop, and fluid departs from the subcutaneous tissues, which therefore contract so that the face alters, the nose becoming sharp, the cheekbones prominent, and eyes sunken and the skin of the fingers becomes wrinkled like that of a washerwoman. ' ' A considerable role is played in the subsequent course of the disease by the depletion of water, with consequent aneuria, low blood pressure, cyanosis, acidosis, etc. The therapeutic effect of saline infusions is said to be astonishing. Animal Pathogenicity. — In animals, cholera never appears as a spontaneous disease. Nikati and Rietsch4 have succeeded in produc- ing a fatal disease in guinea-pigs by opening the peritoneum and injecting cholera spirilla directly into the duodenum. Koch5 suc- ceeded in producing a fatal cholera-like disease in animals by in- troducing infected water into the stomach through a catheter after neutralization of the gastric juice with sodium carbonate. At the same time, he administered opium to prevent active peristalsis. A method of infection more closely analogous to the infection in man was followed by Metchnikoff, who successfully produced fatal dis- ease in young suckling rabbits by contaminating the maternal teat. Subcutaneous inoculation of moderate quantities of cholera spirilla into rabbits and guinea-pigs rarely produces more than a temporary illness. Intraperitoneal inoculation, if in proper quan- tities, generally leads to death. It will be remembered that when working with intraperitoneal cholera inoculations the phenomenon of bacteriolysis was discovered by Pfeiffer. Different strains of cholera spirilla vary greatly in their virulence. The virulence of most of them, however, can be enhanced by re- peated passages through animals. Most of our domestic animals enjoy considerable resistance against cholera infection, though under experimental conditions successful inoculations upon dogs, cats, and mice have been reported. Doves are entirely insusceptible. 4 Nikati und Eietsch, Deut. med. Woch., 1884. 5 Koch, Deut. med. Woch., 1885. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 837 Cholera Toxin. — The absence of the cholera spirilla from the internal organs of fatal cases, in spite of the severe general symp- toms of the disease, points distinctly to the existence of a strong poison produced in the intestine by the microorganisms and absorbed by the patient. It was in this sense, indeed, that Koch first inter- preted the clinical picture of cholera. Numerous investigations into the nature of these toxins have been made, the earlier ones defective in that definite identification of the cultures used for experimentation were not carried out. Pfeiffer,6 in 1892, was able to show that filtrates of young bouillon cultures of cholera spirilla were but slightly toxic, whereas the dead bodies of carefully killed agar cultures were fatal to guinea-pigs even in small quantities. In consequence, he regarded the cholera poison as consisting chiefly of an endotoxin.7 The opinion as to the endotoxic nature of the cholera poison is not, however, shared by all workers. Metchnikoff, Roux, and Salimbeni,8 in 1896, succeeded in producing death in guinea-pigs by introduction into their peritoneal cavities of cholera cultures enclosed in celloidin sacs. Brau and Denier,9 and, more recently, Kraus,10 claim that they have succeeded not only in demonstrating a soluble toxin in alkaline broth cultures of cholera spirilla, but in producing true antitoxins by immunization with such cultures. It appears, there- fore, that the poisonous action of the cholera organisms may depend both upon the formation of true secretory toxins and upon endo- toxins. Which of these is paramount in the production of the disease can not be at present definitely stated. In favor of the great importance of the endotoxic elements is the failure, thus far, to obtain successful therapeutic results with supposedly antitoxic sera. Epidemiology. — Cholera is essentially a disease of man. Endemic in India and other Eastern countries, it has from time to time epidemically invaded large territories of Europe and Asia, not in- frequently assuming pandemic proportions and sweeping over almost the entire earth. Five separate cholera epidemics of appalling mag- nitude occurred during the nineteenth century alone; several of 6 Pfeiffer, Zeit. f. Hyg., xi, 1892. ''Pfeiffer und Wassermann, Zeit. f. Hyg., xiv, 1893. 8 Metchnikoff, Eoux, et Salimbeni, Ann! de Pinst. Pasteur, 1896. 9 Brau et Denier, Comptes rend, de Pacad. des sci., 1906. 10 E. Kraus, Cent. f. Bakt., 1906. 838 PATHOGENIC MICROORGANISMS these, spreading from India to Asia Minor, Egypt, Russia, and the countries of Central Europe, reached even to North and South America. The last great epidemic began about 1883, traveled grad- ually westward, and in 1892 reached Germany where it appeared with especial virulence in Hamburg, and thence, following the high- ways of ocean commerce, entered America and Africa. During this epidemic in Russia alone 800,000 people fell victims to the disease. An important epidemiological fact is the existence of certain endemic foci where cholera is always going on and from which epidemics and pandemics originate. The chief endemic focus seems to be located in lower Burmah, and it is, as yet, an unsolved puzzle as to why the disease should remain smouldering in such regions and spread widely only at certain periods, five, ten or more years apart. During recent years important epidemics have occurred between the years 1879 and 1910. In 1879 an epidemic spread to Europe through Egypt and this outbreak is notable because in 1883 in Egypt, Koch,11 as head of the German Cholera Commission, isolated the cholera spirillum. In 1891 another great epidemic, originating in India, is stated by Cas- tellani as having started on the occasion of a bathing festival held on the Ganges. It spread among pilgrims and reached Europe in 1892, appearing with particular virulence in Hamburg. From there it spread to America and Africa by ocean commerce. During this epidemic it is said that 800,000 people fell victims in Russia alone. Violle records that in 1908, 1909 and 1910 there were a series of epidemics in Russia. In 1908 there were about 30,000 cases with 14,000 deaths; in 1909, there were 21,000 cases with 9,700 deaths, and in 1909 to 1910 there were 130,000 deaths. During the Balkan War in 1912 cholera appeared among the armies. During the late war there were cases of cholera in Galicia in the Austrian Army, and there were outbreaks in Bul- garia, Greece and Turkey, and in Mesopotamia. The prevalence of cholera as an important epidemic disease may be estimated by the following chart of cholera epidemics of the last hundred years which is taken from Violle 's recent work on cholera (1918) to which numerous references have been made. The disease always originates from the dejecta of cholera patients and carriers. At times of epidemic, infection of the water and 11 Koch, Deut. med. Woch., 1883 and 1884. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 839 food supplies naturally plays an important role, and in such epi- demics, as the one in Hamburg, the water supply was primarily responsible. The distribution of the disease here followed definitely EPIDEMICS OF CHOLERA IN VARIOUS PLACES l Place Date Deaths Havana 1833 8,000 Malta.. 1837 4,000 London 1832 4,000 Paris. ... .... 1832 7,000 Basra 1821 5000 Lahore 1845 22,000 Tabriz 1852 12 000 Teheran Bagdad 1852 1852 15,000 2000 Bellary and Mysore, India Province of Bombay 1865 1865 40,000 84,000 Cachemire 1892 5,000 England 1854 20,000 France 1854 140 000 Italy 1854 24,000 Egvpt . 1865 60000 Eevpt 1883 50000 Egypt. . , 1831 150 000 Cairo 1831 36000 Cairo 1902 33000 Russia 1909-10 130 000 Rosetta 1865 2,168 Epidemic of Cholera in Russia In 1908, 30,000 cases 14000 In 1909, 21,000 cases 9,700 the distribution of the infected water supply and the organisms were isolated from the water. This epidemic is one of the classical water epidemics and has served more than any other water epidemic in impressing medical and health authorities with the importance of water supply supervision. In countries like India and Egypt, etc., where water supplies are often taken from collecting tanks not properly supervised, and from individual wells, and where the super- vision of feces disposal is not strict, it is quite natural that dis- tribution by water supplies should be extremely important. Violle adds a number of interesting instances of water transmission which occurred in France in 1885, in which the source was soiled linen 840 PATHOGENIC MICROORGANISMS washed in a stream with distribution of the disease further down- stream in other villages, but not in any of the villages higher up the river. It is also probable that in countries such as India, the custom of throwing dead bodies into rivers may contribute ma- terially to the constant presence of the disease. In endemic centers, it is more than likely that the cholera carrier is a very important factor of distribution. The existence of the carrier is proven beyond doubt, and, as in typhoid, individuals may remain carriers for very long periods. Greig has shown that the organisms may live in the gall-bladders of human beings as in the typhoid carrier state. McLaughlin12 has found as many as 7 per cent of the population of an infected district to be cholera carriers. Again, as in typhoid, distribution of fecal material to food by flies probably plays a very important role, and according to Barber the organisms may live for some time in the intestines in such insects as cockroaches. Whether or not domestic animals can act as distributers of the organisms is uncertain. Violle quotes Haffkine as stating that he had found the spirilla in the intestines of cattle, and that they were found by Hahn in the intestines of cows during cholera epidemics. The importance of this, however, is still quite uncertain. In nature the cholera spirilla may, under favorable conditions, remain alive for considerable periods. In drinking water they have been found alive after several days and they may remain alive for weeks in water supplies. From the investigations of Wernieke,18 Shirnoff14 and others it would appear that under favorable condi- tions the spirilla may remain alive in river water and other natural waters for weeks or even months. In milk and other foods, the longevity of the cholera spirilla seems to depend particularly upon the nature and numbers of other bacteria present and on the produc- tion of an acid reaction. In cholera stools they will remain alive until considerable putrefaction has taken place and, therefore, may be assumed under favorable conditions to live at least one day, or perhaps three days or longer. In cold weather when bacterial growth is more or less inhibited, they may remain alive much longer than this. 12 McLaughlin, quoted from Eosenau 's Preventive Medicine and Hygiene, D. Appleton and Co., New York and London, 1921. 13 Werniclce, Hyg. Eundschau, 1895. uShirnoff, Cent. f. Bakt., 41, 1908. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 841 Cholera Immunization. — One attack of cholera confers protection against subsequent infection. Active immunization of animals may be accomplished by inoculation of dead cultures, or of small doses of living bacteria. In the serum of immunized animals specific bacteriolytic and agglutinating substances are found. The discovery of bacteriolytic immune bodies, in fact, was made by means of cholera spirilla. Both the bacteriolysins and the agglutinins, be- cause of their specificity, are of great importance in making a bac- teriological diagnosis of true cholera organisms. Prophylactic Vaccination. — Active immunization of cholera was one of the first methods of prophylactic vaccination attempted in the bacteriological era of infectious disease study. The work was done by a Spanish bacteriologist, Ferran, who had been a pupil of Pasteur, and as early as 1884 carried out immunization experiments with cholera on guinea.-pigs. Ferran,15 in accordance with the methods prevalent at that time, worked with attenuated cholera cultures and developed a method of attenuation which depended upon room-temperature cultivation on gelatin. He tried this method on human beings in Spain in 1885 with results which seemed to him encouraging. Subsequent to this many different vaccines have been developed. Haffkine16 worked intensely on the subject and observed the results of vaccination on an enormous number of people in India, over a period of more than ten years. Haffkine 's virus has undergone a number of modifications since he first used it. He, too, made use of living cultures, beginning his experiments with attenuation of cholera spirilla by cultivation at temperatures of 40° and over, using, at first, a less virulent and next a more virulent strain. Later, it was found that the cultures attenuated by cultiva- tion at increased temperatures were not necessary, and it appears at the present time that in most places only cultures of a virulence enhanced by passage through guinea-pigs are used. The extensive experimental work in India mentioned above seems to have shown that there is a distinct prophylactic value in the use of Haffkine 's virus. Other observers have made use chiefly of killed cultures. The French vaccine made at the Pasteur Institute consists of broth cultures killed at 50°. Kolle17 grows his cholera spirilla on agar, 15 Ferran, Comptes rend, de 1'acad. des sciences, 1885. "Haffkine, Bull, med., 1892. " Kolle and Schurmann, Kolle and Wassermann Handb., Vol. 4, Second Edition. 842 PATHOGENIC MICROORGANISMS suspending them in salt solution, killing at 56° C. for one hour, then adding one-half per cent carbolic acid. Other observers, like Nicoll and Vincent18 killed without heat, by the addition of carbolic acid. Extracts of the cholera spirilla have also been used in various ways. Strong19 grows cholera organisms on agar, takes them up in salt solution, kills at 60° and then allows the suspensions to stand in the incubator for about five days, subsequently filtering through a Berkefeld candle. This filtrate is used for -inoculation, after its sterility has been determined by culture. Wassermann20 has used materials prepared by precipitation of cultures with alcohol. Cas- tellani21 during the last ten years has prepared what he calls a T. A. B. C., or tetravaccine, which is made by mixing agar cultures of typhoid, paratyphoid "A," paratyphoid "B," and cholera in saline emulsion. The emulsion is killed with one-half per cent carbolic acid, preserved for twenty-four hours in this form at room temperature and then standardized by the usual counting chamber method so that 1 c.c. should contain five hundred thousand typhoid, 250 thousand paratyphoid "A," 250 thousand paratyphoid "B," and two thousand million cholera spirilla. 0.5 c.c. of this is injected, three doses being given within two weeks. This is the vaccine which we used on the Serbian Army during the war. The principle underlying all these procedures seems to us to be the same, in that they consist of introducing, subcutaneously, substances derived from the bodies of cholera spirilla. And since the cholera organisms probably do not live very long after sub- cutaneous introduction, it is not likely that it makes very much difference whether attenuated living cultures, or dead cultures are used. As far as the available statistics show at the present time, cholera vaccination is of distinct value. This has been the judgment of those who have scrutinized Haffkine's immunization experiments, as well as those who have observed more recent army experiences. The following table which we used in our Nelson article, again taken from Violle, will give some idea of the comparisons made 18 Nicoll and Vincent. Cited from Violle loc. cit. 19 Strong. J. of Exp. Med., Vol. 8, p. 229, 1905. 20 Wassermann. Festschr. E. Koch, Jena, 1903. 21 Castellani and Chalmers, Manual of Tropical Medicine, W. Wood & Ho, New York, 1919. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 843 upon vaccinated and unvaccinated individuals' among troops, and in some of the more recent experiments. I EPIDEMIC OF CHOLERA IN THE GREEK ARMY DURING THE SECOND BALKAN WAR (from ARNAUD) Number of Soldiers Cases of Cholera, Per Cent Vaccinated, 2 inoculations 76,652 0.43 Vaccinated, 1 inoculation 21,216 3 . 12 Not vaccinated 14,332 5.7 Morbidity, 1,801 (12.5 per cent) Mortality, 348 (2.5 per cent) II EPIDEMIC OF CHOLERA IN RUSSIA, 1912 (from ABRAMOW) Numbers of Cases of Cholera, Soldiers Per Cent Vaccinated 1,500 5 = 0.3 Not vaccinated Ill RECENT EPIDEMIC OF CHOLERA IN INDIA (from (KATRINE) Cases of Cholera Deaths, Per Cent Not inoculated "I 11 Inoculated / 8'°° 3 IV EPIDEMIC OF CHOLERA IN THE GREEK ARMY DURING THE THE BALKAN WAR (from SAVAS) Number of Deaths, Individuals Per Cent Not vaccinated } 20 Vaccinated once \ 10,000 3 Vaccinated twice J 1 Cholera vaccination naturally is of relative value only, just as this is the case in typhoid vaccination. Vaccination must be repeated certainly every two years and probably more often in the case of armies in the field. 844 PATHOGENIC MICROORGANISMS CHOLERA-LIKE SPIRILLA The biological group of the spirilla, to which the cholera spirillum belongs, is a large one, numbering probably over a hundred separate species. Most of these are of bacteriological importance chiefly because of the difficulties which they add to the task of differentiation, for while some of them simply bear a morphological resemblance to the true cholera vibrio, others can be distinguished only by their serum reactions and pathogenicity for various animals. Additional difficulty, too, is contributed by the fact that within the group of true cholera organisms occasional variations in agglu- tinability and bacteriolytic reactions may exist. Certain strains, too, the six El Tor cultures isolated by Gottschlich, while in every respect similar to true cholera spirilla, are considered as a separate sub-species by Kraus,22 because of their ability to produce hemolytic substances, a function lacking in other cholera strains. Spirillum Metchnikovl — This spirillum was discovered by Gamaleia23 in the feces and blood of domestic fowl, in which it had caused an intestinal disease. Morphologically and in staining reac- tions it is identical with Spirillum choleraeasiaticse. It possesses a single polar flagellum, and is actively motile. Culturally it is iden- tical with Vibrio cholerae except for slightly more luxuriant growth and more rapid fluidification of gelatin. It gives the cholera-red reaction in pepton media. It is differentiated from the cholera vibrio by its power to produce a rapidly fatal septicemia in pigeons after subcutaneous inoculation of minute quantities.24 It is much more pathogenic for guinea-pigs than the cholera vibrio. It is not subject to lysis or agglutinated by cholera immune sera. Spirillum Massaua. — This organism was isolated at Massaua by Pasquale25 in 1891 from the feces of a clinically doubtful case of cholera. Culturally and morphologically it is much like the true cholera vibrio, but in pathogenicity is closer to Spirillum Metchni- kovi, in that small quantities produce septicemia in birds. It pos- sesses four flagella. It does not give a specific serum reaction with cholera immune serum. 22 Kraus, Kraus and Levaditi, "Handbueh," vol. i. p. 186. 23 Gamaleia, Ann. de 1'inst. Pasteur, 1883. 2*Pfeiffer und Nocht, Zeit. f. Hyg., vii, 1889. 25 Pasquale, Giorn, med. de r. escre. ed. B. Marina, Boma, 1891. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 845 Spirillum of Finkler-Prior.26 — Isolated by Finkler and Prior from the feces of a case of cholera nostras. Morphologically it is like the true cholera spirillum, though slightly larger and less uniformly curved. Culturally it is much like the cholera vibrio, but grows more rapidly and thickly upon the usual media. It does not give the cholera-red reaction, nor does it give specific serum reactions with cholera immune serum. Spirillum Deneke.27 — A vibrio isolated by Deneke from butter. Much like that of Finkler-Prior. It does not give the cholera-red reaction. ™FinTder und Prior, Erganz. Hefte, Cent. f. allg. ges. Phys., 1884. 27 Deneke, Deut. med. Woch., iii, 1885. CHAPTER XLIII DISEASES CAUSED BY SPIEOCH^ETES (TEEPONEMATA), CLASSI- FICATION, SYPHILIS AND TEEPONEMA PALLIDUM, EELAPS1NG FEVEES, VINCENT'S ANGINA, YAWS, AND THE SPIROCHAETE PEETENUE, SPIEOCHvETE GALLINAEUM, EAT BITE FEVEE, NON- PATHOGENIC SPIEOCHJETES OF THE HUMAN BODY. THE microorganisms known as spirochsetes are slender, undu- lating, corkscrew-like threads which show definite variations both structurally and culturally from the bacteria as a class. Most im- portant among them are the spirochaete of relasping fever, Spirochaete pallida of syphilis, the spirillum of Vincent, Spirochaete refringens, Spirillum gallinarum, a microorganism which causes disease in chickens, Spirochaete anserina, which causes a similar condition in geese, and several species which have been found as parasites, both in animals and in man, without having definite etiological connection with disease. CLASSIFICATION OF SPIRAL ORGANISMS Classification of the spiral organisms in general is still unsatis- factory because the difficulties of staining and cultivation have made it impossible to apply to these organisms the same exact criteria which can be applied to most species of bacteria. We may say, in general, that the word spirillum should be retained for true bacteria of spiral form in which the cell body is rigid and motility is brought about entirely by flagella. Such, for instance, are the spirillum of Asiatic cholera, the spirillum Metchnokovi, the spirillum Deneke and others. The true spirochaete are probably not true bacteria, and we have no exact criteria upon which we can base their classification with the protozoa. However, the striking parasitism of most of them, and certain features of their immunological relations would suggest that they either belong to, or are very close to protozoa. Schaudinn, the discoverer of the syphilis organism, classified the treponema pallidum with the protozoa on the basis of morphological study. He believed that stained preparations often showed an undulating 846 DISEASES CAUSED BY SPIROCH^TES 847 membrane extending along the long axis of the microorganisms similar to that observed in trypanosomes. He also asserted that most of the spiral forms reproduce by cleavage along the longi- tudinal axis. On the other hand, Laveran,1 Novy and Knapp2 and others maintained a close relationship of these microorganisms to the true bacteria.3 A review of observed facts seems to show that most of these spiral organisms have the power of multiplication by transverse fission. Many of them possess flagella and in some of them definite immune bodies can be demonstrated in the serum of infected sub- jects, similar to those produced by bacteria during infection. In others again, like the treponema pallidum, no true circulating anti- bodies against the virulent parasitic forms can be found. Indeed, in syphilis it seems that immunity exists only so long as the living organisms still persist in the body, an observation which is entirely analogous to that made with certain trypanosomes, and with malaria. Also, with some of them, transmission by an intermediate insect host in which the spirilla undergo multiplication has been definitely shown, a state of affairs which corresponds with conditions in many protozoan infections. Kolle and Hetsch favor a classification mid- way between the protozoa and the bacteria, a view which is probably as correct as any that we have any justification for holding at the present time. Noguchi,4 who has had extensive experimental experience with the spirochaete has suggested the tentative classification which follows : He calls attention to the fact that the term "spirochaeta" was applied first by Ehrenberg in 1838 to a free living, fresh water or marine form of spiral organism which creeps along the surface of an object but does not swim, divides by transverse fission, and probably has nothing to do with the organism to which we now apply this word. Noguchi divides the spiral organisms of the group which we are now considering into : I. Cristispira or Saprospira. — This is a limited group of motile spiral organisms which infest the great crystalline styles of certain 1 Laveran, Comptes rend, de 1'acad. des sei., 1902 and 1903. Novy and Knapp, Jour. Infec. Dis., 3, 190(1. ' Kolle and Hetsch, "Die experimentelle Bakt.," Berlin, 1906. * Noguchi, Jour. Exper. Med., 27, 1918, 575. 848 PATHOGENIC MICROORGANISMS mollusca. The term was first proposed by Gross5 iA 1910. A type of these organisms is found in oysters (Spirochaeta balbianii (Certes,6) 1882). Another genus of the same order, Saprospira, was found by Gross to exist in mussels. None of these are pathogenic for higher animals. They are char- acterized by the presence of a membraneous structure which resembles a crista or ridge which runs spirally along the entire length of the body. The body is chambered, that is, transverse bands seem to show along its entire length. There are no terminal filaments and there seems to be a strong flexible membrane. Reproduction, according to Gross, takes place by multiple transverse fission or sporulation, but Noguchi has failed to confirm the occurrence of sporulation. II. Spironema and Treponema.— This is a large group of parasitic spiral organisms which are commonly spoken of as the "spirochete" in medical nomenclature. The characteristic feature of these is a spiral flexible body with terminal filaments, but no undulating mem- brane. They may apparently multiply by transverse as well as by longitudinal fission. They move by an undulating movement, a few of them, however, retaining their regular curves during motion. Dobell in an address before the Royal Society in 1912 expressed the belief that the word treponema should be used for all of the small parasitic varieties. Noguchi believes with Gonder that the term treponema should be restricted, as was done by Schaudinn,7 to those varieties having great constancy of curves, while spironema should be applied to those with less constant curves, but he, nevertheless, classi- fies them together under the same main heading since he believes they are closely related. In this class belong the Treponema pallidum of syphilis and the Treponema or Spirochcete pertenue of Yaus. The class also includes the organisms of relapsing fever, a number of parasites found in rodents, such as the well known organism which invades apparently normal mice (and was once falsely looked upon as the cause of cancer in mice) and various saprophytic types found in the mouth, intestine and genital mucous membranes, such as the Treponema calligyrum 6 Gross, Mitt. zool. Station Neapel., 1910-13, 20, 41 and 188, Cent. f. Bakt., Orig., 65, 1912, 83. • Certes, Bull. Soc. zool. franc., 7, 1882, 347. 7 Schaudinn, Deut. med. Woch., 43, 1909, 1728, Arb. a. d. k. Gesundhst., 1904. DISEASES CAUSED BY SPIROCH^TES 849 found in smegma, the Treponema microdentium and macrodentium, found in the mouth, especially under the gums, and in the throat. Among the Spironema in this main group Noguchi places the Spironema refringcns8 of smegma, the Spironema vincenti of Vincent's angina, the Spironema recurrent^ of Obermeier,9 the Spironema Duttoni,10 the Spironema Kochi, the Spironema gallinarum and the Spironema Novyi. III. Leptospira. — The types of this class are the Leptospira ictero hcemorrJiagice of Inada and Ido and the Leptospira icteroidis recently isolated by Noguchi from cases of yellow fever, and probably repre- senting the etiological factor of that disease. These organisms are much more easily cultivated than the preceding. They are char- acterized by closely set regular spirals which remain unchanged during a peculiar rotary spinning motion. As described by Noguchi these organisms, while in motion, draw the entire body together into a straight line, except fcfr a hook formation of one or both ends. When one end is extended and straight and the other semicircularly hooked, the organism progresses in the sUree'tibn of the straight portion, appearing to be propelled from the rear by the rotary hook. A specimen with both ends hooked remains stationary in spite of its rapid rotary motions. This description is taken verbatim from Noguchi. In this sort of movement the body assumes wide wavy undulations. So far no terminal or peritrichal flagella have been seen. SYPHILIS AND SPIROCHJETA PALLIDA (Treponema pallidum) The peculiar manifestations of syphilis, its mode of transmission, and the fact that its primary lesion was always located at the point of contact with a preceding case, have always stamped it as unques- tionably infectious in nature. Until very recently the microorgan- ism which gives rise to syphilis was unknown. Many bacteriologists had studied the problem and many microorganisms for which definite etiological importance was claimed had been described. Most of these announcements, however, aroused little more than a sensational interest and received no satisfactory confirmation. A 8 Schaudinn and Hoffmann, Arb. a. d. w. Gesundhst., 22, 1905. 8 Obermeier, Cent, f . d. med. Wiss., 11, 1873. 10 Button and Todd, Brit. Med. Jour., 1905. 850 PATHOGENIC MICROORGANISMS bacillus described by Lustgarten11 in 1884 seemed, for a time, to have solved the mystery. The Lustgarten bacillus was an acid-fast organism very similar to Bacillus tuberculosis, and found by its discoverer in a large number of syphilitic lesions. The observation, at first, aroused much interest and received some confirmation. Later extensive investigations, however, failed to uphold the etiological relationship of this bacillus to the disease but identified it with the smegma bacillus, so often a saprophyte upon the mucous membranes of the normal genitals. In 1905, Schaudinn,12 a German zoologist, working in collabora- tion with Hoffmann, investigated a number of primary syphilitic FIG. 90. — SPIROCH^JTA PALLIDA. Smear preparation from chancre stained by the india-ink method. indurations and secondarily enlarged lymph nodes, and in both lesions discovered a spirochsete similar to, but easily distinguished from, the spirochaetes already known. He failed to find similar microorganisms in uninfected human beings. The microorganism described by him as "Spirochasta pallida" is an extremely delicate undulating filament measuring from four to ten micra in length, with an average of seven micra, and varying in thickness from an immeasurable delicacy to about 0.5 of a micron. It is thus distinctly smaller and more delicate than the spirochaete of relapsing fever. Examined in fresh preparations it is actively motile, its movements consisting in a rotation about the long axis, gliding movements backward and forward, and, occasionally, a bend- ing of the whole body. Its convolutions, as counted by Schaudinn, vary from three to twelve and differ from those observed in many other spirochaetes by being extremely steep, or, in other words, by 11 Lustgarten, Wien. med. Woch., xxxiv, 1884. "Schaudinn und Hoffmann, Arb. a. d. kais. Gesundheitsamt, 22, 1905. DISEASES CAUSED BY SPIROCH^TES 851 forming acute, rather than obtuse, angles. The ends of the micro- organism are delicately tapering -and come to a point. In his iirst investigations, Schaudimi was unable to discover flagella and believed that he saw a marginal undulating membrane similar to that noticed in the trypanosomes. Later observations by this observer, as well as by others, revealed a delicate flagellum at each end, but left the existence of an undulating membrane in doubt. Uncertain, in his later investigations, whether the microorganisms described by him could scientifically be classified with the spirochaste proper, Schaudinn suggested the name of " Treponema pallidum." In the same preparations in which Spirochaeta pallida was first seen, other spirochaetes were present, which were easily distinguished from the former by their coarser con- tours, their flatter and fewer undula- tions, their more highly refractile cell bodies, and, in stained preparations, their deeper color. These microorgan- isms were not found regularly, and were interpreted merely as fortuitous and unimportant companions. To them Schaudinn gave the name of "Spiro- cha3ta refringens." FlG- OI.-SPIBOCHBTA PAL- ml -, . -I . ,. LIDA. Spleen, congenital syph- The epoch-making d i s c o v e r y of /T ,./• ihs. (Levaditi method.) bchaudmn and Hoffmann was soon confirmed by many observers, and the etiological relationship of Spirochaeta pallida to syphilis may now be regarded as an accepted fact. Although our inability to cultivate the microorganism has made it impossible to carry out Koch's postulates, nevertheless in- direct evidence of such a convincing nature has accumulated that no reasonable doubt as to its causative importance can be retained. The spirochaetes have been found constantly present in the primary and secondary lesions of all carefully investigated cases, and, so far, have invariably been absent in subjects not afflicted with syphilis. Schaudinn himself, not long after his original communication, was able to report seventy cases of primary and secondary syphilis in which these microorganisms were found. Spitzer12 found them constantly present in a large number of similar cases. Sobernheim and Tomasczewski14 found the spirochagtes in fifty cases of primary "Spitzer, Wien. klin. Woch., 1905. 14 Sobernheim und Tomasczewski, Munch, med. Woch., 1905. 852 PATHOGENIC MICROORGANISMS and secondary syphilis, but failed to find them in eight tertiary cases. Mulzer,15 who found the microorganisms invariably in twenty cases of clinical syphilis, failed to find them in fifty-six carefully investigated non-syphilitic subjects. The voluminous confirmatory literature which has accumulated upon the subject can not here he reviewed. The presence of these spirochaetes in the blood at certain stages of the disease has been demonstrated by Bandi and Simonelli16 who found them in the blood taken from the roseola spots, and by Lcvaditi and Petresco17 who found them in the fluid of blisters produced upon the skin. In tertiary lesions the spirochaetes have been found less regularly than in the primary and secondary lesions, but positive evidence of their presence has been brought by Tomasczewski/8 Ewing,19 and others who succeeded in demonstrating them in gummata. FIG. 92. — SPIROCH^JTA PALLIDA, Liver, congenital syphilis. (Levaditi method.) Noguehi and Moore20 have recently found the Spirochaeta pallida in the brain of patients dead of general paresis. In congenital syphilis, many observers have found Spirochaeta pallida in the lungs, liver, spleen, pancreas, and kidneys, and, in isolated cases, in the heart muscle. The organisms were always present in large numbers and practically in pure culture. These results more than any others seem to furnish positive proof of the etiological relationship between the spirochaete and the disease. Demonstration of Treponema pallidum. — In the living state the spirochaetes have been observed in the hanging drop or under a coverslip rimmed with vaseline. It is extremely important, in pre- 15 Mulzer, Berl. klin. Woch., 1905, and Archiv f . Dermat. u. Syph., 79, 1906. 16 Bandi und Simonelli, Cent. f. Bakt., 40, 1905. 17 Levaditi et Petresco, Presse med., 1905. 18 Tomasczewski, Munch, med. Woch., 1906. 18 Ewing, Proc. N. Y. Path. Soe., N. S., 5, 1905. 20 Noguehi and Moore, Jour. Exp. Med., xvii, 1913. DISEASES CAUSED BY SPOROCH^TES 853 paring such specimens from primary lesions or from lymph glands, to obtain the material from the deeper tissues, and thus as uncon- taminated as possible by the secondary infecting agents present upon the surface of an ulcer, and also as free from blood as possible. It is best to employ a special device known as a "condenser for dark- field illumination " (Dunkel-Kammer-Beleuchtung). This apparatus is screwed into the place of the Abbe condenser. The preparation is made upon a slide and covered with a cover-slip as usual. A drop of oil is then placed upon the upper surface of the condenser and the slide laid upon it so that an even layer of oil, without air- bubbles, intervenes between the top of the dark chamber and the bottom of the slide. An arc light furnishes the most favorable illumination. In such preparations the highly refractive cell-bodies stand out against the black background, and the motility of the organisms may be observed.21 The dark-field condenser is without question the easiest method of finding the Spirochaeta pallida. Its use is easily learned and the apparatus is sufficiently cheap so that it lends itself to the use of the clinic and the office. With very little practice it is possible to detect the spirochaete in suspension if care is taken that not too much blood or other solid particles are mixed with the preparation. Should it be impossible to obtain the material scraped from syphilitic lesions in a sufficiently dilute condition it is best to emulsify it in a drop or two of human ascitic fluid. EXAMINATION IN SMEARS. — The Spirochaeta pallida can not be stained with the weaker anilin dyes, and even more powerful dyes, such as carbol-fuchsin and gentian-violet, give but a pale and un- satisfactory preparation. The staining method most commonly used is the one originally recommended by Schaudinn and Hoffmann. This depends upon the use of Giemsa's azur-eosin stain employed in various modifications. The most satisfactory method of applying this solution is as follows: Make smears upon slides or cover-slips, if possible from the depth of the lesions, 'as free as possible from blood. Fix in methyl alcohol for ten to twenty minutes and dry. Cover the preparation with a solution freshly prepared as follows: Distilled water 10 c.c. Potassium carbonate 1 : 1,00 . . 0 5-10 gtt. 21 For a critical summary of the various methods of dark-field illumination, the reader is referred to an article by Siedentopf, Zeit. f. wiss. Mikrosc., xxv, 1908. 854 PATHOGENIC MICROORGANISMS Add to this: Giemsa 's solution (fur EomanowsTci Farbung) 10-12 gtt. This staining fluid is left on for one to four hours, preferably in a moist chamber. Wash in running water. Blot. By this method Spirochaeta pallida is stained characteristically with a violet or reddish tinge. A rapid and convenient method for staining such smears consists in the use of azur I and eosin in aqueous solutions as recommended by Wood. The smears are fixed in methyl alcohol as before and are then flooded with the azur I solution. The eosin solution is then dropped on the preparation until an iridescent pellicle begins to form. Satisfactory preparations may be obtained by this method after ten or fifteen minutes of staining. A fairly satisfactory method of staining the treponema pallidum in smear-preparations is that of Fontana.22 For this method, the fol- lowing solutions are necessary : 1. Acetic acid 1 c.c. Formalin 2 c.c. Distilled water 100 c.c. Leave in one minute; wash in water. 2. Phenol 86 per cent (liquefied crystals) 1 c.c. Tannic acid ... * 5 grams Distilled water 83 c.c. Cover preparation with this and steam gently one-half minute; wash. 3. Silver nitrate 0.25 gram Distilled water 100 c.c. Ammonia q. s. Add ammonia drop by drop until the precipitate which first appears goes into solution. Steam one-half minute; wash. Recently a rapid and extremely simple but not very reliable method for the demonstration of Spirochaeta pallida in smears, by the use of India ink, has been described. Smears are prepared in the following way: A drop of the fluid squeezed out of the syphilitic lesion, as free as possible from blood cells, is mixed, on a slide, with a drop of India ink (best variety is "Chin chin" Giinther-Wagner Liquid Pearl ink), and the mixture 22 See Levaditi and BanfcowsTci: Ann de 1'Inst. Past., 1913, XXVII, p. 583. DISEASES CAUSED BY SPIROCH^TES 855 smeared with the edge of another slide as in making blood smears. When the smear dries, which takes about a minute, it may be immediately examined with an oil-immersion lens. The organisms are seen unstained on a black background. DEMONSTRATION OF SPIROCH^TES IN TISSUES. — Ordinary histo- logical staining methods do not reveal the spirochaetes in tissue sections. It is customary, therefore, to employ some modification of Cajal's silver impregnation. The technique most commonly em- ployed is that known as Levaditi's method,2* which is carried out as follows : The fresh tissue is cut into small pieces which should not be thicker than 2 to 4 millimeters. Fix in 10% formalin (4% formaldehyde) for twenty- four hours. Wash in water. Dehydrate in 96% alcohol twenty-four hours. Wash in water. Place in a 3% silver-nitrate solution at incubator temperature (37.5° C.) and in the dark for 3 to 5 days. Wash in water for a short time. Place in the following solution (freshly prepared) : Pyrogallic acid 2-4 grams. Formalin 5 c.c. Distilled water 100 " Leave in this for twenty-four to forty-eight hours at room temperature. Wash in water. Dehydrate in graded alcohols. Embed in paraffin and cut thin sections. The sections may be examined without further staining, or, if desired, may be weakly counterstained with Giemsa's solution or hematoxylin. A modification of this method which has been much recommended is that of Levaditi and Manouelian.'2* The directions given by these authors are as follows : Fix in formalin as in previous method. Dehydrate in 96% alcohol twelve to twenty-four hours. Wash in distilled water. Place in a 1% silver-nitrate solution to which 10% of pyridin has been added just before use. Leave in this solution for two to three hours at room temperature and from four to six hours at 50° C. approximately. Wash rapidly in 10% pyridin. 23 Levaditi, Comptes rend, de la soc. de biol., 59, 1905. 24 Levaditi et Manouelian, Comptes rend, de la soc. de biol., 60, 1906. 856 PATHOGENIC MICROORGANISMS Place in a solution containing 4% of pyrogallic acid to which 10% of C. P. acetone, and 15% (per volume) of pyridin have been added just before use. Leave in this solution two to three hours. Wash in water, dehydrate in graded alcohols, and embed in paraffin by the usual technique. Examined after treatment by either of these methods, the spiro- ehaetes appear as black, untransparent bodies lying chiefly extra- cellularly. They are characteristically massed about the blood- vessels of the organs and only exceptionally seem to penetrate into the interior of the parenchyma cells. Attempts at cultivating Spirochaeta pallida were at first unsuc- cessful. In 1909 Schereschewsky25 reported that he had suc- ceeded in obtaining multiplication of the organisms on artificial media as follows : Sterile horse serum in centrifuge tubes was coagu- lated at 60° C. until it assumed a jelly-like consistency. It was then placed in the incubator at 37.5° C. for three days before being used. The cultures were planted by snipping off a small piece of tissue from a syphilitic lesion, dropping it into such a tube, and causing it to sink to the bottom by means of centrifugalization. The tube was then tightly stoppered with a cork. In such anaerobic serum cultures Schereschewsky claims to have grown the organisms for several generations, though not in pure culture. Miihlens also obtained growth of Spirochaeta pallida in horse serum agar by a method which is very similar to that of Schereschew- sky. The most extensive and convincing work on treponema palli- dum has been more recently by Noguchi. Noguchi26 began his work in 1910 and 1911. His first successful cultivations were made from the syphilis-infected testicles of rabbits, and after many unsuccessful attempts, with slightly varying media and technique, he finally suc- ceeded in the following way: He prepared tubes (20 cm. high and 1.5 cm. wide), containing 10 c.c. of a serum-water made of distilled water, three parts; and horse, sheep, or rabbit serum, one part. These were sterilized by the fractional method in the usual way (15 minutes each day). Into them was then placed a small piece of sterile rabbit kidney or testicle and a bit of the testicle of a syphilitic rabbit, in which many spirochaetes were present. The fluid was then covered with sterile paraffin oil and placed in an 26 Schereschewsky, Deut. med. Woch., N. S., xix and xxix, 1909. 28 Noguchi Jour. Exp. Med., xiv, 1911; xvii, 1913. DISEASES CAUSED BY SPIROCH.ETES 857 anaerobic jar. After ten days at 33.5° C. the spirochaetes had multiplied considerably, in all but one case, together with bacteria. He obtained pure cultures from these initial cultivations after much difficulty, by a number of methods. At first he succeeded only by allowing the spirochgetes to grow through Berkefeld filters, which they did on the fifth day. A better method more recently adopted by him consists in preparing high tubes of three parts of very slightly alkaline or neutral agar to which a piece of sterile tissue has been added. These tubes are then inoculated from the impure cultures with a long pipette. Close to the tissue and along the stab the spirochaetes and bacteria will grow and, after about ten days to two weeks, the spirochastes will have wandered away from the stab and will be visible as hazy colonies. They can then be fished, after cutting the tubes, and directly transplanted to other serum-a gar-tissue tubes prepared as before, and eventually will grow in pure culture. By this method Noguchi has also cultivated pure cultures from lesions in monkeys. The writer, with Hopkins, has successfully applied Noguchi 's method and has found that, after once cultivated artificially,, the treponema pallidum can be obtained in quantity best by cultivation in flasks containing heated or unheated rabbit kidney with ascitic broth and sealed with paraffin. Recently we have been using modifi- cations of a method worked out in our laboratory by Miss Gilbert, in which slanted egg, with or without glycerin, made as for tubercle cultivation, is used instead of kidney tissue. This is put up in high tubes and ascitic broth and paraffin oil added. By this method, large quantities of culture pallida are obtained within two weeks and can be concentrated in large quantities. Animal Pathogenicity. — Until very recently, all experimental inoculation of animals was unsuccessful. During the year 1903 Metchnikoff and Roux27 finally succeeded in transmitting the disease to monkeys. The monkey first used by these observers was a female chimpanzee. At the point of inoculation, the clitoris, there appeared, twenty-six days after inoculation, a typical indurated chancre, which was soon followed by swelling of the inguinal glands. Fifty-six days after the inoculation there appeared a typical secondary erup- tion, together with swelling of the spleen and of the lymph nodes. Similar successful experiments were made soon after this by Lassar.28 "Metchnikoff et Eoux, Ann. de 1'inst. Pasteur, 1903, 1904, and 1905. 28 Lassar, Berl. klin. Woch., xl, 1903. 858 PATHOGENIC MICROORGANISMS Soon after the experiments of Metchnikoff and Roux, successful inoculations upon lower monkeys (macacus) were carried out by Nicolle.29 Since that time, it has been found by various observers that almost all species of monkeys are susceptible. Simple sub- cutaneous injection is not sufficient to produce a lesion. The tech- nique which has given the most satisfactory results consists in the cutaneous implantation of small quantities of syphilitic tissue ob- tained by excision or curetting of primary and secondary lesions. A small pocket is made under the mucous membrane of the genitals or of the 'eyebrows and the tissue placed in this under aseptic precautions. The inoculation may be made directly from the human being, but can also be successfully carried out from monkey to monkey for many generations. Attempts at transmission from ter- tiary lesions have so far been unsuccessful. The spirochaetes can be demonstrated both in the primary lesions of the inoculated animal and in the secondarily enlarged glands. The successful inoculation of rabbits with syphilis has been recently performed by Bertarelli.30 He obtained ulcerative lesions by inoculation upon the cornea and into, the anterior chamber of the eye and was able to prove the syphilitic nature of these lesions by finding the spirochaete within the tissue. In these animals, as well as in the lower monkeys, the disease usually remains localized. In 1907 Parodi showed that syphilitic lesions could be produced by direct inoculation into the testicles of rabbits. This method of inoculation has been subsequently studied by many investigators, especially by Uhlenhuth and Mulzer.31 It is the easiest method of obtaining spirochaete in any quantity from lesions in man. The spirochaate-containing lesions may be either excised or scraped as conditions permit and rubbed up in a mortar with sterile sand, in a few centimeters of sterile human ascitic fluid. This emulsion is then injected directly into the substance of rabbit testicles. A swelling supervenes which is often noticeable after two weeks, and is usually at its height in five to seven weeks. At this time the testicle is much larger than normal, sometimes evenly swollen and sometimes nodular, and of a firm elastic consistency. When taken out at castration it oo/es a sticky fluid, both from testicle and tunica, which is rich in actively motile spirochaetes. By continuous 29 Nicollr, Ann. do 1'inst. Pasteur, 1903. 30 Bertarelli, Cent. f. Bakt., xli, 1906. n Uhlenhuth und Mulzer, Arb. a. d. k. Gesimdh't's Amt., xxxiii, 1909. DISEASES CAUSED BY SPIROCH^TES 859 transinoenlation from one rabbit to another such a strain can be indefinitely carried along. It can be inoculated from rabbits to monkeys and vice versa. This method as well as Noguchi's cultiva- tions have opened a new era of spirochaete investigation. It is stated by some observers that intravenous inoculation of rabbits may be followed by localization in the testis and occasionally gum- matous infections in other parts of the body have been induced after such inoculation by Uhlenhuth, Mulzer, and others. Brown and Pearce in an elaborate series of recent investigations published in Journal of Experimental Medicine in 1920, have succeeded in reproducing almost all types of syphilitic lesions in rabbits by appropriate methods of inoculation. Immunization in Syphilis. — It is a well-known fact observed by clinicians that during active syphilis the patient cannot be superin- fected. That this resistance develops quite rapidly was shown by Metchnikoff and Roux, who found that reinfection of a monkey was possible if attempted within two weeks of the first inoculation, but was unsuccessful if delayed beyond this period. On the basis of this knowledge, Metchnikoff,32 Finger and Land- steiner,33 and others have made attempts to devise some method of immunization. They attempted to attenuate the syphilitic virus by repeated passage through monkeys. These experiments were unsuccessful, the last-mentioned observers finding absolutely no at- tenuation after twelve generations of monkey inoculation. Bertarelli and others have shown that the production of a syphilitic lesion on the cornea of one eye does not protect against an inoculation done on the other. Rabbits that have been inoculated with spirochaete material and that have not developed syphilitic dis- ease can be successfully inoculated on subsequent attempts. The offspring of female rabbits with syphilis of the cornea are, according to Muhlens, not immune. There is no evidence so far that specific therapy or treatment with spirochaete material has had favorable influence upon the disease. Chemotherapy has had results analogous to those obtained in man.34 Attempts at passive immunization have been entirely without success. 8* Metchnikoff, Arch. gen. de m6d., 1905. 33 Finger und Landsteiner, Sitzungsber. d. Wien. Akad. d. Wiss., 1905. 34 Von Prowazelc, "Handbuch der pathogenen Protozoen," i, 1912, Leipzig, Bartsch. 860 PATHOGENIC MICROORGANISMS Investigations carried on in our own laboratory* in the last three years have shown definitely, we think, that immunization of animals with culture pallida produces antibodies, agglutinins, treponemacidal substances, entirely analogous to similar substances produced against bacteria. However, there is a biological change which takes place when treponema pallidum is cultivated. The antibodies produced with the culture pallida have no action whatsoever upon the virulent organisms. The latter, indeed, seem to be entirely insulated against such antibodies and do not induce antibody formation to any great extent, in either the infected animal or man. Both active and passive immunization with culture pallida and the sera produced with them have no effect. We have obtained some evidence, how- ever, that in rabbits a purely local resistance develops in the tissue previously the site of a lesion. The occurrence of a Wassermann reaction was formerly supposed to indicate the existence of specific syphilitic antibodies in the serum of patients. More recent information regarding this reaction seems to show that it depends upon the presence in the serum of syphilitic patients of substances produced indirectly because of the presence of syphilitic infection. It may be a relative increase of globulins or, as Schmidt has suggested, a change in the physical state of the globulins or other substances present in the serum. At any rate it has been found that the fixation of complement in the Wasser- mann reaction does not depend upon the occurrence of a specific antigen-antibody reaction. In the first place the antigens most com- monly used, and successfully so, in the Wassermann reactions, are non-specific lipoidal extracts of normal organs. Again it has been demonstrated that extracts of cultures of the Spirochaeta pallida as well as extractions from the testes of syphilitic rabbits do not furnish an antigen suitable for the Wassermann reaction. This has followed especially from the work of Noguchi,35 of Craig and Nichols,36 and ourselves. This forms a corollary to the other experiments previously mentioned and shows that, what- ever the Wassermann reaction may be, it is not a specific comple- ment fixation in the sense of Bordet and Gengou. It must be admitted, therefore, that our knowledge of syphilis immunity is in * Zinsser and Hopkins, Series of papers, Journ. Exp. Med., 1915 and 1916. 85 Noguchi, Jour. Am. Med. Assoc., 1912. 36 Craig and Nichols, Jour. Exp. Med., xvi, 1912. DISEASES CAUSED BY SPIROCILETES 861 its infancy and that we know very little about the systemic reactions which follow infection with the Spirochaeta pallida. The fact that the syphilitic virus does not pass through a filter has been demonstrated by Klingmiiller and Baermann,37 who in- oculated themselves with filtrates from syphilitic material. Hopkins and the writer have carried out some seventy filtration experiments with culture treponemata at various stages of growth without ever obtaining filter passage. It is our opinion that the assumption of a filtrable stage of the syphilitic virus is entirely un- justified and devoid of valid experimental support. THE SPIRQCHJETES OF RELAPSING FEVER The microorganisms causing relapsing fever were first observed in 1873, by Obermeier,38 who demonstrated them in the blood of patients suffering from this distinct type- of fever. Since his time extensive studies by many other observers have proven beyond question the etiological connection between the disease and the organisms. Morphology and Staining. — The spirochaste of Obermeier is a delicate spiral thread measuring from 7 to 9 micra in length (Novy), and about 1 micron in thickness. While this is 'its average size, it may, according to some observers, be considerably longer than this, its undulations varying from four to ten or more in number. Com- pared with the red blood cells among which they are seen, the microorganisms may vary from one-half to nine or ten times the diameter of a corpuscle. In fresh preparations of the blood, very active corkscrew-like motility and definite lateral oscillation are observed. In stained preparations no definite cellular structure can be made out, the cell body appearing homogeneous, except in de- generated individuals, in which irregular granulation or beading has been observed. Flagella have been described by various ob- servers. Novy and Knapp39 believe that the organisms possess only one terminal flagellum. Zettnow,40 on the other hand, claims to have demonstrated lateral flagella by special methods of staining. 37 Klingmuller und Baermann, Dent. med. Woch., 1904. 38 Obermeier, Cent. f. d. med. Wiss., 11, 1873. 39 Novy and Knapp, Jour, of Infec. Dis., 3, 1906. "Zettnow, Deut. med. Woch., 32, 1906. 862 PATHOGENIC MICROORGANISMS Norris, Pappenheimer, and Flournoy,41 in smears stained by poly- chrome methods, have described long, filamentous tapering ends which they interpreted as bipolar, terminal flagella, never observing more than one at each end. Spores are not found. Cultivation. — Innumerable attempts to induce these microorgan- isms to multiply upon artificial media have been made. Novy and FIG. 93. — SPIROCILETE OF RELAPSING FEVER. (After Norris. Pappenheimer, and Flournoy.) Knapp succeeded in keeping the microorganisms alive and virulent in the original blood for as long as forty days, and call attention to the fact that the length of time for which they may be kept alive depends to a great extent upon the stage of fever at which the blood is removed from the patient. They do not, however, believe that extensive multiplication, or, in other words, actual cultivation, had taken place in their experiments. Norris, Pappenheimer, and Flournoy, on the other hand, have obtained positive evidence of "Norris, Pappenheimer , and Flournoy, Joiir. of Inf. Dis., 3, 1906. DISEASES CAUSED BY SPIROCH^TES 863 multiplication of the spirochaetes in fluid media. They obtained their cultures by inoculating a few drops of spirochsetal rat blood into 3 to 5 c.c. of citrated human or rat blood. Smears made from these tubes, after preservation for twenty-four hours at room tem- perature, showed the microorganisms in greater number than in the original infected blood. A similar multiplication could be observed in transfers made from these "first-generation" tubes to other tubes of citrated blood. Attempts at cultivation for a third generation, however, failed. Noguchi42 has lately successfully cultivated the spirochaete of FIG. 94. — SPIROCH^TE OF RELAPSING FEVER. Citrated normal rat blood. Norris, Pappenheimer, and Flournoy.) (After Obermeier in ascitic fluid containing a piece of sterile rabbit 's kidney and a few drops of citrated blood under anaerobic conditions. Four different, probably distinct varieties of spirochaete have been described in connection with relapsing fever, all of which have been cultivated by Noguchi by means of this method. The first is known as the spirochaete of Obermeier mentioned above. Probably distinct are the Spirochaeta Duttoni of West African Tick fever described by Button and Todd43 in 1905, the Spirochaeta Kochi, and 42 Noguchi, Jour. Exp. Med., xvii, 1913. "Button and Todd, Brit. Med. Jour., 1905. 864 PATHOGENIC MICROORGANISMS the Spirochseta Novyi,44 the organism studied by Norris and Flour- noy and Pappenheimer, and regarded as a different species by them. Pathogenicity. — Inoculation with blood containing these spiro- chaetes produces disease in monkeys, rats, and mice. Attempts to transmit the disease experimentally to dogs, rabbits, and guinea-pigs have so far been unsuccessful. The subcutaneous inoculation of monkeys is followed after from two to four days by a rise of temperature which occurs abruptly as is the case in the disease in man and which may last several days. During this time the spirochaetes can be found in the blood of the animals just as it is found in that of infected human beings. The temperature subsides after a day or more, when it again rapidly returns to normal. As a rule, the paroxysms are not repeated. Occasionally, however, two or three attacks may supervene before immunity is established. In rats, an incubation time of from two to five days occurs. At the end of this time the spirochaetes may be found in large numbers in the blood, and the animals show symptoms of a severe systemic infection. The attack lasts from four to five days, at the end of which time the microorganisms again disappear. Occasionally even in these animals relapses have been observed. Gross patholog- ical changes are not found, with the exception of an enlargement of the spleen. In man the disease caused by the spirochaete of Obermeier and allied organisms commonly known as relapsing fever, is common in Eastern Europe, India, Africa, and most of the warmer countries. It has, from time to time, been observed epidemically in Europe, especially in Eussia, and a few epidemics have occurred in the Southern United States. The disease comes on abruptly, beginning usually with a chill accompanied by a sharp rise of temperature and generalized pains. Together with the rise of temperature, which often exceeds 104° F., there are great prostration and occasionally delirium. Early in the disease the spleen becomes palpable and jaundice may appear. The spirochaetes are easily detected in the blood during the persistence of the fever, w^hich lasts usually from three to ten days. At the end of this time the temperature usually drops as suddenly as it rose, and the general symptoms rapidly disappear. After a free interval of from one to three weeks a relapse may occur, which is usually less severe and of shorter duration than **Novy and Fraenlcel, cited from Noguchi. DISEASES CAUSED BY SPIROCH^TES 865 the original attack. Two, three, or even four attacks may occur, but the disease is not very often fatal. When patients do succumb, however, the autopsy findings are not particularly characteristic. Apart from the marked enlargement of the spleen, which histologic- ally shows the changes indicating simple hyperplasia, and a slight enlargement of the liver, no lesions are found. The diagnosis is easily made during the febrile stage by examination of a small quan- tity of blood under a cover-slip or in the hanging-drop preparation. FIG. 95. — SPIROCHAETE OF RELAPSING FEVER. (From preparation furnished by Dr. G. N. Calkins.) Several types of relapsing fever have been described. In Africa the disease has long been prevalent in many regions and the in- vestigations of Ross and Milne,45 Koch,46 Button and Todd,47 and others have brought to light that many conditions occurring among the natives, formerly regarded as malarial, are caused by a species of spirochaete. Whether or not the microorganisms observed in the African disease are exactly identical with the spirochaete observed by Obermeier is yet a question about which several opinions are held. Dutton and Todd believe that the same microorganism is responsible for both diseases. Koch, on the other hand, believes that the slightly smaller size of the African spirochaete and the milder course of the clinical symptoms indicate a definite difference "Ross and Milne, Brit. Med. Jour., 1904. 44 Koch, Deut. mod. Woch., xxxi, 1905. "Dutton and Todd, Lancet, 1905, and Jour, of Trop. Med., 1905. 866 PATHOGENIC MICROORGANISMS between the two. Animal experiments made with the African or- ganism, furthermore, usually show a much more severe infection than do similar inoculations with the European variety. The spiro- chaete found in the African disease is usually spoken of at present as ' ' Spirochaeta Duttoni." Novy and Knapp,48. after extensive studies with the microorganisms from various sources, have come to the conclusion that, although closely related, definite species differences exist between the two types mentioned above, and that these again are definitely distinguished from similar organisms described by Turnbull49 as occurring in a similar disease observed in India. The mode of transmission of this disease is not clear for all types. :: FIG. 96.-^SpiROCHvETE OF DUTTON, AFRICAN TICK FEVER. (From preparation furnished by Dr. G. N. Calkins.) Dutton and Todd, however, were able to show satisfactorily that, in the case of the African disease at least, transmission occurs through the intermediation of a species of tick. The conditions under which such intermediation occurs have been carefully studied by Koch.50 The tick (Ornithodorus moubata) infects itself when sucking blood from an infected human being. The spirochaete may remain alive and demonstrable within the body of the tick for as long as three days. Koch has shown, furthermore, that they may be found also within the eggs laid by an infected female tick. He succeeded in producing experimental infection in monkeys by 48 Novy and Knapp, loc. cit. "Turnlm!!, Indian Mcd. Gaz., 1905. 50 Koch, Berl. med. Woch., 1906. DISEASES CAUSED BY SPIROCH^TES 867 subjecting the animals to the bites of the infected insects. For the European variety of the disease no such intermediate host has as yet been demonstrated with absolute certainty. It is known, however, that the organism can live in the bodies of bed bugs and it has also been suggested that lice may be the carriers. Lice also are regarded as the transmitting agent of a similar relapsing fever prevalent in North Africa caused by the Spiroschaudinnia, berberi. Immunity. — It has long been a well-known fact that recovery from an attack of relapsing fever usually results in a more or less definite immunity. The blood of human beings, monkeys, and rats which have recovered from an attack of this disease show definite and specific bactericidal and agglutinating substances, and Novy and Knapp have demonstrated that the blood serum of such animals may be used to confer passive immunity upon others. VINCENT'S ANGINA The condition known as Vincent's angina consists of an inflam- matory lesion in the mouth, pharynx, or throat, situated most fre- quently upon the tonsils. The disease usually begins as an acute stomatitis, pharyngitis, or tonsillitis, which soon leads to the forma- tion of a pseudo-membrane, which, at this stage, has a great deal of resemblance to that caused by the diphtheria bacillus. At later stages of the disease there may be distinct ulceration, the ulcers having a well-defined margin and "punched-out" appearance, so that clinically they have often been erroneously diagnosed as syphilis. Apart from the localized pain, the disease is usually mild, but occasionally moderate fever and systemic disturbances have been observed. Unlike diphtheria and syphilis, this peculiar form of angina usually yields, without difficulty, to local treatment. The nature of lesions of this peculiar kind was not clear until Plaut,51 Vincent,52 and others reported uniform bacteriological find- ings in cases of this description. These observers have been able to demonstrate in smears from the lesions a spindle-shaped or fusi- form bacillus, together with which there is usually found a spirillum not unlike the spirillum of relapsing fever. The two microorganisms are almost always found together in tliis form of disease and were r>lPlaul, IViit. mod. Wocli., xlix, LS04. •"- rincait, Ann. do 1'mst. Pasteur, 389(5, and Bull, et mem. do la sue. mod. des hop. de P., 1898. 868 PATHOGENIC MICROORGANISMS regarded by the first observers as representing two distinct forms dwelling in symbiosis. The fusiform bacilli described by Vincent, Plaut, Babes, and others, are from 3 to 10 micra in length, and have a thickness at the center varying from 0.5 to 0.8 micron. From the center they taper grad- ually toward the ends, ending in blunt or sharp points. The length of these bacilli may vary greatly within one and the same smear preparation. They are usually straight, sometimes slightly curved. They do not stain very easily with the weaker anilin dyes, but are readily stained by Loeffler's methylene-blue, carbol-fuchsin, or bet- FIG. 97. — THROAT SMEAR, VINCENT'S ANGINA. Fusiform bacilli and spirilla. ter, by Giemsa's stain. Stained by Gram, they are usually de- colorized, though in this respect the writers have found them to vary. Stained preparations show a characteristic inequality in the intensity of the stain, the bacilli being more deeply stained near the end, and showing a banded or striped alternation of stained and unstained areas in the central body. Their staining qualities in this respect are not unlike those of the diphtheria bacillus, and according to Babes53 the dark areas are to be interpreted as meta- chromatic granules. The bacilli are not motile. The spirilla found in Vincent's angina are usually somewhat longer than the fusiform bacilli, and are made up of a variable number of undulations, shallow and irregular in their curvatures, 88 Babes, in Kolle und Wassermann, 1. Erganzungsband, 1907, DISEASES CAUSED BY SPOROCH^TES 869 unlike the more regularly steep waves of Spirochaeta pallida. They are stained with even more difficulty than are the bacilli and usually appear less distinct in the preparations. The stain, however, is taken without irregularity, showing none of the metachromatism ob- served in the bacilli. By the earlier observers cultivation of these microorganisms was attempted without success. Recently, however, it has been shown that cultivation could be carried out under anaerobic conditions. Tunnicliff54 has cultivated the organisms anaerobically upon slants of ascitic agar at 37.5° C. This observer found that in such cultures, before the fifth day, bacilli only could be found, that after this time, however, spirilla gradually appeared and finally constituted the majority of the organisms in the culture. It appeared to Tun- nicliff from this study that the spirilla might be developed out of the fusiform microorganisms representing the adult form. This, howevr, is an error. The microorganisms of Vincent's angina, when occurring in the throat, are rarely present alone, being usually accompanied by other microorganisms, such as staphylococci, streptococci, and not infre- quently diphtheria bacilli. When occurring together with diph- theria, they are said, by some German observers, to aggravate the latter condition considerably. This frequent association with other microorganisms renders it impossible to decide conclusively that the fusiform bacilli and spirilla are the primary etiological factors in these inflammations. It has been frequently suggested that they may be present as secondary invaders upon the soil prepared for them by other microorganisms. Animal inoculation with these microorganisms has led to little result. Fusiform Bacilli other than those in Vincent's Angina. — Fusiform bacilli morphologically indistinguishable from those found in the angina of Vincent may frequently be found in smears taken from the gums, from carious teeth, and occasionally among the microorganisms in the pus from old sinuses. Several varieties of these bacilli have been described in con- nection with definite pathological conditions. Babes,55 in 1893, observed spindle-shaped bacilli not unlike those described above, but somewhat shorter, in histological sections prepared from tissues from the gums of individuals suffering from scurvy. He found similar bacilli 54 Tunnicliff ', Jour, of Infec. Dis., 3, 1906. 56 Babes, Deut. med. Woch., xliii, 1893. 870 PATHOGENIC MICROORGANISMS in rabbits intravenously inoculated with material from the patients and was ab'le to cultivate the bacilli for sevei al generations. His descriptions, however, of the microorganisms as found in the secondary cultures vary considerably from those of the original findings in the. gums of the patients. His results are not convincing. In uoma, a gangrenous disease of the gums and cheeks, occurring occa- sionally in individuals who have been severely run down by acute infectious diseases or great hardship, Weaver and Tunnicliff have found spirilla and fusiform bacilli in large numbers. The organisms were present not only in smears from the surface, but were also found by histological methods, in large numbers, lying in the tissues beyond the area of necrosis. Here again it is not entirely certain whether these microorganisms were the primary etiological factors or whether they are to be regarded merely as secondary invaders of a necrotic focus. Fusiform bacilli are cultivated with greater ease than formerly supposed; we have found it relatively simple to grow them together with Gram positive cocci in symbiosis in simple broth tubes covered with paraffin oil without the addition of any enriching substance and in similar symbiotic conditions on infusion agar plates under incomplete anaerobic conditions. In such plates they form curious colonies in which the fusiform bacilli and micrococci are intimately commingled. Krumwiede56 has had no difficulty in cultivating them in pure culture in anaerobic plates. SPIROCHJETA PERTENUE In a disease known as ' t Framboesia tropica," or popularly "Yaws," occurring in tropical and subtropical countries and much resembling syphilis, Castellani,57 in 1905, was able to demonstrate a species of spirochaete which has a close morphological resemblance to Spirochseta pallida. The microorganism was found in a large percentage of the cases examined both in the cutaneous papules and in ulcerations. Confirmatory investigations on a larger series of cases were later carried out by von dem Borne.58 The microorganism is from 7 to 20 micra in length with numerous undulations and pointed ends. Examined in fresh preparations, it has an active motility similar to that of Spirochasta pallida. In smears it is easily stained by means of the Giemsa method. Both the clinical similarity between yaws and syphilis, as well as the similarity between the microorganisms causing the diseases, 58 Krumwiede, Jour. Inf. Dis., 1913. " Castellani, Brit. Med. Jour., 1905, and Deut. med. Woch., 1906. Kvon dem Borne, Jour. Trop. Med., 10, 1907. DISEASES CAUSED BY SPIROCH^TES 871 has opened the question as to the identity of the two microorganisms. According to most clinical observers, however, yaws, which is a disease characterized chiefly by a generalized papular eruption, is unquestionably distinct, clinically, from lues, and experiments of Neisser, Baermann, and Halberstadter,59 as well as of Castellani60 himself, have tended to show that there is a distinct difference between the immunity produced by attacks of the two diseases. The disease is transmissible to monkeys, as is syphilis. SPIROCH^TA GALLINARUM An acute infectious disease occurring among chickens, chiefly in South America, has been shown by Marchoux and Salimbeni61 to be caused by a spirochaete which has much morphological similarity to the spirochaete of Obermeier. The disease comes on rather suddenly with fever, diarrhea, and great exhaustion, and often ends fatally. The spirochaete is easily demonstrated in the circulating blood of the animals by staining blood-smears with Giemsa's stain or with dilute carbol-fuchsin. Artificial cultivation of the microorganism has not yet been accomplished. Experimental transmission from animal to animal is easily carried out by the subcutaneous injection of blood. Other birds, such as geese, ducks, and pigeons, are susceptible; mammals have, so far, not been successfully inoculated. According to the investigation of Leviditi and Manouelian,62 the spirochaates are found not only in the blood but thickly distributed throughout the various organs. Under natural conditions, infection of chickens seems to depend upon a species of tick which acts as an intermediate host and causes infection by its bite. The spirochaete, according to Marchoux and Salimbeni, may be found in the intestinal canal of the ticks for as long as five months after their infection from a diseased fowl. In the blood of animals which have survived an infection, ag- glutinating substances appear and active immunization of animals may be carried out by the injection of infected blood in which the spirochaetes havo boon killed, either by moderate heat or by preserva- 09 Neisser, Baermann, und Halberstadter, Miinch. mod. Wodi., xxviii, 1906. 60 Castellani, Jour, of Hyg., 7, 1907. 61 Marchoux et Salimbeni, Ann. de Tinst.. Pasteur, 1903. K Levaditi et Manouelian, Ann. de Pinst. Pasteur, 1906. 872 PATHOGENIC MICROORGANISMS tion at room temperature. The serum of immune animals, further- more, has a protective action upon other birds. It is not impossible that the Spirochaeta gallinarum may be identical with the Spirochaeta anserina previously discovered by Sacharoff.63 This last-named microorganism causes a disease in geese, observed especially in Eussia and Northern Africa, which both clinically and in its pathological lesions corresponds closely to the disease above described as occurring in chickens. The spirochaete is found during the febrile period of the disease in the circulating blood, is morphologically indistinguishable from the spirochaete of FIG. 98- -SPIROCH^ETE GALLINARUM. (From preparation furnished by Dr. G. N. Calkins.) chickens, and can not be cultivated artificially. The similarity is further strengthened by the fact that Spirochaeta anserina is pathogenic for other birds, but not for animals of other genera. Noguchi has succeeded in cultivating Spirochaeta gallinarum by the same method by which he has cultivated the organisms of relapsing fever. Ascitic fluid tubes with a piece of sterile rabbit kidney were inoculated with a few drops of blood containing the spirochaetes and cultivated at 37.5° C. under anaerobic conditions. Spirochaeta phagedenis. — This is an organism cultivated by Noguchi by his ascitic-fluid-tissue method from phagedenic lesions on human external genitals. It is probably a new species. Sacharoff, Ann. de 1'inst. Pasteur, 1891. DISEASES CAUSED BY SPIROCH.ETES 873 Spirochaeta macrodentium. — Cultivated by Noguchi ;64 is believed by him to be identical with the spirochaete found in Vincent 's angina. Spirochaeta microdentium. — A similar organism with wide con- volutions, cultivated by Noguchi from the tooth deposits chiefly in children. It was grown on mixtures of sheep serum water and sterile tissue in a way similar to that employed by him for other organisms of this group. Spirochaeta calligynun, — Cultivated by Noguchi65 from condy- lomata — is probably a new species. Rat-Bite Fever. — Rat-bite fever is a peculiar disease, which, after an incubation period of ten or more days, is characterized by fever, headache and inflammation at the site of the bite, swollen lymph glands, skin eruption and pains. After three to six days the fever ceases and an afebrile period of two or three days ensues.66 After this the fever again occurs. Recently Futaki, Takaki, Taniguchi, and Osumi67 have described a treponema which they have called Treponema morsus muris. It is a spiral organism, somewhat larger than the Treponema pallidum, and is found in the skin, the lymph nodes, and in the blood. They have succeeded in inoculating rats and have cultivated it in Schimamine medium, which consists of 100 c.c. of horse serum in which 0.5 to 0.75 gram of sodium nucleate is dissolved and carbon dioxide passed through the solution until the serum becomes transparent. It is then heated for three days at 60°, and on the fourth day at 65° until it coagulates. This medium is deeply inoculated, but no other anaerobic precautions are taken. "Noguchi, Jour. Exp. Med., xv, 1912. 65 Noguchi, Jour. Exp. Med., xvii, 1913. 66 In connection with Eat-Bite Fever see also Kaneko and Okuda, Journal Exp. Med., vol. xxvi, 1917, p. 363. 67 Jour. Exp. Med., 1916, xxiii, p. 249. CHAPTER XLIV YELLOW FEVER AND THE LEPTOSPIRA ICTEROIDES, WEIL'S DIS- EASE (INFECTIOUS JAUNDICE) AND LEPTROSPIRA ICTERO- HEMORRILEGLZE YELLOW fever is an acute infectious disease which prevails en- demically in the tropical countries of the Western Hemisphere, but occurs also along the western coast of Africa and has exceptionally appeared, in epidemic invasons, in the north temperate United States and Europe. Guiteras, as quoted by Osier, classifies the distribution of the disease into three areas of infection. 1. The area in which the disease is never absent, including tropical South American ports and Havana. 2. The area of periodic epidemics, including sea-ports of the tropical Atlantic in America and Africa. 3. The area of accidental epidemics, extending from parallel 45° north latitude to 35° south latitude. In the United States severe epidemics have frequently occurred in Louisiana, Mississippi, and Alabama, and occasional but severe epidemics have occurred in Philadelphia and Baltimore. The disease occurs spontaneously only in man, and experimental inoculation of lower animals has been successful only in the chim- panzee in a single case reported by Thomas.1 In man afflicted with the malady the clinical picture is one of a rapidly developing fever with severe gastrointestinal symptoms, vomiting of blood, albuminuria, and often active delirium. The mor- tality is usually high, often reaching eighty per cent or more in the severe epidemics. Mode of Transmission. — Until comparatively recent years the mode of transmission of yellow fever was not understood and many erroneous theories were prevalent. It was supposed that yellow fever was contagious, and transmitted from person to person by direct or indirect contact with those afflicted or by fomites. The first to make the definite assertion that yellow fever was transmitted 1 Thomas, Brit. Med. Jour., 1, 1907. 874 YELLOW FEVER AND THE LEPTOSPIRA 875 by the agency of mosquitoes was Carlos Finlay. Finlay,2 as early as 188.1, advanced the theory that mosquitoes were responsible for the transmission of this disease and, furthermore, recognized "Ste- gomyia fasciata" or "Stegomyia calopus" as the guilty species. Finlay 's opinion, although later proved to be correct, was at first based only upon such circumstantial evidence as the correspondence of the yellow-fever zones with the distribution of this species of mosquito and the great prevalence of mosquitoes at times during which epidemics occurred. His theory was, therefore, received with much skepticism and was neglected by scientists until its revival in 1900, when the problem was extensively investigated by a com- mission of American army surgeons. Reed, Carroll, Agramonte, and Lazear were the members of this commission. The courage, self-sacrifice, and scientific accuracy which characterized the work of these men have made the chapter of yellow fever one of the most brilliant in the annals of American scientific achievement. Their work was much facilitated by the experience of Gorgas3 and others, who had demonstrated the absolute failure of ordinary sanitary regulations to limit the spread of yellow fever. They began their researches by investigating carefully the validity of Sanarelli's claims as to the etiological significance of his "Bacillus icteroides. " The results of this work yielded ab- solutely no basis for confirmation. They then proceeded to investigate the possibility of an inter- mediate host. In August, 1900, the commission began its work on this subject by allowing mosquitoes,4 chiefly those of the stegomyia species, to suck blood from patients, later causing the same insects to feed upon normal susceptible individuals. The first nine experiments were negative. The tenth, of wrhich Carroll was the subject, was successful. Four days after being bitten by the infected insect Carroll became severely ill with an attack of yellow fever, by which 2 Finlay, Ann. Roy. Acad. d. Havana, 1881. 'Gorgas, Jour, of Trop. Med., 1903. *Eeed, Carroll, Agramonte, and Lazear, Phila. Med. Jour., Oct., 1900; also Am. Pub. Health Assn. Rep., 1903; Agramonte, N. Y. Med. News, 1900; Reed, Jour, of Hyg., 1902; Reed, Carroll, and Auramontc, Am. Medicine, July, 1901. Boston Med. and Surg. Jour., 14, 1901; Carroll, Jour. Am. Med. Assn., 40, 1903; Carrol, ' ' Yellow Fever ' ' in Mense, ' ' Handbuch der Tropen-Krankheiten, ' ' ii. 876 PATHOGENIC MICROORGANISMS his life was endangered, and from the effects of which he died several years later. On the 13th of September, Lazear, while working in the yellow- fever wards, noticed that a stegomyia had settled upon his hand, and deliberately allowed the insect to drink its fill. Five days later he became ill with yellow fever and died after a violent and short illness. With these experiences as a working basis, the commission now decided upon a more systematic and thoroughly controlled plan of experimentation. In November of the same year, 1900, an experiment station, "Camp Lazear," was established in the neighborhood of Havana, about a mile from the town of Quemados. The camp was surrounded by the strictest quarantine. Volunteers from the army of occupation were called for, and twelve individuals were selected for the camp, three immunes and nine non-immunes. Two of the latter were physicians. The immunes and the members of the commission only were allowed to go in and out. All non-immunes who left the camp were prohibited from re-entering and their places taken by other non-immune volunteers. During December, five of the non- immune inmates were successfully inoculated with yellow fever by means of infected mosquitoes. During January and February five further successful experiments were made. Clinical observations were made by experienced native physicians, Carlos Finlay among them, and the patients, as soon as they were unquestionably ill with yellow fever, were removed to a yellow-fever hospital. This was done to prevent the possibility of the disease spreading within the camp itself. The mosquitoes used for the experiments were all cultivated from the larva and kept at a temperature of about 26.5° C. A further important experiment was now made. A small house was erected and fitted with absolutely mosquito-proof windows and doors. The interior was divided by wire mosquito netting into two spaces. Within one of these spaces fifteen infected mosquitoes were liberated. Seven of these had fed upon yellow-fever patients four days previously; four, eight days previously; three, twelve days previously ; and one, twenty-four hours previously. A non-immune person then entered this room and remained there about thirty minutes, allowing lumself to be bitten by seven mosquitoes. Twice after this the same person entered the room, remaining in it alto- YELLOW FEVER AND THE LEPTOSPIRA 877 gether sixty-four minutes and being bitten fifteen times. After four days this individual came down with yellow fever. In the other room two non-immunes slept for thirteen nights without any evil results whatever. It now remained to show that mosquitoes were the sole means of transmission and to exclude the possibility of infection by contact with excreta, vomitus, or fomites. For this purpose another mos- quito-proof house was constructed. By artificial heating its tem- perature was kept above 32.2° C. and the air was kept moist by the evaporation of water. Clothing and bedding, vessels, and eating utensils, soiled with vomitus, blood, and feces of yellow-fever patients were placed in this house and three non-immune persons inhabited it for twenty days. During this time they were strictly quarantined and protected from mosquitoes. Each evening, before going to bed, they unpacked and thoroughly shook clothing and bedding of yellow-fever patients, and hung and scattered these materials about their beds. They slept, moreover, in contact with linen and blankets soiled by patients. None of these persons con- tracted yellow fever. The same experiment was twice repeated by other non-immunes, in both cases with like negative results. All of the non-immunes taking part in these experiments were American soldiers. Four of them were later shown to be susceptible to yellow fever by the agencies of mosquito infection or blood- injection. The results obtained by the investigations of this commission may be summarized, therefore, as follows: Yellow fever is acquired spontaneously only by the bite of the Stegomyia fasciata. It is necessary that the infecting insect shall have sucked the blood of a yellow-fever patient during the first four or five days of the disease, and that an interval of at least twelve days shall have elapsed between the sucking of blood and the reinfection of another human being. Sucking of the blood of patients advanced beyond the fifth day of the disease does not seem to render the mosquito infectious, and at least twelve days are apparently required to allow the parasite to develop within the infected mosquito to a stage at which reinfection of the human being is possible. The results of the American Commission were soon confirmed by Guiteras5 and by Marchoux, Salimbeni, and Simond.6 These latter 5 Guiteras, Eev. d. med. trop. Jan., 1901, and Am. Med., 11, 1901. * Marchoux, Salimbeni, and Simond, Ann. de Tinst. Pasteur, 1903. 878 PATHOGENIC MICROORGANISMS observers, moreover, confirmed the fact that infection could be experimentally produced by injections of blood or blood serum taken from patients during the first three days of the disease. They showed that blood taken after the fourth day was no longer in- fectious: that 0.1 c.c. of serum sufficed for infection and finally that no infection could take place through excoriations upon the skin. They furthermore confirmed the observation of Carroll that the virus of the disease could pass through the coarser Berkefcld and Chamberland filters, — passing through a Chamberland candle "F" but held back by the finer variety known as "B." The fundamental factors of yellow-fever transmission thus dis- covered, we are in possession of logical means of defense. The most important feature of such preventive measures must naturally center upon the extermination of the transmitting species of mosquito. Stegomyia fasciata or calopus is a member of the group of ' ' Culicidae. " It is more delicately built than most of the other members of the group culicidae, is of a dark gray color, and has peculiar thorax-markings which serve to distinguish it from other species. The more detailed points of differentiation upon which an exact zoological recognition depends are too technical to be entered into at this place. Briefly described, they consist of lyre-like markings of the back, unspotted wings, white stripes and spots on the abdomen, and bandlike white markings about the metatarsi and tarsi of the third pair of legs. The peculiar power of transmitting yellow fever possessed by this species is explained by Marchoux and Simond7 by the fact that Stegomyia fasciata is unique among culicidae in that the female lives for prolonged periods after sucking blood. Among other species — Culex fatigens, Culex confirmatus, and most others — the female lays its eggs within from two to eight days after feeding on blood and rarely lives longer than the twelfth day —the time necessary for the development of the yellow-fever parasite. The limitation of yellow fever to tropical countries8 is explained by the fact that stegomyia develops only in places where high temperatures prevail. The optimum temperature for this species lies between 26° and 32° C. At 17° C. it no longer feeds, and bcomes practically paralyzed at 15° C. In order to thrive, the ''Marchoux and Simond, Ann. de 1'inst. Pasteur, 1906. 8 Otto, in Kolle und Wassermann, "Handbuch," etc., 11, Erganzungsband. YELLOW FEVER AND THE LEPTOSPIRA 879 species requires a temperature never going below 22° C. at night and rising regularly above 25° C. during the day. The females only are dangerous as sources of infection. The insect, like Anopheles, has the peculiarity of feeding chiefly at night. FIG. 99. — STEGOMYIA FASCIATA. (a) Female. (6) Male. (After Carroll.) Experiments done by Reed, Carroll, Agramonte, and Lazear, to ascertain whether the power of infecting was hereditarily transmis- sible from the mosquito to following generations, were negative. A positive result, however, has been reported by Marchoux and Simond.9 This question must still await more extensive research. ETIOLOGY OF YELLOW FEVER Numerous researches have been aimed at the elucidation of the problem of etiology and a large number of different microorganisms for which etiological significance was claimed, have been isolated from dejecta, vomitus and secretions of patients. A few of these claims have only historical importance, but may be mentioned be- cause of the wide interest they aroused among bacteriologists in the past. 9 Marchoux and Simond, Comptes rend, de la soc. de biol., 59, 1905. 880 PATHOGENIC MICROORGANISMS Cornil and Babes,10 in 1883, described chained cocci to which they attributed etiological significance, but their contentions have remained entirely unconfirmed. Sternberg,11 in 1897, described a colon-like organism, "bacillus X," for which he made very con- servative claims, which he himself, later, withdrew. The most active discussion was roused by the announcement of Sanarelli,12 in 1897, that he had discovered, in the blood and tissues of patients dead of yellow fever, a Gram-negative bacillus, which he believed to be the etiological agent of the disease. He based his contention upon the facts that he had isolated the organism from seven cases of yellow fever, had produced symptoms similar to the disease of the human being by the inoculation of pure cultures into dogs, and had obtained agglutination of the bacillus in the serum of convalescent patients. Later he inoculated five human beings subcutaneously with sterilized cultures of this ' ' Bacillus icteroides, ' ' and obtained symptoms which he believed simulated closely those of yellow fever. The claims of Sanarelli at first found much apparent confirmation, but later work by Durham and Myers,13 Otto,1* Agramonte,15 and others has definitely refuted his original claims, and there is to-day no scientific basis for the assumption that the Bacillus icteroides has any etiological relationship to the disease Protozoan incitants, also, have been described by Klebs,16 Schiiller,17 Thayer,18 and others, without bringing conviction or even justifying extensive investigation of their claims. While the earlier etiological investigations, therefore, were in- conclusive, much evidence was adduced which seemed to indicate that the virus was filtrable. Reed, Carroll, Agramonte and Lazear, carried out experiments which they thought demonstrated that the infecting agent was present in the blood of patients during the first three days of the disease, and could pass through the pores of 10 Cornil and Babes, Comptes rend, de 1'acad. des sci., 1883. "Sternberg, Cent. f. Bakt., I, xii, 1897. 12 Sanarelli, Ann. de 1'inst. Pasteur, 1897, and Cent. f. Bakt., I, xxii, xxvii, and xxix. "Durham and Myers, Thompson Yates Laboratory Eeports, 3, 1902. 14 Otto, Vierteljahrsch. f . gericht. Medizin, etc., 27, 1904. 15 Agramonte, N. Y. Med. News, 1900. "Klebs, Jour. Am. Med. Assn., April, 1898. "Schiiller, Berl. klin. Woch., 7, 1906. 18 Thayer, Med. Record, 1907. YELLOW FEVER AND THE LEPTOSPIRA 881 a Berkefeld filter. The filtrability of the virus has recently become doubtful in view of the researches of Noguchi. Eecently, Noguchi19 has carried out investigations which promise to settle the etiological problem in yellow fever conclusively. Noguchi in 1918 carried out extensive studies in the Yellow Fever Hospital at Guayaquil, where he began by observing 172 typical cases of the disease, studying them clinically and patholog- ically. We mention this because the only possible source of error in his investigations seems to us to be that of mistaking of cases of infectious jaundice for yellow fever, which, of course, is possible in view of the clinical similarity between the diseases, as emphasized by Nishi. It is important, therefore, to mention that Noguchi worked on what he considered classical cases of yellow fever in a Yellow Fever Hospital where he was aided by physicians familiar with the disease. He began by injecting blood from these cases in the first week of the disease, into a large number of different animals. The only animals with which he had success, however, were guinea-pigs. With the blood of early cases he inoculated seventy-four guinea-pigs from twenty-seven cases of yellow fever. Of these, eight, representing six cases, came down with symptoms resembling human yellow fever. After an incubation period of three to six days, the guinea-pigs showed a marked rise of temperature, eonjunctival congestion, leucocytosis followed by progressive leuco- penia, and a drop of temperature after a few days. Jaundice was noticed during this period, and hemorrhages from the nose and anus were occasionally observed. At autopsy the tissues were deeply jaundiced and the organs hyperemic. Hemorrhagic spots were found in the lungs and in the intestinal mucous membrane. In the blood, liver and kidneys of the guinea-pigs experimentally infected, Noguchi found the organism which he calls the Leptospira Ictero-Hcemorrhagice. This organism resembles quite closely the causative agent of infectious jaundice. In general, his monkey ex- periments were negative in all ,species except the Marmosets (Midas Oedipus and Midas Geoff royi). The examination of the blood of patients by the dark field in Noguchi 's20 hands never yielded large numbers of organisms. In careful examinations made on twenty-seven cases he found them in three only. He never saw them in urine, but one guinea-pig 19 Noguchi, Jour. Exper. Med., 29, 1919, 547-596. 20 Noguchi, Jour. Exper. Med., 30, 1919, 87. 882 PATHOGENIC MICROORGANISMS inoculated with 10 c.c. of urine came down. Examinations of the organs revealed them in only one case in the kidneys. Working with mosquitoes, Noguchi allowed Stegomyia calopus to bite yellow fever patients during the early stages of the disease. He placed the arm of a patient into a cage containing 200 to 300 mosquitoes hatched from the larvae, and allowed the mosquitoes to feed until the females were full of blood. Twenty-three days after feeding on the patient, the mosquitoes were allowed to feed on guinea-pigs. By this method he claims that he obtained one positive experiment out of six. In this positive experiment the guinea-pigs developed typical symptoms in about fifteen days. Noguchi also cultivated the Leptospira three times directly from yellow fever patients. The medium consisted of a mixture of one part of serum from non-immune persons and three parts of Ringer 's solution, used both in the liquid form and also in the semi-solid condition, by adding small amounts of melted neutral agar. To this about 1 c.c. of citrated blood from the median basilic vein of the patient was added, first being mixed with the semi-solid agar, while this was in the fluid condition at 42° C. This was allowed to solidify by cooling, and the warm Ringer's solution was then poured on the semi-solid portion and about 0.5 to 1 c.c. of the same blood introduced. The culture was then covered with paraffin oil. He also cultivated the organisms from infected guinea-pigs. He described them as follows: The Leptospira is an extremely delicate filament about 4 to 9 micra in length and 0.2 of a micron in width. It tapers gradually toward the extremities and ends in thick sharp points. It is minutely wound at short and regular intervals, each section measuring about 0.25 of a micron. The windings are so placed as to form a zigzag line at angles of 90°. It is not visible by ordinary light, but is easily seen with a dark field. It is actively motile, showing a vibratory motion and some- times twisting parts of the filament. It bores into the semi-solid material and is remarkably flexible. It is difficult to stain with ordinary dyes, but can be fixed by osmic acid and stained with Giemsa or other polychrome stains. In regard to transmission, it is an important fact (Noguchi) that 67 per cent of the wild rats of Guayaquil showed organisms in their kidneys similar in appearance to the leptospira just described; and these inoculated into guinea-pigs produced lesions similar to those produced by the yellow fever blood. YELLOW FEVER AND THE LEPTOSPIRA 883 In regard to the question of the identity of the yellow fever spirochaete with that of Weil's disease, Noguchi states that serolog- ical differentiation could be made. Polyvalent immune sera, one specific for icteroides and the other specific for icterohemorrhagice, showed a high neutralizing power for cultures of the homologous group. He found, however, that the action of the sera is not absolutely specific, since the injection of a sufficient amount of anti-icteroides serum prevents a fatal outcome in guinea-pigs in- oculated with multiple doses of the other organism, and vice versa. Other forms of serum reaction showed the same lack of absolute specificity. Subsequently, Noguchi attempted to protect guinea-pigs against multiple doses by injection of immune sera. He produced polyvalent immune serum by inoculation of a horse, and found that such serum could protect guinea-pigs when administered during the period of incubation, and modified the course of the disease when used in the early stages, but had no perceptible result in the later periods. Subsequent to the experiments of Noguchi recorded above, Noguchi and Kligler21 obtained results of similar experimental sig- nificance in Yucatan. PREVENTION OF YELLOW FEVER Success in the prevention of yellow fever has been one of the important factors in the development of South and Central Ameri- can countries. The conversion of Rio de Janeiro into a healthy port by Oswaldo Cruz, the cleaning up of New Orleans and the work done by Gorgas in Panama indicate the splendid role which an understanding of the relations of transmission in this disease have played in the progress of civilization. Knowing what we do about the Stegomyia and the part played by it in transmission, preventive measures must depend primarily upon the suppression of the mosquito and the isolation of cases from which mosquitoes may acquire the infection. The disease is never, as far as we know, conveyed by direct infection from man to man, or by soiled clothing, etc., and in consequence there is no necessity for care in these matters. The stegmoyia breeds particularly in rather pure water, such as found in rain water cisterns, and fortunately does not, like some other mosquitoes, breed in swamps, ponds and other natural NogucM and Kligler, Jour. Exper. Med., 32, 1920, 601. 884 PATHOGENIC MICROORGANISMS surface waters. Its flying radius is apparently not very large. Unlike the Anopheles, its habits are diurnal, instead of nocturnal. Rosenau states that experience of the epidemic in New Orleans in 1905 showed that this mosquito docs not fly far from its place of birth. We take from the same writer the statement that, in order to hold back the small mosquito, a mesh must be used containing at least twenty strands to the inch. Prevention may, therefore, be summed up as consisting in screen- ing of patients, screening of houses, destruction of mosquitoes within houses by insecticides and fumigation and the painstaking removal of all stagnant waters in the neighborhood of human habitations with especial care to the screening of drinking water cisterns in places where, as in Bermuda, drinking water is collected in rain water receptacles. In places like Bermuda, the Stegomyia fasciata is common, and yet there has been no case of yellow fever, we understand, since 1869. Immunity. — Natural immunity against yellow fever was formerly assumed to exist in the negro race. More recent investigations have not borne out this assumption. The negro soldiers of the American army in Cuba were afflicted equally with the white troops. The relative immunity of dark-skinned races, however, is explained pos- sibly by the fact that the stegomyia prefers to attack light-colored surfaces. A single attack seems to protect against subsequent infection throughout life. Relative immunity was produced by Marchoux, Salimbeni, and Simond,22 by injections of the serum of convalescents, serum heated to 55° C., and of defibrinated blood preserved for eight days in vessels sealed with vaseline. Experimental work is being carried out by Noguchi on both active and passive immunization by the use of his pure cultures of the Leptospira icteroides. These experiments are being actively carried out at the present time, but final results have not yet been achieved. On the whole, however, the promise of this work is great and extremely encouraging. It is especially encouraging in con- sideration of the great similarity between this Leptospira and the one of Weil's disease with which immunization experiments are also full of promise. 22 Marchoux, Salimbeni, and Simond, Ann. parts of salt solution to vaccinate rabbits on the shaven skin of the back. The pulp from this rabbit vaccina- tion is then used for calf vaccination. Actual vaccination of the animals is done as follows: Calves which have been kept under observation for at least a week are thoroughly washed and cleaned and the abdomen is clipped and shaved over an area extending from the ensiform cartilage to the pubic region, including the entire width of the belly and the inner folds of the thighs. It is best to shave the animal a day or two before vaccination so as to avoid fresh scratches and excoriations. Just before actual operation the animal is strapped to a specially constructed operating table in such a way as to allow free access to the shaved area. This area is now thoroughly washed with soap and water followed by alcohol, or, in some institutes, by a weak solution of lysol. If the latter is used, the field of operation must again be thoroughly rinsed with sterile .water. About a hundred small scarifications are made in this area, preferably by crossed scratches, covering for each scarification an area of about 3-4 square centimeters. Into these areas the virus is rubbed, using for each small area a quantity about sufficient to vaccinate three children. Two to three centimeter spaces are left between the lesions. The lesions are then allowed to dry and may be covered with sterile gauze or, as in Vienna, with a paste made up of beeswax, gum arabic, zinc oxid, water, and glycerin. In some institutes the lesions are left entirely uncovered. Ordinarily within about twenty-four hours after vaccination a narrow pink areola appears about the scratches. Within forty-eight 14 Park and Williams, Path. Microorg., N. Y., 1914, p. 569. GENERAL CONSIDERATION OF FILTRABLE VIRUS 897 hours the scratches themselves become slightly raised and papular, and within four or six days typical vaccina vesicles have usually developed. To obtain the vaccine from such lesions, the entire operative field is carefully washed with warm water and soap, followed by sterile water. In some cases two per cent lysol is employed, but must again be thoroughly removed by subsequent washing with sterile water. Crusts, if present, are then carefully picked off and the entire contents of the vesicle, sticky serum, and pulpy exudate removed by the single sweep of a spoon-curette. The curetted masses are caught in sterile beakers or tubes and to them is added four times their weight of a mixture of glycerin fifty parts, water forty-nine parts, and carbolic acid one part.15 German workers prefer a mixture of glycerin eighty parts, and water twenty parts, omitting the use of carbolic acid. The glycerinated pulp is allowed to stand for three or four weeks in order to allow bacteria, which are invariably present, to die out. After preservation for such a length of time, moreover, thorough emulsification is obtained more easily than when this is attempted immediately after curettage. At the end of three or four weeks, the glycerinated pulp is thoroughly triturated, either with mortar and pestle or by means of specially constructed triturating devices. Pulp so prepared should remain active for at least three months if properly preserved in sealed tubes in a dark and cool place. From the serum oozing from the bases of the lesions, after curet- tage, bone or ivory slips may be charged for vaccination with dry virus. The glycerinated pulp is put up in small capillary tubes, sealed at both ends, and distributed in this form. Park states that a calf should yield about 10 grams of pulp (which when made up should suffice to vaccinate 1,500 persons), and. in addition about 200 charged bone slips. The virus may be tested for its efficiency by a variety of methods. Calmette and Guerin inoculate rabbits upon the inner surfaces of the ears and estimate the potency of the virus from the speed of development and extensiveness of the resulting lesions. Guerin16 has estimated the potency of virus quantitatively by a method depending upon the inoculation of rabbits with a series of dilutions. ™Huddleston, quoted in Park, "Pathogenic Bacteria/' N. Y. 1908. 18 Guerin, Ann. de 1'inst. Pasteur, 1905. 898 DISEASES CAUSED BY FILTRABLE VIRUS Beginning with a mixture containing equal weights of glycerin and vaccine pulp, dilutions are made with sterile water ranging from 1 in 10 to 1 in 100. Rabbits are shaved over the skin of the back and 1 c.c. of each of these dilutions is rubbed into the shaved areas. Fully potent virus should cause closely approximated vesicles in a dilution of 1 in 500, and numerous isolated vesicles in a dilution as high as 1 in 1,000. Quantitative estimations of the bacteria in the glycerinated virus should be made by the plating method and the vaccine used only when after several weeks of preservation the numbers of the bacteria have been greatly diminished. In glycerinated pulp the bacteria will often disappear entirely in the course of a month. The vaccine should also be tested for the possible presence of tetanus bacilli, by the inoculation of white mice. Vaccination of human beings is performed by slightly scarifying the skin of the arm or leg with a sharp sterile needle or lancet and rubbing into the lesion potent vaccine virus. The virus was formerly dried upon wood, bone, or ivory slips and moistened with sterile water before the operation. At the present day the glycerinated pulp is almost universally employed. Since the ordinary scarification method has not been universally satisfactory, other methods of vaccination have been suggested. Occasional failure of the scarification method may in part be due to the fact that the glycerinated, ripened virus as used in most countries, has lost considerably in potency. We have seen men in the American Army successfully vaccinated with French virus after two vaccinations with American virus had failed. The French virus used had not been allowed to ripen in glycerine, was reasonably fresh (so-called green virus) and was, therefore, probably more potent. The fact that it still contained staphylococci and other organisms did not in these cases lead to infections of any importance. It must, however, always be dangerous, and the use of green virus, while perhaps more efficient from the point of view of vaccination, does not seem to us to be commendable. It is probably better to attempt modification of the present method by a more efficient in- troduction of the virus. The alternative methods which have been suggested are puncture in which drops of virus are placed on the skin and punctures made through the drops. Scarification or small incisions through the drops have also been recommended. Recent GENERAL CONSIDERATION OF FILTRABLE VIRUS 899 studies by Wright17 have shown that the intracutaneous injection of 0.1 c.c. of the ordinary glycerinated virus diluted with one part of sterile distilled water, resulted in " takes" when the ordinary scarification methods were unsuccessful. He vaccinated 227 negro soldiers in this way, and obtained over 70 per cent successful vac- cinations when only slightly over 8 per cent were obtained on the same series of cases previously, by the incision method. Sternberg, Kinyoun and others have demonstrated that within two weeks after vaccination the blood serum of the vaccinated patients will neutralize vaccine virus if allowed to stand with it in a test tube overnight. That vaccination is of incalculable benefit to the human race is no longer a question of opinion, and opposition to the practice is explicable only on the basis of ignorance. Statistical compilations upon this point are very numerous. It may suffice to select from the voluminous literature a single example, taken from Jiirgensen, which embodies the statistics of death from smallpox in Sweden, during the periods immediately preceding and following the intro- duction of vaccination* In that country the first vaccination was done in 1801. By 1810 the practice was generally in use but not enforced. In 1816 it was legally enforced. The years from 1774 to 1855 can thus be divided into three periods. 1. Prevaccinal period, 1774-1801 (25 years). Deaths smallpox per million inhabitants 2,050 2. Transitional period, 1801-1810 (9 years) 680 3. Vaccination enforced, 1810-1855 (35 years) 169 Prevaccinal period death rate 20.00 per mille. Vaccinal period death rate 0.17 per mille. In considering the benefit of vaccination it must not be forgotten that revaccination is quite as important as the first vaccination, which confers immunity only for from seven to ten years. A child should therefore be vaccinated soon after birth or at least before the eighth month, and the process should be repeated about every seven years thereafter. 17 Wright, Jour. A. M. A. 71, 1918, 654. 900 DISEASES CAUSED BY F1LTRABLE VIRUS RABIES (Hydrophobia, Rage, Lyssa, HundswutJi) Rabies is primarily a disease of animals, infectious for practically all the mammalia, but most prevalent among carnivora, dogs, cats, and wolves. It is said also to occur spontaneously among skunks of the Southwestern United States, and is readily inoculable upon guinea-pigs, rabbits, mice, rats, and certain birds, chicken and geese being especially susceptible. Man is subject to the disease. Infec- tion usually occurs as a consequence of the saliva of rabid animals gaining entrance to wounds from bites or scratches. The disease is more or less widely prevalent in all civilized countries except England, where the careful supervision of dogs, enforcement of muzzling laws, and rigid legislation regarding the importation of dogs, have caused a practical eradication of the disease. A fair estimate of the prevalence of the disease may be ob- tained from the statistics - of animals dyin^ or killed because of rabies in different countries. In Germany, according to Kolle and Hetsch, during the fifteen years ending in 1901, there were 9,069 dogs, 1,664 cattle, 191 sheep, 110 horses, 175 hogs, 79 cats, 16 goats, 1 mule, and 1 fox affected with rabies. In eastern United States the disease is not uncommon. The statistics of the New York Department of Health, for a period of six months ending December 31, 1907, show seventy-four cases of rabies among dogs in New York City and vicinity. Among human beings the disease is no longer common in civilized countries, since early preventive treat- ment is successfully applied in almost all infected subjects. Experimental infection in susceptible animals is best carried out by injections of a salt-solution emulsion of the brain or spinal cord of an afflicted animal, subdurally, through a trephined opening in the skull, but may also be accomplished by injection into the per- ipheral nerves, the spinal canal, or the anterior chamber of the eye, Intravenous and intramuscular injections are also successful, though less regularly so. The time of incubation after inoculation varies with the nature of the virus used, the location of the injection, and the quantity injected. In accidental infections of man and animals the incuba- tion is shortest and the disease most severe when the wounds are GENERAL CONSIDERATION OF FILTRABLE VIRUS 901 about the head, neck, and upper extremities and are deeply lacerated. This is explained by the fact that the poison is conveyed to the central nervous system chiefly by the path of the nerve trunks. This has been experimentally shown by di Vcstca and Zagari18 who inoculated animals by injection into peripheral nerves, and showed that the nerve tissue near the point of inoculation becomes infectious more quickly than the parts higher up; thus the lumbar cord of an animal inoculated in the sciatic nerve is infectious several days before virus can be demonstrated in the medulla. In man, infected with "street virus," that is, with the virus of a dog or other animal not experimentally inoculated, the incubation period varies from about forty to sixty days. Isolated cases have been reported in which this period was prolonged for several months beyond this. The virulence of rabic virus may be markedly increased or diminished by a number of methods. By repeated passage of the virus through rabbits, Pasteur19 was able to increase its virulence to a more or less constant maximum. Such virus which had been brought to the highest obtainable virulence, he designated as "virus fixe." Inoculation of rabbits, dogs, guinea-pigs, rats, and mice with such virus usually results in symptoms within six to eight days. The same animals inoculated with street virus may remain ap- parently healthy for two to three weeks. In dogs and guinea-pigs inoculation usually results first in a stage of increased excitability, restlessness, and sometimes viciousness. This is followed by depression, torpor, loss of appetite, inability to swallow, and finally paralysis. In rabbits the disease usually takes the form of what is known as "dumb rabies," the animals gradually growing more somnolent and weak, with tremors and gradual paralysis beginning in the hind legs. In experimentally infected birds the disease is slow in appearing and may show a course of gradually increasing weakness and progressive paralysis extending over a period of two weeks after the appearance of the first symptoms. In man, the disease begins usually with headaches and nervous depression. This is followed by difficulty in swallowing and spasms of the respiratory muscles. These symptoms occur intermittently, 18 di Vested and Zagari, Ann. de 1'inst. Pasteur, iii. 19 Pasteur's work on rabies. Compt. rend, de 1'acad. des sciences, 1881, 1882, 1884, 1885, 1886, and Ann. de 1'inst. Pasteur, 1887 and 1888. 902 DISEASES CAUSED BY FILTRABLE VIRUS the free intervals being marked by attacks of terror and nervous depression. Occasionally there are maniacal attacks in which the patient raves and completely loses self-control. Finally, paralysis sets in, ending eventually in death. Pathological examination of the tissues of rabid animals and human beings reveals macroscopically nothing but ecchymoses in some of the mucous and serous membranes. Microscopically, how- ever, many abnormal changes have been observed and were formerly utilized in histological diagnosis of the condition. Babes20 has described a disappearance of the chromatic element in the nerve cells of the spinal cord. This observation has been confirmed by others,21 but is no longer regarded as pathognomonic of rabies. The same observer has described a marked leucocytic infiltration which occurs about the blood-vessels of the brain and about the ganglia of the sympathetic system. These changes are not found in animals infected with virus fixe and are present only in animals and human beings inoculated with street virus. In 1903 Negri22 of Pavia described peculiar structures which he observed in the cells of the central nervous system of rabid dogs. While present in all parts of the brain, these " Negri bodies" are most regularly present and numerous in the larger cells of the hippocampus major and in the Purkinje cells of the cerebellum- The presence of these structures in rabid animals and man has been confirmed by a large number of workers in various parts of the world, and the specific association of these bodies with the disease is now beyond doubt. In consequence, the determination of "Negri bodies" in the brains of suspected animals has become an extremely important method of diagnosis — more rapid and ac- curate than the methods previously known. The demonstration of Negri bodies in tissues is carried out as follows: A small piece of tissue is taken from the cerebellum or from the center of the hippocampus major (cornu ammonis), and is fixed for twelve hours in Zenker's fluid. It is then washed thoroughly in water and dehydrated as usual in graded alcohols, embedded in paraffin, and sectioned. The sections are best stained by the method of Mann, as follows: 30 Babes, Virch. Arch., 110, and Ann. de Pinst. Pasteur, 6, 1892. nVan Gehuchten, Bull, de 1'aead. de med. et biol., 1900. "Negri, Zeit. f. Hyg., xliii and xliv. GENERAL CONSIDERATION OF FILTRABLE VIRUS 903 The sections, attached to slides in the usual way, are immersed in the following solution for from twelve to twenty-four hours : Methylene-bluc (Gruebler OO), 1 per cent 35 c.c. Eosin (Gruebler BA), 1 per cent 35 c.c. Distilled water 100 c.c. They are then differentiated in: Absolute alcohol 30 c.c. Sodium hydrate, 1 per cent in absolute alcohol 5 c.c. In this solution blue is given off and the sections become red. After about five minutes the sections are removed from this solution, are washed in absolute alcohol, and are placed in water where they again become faintly bluish. It is of advantage to immerse them, now, in water slightly acidified with acetic acid. Following this they are dehydrated with absolute alcohol and cleared in xylol, as usual. In preparations made in this way, the nerve cells are stained a pale blue, and in their -cytoplasm, lying either close to the nucleus or near the root of the axis-cylinder process, are seen small oval bodies stained a deep pink. The bodies are variable in size, measur- ing from 1 to 27 micra in diameter. They are round or oval, show a more deeply stained peripheral zone which has been interpreted as a cell membrane, and, in their interior, often show small vacuole- like bodies. There may be more than one, often as many as three or four, in a single cell. The rapid demonstration of Negri bodies in smears of brain tissue has recently been advocated by many observers and has been extensively used for diagnosis. It is carried out, according to Van Gieson,23 in the following way : A small pin-head-sized piece of brain tissue from the regions indicated above, is placed on one end of a slide under a cover-glass and the cover is gently squeezed with the finger until the tissue is flattened out into a thin layer. The glass cover is then gently shifted across the slide until the brain tissue is smeared along the entire surface. These smears may be fixed in methyl alcohol and stained by the Giemsa method, as described in the chapter on Spirochaeta pallida. Stained in this way, the Negri bodies are stained light blue, in contrast to Hie darker and more violet cell-bodies. 28 Van Gieson, Proc. of N. Y. Pathol. Soc., N. S., iv, 1906, 904 DISEASES CAUSED BY FILTRABLE VIRUS The smears may also be stained by a method originated by Van Gieson, which gives an excellent contrast stain and reveals more clearly the inner structure of the Negri bodies. Van Gieson 's stain is prepared as follows: Distilled water 10 c.c. Saturated alcoholic solution of rosanilin violet 2 drops. Saturated aqueous solution of methylene-blue diluted one-half with water 2 drops. This method has been modified by Williams and Lowden,24 who add to 10 c.c. of distilled water three drops of saturated alcoholic basic fuchsin and 2 c.c. of Loeffler's methylene-blue. The slides are fixed in methyl alcohol, washed in water, and covered with the freshly prepared stain. The slide is held over the flame until the solution steams and is then rinsed in water and dried. The Negri bodies assume a brilliant red and contain in their interior darkly stained, irregular particles which have been interpreted as chromatin bodies. As to the nature of the Negri bodies opinions are still divided. Their constant presence in rabic brain tissue is unquestioned and their diagnostic significance well established. Cultivation experiments, however, have been uniformly unsuccessful. A number of observers, Negri himself, Calkins,25 Williams and Lowden,26 and others, believe these bodies to be protozoa. The last-named authors base this opinion upon the definite morphology of the bodies, and their staining properties, which in many respects are similar to those of protozoa. These observers also claim that the morphology of the bodies shows a number of regular cyclic changes which are found accompanying different stages of the disease ; these changes correspond, according to these workers, to similar cycles occurring among known protozoa of the suborders of the class Sporozoa. Many pathologists still look upon them as specific degenerations of the nerve cells similar to the changes observed by Babes. It is not possible to decide absolutely from the facts at present at our disposal whether or not the Negri bodies should be regarded as parasites or as specific degeneration products. Their constant presence in rabic animals, and their apparent absence from normal 24 Williams and Lowden, Jour. Inf. Dis., 3, 1906. 25 Calkins, Discussion, Proc. N. Y. Pathol. Soc., N. S., vol. vi, 1906. 56 Williams and Lowden, loc. cit. GENERAL CONSIDERATION OF FILTRABLE VIRUS 905 brains and the brains of animals dead of other diseases, would tend to favor the parasitic view. To us it. would seem that added to this the clear outlines, apparent regularity of structure, and curiously consistent grouping of the darkly staining inclusions would add weight to such an assumption. We have triturated rabic tissue and shaken it up in anti-formin and seen many free Negri bodies apparently enucleated from the cells in consequence. Such com- plete extrusion from the cell also is seen in the ordinary smear preparations. It is at least unlikely that a cell-degeneration area would be expelled from the cytoplasm in so clearly outlined and morphologically unaltered a form. The fact that the virus is filtrable, as shown by Remlinger,27 Poor and Steinhardt,28 and others, would on the other hand seem to contradict the etiological importance of the Negri bodies unless, with some of the observers named, we assumed them to represent a large stage in the life-cycle of a protozoan parasite, which also occurred in smaller forms. It is a curious fact, also, that Negri bodies are scarce or absent in the spinal cord and cerebrum, though these areas are as virulent or more so than the hippocampus and cerebellum. They are small and hard to find in virus fixe, largest and most plentiful in cases in which the incubation period has been prolonged — as with street-virus in- fection. Much can be said on both sides, but in analyzing the present experimental facts, it seems fair to say that neither point of view is certain, though the parasitic nature of the Negri bodies seems very likely. The cultivation of parasites from rabic tissues has of course been attempted by most bacteriologists who have studied the disease since Pasteur. In all attempts, until very recently, either no results were obtained or else the parasites described could be shown to be present because of extraneous contamination. Recently Noguchi announced that he has been able to cultivate the virus by employing a technique similar to his methods of cultivating Treponema pallidum and poliomyelitis virus. Into high tubes filled with ascitic fluid a bit of fresh sterile rabbit kidney and a small piece of rabic virus were placed. The ascitic fluid was covered with sterile oil and the tubes incubated at 37.5° C. After five days' incubation cloudiness ap- peared and with it, minute globoid bodies not unlike those seen in ^Eemlinger, Ann. de Pinst. Past., xvii, 1903. 28 Poor and tfteinltardt, Jour, of Inf. Dis., xii, 1913. 906 DISEASES CAUSED BY FILTRABLE VIRUS poliomyelitis. After several generations large highly refractile bodies with dark central spots appeared in the cultures, and these Noguchi29 regards as possibly the parasites and similar to Negri bodies. Opinions are still divided as to the significance of Noguchi's results. However, whatever may be one's opinion regarding the nature of the peculiar bodies visible in his cultures, he has accom- plished the feat of preserving the virulence of the virus through twenty-one generations on artificial media, a fact which alone would seem to prove that he had successfully cultivated it, even though his " nucleated bodies" do not eventually turn out to be anything more than cell degenerations. The possibility that he may have carried original virus through twenty-one generations and that it has remained virulent for about 100 days at 37.5° C. can not be excluded as yet, but seems very remote. The Specific Therapy of Rabies.— The treatment which is now prophylactically applied to patients infected with or suspected of infection with rabies has been but little altered either in principle or in technical detail since it was first worked out by Pasteur. In principle it consists of an active immunization with virus, attenuated by drying, administered during the long incubation period in doses of progressively increasing virulence. By the repeated passage of street virus through rabbits, Pasteur obtained a virus of maximum and approximately constant virulence which he designated as virus fixe. By a series of painstaking experi- ments he then ascertained that such virus fixe could be gradually attenuated by drying over caustic potash at a temperature of about 25° C., the degree of attenuation varying directly with the time of drying. Thus, while fresh virus fixe regularly caused death in rabbits after six to seven days, the incubation time following the inoculation of dried virus grew longer and longer as the time of drying was increased, until finally virus dried for eight days was no longer regularly infectious and that dried for twelve to fourteen days had completely lost its virulence. The method of active immunization which Pasteur used consisted in injecting, subcutaneously, virus of progressively increasing viru- lence, beginning with that derived from cords dried for thirteen days and gradually advancing to a strong virus. Thus the patient was immunized to a potent virus several weeks before the incubation time 28 Noguchi, Jour. Exp. Med., xviii, 1913. GENERAL CONSIDERATION OF FILTRABLE VIRUS 907 of his own infection had elapsed. Pasteur successfully proved the efficacy of his method upon dogs and finally upon human beings, the first human case being that of a nine-year-old child — Joseph Meister. TECHNIQUE OF RABIES THERAPY. — The technique developed by Pas- teur is still, in the main, followed by those who treat rabies to-day. I. As a preliminary, it is necessary to prepare or obtain virus fixe. This may generally be procured from an established laboratory or may be prepared independently by passing street virus through a series of young rabbits (weighing from 700 to 1,000 gms.). According to Hogyes,30 the passage of the virus through twenty-one to thirty rabbits, in this way, will reduce its incubation time to seven or eight days. Babes claims to obtain a virus fixe more rapidly by passing the virus alternately through rabbits and guinea-pigs. For purposes of inoculation, virus is prepared by emulsifying in sterile salt solution pieces of the medulla or cerebellum of animals dead of a previous inoculation. The brain tissue which is not emulsified may be preserved under sterile glycerin in a dark and cool place for further use. II. Rabbits are inoculated with virus fixe by in- tracranial injection. A small incision is made in the shaved scalp in the median line, and the skin is SPINAL CORD OF RABBIT FOR PURPOSES OF ATTENUATION. retracted. With a small trephine or a round chisel, FIG. 100. — an opening is made in the skull and in the angle OD OF DRYING between the coronary and sagittal sutures. Through this opening about 0.2 to 0.3 c.c. of the virus fixe is injected, either directly into the brain substance or simply under the dura. As soon as a rabbit so inoculated has died it is autopsied. The animal before dissection should be washed in a disinfectant solution — lysol or carbolic acid. The skin is then removed and the animal, lying on its ventral surface, is fastened to a dissecting board. The spinal canal is then laid open with a pair of curved scissors and the spinal cord carefully removed. This is accomplished by cutting across the cord in the lumbar region, and lifting this with a forceps while the nerve roots are divided from below upward. Tlic cord is suspended by a sterile thread within a large bottle *° Hogyes, quoted from Kraus and Levaditi, "Handb., " etc., I. 908 DISEASES CAUSED BY F1LTHABLE VIRUS into the bottom of which pieces of potassium hydrate have been placed. The bottle is then set away in a dark room or closet, the temperature of which is regulated so as to vary little above 25° C. Bacteriological controls as to the sterility of the cord should also be made. After drying, pieces of the cord are prepared for injection. This is done in various ways. At the New York Department of Health 1 cm. of the cord is emulsified in 3 c.c. of sterile salt solution, the dose for injection being usually 2.5 c.c. Marx31 emulsifies 1 cm. of the cord in 5 c.c. of sterile bouillon or salt solution, using 1 to 3 c.c. of this for injection according to the age of the cord. For shipment 20 per cent of glycerin and 0.5 per cent of carbolic acid arc added. The scheme of treatment is also subject to variations according to the individual customs of various laboratories. The following scheme is the routine of the Pasteur Institute in Paris, as quoted in Kraus and Levaditi, ' ' Handbuch f iir Immunitatsf orschung, ' ' Vol. I, p. 713. Day of Treatment MILD CASES Dose MEDIUM CASES Dose SEVERE CASES Dose Days of Drying Days of Drying Days of Drying 1 14 + 13 3 C.C. 14 + 13 3 c.c. A.M. 14 + 13 P.M. 12 + 11 3 c.c. 2 12 + 11 3 C.C. 12 + 11 3 c.c. A.M. 10+ 9P.M. 8+ 7 3 c.c. 3 10+ 9 3 c.c. 10+ 9 3 c.c. A.M. 7 P.M. 6 2 c.c. 4 8+ 7 3 c.c. 8+ 7 . 3 c.c. 5 2 c.c. 5 6+ 6 3 c.c. 6+ 6 3 c.c. • 5 2 c.c. 6 5 Ic.c. 5 2 c.c. 4 2 c.c. 7 5 1 c.c. 5 2 c.c. 3 1 c.c. 8 4 1 c.c. 4 2 c.c. 4 2 c.c. 9 3 Ic.c. 3 1 c.c. 3 1 c.c. 10 5 2 c.c. 5 , 2 c.c. 5 2 c.c. 11 5 2 c.c. 5 2 c.c. 5 2 c.c. 12 4 2 c.c. 4 2 c.c. 4 2 c.c. 13 4 2 c.c. 4 2 c.c. 4 2 c.c. 14 3 2 c.c. 3 2 c.c. 3 2 c.c. 15 3 2 c.c. 3 2 c.c. 3 2 c.c. 16 i 5 2 c.c. 5 2 c.c. 17 4 2 c.c. 4 2 c.c. 18 3 2 c.c. 3 2 c.c. 19 5 2 c.c. 20 4 2 c.c. 21 3 2 c.c. 31 Marx, Deut. med. Woch., 1899, 1900. GENERAL CONSIDERATION OF FILTRABLE VIRUS 909 The treatment at the New York Department of Health is as follows: Day of Treatment MILD CASES Dose MEDIUM CASES Dose SEVERE CASES Dose Days of Drying Days of Drying Days of Drying 1 14 + 13 4 C.C. 10 4 C.C. A.M. 10+9 P.M. 10+9 4 c.c. 2 12 + 11 4 c.c. 9 4 c.c. A.M. 8+7 P.M. 8+7 4 c.c. 3 10+9 4 c.c. 9 4 c.c. 6 4 c.c. 4 8+7 4 c.c. 8+7 4 c.c. 4 4 c.c. 5 6 2 c.c. 6 2 c.c. 3 2 c.c. 6 5 2 c.c. 5 2 c.c. 4 2 c.c. 7 4 2 c.c. 4 2 c.c. 3 2 c.c. 8 3 2 c.c. 3 2 c.c. 2 2 c.c. 9 5 2 c.c. 2 2 c.c. 4 2 c.c. 10 4 2 c.c. 5 2 c.c. 1 2 c.c. 11 3 2 c.c. 4 2 c.c. 4 2 c.c. 12 5 2 c.c. 3 2 c.c. 3 2 c.c. 13 4 2 c.c. 2 2 c.c. 2 2 c.c. 14 3 2 c.c. 4 2 c.c. 4 2 c.c. 15 5 2 c.c. 3 2 c.c. 1 2 c.c. 16 4 2 c.c. 2 2 c.c. 4 2 c.c. 17 4 2 c.c. 3 2 c.c. 18 3 2 c.c. 2 2 c.c. 19 2 2 c.c. 4 2 c.c. 20 3 2 c.c. 21 2 2 c.c. 22 4 2 c.c. 23 3 2 c.c. 24 2 2 c.c. 25 4 2 c.c. 26 3 2 c.c. The severity or mildness of cases is estimated from the depth and degree of laceration of the wounds, also from their location — bites about the face and upper extremities b«ing the most dangerous. During the course of such treatment patients may show trouble- some erythema about the point of injection and occasionally back- ache and muscular pains. Treatment need not be omitted unless these symptoms become excessive. The efficiency of the Pasteur treatment in rabies is no longer problematical. According to Hogyes, 50,000 people have been treated within ten years, with an average mortality of 1 per cent. Although the method described above is the one which is exten- sively used in all established institutes for the treatment of rabies, 910 DISEASES CAUSED BY FILTRABLE VIRUS other methods have been elaborated and used to a slight extent. One of the most important of these is the "dilution method" of Hogyes. This method is carried out as follows : A definite quantity of the spinal cord of a rabbit dead of virus fixe is emulsified in 100 c.c. of normal salt solution. Dilutions of this emulsion are made and the patient is injected at first with a dilution of 1 -.1,000, subse- quent injections being made of gradually increasing concentration until a concentration of 1 :100 is reached. This method, so far as it has been used, has been satisfactory, but it has not yet found exten- sive application. Harris and ShackelP2 describe an improved method of desic- cating rabic virus which consists in placing the material to be dried in the bottom of a vacuum desiccating jar in the upper part of which is a separate dish containing sulphuric acid. The temperature is reduced by placing the jar in a salt and ice mixture, and after thorough solidification of the material has resulted, a rapid vacuum is produced by a Geryk pump to less than 2 mm. of mercury. The virus so dried will retain its virulence for as long as four months, if guarded against moisture. It will be noted that this method cannot be taken as justifying any particular conclusions as to the nature of the rabic virus since the same method has been applied by Swift and others to the maintenance of the virulence of bacteria. Harris33 believes that the attenuation of a rabic cord in the Pasteur method does not depend primarily upon the loss of water, but rather upon the method of extracting the water. Slow desic- cation attenuates the virus, Harris concludes, by reason of the con- centration of salt and other substances in solution of the brain and cord, the action being thus essentially a chemical one. Harris has studied the minimal lethal dose of his rapidly dried material on animals, and has developed with this material a modified method of immunizing animals and human beings against rabies. He prepares suspensions (from the material rapidly dried at zero degrees as above) by emulsifying 10 milligrams in 10 c.c. salt solution. This gives a dilution of 1 milligram to each cubic centi- meter of emulsion, and from this basic suspension dilutions are made. 32 Harris and Shackell, Jour, of Infec. Dis., 8, 1911, 47. 83 Harris, Jour, of Infec. Dis., 13, 1913, 155. GENERAL CONSIDERATION OF F1LTRABLE VIRUS 911 He tests virulence by injecting 0.1 c.c. of the dilution into the brain of a rabbit, by trephining and passing a very fine hypo- dermic needle through the brain to the base or into the lateral ventricle, so that none of the material may escape when the needle is withdrawn. He finds that, by this method, his material after preservation of three weeks, is equivalent to that of fresh cord. After fifty days it is 25 per cent more infective than the same quan- tity of the "one day" cord of the old method. After 200 days its infectivity is exactly equal to that of the "two day" cord of the old method. After 500 days it is two and one-half times as infective as the "three day" cord. The only precaution that is absolutely essential is that in preparing and preserving the diluted material, the presence of moisture must be absolutely prevented. Having prepared this material, Harris thought it worth while to follow the suggestion of Hogyes who has developed a method of immunization dependent upon the dilutions of virulent virus, in- stead of using the Pasteur method of quantitative destruction by drying. Hogyes had treated 10,000 patients by his method without accident, giving from 70 to 220 M. I. D., or minimal infective doses for rabbits, the first day and from 200 to 400 M. I. D. on the second, the M. I. D. being, as in Harris's experiments, determined by injec- tions into the brains of rabbits. Harris has constructed charts from careful experiments in which he tabulates the proportion of infec- tious to non-infectious material in a milligram of desiccated brain after preservation for various periods. Unlike Harvey and Mc- Kenderick (quoted from Harris) who believe that when rabic virus has lost its infectiousness, it no longer has the power of conferring immunity, Harris believes that when all the infectivity of rabic material has been destroyed rapidly by light and various tempera- tures, it still has considerable powers of conferring immunity. It is, of coarse, impossible to determine the degree of immunity conferred by the non-infective portions of desiccated material. Harris begins his treatment with material in which the proportion of living to non-infective material is estimated as being about 1 to 25, that is, material at least six months old. As the treatment continues, he gradually increases until he uses material which con- tains 100 M. I. D. per milligram. In two years of such treatment, he had no accidents either in patients, dogs or rabbits. Attempts to treat active rabies with the sera of immunized animals have so far been unsuccessful. CHAPTER XL VI ACUTE ANTEEIOE POLIOMYELITIS LETHARGIC ENCEPHALITIS THE disease known as acute anterior poliomyelitis has long been recognized as an acute infectious condition, both because of the char- acteristics of its clinical manifestations and of its epidemic occur- rence. For these reasons it was classified with acute infectious dis- eases by Marie and by Striimpell long before any experimental evidence of infection was obtained. Its contagiousness, while not a proven fact, seemed very likely from the evidence of its mode of spreading and has been removed from the sphere of mere conjecture by the careful study of a Swedish epidemic, comprising one thousand cases, made by Wickman.1 While acute anterior poliomyelitis is almost exclusively a disease of childhood, it is assumed by clinicians that it is etiologically closely related to, possibly identical with, certain diseases of the adult, characterized by bulbar paralysis and acute encephalitis. Into this category, also, some observers place the condition known as ' ' Landry 's paralysis. ' ' The basis for the identification of these con- ditions with poliomyelitis lies chiefly in the similarity of the pathol- ogical lesions and upon the fact that the last-named diseases occur most often during the course of poliomyelitis epidemics. The writer some years ago obtained a typical poliomyelitis infection in a monkey with material from a definite case of Landry 's paralysis in a young woman. The most thorough recent clinical study of acute poliomyelitis of which we know is the one by Peabody, Draper and Dochez.2 The incubation period of the disease varies, but appears to be about 10 days. Prodromal symptoms, if they appear at all, come on just before the onset of the disease. They may be very mild, consisting 1 Wiclcman, quoted from Landsteiner and Popper, Zeit. f. Immunitatsforch., ii, 1909. 2 Peabody, Draper and Docliez, Monog. Eock Inst., 4, Jan., 1912. 912 ACUTE ANTERIOR POLIOMYELITIS 913 of slight fever, sweating, drowsiness, pain in the neck and head, and weakness. Intestinal symptoms are not uncommon. Again cases may begin without prodromal symptoms with sudden illness, chill and fever. This may be all that occurs in the so-called abortive cases which in Wickman's studies represent from 25 to 56 per cent of all cases. During the early periods of the disease the cases may show vari- ous types of development. They may simply show signs of general infection, they may resemble influenza, the gastro-intestinal symp- toms may be predominant, and others may show signs of meningeai irritation. The neck may be stiff, but not with the involuntary stiff- ness of meningitis, showing rather a reflex resistance when the at- tempt is made to move the head forward. Peabody, Draper and Dochez state that the best appreciation of the clinical condition in acute poliomyelitis may be had by considering the cases as (1) the abortive cases which never become paralysed,3 the cerebral group which is rare and in which involvement of the upper motor neurons with spastic paralysis is the chief characteristic, and (2) the bulbo- spinal group, which is the largest group, and in which motor neuron involvement and flaccid paralysis occur. In the pre-paralytic stages the leucocytes are apt to be slightly increased in number and there is a definite increase of the poly- nuclears by 10 or 15 per cent. A leucocytosis of 15,000 to 30,000 is stated by the writers mentioned above as distinctly suggestive of the disease, especially if the polynuclears are increased at the ex- pense of the lymphocytes. The study of the spinal fluid is of great value. During the first days of the disease, before the paralysis appears, there is an increase of the cellular contents with a total which may run as high as 500 cells per cubic millimeter, but usually they run about 50 per cubic millimeter. The writers above quoted saw two cases in which there were 999 and 650 cells, respectively. During the second week, of 45 cases seen by them, 8 had over 50, and 23 were above normal. In the later stages of the disease the cell counts come back to normal. During this early stage, most of the cells consist of lymphocytes, rarely showing predominant poly- nuclears. The globulin contents during the early stages are prac- tically normal, or slightly increased. This, however, increases later, as the cell counts drop. The fluid reduces Fehling's solution. 8 For literature, see Landsteiner and Popper, loc. cit. and Wickman, Die Heine- Medinsche Krankheit, Berlin, 1911. 914 DISEASES CAUSED BY FILTRABLE VIRUS ETIOLOGY An important advance in the study of this disease was made in 1908 when Landsteiner and Popper succeeded in transmitting it to two monkeys ( Cynocephalus hamadryas and Macacus rhesus). The transmission was accomplished by intraperitoneal injections of a saline emulsion of the spinal cord of a child that had died during the fourth day of an attack of infantile paralysis — during the stage of acute fever. The first monkey injected became severely ill six days after the injection and died on the eighth day. The second animal became paralyzed seventeen days after the injection and was killed two days later. Cultural experiments with the substance injected were nega- tive, as were also inoculation experiments carried out upon guinea- pigs, rabbits, and mice. The histological lesions produced in the in- oculated monkeys were similar to those occurring in children afflicted with the disease. An attempt to transmit the disease to another monkey with spinal- cord substance of the animal that was killed resulted negatively. Soon after the successful experiments of Landsteiner and Popper, a similar result was recorded by Knoepfelmacher.4 An attempt to trans- mit the disease from monkey to monkey was again negative. Similar positive inoculation results were published, a little later than this, by Flexner and Lewis5 in November, 1909, and by Strauss and Huntoon 6 in January, 1910. Flexner and Lewis, in their work, moreover, succeeded in trans- mitting the disease through several inoculation- generations of monkeys. The same workers7 have ascertained that inoculation may be successfully applied not only by the intraperitoneal route but intra- cerebrally, subcutaneously, intravenously, and by the path of the larger nerves. They also proved that not only the brain and cord of afflicted animals contains the virus, but that this may be found, during the early days of the disease at least, in the spinal fluid, the blood, the nasopharyngeal mucosa, and lymph nodes near the site of inoculation. Landsteiner and Levaditi,8 meanwhile, experimenting with the 4 Knoepfelmacher, Mediz. Klinik, v, 1909. 8 Flexner and Lewis, Jour. Am. Med. Assn., 53, 1909. • Strauss and Huntoon, N. Y. Med. Jour., Jan., 1910. 1 Flexner and Lewis, Jour. Exp. Med., 12, 1909. •Landsteiner and Levaditi, Comptes rend, de la soc. de biol., Nov., 1909, and ACUTE ANTERIOR POLIOMYELITIS 915 virus independently, succeeded in transferring the disease from one animal to others, demonstrated that the virus could pass through the pores of a Berkefeld filter, and showed that the virus was present in the salivary glands — a fact which may prove of great importance in possibly establishing a clew to the mode of contagion among human beings. The same authors, as well as Flexncr and Lewis, were able to show that the virus was preservable under glycerin for as long as ten days and retained its virulence for from seven to eleven days when dried. What has been said concerning infantile paralysis may also be taken to apply to Landry's paralysis. From a typical case of Landry's paralysis in an adult the writer succeeded in obtaining typical poli- omyelitis in monkeys at Stanford University in 1912. The clinical diagnosis in this case was made by Wilbur. The disease in the mon- keys was typical of poliomyelitis and the histological sections showed the typical lesions. According to Flexner and Lewis the virus remains active, when frozen, for as long as forty days, but is extremely sensitive to heat, being destroyed by a temperature of from 45° to 50° C. maintained for thirty minutes. Experiments aimed at the isolation or even morphological detection of a parasite in the virulent material have been entirely without suc- cess until recently. Bacteria which in the past have been isolated from nerve substance and spinal fluid in cases of poliomyelitis can of course be excluded from etiological significance by the recent determination of the filtrability of the virus as determined by Flexner and Lewis, and Landsteiner and Levaditi. Small coccoid forms in smears from the nerve tissue recently described by Proescher9 are of very uncertain significance. The streptococci recently described by Kosenow are, most probably, secondary invaders. The most important contribution which has been made in the solution of this problem is that of Flexner and Noguchi.10 These investigators placed small bits and emulsions of the brain of monkeys, dead of poliomyelitis, in high tubes containing human ascitic fluid together with a piece of fresh sterile rabbit kidney. In all essentials the method was that followed by Noguchi in his culti- vation of Treponema pallidum. It was necessary to use fresh unheated ascitic fluid. Heat sterilization rendered it unsuitable. By this method, after five days opalescence appeared about the 'Proescher, N. Y. Med. Jour., 1913. 10 Flexner and Noguchi, Jour, of Exp. Med., xviii, 1913. 910 DISEASES CAUSED BY FILTRABLE VIRUS pieces of tissue. This increased until the tenth day, when sedimenta- tion began. Microscopical examination by Giemsa's method of stain- ing revealed small globoid bodies measuring from 0.15 to 0.3 micron in diameter, arranged in pairs, short chains, and masses. Similar bodies could later be found in poliomyelitis tissues. Cultures were obtained from glycerinated as well as from fresh virus and from the filtered as well as the unfiltered material. Typical lesions and death have been produced in monkeys with such cultures in a few cases. We have few data which throw light upon possible immunity to the disease. Repeated attacks of the disease in the same human being have not been noted; but this, as Flexner and Lewis point out, may be due to the fact that the epidemics are rare, and individuals once afflicted have passed beyond the susceptible age by the time of the sec- ond epidemic. As a matter of fact, however, these workers have not succeeded in reinfecting monkeys that had recovered. In chickens a disease has been observed similar in many ways to poliomyelitis, but further study has shown this to be a polyneuritis. Of other animals besides monkeys, rabbits only have been success- fully inoculated with this disease. Transmission to these animals was first reported by Kraus and Meinicke 11 and later by Lentz and Hunte- muller. 12 Marks 13 has studied the disease in rabbits thoroughly, and concludes that there is no doubt that the virus can be cultivated through a limited number of generations in rabbits. He was able to transmit to monkeys from rabbit material. The disease, however, does not resemble that of man or monkeys clinically and no definite lesions of the central nervous system are present. The rabbits seem perfectly well for six or seven days, when rapid weakness and death in con- vulsions occur. Animals which have been unsuccessfully injected, even with living virus, do not develop immunity. However, animals that have been successfully inoculated and recovered are, like human beings, thereafter immune. Levaditi and Landsteiner, Roemer and Joseph, and Flexner and Lewis have shown that the serum of recovered monkeys will pro- tect normal animals from fatal doses of the virus. That the same pro- tective power for monkeys has been shown in the serum of human recovered cases, is shown by the same authors and by Anderson and Frost and consequently the intraspinous injection of the serum of 11 Kraus and Meinicke, Deut. med. Woch., xxxv, 1909. 12 Lentz und Huntemiiller, Zeitschr. f. Hyg., Ixvi, 1910. "Marks, Jour, of Exp. Med., xiv, 1911. ACUTE ANTERIOR POLIOMYELITIS 917 recently recovered children into patients in early stages of the dis- ease has recently been advocated and is though well of by a number of observers. This work, however, has not reached completion and final judgment must be withheld. Flexner and Amoss have paid particular attention to the problem of passive immunization and found that in protecting monkeys if the quantity of virus injected into the brain is not too great paralysis can be prevented in some cases and delayed in others, by injecting the serum of recovered monkeys into the subarachnoid space by lumbar puncture. Immunizing sera cannot be produced by treatment of in- susceptible animals with virus, but Flexner and his associates have occasionally succeeded in immunizing actively by injecting, sub- cutaneously, graded doses of crude virus. This method, however, is not very useful, nor is it very safe since some of the animals so treated do not develop a strong immunity and others may become paralyzed. It appears, therefore, that the neutralizing principle, whatever it may be, is present only in animals and man that have recovered from actual infection, and the only method of passive immunization or serum treatment, therefore, available at the present time is that in which the blood serum of individuals who have re- covered from an attack is used. Epidemiological Facts. — To summarize the important epidemio- logical facts in poliomyelitis we may say that the work of Flexner and his group, as well as that of European workers, has shown that the poliomyelitis virus is present in the mucous membranes of the nose and throat, in the excretions from these membranes and in the intestinal contents. It may also be present in the tonsils. It leaves the infected body with the discharges from the nose and throat and the intestines, and when swallowed from the throat can pass into the intestines, resisting the action of the gastric and intestinal secretions. Flexner, Clarke and Dochez 14 have injected monkeys with filtrates from washings of the intestines after feeding monkeys with spinal cord material from infected monkeys and taking the intestinal fluid two hours after feeding. Outside the human body the virus probably can survive for some time, though the exact period is not known. Neustaedter and Thro 15 claim to have been successful in infecting monkeys with dust taken from a sick room. 14 Flexner, Clarke and Dochez, Journ. A. M. A., vol. 59, 1912. 15 Neustaedter and Thro, N. Y. Med. Journal, 94, 1911, p. 613, 813. P18 DISEASES CAUSED BY FILTRABLE VIRUS There is a great deal of evidence to show that poliomyelitis car- riers exist. Experimental evidence of this carrier state has been ad- vanced by Osgood and Lucas 16 who found the virus in monkeys five months after convalescence. We ourselves have seen cases in which it could be definitely proven that they had not been in contact with a preceding case, two of them in country districts where the scantily populated surrounding area could be searched without danger of overlooking a case. It is also likely that at times of epidemic a great many very mild attacks of poliomyelitis may occur, which are mistaken for mild influenza or severe colds, and in connection with the occurrence of a recent case seen by us there were a number of indefinitely diagnosed cases of intestinal disturbance, with fever, in the neighborhood. It is not impossible that such cases may be true poliomyelitis of a mild type without paralysis but capable of passing on the virus. Peabody, Dochez and Draper17 have cited similar cases. The virus probably gets into the new patient by direct and in- direct contact, and can be carried from place to place, perhaps on the feet of flies, a fact which would be indicated by some experiments done by Flexner. In 1911 epidemiologists of the State Public Health Service of Massachusetts established a parallelism between the distribution of poliomyelitis cases and the occurrence of the biting stable fly, Stomoxys. Subsequently, M. J. Rosenau published experiments in which he obtained poliomyelitis infection by allowing infected Stomxys flies to bite monkeys. These observations were confirmed by Anderson and Frost,18 but all subsequent attempts to repeat the experiments have failed. The ordinary manner of infecting of the human being is probably, then, through the nasopharynx. A great many cases begin with intestinal disturbances which may last a few days before the patient is ill enough to go to bed. It is more than likely, therefore, that the virus may also enter the body by ingestion and that infection of food may play a role. The disease is usually present to some extent in crowded centers of the world, in the spring and summer months. The season of greatest prevalence is May to November. Most cases are in children below five, but adult cases do occur. 14 Osgood and Lucas, Journal A. M. A., 57,' 1911, p. 495. 17 Peabody, Draper and Dochez, Monog. Rock. Inst., No. 4, Jan., 1912. 18 Anderson and Frost, Journ. A. M. A., 56, 1911, p. 663. ACUTE ANTERIOR POLIOMYELITIS 919 Although a great many studies have been made to trace the infec- tion of one case to exposure to another, such attempts have failed in most instances, and it seems fairly well established that there is great variation in the susceptibility of individuals to the disease. Whether this depends upon previous mild attacks of the variety spoken of above or whether it is a congenital difference, cannot be stated. ENCEPHALITIS LETHARGICA It is difficult to say whether the disease which we now speak of as Lethargic Encephalitis is identical with the conditions formerly described as "sleeping sickness," "Schlaf Krankheit," etc. Camera- rius, whom we quote from Smith,19 is said to have described an epi- demic disease which occurred in Germany in 1712 which probably represents the same condition. In 1768 and in. 1835 similar epidemics seem to have occurred in the trail of influenza outbreaks, a fact which is of considerable importance in view of the fact that recent interest- in the disease dates from the occurrence of many cases of lethargic encephalitis which followed in the train of the last influenza epidemic. After the epidemic of 1889, relatively few typical cases of what we now speak of as lethargic encephalitis were reported, though nervous complications were apparently very common. During the later stages of the great war epidemic of influenza, cases began to appear in many different places which, at first, were either mistaken for poliomyelitis or undiagnosed before death. We remember ourselves seeing two cases in soldiers during this period in which diagnosis was doubtful and which we now believe to have been lethargic encephalitis. One of the first systematic reports is that of Economo 20 who de- scribed an outbreak of the disease in Vienna in 1917. In 1918 an outbreak occurred in Great Britain, which was studied and reported by Wilson,21 Hall,22 Herringham, and others. (The onset of the disease in America was dealt with in an editorial in the Journal of the American Medical Association, 72, 1919, 414.) In speaking of the distribution of the disease during this last epidemic, Smith states that the first cases occurred in Central Europe in 1917, appeared in France, 19 Smith, U. S. Pub. Health Eeport, No. 6, Vol. 37, February, 1921. 20 Economo, Wien. klin. Woch., 30, 1917, 581. 21 Wilson, Lancet, 2, 1918. 22 Hall, Brit. Med. Jour., 2, 1918, 467. 920 DISEASES CAUSED BY FILTRABLE VIRUS Great Britain and Algeria in late 1917, and early 1918, and reached North America during the latter half of 1918 and in early 1919. The disease spread rapidly throughout the United States, and Smith says that by May, 1919, cases had been reported from twenty of the states. The largest number were reported from Illinois, New York, Louisiana, and Tennessee, a fact which shows the apparent independence of the disease of climatic conditions. The disease spread through the United States from east to west. Smith summarizes the epidemiological features as follows: In almost all outbreaks the appearance of epi- demic encephalitis has been preceded by influenza. Evidence of direct communicability is lacking, since in analyzing approximately 900 people exposed in the immediate families of cases, no secondary cases occurred. The age distribution is entirely different from poliomye- litis, so much so that Smith believes that this alone would distinguish the two diseases. In poliomyelitis over 59 per cent, of the cases occur before the fourth year, and 68 per cent of the cases occur below the age of 5, whereas in epidemic encephalitis 58 per cent, of the cases occurred in individuals 20 years of age and over. As to sex, 60 per cent of the cases of encephalitis occurred in males. Seasonally the apex of the epidemic curve in the United States was reached in March. The relationship of the disease to influenza is vague, but something to be very seriously considered in view of the recent researches of Olitsky with influenza and of Strauss and Loewe on filtrable virus in encephalitis. The onset is usually gradual, with headache, lassitude and grad- ually increasing fever. Occasionally there are vomiting, vertigo and muscular pains. Disturbances of vision may appear early. Following this there may be an acute stage during which the fever rises, vomit- ing may intensify, there may be disturbances of the cranial nerves and great general weakness which gradually lapses into coma. Occa- sionally a case may get well in two or three weeks following this, again the coma may persist for a long time. Paralysis of muscular groups and facial paralysis, ptosis, disturbances of pupil reflexes and other muscular reflexes occur. The spinal fluid shows an increased number of cells in a certain percentages of cases. The leucocytes may be slightly increased, but are usually not very high. The mortality in Smith 's study was 29 per cent. The etiology of the disease has been very carefully worked upon since the last appearance of the disease. A number of bacteria have ACUTE ANTERIOR POLIOMYELITIS 921 been described and isolated from cases, but very probably have no significance. We may dismiss the claims of bacterial causative agents as not in any case sufficiently based on reliable evidence. Of im- portance is a publication by Strauss, Hirschfeld and Loewe in 1919. 23 These workers obtained naso-pharyngeal mucus of fatal cases of the disease, filtered it through Berkefeld candles and injected it sub- durally and intracranially into monkeys and rabbits. In these animals they produced the disease. A monkey (Macacus Cynomolgus) in- jected on April 25th developed by May 2d, lethargy, general malaise, temperature and ptosis of the left lid, but recovered. Similar results were obtained with a Rhesus. Rabbits intracranially injected died in 4 or 5 days with punctate hemorrhages in the brain, intense conges- tion, marked meningitis and mononuclear infiltrations about the ves- sels. They claim to have repeated these experiments many times since their first publication. In 1920 Levaditi and Harvier 24 claimed that they confirmed the experiments of Strauss, Hirschfeld and Loewe, both in monkeys, and in addition assert the susceptibility of guinea pigs. In a later publication of Loewe and Strauss25 they state that the lesions produced in the brains of such experimental animals are similar to those seen in human cases, that is, showing mononuclear perivascular infiltration, small hemorrhages and general congestion, and they add experiments in which they succeeded in transferring the disease with spinal fluid and blood, as well as with material preserved in 50 per cent glycerine for many months. By means of the Noguchi anaerobic tissue-acetic-fluid method, they report the cultivation of minute filtrable coccoid bodies, virtually iden- tical with the globoid bodies described by Noguchi for poliomyelitis. It is not possible at the present time to make definite statements concerning the reliability of these claims. Other observers of great experience with a large amount of material have failed to obtain similar results. The confirmation by Levaditi and more recently by Inmann, of Strauss and Loewe 's experiments, however, would en- courage the hope that they are right. Moreover, the similarity of the disease to poliomyelitis and the general similarity of pathological lesions would incline one to assume the disease to be probably due to a filtrable virus. "Strauss, Hirschfeld and Loewe, N. Y. Med. Jour., 1919, 772; Jour. Infec. Dis., 25, 1919, 378. 2< Levaditi and Earvicr, Compt. Rend, de la Soc. Biol., 83, 1920, 354. 26 Loewe and Strauss, Jour. Infec. Dis., 27, 1920, 250. CHAPTER XL VII MEASLES, SCARLET FEVER, MUMPS, DENGUE FEVER, FOOT AND MOUTH DISEASE MEASLES THE causative agent of measles is unknown to the present day, and it would be a thankless task to review the literature of the many attempts to isolate microorganisms from this disease, none of which has resulted in throwing any light on the etiology. Attempts to produce the disease experimentally have frequently been made, the earliest recorded being those of Home of Edinburgh, published in 1759. 1 Home took blood from the arms of patients afflicted with measles, caught it upon cotton, and inoculated normal indi- viduals by placing this blood-stained cotton on wounds made in the arm. Home claimed that in this way he produces measles of a modified and milder type in fifteen individuals. Home's results, however, while at first accepted, were assailed by many writers and it is by no means certain that the disease produced by him was really measles. A number of other observers after Home attempted experimental inoculation of this disease, and positive results were reported by Stewart of Rhode Island (1799), Speranza of Mantua (1822), Katowa of Hungary (1842), and McGirr of Chicago (1850). The experiments of all these early writers, however, are unsatis- factory, owing to the necessarily unreliable technique of their methods. In 1905 Hektoen2 succeeded in experimentally producing the dis- ease in two medical students by subcutaneous injection of blood taken from measles patients at the height of the disease (fourth day). The experiments were carefully carried out and the symptoms in the sub- jects were unquestionable. They demonstrated that the virus of the disease is present in the blood. Attempts at cultivation carried out with the same blood were entirely negative. It was also 1 Home, "Medical Facts and Experiments," Edinburgh, 1759. 2 Hektoen, Jour. Inf. Dis., ii, 1905. 922 MEASLES, SCARLET FEVER, MUMPS, DENGUE FEVER, ETC. 923 shown by Hektoen's experiments that the virus of measles may be kept alive for at least twenty-four hours when mixed with ascitic broth. Similar experiments were recently carried out by Sellards, both on monkeys and on eight volunteers, but entirely without success. More important than the blood transfer experiments from the point of view of transmission are experiments in which inoculation has been attempted with secretions from the nose and throat. In 1852 Mayer reported the successful inoculation of human beings with mucus from the noses and throats of early measles cases, but complete failure in similar attempts at transfer with skin desquamations following the rash. Anderson and Goldberger3 claimed in 1911 that they were able to produce temperature reactions and mild skin changes in monkeys by the injection of nasal and pharyngeal secretions from early cases. This work has been recently elaborated and brought to more convincing conclusions by Blake and Trask.4 These writers inoculated monkeys (Macacus Rhesus) intratracheally with filtered and unfiltered wash- ings from patients in the early eruptive stages of measles and showed that the lesion which developed in the skin and buccal mucous mem- brane during the course of the monkey infection was histologically almost identical with that found in human measles. They successfully transmitted the infection from monkey to monkey and demonstrated that one attack of experimental measles conferred immunity upon the monkeys. Epidemiology and Prevention. — There are few infectious diseases as common as measles. Crum 5 has collected statistics which show that measles is responsible for about 1 per cent, of all deaths occurring in the temperate zones. In statistical summaries of 22 countries ex- tending over a period of four years preceding 1910, there were over 366,000 deaths attributable to measles of an aggregate population of 32,625,651. All races and ages seem to be susceptible, though children are more often infected, and the discrepancy between adults and chil- dren is probably due merely to the fact that most adults have had the disease before they attain adult life. Whenever young adults from rural districts come together in camps, epidemics will occur quite com- parable and more severe than those occurring among school children and asylum children at an earlier period of life. The disease is com- 8 Anderson and Goldberger, Jour. A. M. A., 57, 1911, 1612. 4 Blake and Trask, Jour. Exper. Med., 33, 1921, 385, 413 and 621. 5 ('mm, Ainer. Jour. Pub. Health, 4, 1914, 289. 924 DISEASES CAUSED BY FILTRABLE VIRUS rnon all over the world and not apparently influenced by climatic conditions. When it appears first among aboriginal populations, it sweeps through them with a violence unknown among more civilized nations with whom the disease has been endemic ,for centuries. Such was the great epidemic in the Fiji Islands in 1874, and similar epidemics have occurred in the South Sea Islands and among American Indians and the negro races. The disease occurs more commonly in cities than in rural districts. Susceptibility of previously uninfected individuals seems to be practically universal. Interesting in this connection are the statistics of concentration camps in the United States during the recent war such as those of Vaughan and Palmer6 made at Camp Wheeler. The population of this camp, like that of many others, was made up of young men from rural communities, many of whom had not had measles before. The sick rate week by week which followed the onset of the epidemic is tabulated by Vaughan and Palmer as follows : For Week Ending Annual Measles Morbidity Rate per 1000 October 19 83 October 26 428 November 2 615 November 9 1760 November 16 2200 November 23 1120 November 30 248 December 7 240 December 14 19 We may assume that the definite exposure to measles of an unin- fected human being will almost invariably result in an attack. Since the disease is probably communicated by the secretions of the nose and throat, reasonable exposure may be taken to imply crowding in sleeping quarters, contact in public vehicles, places of amusement, at meals, at play, in schools and in the ordinary indoor association of work and recreation. Whether or not the disease can be conveyed indirectly to any degree is not certain, but it is very '•Vaughan and Palmer, Jonr. Lab. and Clin. Med., 4, 1919, 647. MEASLES, SCARLET FEVER, MUMPS, DENGUE FEVER, ETC. 925 likely that infection from secretions on toys, food or other objects that are put into the mouth may take place, so long as the secretion is not dried. Judging from what we know or other filtrable virus, more- over, the virus of measles may offer considerable resistance even to drying. One of the most important epidemiological facts is the infectious- ness of the secretions in the early pre-emptive stages. According to Levy of Richmond, the disease may be infectious as long as 4 days before the rash appears and since at this time the patients are rarely very sick, this is the dangerous period for transmission. Uncomplicated measles in itself is not a very fatal disease, but, like influenza, measles seems to bring about a certain susceptibility to various respiratory infections, and measles epidemics are usually ac- companied by many fatal post-measles pneumonias. These pneu- monias may take the form of pneumococcus or streptococcus infec- tions, according to the nature of the most prevalent mouth and throat flora prevailing in the community. The conditions for a fatal measles epidemic, therefore, are fulfilled when measles breaks out in an indus- trial community, a camp, a school, etc., during the cold weather when coughs and colds prevail and when virulent pneumococci and strep- tococci are plentifully scattered about in mucus. The prevention of measles, in crowded communities or groups, is fraught with many difficulties. However, with vigilance and adequate discipline, much can be accomplished. In schools, industrial commu- nities and in military units, the most important procedure in our opinion is constant inspection and early segregation of all individuals with catarrhal colds. In the army it has been the practice of sani- tarians, a practice which we believe we have seen succeed to an un- expected degree, to inspect entire units once a day upon the appear- ance of a case of measles. The entire unit is made to pass an in- specting officer in single file in the morning, a few questions *as to general health are asked, the conjunctive and throats and the skin of the chest and arms are rapidly inspected, and individuals complaining of headache, a restless night, a cold or a cough, or those in whom the conjunctivas are inflamed, or the nose secreting, are made to step out and, on these, temperatures are taken. All those with a temperature of 100° or above are isolated and great care is taken to segregate catarrhal cases from the rest of the population. This method makes it possible to inspect a large group in a very short time and will accomplish far greater results than the mere isolation DISKA.sKS CAUSED liV FILTKABLK V1KIJB of individual suspicious eases which come to the sanitarian of their own accord. Munson 7 has given this method particular attention in the Jinny with astonishingly favorable results. Since the incnliation time of the disease is about two weeks, the exclusion of children from school need not exceed this period. SCARLET FEVER (Scarlatina) The etiology of scarlet fever, like that of measles, is still obscure. Streptococci have been found with striking regularity in the throats of scarlet-fever patients, and a large number of investigations have seemed to furnish evidence for the etiological relationship of these microorganisms with the disease. According to von I/mgelsheim, Crooke as early as 1885 demonstrated the presence of streptococci in the cadavers nf sea Net-fever victims. Uaginsky and Somrnerfeld * in 1900 examined a number of scarlatina cases with reference especially to streptocoecus infection, and reported the presence of streptococci in the heart's blood of eight patients who had died after a very acute and short illness. They expressed the belief that the acuteness of the illness and the rapidity of death in these cases precluded the possi- bility of the streptococci being merely secondary invaders. A large number of other observers have expressed similar opinions, but we can not, as yet, justly conclude that streptococci are actually the etiological agents of this disease. Class" in 1S!M» described a diploeoccus which he cultivated from a large number of scarlat ina patients and with which he was able to pro- duce exanthemata and acute fever in pigs. Subsequent investigations seem to show that (lass was really working with a streptococcus. IVroser,1" working in Kse.herieh's clinic, has recently reported the very favorable influence upon the course of scarlet fever of polyvalent streptococcus antisera. rrhis is not really very strong evidence in favor of the streptococcus etiology of the disease, since then1 is, of COUrse, no doubt that streptococcus infection complicates the disease, 7 Miuixnit, Military Sur^roii, 'Id, 1!H7, (](\(\. " lttifiintil,-ii :IM.| SiHiiHicrfrld, licrl. klin. Worli., I'.MK). 11 r/fi.v.s-. I'liilM. M...I. .lour., iii, 1S1MI. -. (|iifitif>. 13 Mallorii and Medlar, Jour. Mod. Res., 34, 1916. 14 LtiH(lst< -incr, Leraditi and /V DnttH T and llanford, Journ. Kxp. Mod. Vol. 17. ItM.'i. p. f>17. 111 />(/ of an organism which agglutinated in the serum of the patient in a dilution of 1 :200. The organism apparently belonged to the proteus type and was designated by them as " Proteus X2. " Further study with it showed that similar cases also agglutinated this organism. Later another bacillus X19, very similar to the first one, was isolated from another case. The organism is a Gram-negative, motile bacillus which ferments glucose and lactose, coagulates milk with acid formation, liquefies gelatin and in colony appearance resembles the proteus group. Bengston29 of the United States Public Health Service has studied the two organisms ("X2" and "X19" of Weil and Felix) bac- teriologically, and has compared them to laboratory cultures of proteus. She found that they were very slow gelatin liquefiers, that they were extremely slow in digesting coagulated blood serum, but that they did not ferment lactose. In this, the organisms of Weil and Felix which she studied resembled one reported by Fair- ley.30 Agglutination reactions, against proteus sera produced with other strains, were in some cases active against these strains, and conversely sera produced with the Weil-Felix strains were more active against some other proteus organisms. Apparently the agglutination of proteus "X" strains is of dis- tinct value in typhus diagnosis. Fairley observed positive agglutina- tion of these organisms in 97 per cent of the typhus sera which 28 Weil and Felix, Wien. klin. Woch., No. 2, 1916. 29 Bengston, Jour. Infec. Dis., 24, 1919, 428. 30 Fairley, Jour, of Hyg., 18, ]9]9, 203. 946 DISEASES CAUSED BY FILTRABLE VIRUS he examined. He never obtained agglutination with the blood of non-typhus cases, using dilutions of 1 :20 in all the tests. Weil and Felix found that almost all their clinically typical typhus cases agglutinated this organism, whereas very few sera of non-typhus patients and no normal sera showed agglutination in dilutions of 1 :25. Fairley repored that of thirty-five cases examined during the febrile stage, all agglutinated the organisms in dilutions of 1 :20 and 1 :1200 after the fifth day and throughout the second week. The test is carried out by growing the organisms on agar, sus- pending them in salt solution and testing with dilutions of 1 :25 and 1 :50 of the serum of suspected cases. The agglutination titer in true typhus cases may rise as high as 1 :200 or higher by the end of the second week. Bengston states that in one test made on a typhus case there was complete agglutination of the Weil-Felix organisms in diluton of 1 :400, while cultures of the Rawlings typhoid and of Proteus vulgaris were not agglutinated in dilutions of 1 :50. The explanation of this reaction is doubtful. It may be assumed quite definitely that this organism has no etiological relationship to typhus. It is possible that in typhus fever secondary, non-specific agglutinating antibodies for a variety of organisms may be present. We need only call attention to the antibody reactions carried out with the Plotz bacillus and with some of the other organisms for which etiological relationship has been claimed in this connection. Incidentally, the Weil-Felix reaction adds to our scepticism about Plotz 's claims. TRENCH FEVER (WOLHYNIAN FEVER) In 1919 there appeared among the Armies at the front a disease which did not clinically resemble the ordinary well-known febrile diseases. Cases of this condition were seen among British troops by Graham and Herringham31 and on the German front in Poland and Wolhynia similar ones were described by His32 and by WTerner.33 Apparently the disease had been noticed by Gratzer34 as early as 1914. Cases appeared in enormous numbers and because the disease 31 Graham, Lancet, 2, 1915, 703; Herringham, Lancet, 9, 1916, 429. 32 His, Berl. klin. Woch., 53, 1916, 738. 33 Werner, Munch. Med. Woch., 63, 402, 1916. »4 Gratzer, Wien. klin. Woch., 29, 295, 1916. TYPHUS FEVER, TRENCH FEVER, ETC. 947 seemed to arise almost entirely from the front areas was spoken of as Trench fever. The Disease. — Work on the clinical differentiation of the disease was done by a great many army surgeons. An accurate report was made by McNec, Brunt and Renshaw,;i5 by a number of German workers, and finally by a British and by an American commission, the American group organized under Strong, and including as clinician Homer Swift. In a Harvey Lecture by Swift36 printed in the Archives of Internal Medicine, July, 1920, an accurate summary of available facts concerning this disease to date may be found. The disease is sudden in onset with fever, headache and pains in the muscles. The onset resembles that of influenza. In a few days, pain and tenderness of the joints appears, and the temperature shows peculiar remissions which Swift characterizes as being of the " spiky" type. Characteristic of the disease are the bone pains which are not accompanied by any signs of inflammation. There may be continued hyperesthesia. There may be sensory disturbances with increase of the tendon reflexes. The fever curves are very irregular, some show- ing the intermittent "spiky" type referred to above, others develop- ing the typhoid-like ladder type. Another characteristic is the frequency of relapses, in which, after remissions of varying intervals, a second rise of temperature comes on. The relapses may come on after weeks or months. A case of which we have personal knowledge has developed two relapses in the course of two years after the original attack. In other cases Swift states that the manifestations may assume a subacute or chronic form with low grade fever which may continue for months. Transmission and Etiology. — McNee, Brunt and Renshaw, in 1916, succeeded in transferring the disease from man to man by intravenous and intramuscular injections of whole blood. In these early experiments they found that the plasma if entirely free from hemoglobin, was not infectious, but that the red cells contained the virus even after repeated washings. They did not succeed in passing the virus through a Berkefeld filter. In 1917, Werner, whom we quote from Swift, allowed' himself to be bitten by lice that had previously fed on trench fever patients, and is said to have con- KMcNee, Brunt and Kenshaw, Brit. Med. Jour., 1, 1916, 295. "Swift, Harvey Lecture, Harvey Soc., New York, Jan. 10, 1920. 948 DISEASES CAUSED BY FILTRABLE VIRUS traded a mild form of the disease. A similar observation was made by Kuczynski37 oil himself. Davies and Weldon38 in the same year carried out a similar experiment, allowing themselves to be bitten by lice immediately after the lice had fed on trench fever patients. One of them developed trench fever twelve days later. A similar experiment on a volunteer was successfully made by Pappenheimer and Mueller39 in 1917, but in criticising all these experiments Swift believes that the proof brought was not sufficiently conclusive be- cause of inadequate control. In 1918 two commissions were formed for the purpose of studying the disease. The American commission was aided by a British entomologist, Captain Peacock. The first result of these investigations was that McNee's observation about transmission with whole blood was confirmed, but it was found, in contrast to McNee's results, that the plasma as well as the red blood cells was infectious. It seemed that the blood taken as early as the fourth day of the disease was more infectious than that obtained later. Transmission after filtration through Berkefeld filters did not succeed at first, but Swift records that later, when infectious urine was used, filtration was successful. It was found at this time that, as well as the blood, the urine jf patients is also infectious. Careful experiments were made with lice, all of which were reared from eggs and fed on normal subjects, and the non-infectious- ness of these lice was proven by allowing them to feed on eleven different uninfected people. Such lice were allowed to feed several times on trench fever patients and subsequently allowed to feed on twenty-three volunteers, 78 per cent of whom developed trench fever. It did not seem necessary for the lice to be in contact with the skin while feeding, nor was it necessary to produce scarification. In two instances the mere bite of the louse through the meshes of gauze covering the box produced the disease. The incubation time in these louse transmitted cases, varied from fourteen to thirty- eight days, the average being twenty-one. Meanwhile, the British commission found that the excreta of infected lice applied to scari- fied skin could also produce the disease, thus showing that the 87 KuczynsTci, Eeported from Jungmann, Deut. med. Woeh., 64, 1917, 359. 38 Davies and Weldon, Lancet, 1, 1917, 183. "Pappenheimer and Mueller, Amer. Red Cross Committee Report, London, 1918, Oxford Press. TYPHUS FEVER, TRENCH FEVER, ETC. 949 excrement of lice that have bitten trench fever patients may be infectious when it comes in contact with lesions on the skin. Byam40 showed that as late as 300 to 400 days after the onset of the disease, trench fever patients can still infect lice. This is of great importance in appraising the epidemiological possibilities of carriers. Lice could also be infected by patients during the periods of remission. Both the British and the American commission showed that the virus is probably not transmitted through the eggs of the louse. The British commission reported that the headlouse can transmit the disease through its excreta in the same way as the body louse. Other insects, however, did not seem to carry the disease. As to the causative agent, little is definitely known. Toepfer,41 da Rocha-Lima42 and other German observers who have studied Rickettsia bodies in typhus fever were encouraged to undertake similar studies in connection with trench fever because of the similarity of the means of conveyance of the two diseases. These observers, as well as Jungmann43 succeeded in finding Rickettsia bodies in the intestines of lice fed on trench fever patients. Da Rocha4jima comparing lice that had bitten individuals who did not have trench fever with those fed on trench fever patients found that 72 per cent of the insects found on the trench fever patients showed Rickettsia bodies, but 20 per cent of those fed on normal people showed similar ones. Arkwright, Bacot and Duncan44 found« similar Rickettsia bodies in a large number of lice that had fed several times on sixty-four trench fever patients. They found the bodies in only one out of many lots of insects fed on normal people. Their experiments seem to indicate that when Rickettsia bodies appear in the excrement of lice after feeding, these excrements were infectious. It is quite clear that no positive conclusions can be drawn, especially in view of the frequent finding of Rickcttsia-like bodies in lice that have had no connection with this disease. The clue furnished by the finding of Rickettsia, however, must be followed, 40 Byam, et al., Trench Fev?r, Brit. War Office Commit. Eep., Oxford Press, 1919; Bycm, Proc. Roy. Soc. Med., 13, 1919, 19. 41 Toepfer, Munch, mod. Woch., 03, 1916, 1495. 42 da It ocha- Lima, Munch, mod. Woc-h., 04, 1917. "Jungmann, Dent, med. Woch., 64, 1917, 359. "Arkwright, Bacot and Duncan, Proc. Koy. Soc. Med., 13, 1919, 23. 950 DISEASES CAUSED BY F1LTRABLE VIRUS since the possibilities here are the only ones that seem to show great etiological promise at the present time. Prevention of trench fever, like the prevention of typhus, depends upon delousing. ROCKY MOUNTAIN SPOTTED FEVER Rocky Mountain Spotted Fever is a disease which has long existed in the United States. A thorough review of the entire subject has been made by Wolbach45 who states that authentic cases were reported as early as 1873. The disease has been pretty well limited to the mountainous regions, most of the cases being reported from Idaho and Montana. The Disease. — The onset of the disease is usually abrupt, though occasionally it may be preceded by a few days of general malaise. It not infrequently begins with a chill, followed by a rapid rise of temperature which reaches 102° or 104°. The temperature may show morning remissions with rises of one or two degrees in the evening, and gradually increasing, reaching its height during the second week. On the third or fourth day after the onset of the disease, a rash appears first on the wrists, ankles and back, later upon the arms, legs and chest, extending to the forehead and abdomen. It is always least marked on the abdomen, according to Wolbach. It comes out in the course of about thirty-six hours and may also involve the mucous membranes of the mouth and pharynx. The temperature remains up after the appearance of the rash! The rash consists of little red patches about 4 or 5 mm. in diameter which at first disappear on pressure. Like the typhus rash, they become darker red, then purplish and later hemorrhagic in character. Small petechial spots may appear in the center. They fade into pigmented spots later. During recovery desquamation occurs. There may be violent nervous symptoms. The blood picture is not altered materially, the leucocytes slightly increase in number, but the differential count remains approximately normal. The mortality of the disease as estimated from various sources by Wol bach for 1915 and 1916, ranged between 7 and 13 per cent. The incubation time seems to vary between three and nine days. 45 Wolbach, Jour. Med, Kes., 41, 1919, 1. TYPHUS FEVER, TRENCH FEVER, ETC. 951 Epidemiology. — The distribution of the disease follows that of the wood tick, Dermacentor venustus. The disease occury in Idaho, Montana, Nevada, Wyoming, California, Colorado and Washington. Wolbach notes that the distribution of cases in various states seems to be restricted to definite localities. In Idaho, he finds that the cases are particularly frequent in the Snake River Valley and in Montana in the Bitter Root Valley where there seem to be infectious foci. Seasonally the disease occurs almost entirely in the spring. The wood tick named above was associated with the disease first by Wilson and Chowning40 in 1902. RickeUs brought proof of this in 1906,47 showing that the disease could be produced in guinea-pigs by allowing wild ticks of this species to feed upon them. McClintic48 confirmed this and his investigations with those of Ricketts, cen- tralized attention upon this particular species, the Dermacentor venustus. Larvae, fed upon infected ticks, remain infective into the nymph stage and the nymph once infected remains infected into the adult stage. He also showed that eggs from infected females would -produce the disease when injected into guinea-pigs and that both male and female ticks would transmit it. Wolbach 's49 inves- tigations have confirmed most of these points. Guinea-pigs infected by ticks develop a temperature in about three to seven days. Injected with blood from other guinea-pigs, the disease may begin at the end of forty-eight hours. Death, which often follows, occurs on the sixth or seventh day. As the disease progresses there is swelling and reddening of the skin of the scrotum, loss of appetite and general signs of illness. There may be redness and swelling of the eye-lids, ears and paws, and ulcers of the paws may form. On autopsy, there may be edema and hemorrhages of the skin and subcutaneous tissues of the scrotum. Male guinea-pigs show the disease most characteristically because of the scrotal lesions. Rabbits are susceptible, although not regularly so. Foot50 has studied this under Wolbach 's direction. When it does occur in rabbits, the disease is virtually the same as that occurring in guinea-pigs, except that in addition to the other signs of illness, 49 Wilson and Chowning, Jour. A. M. A., 39, 1902. 47 Bicketts, Jour. A. M. A., 46, 1906. 48 McClintic, U. S. Pub. Health and Marino Hosp. Serv., Weekly Bulletin, . 20, 27, 1912. •"• Wolbach, Jour. Med. Research, 41, 1919-1920, 3. , Jour Med., Res., 39, 1919. 952 DISEASES CAUSED BY FTLTRABLE VIRUS the ears are often inflamed, and in white rabbits thrombosed vessels in the ears can be seen. Monkeys are susceptible arid usually die at the end of seven days. Etiology. — The lesions in the blood vessels in animals and man indicate the presence of the causative agent in these locations. Wol- bach49 who studied histological material from this point of view, found within the endothelial cells of the vascular lesions, in smooth muscle cells of the media, as well as occasionally in detached en- dothelial cells present in thrombi extremely minute small "diplo- coccmlike" organisms. These, he states, stain with eosin-methylene- blue. The organisms were best stained with Giemsa solution with which they appear as slender pale blue rods. There was a distinct contrast between the appearance of these organisms and that of accidentally introduced bacteria, in that the shape of the former was vaguely outlined and less sharp than that of the bacteria. Ricketts51 had described similar organisms in the blood of man and guinea-pigs which he described as "lanceolate chromatin-staining bodies" in sets of two, a small amount of eosin-staining substance appearing between the two individuals. The same organism was seen in smears of the intestinal contents of ticks. Wolbach has also seem them in smear preparations made from the eggs of infected ticks. In the case of these bodies, as well as in those described in connection with typhus fever, definite conclusions cannot be reached as yet. A careful analysis of the entire subject will be found in Wolbach 's paper of 1919. The organisms, if they are organisms, probably belong to the class of Rickettsia. They have not been cultivated. The prevention of the disease depends largely upon the preven- tion of tick bites and the suppression of the wood tick, a matter which is very difficult in the countries in which they abound. 51 Eicketts, Jour. A. M. A., 47, 1906, 33 ; Jour. A. M. A., 47, 1906, 358 ; Jour. A. M. A., 47, 1906, 1067; Jour. A. M. A., 49, 1907, 1278; Jour. Infec. Dis., 4, 1907, 141; Jour. A. M. A., 49, 1907, 24; Trans. Chicago Path. Soc., 1907; Jour. Infec. Dis., 5, 1908, 221; Jour. A. M. A., 52, 1909, 379; Medical Record, 76, 1909, 842. TYPHUS FEVER, TRENCH FEVER, ETC, 953 LICE AND DELOUSING The sanitation of typhus fever, of trench fever and of some forms of relapsing fever is so definitely dependent upon processes of louse extermination that a few paragraphs on the habits of lice and the means for their destruction will contain the most important prin- ciples upon which sanitary efforts in the prevention of these diseases must be based. The lice which infest the human body are of two types, the Pediculus humanus which includes the body louse and the head louse, and the Phthyrius pubis, or the pubic or crab louse. It is the first two of these, the body louse and the head louse with which sanitarians are most concerned. For an anatomical description of lice we must refer the reader to Nuttall's comprehensive Monograph in the British Journal of Hygiene of 1917, and to textbooks on medical entomology. The following facts, important for the sanitarian, are compiled from various sources. The ordinary life of a louse is about four to six weeks. The female louse begins to lay eggs about eight or nine days after hatching. It is stated that such lice, well fed, and in normal en- vironment, will lay altogether about 300 eggs at the rate of ten or so a day. It takes about one week to eight days for these eggs to hatch. On this basis, a single generation of lice takes about sixteen to eighteen days. The louse prefers to lay its eggs upon little threads or hairs, more readily upon rougher cloth than upon silk. It was suspected in the early part of the war that silk underclothing gave some protection, but apparently this is not of very much use. The body louse prefers to lay its eggs on the inner surfaces of underclothing and other clothing, preferably along the seams, and on blankets, though most of the louse eggs are probably laid on underclothing. It should not be forgotten that in arranging for disinfestation, the outer clothing, overcoats, blankets, etc., may also be infested. In addition to this, both the head louse and the body louse may lay their eggs on the hairs of the body. The louse, being an habitual parasite on animals and man, prefers to lay its eggs at a temperature little below that of the body, a temperature which is stated as ranging about 30° C. If the temperature is lower than that, it takes them two weeks or more to hatch. According to studies made by British Army sanitarians, they will not hatch below 22° C. and 954 DISEASES CAUSED BY FILTKABLE VIRUS the most favorable temperature for hatching is at 35° C., when the hatching time is very much speeded up and may be less than eight days. It is important to know that louse eggs are destroyed by temperatures slightly above 60° C. But it is not safe to rely upon such low temperatures for disinfestation. While the nits are quite susceptible to temperature, they are much harder to destroy by insecticides than are the adult lice. It is important to remember that many of the insecticide substances which are applied to the body and clothing for the prevention of lousiness may keep lice away but will not kill them when once present. It is also important to remember that, although the adult louse must feed with some regularity in order to thrive and lay its eggs, the eggs may remain alive on clothing, underwear, etc., for a month at least, away from the human body, and may be hatched out when this clothing is put on. Thus, clothing, underwear, blankets, etc., of louse infested dugouts, huts, ships, etc., must be taken care of even if it has not been worn for some time. Although the louse, like the bed bug in the song, has no wings * ' at all," and is not a wanderer, it is astonishing how easily it can pass from one individual to another. Lice may be easily acquired during the examination of a case, in passing through a crowd, or in handling underwear and clothing in laundry work or disinfesting operations. The adult louse feeds about twice a day, and the louse bites, while they may be quite annoying to some individuals, may cause practically no reaction or annoyance in habitually lousy persons. They are apt to leave the body of the sick and usually do leave the body of the dead as soon as possible. When removed from human sources of food, they may die in anywhere from one or two days to a week. Nine or ten days is stated as the probable limit to which the adult may live in clothing that has been hung up or put away. Delousing depends upon early discovery of lousiness in a com- munity, regiment or other unit, personal cleanliness, disinfestation of those who are lousy and disinfestation of clothing, blankets, etc., and quarters. In armies and in communities during the existence of louse-borne diseases, inspection for lousiness of bodies, underclothing, etc., should be carried out at frequent intervals. The individual who attempts to protect himself should inspect his own body and under- clothing on going to bed at night. TYPHUS FEVER, TRENCH FEVER, ETC. 955 The most efficient individual protection against louse infestation consists in frequent baths, preferably hot showers, in which a free use of soap is made, and all the hairy parts of the body very thoroughly soaped. The best type of soap is a soft soap. A British Army preparation which was very useful during the war was made by slowly warming three pounds of soft soap with one-half pint of water and, after removal from the fire, this was mixed with five and one-half pounds of crude paraffin oil. Two and one-half per cent cresol was added to this mixture. This formula is taken from D. G. M. S. Circular Memorandum, No. 16, of the British Army. After bathing, a complete change of underclothing should be made. In the American Army bathing establishments were arranged from ordinary Adrian huts, which were applicable to delousing on a large scale. Bath houses were so arranged that men undressed in an anteroom, tying up their outer clothing into bundles with tags attached and throwing their soiled underclothing into large wire baskets which were immediately taken to the steam sterilizers. They then passed into the shower rooms and came out into a dressing room, into a window of which the outer clothing after sterilization was returned to them, and into which from another window clean underclothing was passed. We have described the arrangement as used in the American Army in an article on army sanitation.52 For the purpose of keeping lice away from the body, naphthalin sprinkled through the underclothing probably has some effect. Kerosene or gasoline when applied to clothing in small quantities may keep insects from lodging in clothing. Various soaps and ointments have been made in which petroleum, kerosene or naphthalin have been used as ingredients, and these have been applied by smearing along the seams of the clothing, under the arm pits, etc. An excellent method for personal prophylaxis has been the spraying of crude creosote oil on the inner and outer clothing. This has been used successfully by Pappenheimer and Mueller. A good way of killing insects that may have wandered into the clothing during hospital or other duties in the course of the day is to drop the clothing into a dress suit bag or other fairly tight container and pouring in an ounce or two of chloroform, closing it for the night. This will not always kill nits. 62Zinsseiv Military Surgeon November, 1918. 956 DISEASES CAUSED BY FILTRABLE VIRUS Much might be written about the various substances that can be applied to the skin and clothing to keep insects away, but none of these means are infallable or sufficiently safe to be relied upon. The best possible method, after all, is the use of shower baths of hot water, the plentiful use of soap, combined with steam disin- festation of the clothing, and clipping of hair and beard, etc. Gaseous Disinfectants for Rooms, Clothing, etc. — It is an im- portant practical fact that formaldehyd, in spite of its powerful action upon bacteria, is a weak insecticide and cannot be relied upon to kill lice, mosquitoes, or fleas. Better than formaldehyd is S02 gas used in quantities of two to three pounds of sulphur per 1,000 cubic feet, with the simultaneous evaporation of water. (Clayton apparatus.) Hydrocyanic acid gas is also very efficient, but of course ex- tremely poisonous and dangerous unless used in a proper way. The most important facts concerning these gases, their generation and application, have been dealt with in a preceding section. For the wholesale disinfestation of clothing, fomites, etc., in connection with louse infested populations, clothing, blankets, etc., the experience of the late war has shown that the most practical systems are those depending upon the application of heat. Heat may be applied as dry heat or moist heat. Our own experience has taught us that the surest and most foolproof method of disinfesting large quantities of material during epidemics is by the use of large autoclave drums placed on trucks such as the Foden-Thresh autoclave lorries, which consist of large autoclaves with steam jackets so arranged that clothing, etc., can be exposed to steam under slight pressure (about five pounds), then the connection between the inner and outer jackets closed and the material dried in the same chamber before removal. The steam is supplied from the motor since such lorries are usually steam driven. In the field, dry heat chambers can be constructed consisting of well sealed huts within which small brick furnaces or tin stoves, with stovepipe arrangements surrounding the walls are used for heating purposes. These dry heat disinfestors are not as uniformly practical or foolproof as are the methods in which steam under pressure is applied, but it may be necessary to use them when other means are not available. Detailed descriptions cannot be given here, but a little ingenuity with attention to proper size in relation to heating apparatus, proper distribution of heat with tin pipes, and TYPHUS FEVER, TRENCH FEVER, ETC. 957 provision for the circulation of air by proper vent holes will yield good results. In the so-called " Canadian" type of hut the heat is applied from below by digging a hole in the floor of the hut which connects with the outside through a small tunnel, in which a furnace, constructed in a variety of ways, can be placed, and a glowing coal fire maintained. According to Bacot53 and others it has been found that nits protected by a single layer of khaki cloth are killed in fifteen minutes at 52° C. The heat of such huts must be carefully controlled, a matter which can be done either by thermometers or, as advised by Bacot, by hanging in various places small tubes containing paraffin or stearin, with a melting point of 60° or over. Excellent methods of applying the various forms of disinfestation by heat are those which were originated by Dr. Richard Strong in Serbia, in which disinfestation trains with a shower bath car, a steam sterilizing car made of a converted refrigerator truck, were drawn by an engine, which supplied the steam for the disinfestors, the hot water for the baths and the motor power. THE RICKETTSIA BODIES In 1910 during their work in Mexico, Ricketts and Wilder observed small ovoid bacterium-like bodies in the intestinal canals of lice which had fed on typhus cases. They described them as showing polar staining, with slightly stained or entirely unstained centers and as having the general appearance of very small 'bacilli. Similar observations were made by Prowazek and by Sergent, Foley and Vialatte in 1913, though the identity of the bodies seen by these observers with those of Ricketts and Wilder was not, at first clear. Most of the original observations were made on typhus material, but subsequently Wolbach saw similar appearances in the endothelial cells and vessel walls of animals infected with Rocky Mountain Spotted Fever which he believed to be probably identical with diplococcus-like structures described in the blood in the same disease by Ricketts a few years earlier. Still later bodies of the same general appearance were noticed in lice taken from Trench Fever cases and in lice collected from the bodies of normal human beings. The peculiar staining properties, frequently intracellular position, minute size and pleomorphic structure of these peculiar bodies suggested to many of these workers the possibility that they 63 Bacot, Brit. Med. Jour., 2, 1917, 151. 958 DISEASES CAUSED BY FILTRABLE VIRUS might represent a group of parasitic, and perhaps pathogenic, or- ganisms not hitherto observed. Von Prowazek did not believe these small bodies to be bacteria, and from the beginning took the position that they were more likely to belong to, or be closely related to the protozoa. Da Rocha-Lima, who has studied them particularly in their relationship to typhus fever, gave the appear- ances which he saw in typhus lice the name of Rickettsia prowazeki. In the course of numerous investigations upon the etiological signifi- cance of these peculiar appearances, especially in connection with Typhus fever, Rocky Mountain Spotted Fever, and Trench Fever, many workers have confirmed the observations of the earlier observers, and while it is quite impossible at the present time to classify them with any degree of certainty either with the bacteria or the protozoa, the various forms described possess sufficient similarity to each other to warrant the establishment of a tentative group. In describing them in a separate section we do not mean to imply that, at the present time, it is absolutely certain that they are parasites. But this seems so likely, and their etiological relationship to the diseases mentioned has been suggested by so many careful investigations, that clearness of treatment at the present time fully justifies such segregation into a separate group. The appearances which are classified together as Rickettsia are minute, ovoid or bacterium-like bodies. They are, as a rule, ex- tremely small, the smallest forms being more minute than the smallest known bacteria, measuring about 0.3 to 0.5 of a micron. Larger forms more bacillary in appearance, may be observed, and it is suggested that the Rickettsia bodies go through a develop- mental cycle. The small forms often appear in the "diplo" form and some German observers have described capsule-like halos around groups of two. They are all very difficult to stain. The ordinary aniline dyes stain them either very faintly or not at all. Prolonged staining with Giemsa gives them a faint reddish blue tinge. They do not retain the Gram stain. They are non-motile. Up to the present time none of the Rickettsia have been cul- tivated, with the exception of one form observed in the sheep louse which grows aerobically on glucose-blood-agar. All of them have an insect host which acts, in the case of the pathogenic Rickettsia, as the transmitting agent, TYPHUS FEVER, TRENCH FEVER, ETC, 959 According to experiments of Rickctts and Wilder, da Rocha- Lima, Sergent and his co-workers, some of the Rickettsia can pass into the egg of the louse and thus be inherited from one generation to the other. Most of them, unlike bacteria and more resembling protozoa, appear to enter the cells of the host as intracellular parasites. The sub-classification of the Rickettsia has been tentatively attempted by Wolbach by whose courtesy we are enabled to insert the following table : Insects Arachnida Acarina Mallophaga Corrodentia Hemiptera Diptera Siphonaptera Wulhaoh's Tentative Classification Melophagus ovinuft (sheep "louse" or "tick") Rickettsia melophagi Noller Psocus Sp.? (dust louse) Unnamed rickettsia . . . 1917 Sikora 1918 Pediculus humanus (human louse) Rickettsia prowazeki Hegler and yon Prowazek. . da Rocha-Lima Rickettsia (rocha-lima?) Weigl, oral statement Rickettsia pediculi Munk and da Rocha-Lima. Rickettsia quintana Munk and da Rocha-Lima. Rickettsia wolhynica Toepfer Cimex (Acanthia) lectularius (bed bug) Rickettsia lectularius . . . . Bacot . . 1914 1916 1920 1917 1917 1916 1921 Culex pipiens (Mosquito, Europe) Unnamed rickettsia Noller, quoted by Sikora. . . 1920 ( Ctenocephalus felis (cat flea) Rickettsia ctenocephali Sikora 1918 Cfenopsylla musculi (mouse flea) Unnamed rickettsia ... . . Sikora . . 1918 Dermacentor venustus (wood tick, U. S.) Dermacentroxenus rickettsi . . .Ricketts... . 1909 Leplus (Tromibidium) akamushi (harvest mite, Japan) Unverified quotation by Sikora Dermanyssu* Sv.f (bird mite, Europe) Unnamed, Noller; quoted by Sikora 1920 1920 In proposing this classification, however, it should be said in justice to Wolbach that he introduces it by stating definitely that a reliable classification of the Rickettsia is impossible at the present time, and that he believes that there have already been included under this heading a number of unrelated forms. He states that the Rickettsia of the sheep louse has little to distinguish it from bacteria and that the Rickettsia seen in connection with typhus fever has peculiarities which separate it from others. The Rickettsia studied by him in connection with Rocky Mountain Spotted Fever resembles somewhat the Rickettsia-prowazeki seen in typhus, and both of them are quite unlike the "morphologically simple " one observed in connection with trench fever. Wolbach summarizes his reasons for constructing a table of classification by saying that he believes it warranted since "they are forms of microorganisms 000 DISEASES CAUSED BY FILTRABLE VIRUS primarily adapted to insect tissues, with occasional representatives pathogenic for animals." For more detailed analysis of the Rickettsia, we refer the reader to the Harvey Lecture and to the articles on typhus investigations in Poland now being prepared for publication by Wolbach, Todd and their associates. Abstracting from the further analysis of the Rickettsia sent us with this table by Wolbach, we may mention the following points. The R. melophagia is not pathogenic and is the only one that has been cultivated upon glucose-blood-agar. The R. cprrodentia of the dust louse is, likewise, not associated with any mammalian host. It lives extracellularly in the stomach of the louse and is apparently non-pathogenic. The R. pediculi quintana and wolhynica are prob- ably identical, according to Wolbach and Todd. They are more uniform in morphology than the typhus one and are easier to stain. They occur extracellularly in the louse's stomach, adhere to the cuticular epithelium, and may invade the epithelial cells. They are transmitted to the egg. Their etiological association with trench fever has been suggested. The R. prowazeki is pleomorphic and seems to be exclusively intracellular in the louse. It seems to be more susceptible to drying and to heat than the preceding ones. It is the one studied by da Rocha-Lima and others connection with typhus fever. The R. lectularius of the bed bug is non-pathogenic, but mor-. phologically resembles R. prowazeki. The ones occurring in mosquitoes and in cat and mouse fleas are non-pathogenic. The Dermacentroxenus rickettsi is the Rickettsia body which has been associated by a number of writers with Rocky Mountain Spotted Fever. Wolbach includes it in the general classification, though he has found many differences between it and the other Rickettsia. Wolbach states that it is less bacterium-like than any of the other Rickettsia and many forms show red and blue staining materials. Unlike the Prowazeki, it does not show the thread-like forms. In the louse he states that the Prowazeki continues to mul- tiply in the gastric epithelium and eventually causes the death of the louse by interfering with digestion. The Dermacentroxenus, however, after multiplying within the nucleus chiefly, floods all the tissues of the tick and then diminishes in numbers, leaving behind in the salivary gland and some other tissues, forms which Wolbach regards as a resistant stage. SECTION V* THE HIGHER BACTERIA, MOLDS AND FUNGI CHAPTER XLIX THE HIGHEB BACTEKIA ( Chlamydobacteriaceoe, TricJiomycetes, Microsiphonales) STANDING midway between the true bacteria and the more complex molds, there are a number of pathogenic microorganisms which offer great difficulties to classification. These forms resemble the hyphomy- cetes in the gross appearance of the cultures, which are dry, tough, wrinkled and sometimes covered with a down of aerial outgrowths. Morphologically they are made up of filaments which often show at the ends chains of round bodies analogous to arthrospores. In the size and structure of their component cells they are, however, far more like the bacteria. The component cells of the filaments are usually about 0.3 micron and rarely more than 1 micron in diameter. They frequently stain unevenly but show no definite nuclei and the round spore-like cells are about the size of micrococci. In the classification of Migula most of these forms have been placed in a rather hetero- geneous group, the Chlamydobacteriaceae. By other authors, notably Lachner-Sandoval,1 Berestnew,2 and by Petruschky,3 the close relation- ship of these forms to the higher hyphomycetes has been emphasized and they have been grouped as a subdivision of the true fungi under the family name of Trichomycetes. * For a careful revision of this Section we are indebted to Dr. J. Gardner Hopkins. * Lachner-Sandoval, "Ueber Strahlenpilze. " Diss. Strassburg, 1898. 2 Berestnew, Eef. Cent, f. Bakt., xxiv, 1898. 'Petruschky, in Kolle und Wassermann, "Handbuch, " etc. 961 962 THE HIGHER BACTERIA, MOLDS AND FUNGI Petruschky proposes the following clear schematization, which, even though possibly defective from a purely botanical point of view, is at least serviceable for the purposes of the bacteriologist. Hyphomycetes True molds Trichomvcetes r 1 Leptothrix Cladothrix Streptothrix Actinomyces Leptofhrix is used to designate those forms which appear as simple threads without branching. Cladothrix is a thread-like form in which false branching may be recognized. By false branching is meant an appearance resulting from the fragmentation of threads. The terminal cell breaks away from the main stem, is set at an angle by the elongation of the thread FIG. 101. — CLADOTHRIX, SHOWING FALSE BRANCHING. itself, and, as both continue dividing, the simulation of true branching is produced. Streptofkrix denotes forms with numerous true branches and spores which usually appear in chains. Actinomyces is of more complicated structure, characterized by the formation of club-shaped ends and the stellate arrangement of its threads. Concerning the use of the last three generic names there lias been much controversy, which has recently been discussed with a full THE H1GHE11 BACTERIA 963 bibliography by Breed and Conn (J. Bacteriol., 1919, iv, 585). The outcome of the matter seems to be that the genus leptothrix may stand as representing filamentous forms without branching, of which our knowledge is very incomplete, and that there are two distinct groups of pathogenic species — Nocardice, which are aerobic, and the actimo- myces, which are anaerobic. The former, at least, includes a large number of related species. FIG. 102. — STREPTOTHRIX, SHOWING TRUE BRANCHING. LEPTOTHRIX Members of the leptothrix group have been observed in connection with inflammations of the mouth and pharynx by Frankel,* Michelson,5 Epstein,6 and others. In many of these cases the organism was identi- fied by morphology chiefly, pure cultures not having been obtained. The disease in none of these cases was accompanied by severe systemic symptoms and it is likely that when found in human beings the organisms may be regarded simply as comparatively harmless sapro- phytes appearing in connection with some other specific inflammation. Cultivation of the Leptothrices is not easy and has been successful only in the hands of Vignal7 and Arustamoff.8 4 Frankel, Eulenburg's ' ' Kealencycl. <1. gesam. Heilkunde, " 1882. 5 Michelson, Berl. kliu. Woch., ix, 1889. 6 Epstein, Prag. med. Woch., 1900. 7 Vignal, Ann. do phys., viii, 1886. 8 Arustamoff, Quoted from Petruschky, loc. cit. 904 THE HIGHER BACTERIA, MOLDS AND FUNGI NOCARDIA Streptothrix, Cladothrix, Oospora, Discomyces. — This genus in- cludes a large group of aerobic organisms which grow in branch- ing filaments made up of bacteria-like units. English medical writers more frequently refer to them as streptothrices, but, as the name Streptothrix is applied to a group of common saprophytjc fungi with coarse filaments, it cannot be properly used for these organisms. Saprophytic varieties of nocardia are numerous and pathogenic strains have also been reported as the cause of varied infections in man and animals. Nocard9 described a member of this group as the etiological factor in a glanders-like disease, "farcin du boeuf," occurring in Guadeloupe, which he called actinomyces farcinica. The first human case was that of Eppinger,10 who cultivated from a brain abscess an organism which he called Cladothrix asteroides on account of the star-like appearance of the young colonies on agar. He also found the organisms in sections of the bronchial lymph-nodes and believed the invasion had occurred through the respiratory tract. Since then a number of fatal systemic infections due to similar organisms have been described by Petruschky,11 Berestnew,12 Flexner,13 MacCallum,14 Norris and Larkin15 and others, and an apparently identical organism was isolated by Musgrave and Clegg in a case of Madura foot. A summary of the various cases up to 1921 has been made by Henrici and Gardner (J. Infect. Dis., 1921, xxviii, 232). In most of these cases the portal of entry was the respiratory tract, but a few began as wound infections. Strains from various cases have been considered by some to be identical, by others to represent a number of closely related species. Morphology. — Morphologically the nocardice show considerable variation. In material from infectious lesions they have most often appeared as rods and filaments with well-marked branching. Occa- sionally the filaments are long and intertwined, and branches have 9 Nocard, Ann. de 1'inst. Pasteur, ii, 1888. 10 Eppinger, Wien. klin. Woch,, 1890. 11 Petruschky, Verhandl. <1. Kongr. f. iiniere Mediz., 1898. "Berestneff, Zeit. f. Hyg., xxix, 1898. 13 Flexner, Jour. Exp. Med., iii, 1896. 14 MacCallum, W. E., Centralbl. f. Bakt., I, O, 1902, xxxi, 529. 15 Norris and Larkin, Proc. of N. Y. Path. Soc., March, 1899. THE HIGHER BACTERIA 965 shown bulbous or club-shaped ends. In Norris and Larkin's case, the young cultures in the first generations seem to have consisted chiefly of rod-shaped forms not unlike bacilli of the diphtheria group, showing marked metachromatism when stained with Loeffler's methylene-blue. They are easily stained with this dye or with aqueous fuchsin. Many strains are acid-fast, but decolorize some- what more readily than do tubercle bacilli. In tissue sections they may be demonstrated by the Gram-Weigert method. Cultivation. — The organism develops slowly on ordinary agar or gelatin plates, forming visible colonies in from two to five days. Later it forms a membrane somewhat adherent to the surface which soon becomes wrinkled. It is at first white but later turns yellow or even a brilliant orange. On broth they grow as a thick pellicle or, occasionally as a flocculent precipitate. Most strains have not liquefied gelatin or altered litmus milk, but liquefying strains have been described. All strains have proved highly virulent for guinea- pigs and somewhat less so for rabbits, producing in the animals lesions indistinguishable from tuberculosis. NOCARDLE IN RAT-BITE FEVER. — In the cases of fatal septicemia following rat bites, Schottmueller16 and Blake17 have recovered nocardias from the blood. It has since been shown, however, that this disease is due to infection by treponemata. Streptothrix of Rosenbach. — A species of nocardia undoubtedly different from the asteroides group has been described by Rosen- bach18 as the cause of an indolent dermatitis of the fingers and toes known as erysipeloid. ACTINOMYCES (Streptothrix Israeli, Kruse; Discomyces bovis, Brumpt ; Colmistrep- tofkrix Israeli, Pinoy) Among the diseases caused by the Trichomycetes or higher bac- teria, the most important is actinomycosis. Occurring chiefly in some of the domestic animals, notably in cattle, the disease is observed in man with sufficient frequency to make it of great clinical importance. In cattle the specific microorganism which gives rise "Schottmueller, Dermat. Wchnschrft., 1914, LVIII, Sup. 77. "Blake, F. G., Jour. Exp. Med., 1916, XXIII, 39. 18 Rosenbach, Arch. f. klin. Chirurg. 1887, xxiv, 346. 966 THE HIGHER BACTERIA, MOLDS AND FUNGI to the disease was first observed by Bellinger19 in 1877. In the following year Israel20 discovered a similar microorganism in human cases. The parasites appear in the pus from discharging lesions as small granular bodies, plainly visible to the naked eye and somewhat resembling sulphur granules, of a grayish or of a pale yellow color. In size they measure usually a frac- tion of a millimeter. Ordinarily they are soft and easily crushed under a cover-slip, but occasionally, especially in old lesions, they may be quite hard, owing to calcification. FIG. 103. — ACTINOMYCES GRANULE CRUSHED BENEATH A COVER-GLASS. Unstained. Low power. Shows radial striations. (After Wright and Brown.) FIG. 104. — ACTINOMYCES GRANULE CRUSHED BE- NEATH A COVER-GLASS. Unstained. The prepara- tion shows the margin of the granule and the "clubs." (After Wright and Brown) . Microscopically they are most easily recognized in fresh prepara- tions prepared by crushing the granules upon the slide under a cover-slip and examining them without staining. They may be rendered more clearly visible by the addition of a drop or two of 20 per cent potassium hydrate. When the granules are calcareous, the addition of a drop of concentrated acetic acid will facilitate ™ Bollinger, Deutsch. Zeit. f. Thiermed., iii, 1877. 20 Israel, Virch. Arch., 74, 1878 and 1879, LXXVIII, 421. THE HIGHER BACTERIA 967 examination. Fresh preparations may be examined after staining with Gram's stain. Observed under the microscope, the granules appear as rosette-like masses, the centers of which are quite opaque and dense, appearing to be made up of a closely meshed network of filaments. Around the margins there are found radially arranged striations which in many cases end in characteristically club-shaped bodies. Inside of the central network there are often seen coccoid or spore-like bodies which have been variously interpreted as spores, as degeneration products, and as separate, cocci fortuitously found in symbiosis with the actinomyces. Individually considered, the central filaments have approximately the thickness of an anthrax bacillus and are, according to Babes,21 composed of a sheath within FIG. 105. — BRANCHING FILAMENTS OF ACTINOMYCES. (After Wright and Brown.) which the protoplasm contains numerous and different sized granules. About the periphery of the granules the free ends of the filaments become gradually thickened to form the so-called actinomycosis ' ' clubs. ' ' These clubs, according to most observers, must be regarded as hyaline thickenings of the sheaths of the threads and are believed to represent a form of degeneration and not, as some of the earlier observers believed, organs of reproduction. They are homogeneous, and in the smaller and presumably younger granules are extremely fragile and soluble in water. In older lesions, especially in those of cattle, the clubs are more resistant and less easily destroyed. They appear only in, the parasites taken from active lesions in animals or man, or, as Wright22 has found, from, cultures to which 21 Bales, Yirch. Arch., 105, 1886. 22 J. H. Wright, Jour. Med. Res., 1905, vii, 349. 968 THE HIGHER BACTERIA, MOLDS AND FUNGI animal serum or whole blood has been added. In cultures from media to which no animal fluids have been added, such as glucose agar or gelatin, no clubs are found. In preparations stained by Gram's method the clubs give up the gentian-violet and take counter- stains, such as eosin. The coccus-like bodies found Occasionally lying between the fila- ments of the central mass, most observers now agree, do not represent anything comparable to the spores of the true hyphomy- cetes. In many cases they are unquestionably contaminating cocci ; in others again they may represent the results of degeneration of the threads. In tissue sections, the microorganisms may be demonstrated by Gram's method of staining or by a special method devised by Mallory.23 This is as follows for paraffin sections : 1. Stain in saturated aqueous eosin ten minutes. 2. Wash in water. 3. Anilin gentian-violet, five minutes. 4. Wash with normal salt solution. 5. Gram's iodin solution one minute. 6. Wash in water and blot. 7. Cover with anilin oil until section is clear. 8. Xylol, several changes. 9. Mount in balsam. Cultivation. — The isolation of actinomyces from lesions may be easy or difficult according to whether the pus is free from con- tamination or whether it contains large numbers of other bacteria. In the latter case it may be almost impossible to obtain cultures. The descriptions of methods of isolation and of cultural character- istics given by various writers have shown considerable differences. The most extensive cultural work has been done by Bostroem,24 Wolff and Israel, and by J. H. Wright, Bostroem has described his cultures as aerobic, but Wolff and Israel25 and Wright26 agree in finding that the microorganisms isolated by them from actinomycotic lesions grow but sparsely under aerobic conditions and favor an :3 Mallory, Method No. 1, Mallory and Wright, "Path. Technique/' Phila. 1908. 24 Bostroem, Beitr. z. path. Anat. u. z. allg. Path., ix, 1890. 25 Wolff und Israel, Virch. Arch., 1891, cxxvi, 4. M J. H. Wright, Jour. Mod. Res., viii, 1905. THE HIGHER BACTERIA 969 environment which is entirely free from oxygen, or at least contains it only in small quantities. The method for isolation recommended by Wright is, briefly, as follows: Pus is obtained, if possible, from a closed lesion and washed in sterile water or broth. The granules are then crushed between two sterile slides and examined for the presence of filaments. If these are present in reasonable abundance, the material is distributed in tubes of glucose agar, which are then allowed to solidify. If these first cultivations show a large number of contaminations, Wright recommends the preservation of other washed granules in test tubes for several weeks, -in the hope that contaminating microorganisms may thus be killed by drying before the actinomyces lose their viability. If cultivation is successful colonies will appear, after two to four days at 37.5° C., as minute white specks, which, in Wright's cultures, appeared most abundantly within a zone situated 5 to 10 millimeters below the surface of the medium. Above and below this zone they are less numerous, indicating that a small amount of oxygen furnishes the best cultural environment. Upon the surface of agar slants, growth, if it takes place at all, is not luxuriant. In alkaline meat-infusion broth growth takes place in the form of heavy, flocculent masses which appear at the bottom of the tubes. Surface growth and clouding do not take place. Milk and potato have been used as culture media but are not particularly favorable. Pathogenicity. — As stated above, actinomycosis occurs spon- taneously most frequently among cattle and human beings. It may also occur in sheep, dogs, cats, and horses. Its locations of pre- dilection are the various parts adjacent to the mouth and pharynx. It occurs also, however, in the lungs, in the intestinal canal, and upon the skin. When occurring in its most frequent location, the lower jaw, the disease presents, at first, a hard nodular swelling which later becomes soft because of central necrosis. It often in- volves the bone, causing a rarefying osteitis. As the swellings break down, sinuses are formed from which the granular pus is discharged. The neighboring lymph nodes show painless, hard swellings. His- tologically, about the filamentous knobs or granules, there is a formation of epithelioid cells and a small round-cell infiltration. In older cases there may be an encapsulation in connective tissue and a calcification of the necrotic masses, leading to spontaneous cure. As a rule, this process is extremely chronic. Infection in the lungs 970 THE HIGHER BACTERIA, MOLDS AND FUNGI or in the intra-abdominal organs is, of course, far more serious. When death occurs acutely, it is often due to secondary infection. The disease is acquired probably by the agency of hay, straw, and grain. Berestnew27 has succeeded in isolating actinomyces from straw and hay which he covered with sterile water in a potato jar and placed in the incubator. After a few days small white specks looking like chalk powder appeared upon the stalks which upon .further cultivation developed a growth which he considered identical with the pathogenic species of the Bostroem type. Typical anaerobic actinomyces have never been isolated except from cases of the disease. Animal inoculation has given conflicting results. Bostroem with his aerobic cultures was unable to produce lesions. Israel and Wolff did, on the other hand, produce nodules resembling those seen in spontaneous infections with their anaerobic cultures, but the lesions were not progressive and healed spontaneously. Wright could produce lesions with anaerobic but never with aerobic strains. He concluded that the anaerobic organism was the true cause of ac- tinomyces, and that Bostroem was probably dealing with a con- tamination. On the other hand Pinoy, Castellani, Brumpt, and others reviewing the subject state that the disease actinomycosis, though usually produced by the anaerobic organism is in some cases caused by aerobic organisms belonging to the genus nocardia. Others have attempted to distinguish between strains by the presence or absence of clubs in the infected tissue, reserving the name actinomyces for those parasites which produce clubs and calling all others strepto- thrices or nocardice. This is an unsatisfactory criterion, however, as MacCallum was able to produce clubs by intraperitoneal injection of his strain which, in every other respect, was a typical nocardia asteroides. B. actinomycetum comitans. — As in other mycoses the isolation of the parasite in this disease is made difficult by the presence in the lesions of numerous bacteria which overgrow primary cultures. The contaminants are frequently pyogenic cocci and saprophytic intestinal bacilli. One unusual type of organism has, however, been found in these lesions with sufficient frequency to deserve mention. Wolf and Israel28 mention the presence in the granules of numerous 27 Berestnew, Bef. Cent. Bakt., 24, 1898. 28 Wolf, M., and Israel, J., loc. cit. THE HIGHER BACTERIA 971 pleomorphic bodies resembling micrococci. Similar organisms have been noted by Klingler-9 and recently in twenty-four cases by Cole- brook"0 011 whose paper the following description is based. In the granules they appear as minute Gram-negative cocco-bacilli fused together into sheets. In culture they grow aerobically or anaerob- ically as minute coherent colonies which on agar are smooth and glistening, in broth, starlike and frequently adherent to the sides of the tubes. They grow on simple media but quickly die out in culture. They form acid but no gas on sugars. Guinea-pigs and rabbits may be killed by large injections but no lesions resembling actinomycosis are produced. It is doubtful if these bacteria play an important role in spontaneous cases of the disease. Actinobacillosis. — A disease of cattle simulating actinomycosis, in which granules with rays occur in the exudates has been described in the Argentine by Ligniercs and Spitz and in England by Griffith.31 Definite filaments were not found in the lesions and cultures yielded a Gram-negative strepto-bacillus. One human case has been reported by Ravaut and Pinoy.32 MYCETOMA (MADURA FOOT) The disease known by this name is not unlike actinomycosis. It is more or less strictly limited to warmer climates and was first recognized as a clinical entity, in India, by Carter.33 Clinically it consists of a chronic productive inflammation most frequently attack- ing the foot, less often the hand, very infrequently other parts of the body. Nodular swellings occur, which break down in their centers, leading to the formation of abscesses, later of sinuses. Often the bones are involved and a progressive rarefying osteitis results. From the sinuses a purulent fluid exudes, in which are found char- acteristic granular bodies. These may be hard, brittle, and black, resembling grains of gunpowder, or may be grayish-white or yellow and soft and grumous. According to the appearance of these granules different varieties of the disease have been described as mycetoma *> Klingler, E., Centr. f. Bact., I, O; 1912, LXIL, 191. » Colebrook, L., Brit. Jour. Exp. Path., 1920, I, 197. 31 Griffith, F., Jour, of Hyg., 1916, XV, 195. *-Ravcmt and Pinoy, Presse Med., 1911, XIX, 49. 33 Carter on Mycetoma, etc., London, 1874. 972 THE HIGHER BACTERIA, MOLDS AND FUNGI with black, white, yellow, or red granules. These different varieties of the disease though clinically similar may apparently be produced by a large number of diffeernt parasites all belonging to the fungi or higher bacteria. We will mention below the few cases from which aspergilli and allied molds have been isolated. In the ma- jority of cases, organisms similar to the nocardia have been found. Brurnpt divides them into two genera: the madurella having septate mycelia and the discomyces which is without septa, but this distinc- tion is not generally recognized. For a discussion of the character- istics of the various strains one should refer to the works of Brumpt and of Castellani. The parasite of the commoner black variety which certainly seems to be a distinct disease has been carefully studied by Wright from whose description the following points are taken. The small, brittle granules observed under the microscope show a dark, almost opaque center along the edges of which, filaments, or hyphse, may be seen in a thickly matted mass. By crushing the granules under a cover-slip in a drop of sodium hypochlorite or of strong sodium hydrate, the black amorphous pigment is dissolved and the structural elements of the fungus may be observed. They seem to be composed of a dense meshwork of mycelial threads which are thick and often swollen, and show many branches. Transverse partitions are placed at short distances and the individual filaments may be very long. Spores were not observed by Wright. In a series of over fifty cultivations on artificial media from the original lesion, Wright obtained growth in a large percentage. In broth, he obtained at first a rapid growth of long hyphae which eventually formed a structure which he compares in appearance to a powder-puff. On agar, growth appeared within less than a week and spread over the surface of the medium as a thick meshwork of spreading hyphae of a grayish color. In old cultures black granules appeared among the mycelial meshes. On potato, he observed a dense velvety membrane, centrally of a pale brown, white at the periphery. Small brown droplets appeared on the growth in old cultures. Animal inoculation with this microorganism has so far been un- successful. CHAPTER L THE PATHOGENIC FUNGI1 THE earliest demonstrations of microorganisms as the causes of disease were the discovery of a fungus in the scutula of favus by Schocnlein in 1839 and Langenbeck's discovery of the thrush para- site in the same year. Later the work of Pasteur and his followers showed the far greater importance of bacteria as disease-producers and the study of these simpler forms has since been given greater attention. We must, however, briefly consider a group of diseases known as the mycoses which are due to infection by the fungi. In the broader sense the term fungi is used to include all thallophyta (plants without stems, roots, or leaves) which are devoid of chlorophyl or its analogues, and which consequently are limited to a saprophytic or parasitic existence. In this sense the fungi form a class of which the bacteria, or fungi which reproduce by simple fission, are the simplest types. In the narrower sense the term is applied only to forms which reproduce by means of spores. The cells of these latter organisms are somewhat larger than those of the bacteria and are usually enclosed in a well differentiated mem- brane. They contain, as a rule, a demonstrable nucleus, granules of various types and often vacuoles. Some fungi are unicellular, as are the bacteria, but most are made up of many cells which are interdependent and show some differentiation in form and function. Typical fungi are made up of cylindrical cells, joined into fila- ments, from which smaller rounded cells called spores are developed. From these two elements, filaments and spores, the fungi build up a structure that differs immensely in complexity in the different species. The unicellular types such as the common yeasts grow in easily dissociated masses like bacteria and each cell combines the functions of absorbing food-stuffs, of building them up into its own substance and of reproducing new individuals. In the molds, which represent simpler multicellular fungi, the filaments lie distinct in 1 This chapter has been rewritten for us by Dr. J. G. Hopkins. 973 974 THE HIGHER BACTERIA, MOLDS AND FUNGI a loose meshwork, without definite arrangement except that certain of them are thrust up vertically and develop spores. In the higher types, of which the mushrooms are familiar examples, certain fila- ments form a cobweb-like net-work which is spread through the soil on which they grow. These serve to absorb and pass on nourish- ment. They connect with other filaments which are welded together to form the firm umbrella-like structure which projects above the ground. This is covered with a tough protective membrane also made up of closely cohering filaments. Along the gills on the under side of the cap are rows of characteristic cells (basidia) on which the spores are born. These different portions are analogous in function to the roots, stems, bark, fruit and seeds of higher plants. The gross appearance, the microscopic structure and especially the type of spores produced are relied on for the identification and classification of the various species of fungi. Consequently, it will be necessary to define some of the morphological terms used before proceeding to a description of the different pathogenic types. MORPHOLOGICAL DEFINITIONS The Thallus. — The entire vegetative portion of a fungus is called the thallus; the individual filaments of which it is composed, hyphae. When the hyphae lie in a loose meshwork without definite arrangement, the mass is termed a mycelium and sometimes this term is applied to the entire thallus, even when it develops a characteristic morphology. In some species the hyphae are continuous tubes with multiple nuclei; in others they are divided by septa into chains of cylindrical cells. Hyphae which have special functions are often differentiated from the rest of the mycelium. In most of the pathogenic fungi the thallus is rudi- mentary and it is not necessary to discuss here the elaborate and somewhat confused terminology used in 'describing the more complex forms. In the species to be considered only the fertile hyphae, i.e., those that give rise to spores are differentiated. They are called sporophores or conidiopJiores, these terms being applied sometimes to one specialized cell, sometimes to a multicellular or branched filament or to a bundle of filaments. A small cell, or even a conical process from a cell, which serves as a point of attachment for spores is called a sterigma; flask-shaped cells of this type are called phialides. Spores. — The term spore in the stricter sense means a rounded reproduc- tive cell analogous in function to the multicellular seed of a higher plant. As a rule, a spore differs in form from the parent cell, and does not divide until it becomes separated from the thallus, then after a latent period it THE PATHOGENIC FUNGI 975 germinates and produces a new thallus. This meaning is quite different from that in which the word is used when describing bacteria. The same term is, however, often loosely used for any rounded cell of a fungus, whether it be a part of the body of the organism, or an encysted resting form, or a true reproductive type. In fact the function of a cell in these rudimentary plants is often hard to define, as one. which seems at first to be merely a component unit of the thallus may if it becomes detached reproduce an entire organism. We may, however, divide all these rounded cells into two classes: (a.) True spores, the sole function of which is reproduction; and (b) Vegeta- tive spores, remembering that the latter may also serve to reproduce the species. Spores differ in their mode of origin and in their arrangement on or within the thallus. Almost every species of fungus produces several types. There are two groups of reproductive spores, one sexually produced, the other asexually. Of the many names given to different forms we will attempt to define only those which it will be necessary to use in this chapter. It will be found that many of these are used in a somewhat varying sense by different writers. REPRODUCTIVE SPORES. — An Oospore is a sexual type produced by the fertilization of a female cell by a differentiated male cell. A Zygospore is a sexual type produced by the fusion of two undifferen- tiated cells. Conidium is a general term applied to all asexual reproductive spores. The term is by some writers restricted to exospores or those formed by a bud which protrudes, from the membrane of the parent cell. Endospore is a general term applied to any spore formed within the membrane of the parent cell. Ascospores are a special class of endospores which are formed in a membrane known as the ascus, the number of spores in the sack being limited to two, four, or eight, and constant for the particular species producing them. The parent cell from which ascospores are produced has originally two nuclei which fuse into one before again dividing to form the ascospores. This fusion is regarded by Dangeard as a rudimentary sexual process. It has also been shown by Harper and others that in certain species the parent cell is the result of the fertilization of a female cell by a differentiated male cell. This is not true of some of the simpler types such as yeasts. Basidiospores are exospores produced on a special type of sporophore, known as a basidium. The number of spores on a basidium is limited and constant for a given species. Like the ascus, the basidium has in certain species been found to have two nuclei which fuse before the spores are produced. VEGETATIVE SPORES. — Thallospore is a general term applied to cells morphologically resembling the above types which are essentially a part of the vegetative portion of the fungus, 976 THE HIGHER BACTERIA, MOLDS AND FUNGI Blastospores are thallospores which develop by budding from the end or side of the parent cell and which may in turn throw out another bud or a mycelial filament without becoming detached, and without any period of latency. The buds of yeast cells are familiar examples. Arthrospores are thallospores formed by the segmentation of a hypha into a chain of cells at first cubical and later rounded. Chlamydospores are single thalospores formed by the concentration of the protoplasm of a hypha into a swollen portion of the filament, the membrane of which becomes thickened. They are purely resting spores and are closely analogous in function to the spores of bacteria. They appear as roughly spherical thick-walled cells much greater in diameter than the hypha3 and are called intercalary if they develop in the course of a continuous filament ; terminal, if at the end of a long hypha or a short lateral branch. CLASSIFICATION The whole subject of the classification of the fungi is in confu- sion and the phylo genetic relationship of the various groups is obscure. Even the identification of a species is often difficult, partly because some simpler types show little that is distinctive in their structure but chiefly because their morphology varies greatly with changed environment. For example, some of the blastomyces which grow in the animal body as round cells reproducing by means of blastospores, when placed on artificial media develop a mycelium and conidia. Many of the higher forms, too, which are parasitic on plants assume on different hosts forms which bear no resemblance to each other. This pleomorphism has made the study of the fungi a difficult field. A species may be observed for years before it exhibits characteristics which show clearly its relationship to certain other species. The modes of spore production are the chief characteristics accord- ing to which the fungi are grouped — especially those modes which seem to represent a sexual process. The various species are classed as those which form oospores, or ascospores, etc. Many, however, produce only conidia and it is generally considered that they are degenerate types which have lost the power of even rudimentary sexual reproduction. Such varieties are often grouped together as the fungi imperfecti. As to relationships among these organisms, there is little agreement and almost every writer has brought forward a new grouping and a new classification. There are, however, certain large groups which are generally recognized, the main characteristics of which are shown in the following table : «J 'o 0! J2 I p p o 02 THE PATHOGENIC FUNGI 'O ^ pg cc i P>5 .a +» J 1 ^ 'o a S3 h ^ s o "oQ 1 o a o S 1 mold. Occasionally in human lesions. ie Yeasts and common | ^ 3n ^2 o "S c8 o> 2 o 02 ^ 3 1 «H O 0 I ^ fl C3 cetes and dermatoph; this group. 1 CO _^H ^ F-H s 1 8 a aj «r £> «H V ^« o 03 o 3 ft CO 3 ^ O . a ° 0> ^ o3 ^ o d CC a « S S o ^ 'eS £ PH A M §H 03 O s CO O S O •S * V ^>X 03 3 ;2 977 M ese Most of the fung s. man included. ^ a • — r g 2 « a c 5 §^ L-1 M I. Jl II 978 THE HIGHER BACTERIA, MOLDS AND FUNGI The Phycomycetes, Ascomycctes and Hyphomycetcs arc the only groups that need be considered here. The two former concern us chiefly because they are found so frequently as contaminants in bacterial cultures and will be briefly discussed. PHYCOMYCETES Members of the genus Mucor belonging to this order frequently appear in agar plates which have been opened. They develop as a mesh of delicate white filaments completely filling the plate and press- FIG. 106. — MUCOR MUCEDO. Single-celled mycelium with three hyphse and one developed sporangium. (After Kny, from Tnvel.) ing against the cover. The sporangia can be seen with the naked eye as black dots scattered through the growth. On opening such a plate the meshwork quickly collapses and forms a white speckled feltwork over the medium. Microscopically the mycelium consists of branching tubular fila- ments with or without septa. The usual mode of reproduction is by asexual spores. These develop in the tip of a hypha which enlarges to form a spherical or pear-shaped capsule, the sporangium. The septum 'which divides it from the supporting hypha bulges into the sporangium, forming the columella. Within the capsule innumerable THE PATHOGENIC FUNGI 979 sporangiospores develop which are freed by its rupture. Sexual reproduction, which is less frequent, consists in the fusion of the tips or lateral processes of two neighboring hyphae, which form a large spore covered with a warty membrane, known as a Zygospore. No exospores are formed by the mucors but chlamydospores are numerous. FIG. 107. — MUCOR MUCEDO. 1. Sporagium, c. columella, m. sporangium capsule sp. spores. 2. Columella, after bursting of sporangium. 3. Poorly developed sporangia. 4. Germinating spore. 5. Emptying of sporangium. Brefeld.) (After The common laboratory contaminants are Mucor mucedo a constant in- habitant of horse dung, and Mucor pusillus, which can usually be obtained by allowing a piece of moistened bread to stand in a covered Petri dish. Mucor corymbifer (Lichteimia corymbifera) differs from the preceding species in having pear-shaped instead of spherical sporangia born in loose clusters on hyphae which are not raised above the surface of the medium. This species is pathogenic for rabbits, and has been reported as the cause of inflammations of the auditory canal and of other infections in man. ASCOMYCETES In this group are included all fungi which form ascospores. The majority have a mycelium made up of septate filaments and repro- duce by means of conidia which are frequently born on characteristic 980 THE HIGHER BACTERIA, MOLDS AND FUNGI structures. In the lower types met with as laboratory contaminants ascospore formation is rarely observed. The Yeasts. — The simplest forms of ascomycetes are the exoasci of which the yeasts or saccharomycetes are familiar examples. These develop on laboratory media as moist masses of separate round or oval cells usually from 10 to 20 microns in diameter. These send out buds (blastospores) which gradually assume the size of the FIG. 108. — MUCOR MUCEDO. Formation of zygospore. 1. Two branches coalesc- ing. 2 and 3. Process of conjugation. 4. Ripe zygospore. 5. Germination of zygospore. 6 and 7. Mucor erectus. Azygo sporulation. No two branches meet, but form spores without conjugation. 8 and 9. Mucor tenuis. Azygo sporulation. The spores grow out from side branches without sexual union. (1-5 after Brefeld; 6-9 after Banier, from Tavel.) parent cells from which they quickly separate. When grown on a dry surface some of the cells divide into ascospores which remain for a while enclosed in the membrane of the parent cell OF ascus. Typical yeasts are unicellular but in some species the cells succes- sively produced by budding adhere to the parent cells and form mycelial filaments, which consist of chains of round or oval units. In other cases the individual cells of such a chain elongate and form a hypha with cylindrical elements. THE PATHOGENIC FUNGI 981 The yeasts have been studied most extensively in connection with fermen- tation and are industrially of great importance in the production of wine and beer. Much of our knowledge of the life-processes of bacteria is based on these early investigations of the yeasts. Although Schwann, as early as 1837, recognized the fact that many fermentations could not occur without the presence of yeast, it was not until considerably later that the study of such fermentations was put upon a scientific basis. The typical fermen- A FIG. 109. — YEAST CELLS. Young culture unstained. (After Zettnow.) tative action consists in the transformation of sugar into ethyl alcohol accord- ing to the following formula': C6H12O8 = 2 C2H5OH -f 2 CO2. The enzyme by which this fermentation is produced is known as "zymase," and is, according to Buchner, in most cases, an endo-enzyme which may be procured from the cell by expression in a hydraulic press. In addition to this, however, the various yeasts also produce other ferments by means of which they may split higher carbohydrates, such as saccharose, maltose, and even starch, and prepare them for action of the zymase. The manner in which this is accomplished, and the by-products which are formed during the process, vary among different species, and it is for this reason that the employment of pure cultures is of such great importance in the wine and beer industries where differences in flavor and other qualities may be directly dependent upon the particular species of yeast employed for the 2 Pasteur, Etudes sur la biere, Paris, 1876. 982 THE HIGHER BACTERIA, MOLDS AND FUNGI fermentation. It is due to the work chiefly of Pasteur12 and of Hansen3 that the beer and wine industries have been carried on along exact and scientific lines. Many observers include in the yeast family certain pathogenic fungi causing thrush and blastomycosis. These will be described in the section on the hyphomycetes. THE MOLDS. — The powdery molds which so frequently appear in Petri dish cultures and which even grow through cotton plugs and FIG. 110. — PENICILLIUM GLAU- FIG. 111. — ASPERGILLUS GLAUCUS. m. Mycelial threads, s. Sterigmata. r. Ascospore. p. Germinating cond'lum. A. Ascus. (After de Bary.) CUM. A. Showing septate mycelia. B . End of a hyp ha — branching into two coni- diophores, from which are given off the sterigmata. From the ends of these are developed the round conidia. (After Zopf .) invade tube cultures also belong to ihu ascomycetes. The two commonest genera have characteristic conidiophores which make their identification easy. 'Hansen, Prac, Studies in Fermentation, London, 1896, THK PATHOGENIC FUNGI 983 In the Penicillium glaucum (or crustaceum) the fertile hyphae show numerous branches toward their upper extremities and terminate in a radiating group of flask-shaped cells (phialides) from the tips of which chains of conidia develop — the structure somewhat resembling a broom. Typical cultures have a dusty green color. The Aspergilli are equally common and troublesome contaminants. They' appear on culture media as a white feltwork often thickly dotted with black points becoming in older cultures diffusely black or yellow or green. The conidia are born on hyphae which terminate in a large rounded head from whicli phialides project in all directions. From the tips of these extend chains of conidia, often so densely packed together that the supporting structure is hidden and the whole appears as a spherical mass of pigmented spores. These mold are active producers of oxalic and other organic acids. The Sterigmocystis differs from the aspergillus in that secondary phialides each bearing a chain of spores project from each primary phialide. Pathogenicity. — Many species of these molds are pathogenic for labora- tory animals if the spores are injected intravenously. A number of human infections have also been ascribed to them. So-called pulmonary asperigillosis is a condition clinically resembling tuberculosis, in which an aspergillus, usually the species fumigatus, is found abundantly in the sputum. In most cases the fungus merely invades tissue previously infected with tuberculosis, in other cases (notably in the infection which has been described in the pigeon feeders of Paris by Dieulafoy and by Chantemesse and Widal) the disease is apparently primary and spontaneous cure may result. Madura foot, a disease which will be referred to in discussing the actinomyces group, has been in certain cases attributed to infection with these molds. Pinta or Carate, a disease of tropical America characterized by super- ficial colored patches on the skin is thought to be caused in some cases by aspergilli, in others by a species of trichophyton. Aspergilli have also been reported as infecting the eye, nose, auditory canal and wounds in various regions. HYPHOMYCETES (Fungi imperfecti) Most of the fungi which have definitely been shown to be the cause of human disease are rudimentary types in which one can detect no distinctive reproductive processes. This makes impossible their classification in the well defined groups of fungi. The thallus is, as a rule, without characteristic structure and reproduction is by means of simple conidia. Certain types do show close analogy to saprophytic fungi with specialized spores. For example, some 084 THE HIGHER BACTERIA, MOLDS AND FUNGT strains of blastomycetes grow as a mass of round cells developing by means of blastospores, which indicates a close relationship to the true yeasts, and although ascospore formation has never been observed Brumpt and others group them among the ascomycetes. , Other strains producing the same type of disease are considered by all to be hyphomycetes. . As the botanical classification of these organisms is still the subject of controversy it has seemed clearer for the purposes of this chapter to arrange them according to the diseases which they produce. For discussion of their botanical relationships as well as for an account the numerous species which have been described, one is referred to the works of Brumpt,4 Castellanni,5 and Plant.8 Only the more important types can be mentioned here. BLASTOMYCOSIS Busse7 in 1894 reported a case of fatal, generalized yeast infec- tion beginning in an abscess of the tibia. Pus from the lesions contained numerous giant cells in which he observed round or oval, double-contoured bodies surrounded by a wide capsule. They varied in size from that of a red corpuscle to that of a liver cell. Many showed small cells projecting as buds from the larger parasites. The parasite grew readily on ordinary media in budding forms, often surrounded by a capsule as in the tissues. It was pathogenic for laboratory animals especially for white mice. In glucose solu- tions it produced carbon dioxide and alcohol. In 1896 Gilchrist8 described a similar organism isolated from a patient with a severe chronic cutaneous disease which he described as pseudo lupus vul- garis. This organism also was found in giant cells, was capsulated, and showed budding forms. In culture, however, it produced mycelia and conidia and did not ferment glucose. Oxalic acid crystals were seen in the cultures. In the same year Curtis9 in 4 Brumpt, Precis de Parisitologie, Masson et Cie, Paris. 6 Chalmers and Castellani, Handbook of Tropical Medicine. 6 Plant, H. C., Handb. d. Path. Microorg., Kolle u. Wassermann, vol. V. •'Busse, Centralbl. f. Bakteriol., I, 1894, xvi, 175. 8 Gilchrist, Bull. Johns Hopkins Hosp., 1896, vii. • Curtis, Ann. de 1'Inst. Pasteur, 1896, x. THE PATHOGENIC FUNGI 985 France isolated a similar parasite from a myxornatous tumor of the leg. Numerous cases of infection with yeast-like organisms have since been described. In a case reported by Zinsser10 the lesion was an abscess of the back involving the spine. The parasite corresponded morphologically to Busse's. Animal inoculation in rabbits and guinea-pigs proved positive and the organism seemed to show a selective preference for the lungs and spleen. In the lungs of the animals, especially, lesions were found with surprising regularity even when the inoculation was made intraperitoneally. Clinically there are two distinct types of this disease, the blasto- mycetic dermatitis of Gilchrist and the systemic blastomycosis. The former is by far the more common. Of the latter a collection of forty-seven cases was made in 1916. " The portal of entry in many of these has seemed to be the respiratory tract, the earlier lesions being in the lungs. In other cases the general infection has followed a cutaneous lesion. The skin and subcutaneous tissues show the most numerous secondary lesions but the bones, liver, spleen, kidney and brain have each been involved in several instances. BLASTOMYCES HOMINIS (Sacchnromyces hominis, Busse; Cryptococcus Gilchristi, Vuillemin; Zymonema Gilchristi, de Beurmann and Gougerot; Mycoderma dermatitis, Brumpt ; Oidium Hektoenii, Ricketts) The parasites causing this disease probably represent a group of allied organisms rather than an individual species. As seen in the tissues or exudates they have shown a marked similarity in all cases. In the abscesses of the generalized form they are very numerous, in the cutaneous form somewhat more difficult to find and are best sought in the small epidermal abscesses which border the lesion. In an unstained moist film of pus spread out between a cover-slip and slide they appear as round or occasionally oval highly refractive bodies containing granules of various sizes and often vacuoles and surrounded by a hyalin capsule. They vary greatly in diameter usually from 10 to 20 microns. Budding forms 10 Zinsser, Proc. New York Path. Soc., 1907, vii. 11 Wade and Bel, Arch. Int. Med., 1916, xviii, 103. 986 THE HIGHER BACTERIA, MOLDS AND FUNGI and pairs united in a figure eight can usually be found. They stand out more definitely if the pus is cleared by mixing with a 10 per cent potassium hydrate solution. They stain irregularly. As a rule with the stronger aniline dyes they are overstained so that the details are obscured. With hemotoxylin eosine the capsule usually remains unstained and the body takes a pale stain with deep blue granules but in some cells the capsule may stain deeply. Thionine, polychrome methylene blue, and Wright's blood stain have been recommended. In tissue sections stained with hemotoxylin and eosin, or better with thionine, or methylene blue the clean cut, circular parasites are easily recognized, lying, as a rule, within multinuclear giant cells. Isolation. — The isolation of the organism is rendered difficult by the frequent presence in the lesions of bacteria which develop more rapidly than the fungus. Gilchrist and Zinsser encountered Gram-positive cocci, others diphtheroid bacilli. No special methods for facilitating isolation have been devised but success will often attend painstaking and repeated plating of the cultures in high dilution. The most favorable medium is glucose agar and the organisms develop well at room or at incubator temperature. Cultural Characteristics. — On agar or glucose agar the colonies appear after two to four days as minute glistening white hemispherical spots which are not unlike colonies of staphylococcus albus. In older cultures the appear- ance of different strains shows marked variations, some remaining smooth and pasty, others changing to a tough wrinkled membrane firmly adherent to the agar, and still others becoming covered with white aerial hyphae. All become brown with age. In agar stab cultures the organisms show their preference for a well oxygenated environment by growing but slightly along the course of the stab and by heaping up a thick creamy layer on the surface of the medium. Most strains fail to liquefy gelatine. In broth cultures the medium remains clear, the organisms growing as a stringy sediment, as a pellicle, or as tufted masses in the depth of the medium. On blood serum, potato and bread growth is easily obtained. Some strains ferment carbohydrates but most do not. Morphology. — In freshly isolated cultures the growth consists of large round cells with blastospores like those seen in the tissues. Capsules are often formed. Most strains sooner or later develop coarse, irregular, branching mycelial filaments. These are divided by septa and produce chains of arthrospores and terminal or lateral conidia. Hamburger1'- in a careful culture study of four strains 12 Hamburger, Jour. Inf. Dis., 1907, IV, 201. THE PATHOGENIC FUNGI 987 from systemic infections was impressed with the effect of the tem- perature of incubation on the morphology of the organism. All of his strains grown in the incubator tended to multiply by budding, while at room temperature all were filamentous, and two produced aerial hyphae. Pathogenicity.— All strains which have been tested have proved pathogenic for laboratory animals. White mice seem the most susceptible. Classification. — Cultures from the various cases have shown little uni- formity. It is impossible to be certain that they represent distinct species as they are somewhat pleomorphic and change their morphology with varying cultural conditions and after long preservation on artificial media. The most serious attempt to systematize their varying char- acteristics is that of Ricketts.13 He considered the seventeen strains on which he based his report all to be closely related species of one genus. He described three main types under which in spite of minor differences he grouped the various strains. His classification, is not however, satisfactory. Stober14 has observed the three types of agar growth to occur successively in the same strain. A definite classification of these organisms is as yet impossible but there seem to be two types which the various strains more or less closely resemble. I. The fermenting type described by Busse and called Cryptococcus hominis by Vuillemin, Brumpt and Castellani. These organisms resemble the true yeasts in their morphology and zymogenic properties. II. The non-fermenting type described by Gilchrist and called Cryptococcus Gilchristi by Vuillemin, Cryptococcus dermatitidis by Castellani and Mycoderma dermatitis by Brumpt. These types usually produce mycelium and reproduce by means of thallospores and conidia. They are related to the sporothrices and trichophyta described below. There is no correlation between the type of organism and the type of lesion it produces. COOCIDIOIDAL GRANULOMA There is a group of cases resembling closely systemic blastomy- cosis, the first observation of which was that of Wernicke and Posadas.15 "Eicketts, J. Med. Res., 1901, vi, 373. 14 Stober, Arch. Int. Med., 1914, xiii, 509. 15 Wernicke , E., Centralbl. f. Bakteriol., I, 1892, xii, 859. 988 THE HIGHER BACTERIA, MOLDS AND FUNGI Their case presented numerous pea sized cutaneous tumors and clinically resembled mycosis fungoides. In the lesions they dis- covered intracellular spherical hyalin bodies which they thought to be protozoan cysts. These cysts were surrounded by hyalin capsules and some of the larger forms contained a great number of the daughter cysts. Later Rixford and Gilchrist16 reported a similar case and since then a number of other cases have been reported, most of them from the San Joaquin Valley, California. Ophuls described three clinical types: (1) with primary cutane- ous lesions, and later generalization; (2) with primary pulmonary lesions and later generalization but no skin lesions; (3) with primary pulmonary lesions and secondary subcutaneous lesions. The disease runs a more acute and severe course than blastomy- cosis and of twenty-four cases collected by MacNeal and Taylor, but two recovered. The cutaneous lesions consist of large rather painless granulomatous abscesses; there is usually marked lympha- denitis and the lungs, bones, liver, kidney, and meninges have been found to be involved. Histologically, the lesions both in man and in experimentally infected animals very closely resemble those of tuberculosis. COCCIDIOIDES IMMITIS (Oidium coccidioides, Ophuls ; Mycoderma Immite, Brumpt) The distinction between this parasite and that of blastomyces is recognized by Ophuls,17 Wolbach18 and MacNeal,19 but no typical cases have been described by Europeans. The parasite seen in the tissues resembles the blastomyces, but does not show buds and repro- duces by the formation of endopores. These appear as a mass of minute round bodies— each of which may be capsulated — within the membrane of the parent cell. The parasites vary greatly in size and some are much larger than those usually found in blastomycosis, reaching 50 microns in diameter. Cultures. — The colonies appear in surface plants in from two to seven days as small slightly raised disks distinctly penetrating the media. Older cultures become covered with a dusty white layer "Bixford and Gilchrist, Johns Hopkins Hosp. Reports, 1896, I. "Ophuls, W., J. Exper. M., 1905, vi, 443. 18 Wolbach, J. M. Res., 1904, N. S. viii, 53. "MacNeal, W. J., and Taylor, E. M., J. M. Res., 1914, xxv, 261. THE PATHOGENIC FUNGI 989 of aerial hypha. The cultures become brown with age. In broth they grow as a fluffy mass which sinks to the bottom. Sugars are not fermented. Gelatin is slowly liquefied and milk slowly pep- tonized. Morphology. — In culture the spherical bodies immediately throw out filaments 2 to 8 microns in diameter which are branched and septate. In older cultures they develop large chlamydospores which resemble the forms seen in culture and also conidia which are usually arranged in chains at the end of the hyphae. In anaerobic cultures in Noguchi's ascitic agar, rabbit kidney medium, MacNeal and Taylor observed the formation of endospores like those found in the lesions. The organism is pathogenic for dogs, rabbits and guinea-pigs. Cooke20 has studied the immune reactions in a human case of this disease and found precipitins which reacted up to a 1 :160 dilution of the serum against extracts of the cultures. The serum did not react with extracts of cultures of blastomyces. He was unable to obtain complement fixation or positive skin reactions with his extracts. t THRUSH Thrush is a localized disease of the mouth, occurring most fre- quently in children suffering from malnutrition, but also in cachectic adults. It is characterized by the development of creamy patches on an area of catarrhal inflammation, usually on the tongue. MONILIA ALBICANS (Oidium albicans, Robin; Saccharomyces albicans, Rees; Endomyces albicans, Vuillemin) The microorganism which causes thrush was first described by Langenbeck in 1839. It is found abundantly in the false membrane covering the lesion where it appears as a mass of simple or branched mycelial filaments made up of irregular units of about four by twenty microns. Oval cells of somewhat larger diameter are also found, attached to the ends of the filaments, or lying free and throwing out buds. 20 Cooke, J. V., Arch. Jjjt, MecL, 1915, xv, 479. 990 THE HIGHER BACTERIA, MOLDS AND FUNGI The parasite grows readily on all ordinary media. On agar it forms a creamy layer, in broth a flocculent deposit. It forms gas on certain sugars. Most strains do not liquefy gelatin or clot milk. The mor- phology of the organism has been most thoroughly studied by Roux and Linoissier. Commonly it develops as a mass of oval cells reproducing by budding but often, especially in fluid media forms filaments like those seen in the lesions, with terminal or lateral spores. Large globoid chlamy- FIG. 112. — THRUSH. Oidium albicans, unstained. (After Zettnow.) dospores at the end of short lateral branches are also found. These charac- teristics have led most authors to call the parasite a monilia. Monilia is a rather ill-defined genus of hyphomycetes in which are included yeast-like organisms which reproduce by blastospores but which also form filaments of short irregular units which are easily detached. The filaments give rise to large oval cells fre- quently appearing as short terminal chains which are called by some conidia, by others, blastospores. Castellani includes in this genus only organisms which ferment sugars with the formation of gas. Several authors have reported the presence of ascospores in cul- tures of the thrush fungus and have classed it with the saccharom- yces. Vuillemin found also endospores within the mycelial filaments. It seems probable that the disease is not always caused by the same organism. Fischer and Brebeck described two varieties, a large- spored type with endospores which liquefied gelatine, and a small- spored type, without endospores which did not liquefy. Castellani concludes that several species of moniliae and also members of other genera may cause thrush. On the other hand Fineman21 studied five strains from thrush and found them identical. They were however indistinguishable from eight strains found incidentally nFineman, B. C., Jtnir. Inf. Pis., 1921, XXVIII, 185. THE PATHOGENIC FUNGI 991 in throat cultures from diphtheria suspects. All produced acid and gas on dextrose, levulose and maltose ; acid only on galactose and saccharose; and failed to ferment lactose and mannite.22 SPRUE Sprue is an important disease of subtropical countries which is characterized by progressive wasting with profuse anemia and a white frothy diarrhea. There is an inflammation of the entire intes- tinal tract and the lesions on the tongue are frequently characteristic. MONILIA PSILOSIS Ashford23 has made an elaborate study of cases occurring in Porto Rico. He isolated from the tongue and stools of two hundred patients an organism which he calls monilia psilosis. This is ap- parently identical with the monilia enterica of Castellani. Ashford 's monilia is a large round organism 4 to 7 microns in diameter with a granular and usually vacuolated protoplasm. On Sabouraud's agar it grows as a faintly greenish creamy elevated mass with mycelium which usually penetrates the medium. In gelatin it invariably produces hyphse which spread laterally from the stab and give the growth an appearance which Ashford describes as that of an inverted Christmas tree. Monilia Psilosis produces acid and gas on dextrose, levulose, maltose and usually on saccharose but does not ferment lactose nor mannit. The fungus turns milk faintly alkaline without further change and does not liquefy gelatin. On passage through laboratory animals (rabbits and guinea- pigs) it produces a systemic mycosis and gradually increases in virulence. With these passage strains Ashford could produce stomatitis and diarrhea by feeding. The etiological relationship of moniliae to sprue is not univer- sally accepted. Castellani has isolated six different species from the stools typical cases. He believes that they are not the primary cause of the disease, though they may be responsible for the frothy diarrhea. - Eoux and Linoissier, Arch, de m&l. exp. et (Tanat. path. 1890, IT, 62; Compt. rend, de Tacad. d. sc., 1889, 109, 752. ~* Ashford, B. K., Am. J. Med. Sci., 1917, cliv, 159. 992 THE HIGHER BACTERIA, MOLDS AND FUNGI OTHER YEAST-LIKE PARASITES Other diseases caused by members of the yeast family have been reported by a number of observers. Tokishige24 found a very minute type in a skin disease, pseudo glanders, occurring among horses in Japan. Brumpt states that similar cases have been found in many countries and that the infection may attack man. According to Mesnil,25 the Jiistoplasma capsulatus of Darling,26 which he found in three cases of splenomegaly in Panama, is closely related to the above parasite and should be classed as a cryptococcus. The fact that blastomycetes have frequently been found in tumor tissue has led several Italian observers27 to assume an etiological relationship between these microorganisms and malignant growths. Absolutely no satisfactory evidence in favor of such a belief has been advanced, however, and the yeasts in these conditions must be regarded as purely fortuitous findings. In considering the possible origin of blastomycetic infections in animals and man, it is, of course, important that we should have some knowledge as to the pathogenic properties of yeasts met with and handled in daily life. Rabinowich28 has investigated the pathogenic properties of fifty different species of yeast and among them found only seven varieties that had any pathogenicity for rabbits, mice and guinea-pigs. In most of those successfully inoculated the disease produced in labora- tory animals had but very little resemblance to blastomycetic infec- tious conditions observed in man. SPOROTRICHOSIS Sporotrichosis is a chronic infection usually limited to the skin, the subcutaneous tissues, and lymphatics, occasionally involving the muscles, bones and joints — and exceptionally the viscera. The lesions resemble closely syphilitic gummata and in typical cases occur in 24 Tokishige, Centralbl. f . Bakteriol., 1896, i, 19. 25 Mesnil, Quoted by Brumpt, loc. cit. M Darling, S. T., J. Exper. Med., 1901, 11, 515. 27 San Felice, Centralbl. f . Bakteriol., I, 1902, xxxi, Ztschr. f . Hyg., 1895, xxi, 1895, xviii. ** Eabinowich, Ztschr. Hyg., 1895, xxi. THE PATHOGENIC FUNGI 993 a chain extending up the arm, connected by thickened lymphatic vessels. They slowly soften and ulcerate. The first cases described were those of Schenck29 and of Hektoen and Perkins30 in this country. It is a rare infection here, but twenty-eight cases being reported up to 1912.31 In France and Switzerland it is relatively common and our present knowledge of the disease is based chiefly on the work of de Beurmann and Gougerot.32 It may be caused by several species of hyphomycetes belonging to the genus sporotrichum. SPOBOTRICHUM It is difficult and often impossible to demonstrate the parasites by direct examination. When found in smears of the pus, or in sections of the lesions, they appear as oval or cigar-shaped cells varying in length from 10 to 2 and in breadth from 3 to 1 microns. These are frequently within large mononuclear phagocytes. The parasites may be demonstrated by clearing the pus with 40 per cent NaOH or by staining with thionine or other basic stains. They are Gram-positive. Cultural Characteristics. — As a rule the organism can be found only by making cultures from the pus or from the bloody fluid aspirated from firmer lesions. Tubes of Sabouraud's test medium or of 4 per cent glucose agar should be heavily inoculated on the surface and incubated at room temperature. Taylor33 recommends glycerine-glucose-agar to which acetic or citric acid (1-1500) is added. The colonies appear in four days or more as minute gray flecks, soon surrounded by a delicate fringe. The centers as they enlarge become raised and wrinkled and darken to a buff, or chocolate, or in some species to a black color. In flask cultures they may attain a diameter of 10 cm. and more. At the periphery is a smooth flat zone with delicate radiating outgrowths, which, if they reach the side of the tube, grow upward along- the dry glass. The surface is usually hard and glistening but in old cultures may show hairy or powdery outgrowths from the surface. The sporothrix grows on media of very simple composition but more 29 Schenck, B. R., J. Hopkins Hosp. Bull. 1898, ix, 286. 80 Hektoen, L., and Perkins, C. F., Jour. Exp. Med., 1900, V, 77. 81 Hamburger, W. W., Jour. Am. Med. Assn., 1912, LIX, 1590. (Bibliography.) 82 de Beurmann and Gougerot, Les Sporotrichoses, Felix Alcan.,, Paris, 1912. u Taylor, Kenneth, Jour. Am. Med. Assn., 1913, LX, 1142. 994 THE HIGHER BACTERIA, MOLDS AND FUNGI luxuriantly on those containing sugar. On glucose broth it forms a thick membrane. It liquefies glucose gelatine but does not clot or digest milk. On hexoses it forms lactic acid but does not ferment mannite or dulcite. Species vary in their action on disaccharides. Morphology. — This is best studied in hang drop cultures. The growth is made up of interlacing, branching septate hyphae. These are delicate (about 2 microns) and of uniform diameter. The spores FIG. 113. — SPOROTRICHUM BEURMANNI IN HANG DROP (Hopkins). are oval or pear-shaped (about 3 by 4 microns) and are found on any part of the hyphse to which they are usually attached by short sterigmata. In de Beurmann's species they are very numerous and form thick clusters at the tips of the hyphae or sheaths along their course. Chlamydospores are also found. Species. — Sporotrichum Schencki is the species found in most American cases. The cultures are white or slightly brownish. They ferment lactose but not saccharose. The spores are not numerous and have no sterigmata. Sporotrichum Beurrnanni is the commonest species in France. The cul- tures soon darken to a chocolate brown. They ferment saccharose but not THE PATHOGENIC FUNGI 995 lactose. Spores are very numerous and often provided with short sterigmata. Five other pathogenic varieties are listed by de Beurmann and there are many saprophytic species. Wolbach34 has described a species isolated from an arthritis of the knee which he named Sp. Councilmanni. Pathogenicity. — Spontaneous infections due to the sporotrichum Beurmanni have been observed in rats, dogs and horses. Experi- mentally cultures have been shown to be virulent for rats and other laboratory animals. The pathogenic species seem capable also of saprophytic existence. De Beurmann recovered his species from the normal throats of patients with sporotrichosis and of others who had recovered from the disease. Gougerot found in oat grains, and in other plants sporothrices which he regarded as identical with the human species and which were virulent for rats. Immunity. — Widal and Abrami35 found that suspensions of sporothrix spores were agglutinated by the sera of patients in dilutions 1-200 or above and have used this reaction in diagnosis. They also obtained fixation of complement. Others report that patients give skin reactions with culture extracts. In human cases there is no evidence of the development of an immunity but in animals successful immunization, both active and passive, has been reported. DISEASES OP THE RINGWORM GROUP There is a group of common and relatively trivial fungus infec- tions called dermatomycoses. In it are included a number of dis- eases clinically distinct, all of which are, however, characterized by the fact that the invasion of the parasite rarely penetrates deeper than the epidermis and its appendages — the hair and nails. The fungi causing these infections are known as dermatophytes. They are filamentous organisms resembling in structure the hyphomycetes, in which group they are usually placed. Brumpt, Castollani and others prefer to group them with the asco- mycetes. This decision is based partly on their general structural resem- blance to these higher fungi but chiefly ou the observation of Matruchot 31 irolbach, Sisson and Meier, Jour. Mecl. Res., 1917, CXXXVI, 337. 35 Quoted by de Beurmann and Gougerot, loc. cit. 996 THE HIGHER BACTERIA, MOLDS AND FUNGI and Dassonville of aseospore formation in Eidamella spinosa, a parasite isolated from a dermatomycosis of the dog. In no other member of the group, however, have ascospores been found. The fact is that ringworm-like diseases may be caused by various fungi which show little resemblance to each other and that the dermatophytes as a group are denned not by any common botanical characteristics but by the type of lesions they produce. Our knowledge of the dermatophytes dates from Schoenlein's discovery of the favus fungus in 1839. Two years later Gruby discovered the fungus of ringworm and distinguished between the large and small spored types. Since then many workers have con- tributed to our knowledge of these parasites but by far the most thorough and extended investigation has been made by Sabouraud.38 Cultural Characteristics. — The more important varieties of dermatophytes have been cultivated and show certain common char- acteristics. All of them grow as leathery masses of closely inter- woven hyphse. From a point of inoculation on solid media they spread out symmetrically over the surface at the same time sending down numerous short branches which penetrate the substrate and bind the growth firmly to it. As the membranous disc extends peripherally the central mycelium continues to grow increasing in thickness and forcing the less adherent portions upward. This produces on the surface a series of humps and ridges, with cor- responding hollows and grooves on the under side, which often form striking geometrical patterns. The surface may be smooth and hard but most species sooner or later develop a duvet — a covering of aerial hyphse which according to their length give the surface a powdery or velvety or hairy appearance. The majority develop a yellow or brown pigment and some are characterized by brilliant red and violet colors which appear late and are most marked in those portions of the membrane which are raised above the surface of the medium. The rapidity of growth varies greatly in the different species but their evolution is always a matter of weeks. Some varieties such as the microspora attain a diameter of ten centimeters or more. Others do not extend beyond one or two centimeters from the center, but may pile up a mass a centimeter in thickness. 86 Sabouraud, E., Les Teigncs, Masson et Cie., Paris, 1910. THE PATHOGENIC FUNGI 097 On broth they usually form a thick membrane which spreads over the surface, but if the fragment planted sinks it develops 'only a delicate mesh of filaments in the bottom. On potato the growth is less vigorous and often moist. Gelatin is liquefied by most strains. Glucose and mannite are usually completely consumed but no gas is formed and the medium is not acidified. Lactose, saccharose and maltose are less favorable to growth. Like many other fungi the dermatophytes grow well on the simpler synthetic media without peptone or protein, if glucose be added. The optimum reaction is somewhat more acid than that for bacteria, about PH 7.0; but they develop readily throughout a range of reaction from 4.0 to 8 and above. Although obligate aerobes, they will grow feebly with scanty oxygen supply as in the depth of a broth culture. They develop well at room temperature and most species also in the incubator. Morphology. — Morphologically as has been said there is little that characterizes the ringworm fungi as a group. In the infected hairs or skin, they appear as masses of spores or as filaments, the latter often consisting of chains of spore-like cells. In culture the growth is made up of branching hyphae which are always septate but may be coarse or delicate, straight or crooked, cylindrical or irregularly bulging. Under favorable conditions most species pro- duce conidia. Chlamydospores are far more common and arthro- spores are also found. From the common molds which they some- what'resemble these parasites are distinguished by the absence of ascospores or of the specialized conidiophores such as are found in the aspergilli and penicillii. These characteristics do not, how- ever, serve to distinguish them from other hyphomycetes. Individual species do develop in culture peculiar spores and mycelial structures which help to distinguish them. The following are some to which Sabouraud has given descriptive names : Morphological Definitions. — Clubs. — These are swollen mycelial tips which vary greatly in size. They are not markedly differentiated from the hyphae which bear them but when occurring in great numbers as in the achorion of favus they present a striking and characteristic appearance. Fuseaux. — These are large elongated chambered bodies considered as chlamydospores by some, by others as specialized conidia. Sabouraud applies this name to two different types, one a lenticular spore with a tapering pointed tip and thick doubly contoured wall, covered with warty or hairy outgrowths, the other a blunt club-shaped spore with thin smooth walls. Both are divided into segments by parallel transverse septa. Conidia. — These are irregular in shape, size, and arrangement. In the 998 THE HIGHER BACTERIA, MOLDS AND FUNGI microspora they are attached to the fertile hyphae by a flattened facet so that they resemble abortive branches (Aeladium type). In the tricophyta they are more frequently attached by a pointed tip (Botrytis type). Often they are scattered irregularly along the hyphae, but occasionally show a characteristic arrangement. Groups of conidia formed along the sides of an unbranched terminal hypha Sabouraud calls thyrses, larger groups born on branched conidiophore clusters (grappes}. Pectinate bodies are swollen and usually curved ends of hyphae which give off a row of abortive branches from one side — the structure vaguely resembling a comb. Spirals. — These are simply convoluted hyphae which may take all the forms of a tendril from a spirillum-like form to a close set coil. Nodular organs. — These are chains of large rounded cells knotted together into small dense masses suggesting the sclerotia of higher fungi. Pleomorphism. — The gross appearance and microscopic structure of the dermatophytes varies greatly with the cultural conditions. After a period of growth on sugar containing media they seem to undergo a permanent change, ceasing to produce conidia and developing a thick covering of very long aerial hyphae. This gives to these altered cultures a smooth white downy appearance. Sabouraud refers to cultures of this type as Pleomorphic using the term here in a special sense. The altered forms of different species resemble each other closely so that the identification of a culture by its gross or microscopic appearance is often almost impossible once this change has taken place. Pathogenicity. — The ringworm fungi are found with such regularity and in such profusion in many of the human lesions as to leave little doubt as to their causative relationship to the disease. Many of the same species are found, too, in similar lesions of domestic animals. These latter will also induce lesions in guinea- pigs with more or less regularity if culture fragments are inserted in the skin. Such experimental lesions are often transitory but resemble somewhat the spontaneous disease. Other species such as the Microsporon Audouim and Epidermophyton inguinale, mentioned below, although found abundantly in characteristic human lesions, have not been found in animals and are innocuous when experi- mentally injected. Concerning the power of the dermatophytes to produce systemic disease we have little information. Their entire lack of invasive power in the spontaneous infections is quite striking. The isolated reports of Sabrazes THE PATHOGENIC FUNGI 999 and of Stavino on the production of tuberculoid lesions by intravenous injections of cultures of these fungi have little significance in view of the fact that many types of foreign bodies produce similar results when so introduced. Immunity. — Jadassohn37 has noted that patients who have re- covered from deep ringworm infections seem immune from subse- quent attacks. A number of experimental observations especially those of Bloch and Massini88 and of Kusunoki39 bear this out. It appears that animals which have been infected with the more virulent types which produce suppurative lesions resist reinoculation and that the protection is valid against other species than those first injected. On the other hand both clinical and experimental observations show that infections with less virulent species induce no immunity. All attempts to immunize animals by the injections of killed cultures and extracts have failed. The reports40 on the favorable therapeutic action of vaccines are inconclusive on account of the variable course of the disease. Plato41 was able to produce in patients with suppurative ringworm both cutaneous and generalized reactions by the injection of extracts of trichophyton cultures. Skin reactions have also been obtained in animals and seem to be nonspecific in regard to the species of fungus concerned but are, as a rule, seen only in animals with those more severe infections which confer immunity. Kolmer and Strickler42 report that serum from ringworm cases gives comple- ment fixation with extracts of cultures but that the reactions were again not highly specific as to species. Methods of Examination. — The demonstration of the spores and mycelium in lesions is most readily made by placing the infected hairs or scrapings from the skin in caustic soda or potash and covering with a cover slip. The alkali renders the hair and epidermis transparent but does not attack the fungus and renders it easily visible. (A satisfactory solution for this purpose is a mixture of equal parts 30 per cent aqueous sodium hydroxide and glycerin.) It is possible to stain the parasites with borax methylene blue and by modifications of Gram's method, but none of the staining methods suggested 37 Quoted by Sabouraud. 38 Bloch, B., and Massini, E., Ztschr. f. Hyg., 1909, Ixxiii, 68. 39 Kusunoki, F., Arch. f. Dermatol. u. Syph., 1912, cxiv, 1. 4(1 Strickler, Jour. Am. Med. Assn., Ixv, 225. "Keported by Neisser, Arch. f. Dermatol. u. Syph., 1902, lx, 65. ** Kolmer and Strickler, Jour. Am. Med. Assn., 1915, 1000 THE HIGHER BACTERIA, MOLDS AND FUNGI have proved as satisfactory for diagnosis as the simple treatment with caustic soda. The process of clearing may be hastened by gently heating the slide. Cultivation. — Cultures may be obtained from the lesions by distributing fragments of the infected epidermis or hairs over the surface of agar slants. Four per cent glucose agar or Sabouraud's test medium mentioned below are suitable for this purpose but cultures can also be obtained on plain agar. The chief difficulty is to avoid overgrowth of the parasites by bacteria and molds. The ordinary plating procedures are inapplicable. Skin fragments may be soaked for a few minutes, and hairs for hours, in 95 per cent alcohol before planting without killing the fungi. Increasing the acidity of the media to PH 5.0 ; adding to it 1 part in 80,000 of methyl violet, or 1-200,000 brilliant green are of some assistance. Any of these procedures inhibit the bacteria considerably but they are of little avail against the molds. Main reliance must be placed on making a large number of plants from each case. As the gross appearance of the cultures varies greatly with the cultural conditions, Sabouraud has suggested a standard test medmm on which cultures should be planted for the purpose of identification. This has the following composition: ( Sabouraud 's Test Medium) Maltose brute de Chanut 40 gm. Peptone granulee de Chassaing 10 gm. Distilled water 1000 gm. Agar agar 18 gm. The mixture is dissolved in the autoclave, filtered through paper, poured into Erlenmeyer flasks to a depth of about 1 cm. and sterilized once in the autoclave, allowing the temperature to rise slowly to 120 degrees. Sabouraud does not define the reaction of the medium and it cannot be exactly reproduced except by employing the brands of reagents which he suggests. It has, however, a reaction of about PH 5.5 and similar though not identical results can be obtained by employing domestic peptone and malt extract and adjusting the reaction to this point. In order to prevent the pleomorphic changes in the cultures, Sabouraud recommends that stock strains should be preserved on a medium of the following composition: (Habourand 's Conservation Medium) Peptone granulee (10 to 50) usually 30 gm. Agar agar 18 gm. Distilled water 1000 gm. THE PATHOGENIC FUNGI 1001 The study of the morphology of cultures is made difficult by the fact that in detaching pieces for observation from the tough mass of growth the spores are usually detached and characteristic mycelial structures deformed or broken up. Certain points can be made out by examining such fragments mounted in water in an unstained condition but more information can be gained from the study of hang-drop cultures. These are made by placing a large drop of 4 per cent maltose broth on a sterile cover slip inoculating it with a fragment of the culture and inverting over a hollow slide with a deep concavity. This may be sealed with sterile oil or petrolatum. FAVUS Favus is a disease usually limited to the scalp but which occasion- ally attacks the glabrous skin and the nails. It is characterized by the formation at the mouths of the hair follicles of small yellow cup- shaped crusts known as scutula. It is found in adults as well as in children and in this country occurs chiefly among immigrants from eastern and southern Europe where it is endemic. ACHORION SCHOENLEINI Favus of the scalp is caused by one species of fungus the Achorion Schoenleini. A scrutulum when crushed in alkali is found FIG. 114. — ACHORION SCHOENLEINII. Section of favus crust. Stained by Gram (After Fraenkel and Pfeiffer.) to be composed almost entirely of the fungus. The central portion is made up of rounded sporelike bodies of varying size without definite arrangement. Toward the periphery similar elements are 1002 THE HIGHER BACTERIA, MOLDS AND FUNGI seen strung out in filaments, and mixed with them hyphae of thicker elongated elements with irregular contours. Within the diseased hairs are filaments, sometimes of cubical, sometimes of elongated elements. They differ from those found in the hairs of ringworm, chiefly in that cells of different sizes and forms are found in the same case. The isolation of the achorion is rendered difficult by its frequent ass'ocia- tion in lesions with pyogenic cocci and molds. It develops slowly on agar and the growth attains a maximum diameter of 2 to 3 cm. in three to four weeks. It forms a remarkably tough brownish membrane with deep irregular folds the general outline being rounded upward toward the center. The FIG. 115. — ACHORION SCHOENLEINI (Eight Weeks Culture on Sabouraud's Test Medium ^ Natural Size. Hopkins.) surface is waxy at first and later shows a whitish powdery duvet. In most strains however after long cultivation on artificial media subcultures grow more rapidly, attain a larger size and become covered with a white velvety layer of aerial hyphae (pleomorphism). On gelatin a small surface growth quickly fluidifies the entire tube but the achorion utilizes sugars slightly if at all. Microscopically the growth is made up of crooked hyphae of irregular contour often made up of chains of oval cells. Pear- shaped conidia may be found scattered on the sides of the more delicate filaments but never in clusters. Chlamydospores are always numerous sometimes attached singly to the clubbed tips of hyphae but more often occurring in chains in their course. These chains may be knotted into a small nodular mass. About the periphery of a culture are numerous clubs either pear- shaped or notched at the tip. These may occur singly (tetes de clous) or in clusters (chandeliers fa viques). Inoculation of fragments of scutula into the skin of guinea-pigs THE PATHOGENIC FUNGI 1003 produces transitory ringworm like lesions. With some culture strains similar results have been obtained with others inoculation has been unsuccessful. Other Species of Achoria. — Fungi have also been cultivated from favus-like lesions in animals. The Achorion Quinckeanum which frequently infects house mice has occasionally been found in human cases of favus of the body. In culture these so-called achoria show little in common with Schoenlein's parasite but resemble somewhat trichophyta of the gypseum group (V. infra). Plant concludes that there is no reason for placing them in this genus except that they form scutula in lesions. RINGWORM OR TINEA The common form of this disease is tinea of the scalp which affects only children. It is highly contagious and in children's schools and institutions may assume epidemic proportions. The infection begins in the epidermis where it is often transitory but the parasite soon invades the hairs and there remains. There is usually slight evidence of inflammation but some species of the ringworm fungi cause suppuration in and about the hair follicle. This may result in the formation of large indolent subcu- taneous abscesses known as kerions. Tinea of the body may occur secondarily to scalp lesions in children or as a primary infection at any period of life. The lesions often assume a circular form — the disease progressing at the per- iphery and clearing at the center. There may be only slight thicken- ing and desquamation of the epidermis but usually there are super- ficial vesicles which quickly dry into crusts, and occasionally follicular pustules and kerions. Some species invade the nails. Ringworm is also a common disease in domestic animals and human cases can often be traced to infection from these sources. The fungi of animal origin when they infect man either directly from a diseased animal or indirectly from another human case, usually produce more inflammatory lesions than do those species which affect man only. MICROSPORON The small sporod ringworm fungi are in this locality the com- monest cause of ringworm of the scalp. The species of animal 1004 THE HIGHER BACTERIA, MOLDS AND FUNGI origin also attack the glabrous skin especially in children. In the epidermis of the diseased scalp they appear as curved branching hyphae made up of elongated elements. The stumps of the diseased hairs are covered with a mosaic of small round spores of uniform diameter (about 2 microns) which completely envelops the hair. If the hair is crushed, mycelial threads are also seen which grow along the medulla. From these central filaments branches project out through the cortex and give rise to the sheath of spores. Cultures. — These parasites are easily cultivated. They grow rapidly on agar producing large flat colonies which from the first are covered with a duvet of aerial hyphaB. At the center is a raised papilla and from this folds in the membrane radiate out. The color of the duvet varies from snow-white to deep buff and of the mem- Fio. 116. — MICROSPORON LANOSUM (Six Weeks Culture on Sabouraud's Test Medium \ Natural Size. Hopkins). brane from buff to brilliant orange or even a russet brown. The pigment, which is well developed only on glucose-containing media, is diffusable. Gelatin is very slowly liquefied. Morphology. — In young cultures, long straight coarse trunks radiate out from the center giving off frequent branches which later form an inextricable tangle of threads running in all directions. Some terminal branches bear conidia attached to their sides by a flattened facets (Acladium type). They also form chlamydospores, and fuseaux. Sabouraud describes eleven species of microspora which he divides into two groups : one affecting man only and one affecting both animals and man. Microsporon Audouini. — This is the type species of the former group. In gross appearance the cultures are distinguished by their slower growth, THE PATHOGENIC FUNGI 1005 their velvety white or faintly buff duvet and less active pigment production. Microsporon Audouini produces only occasional and atypical fuseaux but numerous chalmydospores which are frequently sub-terminal the hypha pro- jecting beyond them like a spine. It also produces typical pectinate bodies. Animal inoculation has been unsuccessful. Closely allied to this species are: Microsporon umbonatum, M. tardum, and M. velveticum. Microsporon lanosum (M. Audouini, var. cams). — This is the type species of the microspora of animal origin. It produces ringworm in the dog. The cultures grow very rapidly and on glucose are deeply pigmented. The duvet is long, shaggy, and in older growths assumes a dark tan color. The upper FIG. 117. — FUSEAUX OF MICROSPORON LANOSUM X200. surface is covered with a thick crop of typical lenticular fuseaux which alone serve to identify cultures as belonging to this group. Experimental lesions in the guinea-pig can be produced by the inoculation of cultures. Other members of this group are: Microsporon felineum, M. equinum, M. fulvum, M. villosum, M. pubescens, and M. tomentosum. TRICHOPHYTON The trichophyta, like the microspora, may produce ringworm of the scalp but they also produce lesions on the glabrous skin and nails. The genus is defined chiefly by the fact that its members produce lesions without scutula and appear in infected hairs as chains of spore-like elements. This alignment of the so-called spores (which are in reality short mycclial elements) distinguishes them from those of the preceding genus. In most trichophyta, too, the spores are large but in a few they are of about the same size as those of the microspora (3 microns). In culture most species produce conidia attached to the hyphae by pointed tips and frequently 1006 THE HIGHER BACTERIA, MOLDS AND FUNGI arranged in clusters. A few species (Tr. violaceum) which bear no conidia are, however, included in the genus. On artificial media the growths are much buckled and raised as compared to the flat cultures of the microspora. The cultures, however, vary so greatly among themselves in appearance that no description can be given which would apply to the genus as a whole. Classification. — Sabouraud lists thirty species. His classification is based primarily on the appearance of the parasite in the infected hairs. Such a scheme has the disadvantage that it groups together forms which have diverse cultural characteristics and separates others which culturally are similar. He divides them first into three divisions : I. The Trichophyton endothrix in which the fungus is found only within the medulla of the hair. II. The Trichophyton neo-endothrix in which some infected hairs show also a few filaments on their surface. III. The Trichophyton ectothrix in which besides invading the substance of the hair the fungus proliferates actively on its surface. Of this third division there are two groups : A. The microid endothrices, of which the spore-like elements about the infected hairs are from 3 to 4 microns in diameter. They are again divided into two sub-groups, differing culturally: (a) The Gypseum Group and (b) The Niveum Group. B. The megalospora, of which the rounded elements are from 6 to 8 microns in diameter. These Sabouraud again divides into: (a) A group forming downy cultures, and (b) A group in which the cultures are faviform. The typical members of the Endothrix Group (Tr. crateriforme and Tr. acuminatum) form small raised colonies less than 4 cm. in diameter. They are white or slightly yellowish and are covered from the first with a short powdery or velvety duvet but show little tendency to pleomorphic change. They produce numerous conidia in thyrses or small clusters but show no other characteristic structures. Sabouraud describes many variants of these species which differ in the contour of their colonies or in pigment production. They are frequently found in mild ringworm of the scalp in France but so far we have not found them in New York. Trichophyton violaceum, which is also an endothrix, is a very different organism. It develops slowly, forming small wrinkled colonies with a hard glistening surface, which slowly develop a black-violet color. The mycelium is made up of short crooked elements and no conidia are formed. It is found with relative frequency in tinea of the scalp, body and nails. A variant (Tr. glabrum) differs only in the absence of pigment. Trichophyton cerebriforme, the type of the Neo-Endothrix, Group, re- sembles in culture the Tr. crateriforme. THE PATHOGENIC FUNGI 1007 The Gypseuni Group (Tr. asteroides, radiolatum, etc.) grow more vigor- ously forming large colonies up to 10 cm. in diameter. The surface is powdery or plaster-like, but the parasites soon become pleomorphic and produce a long velvety duvet. They produce conidia in large clusters, rudi- mentary fuseaux, and numerous spirals. The lesions are inflammatory, with folliculitis and kerion formation. They cause ring-worm in horses. The Niveurn Group resemble the pleomorphic forms of the gypseurn group. They form conidia only. Trichophyton rosaceum (the type of the Downy Megalospora) forms a colony of medium size resembling a folded disc of white velvet. The deep portion develops a crimson or violet pigment which gives a rose tint as seen through the white duvet. It forms long thyrses and rudimentary fuseaux. The Faviform Strains (Tr. ochraceum, Tr. album, etc.) resemble culturally the Achorion Schoenleini. They are grouped with the trichophyta chiefly because they form no scutula in lesions. A description of all the individual species would exceed the scope of this chapter. ECZEMA MARGINATUM AND POMPHOLYX Eczema marginatum or ringworm of the groin is a common derma- tosis. In the lesions Castellan! and Sabouraud found a fungus to which the latter gave the name Epidermophyton inguinale. More recently Ormsby and Mitchell43 and others in this country have found the same fungus in eczematous lesions of the hands and feet and also in the vesicular eruptions in these regions formerly called pompholyx. Sabouraud has also found various species of trichophyta in palmar eczemas. EPIDERMOPHYTON INGUINALE (Trichophyton cruris) In the epidermis the parasite is seen as long interlacing filaments made up of oblong or oval cells with double contours. It develops slowly in culture as a greenish buff colony with folds radiating from a central or slightly eccentric peak, attaining a diameter of perhaps two centimeters in a month. The surface at first is dry and powdery but on sugar media it quickly becomes pleomorphic, developing a 43 Ormsby, 0. 8., and Mitchell, J. H., Jour. Am. Med. Assn., 1916, LXVII, 711. 1008 THE HIGHER BACTERIA, MOLDS AND FUNGI thick white duvet. Related varieties are also found which form huge crateriform colonies. The cultures form no conidia but are readily identified by the innumerable blunt fuseaux which are born on aerial hyphge. These are often in clusters which have been compared to a hand of bananas. Older cultures show also many intercalary chlamydospores. The fuseaux are not found in cultures which have become downy. t.>&% V?- FIG. 118. — FUSEAUX OF EPIDERMOPHYTON INGUINALEX200. Inoculations of epidermophyton cultures into men, dogs and guinea-pigs have been uniformly unsuccessful. TINEA VERSICOLOR (Pityriasis versicolor, Chromophytosis) Tinea versicolor appears as large fawn-colored patches on the covered parts of the body. These show slight superficial scaling but no evidence of inflammation. The causative fungus is found abundantly in the horny epidermis where it seems to grow as a saprophyte. THE PATHOGENIC FUNGI % 1009 Microsporon furfur (Malassezia furfur). This fungus bears little resemblance to the microspora which cause ring-worm. It appears as septate filaments of very irregular contour 3 to 4 microns wide. They are usually unbranched but interlace, forming a meshwork in which are found masses of spore-like bodies. Most attempts to isolate the organism have failed. A few investigators44 have reported successful cultures but their work lacks confirmation. ERYTHRASMA Erythrasma is a superficial infection of the epidermis which produces round scaling patches usually located in the axillae or groins. There is no evidence of inflammatory reaction except a hyperemia which gives the lesions a characteristic red color. A parasite is found in the epidermis which appears as minute twisted threads which are easily broken up into elements about the size of bacilli. Cultures have been described by Michele and by Ducrey and Reale but according to others the organism cannot be cultivated. It has been given various names: Microsporon minutissimum, sporo- tJirix minutissimum, and nocardia minutissimum. "Kotjar, Kef. Baugartens Jahresbericht, 1892. SECTION VI BACTERIA IN AIR, SOIL, WATER, AND MILK CHAPTER LI BACTERIA IN THE AIR AND SOIL BACTERIA IN THE AIR BACTERIA reach the air largely from the earth's surface, borne aloft by currents of air sweeping over dry places. Their presence in air, therefore, is largely dependent upon atmospheric conditions; humidity and a lack of wind decreasing their numbers, dryness and high winds increasing them. Multiplication of bacteria during transit through the air probably does not take place. Apart from these considerations the presence of bacteria in air also depends upon purely local conditions prevailing in different places. They are most plentiful in densely populated areas and within buildings, such as theaters, meeting halls, and other places where large numbers of people congregate. On mountain tops, in deserts, over oceans, and in other uninhabited regions, the air is comparatively free from bacteria. A classical illustration of this fact is found in the experiments which Pasteur carried out in his refutation of the doctrine of spontaneous generation. Tyndall also, in working upon the same subject, demonstrated this fact. From the surface of the ground and other places where bacteria have been deposited, they reach the air only after complete drying. It is a fact of much importance, both in bacteriological work and in sur- gery, that bacteria do not rise from a moist surface. From dry surfaces they may rise, but only when the air is agitated either by wind or by air-currents produced in other ways. In closed rooms, therefore, even when bacteria are plentiful and the walls and floors are perfectly dry, there is little danger of the inhalation of bacteria unless the air is agitated in some way. The most favorable condi- 1010 BACTERIA IN THE AIR AND SOIL 1011 tions for the occurrence of many bacteria in air are the existence of a prolonged drought followed by a dry wind. Under such condi- tions, even the dark places and unlighted corners of streets and habitations are thoroughly dried out, and bacteria are taken up and carried about together with particles of dust. At such times the dangers from inhalation are much multiplied. By experiments made in balloons, it has been found that bacteria are plentiful below altitudes of about fifteen hundred feet and may be present, though much reduced in numbers, as high up as a mile above the earth's surface. The species of bacteria found in the air are, of course, subject to great variation, depending upon locality. Molds and spore-forming bacteria, being more regularly resistant to the effects of sunlight and drying than bacteria possessing only vegetative forms, are naturally more generally distributed. Out of air thus laden with bacteria, they may again settle when the wind subsides and the air becomes quiescent. The process of settling, however, is extremely slow, since the weight of a bacterium is probably less than a billionth of a gram, and it may be held in suspension in air for considerable periods. Rains, snow, or even the condensation of moisture from a humid atmosphere, hastens this process considerably and large quantities of bacteria may settle out from air, in a comparatively short time, in ice chests, in operating rooms, or in other places in which much condensation of water vapor takes place. The importance of the air as a means of conveying disease is still a problem upon which much elucidation is needed. The im- portance of fhis manner of conveyance in smallpox, in measles, in scarlet fever, and in other exanthemata, can not be denied. As regards the diseases of known bacterial origin, conveyance by air is of importance in the case of tuberculosis, where infection by inhalation may take place, and in the case of anthrax, where inhaled anthrax spores may give rise to the pulmonary form of the disease. The importance of air conveyance for any great distance in pneu- monia, in influenza, in diphtheria, and in meningitis is by no means clear and requires much further study. The expulsion of bacteria from the lungs and naso-pharynx does not take place during simple expiration, since an air-current passing over a moist surface is not sufficient to dislodge microorganisms. Expulsion of bacteria in these conditions must take place together with small particles of moisture carried out in sneezing, coughing, or any forced expiration. 1012 BACTERIA IN AIR, SOIL, WATER, AND MILK The bacteria thus discharged arc then subject to the process of drying and often are exposed to direct sunlight for a considerable period before they are again taken up in the air. The methods of estimating the bacterial contents of the air are not entirely satisfactory. The simple exposure of uncovered gelatin or agar plates for a definite length of time, and subsequent estimation of the colonies upon the plates, yield a result which is variable according to the air-currents and the degree of moisture in the atmosphere, and furnish no volume standard for comparative results. The methods which are in use at the present time depend upon the suction of a definite quantity of air by means of a vacuum-pump through some substance which will catch the bacteria. One of the first devices used for this purpose was that of Hesse, who sucked air through a piece of glass tubing, about 78 cm. long and about 3.5 cm. in diameter, the inner surface of which had been coated with gelatin in the manner of an Esmarch roll tube. This method is not efficient, since a large number of the bacteria may pass entirely through the tube without settling upon the gelatin. One of the most satisfactory methods at present in use is that in which definite volumes of air are sucked through a sand-filter. Within a small glass tube, a layer of sterilized quartz sand, about 4 cm. in depth, is placed. The sand is kept from being dislodged by a small wire screen. After the air has been, sucked through the filter the sand is washed in a definite volume of sterile water or salt solution, and measured fractions of this are planted in agar or gelatin in Petri plates. The colonies which develop are counted. Thus, if two liters of air have been sucked through the filter, and the sand has been washed in 10 c.c. of salt solution, and 1 c.c. of this is planted, with the result of fifteen colonies, then the two liters of air have contained one hundred and fifty bacteria. BACTERIA IN SOIL Besides the normal bacterial inhabitants of the soil, bacteria reach the soil from the air, in contaminated waters, in the dejecta, excreta, and dead bodies of animals and human beings, and in the substance of decaying plants. It is self-evident, therefore, that the distribution of bacteria in soil depends largely upon the density of population and the use of the soil for agricultural or other BACTERIA IN THE AIR AND SOIL 1013 purposes. Thus, bacteria are most plentiful in the neighborhood of cess-pools or in manured fields and gardens. Such conditions, how- ever, may be regarded as abnormal. Even in uncultivated fields there is a constant bacterial flora in the soil which is of great importance in its participation in the nitrogen cycle, a phase of the bacteriology of soil which has been discussed in detail in another section. There are, thus, regular and normal inhabitants of the soil which fulfill a definite function and may be found wherever plant life flourishes. In addition to these, innumerable varieties of sapro- phytes and pathogenic germs may be present, which vary in species and in number with local conditions. Numerous investigations into the actual numerical contents of the soil have been made. Houston1 found an average of 1,500,000 bacteria per gram in garden soil, and about 100,000 bacteria per gram in the arid soil of uncultivated regions. Fraenkel,2 in studying the horizontal distribution of bac- teria in the 'earth, has found that they are most numerous near the surface, a gradual diminution occurring down to a depth of about two yards. Beyond this, the soil may be often practically sterile. Pathogenic bacteria may at times be found in the surface layers, and these are often of the spore-bearing varieties. Most important among them from the medical standpoint are the bacillus of tetanus, of malignant edema, and the Welch bacillus. If a guinea-pig is inoculated subcutaneously with an emulsion of garden soil, death will result almost invariably with enormous bloating and swelling of the body due to gas production. This is due to the fact that the spore-bearing, gas-producing anaerobic bacilli are commonly present and are actively pathogenic for these animals. The frequent occurrence of tetanus in persons sustaining wounds of the bare feet and hands in fields and excavations, is a matter of common knowl- edge. Anthrax, also, may be easily conveyed by soil in localities where animals are suffering from this infection. It is not probable that pathogenic germs which are not spore-bearers survive in the soil for any great length of time. Unless the soil is specially pre- pared by the presence of defecations or other organic material, the nutrition at their disposal is not at all suitable for their needs, since rapid decomposition of organic materials by saprophytes is always 1 Houston, Report Mod. Officer, Local Govern. Bd.. London, 1897 2 Fraenkel, Zeit. f . Hyg., ii, 1887. 1014 BACTERIA IN AIR, SOIL, WATER, AMD MILK going on in the upper layers. Furthermore, in the deeper layers the conditions of temperature and possibly oxygen supply are not at all favorable for the growth of most pathogenic bacteria. Within a short distance from the surface the temperature of the soil usually sinks below 14° or 15° C.' An interesting series of experiments by Fraenkel3 have demonstrated this point. This investigator buried freshly inoculated agar and gelatin cultures of cholera spirilla and of typhoid and anthrax bacilli at different levels, and examined them for growth after two weeks had elapsed. The anthrax bacilli hardly ever showed growth at a depth below about two yards, and cholera and typhoid developed colonies at these depths only during the summer months. Under natural conditions it must be remem- bered that, at these levels, suitable nutritive material is not found. A consideration of practical importance in this connection is the possibility of infection by means of buried cadavers. An elaborate series of experiments has been carried out upon this subject in Germany, with results which demonstrate that the danger from the burial of persons dead of infectious diseases was formerly much exaggerated. Experiments4 usually failed to reveal the presence of cholera and typhoid bacilli within two to three weeks after burial, and tubercle bacilli were never found after three months had elapsed. It was only in the case of sporulating microorganisms, such as the anthrax bacillus, that the living incitants could be found for as long as two years after burial. The dangers of infection of human being through the agency of soil, therefore, are chiefly those arising from the spore-bearing bacteria which are able to remain alive in spite of. the unfavorable cultural conditions. It has been found by some observers,5 however, that, under special conditions, non- sporulating bacteria, more especially the typhoid bacillus, may remain alive in soil for several months. Although these bacteria, as well as those of cholera, diphtheria, etc., can not proliferate under the conditions found in the soil, the fact that they can remain viable for such prolonged periods in the upper layers suggests the possibility of danger from the use of unwashed vegetables, such as lettuce or radishes or other soil and sewage contaminated food products. The examination of soil for colon bacilli, while demon- 3 Fraenkel, Zeit. f . Hyg., xi, 1887. • 4Petri, Arb. a. d. kais. Gesundheitsamt, vii. B Firth and Horrocks, Brit. Med. Jour., Sept., 1902. BACTERIA IN THE AIR AND SOIL 1015 strating the presence or absence of manure or sewage contamination, has no practical value, since colon bacilli are found in the dejecta of animals. Examination of specimens of soil for their numerical bacterial contents is extremely unsatisfactory because the bacteria there found can hardly ever all be cultivated together under one and the same cultural environment. A large number arc anaerobic, others again thrive at low temperatures, while again another class may require unusually high temperatures. When such examinations are made, however, specimens of the soil from the surface layer may be taken in a sterile platinum spoon. When taken from the lower levels, a drill, such as that devised by Fraenkel, may be used. This consists of an iron rod the lower end of which is pointed. Just above the point a movable collar is fitted. This collar has a slit-like opening. The rod beneath the collar has a deep longitudinal groove corresponding to the slit in the collar. A flange on the collar permits opening and closing of the groove while the instrument is below the ground. The drill is forced into the earth to the desired depth, the groove is opened and earth is forced into the chamber by twist- ing the rod. In the same manner the groove may be closed. The soil obtained in this way is taken out of the chamber and a definite quantity, say one gram, is dissolved and washed thoroughly in a measured volume of sterile water or sterile salt solution. Fractions of this are then mixed with the culture medium, plated, and cul- tivated aerobically or anaerobically as desired. CHAPTER LII BACTERIA IN WATER ALL natural waters contain a more or less abundant bacterial flora. This fact, combined with our knowledge that the incitants of several epidemic diseases and a number of minor ailments of a diarrheal character are water borne, gives the bacteriological in- vestigation of water a place of great importance in hygiene. In nature, there are very few sources of water supply which do not contain bacteria of one or another description. While pathogenic bacteria are usually not present except in those waters which are directly contaminated from human sources, a thorough understand- ing of the quantitative and qualitative bacterial contents of all natural waters is necessary in order that we may intelligently gather comparative data as to the fitness of any given water for human consumption. The gross appearance of water is rarely, if ever, an indication of its danger. The turbid waters of running streams in sparsely populated agricultural districts may be safe, while perfectly clear well waters subjected to the dangers of contamination from neigh- boring sinks or cess-pools may contain large numbers of pathogenic germs. The diseases which are known to be more directly connected with water supply are typhoid fever and cholera. Typhoid germs discharged from the bowel or from the urine of typhoid patients or convalescents may be carried by the sewage or from the neighboring soil into a river or lake and lead to infection of the population deriving its drinking water from this source. There are a great many investigations on record in which severe typhoid epidemics have been traced to such sources. In the case of cholera, where the germs are discharged from the bowels in enormous numbers, conveyance of the disease by water is even more apparent, and the discoverers of the cholera germ themselves, in their early work in Egypt and India, were able to isolate the bacteria from contaminated water supplies. 1016 BACTERIA IN WATER 1017 In regard to the less clearly understood diarrheal diseases, dysen- tery, cholera infantum, etc., the direct relation to water supply has not been so definitely proven, and can be deduced only from the diminution of such infections after the substitution of pure water for the previously used impure supply. It is thus seen that water bacteriology is one of the most important branches of the science of hygiene, and has led, and is constantly leading, to enormous diminution of the death rate in all communities where an intelligent study of the conditions has been made. The bacterial purity of natural waters, although dependent upon special and local conditions in relation to possible contamination, differs widely, according to the source from which such waters are derived. Rain water and snow water are usually contaminated with bac- teria by the dust which they gather on their way to the ground, and are especially rich in bacteria when taken during the first few hours of a rain or snow storm when the air is still dusty and filled with floating particles. During the later hours of prolonged storms, rain water and snow water may be comparatively sterile. Miquel,1 who made extensive experiments in France on the bacterial contents of rain water, found that in country districts, where the air is less dusty, rain water contained an average of about 4.3 bacteria to the cubic centimeter. The bacterial counts of snow water are usually somewhat higher than those of rain. The waters of streams, ponds, and lakes are usually spoken of as surface waters, and these of all natural supplies contain the largest number of bacteria. In each case, of course, the quantitative and qualitative bacterial flora of such waters is intimately dependent upon the conditions of the surrounding country, the density of the population, and the relation of these waters to sewage. It is also, and to no less important degree, dependent upon weather conditions, the influence of light and temperature, and the food supply contained within the waters in the form of decayed vegetation. In all such surface waters there is constantly going on a process of self-purifica- tion. The chief factor in this process is sedimentation. In stagnant ponds and lakes with but sluggish currents there is a constant sedimentation of the heavier particles, which gradually but steadily 1 Miquel, Revue d'hyg., viii, 1886. 1018 BACTERIA IN AIR, SOIL, WATER, AND MILK leads to a diminution of the number of bacteria in the upper layers of the water. In rivers where sedimentation is to a certain extent prevented by rapidity of Current, the effectiveness of such sedimen- tation is, of course, entirely dependent upon the speed of the current. The influence of light in purifying surface waters is important chiefly in ponds, lakes, and sheets of water which expose a large surface to the sunlight, and where the surroundings are such that the sun has free access throughout the day. According to the researches of Buchner,2 the bactericidal effects of light penetrate through water to a depth of three feet. The effects of temperature in purifying surface waters under natural conditions are probably not great. There is, however, a general tendency toward diminution of the bacterial flora as the temperature of such waters becomes lower. The presence of protozoa in natural waters as purifying agents has recently been emphasized by Huntemiiller,3 who claims that these organisms by phagocytosis greatly diminish the number of bacteria in any given body of water. It is self-evident that the number of bacteria in any of these waters is never constant, since all factors which tend to a diminution or increase in volume, such as drying up of tributary streams or the occurrence of heavy rains, would lead to differences of dilution which would materially change numerical bacterial estimations. The influence of rains, furthermore, may be a twofold one. On the one hand, heavy rain-falls, by wash- ing a large amount of dirt into the rivers and lakes from the sur- rounding land, have a tendency to increase the bacterial flora. This influence would be especially marked in all bodies of water which are surrounded by cultivated land where manured fields and graz- ing-meadows supply a plentiful source of bacteria. On the other hand, in regions where arid, uninhabited lands surround any given river or lake, the rain would carry with it very little living con- tamination and would act chiefly as a diluent and diminish the actual proportion of bacteria in the water. Another and extremely important source of water supply is that spoken of technically as "ground water/' The "ground waters" include the shallow wells employed in the country districts, springs, and deep or artesian wells. The shallow wells that form the water 2 Buchner, Arch. f. Hyg., xvii, 1895. 8 HuntemiiUer, Arch. f. Hyg., liv, 1905. BACTERIA IN WATER 1019 supply for a large proportion of farms in the eastern United States are usually very rich in bacteria and are by no means to be regarded as safe sources, except in cases where great care is observed as to cleanliness of the surroundings. In such wells the filtration of the water entering the well may be subject to great variation accord- ing to the geological conditions of the surrounding ground. The proximity of barns and sinks may lead to dangerous contamination of such waters. Examinations by various bacteriologists have shown that such wells frequently contain as many as five hundred bacteria to the cubic centimeter. Perennial spring waters are usually pure. Examinations by the Massachusetts State Board of Health4 in 1901 showed an average of about forty bacteria per cubic centimeter. As sources of water supply for general consumption, however, springs can hardly be very important because of the insignificant quantities usually derived from them. Of much greater practical importance are deep artesian wells, which, under ordinary conditions, are largely free from bacterial contamination. Quantitative Estimations of Bacteria. — The quantitative estima- tion of bacteria in water is of necessity inexact, because of the difficulty of always securing fair average samples from any large body of water, and because of the large variations in cultural re- quirements of the flora present in them. All these methods depend upon colony enumeration in plates of agar or gelatin, preferably of both. For the sake of gaining some basis of comparison for results which, at best, can never be entirely accurate, an attempt has been made by the American Public Health Association5 to standardize the methods of analysis. Water for analysis should always be collected in clean, sterile bottles, preferably holding more than 100 c.c. If w^ater is to be taken from a running faucet or a well supplied with piping, it is important that it should be allowed to run for some time before the sample is taken, in order that any change in bacterial content occurring inside of the pipes may be excluded. It is obvious that in water pipes through which the flow is not constant, bacteria may 4 Mass. State Bd. of Health, 33d Annual Eeport for 1901. 5 Fuller, Trans. Amer. Public Health Assn., xxvii, 1902. Eeport of Com. on Standard Methods of Water Analysis, Jour. Inf. Dis., Suppl. 1, 1905. 1020 BACTERIA IN AIR, SOIL, WATER, AND MILK find favorable conditions for growth and such a sample would not represent fairly the supply to be tested. When the water is taken from a pond, lake, or cess-pool, the bottle may be lowered into the water by means of a weight, or may be plunged in with the hand, great care being exercised not to permit contamination from the fingers to occur. After the water has been collected it is important to plate it before the bacteria in it have a chance to increase. The changes taking place during transportation, even when packing in ice has been resorted to, have been found by Jordan and Irons6 to be considerable. It is imperative, therefore, that plating of the water, if possible, shall not be delayed for longer than one or two hours after collection. Bacteriological Examination of Water. — In describing the methods of bacteriological examinations of water, we adhere strictly to the recommendations of the Committee on Standard Methods of the American Public Health Association,7 taking the following paragraphs with slight changes from their report of 1915 : It should be remembered that quantitative estimations of bacteria in water are of most value when repeatedly done and a " normal" for the particular water supply has been established, so that devia- tion from this "normal" can be easily recognized. Single isolated determinations may easily lead to error. The following paragraphs are taken without change from the Public Health Association 's report : "Since gelatine does not give the total number of bacteria in the water, the committee has thought it wise to use agar incubated at 37° C. as a standard medium. This admits of counts in one day instead of two, and gives results on the kind of bacteria growing at blood temperature and therefore more nearly related to pathogenic types'. "Media. — The standard medium for determining the number of bacteria in water shall be nutrient agar. All variations from this medium shall be considered special media. If any medium other than standard agar is used, this fact shall be stated in the report. "For general work the standard reaction shall be +1 per cent, but for long continued work upon water from the same source the optimum reaction shall be ascertained by experiment and thereafter adhered to. If the reaction used, however, is different from the standard, it shall be so stated in the report. • Jordan and Irons, Eeports of the Amer. Pub. Health Assn., xxv, 1889. 7Amer. P. H. A., Stand. Meth. Exam. Water and Sewage, 1915. BACTERIA IN WATER 1021 "Procedure. — Shake at least twenty-five times the bottle which contains the sample. Withdraw 1 c.c. of the sample with a sterilized pipette and deliver it into a sterilized Petri dish, 10 cm. in diameter. If there be reason to suspect that the number of bacteria is more than 200 per c.c., mix 1 c.c. of the sample with 9 c.c. of sterilized tap or distilled water. Shake twenty-five times and measure 1 c.c. of the diluted sample into a Petri dish. If a higher dilution be required proceed in the same manner, e.g., 1 c.c. of the sample to 99 c.c. of sterilized water, or 1 c.c. of the once diluted sample to 9 c.c. of sterilized water, and so on. In the case of an unknown water or a sewage it is advisable to use several dilutions for the same sample. To the liquid in the Petri dish add 10 c.c. of standard agar at a temperature of about 40° C. Mix the medium and water thoroughly by tipping the dish back and forth, and spread the contents uniformly over the bottom of the plate. Allow the agar to cool rapidly on a horizontal surface and transfer to the 37° C. incubator as soon as it is hard. Incubate the culture for twenty-four hours at a temperature of 37° C. in a dark, well-ventilated incubator where the atmosphere is practically saturated with moisture.8 After the period of incubation place the Petri dish on a glass plate suitably ruled and count the colonies with the aid of a lens which magnifies at least five diameters. So far as practicable the number of colonies upon the plate shall not be allowed to exceed 200. The whole number of colonies upon the plate shall be counted, the practice of counting a fractional part being resorted to only in case of necessity. "It will be found advantageous to use Petri dishes with porous earthen- ware covers in order to avoid the spreading of colonies by the water of condensation.9 "Expression of Results. — In order to avoid fictitious accuracy and yet to express the numerical results by a method consistent with the precision of the work the rules given below shall be followed : ''Numbers of Bacteria per c.c. From 1 to 50 Recorded as found 51 " 100 101 " 250 251 " 500 501 " 1,000 1,001 " 10,000 10,001 " 50,000 50,001 " 100,000 100,001 " 500,000 500,001 " 1,000,000 1,000,001 " 10,000,000 to the nearest 5 10 25 50 100 500 1,000 10,000 50,000 100,000" Qualitative Bacterial Analyses of Water. — Of far greater im- portance than quantitative analysis is the isolation of bacteria either 8 Whipple, Tech. Quar., 1899, 12, p. 276. 9 Hill, Jour. Med. Res., 1904, N.-S., 8, p. 93. 1022 BACTERIA IN AIR, SOIL, WATER, AND MILK distinctly pathogenic, such as the cholera spirillum and the typhoid bacillus, or of other species probably emanating from contaminating sources, such as a B. coli. Unfortunately there are no reliable methods by which typhoid and cholera germs can be isolated from water with any degree of regularity or certainty. Although fre- quently the isolation of such organisms is possible, a negative result in these cases is by no means eliminative of their presence. The isolation of typhoid bacilli from water is very difficult, chiefly because of the great dilution which contaminations undergo upon entering any large body of water. The difficulty of isolating typhoid bacilli, even from the stools of infected patients, makes it clear that such difficulties are enhanced when a considerable dilution of the excreta takes place. Furthermore, water is by no means a favorable medium for the typhoid bacillus. Russell and Fuller10 have shown that typhoid bacilli may die in water within five days, and it is unquestionable that the rate of increase of these bacteria is by no means equal to that of many other microorganisms for which pol- luted water at the temperature encountered in streams and lakes forms a much more favorable medium. It is thus clear that even in infected waters the number of typhoid bacilli encountered can never be very great.11 A large number of methods for the isolation of the typhoid bacillus from water have been devised. Most of the media used are identical with those employed for the isolation of these bacteria from the stools. These media have been discussed in the chapter dealing with the typhoid bacillus. Success is rendered more likely if 10 c.c. of the water is first planted into lactose-bile in fermentation tubes holding 40 c.c. After forty-eight hours at 37.5° there will be an enrichment of typhoid bacilli which can be then isolated by plating in the usual manner on Endo 's medium, Conradi Drigulski or any of the other usual differential media. A method which has proved useful in the hands of Adami and Chapin12 is one which depends upon the phenomenon of agglutina- tion. They attempt to agglutinate the bacilli out of liter samples of water by adding powerful agglutinating serum. Vallet and others have attempted to precipitate typhoid bacilli out of water by chemical means. To two liters of water add 20 c.c. ™Eussell and Futter, Jour. Inf. Dis., Suppl. 2, 1908. 11 Laws and Anderson, Rep. of Med. Officer, London County Council, 1894. J2 Adami and Chapin, Jour. Med. Res., xl, 1904. BACTERIA IN WATER 1023 of a 7.75 per cent solution of sodium hyposulphite and 20 c.c. of a 10 per cent solution of lead nitrate. When the precipitate has settled, the clear supernatant fluid is decanted and the precipitate dissolved in a saturated sodium hyposulphite solution. This clear solution is then plated. Willson13 has modified this method by adding to the water 0.5 gm. of alum to each liter. The supernatant fluid is removed and the precipitate plated. The isolation of the vibrio of cholera is less difficult than that of B. typhosus, primarily because of the much greater numbers of these microorganisms discharged into sewage. The number of cholera spirilla in the excreta of cholera patients is enormously higher than is that of B. typhosus in the stools of typhoid-fever patients. It is not infrequent, therefore, that the source of a cholera infection may be directly traced to the water supply. Koch,1* the discoverer of the cholera vibrio, has indicated a method which has frequently found successful application. To 100 c.c. of the infected water are added one per cent of pepton and one per cent of salt. This mixture is then incubated at 37.5° C., and after ten, fifteen, and twenty hours, specimens from the upper layers are examined microscopically and are plated. The scum from the surface of such a medium may be plated on the starch agar of Stokes and Haechtel,15 on which colonies of intestinal spirilla will appear pink and spreading. Because of the great difficulties outlined above in isolating specific pathogenic germs from polluted waters, bacteriologists have at- tempted to form an approximate estimation of pollution by the detection of other microorganisms which form the predominating flora of sewage. Chief among these is B. coli. The isolation and numerical estimation of B. coli in polluted water has been for a long time one of the criteria of pollution. This so-called colon test, however, should always be approached with conservatism and should never be carried out qualitatively only. Careful quantitative es- timation should be made in every case. B. coli in water is by no means always the result of human con- tamination, since this bacillus is found in great abundance in the 13 Willson, Jour, of Hyg., v, 1905. "Koch, Zeit. f. Hyg., xiv, 1893. 15 Stokes and Haechtel, see Eeport 1915 A. P. H. A., on Water Analysis. The medium is an agar with 5.5 grams agar, 5.0 meat extract, 10 Pepton and 8.5 NaCl to liter to which is added 10 grams of soluble starch. 1024 BACTERIA IN AIR, SOIL, WATER, AND MILK intestines of domestic animals. According to Poujol, B. coli does not even always point to fecal contamination, since this author was able to find the bacillus in the water of a number of wells where no possible contamination of any sort could be traced. Prescott18 explains this, as well as similar cases, by the fact that organisms of the colon group may occasionally be parasitic upon plants. The opinions of hygienists are widely at variance as to the value of the colon test. While the discovery of isolated bacilli of the colon group may therefore be of little value, it is nevertheless safe to follow the opinion of Houston,17 who states that the discovery of B. coli in considerable numbers invariably points to sewage pollu- tion, and that the absolute absence of B. coli is, as a rule, reliable evidence of purity. Rosenau states that a ground water should be condemned even if only a few colon bacilli are found, for, as he put it, ' * these bacteria have no business in a soil-filtered and properly protected well or spring-water." Surface waters, however, may easily contain a few colon bacilli without necessarily having been exposed to contamina- tion by human forces. The limit of safety, Rosenau states, is one colon bacillus per c.c. If more are present the water should be regarded as suspicious. If more than 10 per c.c. are found the water must be regarded as dangerous and unqualifiedly condemned. Presumptive Colon Tests. — For this purpose, a large number of methods have been devised. In examining sewage or other polluted waters in which the number of colon bacilli is comparatively large, the direct use of lactose litmus agar plates yields excellent results. The method advised by the American Public Health Association is as follows : "Add the quantities of water or sewage to be tested to fermentation tubes holding at least forty cubic centimeters Q£ lactose bile,18 incubate at 37° C. and note the production of gas. The standard time for observing gas produc- tion is forty-eight hours. Small numbers of somewhat attenuated B. coli may require three days to form gas. Attenuated B. coli does not represent recent contamination and all B. coli not attenuated grows readily in lactose bile. No other organism except B. Welchii gives such a test in lactose bile. B. Welchii is of rather rare occurrence in water, is of fecal origin, is almost invariably accompanied by B. coli, and the sanitary significance is the same. "Prescott, Science, xv, 1903. 17 Houston, Kep. Medical Officer, Local Gov. Board, London, 1900. 18 Prescott, Science, xvi, 1902. BACTERIA IN WATER 1025 "A comparison of the positive results obtained in the various dilutions of the water or sewage planted into the lactose bile gives a good idea of the relative amount of contamination in the various samples examined. "Quantities of Water Tested. — For ordinary waters, 0.1, 1.0 and 10.0 c.c. shall be used for the colon test. For sewage and highly polluted surface waters, smaller quantities shall be used; and for ground waters, filtered waters, etc., the quantities shall be larger, if necessary to obtain positive results. The quantities shall vary preferably in the tenfold manner in- dicated. Single tests with quantities which give ordinarily a positive result or ordinarily a negative result are in themselves of but little account for quantitative determinations. The range in quantities studied shall be sufficient to allow the quantities needed for both a positive and a negative result to be recorded for each sample. When this is done, the results of several tests allow an approximate estimate of the number of B. coli per c.c." The identification of colon bacilli so obtained should then be undertaken. The following table, again taken from the report of the A. P. H. A., will be of help : 1026 BACTERIA IN AIR, SOIL, WATER, AND MILK + + I + I I | }* 'a PQ ~ £ PQ + PQ 11 § a ffl'M • — i a — + + + i i .1 22§§§ + + + i i + i i A. r^H + + + + + | | '3 ~ ^ w v. I • i"J ! ++ 1 ' i '3 « >. « •<1 PQ O Q + + I + I | + + + I I | 'S ~ ^ ^ ^ I CHAPTER LIII BAGTEEIA IN MILK AND MILK PRODUCTS, BACTERIA IN THE INDUSTRIES BACTERIA IN MILK THE universal use of cows' milk as a food, especially for the nourishment of infants, has necessitated its close study by bac- teriologists and hygienists. It furnishes an excellent culture medium for bacteria and is, therefore, pre-eminently fitted to convey the germs of infectious disease. The many changes which take place in milk, furthermore, and which add or detract from its nutritive value, are due largely to bacterial growth and have been elucidated by bacteriological methods. Within the udder of the healthy cow, milk is sterile. If pyogenic or systemic diseases of bacterial origin exist in the cow, the milk may, under certain circumstances, be infected even within the mam- mary glands. In the milk ducts and in the teats, however, even in perfectly healthy animals, a certain number of bacteria may be found. For this reason, even when all precautionary measures are followed, the milk as received in the pail is usually contaminated. As a matter of fact, the anatomical location of the udder and the mechanical difficulties of milking make it practically impossible to collect milk under absolutely aseptic conditions, and, under the best circumstances, from 100 to 500 microorganisms per c.c. may usually be found in freshly taken milk. Withdrawn under conditions of ordinary cleanliness, the bacterial contents of milk are considerably higher than this. After the process of milking, in spite of all prac- ticable precautions, the chances for the contamination of milk are considerable ; but even could these be eliminated, the bacterial con- tents of a given sample would ultimately rapidly increase because of the rich culture medium which the milk provides for bacteria. Whether large increases shall take place or not depends, in the first place, upon the temperature at which milk is kept, and, in the second place, upon the length of time which intervenes before 1027 1028 BACTERIA IN AIR, SOIL, WATER, AND MILK its consumption. Though fresh milk possesses slight bactericidal powers,1 these are by no means sufficient to be of practical im- portance in the inhibition of bacterial growth. Kept at or about freezing-point, the bacterial contents of milk do not appreciably increase. At higher temperatures, however, a rapid propagation of bacteria takes place which, especially during the summer months, speedily leads to enormous numbers. In a case reported by Park,2 where milk, containing at the first examination 30,000 microorgan- isms per cubic centimeter, was kept at 30° C. (86° F.) for twenty- four hours, the count at the end of this time yielded fourteen billions of bacteria for the same quantity. It is of much importance, therefore, that the cleanliness of dairies, of cattle, and in the handling of milk should be reinforced by the utmost care in chilling and icing during shipment and before sale. Because of its great importance, especially for the health of the children in large cities during the summer months, the milk question has, of recent years, received much attention from health officers. In the city of New York, the question has been made the subject of many careful studies by Park3 and his associates. Commissions, working in Chicago,4 Boston,5 and other large towns, have placed the sale of milk under more or less exact bacteriological supervision. Park has determined that the milk, as sold in New York stores during the cold weather, not infrequently averages seven hundred and fifty thousand bacteria per cubic centimeter; during the hot summer months, the bacterial contents of similar milk not infre- quently average one million and more, for the same quantity.6 In consequence of these and other researches, large dairies have intro- duced bacteriological precautions into their method of milk produc- tion. They have attempted the reduction of the bacterial contents of milk by scrupulous cleanliness of the barns and of the udders and teats of the cow, by the elimination of diseased cattle, by sterilization of the vessels in which the milk is received, and of the hands of the milker; also by the immediate filtering and cooling of the milk and the packing of the milk cans in ice, where they 1 Eosenau and McCoy, Jour. Med. Ees., 18, 1908. *Park, W. H., "Pathogenic Bacteria," New York, 1905, p. 463. 3 Park, Jour, of Hygiene, 1, 1901. 4 Jordan and Hcinemann, Rep. of the Civic Federation of Chicago, 1904. 5 Sedgwick and Batchelder, Bost. Med. and Surg. Jour., 126, 1892. *Escherich, Fort. d. Medizin, 16 and 17, 1885. BACTERIA IN MILK 1029 remain until delivered to the consumer. In consequence of such measures, it is possible for cities to be supplied with milk containing no more, and often less, than fifty thousand bacteria to the cubic centimeter. A standard of cleanliness has been established in various towns by the introduction of the so-called "certified milk," which, by the New York Milk Commission, is required to contain no more than thirty thousand bacteria per cubic centimeter. Great stress is laid upon such numerical counts simply in that they are ap- proximate estimates of cleanliness. Most of the bacteria, however, contained in milk are non-pathogenic, and numbers much larger than the maximum required for certified milk may be present with- out actual disease or harm following its consumption. The various species of bacteria which may be found in milk include almost all known varieties. Whether there are special, so-called milk bacteria or not is a question about which investigators have expressed widely differing opinions. It is probable that many of the species, formerly regarded as specifically belonging to milk, are there simply by virtue of their inhabitual presence in fodder, straw, or bedding, or upon cattle. It is likely, furthermore, that some of these species are found with great regularity because of their power to outgrow other species under the cultural conditions offered them in milk. Under normal conditions, milk always undergoes a process which is popularly known as souring and curdling. This is due to the formation of lactic acid from the milk sugar and is the result of the enzymatic activities of several varieties of bacteria commonly found in milk. Most common among these bacteria is the so-called Bacillus lactis aerogenes, an encapsulated bacillus closely related to the colon- bacillus group. (See page 637.) The transformation of the lactose into lactic acid may occur either directly by hydrolytic cleavage : C12 H220U + H20 = 4 C3 H6 0,j or indirectly through a monosaccharid, C12 H22 Olt + H2 O = 2 G6 H12 06 = 4 C3 H6 O8. Other microorganisms which may cause lactic-acid fermentation in milk are the so-called Streptococcus lacticus, the common pyogenic streptococcus, the Staphylococcus aureus, Bacillus coli communis and communior, and many other species. Most commonly concerned in 1030 BACTERIA IN AIR, SOIL, WATER, AND MILK lactic-acid production, however, according to Heinemann,7 are the two first-mentioned, Bacillus lactis aerogenes and Streptococcus lacticus. The secret of the regularity with which some of these bacteria are present in sour milk is probably found in the ability of these varieties to withstand a much higher degree of acidity of the culture medium than other species. In consequence, they are able to persist and develop when cultural conditions are absolutely unsuited to other bacteria. Consequent upon acidification of the milk by lactic-acid forma- tion, there is coagulation of casein. Casein precipitation, however, may also be due to a non-acid coagulation caused by a bacterial ferment. Casein precipitated in this way may be redissolved by a bacterial trypsin or casease, produced by the same or other bac- teria, the milk again becoming entirely liquid, transparent, and of a yellowish color. The casein precipitated by lactic-acid formation, however, is never thus redissolved, because the high acidity does not permit the proteolytic ferments to act.8 Butyric-acid fermentation in milk, a common phenomenon, is also an evidence of bacterial growth. As a rule, it is produced by the anaerobic bacteria, and is a process developing much more slowly than other fermentations. A large number of bacteria have been described which are capable of producing such changes, the chemical process by which they are produced being, as yet, not entirely understood. It is probable that the process takes place after hydrol- ysis of the dissaccharid somewhat according to the following formula : C« H12 Q6 — C4 H8 O2 + 2 CO2 -f- 2 H2. Special bacteria have been described in connection with this form of milk fermentation,9 most of them non-pathogenic. It is unques- tionable, however, that many of the well-known pathogenic bacteria, such as Bacillus aerogenes capsulatus, Bacillus cedematis maligni, possess the power of similar butyric-acid formation. While less commonly observed in milk, because milk is rarely kept long enough to permit of the action or development of these enzymes, the butyric- acid fermentation is of importance in connection with butter, where it is one of the causes producing rancidity. 7 Heinemann, Jour, of Inf. Dig., 3, 1906. 6 Conn, Exper. Stat. Rep., 1892. 9 Schattenfroh und Grasberger, Arch. f. Hyg., 37, 1900. BACTERIA IN MILK 1031 Alcoholic fermentation may take place in milk as a result of the activities of certain yeasts. Upon the occurrence of such fer- mentations depends the production of kefyr, koumys, and other beverages which have been in common use for many years, especially in the region of the Caucasus. The characteristic quality of these beverages is contributed by the feeble alcoholic fermentation pro- duced by members of the saccharomyces group, but side by side with this process lactic-acid formation also takes place. Beijerinck,10 who has carefully studied the so-called kefyr seeds, used for the production of kefyr in the East, has isolated from them a form of yeast similar in many respects to the ordinary beer yeast, and a large bacillus to which he attributes the lactic-acid formation. Occasional but uncommon changes which occur in milk lead to the formation of the so-called " slimy milk," yellow and green milk, and bitter milk. These may be due to a number of bacteria. A microorganism which is commonly found in connection with the slimy changes in milk is the so-called Bacillus lactis viscosus. Ac- cording to the researches of Ward,11 this microorganism is frequently derived from water and it is the water supply which should attract attention whenever such trouble occurs in dairies. The so-called blue, green, and yellow changes are usually due to chromogenic bacteria, such as Bacillus cyanogenes, Bacillus prodigio- sus, and others. ' * Bitter milk, ' ' a condition which has occasionally been observed epidemically, is also the consequence of the growth of microorgan- isms. Conn,12 in 1891, isolated from a specimen of bitter cream a diplococcus which occasionally forms chains and which in sterilized milk develops rapidly, producing an extremely bitter taste. The organism of Conn differs from a similar diplococcus described by Wagmann13 in that it possesses the ability of producing butyric acid. Milk in Relation to Infectious Disease. — As a source of direct infection, milk is second only to water, and deserves close hygienic attention. A large number of infectious diseases have been traced to milk, although the actual proof of the etiological part played by it in such cases has often been difficult to adduce and has neces- sarily been indirect. Nevertheless, even when indirect proof only 10 Beijerinck, Cent, f. Bakt,, vi, 1889. "Ward, Bull. 1«5, Cornell Univ. Agri, Exp. Stat., 1899, 12 Conn, Cent. f. Bakt,, ix, 1891. " Wagmann, Milchztg., 1890. 1032 BACTERIA IN AIR, SOIL, WATER, AND MILK has been brought, it has been sufficiently convincing to necessitate the most careful investigation into milk supplies whenever epidemics of certain infectious maladies occur. Typhoid-fever epidemics have been frequently traced to milk infection, and, in this disease, milk is, next to water, the most frequent etiological factor. Schiider,14 in an analysis of six hundred and fifty typhoid epidemics, found four hundred and sixty-two attributed to water, one hundred and ten to milk, and seventy-eight to all other causes. Trask15 compiled statistics of one hundred and seventy-nine typhoid epidemics supposed to have been caused by milk, in various parts of the world. In all such epidemics the origin of infection was generally traceable to diseased or convalescent persons employed in dairies, to contaminated well water used in washing milk utensils, or to the use of cans and bottles returned from dwellings where typhoid fever had existed. Actual bacteriological proof of the in- fectiousness of milk by the isolation of Bacillus typhosus is rare, but has been accomplished in isolated instances. In the case of one epidemic, Conradi16 isolated the bacillus from the milk on sale at a bakery at which a large number of the infected individuals had purchased their milk. The examination of market milk at Chicago, through a period of eight years, revealed the presence of typhoid bacilli but three times. In spite of the few cases in which actual bacteriological proof has been brought, it is not unlikely that careful and systematic researches would reveal a far greater number, since many writers have shown that typhoid bacilli may remain alive in raw milk for as long as thirty days,17 and may actively proliferate in the milk during this time. One peculiarity of epidemics which may aid in arousing the suspicion that they have originated in milk is that, in such cases, women and children are far more frequently attacked than men.18 A feature which adds considerably to the dangers of milk infec- tion is the unfortunate absence of any gross changes, such as coagula- tion, by the growth of typhoid bacilli. 14 Schuder, Zeit. f. Hyg., xxxviii, 1901. ™ Trask, Bull. No. 41, TI. S. Pub. Health and Mar. Hosp. Serv., Wash. 16 Conradi, Cent, f . Bakt., I, xl, 1905 17 Heim, Arb. a. d. kais. Gesundheitsamt, v. 18 Wilckens, Zeit. f. Hyg., xxvii, 1898. BACTERIA IN MILK 1033 Scarlet fever,19 though as yet of unknown etiology, has in many cases been traced indirectly to milk infection. Trask has collected fifty-one epidemics of scarlet fever presumably due to milk. In one epidemic occurring in Nor walk, Conn.,20 twenty-nine cases wer"e distributed among twenty-five families living in twenty-four different houses. The individuals affected did not attend the same school, and were of entirely different social standing, the only factor com- mon to. all of them being the milk supply. Diphtheria has been frequently traced to the use of infected milk. In most of the epidemics reported as originating in this way, the proof has been necessarily indirect. In two out of twenty-three epidemics reported by Trask, however, Bacillus diphtherias was isolated from the milk directly. The ability of the Klebs-Loeffler bacillus to proliferate and remain alive for a long while in raw milk has been demonstrated by Eyre21 and others. Whether or not cholera asiatica may be transmitted by means of milk has been a disputed question. Hesse22 claims that cholera spirilla die out in raw milk within twelve hours. This statement, however, has not been borne out by other observers.23 Unquestion- able cases of direct transmission of cholera by means of milk have been reported by a number of writers, notably by Simpson.24 The relation of milk to the diarrheal diseases of infants has, of late years, received a great deal of attention. In large cities, during the summer months, numerous cases of infantile diarrhea among bottle-fed babies occur, which, in many instances, are attributed to feeding with contaminated milk. Park and Holt,25 who have made extensive researches upon this question in New York City, have come to the conclusion that the harmful effects of contaminated milk upon babies can not be ascribed to any given single microorganism in the milk. Specifically toxic properties were found by these writers for none of the one hundred and thirty-nine different species of bacteria isolated from unsterilized milk. It is unlikely, therefore, that the diarrheal diseases among babies have a uniform bacteriolog- 18 Trask, loc. cit. 20 Herbert E. Smith, Eep. Conn. State Bd. of Health, 1897. 21 Eyre, Brit. Med. Jour., 1899. 22 Hesse, Zeit. f. Hyg., xvii, 1894. 2*Basenau, Arch. f. Hyg., xxiii, 1895. 24 Simpson, Indian Med. Gaz., 1887. 25 Park and Holt, Arch, of Fed., Dec., 1903. 1034 BACTERIA IN AIR, SOIL, WATER, AND MILK ical cause. Whether or not these diarrheal conditions depend en- tirely upon the bacterial contents of milk or, in a large number of cases at least, upon the inability of the child to digest cow 's milk because of chemical conditions, must be left undecided. Park and Holt, in analyzing their extensive data, conclude that milk con- taining "over one million bacteria to the cubic centimeter is cer- tainly harmful to the average infant." The significance of the presence of streptococci in milk, as an element of danger, has recently received much attention in the literature. Heinemann,26 who has made a careful comparison of Streptococcus lacticus (formerly spoken of as Bacillus acidi lactici [Krusel]), with other streptococci, has shown that, essentially, this streptococcus does not differ from streptococci from other sources, and is practically indistinguishable by cultural methods from Strep- tococcus pyo genes. Similar comparisons made by Schottmuller,27 Miiller,28 and others have led to like results. Since streptococci may be found in milk from perfectly normal cows and are almost regularly associated with lactic-acid fermentation, it is unlikely that these microorganisms hold ordinarily any specific relationship to disease. Recently, however, a number of epidemics of sore throat caused by streptococci have been traced to milk upon reasonably reliable evidence. Accounts of such epidemics in Chicago and in Baltimore have been published by Capps and Miller29 and by Hamburger.30 The presence of pus cells and leucocytes in milk, together with streptococci, was also formerly regarded as of great importance. Enumerations of leucocytes in milk were first made by Stokes and Weggefarth.31 Their method of enumeration consisted in centri- fugalizing a definite volume of milk, spreading the entire sediment over a definite area on a slide, and counting the leucocytes found in a number of fields. Calculations from this may then be made as to the number of leucocytes per cubic centimeter. This method, and modifications of it, have been used by a large number of observers, but the value of the conclusions drawn from them has been much exaggerated. Normal milk may contain leucocytes in 26 Heinemann, Jour. Inf. Dis., 3, 1906. "Schottmuller, Munch, med. Woch., 1903. 28 Miiller, Arch, f . Hyg., Ivi, 1906. 29 Capps and Miller, Jour. A. M. A., June, 1912, p. 1848. 30 Hamburger, Bull, of the Johns Hopk. Hosp., xxiv, Jan., 1913. n Stokes and Weggefarth, Med, News, 91, 1897. BACTERIA IN MILK 1035 moderate numbers, and importance may be attached to such leu- cocyte counts only when their number largely exceeds that present in other specimens of perfectly normal milk. Whenever such high leucocyte counts are found, of course, a careful veterinary inspection and examination for pyogenic disease should be made. Foot-and-mouth disease, an infectious condition prevailing among cattle, characterized by a vesicular rash on the mouth and about the hoofs, has, in a number of cases, been definitely shown to be trans- mitted to man through the agency of milk. Notter and Firth32 reported an epidemic occurring among persons supplied with milk from a single dairy in which foot-and-mouth disease prevailed among the cows. In this epidemic, two hundred and five individuals were affected with vesicular eruptions of the throat, with tonsillitis and swellings of the cervical lymph nodes. Similar cases have been reported by Pott.33 Although anthrax has never been definitely shown to have been conveyed by milk, Boschetti34 succeeded in isolating living anthrax bacilli from a sample of milk two weeks after its withdrawal from the cow. Milk and Tuberculosis. — The question of the conveyance of tuber- culosis by means oi milk is a subject which, because of its great importance, has been extensively investigated by bacteriologists. A large number of observers have succeeded in proving the presence of tubercle bacilli in tho milk of tuberculous cows by intraperitoneal inoculation of rabbits and guinea-pigs with samples of milk. Such positive results have been obtained by Bang,35 Hirschberger,36 Ernst,37 and many others. A number of these observers, notably Ernst, have shown that tubercle bacilli may be present in the milk without tuberculous disease of the udders. In an examination of the milk supply of Washington, D. C.,38 6.72 per cent of the samples contained tubercle bacilli. The path of entrance of the bacilli from the cow into the milk 32 Notter and Firth, quoted from Harrington, ' ' Theory and Practice of Hygiene. ' ' 33 Pott, Munch, med. Woch., 1899. 34Hox<>lictti, fliorn. mod. vet., 1891. M #«./»//, Dent. /fit. f. Tierchem., xi, 1884. "Hirwlibrruc); Deut. Arch. f. klin. Med., xliv, 1889. 37 Ernst, H. C., Amer. Jour. Med. Sei., xcviii, 1890. 38 Anderson, Bull. No. 41, U. S. Pub. Health and Mar. Hosp. Serv., Wash., 1908. 1036 BACTERIA IN AIR, SOIL, WATER, AND MILK has long been a subject of controversy. That the bacilli may easily enter the milk, when tuberculous disease o'f the udder is present, stands to reason and is universally conceded. It is now believed also, on the basis of much experimentation, that in systemically infected cows tubercle bacilli may pass through the mammary glands into the milk, without evidence of local disease in the secreting gland. An experiment performed by the Royal British Tuberculosis Commission39 illustrates this point. A cow, injected subcutancously with tubercle bacilli behind the shoulder, began to discharge tubercle bacilli in the milk within seven days after inoculation and continued to do so until death from generalized tuberculosis. Milk may become indirectly contaminated, furthermore, with tubercle bacilli emanating from the feces of cows. It has been shown that tubercle bacilli are present in the feces of cattle so early in the disease that diagnosis can be made only by a tuberculin test.40 Whether or not contaminated milk is common as an etiological factor in human tuberculosis, must be considered at present as an unsettled question. Behring, at the Congress of Veterinary Medicine, at Cassel, in 1903, advanced the view that pulmonary tuberculosis in adults may be a late manifestation of a milk infection contracted during infancy. He stated as his own opinion, moreover, that most cases of tuberculosis in man are traceable to this origin. The problem is as difficult of solution as it is important. In bottle-fed infants, infection by means of milk unquestionably occurs with considerable frequency. Smith,41 Kossel, Weber, and Huess,42 and others, have isolated tubercle bacilli of the bovine type from the mesenteric lymph nodes of many infected children. Animal experimentation has, furthermore, revealed that lesions in the mesenteric nodes, as well as later in the bronchial lymph nodes, may occur as a consequence of feeding tubercle bacilli, without any demonstrable lesions in the intestinal mucosa. It is thus certain that infection by the ingestion of tuberculous milk may occur, especially among young children who, as is well-known, are comparatively susceptible to bacilli of the bovine type. Whether or not such infection will 30 Quoted from Mohlcr, P. TT., and Mar. Hosp. Serv. Bull. 41, 1908. 40 KcJirocdcr and Cotton, Bull. Bureau Animal Industry, Wash., 1007. 41 8 milk, Trans. Assn. Ainer. Physic., 18, ]JM):'.. 4- Kossel, Weber, and Huess, Tubcrkul. Arb. a. d. kais. Gesimdheitsamt, 1904, 1905, Hft. 1 and 3. BACTERIA IN MILK 1037' account for many cases of tuberculosis in adults is a question which, for final solution, will require much more investigation. The sole reliable method of approaching it lies in determining the type, human or bovine, of the bacilli present in a large number of cases. Experience thus far seems to indicate that the bovine type is com- paratively rare in the pulmonary disease of adults. The value of the tuberculin reaction for diagnosis, and the elimination of all cattle showing a positive reaction, for the preven- tion of tuberculosis, can not be overestimated. The failure of the test in diseased animals is rare, and an accurate diagnosis can be established in over 90 per cent of diseased animals.43 The assertion that the cattle are permanently injured by tuberculin injections is without scientific basis. If this test were conscientiously carried out, and infected cattle condemned, the dangers from bovine bacillus infection would be practically eliminated, for there are but few instances in which science has been able to furnish such definite information for absolute protection. It is needless to say, however, that the carrying out of such precautions is subject to great expense and great difficulties of organization. Dairy inspection is practiced in the vicinity of many of our larger cities, and the movement is daily gaining ground. Until fully estab- lished, however, upon a financial basis which brings the best products within the means of the poorer classes, other inexpensive measures to render milk safe must often be resorted to. Sterilization by high temperatures is objected to by pediatricians because of the physical and chemical changes produced in the milk which are said to detract from its nutritive value. The development of scurvy and rickets in infants has often been attributed to the use of such milk. These objections, however, do not apply to the use of milk which has been subjected to the process of ' 'pasteurization." By this term is meant the heating of any substance to 60° C. for twenty to thirty minutes. The process, first devised by Pasteur for the purpose of destroying germs in wine and beer in which excessive heating was supposed to injure flavor, brings about the death of all microorganisms which do not form spores — in other words, of all the bacteria likely to be found in milk which can give rise to infection per os. At the same time the chemical and physical constitution of the milk is not appreciably "Hohler, loc. cit. 1038 BACTERIA IN AIR, SOIL, WATER, AND MILK changed, at least not to an extent which renders it less valuable as a food. Statistics by Park and Holt have shown strikingly the advantages of pasteurized over raw milk in infant feeding. Of fifty-one children fed with raw milk during the summer months, thirty-three had diarrhea, two died, and only seventeen remained entirely well. Of forty-one receiving pasteurized milk, but ten had diarrhea, one died, and thirty-one remained entirely well throughout the summer. The actual diminution of the living bacterial contents of milk by pasteurization is enormous, the milk so treated often containing not more than one thousand, usually less than fifteen thousand, living bacteria to each cubic centimeter. Methods of Estimating the Number of Bacteria in Milk. — In es- timating the number of bacteria in milk, colony counting in agar or gelatin plates is resorted to. Great care must be exercised in obtaining the specimens. If taken from a can, the contents of the can should be thoroughly mixed, since the cream usually contains many more bacteria than the rest of the milk. The specimen is then taken into a sterile test tube or flask. If the milk is supplied in an ordinary milk bottle, this should be thoroughly shaken before being opened, and the specimen for examination taken out with a sterile pipette. Dilutions of the specimen can then be made in sterile broth or salt solution. If an initial dilution of 1 :100 is made, quantities ranging from 1 c.c. to 0.1 c.c. of this will furnish 0.01 c.c. to 0.001 c.c. of the milk, respectively. Inoculation of properly cooled tubes of melted neutral agar and gelatin, with varying quantities of these dilutions, are then made and plates poured. After twenty-four to forty-eight hours at room temperature or in the incubator, colony counting is done, and the proper calculation is made. In samples in which few bacteria are expected, direct transference of 1/20 or 1/40 of a c.c. of the whole milk into the agar may be made. This method saves time but is less accurate. Direct Methods of Counting Bacteria. — Direct methods of count- ing bacteria in milk have recently been advised, the one most extensively tried being that of Prescott and Breed. By this method a capillary tube is marked to measure accurately 0.01 c.c. This amount of the milk is spread over a square cm. on a microscope slide. It is dried in the air and fixed with methyl alcohol, after which the fatty constituents can be dissolved with xylol. It can then be stained lightly with the Jenner stain. The bacteria are counted under an oil immersion lens, the tube length and magnification being so BACTERIA IN MILK 1039 arranged that the microscopic field covers 1/50 sq. mm. A standard- ized eyepiece micrometer may be used. The average number of bacteria found in such fields may be multiplied by 5,000 to give the number of bacteria contained in 0.01 c.c. of milk. This method nas not yet displaced the one of plating and does not promise to do so for some time. For the isolation of special pathogenic bacteria from milk, no rules can be laid down, since, in every case, the method adapted to the particular organism sought for must be chosen. Tubercle bacilli can be isolated from milk with success only by guinea-pig injection. The milk is centrifugalized and 5 c.c. of the sediment, together with some of the cream that has risen to the top, is intraperitoneally or subcutaneously injected. The control of milk in the market depends upon careful regula- tions, which must include care of cattle, dairy inspection and bac- teriological control of the delivered milk. This is a subject which is too extensive to touch upon in a book of this kind. However, a general idea of the methods employed may be obtained by studying the accompanying table, which is taken from the New York City Department of Health Regulation for the Sale of Milk and Cream. Bacteria and Butter. — Butter is made from cream separated from milk either by standing or by centrifugalization. After this, the cream is agitated by churning, which brings the small fat-globules into mutual contact, allows them to adhere to each other and form clumps of butter. It has been a matter of common experience, how- ever, that unless the cream is allowed to "ripen" for a considerable period before churning, the resulting butter lacks the particular quality of flavor which gives it its market value. The interval of ripening, at first a necessity upon small farms where cream must be collected and allowed to accumulate, has now been recognized as an essential for the production of the best grades of butter, and it has been shown that the changes taking place in the cream during this period are referable to the action of bacteria. Cream, which before the ripening process contains but 50,000 bacteria to each cubic centimeter, at the end of a period of " ripening" will often contain many millions of microorganisms. At the same time, the cream becomes thick and often sour. The species of bacteria which take part in this process and which, therefore, must determine to a large extent the quality of the end product, are various and, as yet, incompletely known. Usually some 1040 BACTERIA IN AIR, SOIL, WATER, AND MILK REGULATIONS GOVERNING THE GRADES AND DESIGNATION OF MILK The following classifications apply to milk and cream. The regulations regarding GRADES OF MILK OR CREAM WHICH MAY BE SOLD IN THE CITY OF NEW YORK DEFINITION TUBERCULIN TEST AND PHYSICAL CONDITION BACTERIAL CONTENTS GRADE A Milk or Cream (Raw) Grade A milk or cream (raw) is milk or cream produced and handled in accord- ance with the minimum requirements, rules and regulations as herein set forth. 1. Only such cows shall be ad- mitted to the herd as have not reacted to a diagnostic injec- tion of tubercu- lin and are in good physical condition. 2. All cows shall be tested annually with tuberculin and all reacting ani- mals shall be ex- cluded from the herd. Grade A milk (Raw) shall not con- tain more than 60,000 bacteria per c.c. and cream more than 300,000 bacteria per c.c. when delivered to the consumer or at any time prior to such delivery. Milk or Cream (Pasteurized) Grade A milk or cream (pasteurized) is milk or cream handled and sold by dealers holding permits therefor from the Board of Health, and produced and handled in accordance with the requirements, rules and regulations as herein sot forth. No tuberculin test required but cows must bo healthy as dis- clossd by phys- ical examination made annually. Grade A milk (pasteurized) shall not contain more than 30,000 bacteria per c.c. and Cream (pasteurized) more than 150,000 bacteria psr c.c. when delivered to the consumer or at any time after pasteurization and prior to such delivery. No milk supply averaging more than 200,000 bacteria per c.c. shall be pasteurized for sale under this desig- nation. GRADE B Milk or Cream (Pasteurized) Grade B milk or cream (pasteurized) is milk or cream produced and handled in accordance with the minimum require- ments, rules and regulations herein set forth and which has been pasteurized in accordance with the requirements and rules and regulations of the Department of Health for pasteurization. No tuberculin tests required but cows must be healthy as dis- closed by phys- ical examination made annually. No milk under this grade shall con- tain more than 100,000 bacteria per c.c. and no cream shall contain more than 500,000 bacteria per c.c. when delivered to the consumer or at any time after pasteurization and prior to such delivery. No milk supply averaging more than 1,500,000 bacteria per c.c. shall be pasteurized in this city for sale under this designation. No milk supply averaging more than 300,000 bacteria per c.c. shall be pasteurized outside of this city for sale under this designation. GRADE C Milk or Cream (Pasteurized) (For cooking and manu- facturing pur- poses only.) Grade C milk or cream is milk or cream not conforming to the requirements of any of the subdivisions of Grade A or Grade B and which has been pasteurized according to the requirements and rules and regulations of the Board of Health or boiled for at least two (2) minutes. No tuberculin test required but cows must be healthy as dis- closed by phys- ical examination made annually. No milk of this grade shall contain more than 300,000 bacteria per c.c. and no cream of this grade shall con- tain more than 1,500,000 bacteria per c.c. after pasteurization. NOTE — Sour milk, buttermilk, sour cream, kumyss, matzoon, zoolac and similar products shall not be made the process of souring. Sour cream shall not contain a less percentage of fats than that designated for cream. No other words than those designated herein shall appear on the label of any container containing milk or cream The tsrm "certified" milk is usually defined for each region by a special commission of the County Med. Soc., County, N. Y.: CERTIFIED MILK must have every characteristic of pure, clean, fresh, wholesome cow's milk. The milk must Nothing must be added to the milk and nothing taken awav. CERTIFIED MILK shall not contain less- than 4 ner cent of butter fat. * Table taken from Rules and Regulations of N. Y. City Department of Health, 1914, applying to sale of milk BACTERIA IN MILK 1041 AND CREAM WHICH MAY BE SOLD IN THE CITY OF NEW YORK * bacterial content and time of delivery shall not apply to sour cream NECES- SARY SCORES FOR DAIRIES TIME OF DELIVERY BOTTLING LABELING PASTEURIZA- TION PRODUC- ING Unless other- wise specified in Equip. 25 Shall be de- livered within the permit this milk or cream Outer caps of bottles shall be white and shall contain the words Grade A, Raw, Meth. 50 36 hours after shall be delivered in black letters in large type, and shall production. to the consumer state the name and address of the dealer. Total 75 only in bottles. Equip. 25 Meth. 43 Total 68 Shall be de- livered within 36 hours after pasteurization. Unless other- wise specified in the permit this milk or cream shall be delivered to the consumer only in bottles. Outer caps of bottles shall be white and shall contain the words Grade A in black letters in large type, date and hours be- tween which pasteurization was com- pleted; place where pasteurization was performed; name of the person, firm or the corporation offering for sale, selling or de- Only such milk or cream shall be regarded as pasteurized as has been sub- jected to a tem- perature averag- livering same. ing 145° Fahr. for not less than 30 minutes. Outer caps of bottles containing milk and tags affixed to cans containing milk or cream shall be white and marked Only such "Grade B" in bright green letters in milk or cream Equip. 20 Milk shall be delivered within large type, date pasteurization was com- pleted, place where pasteurization was shall be regarded as pasteurized as Meth. 35 36 hours and May be deliv- performed, name of the person, firm or has been sub- cream within 48 ered in cans or corporation offering for sale, selling or de- jected to a tem- Total 55 hours after pas- teurization. bottles. livering same. Bottles containing creams shall be labeled with caps marked "Grade perature averag- ing 145° Fahr. for B" in bright green letters, in large type and shall give the place and date of bot- not less than 30 minutes. tling and shall give the name of person, firm or corporation offering for sale, selling or delivering same. Only such milk or cream Shall be de- Tags affixed to cans shall be white and shall be regarded livered within May be deliv- shall be marked in red with the words as pasteurized as Score 40 48 hours after ered in cans only. "Grade C" in large type and "for cook- has been sub- pasteurization. ing" in plainly visible type, and cans shall have properly sealed metal collars, painted jected to a tem- perature averag- red on necks. ing 145° Fahr. for not less than 30 minutes. from any milk of a less grade than that designated for "Grade B" and shall be pasteurized before being put through or milk or cream products except the word "certified" when authorized under the State laws. sanction by State Law. The following is the definition of certified milk given by the Milk Commission of Kings be in its natural state, not having been heated and without the addition of coloring matter or preservatives, and cream. 1042 BACTERIA IN AIR, SOIL, WATER, AND MILK variety of lactic-acid bacilli is present and these, as in milk, outgrow other species and, according to Conn,44 are probably essential for ' ' ripening. ' ' It would be of great practical value, therefore, if definite pure cultures of the bacteria which favor the production of agreeable flavors could be distributed among dairies. In Denmark this has been attempted by first pasteurizing the cream and then adding a culture of bacteria isolated from " favorable " cream. These cul- tures, delivered to the dairyman, are planted in sterilized milk, in order to increase their quantity, and this culture is then poured into the pasteurized cream. In most cases, these so-called " starters" are not pure cultures, but mixtures of three or more species derived from the original cream. Adverse accidents in the course of butter-making, such as "sour- ing" or "bittering" of butter, are due to the presence of con- taminating, probably proteolytic, microorganisms in the cream during the process of "ripening." As a means of transmitting infectious disease, butter is of im- portance only in relation to tuberculosis. Obermuller,45 Rabino- witch,46 Boyce,47 and others, have repeatedly found tubercle bacilli in market butter, and Mohler,48 Washburn, and Rogers have recently shown that these bacilli could remain alive and virulent for as long as five months in butter kept at refrigerator temperature. The acid-fast butter bacillus, described by Rabinowitch as similar to the true Bacillus tuberculosis, shows decided cultural and mor- phological differences from the latter. Bacteria and Cheese. — The conversion of milk products into cheese consists in a process of protein decomposition which, by its end products, leucin, tyrosin, and ammonia compounds, largely determines the cheese-flavors. The production of cheese, therefore, is due to the action of proteolytic bacterial enzymes49 and the variety of a cheese is largely determined by the microorganisms which are present and by the cultural conditions prevailing. The sterilization "Conn, "Agricultural Bacteriology," Phila., 1901. 45 Obermuller, Hyg. Eundschau, 14, 1897. *• Rabinowitch, Zeit. f. Hyg., xxvi, 1897. 47 Boyce and Woodhead, Brit. Med. Jour., 2, 1897. 48 Mohler, U. S. P. H. and Mar. Hosp. Serv. Bull. 41, 1908. 49 Freudenreich, Koch's Jahresbericht, etc., 135, 1891. BACTERIA IN MILK 1043 of cream, or the addition of antiseptics, absolutely prevents cheese production. The organisms which arc concerned in such processes have been extensively studied and attempts have been made, with moderate success, to produce a definite flavor with pure cultures. In the production of cheese the two varieties, hard and soft cheeses, depend not so much upon the bacterial varieties as upon the differences in the treatment of the curds before bacterial action has begun. In the former case, a complete freeing of the curds from, the whey furnishes a culture medium which is comparatively dry and of almost exclusively protein composition; in the latter, reten- tion of whey gives rise to cultural conditions in which more rapid and complete bacterial action may take place. The holes, which are so often observed in some of the hard cheeses, are due to gas production during the process of "ripening." As to the varieties of microorganisms present in various cheeses, much careful work has been done. Duclaux50 attributed the "ripen- ing" of some of the soft cheeses to a microorganism closely related to Bacillus subtilis. V. Freudenreich51 in part substantiated this, but laid particular stress upon the action of Oidium lactis, a mold, and upon several varieties of yeast. Conn,52 more recently, in a bacteriological study of Camembert cheese, has demonstrated that the production of this cheese depends upon the united action of two microorganisms, one an oi'dium, like the Oidium lactis of Freuden- reich, which is found chiefly in the interior softened areas, the other a mold belonging to the penicillium variety, found in a matted felt- work over the surface and penetrating but a short distance. In spite of the scientific basis upon which the work of these men and of others has seemed to place cheese production, attempts at uni- formity in cheese production have met with almost insuperable obstacles because of the presence of a variety of adventitious micro- organisms which, depending in species and proportion upon the local conditions under which the various cheeses have been produced, have added minor characteristics of flavor which have determined market value. Occasional failure of good results in cheese produc- 60 Duclaux. "Le Lait," Paris, 1887. 61 V. Freudenreich, Cent, f . Bakt., II, i, 1895. *-C&nn, Bull. Statis. Agri. Exp. Stat. 35, 1905. 1044 BACTERIA IN AIR, SOIL, WATER, AND MILK tion53 is due to contamination with other chromogenic or putrefactive bacteria. In its relationship to the spread of infectious disease, cheese is relatively unimportant except in regard to tuberculosis. Typhoid and other non-spore forming pathogenic germs can not survive the conditions existing during cheese-ripening for any length of time. Tubercle bacilli, both of the human and bovine types, have been found in cheese by Harrison54 and others, and Galtier has shown experimentally that tubercle bacilli may remain alive and virulent in both salted and unsalted cheese for as long as ten days. THE LACTIC-ACID BACILLI AND METCHNIKOFF'S* BACTERIOTHERAPY A problem which has occupied clinical investigation for many years is that of gastrointestinal autointoxication. There are a number of conditions occurring in man, in which symptoms pro- foundly affecting the nervous system, the circulation, and, in a variety of ways, the entire body, can be clinically traced to the intestines, and can, in many cases, be relieved by thorough purgation and careful diet. In some of these conditions, specific microorgan- isms can be held accountable for the diseases (B. enteritidis, B. botulinus, etc.). In other cases, however, etiological investigations have met with but partial success because of the large variety of microorganisms present in the intestinal tract and because of the complicated symbiotic conditions thereby produced. Intestinal putrefaction, recognized as the cardinal feature of such maladies, has been attributed to Bacillus proteus vulgaris,55 to Bacillus aerogenes capsulatus, to Bacillus putrificus,56 and to a number of other bacteria, but definite and satisfactory proof as to the etiological importance of any of these germs has not yet been advanced. The fact remains, however, that, whatever may be the * See also B. Acidophilus, etc., in another section of this book. M Beijerinck, Koch's Jahresber, etc., 82, 189. "Harrison and Galtier, quoted from Mohler, U. S. Pub. H. and Mar. Hosp. Serv., Hygiene Lab. Bull. 41, 1908. 65 Lesage, Eev. de med., 1887. 64 Tissier, Ann. de 1 'inst. Pasteur, 1-905. BACTERIA IN MILK 1046 specific cause, the disease itself, a grave and often fatal affliction, may be clinically traced, in a number of cases, to the absorption of poisons from the intestinal canal, and it is more than likely that these poisons are the products of bacterial activity. Reason dictates, furthermore, that the bacteria primarily responsible for the produc- tion of these toxic substances do not belong to the varieties which attack carbohydrates only, but must belong to that class of aerobic and anaerobic germs which possess the power of breaking up proteins —in other words, the bacteria of putrefaction. On the basis of the mutual antagonism existing in culture be- tween many acid-producing bacteria and those of putrefaction — a phenomenon recognized by some of the earliest workers in this field, many investigators have suggested the possibility of combating intestinal putrefaction by adding acid-forming bacteria together with carbohydrates to the diet of patients suffering from this condi- tion. The first to suggest this therapy was Escherich57 who proposed the use, in this way, of Bacillus lactis aerogenes; with the same end in view, Quincke,58 a little later, suggested the use of yeasts — Oi'dium lactis. The reasoning underlying these attempts was mean- while upheld by experiments carried out both in vitro and upon the living patient. Thus Brudzinski5^ was able to demonstrate that Bacillus lactis aerogenes, in culture, inhibited the development of certain races of the proteus species and succeeded in obtaining markedly favorable results by feeding pure cultures of Bacillus lactis aerogenes to infants suffering from fetid diarrhea. Similar experiments60 carried out with the Welch bacillus (aerogenes cap- sulatus) and Bacillus coli, however, had no such corroboratory results, since this anaerobe possesses a considerable resistance against an acid reaction. In considering the difficulties of the problems in- volved in this question, it occurred to Metchnikoff61 that much of the practical failure of therapy, based upon the principles stated above, might be referred to insufficent powers of acid production on the part of Bacillus coli, Bacillus lactis aerogenes, and other germs 57 Escherich, Therapeut. Monatshefte, Oct., 1887. **Quincke, Verhandl. des Congress f. Inn. Med., Wiesbaden, 1898. 69 Brudzinski, Jahrbuch f. Kinderheilkunde, 52, 1900 (Erganzungsheft). 80 Tissier and Martelly, Ann. de 1 'inst. Pasteur, 1906. n Metchnikoff, "Prolongation of Life/' G. P. Putnam's Sons, N. Y.; also .in ' ' Bacteriotherapie, " etc. "Bibliotheque de therapeutique, " Gilbert and Carnot, Paris, 1909. 1046 BACTERIA IN AIR, SOIL, WATER, AND MILK \ previously use'd. In searching for more powerful acid producers, his attention was attracted to Bacillus bulgaricus, isolated from milk by Massol62 and Cohendy63 in 1905. This bacillus, according to the researches of Bertrand and Weisweiller,6* produces as much as 25 grams of lactic acid per liter of milk. In addition to this, it manufactures, from the same quantity of milk, about 50 cen- tigrams of acetic and succinic acids and exerts no putre- factive action upon proteins. Added to these characters, it is especially adapted to thera- peutic application by its com- plete lack of pathogenicity. The administration of the bacillus to patients suffering from intestinal putrefaction, first suggested by Metchnikoff in 1906, has, since that time, been extensively practiced and often with remarkable success. In spite of sharp criticism, especially by Luersen and Kiihn,65 who deny much of the antiputrefactive activity of the bacillus, the treatment of Metchnikoff has found many adherents, upon the basis of purely clinical experi- ment. It is not possible to review completely the already extensive literature. Among the more valuable contributions may be mentioned the articles by Grekoff,66 by Wegele,67 and by Klotz.68 In Metchni- koff's experiments and in the work of his immediate successors, the FIG. 119. — BACILLUS BULGARICUS. ™Massol, Eevue medicale de la Suisse romande, 1905. 63 Cohendy, Comptes rend, de la soc. de biol., 60, 1906. "Bertrand and Weisweiller, Ann. de Pinst. Pasteur, 1906. 85 Luersen and Kuhn, Cent. f. Bakt., II, xx, 1908. "Grckoff, " Observations cliniques sur 1'effet du lact. agri.," etc., St. Peters- burg, 1907. «7 Wcyele, Deut. med. Woch., xxxiv, 1908. "Klotz, Zentralbl. f. innere Med., 1908. BACTERIA IN ILKM 1047 bacillus was used cither in milk culture or in broth in which it was induced to grow in symbiosis with other microorganisms. BACTERIOLOGICAL EXAMINATION OF OYSTERS On account of the danger of the transmission of typhoid fever by oysters which have been bred or stored in contaminated water, standard methods69 have been devised for the estimation of the bacterial content of oysters. These are similar in principle and method to those used for the examination of water, and a most important index of sewage contamination and consequent danger of typhoid infection is the number of colon bacilli present in the shell fish. The shell liquor is used for examination, and in examining oysters in the shell the following procedure is followed : Five oysters having deep bowls and closed shells are selected. Lips of the shell are sterilized in the flame or by burning with alcohol. The liquor is obtained by opening the shell with a sterilized knife, or better, by drilling a hole through the flame surface with a sterilized gimlet. For determining the total number of bacteria the shell liquor is with- drawn with a sterilized pipette, diluted with 1 per cent salt solution, and placed in agar. More important, however, is the presumptive colon test, which is carried out by inoculating three lactose bile tubes with 1.0 c.c., 0.1 c.c., and 0.1 c.c., respectively, from each of the five oysters. The tubes are incubated for three days, and the development of over 10 per cent of gas in the closed arm is con- sidered a positive reaction. The score is recorded as the approximate number of colon bacilli contained in the 5.55 c.c. of shell liquor from the five oysters, and is estimated in the following way: A positive reaction in a tube inoculated in 1 c.c. is recorded as 1.0, a positive reaction in 0.1 c.c. is 10, and in 0.01 is recorded as 100. The sum of these figures is the score for the batch of oysters from which the five have been taken. In examining shucked oysters a well-mixed sample of oysters and the surrounding fluid are put in a sterilized vessel and lactose bile tubes inoculated in triplicate with 1.0 c.c., 0.1 c.c., 0.01 c.c., 0.001 c.c. of the liquor. No definite standard score has been adopted, but the United States Pure Food Board has condemned unshucked stock having a score of 32 or higher. 69Amer. Jour. Pub. Health, 1913, ii, 34. SECTION VII PATHOGENIC PROTOZOA FREDERICK F. RUSSELL, M.D. INTKODUCTION IN the practice of his profession the physician requires a knowl- edge of the pathogenic protozoa found in man and the domestic animals and of their closely related non-pathogenic forms. Quite commonly in the diagnosis of fevers it is necessary to examine the blood of the same patient for both malaria and bacteria, therefore a working knowledge of the principal pathogenic protozoa is essen- tial. In this work it will be possible to describe the forms only of medical interest, and the reader is referred to other works for further information. The protozoa are unicellular animal organisms that occur singly or in temporary colonies. The functions of the animals are carried out by the protoplasm of the single cell, parts of which may be differentiated for special purposes and are then called organete. CLASSIFICATION OF THE PROTOZOA CLASS I. SARCODINA (Rhizopoda) . — The body is naked or encased and the animal moves by means of protruding temporary prolongations of the body called pseudopods. They possess one or many nuclei and reproduce by fission or multiplication in a cyst. Order I. Amcebce (Lobosa). — Naked or with a simple shell, the pseudopia are lobose or finger-shaped, the nucleus is usually single and there is sometimes a contractile vacuole. Example, the amcebce. CLASS II. MASTIGOPHORA (Flagellata). — They possess flagella for locomotion and for obtaining food; they may be naked or fur 1048 INTRODUCTION 1049 nished with a membrane ; many forms possess nucleus, contractile vacuole and a small groove spoken of as the cytostome. Examples, the trypanosomes and intestinal flagellates. CLASS III. SPOROZOA. — They live parasitically in the tissues of other animals, ingesting food by osmosis; they have no cilia in the adult stage but may form pseudopoda, one or more nuclei, no contractile vacuole, reproduction by spores. They are divided into two subclasses, telesporidia and neosporidia. Examples, gre- garinida, coccidiidea,, hemosporidia, sarcosporidia, etc. CLASS IV. INFUSORIA (Ciliata). — The body is generally uniform in shape, with cilia and contractile vacuole, and usually with macro- and micronucleus. Examples, pammecium, balantidium. CHAPTER LIV CLASS I— SARCODINA (RHIZOPODA) THE AMOEBA THESE organisms belong to the order Amcebina (Ehrenberg). They are characterized during the vegetative stage by a semifluid consist- ence, permitting rapid changes of form, amoeboid movements, and progression by means of pseudopods. There is no internal skeleton and the protoplasm is naked and may be differentiated into endo- and ectoplasm, and in some cases a contractile vacuole is present. All forms possess one or more nuclei. Multiplication takes place by divi- sion into two or more daughter cells. Fertilization possibly takes place by the conjugation of two gametes. . Since some flagellates possess an amoeboid stage, it is necessary to know most of the life cycle of an organism before classifying it as an ameba. The protoplasm varies greatly in its consistency, depending on the species as well as the stage of the life cycle, and the environ- ment and food supply. Most amoeba, including all the parasitic forms (entamoebae), possess a single nucleus, yet Amoeba diploidea and Amoeba binucleata always have two, and the other species may show more. The nucleus of all types possesses a karyosome. The nucleus is well developed and in it may be followed either a simple or typical mitosis. The cytoplasm is usually at some stage divided into a granular endo- plasm and a clear or hyalin ectoplasm, the latter forming the pseu- dopods by which the animal moves from place to place. Until recent years all amoeboid organisms were placed in the genus Amoeba, but Schaudinn established a genus, the Entamoeba, for the parasitic species which have many points of difference from the free living varieties. Of the free living forms, the easiest to study is the Amoeba proteus (Pallas), a very large organism, 200 microns in diameter, found frequently in stagnant water; it, however, has no direct importance in medicine. Another group of free living amoeba) is of some interest, because of the confusion they have caused in the study of parasitic amoeba; they are the so-called "limax amcrbse," 1050 SARCODINA 1051 which have been cultivated on agar, and for this genus Chatton (1912) has proposed the name Valilkampfia. They are small organisms, 5 to 30 microns in diameter, provided with fingerlike or spinous pseu- dopodia, and characterized by a nucleus witli a large karyosome and a single nucleated resistance cyst in which no multiplication occurs. They have repeatedly been cultivated from human dysenteric stools, from the air, and apparently from liver abscess pus. It has been shown, beyond doubt, that they are harmless to man, and that they pass through the intestinal tract with food and water in the cyst FIG. 120. — ENTAMBGEA HISTOLYTICA. Vegetative form showing histolytica type of nucleus. Stained with iron hematoxylin. (Army Medical School Collection, Washington, D. C.) form. While they will develop in cultures at body temperature, a better growth is obtained at the temperature of the room. Since the true parasitic amoeba? have never been cultivated on artificial media, the Vahlkampfia may be dismissed with the statement that they are not pathogenic. The genus Entamoebse includes all human parasitic forms, and is characterized, among other things, by the absence of a contractile vacuole, which is always present in Amo?ba and Vahlkampfia. The 1052 PATHOGENIC PROTOZOA species of importance to physicians are Entamoeba liistolytica, En- tamceba coli and Entamceba gingivalis. Leidy of Philadelphia established the genus endamceba which may possibly be closely related to or identical with the genus entamocba, for the large amoeba which is parasitic in the cockroach, and called it Endamceba blattce; this genus presents ; some points of resemblance to those present in man, but its life history has not been sufficiently studied by our present methods and the merging of the two at this period seems scarcely justifiable. The nomenclature of the human parasitic entamoebae is shown in the following table which has been taken from Dobell (1919) : SYNOPSIS OF GENERA AND SPECIES OF AMCEB.^E LIVING IN j MAN Genus I. ENTAMCEBA (Casagrandi and Barbagallo, 1895. (nee Endamoeba Leidy, 1879.) Synonyms : Poneramceba Liihe, 1908. •j Chatton and Lalung-Bonnaire, 1912. Loschia [ Proctambceba Alexeieff, 1912 (Amoeba (pro parte), Endamceba, Entameba, Endameba, En-, tamoba, Auctt.) Type: E. coli (Grassi) Casagrandi and Barbagallo. Species in Man: E. coli (Grassi) Casagrandi and Barbagallo. E. Jiistolytica Schaudinn (emend. Walker). E. gingivalis (Gros) Brumpt. Genus II. ENDOLIMAX Kuenen and Swellengrebel, 1917. Only species, hence type: E. nana (Wenyon and O'Connor) Brug. Genus III. IODAMCEBA nov. gen. Only species, hence type: I. butschlii (Prowazek) Dobell. Genus IV. DIENTAM(EBA Jepps and Dobell, 1918. Only species, hence type: D. fragilis Jepps and Dobell, SARCODINA 1053 ENTAMCEBA HISTOLYTICA (EntamoBba tetragena [Viefeck], Entamceba africana [Hartmann] Entamceba nipponica [Koidzumi, pro parte], Entamoeba tropicalis [Lesage, pro parte] ) It has long been customary to say that amoebse as a cause of disease were first described by Lambl of Prague, in 1860, who found them present in the stools from a case of severe diarrhea in a child, but some zoologists, Leuckart (1863), Grassi (1888) and Dobell (1919) believe that the organisms he described were degenerated trichomonads. FIG. 121. — ENTAMCEBA HISTOLYTICA. Vegetative form, simple division. (X 1300.) (Army Medical School Collection, Washington, D. C.) In 1870 Lewis and Cunningham found amoebae in 20 per cent of the stools of cholera patients, but attached no pathogenic importance to them. The first accurate description we owe to Loesch of Petrograd, who in 1875 studied an undoubted case of amosbic dysentery with relapses, and he named the organism Amoeba coli. He was further successful in reproducing the disease in a dog, and thus began its experimental investigation. Not much progress was made until Kartulis in Egypt began, in 1886, the publication of a long series of studies which has continued up to the present time, and because of the rich clinical and pathological material at his disposal his work has been of the greatest value. In 1890 Osier published the first paper 1054 PATHOGENIC PROTOZOA in America. He was followed by Musser and Stengel and, in 1891, by Dock, and Councilman and Lafleur. The work of the last two authors was especially complete and firmly established the entity of this disease in America. In 1902 Jiirgens differentiated the pathogenic amoeba from the harmless, and in 1903 the work of Schaudinn ap- peared. This author, who was a zoologist by training, showed clearly that there were two forms of parasitic amoebae and he followed out most of the details in their life history, renaming them Entamoiba histolytica and Entamozba coli. Schaudinn accepted the name for the genus proposed by Casagrandi and Barbagallo (1895) but named his pathogenic species histolytica and the non-pathogenic coli. These names are still in use although the work of later investigators has shown that many of his observations were erroneous. Our present knowledge of this organism we owe to the work of Craig, Whitmore, Walker, Sellards, Darling, Dobell and others. CLINICAL DYSENTERY Dysentery as a disease has been known from the earliest times and references are found to it in Sanscrit and Egyptian literature and in early Greek and Roman writings. Until recent years its etiology was obscure, but we now recognize two separate forms, bacillary and amoebic ; the former has already been described under the dysentery bacilli. Amoebic dysentery is a distinct clinical entity, and runs a course quite different from the bacillary form. It begins gradually, and in some cases is chronic in character from the start. Usually there is no rise in temperature nor any great change in weight or health until the disease has existed some time. The bowel movements become gradually more frequent and the fecal matter is accompanied by larger and larger amounts of mucus and blood. As the disease progresses and more and more of the colon is involved the amount of blood and mucus increases until the stool contains little else. The colicky pains increase in frequency and severity and there is added tenesmus and finally nausea and vomiting. The patient loses flesh and strength and when the stools increase to twenty and thirty daily, becomes bed-ridden. The abdomen is con- cave and tender on pressure, especially over the colon. The course of the disease, if untreated, tends to progress with periods of remis- sion, and spontaneous cure probably does not occur. Bacillary SARCODINA 1055 dysentery, it will be remembered, is a disease with a short incuba- tion period and an acute onset; after two or three days' illness the bacillary ease is confined to bed, is pale, weak, emaciated and presents every evidence of profound toxemia ; an amoebic case, sick the same length of time, will be up and about and perhaps will not have applied for treatment. Complications. — A common and most dangerous complication is abscess of the liver. The amoebae travel from the ulcers in the colon by way of the lymphatics to the liver and there set up a liquefying necrosis of the parenchyma. The liquefied portion contains a red- dish or chocolate-colored fluid, which is not pus in the ordinary- sense, although it may become a pus-containing abscess if secondary bacterial infection occurs. Liver abscesses may be single, but are much more often multiple, and at times the whole liver may be riddled with large and small abscess cavities; both right and left lobes may be involved. If surgical interference be withheld, the abscess increases in size, approaches the surface, and finally ruptures into the lung through the diaphragm or into the peritoneal cavity. A few cases of amoebic abscess of the brain have been reported. (Kartulis, 1904.) At autopsy the lesions are found in the colon, principally at the sigmoid flexure and in the cecum, though in chronic cases the whole colon is involved, showing ulcers with undermined edges, swollen solitary follicles and a hemorrhagic-catarrhal inflammation of the mucous membrane. The ulcers, readily differentiated from those caused by the tubercle bacillus, are of all sizes, shallow or deep, and are characterized by irregular margins and undermined edges. Fresh smears made at autopsy will show vegetative amoebae. In chronic cases, the colon is a mass of scars and ulcers and acutely inflamed, swollen and thickened mucous membrane resting on a hypertrophied submucosa. The severe and chronic forms of the disease are now as rare as they were formerly common as a result of the present specific treatment with emetin. Geographical Distribution. — Although amoebic dysentery is classed among the tropical diseases, it is by no means confined to the tropics. In the United States, for example, it is endemic as far north as Baltimore and Washington, and cases are not very infre- quent in the northern tier of states; hence one must examine the stools for amoebae in dysenteric cases regardless of the location of the patient's home. 1056 PATHOGENIC PROTOZOA Diagnosis. — While the "history of the case may suggest amoebic infection, the diagnosis can only be made with certainty by micro- scopic examination of the stool. For this purpose the examination should be made as soon after the stool is passed as possible; and in this disease it is usually practicable to have the patient come to the hospital, clinic or office and pass a stool there. It may then be examined immediately. If this is impracticable, the stool may be kept warm and sent to the laboratory in a small glass jar inside a tin pail partly filled with water at body heat; a little cloth or absorbent cotton will hold the hot water and prevent splashing during transit. The stool will show bloody mucous masses, and small drops of this are placed on slides, protected with a cover glass and ringed with warm vaseline to prevent evaporation. The prepara- tion, to be of value, must be thin, and the bloody mucus may be diluted with salt solution if necessary. Except in hot weather, the slide should be examined on a warm stage, or the slide may be warmed by placing heated coins on it, near the cover glass. At least half a dozen slides should be examined before reporting a negative result. Since the entamoebae degenerate and die soon after the stool is passed, it is particularly important, when studying the life history, to use only the very freshest material. Dobell1 believes that most of the mistakes which have been made in studying the life history of these organisms have been caused by the examination of de- generated or dead parasites, in which both nucleus and cytoplasm may have been abnormal. For the study of living amoebae and cysts it is helpful to mix a particle of stool in a drop of salt solution on one slide and in a drop of iodine solution on another. (Iodine should be used as a strong aqueous solution, in potassium iodide— the stronger the bet- ter. Dobell.) The iodine penetrates the cyst wall, and if glycogen be present in the vacuoles, gives it the characteristic color; it also acts as a fixative and renders the nuclei easily visible, so that they may be counted and the details of structure made out fairly well. Stained preparations are not difficult to prepare, although the process requires some time and care. As in most zoological work, wet, rather than dry, fixation is used. Thin smears are made on cover glasses or slides and before they can dry are covered with or immersed in Schaudinn's fluid. This is a mixture of two parts 1 Dobell, Clifford, The Amoebae Living in Man, London, 1919. SARCODINA 1057 of a saturated solution of bichlorid of mercury in normal salt solution and one part of absolute alcohol. The mercuric solution is prepared by adding to boiling normal salt solution a little more mercury than will dissolve; on cooling, some of the bichlorid crystallizes out. At no stage of the process must the preparation become dry or the smear is worthless. 1. Fix in hot (60° C.) Schaudinn's fluid, five to ten minutes. 2. Harden in 70 per cent alcohol ten to thirty minutes, then wash in 70 per cent alcohol to which a few drops of tincture of iodin have been added until it is distinctly colored — ten minutes; store in 70 or 80 per cent alcohol until ready to stain. FIG. 122. — ENTAMCBBA HISTOLYTICA. Motile forms showing ingested blood cells and clear rectoplasm. (Army Medical School Collection, Washington, D. C.) 3. The Rosenbusch hematoxylin is quite satisfactory. Transfer the slides to distilled water and change several times until they are free from alcohol, then immerse in 3.5 per cent iron-alum solution for from half an hour to overnight. 4. Stain in the following solution, after rapid washing in distilled water : (a) 1 per cent hematoxylin in 95 per cent alcohol. (6) Saturated aqueous solution of lithium carbonate. Solution (&) is added to solution (a) until the mixture is a cherry red, four or five drops of lithium to 10 c.c.-of hematoxylin is sufficient. The solution is either pipetted onto the slides or they are im- mersed in it. Stain from twenty minutes to overnight. 5. Wash thoroughly in distilled water. 6. Differentiate with a weak iron-alum solution (three parts of 1058 PATHOGENIC PROTOZOA distilled water to one of the iron-alum solution is satisfactory), until the slide under the microscope shows the structure of the nucleus; the examination is made in water under a cover glass. 7. When the differentiation is complete the slide is washed in distilled water and passed through graded alcohols, 80, 95 and absolute into xylol and xylol-balsam. This stain is permanent. Romanowski stains on dried smears may be used, but are not so good. In fresh specimens Entamoeba histolytica presents the following appearance: the vegetative forms are pale, unstained with bile, and are seen to be large bodies, 20 to 30 microns in diameter, consisting of endo- and ectoplasm, and often showing a delicate nucleus and also many inclusions in the digestive vacuoles, principally red blood cells. The organisms for several hours after the stool is passed remain actively motile, pushing out clear, glass-like pseudopods, into which the granular endoplasm pours as the amoeba progresses across the field. Even when there is no progression the pseudopods are protruded or retracted first in one then in another direction. There is usually, during motion, a distinct separation of the clear ectoplasm from the granular endoplasm, and the latter, in acute cases es- pecially, contains many red blood cells, occasional examples show- ing as many as twenty or thirty. The presence of red blood cells either entire or partly digested is characteristic of Entamoeba his- tolytica. The amoeba is sometimes greenish, and it is supposed that this color is due to hemaglobin liberated from the ingested red cells. The pseudopods of this species are clear, glassy and evidently viscid and dense and have given it its name "histolytica," since Schaudinn states that he saw the amoeba penetrate the mucous membrane, the pseudopods dissecting apart the epithelial cells. It is much more probable however, that the parasite secretes a strong ferment, which first softens and then dissolves the tissue cells. The nucleus, when the endoplasm is packed with inclusions, may not be visible, but further search will reveal amoebae showing a nucleus. It is vesicular, with a delicate limiting membrane, and as it is highly refractile, may appear as a clear bright spot. As the specimen grows older the amoebae lose much of their motility and the nucleus may become clearly visible, revealing small chromatic dots or masses adherent to its inner surface and a small central karyosomo. The motile amoebae cannot be confused with anything else, but when in the resting stage they have been mistaken for swollen and SARCOD1NA 1059 edcmatous epithelial cells. A little attention to the nucleus will prevent this error, since the tissue-cell nucleus is large, distinct, and entirely different from the nucleus of an amoeba. In specimens stained with hematoxylin the finer details, especially in the nucleus, may be studied, but stained preparations are never necessary for clinical diagnosis. In smears from fresh cases vegeta- tive forms only are found, later many degenerative forms appear and during convalescence only cysts may be seen. In stained speci- mens there is rarely any separation of ectoplasm and endoplasm, but the nucleus is always visible. The cytoplasm is granular and has a coarse honey-combed appearance. The nucleus shows a dis- FIG. 123. — ENTAMCEBA HISTOLYTICA. (Army Med. School Collection, Wash- ington, D. Cj FIG. 124. — ENTAMCEBA HISTOLYTIC.— (X 1150) Cyst, showing four nuclei, two of which are very distinct, and large chromatoid body. (Army Med. School Collection, Washington, D. C.) tinct, though delicate, limiting membrane, on the inner surface of which are few or many chromatin dots. In the center is a small karyosome, which may show a central body or centriole. The outer zone of the nucleus has a honey-comb structure, in which are im- bedded granules of chromatin. Multiplication in the vegetative stage is by division into two daughter cells ; in the intestinal contents, it is difficult to find forms which are undergoing division, but a few have been described and pictured. In experimental dysentery, in the cat, however, all stages of the process may be followed by removing the intestine 1060 PATHOGENIC PROTOZOA from an infected animal which has been killed and making serial sections of the ulcers. In such sections it can be seen that the chromatin of the nucleus migrates from the nuclear membrane to- ward the center, and the nucleus elongates, being first oval and later spindle shaped; although the chromatin dots and threads and the achromatic fibers can be readily seen in the well stained speci- mens they do not show the usual typical figures of a typical mitosis, but are arranged in an atypical and irregular manner. The elongated nucleus becomes constricted in the middle and a little later divides and the cytoplasm soon follows, leaving two daughter cells. Degenerative Forms. — These are extremely common in stale stools, in cases during convalescence, or under active treatment, and also in experimental dysentery in the cat, and they have led to much confusion in the past. The nucleus breaks up into fragments and chromatin masses are extruded into the cytoplasm in irregular forms, and parts of the cells are apparently budded off. At one time the budding process was looked upon as normal by Schaudinn and his followers, but there is now little doubt that both spores and buds are degenerative changes and that the animal multiplies only by binary fission in the vegetative forms or by the development of four nuclei in the cysis. Cyst Formation. — The encystment follows the general rule in that under suitable conditions, an amoeba comes to rest, ejects all food particles from the cytoplasm which becomes finally granular, and round, and then secretes a cyst wall, and in this condition •passes out of the body with the feces. The cyst of Entamoeba his- tolytica was first described by Quincke and Roos (1903), and again by Huber (1903), but without making any real impression on the medical or zoological opinion of the day. They were redis- covered by Viereck (1907) and called by him Entamoeba tetra- gena, and for a time was believed to be a new species. In fact, Hart- mann described a vegetative stage of Entamoeba tetragena as Ent- amoeba africana, afterwards accepting the name "tetragena," but it is now apparent that tetragena is merely the end, or cyst stage, of Entamceba histolytica, which had formerly been overlooked by Schaudinn and his followers. Cysts are not easily found in all cases, and it is possible that when treatment is vigorous they never develop. They are, without doubt, the form in which 'the parasite leaves the body to infect new victims; because of their heavy cyst wall they are quite resistant. The protoplasm of the cyst and the precystic SARCODINA 1061 stage is granular, but shows no vacuoles nor cell inclusions. The nucleus undergoes division by mitosis first into two, and then four small ring-like nuclei, and the presence of these four nucleated cysts is pathognomonic of the disease. They may be found most abundantly, not in the small amount of mucus which may adhere to the formed feces, but in surface scrapings from the fecal mass. In addition to the four small ring-like nuclei, the cysts contain few or many clumps of chromatin; these in total mass may be many times greater than the nucleus, and it is impossible, therefore, that FIG. 125. — ENTAMGEBA COLI. (Army Merl. School Collection, Washington, D. C.) they are simply extruded from the nucleus ; evidently, the chromatin grains, while in the cytoplasm, increase in size and number. In hematoxylin stains no structure in these masses is discernible and their function is unknown ; it is possible that they are merely reserve food material; after a time they disappear and one finds cysts quite free of them. The presence, however, of many large chromatin masses in the cysts is quite characteristic of Entamoeba histolytica. These masses, which stain deeply with iron-haematoxylin, have been given various names: chromatin, chromatoid masses, chromedia, crystalloids, inclusions, etc. 1062 PATHOGENIC PROTOZOA Fertilization inside the cyst has not been demonstrated, nor has any other sexual process or conjugation been shown to occur. In size the cysts from different patients vary considerably, so much so that well recognized races or pure lines occur : Dobell and Jepps (1918) describe five such races, the cysts of which have average diameters of 6.6 microns, 8.3 microns, 11.6 microns, 13.3 microns and 15.0 microns. The Dysentery Carrier. — Both convalescent and healthy contact FIG. 126. — ENTAMCEBA COLI. Typical nucleus. (Army Med. School Collection, Washington, D. C.) carriers are known, and recent experiments have shown that they are not infrequent, even in the absence of cases of chemical dysen- tery. To explain the carrier state, it is of course necessary to predicate some insignificant and silent lesions in the colon of the apparently healthy man. There the first part of the life history is lived through and the parasites which come to lie in the lumen of the intestine encysts and are found in the feces. The treatment of amoebic dysentery, to be effective, must be radical and persistent, and may be compared to the treatment of malaria with quinine. For many years the English in India, with a few SARCODINA 1063 followers in other parts of the world, had treated dysentery with ipecac in massive doses, with wonderful results in some cases and failure in others. The treatment was quite disagreeable and not entirely satisfactory. Vedder, in 1911, examined the various al- kaloids of ipecac and found that emetin was strongly amoebacidal, and he recommended its use for this disease. Rogers, in India, following out this suggestion, soon reported excellent results, and the drug is now accepted as a specific. It is administered hypo- dermically in 1/3-grain doses three times a day at first, then twice and later once daily until a total of ten grains has been administered (Vedder). In addition, the patient is put to bed and placed on a milk diet. During convalescence large doses of bismuth subnitrate, a heaping teaspoonful suspended in water or milk, may be given (Decks). Relapses are of course treated in the same manner as primary infections. As a result of the emetin treatment and exact diagnosis the clinical picture of amoebic dysentery has completely changed, and we no longer see the weak and emaciated dysenteries who formerly crowded the wards of tropical hospitals. Epidemiology. — One significant fact appears in the epidemiology of the disease — it always occurs sporadically and never in explosive epidemics such as we see in water-borne diseases, like typhoid and cholera ; house epidemics are, on the contrary, not uncommon. This fact points to the importance of contact; and flies, as the chief agents in its spread. Buxton2 and others have examined the drop- pings and intestinal contents of flies caught in latrines. Buxton found 0.3 per cent of a thousand flies harboring E. histolytica cysts, so that there remains little doubt but that the house fly is one of the principal carriers of dysentery. Extreme cleanliness among the servants and in the kitchen will prevent the transfer of histolytica cysts from the ill to the well. The disease has disappeared from the Panama Canal Zone, where it formerly was common, since the intro- duction of good water and sewer systems and better hygienic conditions. ENTAMffiBA COLI (grass! 1879) casa grand! et Barbagallo 1895 This is a harmless parasite of man, and its presence in stools, at one time, gave rise to much confusion, and in the minds of many, 2 Buxton, P. A., The Importance of the House Fly as a Carrier of E. histoly- tica. Brit. Mecl. Jour., London, 1920, 142. 1064 PATHOGENIC PROTOZOA threw doubt upon the existence of a form of dysentery due to amoeba, since it was found not infrequently in healthy individuals. Schau- dinn found it present in the stools of 50 per cent of the persons examined in East Prussia, in Berlin in 20 per cent, and in Istria in 60 per cent. Craig, and Craig and Ashburn found it present in 176, or 58 per cent, of 307 examinations of healthy American soldiers. Craig was able to follow some individuals for four to six years, during which time they constantly showed Entamoeba coli in the feces, yet never developed dysentery. The organism seems to be FIG. 127. — ENTAMCEBA COLI. Small precystic form. (Army Med. School Collec- tion, Washington, D. C.) found iii all countries, regardless of climate. Its recognition and separation from histolytica we owe to Schaudinn, Jurgcns, Craig and others. In size it varies from ten to forty microns, the average being between twenty and thirty. The ecto'plasm is never seen except during movement, and it is then hyaline, and only slightly refractile, and much more fluid than in histolytica. The digestive vacuoles rarely, if ever, contain red blood cells, but are filled with cocci and bacilli, a form of food not seen in healthy histolytica. In general, the vacuoles are larger and more numerous in coli than in histolytica, and the motility is feebler. In fresh specimens the nucleus is rather SARCODINA 1065 easier to find than in histolytica, and is distinctly outlined by a heavy, double-contoured membrane. The nucleus, as in all amoeba, is vesicular, and shows a small eccentric karyosome and dots of chromatin on the nuclear membrane and imbedded in the nuclear network. Multiplication in the vegetative stage is by binary fission of the nucleus and the cytoplasm, resulting in two daughter cells. FIG. 128. — ENTAMCEBA COLI. CYST showing a large vacuole. (Army Med. School Collection, Washington, D. C.) Cyst Formation. — This is characteristic of the species, and it furnished one of the principal reasons for the separation of coli and histolytica. Before encysting the animal frees itself of all inclusions and becomes clear, transparent, and assumes a spherical form, and secretes a cyst wall. The nucleus divides first into two, then four, and finally eight daughter nuclei ; there is a large vacuole containing glycogen which reaches its maximum size in the double nucleus stage ; it later disappears and is not seen in the mature cyst. Schaudinn described a complicated autogamy in the cyst, yet later researches by Hartmann and Whitmore show nothing more than 1066 PATHOGENIC PROTOZOA repeated binary division of the nucleus. The normal number of nuclei in a coli cyst is eight, yet occasionally cysts are seen in which division has gone on until there are as many as sixteen. In size the cyst measures 10 to 30/x or more. Cats or human beings may be parasitized by feeding material containing coli cysts, and in nature, as the cysts are the resistant forms of the parasite, the infection is probably transmitted from one host to another by means of them. No disease, however, results, though the amoebae continue to be present in the stools for years. FIG. 129.— ENTAMCBBA COLI. Cyst showing eight nuclei. (Arch, fur Protisten- kunde, 1912, xxiv.) • It is possible that fertilization takes place between the young amoebae (gametes?), which are liberated when the cyst dissolves in u new host, as is the case with Entamoeba blattas. ENTAMCEBA GINGIVALIS (Gros 1849, emend, von Prowazek 1904) This amoeba is found in the human mouth both in health and disease. It has been described at different times under various names (bucalis, dentalis) by Gros, Steinberg, von Prowazek, Lewald, Smith and Barrett, Chiavaro and Craig, and quite recently has been sug- gested as the cause of pyorrhea alveolaris by Smith and Barrett and Bass and Johns. It is widely distributed, and has been reported from all quarters of the world. The organism is easily found in the tartar at the base of the teeth, in cavities in the teeth, and even at the gum margin in healthy mouths. It varies in size from seven to thirty-five microns, averaging SARCODINA 1067 between twelve and twenty (Craig). Motility is well marked, though it is not so active an organism as histolytica, the pseudopods, mostly short and blunt, being formed of the clear, slightly refractile ectoplasm. The endoplasm is granular, contains the nucleus and many food vacuoles containing nuclei of leucocytes and granular matter, and possibly a few red blood cells. Although Dobell and others main- tain that red cells are never found. The nucleus is small, and in fresh specimens is usually invisible. In structure, it is not unlike the nuclei of coli and histolytica; the chromatin granules form a compact and distinct ring obscuring the nuclear membrane. The karyosome is small but distinct and is either central or slightly excentric and is surrounded by a clear achromatic halo. There is no chromatin visible between the karyosome and the nuclear margin. Before encysting the parasites are much reduced in size, and the cytoplasm frees itself from all inclusions and becomes clear, spherical and immobile. The cysts are small, eight to ten microns, circular and definitely outlined, sometimes with a double contour, and in stained specimens the nucleus is always visible. It is small, averaging only three microns (Craig), making it smaller than in histolytica and coli. The limiting membrane, while not heavy, is distinct, and encloses a nuclear body having very little chromatin other than the small centrally located karyosome. Multiplication occurs only in the vegetative state and by binary fission. Cyst Formation. — Cysts are rarely observed, and then in small numbers ; the cyst wall is not heavy, but may show a double contour ; the protoplasm is clear, free from all inclusions and vacuoles and shows a single small nucleus, but without any signs of multiplication. It is apparent, therefore, that the cyst is a protective stage and has nothing to do with reproduction, which occurs in the vegetative state only. In this respect it resembles the Vahlkampfia. The find- ing of cysts is so rare that many investigators doubt their existence altogether, and suggest that cysts when found may pertain to some other organism accidentally present in the mouth. Further studies are necessary. Transmission of the infection could occur directly by contact from person to person by kissing, and a cyst stage is not, .therefore, essential to the survival of the parasite. Although the organism is almost constantly present in pyorrhea alveolavis it is also found in healthy mouths, and in the absence of all experimental proof, it is doubtful if the organism is of patholog- 1068 PATHOGENIC PROTOZOA ical importance. Emetin has a decided effect upon many cases of pyorrhea alveolaris, and under that treatment alone the disease may disappear ; the nature of its therapeutic action is not yet clear, and does not necessarily indicate any etiological relationship. ENDOLIMAX NANA (Wenyon and O'Connor, 1917) Burg, 1918 (Amoeba Umax, Wenyon, 1916. Entamceba nana, Wenyon and O'Con- nor, 1917. Endolimax intestinal™, Kuenen and Swellengrebel, 1917.) This organism was first described in 1917 by Wenyon and 0 'Con- nor, by Swellengrebel and Mongkoe Winoto, by Kuenen and Swel- lengrebel and by Dobell and Jepps. The last mentioned authors believe it to be the commonest inhabitant of the human bowel. It is agreed by all investigators that it is not pathogenic and that its principal importance is due to the possibility of confusing it with E. histolytica. In the vegetative stage it is quite small, usually measuring 6 to 12 microns in diameter. It usually contains food vacuoles filled with bacteria but no blood cells and never contractile vacuoles. Its movements are sluggish, resembling those of E. coli, the differentiation between ecto- and endoplasm is not clear cut and the pseudopods are few and blunt. Outside the body all move- ment soon ceases. The nucleus is seen with difficulty in the living specimen but when the organisms are properly fixed and stained with iron haematoxylin the nucleus becomes the most characteristic feature of the parasite. It is vesicular and 1 to 3 microns in diameter averaging between 2 and 2.5 microns. The karyosome is large and pleomorphic. It consists of a principal mass, which is excentrically located and provided with few or many lobes, which are often almost detached from the main body, being connected only by a narrow isthmus. It is necessary to have well differentiated stains" or the details of structure will be overlooked. If the stool is not fresh the amoebae may degenerate and the nucleus present quite a different appearance; the segments of the karyosome can no longer be distinguished and the total mass may come to lie on the outer ring of cliromatin granules, giving rise to a signet ring appearance. It is best therefore to obtain the fresh- est possible material for study, SARCODINA 10G9 Cysts. — Before encystment the parasite extrudes all food par- ticles and the protoplasm becomes clear, all movement ceases and the parasite assumes a round or oval form and the secretion of a cyst wall begins. The mature cyst has four nuclei, whose internal structure resembles that of the vegetative form; that is, there is a karyosome made up of a central mass and more or less detached lobules of chromatin. There are no chromatoid rods in the cytoplasm as in histolytica but there are granules which give the staining reactions of volutin. In iodin solution the cysts are stained yellow ; in some may be noted masses of glycogen, but this substance is a b c d FIG. 130. — ENDOLIMAX NANA. This figure contains figures Nos. 23, 25, 26 and 27 of plate No. 2, Dobell's "Amoebae Living in Man." The first figure on the left represents an active amoeboid form. The next three show uninucleate, binu- cleate and quadrinucleate (mature) cysts, respectively. Plate 11 of "Amoebae of Living Man" by Clifford Dobell, published by John Bale, Sons & Danielsson, London. commoner in the vegetative, precystic and young cysts than in those which are mature. Pathogenicity. — It is not believed that the parasite causes any disease ; it may however be found in connection with histolytica and thus cause confusion. No treatment so far given has had any influence on the organism. IODAMCEBA BUTSCHLII (Prowazek, 1912, Emend. Dobell, 1919) (Entamceba butschlii, Prowazek, 1912. Iodin Cysts or I. Cysts, Wenyon, 1916. Pseudolimax, Kuenen and S\v ellengrebel, 1917.) "Wenyon in 1915 described quite briefly some spherical bodies containing an inclusion which stained with iodin solutions. Later he called these I or iodin cysts and under that name they have been recognized by many as not very rare. They are always found 1070 PATHOGENIC PROTOZOA in connection with some other amoeba and never alone, but it is not believed that they have any pathological importance. Until recently the vegetative stage had not been recognized, but in 1919 Dobell published a complete description, and identified the parasite with the one incompletely described by Prowazek as En- tamoeba biitschlii in 1912. In size the organism is small, averaging 9 to 13 microns, and it resembles E. coli in general appearance except that the nucleus is almost if not quite invisible, particularly in organisms with food vacuoles filled with bacteria and granular matter. In stained specimens the structure of the nucleus serves to dis- tinguish it from other intestinal amoebae. The nucleus is vesicular and contains a relatively large karyosome, one-third to one-half the diameter of the nucleus. Between the karyosome and the well developed nuclear membrane lies a row of granules of peripheral chromatin which Dobell has succeeded in counterstaining with eosin in well differentiated hematoxlyn preparations. It is the presence of this large karyosome and the layer of peripheral chromatin dots that permit the differentiation of the organism from E. coli; the two organisms are alike in their food habits. The cysts are peculiar and quite unlike those of any other intestinal amcebas. In shape they are irregular although some are round or oval. In measuring them Dobell averaged the two prin- cipal dimensions and found that the average size is 9 or 10 microns, with extreme of 6 to 16 microns. The nucleus of the cysts is single, and is distinguished by having the karyosome, which has already been described, pass to the periphery and come to rest against the nuclear membrane, giving rise to a well marked signet ring appear- ance. Peripheral granules of chromatin may still be made out in well stained specimens. Occasional cysts may have two nuclei but they may be interpreted in the same way that we interpret more than the usual number of nuclei in E. histolytica and coli, as an abnormality. The iodin masses in the cysts which give this organism its name are well brought out by staining the fresh specimens in iodin solu- tion. As a rule the glycogen mass is single, large and has well defined borders, but its appearance varies with its age ; the precystic amoeba shows merely a diffuse brown stain, older specimens appear as above, although occasional specimens may show more than one iodin staining mass. Throughout the cytoplasm may be seen granules SAUCODINA 1071 of volutin and the granules increase in visability as encystment proceeds. The cysts, like those of other amoebae, do not withstand drying but they remain alive in feces or water for two or three weeks without undergoing any further development. 35 37 39 40 41 42 FIG. 131. — IODAMCEBA BuTSCHLii. Taken from Dobell as above. The numerals on the plate are Dobell's figure numbers. Nos. 35 and 36 represent precystic amoebae. Observe changes taking place in nuclear structure, freedom from cytoplasmic inclusions, etc. No. 37 is an organism just encystic, volutin granules, pink in cytoplasm, small clear space representing glycogen. No. 39 is a mature cyst, large red glycogen mass. Nos. 40, 41 and 42 are mature cysts showing typical structure of nucleus, volutin granules and glycogen vacuole. Nos. 41 and 42 are cysts of irregular shape often formed by this species and not artifacts. Plate 11 of " Amoebae of Living Man" by Clifford Dobell, published by John Bale, Sons & Danielsson, London. This parasite has been found in the healthy as well as in dysen- tery cases and there is no evidence that it has any pathogenic power, it disappeared under the administration of cmctin and in this point alone resembles E. histolytica. 1072 PATHOGENIC PROTOZOA DIENTAMCEBA FRAGILIS (Jepps and Dobell, 1918) This is the only species of the genus and was discovered and described by Dobell and Jepps in 1918, although it is probable that Wenyon saw but did not describe it in 1909. Only eight cases of proved infection are known, although it is probable that it is commoner than this would lead one to suppose since the organism dies quickly and the degenerated forms are soon unrecognizable. It is quite small the average size being 8 or 9 microns with extremes of 3.5 to 12. The eeto- and endoplasm is sharply differentiated and the pseudopods, consist almost entirely of ectoplasm. The cytoplasm contains food vacuoles filled with bacteria but no red cells are ever seen; the organism is probably, therefore, a pure saprophyte and without pathological importance. The nucleus is characteristic and is typically double, although a few examples with only one nucleus have been seen. The nucleus varies in size with the size of the cell; its nuclear membrane is delicate and free from chromatin particles. All the chromatin of the nucleus is accumulated in, the central karyosome where it consists of a number of granules of varying distinctness all imbedded in a linin network. The discoverers believe that the cell grown to full size and then divides into two uninucleate daughter cells, which increase in size and the single nucleus divides into two. Up to the present time no cysts have been found, and in this it possibly resembles E. gingivalis, in which the presence of cysts is still doubtful. CHAPTER LV CLASS II— MASTIGOPHOKA (DIESING) SUB-CLASS— FL AGE LL AT A (COHN EMEND. BUTSCHLI) ORDER I— POLYMASTIGINA (Blochmann) THESE are flagellates, possessing three to eight flagella. Technic of the examination for intestinal flagellates: It is not necessary to administer a cathartic unless motile vegetative forms are desired; cysts are found in formed stools. Small particles of feces are mixed with a drop of water, or dilute stain and the slide is examined with a high, dry lens, after a cover glass has been applied. The stains most used are Gram's iodin and dilute eosin; the iodin besides staining the parasite, colors the iodophilic inclusions. Thin smears may be stained with iron hematoxylin and with eosin methylene blue mixtures. The flagellates may be cultivated on media used for the cultural amoebae. GENUS 1.— Trichomonas (Donne, 1837).— These have pyroform (pear-shaped) bodies, rounded in front and tapering to a point behind, provided with three long flagella, often matted together at the anterior end. An internal supporting structure, known as the axial filament or axostyle, is present. There is an undulating mem- brane bordered by a trailing flagellum that begins anteriorly and runs obliquely backwards. Trichomonas vaginalis (Donne). — The organism is fifteen to twenty-five microns long and seven to twelve wide; it is provided with three flagella and an undulating membrane. It is found in the vaginal secretion only when it is acid, and in three instances it has been transmitted to the male. Trichomonas intestinalis (R. Leuckard, 1879).— This parasite is practically indistinguishable from Trichomonas vaginalis. It occurs in the small intestine and appears in the stools during diarrheal attacks, but is probably non-pathogenic. It is readily found in the intestine and colon of mice and guinea-pigs. In fresh specimens 1073 1074 PATHOGENIC PROTOZOA (protected with a cover glass and vaseline) it is actively motile, but the undulating membrane is difficult to detect until the move- ment has slowed down. The presence of cystic forms has been questioned, and two quite different forms have been called resistance or dauer cysts. The earlier one, described by Ucke (1908), Bohne and von Prowazek (1908), and Benson (1910), is a fairly large body, showing a double contour and a central homogeneous mass, perhaps food material, and an outer ring-like body containing two or more nuclei. Brumpt and Alexieff believe this form to be a fungus, having no relation to the trichomonad, and have called it ' ' Blast ocystis hominis. ' ' Lynch1 agrees with these authors, and describes an altogether different body as the re- sistant form. It is six by eight microns in size and perfectly symmetrical in shape. The wall is distinct, and there is a clear space between it and the body of the parasite. The nucleus, undulating membrane and flagella remain visible in FIG. 132\-TmcHOMONAS IN- the t but L h was unable to detect TESTINALIS. (After Brumpt, . . "Precis de Parasitologie," an*v change m the parasite indicating m- 1914 ed.) tracystic multiplication. Infection takes place probably by con- tact, and, as in typhoid fever, food, fingers and flies carry the resistant forms from one individual to another. Among the natives of tropical countries infection is almost universal, but the parasites are rarely seen in the large cities of the North. GENUS 2. — Tetramitis mesnili (Wenyon, 1910). — Macrostoma mes- nili, Ckilomastix mesniU, Fanapapea intestinalis. This organism, first described by Wenyon, from a native of the Bahamas, differs from trichomonas by the possession of a deep groove or cystostome, in which is found the undulating membrane. It is present in diarrheal discharges, but its pathogenicity is doubtful. GENUS 3. — Giardla intestinalis (synonym, Lamblia intestinalis). — The giardia are bilaterally symmetrical, pear-shaped organisms, 1 Lynch, Kenneth M., Jour. Parasitol., Urbana, 1916, iii, 28. MASTIGOPHORA 1075 provided with a sucking disk anteriorly. There are eight pairs of fiagella, the two posterior ones being continuations of longitudinal axostyles. The nucleus is first dumb-bell shaped and later divided into two separate nuclei. Cysts are found, and according to Scliau- dinn conjugation occurs in them with the development of four nuclei. The young parasites attach themselves to the surface of epithelial cells of the small intestine by the sucking disk, but even when present in large numbers do not produce any characteristic symptoms. Giardia infestation is quite common in children in the United States, and not uncommon in adults. Maxcy* found 20 per FIG. 133. — LAMBLIA INTESTINALIS. Cyst formation. (After Doflein, " Lehrbuch der Protozoenkunde . " ) cent of children infested and Kofoid found 6 per cent of young healthy soldiers harboring the parasite. The same or like parasites are present in mice, rats, dogs, cats, and sheep. Transmission is by contact, as in trichomonad infections. ORDER II— PROTOMONADINA The Protomonadina, another order of the Flagellata, have less than three flagella, and are divided into the Cercomonadidce, Bodonidce and the Trypanosomidce. GENUS 1. — Cercomonadidae — Cercomonas hominis (Davaine, 1854). — As originally described, this organism has a pear-shaped body, * Maxcy, Kenneth F., Johns Hopkins Hospital Bull., 1921, 32, 166. 1076 PATHOGENIC PROTOZOA drawn out to a point posteriorly, is armed with a single flagellum in front, and has no undulating membrane. It is a doubtful species and of no present importance. GENUS 2. — Bodonidae — Prowazekia (Hartmami and Chagas). — These organisms, the only examples of the Bodonidce of medical inter- est, are of some importance, since they have been cultivated from human feces on agar plates. The genus was founded for Prowazekia cruzi, a species discovered in human feces in Brazil. Other species are urinaria, asiatica, parva, weinbergi and javanensis. There are two flagella, arranged in the heteromastigote manner, that is, one flagellum projects forward and one trails behind. There is no undulating mem- brane, but in stained specimens a second nucleus is seen, the kineto- nucleus or blepharoplast. They are also found in water, and are probably not the cause of any disease. GENUS 3. — Trypanosomidse. — History of the genus. — In 1841 Val- entine discovered the first hemoflagellate in the blood of a trout now known as Trypanoplasma valentini* and the following year Gruby described a flagellate in frog's blood and named it a ' ' trypanosome. ' ' It was not until 1878 that Lewis discovered the rat trypanosome, Trypanosoma lewisi. The first pathogenic member of the genus was noted by Evans in 1889 in the blood of Indian horses sick with surra, Trypanosoma evansi. Bruce in 1894 described the trypanosome of Nagana, Trypanosoma Irucei a horse disease of Zululand, and also demonstrated its transmission by the tsetse fly, glossina palpalis. In 1894 to 1899, Rouget, Schneider and Buffard found the trypanosome of Dourine, or "mal de coit," among Algerian horses. Elmassian described, in 1901, the South American horse disease, "mal de caderas," and discovered the parasite, Trypanosoma equinum. Since this time a large number of new species have been discovered, the more important of which will be described. Morphology. — The morphology of the trypanosomes, while subject to many variations in detail, is still uniform as to the characteristics of the genus, so that there is little difficulty in immediately recognizing the parasite. The body is long and sinuous, tapering anteriorly to a fine point called the flagellum; the posterior end is never so delicate and is often quite blunt. All contain two nuclei, the larger being called the trophonucleus and the smaller the kinetonucleus. The trophonucleus is usually located midway in the length of the body, * Gauthier, M. C. E. Acad. Sci., 1920, 170, 69. MASTIGOPHORA 1077 and the kinetonucleus behind it, often at the posterior extremity. The flagellum arises from a centriole (blepharoplast), which is located close to or in the kinetonucleus, and quickly reaches the surface of the body, when it turns forward and forms the border of the undulating membrane, a thin fold of periblast running the entire length of the body, and is often continued further forward as delicate filament. During life the undulating membrane has a constant wave-like motion. Transmission from one animal to another is usually by means of some blood-sucking invertebrate. Two possible forms of transmission have been recognized, the direct and indirect or cyclical; the direct form is used in the laboratory when transferring blood with a hypo- dermic syringe from an infected animal to a healthy one, and it also occurs in nature, although not so frequently as the second. Dourine, or mal de coit, is the best example of the natural direct method. The cyclical method is exemplified in the transmission of Trypanosoma leAvisi by the rat flea, Ceratophyllus fasciatus, in which insect the trypanosome passes through a complicated life cycle. Whether the parasite in the insect ever passes from parent to offspring is still doubtful. Among fishes, reptiles and amphibians the parasites are carried by leeches, in whose intestinal tract they undergo a cycle of development. Just as in malaria, there is usually an alternation of hosts, from invertebrate to vertebrate, a part of the life cycle being passed in each. In the blood of the vertebrate is found the fully developed trypano- some, and in the intestinal tract of the invertebrate, crithidial. and trypanomonad types, which are characterized by having the kineto- nucleus placed in front of or close beside the trophonucleus and by having a rudimentary undulating membrane. Cultivation. — In 1903 Novy and MacNeal2 first obtained pure cul- tures of trypanosomes on artificial media. The medium devised by them is prepared by equal parts of nutrient agar and defibrinated rab- bit blood. After the agar has been melted and cooled to about 50° C. an equal quantity of rabbit blood is added, mixed and allowed to cool. "The tubes thus prepared are allowed to set in an inclined position, after which they are at once inoculated. It is essential that the surface of the medium be moist and soft, and if this is not the case, the tubes should be placed in an upright position until some water of condensation accumulates at the bottom. The initial 2 Novy and MacNeal, Contrib. Med. Eesearch (Vaughan), Ann Arbor, 1903, p. 549. 1078 PATHOGENIC PROTOZOA culture usually requires a week or more, although not infrequently fairly rich growths may be obtained in three or four days" (Novy). Trypanosoma rotatorium. — Gruby described and named this hemoflagellate in 1843, and it is, therefore, the type species of the genus. The organism is widely distributed throughout the world, and is found in Rana esculenta, Rana temporaria and Hyla arborea: the organisms are, however, not very numerous in any single frog. It is most often found during the spring and summer months, rarely in winter. Morphology. — Both body and undulating membrane are broad, the cytoplasm is granular, and toward the straight side shows striae, probably indicating the presence of myonemes. The trophonucleus FIG. 134.— TRYPANOSOMA ROTATORIUM IN BLOOD OF FROG. (After MacNeal "Pathogenic Microorganisms," published by P. Blakiston's Son & Co.) (s large, lies near the middle, of the body and near the undulating membrane; the kinetonucleus is smaller, lies posteriorly and stains deeply; the flagellum which originates near the kinetonucleus turns forward, forming the border of the undulating membrane, and is continued forward as a short flagellum. The posterior end is usually drawn out to a stubby point. The fully developed organism is large, being 40 to 80 microns long by 5 to 40 wide. One striking thing about this parasite is its tendency to pleomorphism. Multiplication in the blood stream of the frog is by binary fission ; in addition, a form of multiple division occurs in the viscera, pre- ceded, according to Machado, by conjugation of sexually differen- tiated forms. The merozoites liberated from the mother cell are small trypanosomes, which in turn grow to large size, thus explaining the pleomorphism of the parasite. MASTIGOPHORA 1079 Cultures have been obtained by Lewis and Williams on the blood a gar of Novy and MacNeal in which a great variety of forms may be seen ; the method of transmission is imkown, but the infection is probably conveyed by leeches. Many other trypanosomes have been found in fishes, frogs, and reptiles all over the world. Trypanosoma lewisi (Kent). — This, one of the longest known and commonest forms, has been studied more completely than any other organism of its class. It occurs in a large proportion of rats through- out the world, twenty-five to one hundred per cent being infected, and since it is non-pathogenic, it is a convenient organism for research. It may be passed fr^m wild to white rats without difficulty, by in- oculating the latter with a small quantity of citrated blood contain- ing the organisms. At first the para- sites are few, but after the lapse of three or four days, lafcge numbers may be found; the condition of rapid multiplication lasts from eight to fourteen days, and is succeeded by a period of a month or more, during which time the parasites gradually diminish in number, finally disappearing completely, rendering the animal immune from further infection, the immunity being com- plete. The serum of an immune rat has a certain protective power, and when inoculated simultaneously with blood containing trypano- somes, may prevent the infection. No other animals are susceptible. The blood should be examined in both fresh and stained speci- mens. In fresh specimens, because of the rapid, lashing movements of the parasite, the organisms are particularly easy to find. The details of structure, however, do not appear except in spreads stained with some of the modifications of the Romanowski stain, such as Wright's or MacNeal 's. In the adult stage the organisms are quite uniform in size and shape, being 27 or 28 microns long and 1.5 to 2.0 microns broad; the posterior end is long, tapering and pointed; the kinetonucleus FIG. 135. — TRYPANOSOMA LEWISI. (After Doflein and Minchin. Mac- Neal, " Pathogenic Microorgan- isms," published by P. Blakiston's Son & Co.) 1080 PATHOGENIC PROTOZOA oval and flattened; the trophonucleus is located near the anterior end, and the undulating membrane, while distinct, is relatively narrow. The endoplasm is finely granular, and by careful focusing the bodywall or periblast may be seen. Multiplication in the rat is rapid, and many young forms are seen; these are smaller, stain more deeply, and vary much more in size than the adults. Dividing forms are common, the division being longitudinal and unequal, the parent retaining the flagellum. Mul- tiple division also occurs, resulting in the production of rosettes, whose structure suggests that repeated longitudinal division has occurred without the separation of the daughter cells. The insect hosts are two : the rat flea, Ceratopliyllus fasciatus, and the rat louse, Hcematopinus spinulosus; the former being the right host and the latter the wrong one, since in it development is incom- plete. Minchin and Thompson3 have studied the cycle in the flea, which is briefly as follows: When the injected blood and parasites reach the midgut of the flea, the trypanosomes lose their flexibility and become more or less rigid, and are able to penetrate the outer wall of the epithelial cells of the stomach. Once inside the cell, the parasite folds upon itself and grows to large size; the nuclei multiply, the body becomes spherical and divides up within its own periblast into six or eight daughter cells, all actively moving within their common envelope. This becomes tense and finally bursts, liberating the young trypanosomes within the epithelial cell, through whose wall they soon escape into the lumen of the stomach. This form of multiplication may be several times repeated, after which the young trypanosomes pass down the intestine to the lower end to begin the rectal phase. There the parasites in large numbers are found attached to the epithelial cells by their flagella. Rapid mul- tiplication takes place by repeated fission and the parasite becomes crithidial in form, that is, it loses its undulating membrane, becomes short .and stubby, and the kinetonucleus moves forward close to or in front of the trophonucleus. Ultimately some change back to minute trypanosomes,' and these, when regurgitated or passed in the feces, serve to infect the next victim. The rectal phase, when once established, lasts for several months or perhaps indefinitely, making every infected flea a chronic carrier. Trypanosoma evansi. — Surra is a disease of horses and mules, camels, elephants, buffaloes, and dogs, which prevails in India and 8 Minchin and Thompson, Quarter. Jour. Micr. Sc., Lond., 1915, Ix, 463. MASTIGOPHORA 108 1 other parts of Asia, and also in the Philippines and Northern Aus- tralia. The Philippine outbreak was traced to animals returned from China after the Boxer outbreak; for at that time American troops came into contact with native Indian troops and their animals. The trypanosome causing the disease was discovered by Evans in 1880. The clinical course of the disease is marked by an irregular recurring fever, with many remissions, during which the parasite cannot be demonstrated in the blood, although it is not difficult to find during the febrile period. The animal is anemic, weak, emaciated, and may show an ecchymotic eruption on the abdomen. The course of the disease may be either short or long, but leads almost invariably to death. In camels it lasts from two to four years, often without symptoms until near the end, and these animals prob- ably act as chronic carriers. Morphology. — Morphologically, the parasite is very like the Tryp- anosoma brucei of Nagana, yet, as a rule, the trophonucleus lies nearer the anterior end than in brucei, although it may be impossible to distinguish in smears between the two. The disease is carried by biting flies, tabanidoe, and stomoxys, and also by fleas. Trypanosoma brucei. — Nagana is a well-known horse and animal disease of Africa, which causes an enormous economic loss and has greatly interfered with the development of the country. The parasite was discovered by Bruce in 1895. Among the natives it is known as tsetse fly disease, and investigation has incriminated Glossina mor- sitans as the carrier. Clinically, the disease in horses is much like the Surra of India ; the native name for the disease, nagana, means weak- ness. Nearly all the larger animals are susceptible to either natural or artificial infection, yet man is apparently immune., Morphology. — Morphologically, it resembles closely most of the other pathogenic trypanosomes, and Minchin makes it the type of a group of pathogenic trypanosomes, all closely resembling one another and possibly descended from one common ancestor : the group consists of brucei, gambiense, evansi, equiperdum, rhodesiense, and hippicum. The organism is less slender than Icwin and has a wider undulating membrane. The posterior end is relatively short, the trophonucleus lies in the middle of the body and the kinetonucleus at the extreme posterior end ; a vacuole is placed just in front of the latter. In length the parasite measures twenty-five to thirty-five microns and is one and 1082 PATHOGENIC PROTOZOA a half to two and a half microns in width ; multiplication in the blood stream is by binary fission. Transmission is by means of the tsetse fly, Glossina morsitans, and perhaps Glossina pallidipes and Glossina fusca. The fly may transmit the disease directly after infection, acting as a mere mechanical carrier, but it is more probable that a cyclical development of the parasite takes place in the fly, after which it remains infectious for D FIG. 136. — THE MOST IMPORTANT TRYPANOSOMES PARASITIC IN VERTEBRATES. A, Tr. lewisi; B, Tr. evansi (India); C, Tr. evansi (Mauritius); D, Tr. brucci; E, Tr. equiperdum; F, Tr. equinum; G, Tr. dimorphon; H, Tr. gambiense. (X1500.) (From Doflein after Novy. MacNeal, " Pathogenic Microor- ganisms," published by P. Blakiston's Son & Co.) a long period. It has been shown that after the first few hours the fly is not infectious again until the lapse of eighteen days, when its bite once more conveys the disease, and trypanosomes may be found in the intestinal canal, the body cavity, the salivary glands and in the proboscis. Studies of the cycle in the fly show that only about five per cent of the flies permitted to feed on sick animals become chronic carriers. The work of Bruce and others has shown that the trypanosomes are more or less harmless parasites of the big game animals of Africa, which therefore are believed to act as a reservoir, from which the disease is transferred to the domestic MASTIGOPHORA 1083 animals by the tsetse fly. The distribution of Glossina is not uni- form, as they are only present in certain definite areas called fly belts. Since the disease does not spread in the absence of the larger wild animals, it has been proposed that all big game be exterminated as a prophylactic measure. Mice and rats are sus- ceptible and die in six to fourteen days after inoculation; guinea- pigs are more resistant, and may show one or more relapses within two to ten weeks. It has not been possible to immunize larger animal's, although a certain degree of success has been obtained with the smaller animals used in the laboratory. Cultures have been grown on artificial media, yet not so readily as with lewisi and avian trypanosomes. The medium recommended by MacNeal contains the extractives of one hundred and twenty-five grams of meat, ten of pepton, five of salt, and twenty-five of agar to the liter ; to this is added twice its volume of warm, defibrinated rabbit 's blood. The blood agar slants should be soft and moist when inoculated. Filtrates from cultures are not toxic, the toxin ap- parently being liberated, according to MacNeal, from the body of the disintegrating trypanosome. Trypanosoma hippicum (Darling). — The disease caused by this trypanosome in horses and mules has been known in Panama for many years under the name of "Murrina de caderas" or "Der- rengadera de caderas," the latter term being used when paralysis of the posterior extremities is the dominant symptom; both names indicate a weakness of the hind quarters. The symptoms are weak- ness, emaciation, and, sooner or later, conjunctivitis and subcon- junctival ecchymosis, and anemia. The horses and mules affected are obviously weak, and while in the stall, pull back on the halter, or stand with straddling hind legs. The incubation period in animals used for experiment is less than a week, a few animals lose weight rapidly and die within a few days, others live for several weeks. Treatment, including the use of arsenical preparations, is without effect, and all infected animals should be destroyed. The disease is apparently transmitted directly by flies, which carry blood and serum from ulcers and abrasions on infected to healthy animals. Morphology of the Parasite. — The trypanosome is sixteen to eighteen microns long and two microns wide. The kinetonucleus is about two microns from the posterior end, the trophonucleus 1084 PATHOGENIC PROTOZOA about eight to ten microns from the same point. The posterior end is blunt and the cytoplasm usually contains numerous basophile granules; the undulating membrane is well developed and a chromatin filament runs from the kinetonucleus to the tip of the flagellum. The large kinetonucleus distinguishes this organism from Trypanosoma equinum. PatJwlogical Anatomy. — Aside from the emaciation, edema of the belly wall, conjunctivitis and subconjunctival ecchymosis, there is usually excessive fluid in the body cavities, an enlarged spleen, and, what is more characteristic, small petechial spots on the capsule of the spleen and in the cortex of the kidney and in the endo- and pericardium and occasionally on the pleura! surfaces. Prophylaxis consists in the destruction of all infected animals; the protection of wounds and ulcers in otherwise healthy animals by dressings and, wherever possible, the use of fly screens about the stables. Trypanosoma equiperdum (Do urine). — This organism is the cause of dourine, a disease of horses and donkeys, which is usually transmitted by coitus, but may be carried by biting flies, stomoxys. The organism was first described by Rouget in 1894; it resembles brucei in many ways and produces a progressive, fatal disease of great economic importance. Formerly it was present throughout the greater part of Europe, but is now almost limited to the shores of the Mediterranean. From time to time it has been introduced into the United States and Canada by blooded French stallions and has spread into parts of the Northwest. The clinical course may be divided into a stage of edema, lasting about a month, during which there is a painless, soft swelling, limited to the genitalia and the belly wall. This is followed by the stage of eruption, during which plaques, or round edematous areas, are found under the hide on the flanks and hind quarters, and sometimes on thighs, shoulders and neck; this stage is short, lasting about a week. It is followed by the third stage of paralysis and anemia ; the animal loses flesh and strength, develops superficial ulcers, conjunctivitis, keratitis, and ultimately paralysis, leading to death in two to eighteen months. The trypanosome is found most readily in the serous exudate from the ulcers, as it is infrequent in the peripheral circulation; in this respect it resembles the treponema of lues. The organism is about twenty-five microns in length and possesses a clear cyto- MASTIGOPHORA 1085 plasm, free from granules, except when propagated in white mice, when they are plentiful. Diagnosis by Complement Fixation. — E. A. Watson,4 of Canada, has shown that it is possible not only to diagnose the disease when the clinical signs are clear, but also to determine the existence of its non-clinical, obscure and latent forms. Horses may tolerate an infection for one to three years, during which time they arc capable of conveying the disease and yet remain normal in health and FIG. 137. — DOURINE. Showing swelling of genitalia and plaques on the skin. (After Kolle and Wassermann, "Handbuch der Pathogenen Mikro-organismen," 2te Aufl., 1913.) general appearance, and this method of diagnosis is, therefore, invaluable. Watson obtains the antigen by inoculating a large number of white rats with Trypanosomv, equiperdum, collecting their blood when teeming with trypanosomes, and separating them from the erythro- cytes and plasma by washing and centrifuging. Each of ten to twenty rats receive 0.3 c.c. of blood rich in trypanosomes intraperi- toneally, and at about the end of the third day, when the organisms are very numerous, the rats are bled into citrate solution. By . A. Watson, Parasitology, Cambridge, Eng., 1915, VIII, 156. 1086 PATHOGENIC PROTOZOA repeated washing the organisms may be separated, as a pure white layer overlying the erythrocytes. This mass of organisms is killed and preserved by a formalin-glycerin mixture, after which its an- tigenic strength is standardized by titration in the usual way. The test, a pure culture of trypanosomes being used as antigen, is specific and is not positive in any .other disease of horses. Trypanosoma avium. — This parasite was first described by Dani- lewski in 1885. In 1905 .Novy and .MacNeal5 .found trypanosomes in 8.8 per cent of 431 American birds. Although there are doubt- less several species, the most common is Trypanosoma avium, a para- FIG. 138.— TRYPANOSOMA AVIUM IN BLOOD OF COMMON WILD BIRDS. (After Novy and MacNeal. MacNeal, " Pathogenic Microorganisms," published by P. Blakiston's Son & Co.) site twenty to seventy microns long and four to seven microns wide. They are found in the blood over long periods of time and do not appear to be pathogenic. Cultures are easily made and kept alive for long periods by weekly transfers. The mode of transmission is unknown. This was the parasite which was confounded in 1904 by Schaudinn with developmental stages in the life cycle of Hemorproteus noctuce and Hemorproteus ziemani with resulting confusion in the study of trypanosomes and hemocytozoa, and it is only recently that the error has been generally acknowledged. Trypanosoma gambiense (Sleeping Sickness). — Two names have been given to the disease caused by this parasite, both of which are 9 Novy and MacNeal, Jour, Infect. Dis., Chicago, 1905, ii, 256. MASTIGOPHORA 1087 now recognized as stages in one and the same infection, human tryp- anosomiasis : they were trypanosome fever, and sleeping sickness. It is a chronic infection characterized by fever, lassitude, weakness, wasting, and, in its terminal stages, by a protracted lethargy. Sleeping sickness and trypanosome fever had long been known in tropical Africa, and the disease at present is widespread and the FIG. 139. — TRYPANOSOMA AVIUM IN CULTURE ON BLOOD AGAR. (X1500). (After Novy and MacNeal. MacNeal, " Pathogenic Microorganisms," published by P. Blakiston's Son & Co.) cause of tremendous mortality. It is estimated that one hundred thousand deaths occurred during the ten years ending in 1910. It is endemic in the lake region of Central Africa, and in the Congo basin. It was early introduced into Martinique in the West Indies, but did not spread and has now died out. Button and Todd found the parasite in 1901 in the blood of an Englishman in Gambia, who died after a febrile illness of two years' 1088 PATHOGENTC PROTOZOA duration ; Castellan! in 1903 found the parasite in the cerebro-spinal fluid of well-marked eases of sleeping sickness occurring among natives of Uganda. Clinical Signs. — The disease begins with slight febrile attacks, headache and increasing weakness, emaciation, swelling of the eyelids and enlargement of the lymph nodes. The temperature increases, edema of the extremities appears and the spleen enlarges. During the last stages nervous symptoms predominate and the patient sleeps day and night, but may have periods of excitement or convulsions, yet finally sinks into deep coma and dies of exhaustion. FIG. 140. — TRYPANOSOMA GAMBIENSE. Calkin, "Protozoology." Etiology. — The disease is transmitted by the bite of the tsetse fly, Glossina palpalis, which is apparently able to transmit the infection mechanically immediately after biting an infected host, yet in most flies the trypanosomes disintegrate and disappear from the intestinal tract within four or five days. In from five to ten per cent of the flies, however, the trypanosomes multiply in the intestinal tract, and after eighteen to fifty-three days they again become infectious and remain so for a long period, the parasites being found regularly in the salivary glands and in the proboscis. It is possible that the disease is transmitted in other ways than by Glossina palpalis; blood-sucking insects, such as stomoxys, MAST1GOPHORA - 1089 anopheles, mansonia and perhaps fleas, may act as mechanical carriers. It is also possible that the disease is transmitted by coitus. Without some such explanation it is difficult to understand certain house epidemics which have occurred outside the fly belts. The animal host of the Trypanosoma gambiense is believed to be the big game animals, particularly the antelope. FIG. 141. — TSETSE FLY (GLOSSINA PALPALIS). (From Rosenau, " Preventive Medicine and Hygiene.") Morphology. — The organism belongs to the brucei group, and its differentiation on morphology is difficult, yet, on the average, the posterior end is somewhat more pointed than the brucei. In length it varies from fifteen to thirty microns, and in thickness from one to three microns. In fresh preparations the motility is not marked; both plump and slender forms are found in the blood, but in the cere- bro-spinal fluid slender forms only are seen. Cultures on blood agar have been made by Thompson and Sinton, yet they died out after a few weeks, and were never virulent. The pathogenicity varies somewhat with the strain used, but apes are easily infected. In white rats there may be two or three relapses before death occurs, while when inoculated with brucei death follows within two weeks. Pathogenicity. — Although cultures vary greatly in virulence, it is possible to infect rats, dogs arid monkeys with a fatal trypanosomiasis ; cattle, sheep and goats continue to show a few parasites for months !090 PATHOGENIC PROTOZOA after inoculation but without sickening. In no animal, however, is it possible to reproduce the sleeping sickness stage as it occurs in man. Trypanosoma rhodesiense. — This species was established by Stephens and Fantham.6 It is transmitted by the Glossina morsitans, a fly which is widespread over large tracts of country, independently of the presence of water. It is becoming generally recognized that there are two forms of sleeping sickness, one of which is caused by this trypanosome. This form of the disease is more acute and is less amenable to treatment; the trypanosome is also more virulent for animals and may be differentiated from gambiense on its morphology. As both parasites are found in the antelope, the prophy- laxis is the same. Bruce7 is of the opinion that rhodesiense and brucci are identical, but Taute and Huber,* by inoculating themselves and 129 natives with the blood of naturally infected animals with out reproducing the disease, seem to have shown that the parasites are not identical. Diagnosis. — When the disease is well developed in an endemic area, the diagnosis is easily made. During the early stages the exami- nation of the cerebro-spinal fluid, puncture fluid from the lymph nodes and the peripheral blood may all show the trypanosome; since the parasites are scarce the use of the thick film method of Ross may be necessary. When direct examination is unsuccessful, enrichment in the blood of susceptible animals, rats and mice will establish the diagnosis. Treatment. — Treatment is based upon the observation of Bruce and Lingard, that arsenious acid is trypanocidal. The best results have been obtained with atoxyl, in half gram doses, repeated at inter- vals of ten days or more for not less than four months. Light cases become trypanosome free and are apparently cured, yet many relapse on cessation of treatment. Well marked cases may show improvement yet ultimately grow worse and die. Better results are obtained when atoxyl and tartar emetic are both used and also when the treatments are repeated every six months. The successful treatment of an intract- able case of gambiense infection with stibenyl (the sodium salt of p-acetylaminophenyl-stibinic-acid) has been reported by Manson-Bahr 'Stephens and Fantham, Proc. Eoy. Soc., 1910, Ser. B., Ixxxiii, 28. 1 Bruce, Bull. Trop. Dis., 1916, vii, 68. * Taute, M., and Uulxr, /•'., Abstracted in. Trouieal Diseases. Bull. London, 1930. MAST1GOPHORA 1091 •(Brit. Med. Jl., 1920, 235). Other arsenical preparations have been used but none are entirely successful. Salvarsan drives the parasite from the peripheral blood but not from the cerebro-spinal fluid. The prognosis as to ultimate recovery is unfavorable. The most hopeful cases are those which are brought under treatment in the earliest stage of the disease. Prophylaxis. — Prophylaxis is quite complicated and is carried out along several different lines. Infected fly belts are depopulated, the inhabitants being removed to a fly-free district where they may be treated at hospital stations. The fly breeding may be greatly diminished by clearing off the forest and brush, especially along the river courses, since the glossina needs abundant moisture for its propa- gation. Since the fly bites only during the day, all traveling in infected districts is best done at night. A prophylactic measure of prime importance is the search for and the treatment of the cases, which are also carriers. While the treatment may not cure, it does bring about improvement and lessens the number of heavily infected carriers. Trypanosome cruzi, Chagas (Schizotrypanum cruzi). — This para- site, which differs from all other trypanosomes, is the cause of a FIG. 142. — SCHIZOTRYPANUM CRUZI IN HUMAN BLOOD. (From Doflein after Chagas. MacNeal, "Pathogenic Microorganisms," published by P. Blakis- ton's Son & Co.) form of human trypanosomiasis occurring in Brazil. It is trans- mitted by a bug, Triatoma magista (Conorkinus megistus), in which the parasite passes part of its cycle of development. Brumptf believes the infection is transmitted by the dejecta of the bug as well as by its bite, and thinks the first method is probably the more usual one. In the human being, multiplication takes place in endothelial cells, lymphocytes and parenchymatous cells of the viscera ; and also in the skeletal and heart muscles. While in this stage the parasite | Brumpt, Bull, Acad. Med., Paris, 1919, 81, 251. 1092 PATHOGENIC PROTOZOA FlG. 143. — SCHIZOTRYPANUM CRUZI DEVELOPING IN TISSUES OF GlJINEA-PIG. 1. Cross-section of fibers of striated muscle containing Schizotrypanum cruzi; 2, Section of brain showing cyst in a neuroglia cell containing chiefly flagellated forms; 3, Section through suprarenal, fascicular zone; 4, Section of brain showing neuroglia cell filled with round forms. (After Low and Vianna. MacNeal, "Pathogenic Microorganisms," published by Blakiston's Son & Co.) MASTIGOPHORA 1093 has no flagellum and resembles the leishmania; only after escape into the blood does it take 011 the trypanosome form. Guinea-pigs, rats, mice and monkeys are susceptible; the bed- bug, cimex, is also capable of transmitting the disease. Cultures were obtained by Chagas and proved virulent for animals. The human disease is found both in children and adults and is regularly fatal. It is characterized by an irregular fever, severe anemia, swelling of the lymph nodes, edema and disturbance of the nervous system. Two quite distinct forms are recognized, the acute, which is usually found in young children, and the chronic, found in adults. The acute form is characterized by fever, a myxe- dematous swelling of the face and neck or even the whole body, and the presence of trypanosomes in the circulating blood. The nervous system may be involved in the acute cases, in which event they end fatally. The chronic form has no trypanosomes in the blood, but has foci, or cysts in the heart, the voluntary muscles or in the viscera or the nervous system. Disturbances of almost any part of the body may result. While the foci may be generalized they do not follow the blood vessels. Leishmania. — This genus was founded by Boss in 1903 for the Leishman-Donovan and Wright bodies found in kala-azar and Delhi boil, to which Nicolle added another in 1909, the parasite of infantile splenomegaly. Leishman, Donovan and Wright, working independ- ently, described the first two parasites in 1903, and, although they have received various names, leishmania is now the accepted term. Rogers, Calkins and others, however, class them as herpetomonads, because of the elongated, flagellated form all take in cultures on the Novy-MacNeal-Nicolle blood agar medium. It is, however, best to consider them as a separate genus, because of their natural parasitic habits in human beings. Leveran, Fantham and others have shown that it is possible in the laboratory to induce the herpetomonads parasitic in the intestine of various insects to become parasitic in various vertebrates. Leishmajiia donovani (Kala-azar). — This parasite is the cause of kala-azar, a disease characterized by irregular fever, weakness, anemia, cachexia and a remarkable enlargement of the spleen, and occasionally of the liver. It is chronic, progressive and frequently fatal, the mortality being about 80 to 90 per cent. This disease is common in tropical Asia and in northeastern Africa. Morphology. — The parasite is intracellular, and is found prin- 1094 PATHOGENIC PROTOZOA cipally in the endothelial cells of the spleen and liver, and in the bone marrow. It is oval, two to four microns in diameter, finely granular and occasionally vacuolated. It contains a large, round nucleus and a smaller blepharoplast which is oval or rod shaped; a third body, a slender short thread, may sometimes be recognized, which is presumably the undeveloped flagellum. Stained specimens 4^ ; FIG. 144. — LEISHMANIA DONOVANI. (Army Med. School Collection, Washington, D. C.) of blood, spleen and liver pulp, and bone marrow, usually show large endothelial cells or leucocytes closely packed with parasites, one to two hundred to a single cell. Multiplication in the body is by simple division, and incompletely divided pairs of organisms are frequently seen. Cultures have been obtained in citrated blood and on the usual N. N. N. medium. When fully grown the cultural organisms are typical herpetomonads (leptomonads) ; the cell body elongates and the rudimentary whip develops into a true flagellum. Both dogs and monkeys are susceptible to artificial inoculations. MASTIGOPHORA 1095 The parasite is probably transmitted by some insect, either cimex (Rogers), or by the dog flea, Ctenocephalus canis (Wenyon), or a plant-feeding bug, Conorhinus, which occasionally sucks blood. Animal Pathogenicity. — Wenyon in 19138 inoculated a dog with splenic emulsion from a man who died in London of kala-azar con- tracted in Calcutta. The parasite has been successfully carried through five animals, and in 1915 an examination of the bone marrow showed not only typical leishmania, but also a few large, well-marked leptomonad forms. Similar forms were described by Escomel in 1911, from South American dermal lesions. Monkeys may also be infected. Leishmania tropica (Delhi or Aleppo boil) is the organism found in a local skin affection variously termed Delhi boil, Aleppo boil or tropical ulcer. While it is probably transmitted by some insect, there is as yet no definite proof. The incubation period is about two months, while the disease, once manifest, lasts twelve to eighteen months and is followed by immunity for life. The parasite, which was first described by J. H. Wright,9 shows minor differences from leishmania donovani, particularly a variable morphology, all gradations, from the usual oval to elongated narrow forms with pointed ends, being found. Cultures may be obtained on the N. N. N. blood agar, which develop into leptomonads, as with Leishmania donovani. Dogs and monkeys are susceptible to artificial inoculation, and it is possible that in nature the disease is carried from dogs to human beings by some insect. Leishmania infantum (Infantile Splenomegaly) was described by Nicolle in 1909 from cases of infantile splenomegaly occurring in Northern Africa. It is, however, not limited to this region, but occurs throughout the whole Mediterranean District. Javarone10 described 110 cases observed in Naples from 1913 to 1920. The disease resembles kala-azar in all respects, except that the patients are young children, and it is possible it is the same disease. With- out treatment the disease, like kala-azar runs a progressive course almost always leading to death. The parasites are found in abun- dance in the liver, spleen and bone marrow at autopsy and may be cultivated in the usual way on the N. N. N. blood agar. 8 Wenyon, Jour. Trop. Med. and Hyg., London, 1915, xviii, 218. 9 Wright, J. H., Jour. Med. Res. Bost. 1903, x, 472. 10 Javarone, N., Infantile Leishmaniasis hi Naples and Neighborhood, Tropical Diseases Bulletin, London, 1920, 16, 454, 1096 PATHOGENIC PROTOZOA Animal Pathogenicity. — The disease occurs naturally in African dogs, and they are probably the source of infection, the parasite being carried by a flea or some other insect. Dogs, monkeys and guinea-pigs are susceptible to artificial inoculation. Treatment. — The use of tartar emetic in the treatment of kala-azar is due to the success of the treatment of infantile leishmaniasis intro- duced by Di Christina and Caronia. In kala-azar it has now been L r* -.>*' J FIG. 145. — LEISHMANIA INFANTUM. (Army Med. School Collection, Washington, B.C.) used successfully by many and when properly given is without danger and gives a large number of recoveries. The untreated disease has a mortality of about 90%. Dobbs- Price11 has reported 2000 injections with 67% of recoveries. He used a 1%% solution of sodium antimony tartrate, in a weekly dosage increasing from 1 to 8 c.c. 11 Dodds-Price, J., Kala-Azar in Europeans in the Nowgong District of Assam — Indian Medical Gazette, 1920, Vol. 55, No. 3, pp. 87-89. MASTIGOPHORA 1097 Since intravenous treatment of children with any drug is difficult, a search has been made for preparations which can be given into the muscles, without producing pain or necrosis. Some promising results have been obtained by Spagnolio and Manson-Bahr,12 with acetyl-p- amino-phenyl-stibiate of sodium, and "stibenyl" a related drug. The drug is dissolved in distilled water and "stibenyl" according to Manson-Bahr, may be given in doses up to 0.6 gram for an adult. The treatments are given weekly over a period of 4 or 5 months with resulting cure. The time of treatment may be shortened by using the intravenous route when possible. The prognosis, as a result of the new treatment with antimony, may now be considered good, particularly in acute cases. Two other forms of dermal leishmaniasis have been described ; the first, due to Leishmania braziliensis, occurs in many parts of South America. The parasite is morphologically identical with Leishmania tropica. Since the disease is always contracted in the virgin forest, one name for the affection is forest yaws ; uta and espundia are prob- ably different clinical forms of the same disease. The transmitting insect cannot well belong to the household vermin or domestic insects ; sylvan insects such as the ixodides, tabanides, simulids, mosquitoes and ConorJiinus are all suspected of being carriers. The second form is called Leishmania nilotica (Brumpt, 1913). and is found in non-ulcerating keloid nodules in Egyptian negroes. Morphologically, the parasite is indistinguishable from Leishmania tropica. 12 Spagnolio, Giuseppe, Tropical Diseases Bulletin, London, 1920, 16, 455, and Manson-Bahr, Philip, Brit. Med. Jour. 1920, Aug. 14, 235. CHAPTER LVI CLASS III— SPOROZOA1 SUB-CLASS— TELOSPORIDIA HEMOSPORIDIA THE Hemosporidia and Sarcosporidia are the only members of this order of medical interest. The hemosporidia belong to the sub-class Telosporidia of the Sporozoa, because spore formation begins at the end of the life cycle. The systematists have not yet agreed upon the proper classification of this group of parasites; consequently the older arrangement will be followed. They are, like the coccidia, parasites of cells, at least during the schizogenous cycle ; all change hosts to some insect for the sporogenous cycle. As the name implies, they live in blood cells and are rapidly growing ameboid bodies, which, beginning as sporozoites, penetrate the host cells and develop into trophozoites. These grow rapidly to adult segmenting parasites, in which case they are called schizonts, or to sexual forms, or gametes, when they are termed sporonts. In the course of their development, most species produce melanin from the destruction of the hemaglobin. The nucleus, which is readily stained, is single and posesses a karyosome ; the mature schizont divides into many small forms called merozoites, and these, when freed by the rupture of the degenerated erythrocyte, escape into the blood plasma, and if not phagocyted, penetrate other erythrocytes and repeat the asexual or schizogenous cycle. The pigment and undivided portion (restkorper) of the cyto- plasm of the mother cell accumulate in the bone marrow, spleen and other viscera. After a number of cycles of asexual multiplication have been lived through, a new development takes place and sexual forms begin to appear in the circulation. These grow to large size, yet show no indi- cation of division into merozoites and were at one time considered 1 For classification, see page 1049. 1098 SPOROZOA 1099 degeneration forms. Two varieties may be distinguished, one with a dark staining cytoplasm and fine granular melanin, and the other witk light staining, hyaliii cytoplasm and coarse pigment; the former, loaded with reserve food material, is the female or macrogametocyte ; the latter, the male or microgametocyte. The gametes do not develop further until taken into the digestive tract of the insect host. For purposes of study, however, the liiicrogametocytes may be made to exflagellate on the slide, dampened a little by breathing upon it, to stimulate the condition in the insect host. In such a preparation, the flagella, or microgametes, -may be seen actively moving inside the cell body, whose wall they ultimately rupture, and all, four to eight, escape and whip about until they come in contact with a macrogametocyte, when one microgramete enters through the micropyle and finally fuses with the female nucleus. Hemoproteus columbae (Halteridium). — This parasite of the red blood cells of doves was described in 1891 by Celli and Sanfelice. It is widely distributed in nature and has been reported from Europe, Asia and North and South America. The organism is found within the cytoplasm of the erythrocyte; the nucleus, which is not regularly displaced, is surrounded by the growing parasite like a halter, and for this reason it was named halteridium by Labbe. It is sluggishly ameboid and produces an abundance of melanin, and when the blood is drawn the ripe male sporonts, the microgametocytes, rupture easily, liberating the active flagella, or microgametes. Under favorable circumstances the fertilization of the macrogametocyte by the microgametes may be observed on the slide, and it was while work- ing with this parasite that Macallum first followed out the whole process of fertilization in the hemosporidia and gave the proper explanation of the flagellate stage seen in the malarial parasite. In the blood of the dove this parasite is usually seen as a large or small crescent, partly encircling the nucleus; the gametes are readily recognized by the usual marks, that is, the female, or macrogametocyte, is rich in reserve material and the stained specimen takes a deep color ; the male, or microgametocyte, being poor in reserve material stored in the cytoplasm, appears relatively pale in stained specimens. The invertebrate host of the parasite is Lynchia maura (Bigot), or Lynchia lividocolor, a biting hippoboscid fly of louse-like habits which lives in the nest and in the plumage. The cycle in the fly has 2 Adie, Helen, Indian Jour. Med. Research, Calcutta, 1915. 1100 PATHOGENIC POKTOZOA been successfully worked out by Adie,2 who has demonstrated the ookinetes, zygotes and oocysts in the lower portion of the midgut. As FIG. 146. — H^MOPROTEUS COLUMB.E. la to 3a, Development of female parasite in blood of dove; Ib to 36, Development of male parasite in blood of dove; 4taf 46, 56, 6 to 12, Development in the digestive tube of the fly (Lynchia) ; 13 to 20, Development of the parasite inside leucocytes in the lung of the dove. (After Avagao. MacNeal, "Pathogenic Microorganisms," published by P. Blakiston's Son & Co.) the oocyst grows, it stands out from the gut wall and finally shows the striations indicative of the presence of sporozoites; after rupture of SPOROZOA 1101 the mature cyst, these collect in large numbers in the salivary glands and ducts. The life history of the parasite is seen to be like that of proteosoma and malaria, except that the asexual or schizogenous cycle appears to be lacking. Proteosoma (plasmodium) prseoox. — This parasite is a typical representative of the sporozoa, and is interesting historically, since it was the one with which Ross worked in 1898, when he first demonstrated the part played by the mosquito in "bird malaria." Grassi and Feletti described the parasite in 1890 under the name of hemameba precox. It is widely distributed geographically, and is common in the blood of small birds, sparrows, robins and larks. It can be propagated in the laboratory in the blood of canaries with- FIG. 147. — PROTEOSOMA PKECOX IN BLOOD OF FIELD LARK. A. Young parasite in blood cell; 5, Half -grown parasite which has pushed aside nucleus of blood cell; C, Parasite with clump of pigment and many nuclei; D, Division into many merozoites. (After Doflein and Wasielewski. MacNeal, "Pathogenic Micro- organisms," published by P. Blakiston's Son & Co.) out great difficulty; sparrows, however, do not long survive in cap- tivity unless kept in round glass jars, where they cannot injure them- selves by dashing against the walls. The blood for examination is obtained from the cephalic wing vein, close to the body, which is nicked with a razor, and the blood taken up in a capillary glass tube containing a little citrate solution. To inoculate a new bird, it is sufficient to inject a small quantity of citrated blood from an infected canary into the breast muscles of the new bird, transferring to a new host at intervals of a month or less. Because it is not difficult to keep on hand, this organism may be used for class study in localities where malarial cases are infrequent. There is no apparent reason for placing it in a different genus from the malarial parasites. The entire asexual cycle, schizogony, may be studied in the periph- eral circulation, as in quartan malarial fever. In nature it is transmitted by both culex and stegomyia (Aedes 1102 PATHOGENIC PROTOZOA calopus), and its development is briefly as follows: The bird is inoculated by the mosquito with spindle-shaped young forms known as sporozoites. These possess the power of ameboid motion, and rapidly penetrate into an erythrocyte, in which they grow quickly ; they con- stantly move about inside the cell until nearly full grown, and are during this stage called trophozoites. The substance of the ery- throcyte is rapidly consumed by the parasite and a dark pigment, melanin or hemozoin, is formed from the destroyed hemaglobin. The mature parasite divides into many small forms called merozoites. and these, when freed by the rupture of the degenerated erythrocyte, escape into the blood plasma, and if not phagocyted, penetrate other erythrocytes and repeat the asexual or schizogenous cycle. The pig- FlG. 148. MlDGUT OF CULEX MOSQUITO, COVERED WITH OoCYSTS OF PROTEOSOMA PRECOX. V, VASA MALPIGHII. (After Doflein and Ross. MacNeal, "Path- ogenic Microorganisms," published by P. Blakiston's Son & Co.) ment and undivided portion (restkorper) of the cytoplasm of the mother cell accumulate in the bone marrow, spleen and other viscera. MALARIA This is one of the most common and widespread of preventable human diseases, and in some localities is the cause of a greater mor- tality and morbidity than tuberculosis. It is caused by one or more of the three forms of the malarial plasmodium. As a rule the infec- tions are simple, yet in the tropics it is not uncommon to find two or even three species of plasmodia in the same patient, and this condition is called a mixed infection. History. — The disease under various names, as chills and fever, Roman fever, Chagres fever, has been known since the greatest antiquity. The cause was not discovered until 1880, when Laveran, a French military surgeon stationed in Algeria first saw the organism and described it as the cause of malaria. He saw and described ;oof SPOROZOA 1103 only the pigmented trophozoite, but also the crescentic gametocytes and flagellating microgametes, and, because of the activity of the flagella, called the parasite Oscillaria malaria, a name afterwards given up. Later, in 1885, Celli and Marchifava described the parasite with greater accuracy and named it Plasmodium malarias, a poor name, since it describes merely a condition assumed by some fungi and mycetozoa, yet, according to the rules of zoological nomenclature, it must stand. In the same year, Golgi described the quartan parasite and in the following year demonstrated the relation of the various stages of the life,cycle of the tertian parasite to the temperature .curve. Even in antiquity many had noted the curious distribution of malaria, and its intimate relation to swamps and marshy places. Manson, who had already shown the role played by an infected mos- quito in transmitting filarial disease, in 1894, suggested that the epidemiology of the disease could best be explained by the hypothesis that it was conveyed by the bite of some blood-sucking insect, probably the mosquito. For years the interpretation of the flagella was a subject of con- troversy. They were regarded as degeneration products by some and as living elements by others. In 1897, MacCallum, working with halteridium, was able to show that they were, in fact, spermatozoa, as he saw them penetrate and fertilize the macrogametes, or large spherical forms without flagella. In 1897, Ross, of the British- Indian Medical Service, described the beginning of the sporogenous cycle in what he called a dapple-winged mosquito, which we now recognize as an anopheline. Following out further Manson 's hypothesis, he was able the same year after long and laborious research to clear up the method of transmission of bird malaria, proteosoma, an analogous disease. Grassi and Bignami and Bastianelli, in 1898, succeeded in demonstrating the complete life cycle of the human form of malaria in the anopheles mosquito. Geographical Distribution. — The disease is found in a belt round the world extending from 40 degrees S. latitude to 60 degrees N. ; it is, however, not equally distributed throughout this zone, and even in the tropics there are many malaria-free areas, principally in the regions of higher altitudes, since the special home of malaria is in the low- lying, swampy and torrid coastal districts and river basins. Islands at a distance from the main land may be entirely free. Malaria reaches its maximum intensity in the tropics, where the anopheline mosquitoes breed continuously throughout the year- and new infec- 1104 PATHOGENIC PROTOZOA tions may occur at any time ; while in the sub-tropics and temperate regions it is a seasonal disease, appearing soon after the onset of hot weather with its new crop of anophelines and continuing until the first cold weather which destroys most of the infected mosquitoes. It is possible that the disease may be carried over from season to season by the hibernating mosquito although definite proof of the importance of this is lacking. It is carried over to the next season by the human carrier, in whom the disease may be latent or who may have suffered from clinical relapses throughout the year. Modern times have seen it disappear from many regions where it was formerly N$^!c^0 FIG. 149. — PLASMODIUM VIVAX (Army Med. School Collection, Washington, D. C.) FIG. 150. — PLASMODIUM VIVAX. (Gamete.) (Army Med. School Collection, Washing- ton, D. C.) endemic, because of increased cultivation of the soil and better surface drainage, as, for example, in England and the Ohio river valley. In the registration area of the United States there were 1565 deaths from malaria in 1913 ; in Italy, up to 1900, the average number of deaths from this cause annually was 16,000. One cannot obtain a true picture of the importance of the disease, however, from mortality statistics, since it is not often fatal, and the morbidity is out of pro- portion to the mortality. In many villages, where it is endemic, one- third to one-half the population may have parasites in the blood, most of them without clinical symptoms, yet they are not able to work and the children remain undeveloped and backward. Much of the illness attributed to hookworm infection is, in reality, due to latent malaria. SPOIIOZOA 1105 During the first part of the acute attack the predominant forms are sehizonts, but later in the course of the disease, as a result of increasing immunity or of treatment, the sporogenous cycle becomes evident. The trophozoites, in increasing numbers, develop into sporonts rather than schizonts. These forms do not develop a vacuole but increase in size and remain round or oval bodies. When mature they occupy most of the red cell but show no sign of nuclear change or of segmentation and they are then called gametocytes. The sex can be distinguished in well stained specimens by remembering that the micro gametocyte, or male form, is rich in nuclear chromatin, of which the flagella or spermatozoa will be formed after the game- tocyte has been ingested by the proper mosquito. The macro game- tocyte, or female form, is distinguished, on the other hand, by the store of nutrient material in the cytoplasm, causing it to stain deeply. After ingestion by a susceptible mosquito the red cells undergo dissolution and the contained gametocytes are liberated. The micro- gametocyte sends out several micro gametes (flagella) one of which fertilizes a macrogamete by penetrating the cytoplasm and uniting with its nuclear chromatin. The fertilized cells change its shape from round to ovoid and becomes motile and is called a traveling vermicule, or ookinette. It travels to the stomach wall which it penetrates and comes to rest on its outer surface and is then called an ob'cyst or zygate. The cyst or zygate increases in size with each successive segmentation of its nucleus until it is ripe. It then ruptures and discharges a mass of sporozoites into the body cavity. These wander to all parts of the body of the mosquito but many reach the salivary glands and later the saliva, and when next the mosquito bites they are injected into the new host. In the state of Mississippi Bass has shown that the age distribution is such that 23 per cent of the population under 20 years of age showed parasites in the blood, whereas only 19 per cent of persons over 20 years were infected, and that the five-year period from 5 to 9 years of age showed the greatest number of infections. Eace and color are important, and negroes showed 36 per cent more infections than whites, and the high point is reached much earlier in black than in white children. (South Med. Jour., 1919, 12, 456.) The parasites belong to the class of hemosporidia, and are closely related to the coccidia, which are parasites of epithelial cells, while 1106 PATHOGENIC PROTOZOA the plasmodia are parasitic on red-blood cells. There are two divisions of the life cycle; that which occurs in man, the endogenous, asexual or schizogenous, and that which occurs in the mosquito, the exogenous, sexual or sporogenous ; for this reason the mosquito is the definitive and the man the intermediate host. Although the life cycle of the parasite varies in details in the different forms of malaria, certain stages are common to all, and in general the life cycle is usually described as follows: an infected mosquito bites a warm blooded animal, often a human being, in order to obtain its meal of blood. As it bites, it infects the wound with its saliva which contains sporozoites coming from the salivary glands. The sporozoites, in the new host, soon gain access to the blood and attach themselves to the erythrocytes upon which they become parasitic. In shape they are long and slender spindles, and when stained with the usual eosin-methylene blue dyes, show a blue cytoplasm and a compact dot of red nuclear chromatin in the center. When the sporozoite escapes the phagocytes, and succeeds in es- tablishing itself on a red cell it soon changes its shape to the ring form and grows rapidly and during this growing stage is known as a trophozoite. This last named form may develop in either of two ways: in the asexual or schizogenous cycle when it is called a schizont, or in the sexual or sporogenous cycle, when it is called a sporont. The schizont goes on to full development in the human host ; the sporont cannot complete its cycle until taken into the stomach of a suitable anopheline mosquito, capable of conveying malaria. The trophozoit which ends its life as a schizont grows rapidly at the expense of the red cell and develops a characteristic vacuole which increases the area of the parasite in contact with the host cell. When mature its nucleus undergoes mitotic changes and the parasite divides into a more or less definite number of segments called merozoites ; these when liberated by the disintegration of the host cell, attack new erythrocytes and develop during the second and subsequent generations in the same manner as the sporozoite. There are three well-recognized forms of the plasmodia, (1) Plas- modium vivax (Grassi and Filetti), causing tertian fever (also called "benign tertian"); (2) Plasmodium malarice (Laveran), causing quartan fever ; (3) Plasmodium falciparum (immaculatum) (Welch), causing the tropical form of malaria, the so-called aestivo-autumnal or subtertian. As the details of development cannot be made out SPOROZOA 1107 easily in fresh specimens, the following description applies to those stained with some form of the Romanowski stain. Plasmodium vivax. — The parasite of tertian fever has a life cycle lasting forty-eight hours arid is easily recognized only when full grown, that is, twenty-four to forty-eight hours after the chill. While a diagnosis may be made on younger forms, it is not so readily made. As its name implies, the Plasmodium vivax is actively ame- boid, and pseudopods and irregular outlines characterize the well- grown parasite; the infected erythrocyte is swollen, often to twice FIG. 151. — PLASMODIUM VIVAX, AN ATYPICAL MACROGAMETOCYTE. Form interpreted by Schaudinn as undergoing partheno- genesis. (Army Med. School Collection, Washington, D. C.) FIG. 152. — PLASMOD'IUM VIVAX (Army. Med. School Collection, Washington, D. C.) • its normal size, the hemoglobin is pale and, especially in spreads in which Hanson's stain has been used, it is so much paler than in the surrounding cells that the infected cell stands out clearly. The part of the cell unoccupied by the parasite is stippled, that is, dotted with reddish granules called Schuffner's dots, and, as the swollen red cell and Schuffner's dots are found in no other form of malaria, their presence is pathognomoriic of tertian. The youngest form, the free merozoite, is rarely seen, but young comet-like forms composed of a particle of red chromatin and a little blue cytoplasm may readily be detected at the height of the fever; that is, a few hours after the chill and sporulation. The round, young schizont as it grows develops early a central vacuole 1108 PATHOGENIC PROTOZOA and assumes the shape of a signet ring, the red chromatin dot being the stone. This small tertian ring grows rapidly as the fever sub- sides, and at the same time the infected cell increases in size. Twenty-four hours after the chill the ring has grown so much that it is referred to as the large tertian ring, and its tendency to irregularities of shape and ameboid form becomes apparent, and fine granules of pigment, called melanin or hemozoin, begin to be visible. After thirty-six hours the rings will all have grown into large ameboid forms. After about forty hours the parasite occupies almost the entire cell and the pigment begins to collect in masses FIG. 153.— PLASMODIUM VIVAX. (Army Med. School Collection, Washington, D. C.) toward the center. Soon after the first signs of segmentation appear, which becomes more and more distinct until fifteen to twenty separate segments or merozoites are seen, each composed of nucleus and cytoplasm. The pigment of the adult parasite and the unused portion of the cytoplasm are cast off after segmentation as a rest- korper, which is promptly phagocyted and such masses accumulate in the spleen, bone marrow and viscera. With rupture of the erythrocyte, at the time of the chill, the merozoites are set free, and if not phagocyted, immediately attack new erythrocytes and the asexual or schizogenous cycle is repeated, until treatment or Jncreasing immunity halts or alters the cycle. SPOROZOA 1109 In practice it is not unusual to find parasites of different ages in the same film, as some individuals seem to develop in advance of others; in this case, however, there will not be much difference in their appearance. When extreme difference of age is noted in films it is probable that there have been several different inoculations, producing double or triple infections with quotidian or irregular fever curves, and such cases are not uncommon. As all the forms so far described belong to the schizogenous cycle, they may be called schizonts, or trophozoites of the schizo- I FIG. 154. — PLASMODIUM VIVAX. (Army Med. School Collection, Washington, D.C.) geiious cycle. The sporogenous cycle begins in man and is com- pleted in the mosquito. The earliest sexual forms noted were the so-called "spheres," large adult parasites, first seen in wet prepara- tions, which did not segment with the schizonts. They are now called gamctocytes and after the disease has lasted some time are found in films made at all stages of the fever; that is, they are incapable of further development until taken into the stomach of the mosquito. The possibility of parthenogenesis will be referred to later. In appearance they are round or oval, and m this fever may be twice the si/e of the red cell. As a rule a narrow margin of red cell is visible after Romaiiowski stains, although the gamete 1110 PATHOG U N 1 C PK( >T< >Z( ) A may lie free in the plasma. Unlike the schizonts, the gamctocytcs have the pigment uniformly distributed throughout the body and there is no indication of segmentation. The young sporonts are distinguished from schizonts by the absence of the vacuole, and, when a little older, by a larger amount of hemozoin. Flasmodium malarias. — The quartan parasite has a life cycle of seventy-two hours, or twenty-four hours longer than the tertian, and the paroxysms come on every third day, or, according to the Italian method of reckoning time, on the fourth day. The young rings of the plasmodium mala-rice are indistinguishable from young tertian rings, but the diagnosis may be made on older forms. The bleach- FIG. 155. — PLASMODIUM M A L A"R i m . FIG. 156— PLASMODIUM MALARLE. (Army (Army Med. School Collection, Wash- Med. School Collection, Washington, ington, D. C.) D. C.) ing, enlargement and stippling of the erythrocyte character- istic of tertian is never found in quartan fever, the infected ery- throcyte being almost normal in appearance. The well-grown quartan parasite does not show amoebic changes but assumes a band form, more or less wide, stretching across the red cell from border to border; with increasing age the band widens until the parasite is nearly square and the hemozoin accumulates toward the center. Segmentation gives rise to almost symmetrical "daisy" forms, show- ing six to eight or, rarely, fourteen merozoites. Parasites of dif- ferent ages may be found, as in tertian, and it is characteristic of quartan fever that examples of all stages of the life cycle may be found at the proper time in the peripheral circulation. Gametocytes differ from tertian mainly in size, since they are never larger than SPOROZOA 1111 the normal erythrocyte until after the latter has raptured, but when free in the plasma it is practically impossible' 1o distinguish them from tertians. Plasmodium falciparum. — The parasite of a^stivo-autumnal fever, I'lfisnifHlinm falciparum, differs considerably from the two forms al- ready described; the life cycle varies between twenty-four and forty- eight hours, and, at least in new infections, only ring forms are found in the peripheral blood, although at a later stage crescentic gametocytes may be present. The youngest aestlvo-autumnal rings, found at the height of the fever, are more delicate than the younp; tertians. As the temperature falls the rings increase in size, but FIG. 157. — PLASMODIUM MALARLE. (Army Med. School Collection, Wash- ington, D. C.) FIG. 158. — PLASMODIUM FALCIPARUM. (X 150.0) (Army Med. School Col- lection, Washington, D. C.) without change of form; the growth is not uniform, but occurs as a thick crescentic swelling on the convex surface of the ring, and occasionally more than one such swelling is present. The large aestivo-autumnal ring, found after the febrile paroxysm has passed, occupies one-third to one-half the red cell, which is never swollen nor stippled, as in tertian, and the parasite is never band-like, as in quartan. Segmenting parasites are almost never seen in the peripheral blood in aestivo-autumnal fever, though in tertian they are common and in quartan numerous. If, however, films are prepared at autopsy from the spleen, liver, bone marrow and brain, enormous numbers of segmenting forms, together with all other stages of the parasite, may be found. The full grown segment cr occupies one- third to one-half the cell and shows a collection of hemozoin in large 1112 PATHOGENIC PROTOZOA blocks in the center. The merozoites vary in number from eight to twenty-five. In addition to the small and large rings the per- ipheral blood shows, after the fever has lasted sufficiently long, the sexual forms or gametocytes. The infected erythrocyte is never stippled nor swollen, but, on the contrary, may appear shrunken. Both the micro- and macrogametocytes in aestivo-autumnal fever are crescentic in shape, their length being about one and one-half and the width about one-half that of an erythrocyte; the pigment is collected toward the center, which is rather paler in stained speci- mens than the poles. At first sight the gametocytes appear to lie free in the plasma, yet in stained specimens a rim or rib of the pale red cell may be seen on the con- cave side. When liberated from the erythrocyte the gametocyte becomes first spindle-shaped and finally oval or round. The male crescent is short and broad, and the female relatively long and slender. The Finer Structure of the Plas- modia. — The finer details, which are only hinted at in fresh speci- mens and in those stained with Man- son 's stain, can be studied to ad- vantage in those stained with some one of the many modifications of the Romanowski stain, such as that of Wright, Hastings, MacNeal or Giemsa. The tertian parasite, which lies in a red cell, may be seen to be divided into a blue cytoplasm and a brilliant red nucleus, and it would be well for the novice to remember that these three conditions must be satisfied before the diagnosis of malaria can be made ; the principal stumbling-block is the blood platelet, often found overlying a red cell, but it, although possessing a ragged blue cytoplasm, has always a relatively large purple nucleus. The chromatin of the young rings is usually present as a single dot, but two such dots are frequently seen. In older forms of the tertian and quartan parasites the various changes found in mitosis may be followed in nucleus. The whole schizogenous cycle may be followed by FIG. 159. — PLASMODIUM FALCIPARUM. (X1500.) (Army Med. School Col- lection, Washington, D. C.) the taking blood smears from a single case of malaria at intervals of SPOROZOA 1113 three or four hours for forty-eight hours for tertian and sestivo- autumnal, and for seventy-two hours for quartan. Two forms of sporonts or gametocytes may be seen; in one the quantity of chromatin is large and the cytoplasm pale blue ; while in the other the reverse is found, the nuclear chromatin is com- paratively small in quantity and the cytoplasm, being rich in nutrient material, stains deeply. The first form, with abundant chromatin, is the male, or microgametocyte, and the latter the FIG. 160. — PLASMODIUM FALCIPARUM, MALE CRESCENT. (Army Med. School Col- lection, Washington, D. C.) female, or macrogametocyte. The differentiation between schizont and sporont may be made while the parasites are still quite young, since the schizont is characterized by the presence of a nutrient vacuole, and the sporont, of equal age, while lacking the vacuole, shows a greater amount of hemozoin, which is never concentrated in the center of the parasite but is scattered equally throughout the body. The cytoplasm of the sporont is less fluid than that of the schizont and shows no tendency to ameboid motion. The chromatin is relatively large in amount and, although broken up more or less 1114 PATHOGENIC PROTOZOA into granules and threads, shows no real tendency to segment or disperse, but remains a compact mass. The quartan parasite, when stained with Wright's or a similar preparation, shows quite regular and symmetrical segmentation, usually into eight merozoites. The distinction between schizont and sporont and between male and female gametocytes may be made on the same grounds as in tertian. In aestivo-autumnal fever the chromatin dot in the young ring is often doubled, or even trebled, and in general is large and stains brilliantly. The adult and half-grown gametes may be differentiated into male and female by the criteria already given. The Examination of Fresh Blood. — Directions have already been given (Chap. LIX) for making wet preparations and if, by ringing the cover-glass with vaseline, drying be prevented, the preparations will keep and may be studied for hours. In tertian fever the young ring forms are at first difficult to detect, unless the amount of light going through the microscope be cut down to the minimum. As the parasite grows older, an increasing number of hemozoin granules appear, and since they are in constant motion the parasite is readily detected. Its cytoplasm is delicate, and with very young parasites is difficult to distinguish from the red cell itself; older parasites, however, develop pseudopods, which are constantly projected and retracted, and the entire organism shows active movements, render- ing it easy to see. The pigment continues to increase, and in the gametes is abundant and in constant motion ; the gametes, however, fail to show any ameboid changes, and the protoplasm is stiff and rigid with a regular, unbroken margin. At times a clear refractile spot is seen, which is the nucleus. The infected erythrocyte is pale and swollen. Even in unstained preparations the sexes may be distinguished; the microgametocyte is about the size of a red cell, the cytoplasm is hyalin, and after the preparation has been made ten to twenty minutes the flagella, or microgametes, may be seen thrashing about in the parasite. After repeated attempts four to eight microgametes rupture the cell and emerge. The macrogamete is larger than a red cell and is finely granular and no exflagellatioii of microgametes occurs. In quartan malaria the differences already described in stained blood may be easily followed. In aestivo-autumnal fever the diagnosis with fresh blood is much more difficult in new infections because of the relative scarcity of SPOROZOA 1115 the parasites in the peripheral blood and the exceedingly small size of the young rings, the absence of hemozoin in them, and the very slight ameboid motion. The older rings arc larger, contain some pigment and are more easily seen. The infected crythrocyte is never pale nor swollen, but, 011 the contrary, may be shrunken and brassy in color. The crcscentic gametocytes are readily detected, and the sexes may be differentiated by their shape and the hyaline or granular character of the cytoplasm. Incubation Period of the Malarial Fevers. — Two methods have been used to determine this point — the injection of infected malarial blood, and biting experiments with infected anophelines. By the first method the incubation period was eighteen days (the longest) for quartan, three days (the shortest) for aestivo-autumnal, and ten days for tertian. By the second method aestivo-autumnal was nine to twelve days, tertian fourteen to .nineteen days, and quartan eighteen to twenty-one days. Since aestivo-autumnal is the only parasite which can complete its cycle in twenty-four hours, the short incubation period is easily understood; on the other hand, the long life cycle of quartan, seventy-two hours, explains its slower develop- ment. Clinical descriptions of the malarial fevers may be found in the standard text-books on medicine, and it is only necessary here to refer briefly to the various forms found in practice. The classical malarial fever consists of a series of paroxysms, following one an- other with a definite periodicity, daily, every other day, or every third day. Each paroxysm is ushered in by a pronounced chill, which is sometimes preceded by malaise, headache and lassitude. The chill lasts from ten minutes to an hour or more, and the patient wraps himself up in heavy blankets. During the chill the tempera- ture begins to rise and within a few hours reaches its high point, 103° to 106°, and then falls slowly to normal during the next few hours. The decline of the fever is accompanied by a profuse per- spiration. Successive paroxysms may occur at exactly the same hour of the day, or may anticipate, febris anteponens, or be delayed an hour or more, febris postponens. The sequence of events, therefore, in a typical malarial paroxysm is malaise, chill, fever and sweat, followed by a period of apparent well-being. There, is, of course, a typical symptomatology and clinical course in the various forms of malarial fever, but it must not be forgotten that there are many atypical cases, and that malaria is a protein 1116 PATHOGENIC PROTOZOA disease, mimicking many other infections, and that without a proper examination of the blood that a correct diagnosis is frequently im- possible. Pernicious malaria is often mistaken for typhoid, for gastric, renal or cerebral disease, and malaria in children may end fatally without presenting any of the classical symptoms. Blood examinations in all febrile cases in malarious districts are therefore necessary to a correct diagnosis. The most important sequel is marked secondary anaemia, which comes on rapidly, even in cases so mild that their nature is unsus- pected; in severe cases the red cell count may drop to three, two or even one million red cells per cubic millimeter. The occurrence of anemia, in malarious regions, should always lead one to suspect this disease. It disappears rapidly under proper quinine treatment but recovery can be hastened by the administration of iron and arsenic. 1. Tertian malaria is distinguished by a chill and fever occurring every other day, the patient feeling quite well on fever-free days. A double tertian infection occurs not infrequently, giving a daily, or quotidian, chill and fever with no free day. 2. Quartan fever, which is relatively rare, gives a chill and fever every third day, with two fever-free days. In this disease also there may be double or even triple infections, giving a quotidian or irregular type of fever. 3. ^Estivo-autumnal fever (subtertian, or malignant tertian) shows an irregular temperature curve, the cycle varying from twenty-four to forty-eight hours. By some authors this type of malaria is divided into two forms, quotidian and tertian, the former giving a minute ring, the latter a larger one. As multiple infections are common the resulting fever curve may be irregular or continuous and the chill entirely absent. In contrast to the regular intermit- tency of tertian and quartan this form is often remittent, the tem- perature curve never dropping to normal. 4. Mixed infections with any two of the above fevers are often found in bad malarial regions in the tropics. 5. Latent malaria is also not infrequent, in which the patient, having no symptoms of the disease, consults a physician for some other reason. In a region where the disease is very prevalent, Bass* has shown * C. C. Bass, South. Med. Journ., 1919, 12, p. 460. SPOROZOA 1117 that almost half (45 per cent) the infections are latent, give rise to no symptoms and have negative histories. 6. The carrier state is found among natives or persons long resident in malarial regions, and, aside from the presence of a large spleen and some secondary anemia, may present no symptoms. It is particularly common among native children, tramps and vaga- bonds. It is not uncommon to find fifty to one hundred per cent of the children in a native village harboring the parasite. THE DEVELOPMENT OF THE HUMAN MALARIAL PARASITE IN THE MOSQUITO (Sexual half of the life cycle, Sporogonie) For this stage to be successful the mosquito must bite a malarial patient with gametes in his blood, for if the patient be one in the first stage of the disease, with only schizonts in his blood, no infec- tion of the mosquito will take place, since the schizonts all perish in its stomach. On the contrary, if the mosquito takes blood from a person who has been ill with malaria for some time, or from an apparently healthy carrier, the schizonts die as usual, but the gametes find in the mosquito stomach for the first time conditions suitable for their further development. The various stages may be studied by causing suitable species of anophelin.es to bite persons with many gametes in their blood, and then dissecting the stomach and observing the changes which take place there. The development is visible in unstained specimens with high, dry lenses. Since there is no essential difference in the development of the three forms of malaria in the mosquito, they will be considered together. The first stage has already been described in discussing the appearance and behavior of gametes in fresh blood. In the mosquito the process may be followed further; the macro- gamete, freed from its enveloping red cell, projects a little mound on its surface, and this apparently attracts the microgametes to its neighborhood. Into this microphyle one, but never more, of the flagella penetrates, following which the mound is instantly retracted. The fertilized macrogamete, now called a "zygote," soon develops the power of vermicular motion, then being called an ookinet, and travels to the wall of the stomach, and, like the coccodia, penetrates an epithelial cell and there encysts, making the oocyst. This grows HIS PATHOGENIC PROTOZOA rapidly and soon escapes from its host cell and conies to lie in the outer layers of the stomach wall, and as it grows projects into the body cavity of the mosquito. The nucleus divides repeatedly, always accompanied by some of the cytoplasm, forming numerous sporo- blasts, and these, in turn, subdivide into innumerable sporozoites; these last escape, with the rupture of the oocyst, into the body cavity. From there they pass to all parts of the mosquito, but especially, perhaps because of chemotaxis, to the salivary glands and ducts, and when next the mosquito bites a warm-blooded host the sporozoites enter the blood stream and start life anew. Cultivation of the Malarial Parasites in Vitro.— Bass and Johns in 1911 announced the cultivation of a few generations of plasmodium vivax in vitro under strict anaerobic conditions. Ten c.c. or more of blood from a malarial patient is defibrinated and distributed in small test tubes in one c.c. quantities and to it is added one per cent of a fifty per cent solution of glucose. The red cells settle so that they are covered with one-half cm. of serum; the parasites grow in a thin layer near the top of the cell mass; beneath this they die, or are phagocyted. The optimum temperature is 19° to 40° C. Bass states that he has cultivated all three species of plasmodia by destroying the complement by heating one-quarter to one-half hour at 40° C. Under strict anaerobiasis it was possible to transfer the cultures and to keep them alive for twenty days. Transmission. — Transmission is solely by various species of anopheles mosquitoes. Although there are fifty or more recognized species, only sixteen have been proved malarial carriers. The more important are Anopheles quadrimaculatus in the United States, Anopheles albimanus in the American tropics, Anopheles maculipennis in Europe, Anopheles senensis in India, and Anopheles costalis in Africa. Description of the Mosquito. — It is impractical to give more than a hasty description of mosquitoes, and the reader is referred to larger works on the subject (Howard, Dyer and Knab; Theobald or "Medical Entomology," Patton and Oragg, London, Madras and Calcutta, 1913). Malaria at Home and Abroad, S. P. James, 1920. It may be noted, however, that the Culicidw, or mosquitoes, belong to the Dipt era, or two-winged insects. They pass through four distinct, stages in their development, the egg, larval, pupal and adult or imago stage. The eggs are invariably laid in water where, if the temperature is warm, they hatch in one to four days. Anopheles eggs are single SPOROZOA 1119 and oval, supported on the surface of the water by ornamental air cells; those of culex are cemented together when laid in raft-like masses. The larva are aquatic and die quickly out of water. They are both bottom and top feeders, eat voraciously, consuming alga? and other vegetable matter, and some varieties are cannibalistic. The larvae are provided with a breathing tube or respiratory siphon pro- jecting upward from the dorsal surface at the caudal end, and in breathing this is thrust upward to the surface of the water and the larva hangs suspended from the surface film. In the anophelines the breathing tube is short and its angle with the body is such that the larva lies parallel with the surface; with culex and other genera the body lies at an angle with the surface. The larval stage lasts about six to fourteen days, depending upon the temperature and food sup- ply, and is followed by the pupal stage, during which no feeding occurs; the pupa, however, needs air and is provided with a short respiratory tube at each side of the head. The habit of coming to the surface of the water to obtain air, which obtains in all mosquitoes except the Mansonia, gives a point of attack in combating them, since a layer of mineral oil on the surface of the water occludes the respira- tory siphon and so kills them. The adult, or imago, emerges from the pupa when the latter is one to three days old; the pupal case ruptures along its dorsum and the emerging imago rests on the floating pupal case until its wings are dry. Since this is a critical stage in its life history and demands quiet water, it is evident that the least wave action is fatal to the mosquito. The nature of the breeding place is characteristic, to a certain extent, of each genus of culicidce; stegomyia (cedes calopus), the car- rier of yellow fever, for example, is strictly domestic and breeds in water jars, tin cans, old beer bottles and other artificial collections of water, ivyeomyia breeds exclusively in the fluid at the base of the leaves of air plants (bromeliads) • the anophelines, while domestic to the extent that they live near human habitations, require natural collections of water for breeding places, such as sheltered spots along the overgrown banks of streams, temporary puddles and even in water in the footprints of man and animals. A proper classification of mosquitoes requires considerable train- ing, but the following points will suffice to separate the anophelines from other mosquitoes. On either side of the proboscis, as seen 1120 PATHOGENIC PROTOZOA FIG. 161. — COMPARISON OF CULEX (LEFT) AND ANOPHELES (RIGHT). Eggs; larvae (note position) ; position of insects at rest; wings; heads showing antenna and palpi. (After Kolle and Hetsch. Jordan, "General Bacteriology," Saunders.) SPOROZOA 1121 under a hand lens, are two pairs of organs; next to the proboscis are the palpi, and outside of these the antennae ; the latter serve to distinguish the sexes, the antennae of the male being heavily ornamented with a bushy, hairy investment (plumose) ; the female antennae, on the contrary, are provided with relatively few, short hairs, arranged in rings at the joints (pilose). In the anophelines the palpi in both sexes are long, at least as long as the probocis, while in all other mosquitoes they are short in the female or in both sexes. This is the principal differential point. The wing mark- ings are of some help, since anopheline wings are almost always spotted. Quite characteristic also is the position assumed by both genera while at rest; among the anophelines the head, thorax and abdomen are all in a straight line and the insect makes an angle with the surface upon which it rests; while the culicidae are hump- backed, the thorax and head are bent on the abdomen so that the latter lies parallel to the surface. By these characteristics it is easy to identify a mosquito as an anopheline, but the further classifi- cation into species is less simple, and works on entomology must be consulted. The life history of the anophelines is not yet completely known ; they fly and bite at dusk and dawn and during the night, and thus differ from stegomyia and most other culicidae, which are day-time biters. Their breeding places have already been described ; of special importance are the temporary collections of water in which the larvae, unhampered by their natural enemies, quickly reach maturity in large numbers. With abundance of food and warm weather, the larva? may require 110 more than ten days to complete their three molts. The female alone sucks blood, the male living on fruit and vegetable juices. Egg-laying does not take place until after a meal of blood, and it is possible that the female journeys fairly long distances to obtain this food, and that the first flight, from breeding place to human habitations, may be longer than subsequent flights. In general the flight is short, not over three hundred yards, and Gorgas found, in Panama, that a clearing of that width about houses gave ample protection. The incubation period of the parasite in the mosquito is about twelve days, after which the insect remains a carrier during the rest of its life, and as the health of the mosquito is unaffected, this may be for two months or more. 1122 PATHOGENIC PROTOZOA Epidemiology. — Since malaria is conveyed solely by the bite of an infected anopheline, the epidemiology is comparatively simple. In practice, nevertheless, the prevention of the disease is extremely difficult; but it is the same for all forms of malaria. It must be attacked from all possible angles and the following are the main points to be observed : 1. Screened houses afford, perhaps, the simplest form of protec- tion, and in beginning work in a new, badly infected place, should be the first thing provided, since they afford a place of security in an otherwise dangerous area, where the workers may take refuge until the situation is under control. This method alone has given magnificent results in Italy (Celli) since it was first used experi- mentally by Sambon and Low in the Roman Campagna. The screens, to be durable, must be of bronze and not iron, and of a fine mesh (20 strands to the inch), and should .be placed, not on windows and doors, but on the outside of porches and balconies ; doorways should have screened vestibules. In default of metallic house screens, bed nets may be used, although they are not very satisfactory, since one must retire at dusk to be protected. In default of both screens and bed nets, something may be accomplished, temporarily, by daily mosquito catching, and in Panama the method has given remarkable results. A native, armed with a small acetylene lantern and a few catching bottles, soon becomes expert, arid can capture each day all the mosquitoes in a number of dwellings. In this way very few anophe- lines escape capture long enough to become infective for man. Chloroform catching bottles are easily prepared by packing a half ounce of small rubber bands, cut up finely, into the bottom, and pouring in as much chloroform as the rubber will absorb and covering it over with dry blotting paper. The wide-mouthed catch- ing bottle is uncorked and inverted over the resting mosquito, which is killed by the chloroform. A convenient trap bottle has been described by La Prince. Below the cork is placed a funnel trap, making it possible to pass from one spot to another without waiting for the chloroform to act upon the mosquito. 2. The second measure is the prevention of mosquito breeding; this is a large but not a hopeless undertaking, if carried out intel- ligently. In the first place, it is to be remembered that compara- tively few mosquitoes are disease carriers, and that measures need be directed against them only. It was first shown, for example, by SPOROZOA 1123 Gorgas, in Havana, that stegomyia could be practically exterminated by doing away with water in artificial containers in the neighbor- hood of dwellings; this so reduced their numbers that when a case of yellow fever was introduced into a community the disease would not spread. In the same way malaria-carrying mosquitoes may be fought, without regard to the presence of non-disease carriers. An accurate mosquito survey is first made, both by examination of the catch of adults and by a hunt for larvae in collections of water throughout the district. A puddle or stream is examined by dipping with a white saucer or a long-handled dipper, along the margins; after a little practice it is possible to obtain any larvae which may be present, and to decide by their appearance and behavoir whether they are anophelines. It is, of course, unnecessary to waste time and money destroying collections of water free from anophelines. When the breeding places have been located, they may be destroyed by the use of oil or larvacide, by draining or by filling. The use of oil and larvacides, while usually a temporary measure, is, nevertheless, of great importance ; any light fuel oil may be used by spraying, by mopping the sides of ditches or margins of ponds, or by a drip barrel at the head of a water course. Since in the malarial season the mosquito develops rapidly the oil must be applied regularly once a week. It is better in the long run to begin in the center of the settlement, with permanent improvements, working outwards as money becomes available, and not to depend on oiling except as a temporary measure. Wherever possible, the swampy areas and pools must be drained, and for this purpose both agricultural tile and open ditches may be used; in the tropics it is necessary to line the latter with concrete to prevent overgrowth by rank vegetation and to protect the banks against caving. Where, because of the lay of the land, drainage is impracticable the area may be filled, or sometimes flooded, or irrigated with sea water, in which most anophelines do not survive. 3. The infection is kept alive in a community by human carriers, and these are especially common among natives and the poor and ignorant, and especially among the native children. Newcomers should not live within five hundred yards of the dwellings of these classes, as infected anophelines can easily travel shorter distances. Only when it is possible to protect the natives also by these measures is it safe to live among them; and that this is possible has been shown many times, particularly in Panama. 1124 PATHOGENIC PROTOZOA 4. The proper treatment and cure of all cases will not only prevent relapses and the carrier state (malarial cachexia), but is one of the most important means of exterminating the disease, since every neglected case becomes a focus for new mosquito and in conse- quence new human infections. It is just as important a part of the prevention of the disease as any other single measure, and in general has been given too little consideration. Between 50 and 90 per cent of all malarial attacks are relapses and not new infections, and these cases can be cured and the sick rates be reduced accordingly by giving proper and adequate treatment during the acute attack and during convalescence. This means, in effect, that the education of the physicians and of the community in general must be under- taken by the health authorities. It is not sufficient merely to obtain the support of the physicians in any given locality, for perhaps not more than 25 per cent of all cases of malaria ever consult a physician. Many now buy various advertised remedies most of which are of little value, but so far as they are curative, the good effect is without doubt due to the quinine which they contain. The general public can be influenced to buy and take quinine in adequate doses over a considerable period by the educational division of the Health Depart- ment. Bass in Mississippi has obtained excellent results by using and advocating the use of a standard treatment of ten grains a day, after the acute attack is past, for a period of eight weeks. While this will not cure all cases, it is a great improvement upon the system of treatment commonly used. To provide as far as possible for the continuation of the treatment for the regular period of eight weeks, the standard package sold and recommended by the local health authorities contains enough quinine for the complete treatment, and it is impossible to buy a part of it at the official price. The immediate effect upon the morbidity and mortality rates of adequate treatment of malarial attacks is immediate and very satisfactory. It is not so much a question of educating the public to take medicine for their illness, they already do that in some form or other, as it is to educate them to take quinine in proper doses. The influence upon the amount of malaria in a community of proper treatment of all cases is difficult to estimate, because of the shifting of population in malarious regions, but it promises to diminish the number of carriers and correspondingly the number of foci of infection. SPOROZOA 1125 5. Quinine prophylaxis is an unsatisfactory measure which must be used by travelers, explorers and troops. There is no method of using quinine which will entirely prevent malaria when the chances for infection are many; the following' methods have all been used: (1) The so-called gram prophylaxis, in which one gram of quinine is taken intermittently every tenth day as the minimum to every fourth day as the maximum. The gram may be taken in a single dose at bed-time, or in four divided doses during the day-time. (2) The double gram prophylaxis, in which the dose is taken on two successive days, as, for example, on the tenth and eleventh, or on the fifth and sixth. (3) The half gram prophylaxis, as proposed by A. Plehn, was 0.5 gram every fifth day; experience has shown that it is of little value. (4) A daily dose of 0.4 to 0.8 gram gives better results than any other method, since the patient suffers less from cinchonism than when larger doses are taken intermittently, and takes his quinine more faithfully. The size of the dose depends on the form of fever present and the number of chances for infection; 0.4 gram will often protect against tertian and quartan, while even 0.8 may fail to prevent aestivo-autumnal. Latent infections and relapses among "prophylacticers" are common and black water fever is not an infrequent sequel. It is well to remember that quinine is rapidly absorbed from the intestinal tract and also as rapidly excreted, and that so far as possible the doses should be properly timed. It is probable that quinine is already in the blood within half an hour of the time it is taken, and that the whole dose is absorbed within six hours and that most of it has already been excreted in the same period. To provide quinine in the blood in sufficient con- centration to be effective in killing the sporozoites injected by the mosquito, it is most reasonable to take one dose of five grains an hour before sunset, and a second dose, if one is compelled to be out, just before midnight. Ten grains taken in this way will provide against infection at dusk and at dawn, the two principal periods during which the anophelines bite, better than one dose at bed time, of the same size. 6. Personal prophylaxis by means of head nets, gloves, suitable clothing, and the use of essential oils, such as citronella, on exposed parts of the body, is helpful in emergencies. 7. The protection of human beings by means of animal barriers has been recommended by several writers. The subject has been 1126 PATHOGENIC PROTOZOA reviewed by Roubaud,* who has been much impressed by the prac- tical disappearance of malaria from France without a corresponding diminution of anophelines capable of acting as carriers. In La Vendee, a Department of France, are regions where there are many swamps and few cattle or domestic animals and it is in such regions that human malaria remains; in other regions, abundantly supplied with domestic animals the disease has practically disappeared al- though anophelines still may be found in the animal shelters. He believes that the insects prefer the blood of cattle and horses to that of human beings, and that a sufficient number of animals will protect the human inhabitants against infection in an otherwise malarious locality. The method is of sufficient importance to merit further study and trial. In conclusion, it may be affirmed that any district, no matter how notorious, may be freed from malaria, if necessity demand it and money be forthcoming, by means of the above measures. Pathology. — The pathological features of the disease are quite definite ; at autopsy there is evidence of some secondary anemia, due to the destruction of enormous numbers of erythrocytes ; the hemo- zoin, in well-marked cases, accumulates in the viscera until they are chocolate or slate colored; the spleen is enlarged and friable, and the liver and kidneys may show cloudy swelling. In smears prepared from the spleen, liver, kidneys, brain and bone marrow, parasites and hemozoin will be found, although the former may not be numerous. The distribution of the parasites is usually unequal, but they are often present in the spleen and brain in greatest numbers. The origin of the pigment has already been described; it is phagocyted and accumulates in the viscera, but after a time dis- appears in some unknown way ; its presence, therefore, is an indica- tion of malaria in recent years. It must be distinguished from hemosiderin, a yellowish pigment found in the viscera after extensive destruction of red blood cells. Hemozoin is soluble in alkalis and insoluble in acids, water, chloroform, alcohol and ether, while hemo- siderin is insoluble in acids and alkalis but soluble in alcohol. Both contain iron, yet the former (hemozoin) does not give a Berlin blue reaction, while it is present with the latter. * Roubaud, E., Les Conditions