LIBRARY OF fJULdSkA '^ngi/Haut Cti/igo, W.C. Africa oks are gladly lent, but please return w \ Culture of glanders bacillus upon cooked potato (Loffler). A TEXT-BOOK UPON THE PATHOGENIC BACTERIA FOR STUDENTS OF MEDICINE AND PHYSICIANS BY JOSEPH McFARLAND, M.D. Professor of Pathology in the Medico-Chirurgical College, Philadelphia; Pathologist to the Medico-Chirurgical Hospital, Philadelphia ; Fellow of the College of Physicians of Philadelphia, etc. WITH 14-2 ILLUSTRATIONS Third Edition, Revised and Enlarged PHILADELPHIA W. B. SAUNDERS & COMPANY 1900 Copyright, 1900, by W. B. SAUNDERS & COMPANY. aR f4 Has * IQOO ELECTROTYPEO BY WESTCOTT & THOMSON, PHILADA. PRESS OF W. B. SAUNDERS & COMPANY. TO MY HONORED AND BELOVED GRANDFATHER Mr. JACOB GRIM WHOSE PARENTAL LOVE AND LIBERALITY HAVE ENABLED ME TO PURSUE MY MEDICAL EDUCATION THIS BOOK IS AFFECTIONATELY DEDICATED PREFACE TO THE THIRD EDITION. Since this work first appeared, extensive progress has been made in the subjects of which it treats, and consid- erable valuable literature has been added, making it necessary to materially increase the size of the book. The gradual but steady specialization that has taken place in bacteriology, especially in relation to the public health and industries, has given us so much information upon the infectious diseases of man, their etiology, diag- nosis, and treatment ; the infectious diseases of the lower animals and their danger to man ; the proper source and preparation of water for public use; the disposal of sewage ; the protection of the consumer against polluted milk ; the means of artificially ripening cream and flavoring butter ; and the protection of canned goods from contamination during manufacture, that it has become a serious problem to know how much can safely be left out, and just what must be put in, a text-book. That author is indeed to be congratulated who can satisfy his readers in a limited space ! In preparing this edition the primaiy object of the book has not been abandoned, but the endeavor has been made to carry out as consistently as possible the original plan of a work upon Pathogenic Bacteria. Some bacteria harmless in themselves have been con- sidered because of their confusing resemblances to patho- genic forms. As nearly as possible, space has been ap- portioned according to the importance of the subjects dealt with. The matter upon Infection and Immunity has been entirely rewritten, and is presented in what is hoped will 8 PREFACE TO THE THIRD EDITION. prove to be a lucid manner. Considering that we know- little about infection and nothing about immunity, there is no other course left than to consider the theories that have been advanced and to refute them one by one. Other new chapters appear here and there, and much of the substance of the book has been rewritten and altered. The principal changes will be found where the advances have been most rapid — that is, under Tuberculosis, Diph- theria, Tetanus, Plague, etc. Many of the changes have been in accord with sugges- tions made in the numerous "reviews," though in this particular no attempt was made to please everyone, or the work would have been doubled in size and its character entirely changed. While acknowledging that foot-note references are not ideal, they are less objectionable than references inserted in the text, as they do not interrupt its continuity, and are more likely to be observed than when appended at the end of the chapter or in the form of a general bibli- ography at the end of the book. All the new matter con- tains foot-note references to the original writings, and, where possible, all important subjects are now accom- panied by references to the original communications, ex- perience having shown that many students prefer to con- sult the authorities when references are at hand. Experience seems to indicate that the chapters upon the Technic of Bacteriology suffice for the average stu- dent ; and while the work is not intended for a laboratory guide, much new matter upon manipulations has been added. In criticising the book, one should not forget that it is upon The Pathogenic Bacteria, and is written for students and practitioners of medicine, and should not condemn it if it does not contain matter outside of its logical limitations. JOSEPH McFARLAND. August, 1900. > PREFACE. The following pages are intended to convey to the reader a concise account of the technical procedures necessary in the study of bacteriology, a brief descrip- tion of the life-history of the important pathogenic bacteria, and sufficient description of the pathological lesions accompanying the micro-organismal invasions to give an idea of the origin of symptoms and the causes of death. The work being upon Pathogenic Bacteria, it does not cover the whole scope of parasitology, and the parasites of higher orders are all omitted. Malaria and amebic dysentery are omitted as logically as tape-worms and pediculi. The higher fungi are also omitted, both because they are not bacteria and because their proper consideration would make a small book in itself. In leaving out the non-pathogenic bacteria of course a stumbling-block was encountered. The Sarciua ven- triculi, for instance, may be a cause of dyspepsia, yet can scarcely be regarded as pathogenic, and, together with other similar bacteria of questionable deleterious operation, has been omitted ; on the other hand, it has been thought advisable to include and describe somewhat at length a long list of spirilla similar to, and probably closely allied with, the spirillum of cholera, yet not the cause of any particular diseased condition. 9 IO PREFACE. The aim has been to describe only such bacteria as can be proven pathogenic by the lesions or toxins which they engender, and, while considering them, to mention as fully as is necessary the species with which they may be confounded. The book, of course, will find its proper sphere of usefulness in the hands of medical students ; its pages, however, will be found to contain much that will be of interest and profit to those practitioners of medicine who graduated before modern science had thrown its light upon the etiology of disease. In writing this work the popular text-books have been drawn upon. Hiippe, Fliigge, Sternberg, Frankel, Gunther, Thoinot and Masselin, and others have been freely consulted. The illustrations are mainly reproductions of the best the world affords, and, being taken from the great stand- ards, are surely superior to anything new covering the same ground. Credit has carefully been given for each illustration. J. McF. Philadelphia, Feb. i, 1896. CONTENTS. » PART L— GENERAL CONSIDERATIONS. PAGE Introduction 17 CHAPTER I. Bacteria 29 CHAPTER II. Biology of Bacteria 43 CHAPTER III. [nfbctiqm and Intoxication 62 CHAPTER IV. Immunity and Susceptibility 90 CHAPTER V. Methods of Observing Bacteria 144 CHAPTER VI. Sterilization and Disinfection 163 CHAPTER VII. Cultivation of Bacteria ; Culture-media 183 CHAPTER VIII. Cultures, and their Study 202 CHAPTER IX. The Cultivation of Anaerobic Bacteria 216 11 12 CONTENTS. CHAPTER X. PAGE Experimentation upon Animals 221 CHAPTER XI. The Recognition of Bacteria . 227 CHAPTER XII. Bacteriologic Examination of the Air 228 CHAPTER XIII. Bacteriologic Examination of Water 233 CHAPTER XIV. Bacteriologic Examination of Soil 238 PART II —SPECIFIC DISEASES AND THEIR BACTERIA. A. THE PHLOGISTIC DISEASES. I. THE ACUTE INFLAMMATORY DISEASES. CHAPTER I. Suppuration; Gonorrhea; Mumps 246 CHAPTER II. Cerebrospinal Meningitis 274 CHAPTER III. Pneumonia 279 II. THE CHRONIC INFLAMMATORY DISEASES. CHAPTER I. Tuberculosis 292 > CONTENTS. 13 CHAPTER II. PAGE Leprosy 336 CHAPTER III. Glanders 344 CHAPTER IV. Syphilis 351 CHAPTER V. Actinomycosis 356 CHAPTER VI. Mycetoma, or Madura-foot 363 CHAPTER VII. Farcin du Pkeuf 367 CHAPTER VIII. Rhinoscleroma 370 B. THE TOXEMIAS. CHAPTER I. Tetanus 371 CHAPTER II. Hydrophobia, or Rabies 384 CHAPTER III. Diphtheria 390 CHAPTER IV. Cholera, and Spirilla Resembling the Cholera Spirillum . . . .421 14 CONTENTS. C. THE BACTEREMIAS. CHAPTER I. PACE Anthrax 455 CHAPTER II. Typhoid Fever 466 CHAPTER III. Bacilli Resembling the Typhoid Bacillus 504 CHAPTER IV. Yellow Fever 518 CHAPTER V. Chicken-cholera 527 CHAPTER VI. HOG-CHOLERA I 53I CHAPTER VII. SWINE-PLAGUE 538 CHAPTER VIII. Typhus Murium 541 CHAPTER IX. MOUSE-SEPTICEMIA 544 CHAPTER X. Relapsing Fever 549 CHAPTER XI. Bubonic Plague 551 CHAPTER XII. Tetragenus 563 CONTENTS. 15 CHAPTER XIII. PAGB Influenza 566 CHAPTER XIV. Measles 571 CHAPTER XV. Malta Fever 573 D. MISCELLANEOUS. CHAPTER I. Symptomatic Anthrax 575 CHAPTER II. Malignant Edema 581 CHAPTER III. Gaseous Edema 585 CHAPTER IV. Bacillus Proteus Vulgaris 594 CHAPTER V. Whooping-cough 598 INDEX 603 PATHOGENIC BACTERIA. PART I. GENERAL CONSIDERATIONS. INTRODUCTION. Biology, chemistry, medicine, and surgery, in the progress of their evolution, have contributed little by little to the growth of a new branch of learning whose subsequent development has been of inestimable impor- tance to each. Indeed, bacteriology illustrates the old adage, " The child is father of the man," for while it is the offspring of the medicine of the past, it has estab- lished itself as the dictator of the medicine of the present and future, especially in the management of the infectious diseases. I. BIOLOGIC CONTRIBUTIONS; THE DOCTRINE OF SPON- TANEOUS GENERATION. Among the early Greeks we find that Anaximander (43d Olympiad, 610 b. c.) of Miletus held the theory that animals were formed from moisture. Empedocles of Agrigentum (450 b. c.) attributed to spontaneous genera- tion all the living beings which he found peopling the earth. Aristotle (b. c. 384) is not so general in his view of the subject, but asserts that "sometimes animals are formed in putrefying soil, sometimes in plants, and some- times in the fluids of other animals." Three centuries later, in his disquisition upon the Pythagorean philosophy, we find Ovid defending the 2 17 1 8 PATHOGENIC BACTERIA. same doctrine, while in the Georgics Virgil gives di- rections for the production of bees. Not only was the doctrine of spontaneous generation of life current among the ancients, but we find it persist- ing through the Middle Ages, and descending to our own generation to be an accidental but important factor in the development of a new branch of science. In 1542, in his treatise called De Subtilitate, we find Cardan as- serting that water engenders fishes, and that many ani- mals spring from fermentation. Van Helmont gives special instructions for the artificial production of mice, and Kircher in his Mundas Subterraneus (chapter " De Panspermia Rerum ") describes and actually figures cer- tain animals which were produced under his own eyes by the transforming influence of water on fragments of steins from different plants.1 About 1686, Francesco Redi seems to have been the first to doubt that the maggots familiar in putrid meat arose de novo: "Watching meat in its passage from freshness to decay, prior to the appearance of maggots, he invariably observed flies buzzing around the meat and frequently alighting on it. The maggots, he thought, might be the half-developed progeny of these flies. Placing fresh meat in a jar covered with paper, he found that although the meat putrefied in the ordinary way, it never bred maggots, while meat in open jars soon swarmed with these organisms. For the paper he sub- stituted fine wire gauze, through which the odor of the meat could rise. Over it the flies buzzed, and on it they laid their eggs, but the meshes being too small to per- mit the eggs to fall through, no maggots generated in the meat ; they were, on the contrary, hatched on the gauze. By a series of such experiments Redi destroyed the belief in the spontaneous generation of maggots in meat, and with it many related beliefs." In ,1683 Anthony van Leeuwenhoek, justly called the " Father of microscopy," demonstrated the continuity of 1 See Tyndall : Floating Matter in the Air. INTRODUCTION. 19 arteries and veins through intervening capillaries, thus affording ocular proof of Harvey's discovery of the circu- lation of the blood; discovered bacteria, seeing them first in saliva, discovered the rotifers, and first saw the little globules in yeast which Latour and Schwann subse- quently proved to be plants. Leeuwenhoek involuntarily reopened the old contro- versy about spontaneous generation by bringing forward a new world, peopled by creatures of such extreme minuteness as to suggest not only a close relationship to the ultimate molecules of matter, but an easy transition from them. In succeeding years the development of the compound microscope showed these minute organisms to exist in such numbers that putrescent infusions, both animal and vegetable, literally teemed with them, one drop of such a liquid furnishing a banquet for millions. Abbe Lazzaro Spallanzani (1777) filled flasks with organic infusions, sealed their necks, and, after subjecting their contents to the temperature of boiling water, placed them under conditions favorable for the development of life, without, however, being able to produce it. Spallan- zani' s critics, however, objected to his experiment on the ground that air is essential to life, and that in his flasks the air was excluded by the hermetically-sealed necks. Schulze (1836) set the objection aside by filling a flask only half full of distilled water, to which animal and vegetable matters were added, boiling the contents to destroy the vitality of any organisms which might al- ready exist in them, then sucking daily into the flask a certain amount of air which had passed through a series of bulbs containing concentrated sulphuric acid, in which it was supposed that whatever germs of life the air might contain would be destroyed. This flask was kept from May to August; air was passed through it daily, yet with- out the development of any infusorial life. It must have been a remarkably germ-free atmosphere in which Schultze worked, for, as was shown by those 20 PATHOGENIC BACTERIA. who repeated his experiment, under the conditions that he regarded as certainly excluding all life, germs can readily enter with the air. The term "infusorial life" having been used, here it is well to observe that during all the early part of their recognized existence the bacteria were regarded as ani- mal organisms and classed among the infusoria. Tyndall, stimulated by the work of Pasteur, con- clusively proved that the micro-organismal germs were in the dust suspended in the atmosphere, not ubiquitous in their distribution. His experiments were very ingen- ious and are of much interest. First preparing light wooden chambers, with one large glass window in the front and one smaller window in each side, he arranged a series of test-tubes in the bottom, half in and half out of the chamber, and a pipette in the top, working through a rubber diaphragm, so that when desired the tubes, one by one, could be filled through it. The chamber was first allowed to stand until all the contained dust had settled, and was then submitted to an optical test to de- termine the purity of its atmosphere, a powerful ray of light being passed through the side windows. When viewed through the front window, this ray was visible as long as there were particles suspended in the atmo- sphere to reflect it. When the dust had completely settled and the light ray was invisible because of the purity of the atmosphere, the tubes were cautiously filled with urine, beef-broth, and a variety of animal and vegetable broths, great care being taken that in the manipulation the pipette should not disturb the dust. Their contents were then boiled by submergence in a pan of hot brine placed beneath the chamber, in con- tact with the projecting ends of the tubes, and allowed to remain undisturbed for days, weeks, or months. In nearly every case life failed to develop after the purity of the atmosphere was established. The following extracts from Tyndall' s work 1 will illus- 1 Op. cit. IN TROD UCTION. 2 1 trate how slowly the doctrine of spontaneous generation was abandoned : u At a meeting of the Pathological Society of London, held April 6, 1875, the ' germ theory ' of disease was formally introduced as a subject for discussion, the debate being continued with great ability and earnestness at sub- sequent meetings. The conference was attended by many distinguished medical men, some of whom were profoundly influenced by the arguments, and none of whom disputed the facts brought forward against the theory on that occasion." "The leader of the debate, and the most prominent speaker, was Dr. Bastian, to whom also fell the task of replying on all the questions raised." "The coexistence of bacteria and contagious disease was admitted; but, instead of considering these organisms as probably the essence, or an inseparable part of the es- sence, of the contagium, Dr. Bastian contended that they zvere patJiological products spontaneously generated in the body after it had been rendered diseased by the real con- tagium" " The grouping of the ultimate particles of matter to form living organisms Dr. Bastian considered to be an operation as little requiring the action of antecedent life as their grouping to form any of the less complex chem- ical compounds." "Such a position must, of course, stand or fall by the evidence which its supporter is able to produce, and accordingly Dr. Bastian appeals to the law and testimony of experiment as demonstrating the soundness of his view." " He seems quite aware of the gravity of the matter at hand; this is his deliberate and almost solemn appeal : ' With the view of settling these questions, therefore, we may carefully prepare an infusion from some animal tissue, be it muscle, kidney, or liver; we may place it in a flask whose neck is drawn out and narrowed in the blowpipe flame; we may boil the fluid, seal the vessel during ebullition, and, keeping it in a warm place, may await the result, as I have often 22 PATHOGENIC BACTERIA. done After a variable time the previously heated fluid within the hermetically-sealed flask swarms more or less plentifully with bacteria and allied organisms, even though the fluids have been much degraded in quality by exposure to the temperature of 212 ° F., and have in all probability been rendered far less prone to engender independent living units than the unheated fluids in the tissues would be.' " These somewhat lengthy quotations are of great in- terest, for they show exactly the state of the scientific mind at a period as recent as twenty-five years ago. II. CHEMICAL CONTRIBUTIONS; FERMENTATION AND PUTREFACTION. As in the biologic world the generation of life was an all-absorbing problem, so in the world of chemistry the phenomena of fermentation and putrefaction were inex- plicable so long as the nature of the ferments was not understood. Cagniard Latour and Schwann in the year 1837 suc- ceeded in proving that the minute oval bodies which had been observed in yeast since the time of Leeuwenhoek were living organisms — vegetable forms — capable of growth. While yeast was looked upon as an inert substance in the act of fermenting, it was impossible to under- stand how it could impart fermentation to other sub- stances; but when it was learned by Latour that the essential element of yeast was a growing plant, the phenomenon became a perfectly natural consequence of life. Not only the alcoholic, but also the acetic, lactic, and butyric fermentations have been shown to result from the energy of low forms of vegetable life, chiefly bacterial in nature. Prejudice, however, prevented many chemists from accepting this view of the subject, and Liebig strenuously adhered to his theory that fermenta- tion was the result of internal molecular movement which a body in the course of decomposition communicates to INTRODUCTION. 2$ other matter in which the elements are connected by a very feeble affinity. Pasteur was the first to declare and prove that fermen- tation is an ordinary chemic transformation of certain substances, taking place as the result of the action of living cells, and that the capacity to produce it resides in all animal and vegetable cells, though in varying degree. In 1862, he published a paper "On the Organized Corpuscles existing in the Atmosphere," in which he showed that many of the floating particles which he had been able to collect from the atmosphere of his laboratory were organized bodies. If these were planted in sterile infusions, abundant crops of micro-organisms were obtainable. By the use of more refined methods he repeated the experiments of others, and showed clearly that "the cause which communicated life to his infu- sions came from the air, but was not evenly distributed through it." Three years later he showed that the organized cor- puscles which he had found in the air were the spores or seeds of minute plants, and that many of them possessed the property of withstanding the temperature of boiling water — a property which explained the peculiar results of many previous experimenters, who failed to prevent the development of life in boiled liquids enclosed in hermetically-sealed flasks. Chevreul and Pasteur (about 1836) proved that animal solids did not putrefy or decompose if kept free from the access of germs, and thus suggested to surgeons that the putrefaction which occurred in wounds was due rather to the entrance of something from without than to some change within. The deadly nature of the discharges from these wounds had been shown in a rough manner by Gaspard as early as 1822 by injecting some of the material into the veins of animals. 24 PATHOGENIC BACTERIA. III. MEDICAL AND SURGICAL CONTRIBUTIONS; THE STUDY OF THE INFECTIOUS DISEASES. Probably the first writing in which a direct relationship between micro-organisms and disease is indicated is that by Varro, which says : " It is also to be noticed, if there be any marshy places, that certain minute animals breed [there] which are invisible to the eye, and yet, getting into the system through mouth and nostrils, cause serious disorders (dis- eases which are difficult to treat) " — a doctrine which, as Prof. L,amberton, to whom I am indebted for the extract, points out, is handed down to us from uthe days of Cicero and Caesar, ' ' yet corresponds closely to the ideas of malaria which we entertain at present. Surgical methods of treatment depending for their suc- cess upon exclusion of the air, and of course, incidentally if unknowingly, exclusion of bacteria, seem to have been practised quite early. Theodoric of Bologne about 1260 taught that the action of the air upon wounds induced a pathologic condition predisposing to suppuration. He also treated wounds with hot wine fomentations. The wine was feebly antiseptic, kept the surface free from bac- teria, and the treatment was, in consequence, a modifica- tion of what in later centuries formed antiseptic surgery. Henri de Mondeville in 1306 went even further than Theodoric, whom he followed, and taught the necessity of bringing the edges of a wound together, covered it with an exclusive plaster compounded of turpentine, resin, and wax, and then applied the hot wine fomenta- tion. In 1671 Kircher wrote a book in which he expressed the opinion that puerperal purpura, measles, and various other fevers were the result of a putrefaction caused by worms or animalculse. His opinions were thought by his contemporaries to be founded upon too little evidence, and were not received. Plencig of Vienna became convinced that there was an undoubted connection between the microscopic animal- INTRODUCTION. 25 cules exhibited by the microscope and the origin of dis- ease, and advanced this opinion as early as 1762. Un- fortunately, the opinions of Plencig seem not to have been accepted by others, and were soon forgotten. In 1704 John Colboch described "a new and secret method of treating wounds by which healing took place quickly, without inflammation or suppuration." Boehm succeeded in 1838 in demonstrating the occur- rence of yeast plants in the stools of cholera, and con- jectured that the process of fermentation was concerned in the causation of that disease. In 1840 Henle, considering all the evidence that had been collected, determined that the cause of the infec- tious diseases was to be sought for in minute living organisms or fungi. He may be looked upon as the real propounder of the Germ Theory of Disease, for he not only collected facts and expressed opinions, but also investigated the subject ably. The requirements which he formulated in order that the theory might be proved were so severe that he was never able to attain to them with the crude methods at his disposal. They were so ably elaborated, however, that in after years they were again postulated by Koch, and it is only by strict conformity with them that the definite relationship between bacteria and disease has been determined. Briefly summarized, these requirements are as follows : 1. A specific micro-organism must be constantly asso- ciated with the disease. 2. It must be isolated and studied apart from the disease. 3. When introduced into healthy animals it must pro- duce the disease. Pollender (1849) and Davaine (1850) succeeded in demonstrating the presence of the anthrax bacillus in the blood of animals suffering from and dead of that dis- ease. Several years later (1863) Davaine, having made numerous inoculation-experiments, demonstrated that this bacillus was the materies morbi of the disease. The 26 PATHOGENIC BACTERIA. bacillus of anthrax was probably the first bacterium shown to be specific for a disease. Being a very large bacillus and a strong vegetative organism, its growth was easily observed, while the disease was one readily communicated to animals for experimental purposes. In 1873, Obermeier observed that actively motile flex- ible spiral organisms were present in large numbers in the blood of patients in the febrile stages of relapsing fever. Klebs, who was one of the pioneers of the germ theory, published in 1872 his work upon septicemia and pyemia, in which he expressed himself convinced that the causes of these diseases must come from without the body. Billroth strongly opposed such an idea, asserting that fungi had no especial importance either in the proc- esses of disease or in those of decomposition, but that, existing everywhere in the air, they rapidly developed in the body as soon as through putrefaction a " Faulniss- zymoid," or through inflammation a " phlogistische- zymoid," supplying the necessary feeding-grounds, was produced. In 1875 the number of scientific men who had entirely abandoned the doctrine of spontaneous generation and embraced the germ theory of disease was small, and most of those who accepted it were experimenters. A great majority of medical men either believed, like Billroth, that the presence of fungi where decomposition was in progress was an accidental result of their universal dis- tribution, or, being still more conservative, retained the old unquestioning faith that the bacteria, whose presence in putrescent wounds as well as in artificially prepared media was unquestionable, were spontaneously generated there. Before many of the important bacteria had been dis- covered, and while ideas upon the relation of micro- organisms to disease were most crude, there were sug- gested some practical applications that produced greater agitation and incited more observation and experimen- INTRODUCTION. 2J tation than anything suggested in surgery since the introduction of anesthetics — namely, antisepsis. "It is to one of old Scotia's sons, Sir Joseph Lister, that the everlasting gratitude of the world is due for the knowledge we possess in regard to the relation existing between micro-organisms and inflammation and suppura- tion, and the power to render wounds aseptic through the action of germicidal substances." ' Lister, convinced that inflammation and suppuration were due to the entrance of germs from the air, instru- ments, fingers, etc., into wounds, suggested the employ- ment of carbolic acid for the purpose of keeping sterile the hands of the operator, the skin of the patient, the surface of the wound, and the instruments used. He finally concluded an operation by a protective dressing to exclude the entrance of germs at a subsequent period. Listerism originated in 1875, and when Koch pub- lished his famous work on the Wundinfcctionskrank- heiten (traumatic infectious diseases), in 1878, it spread slowly at first, but surely in the end, to all departments of surgery and obstetrics. From time to time, as the need for them was realized, the genius of the investigators provided devices which materially aided them in their work. Some of these have been indispensable throughout all subsequent in- vestigations and have made possible many discoveries that must otherwise have failed. Among them may be mentioned the improvement of the compound microscope, the use of sterilized culture fluids by Pasteur, the introduc- tion of solid culture-media and the isolation methods by Koch, the use of the cotton plug by Schroeder and van Dusch, and the introduction of the anilin dyes by Weigert. It is interesting to note that after the discovery of the anthrax bacillus by Pollender and Davaine in 1849 there was a prolonged period during which no important patho- genic organisms were discovered, but during which the technic was being elaborated. This was again followed 1 Agnew's Surgery, vol. i. chap. ii. 28 PATHOGENIC BACTERIA. by a period during which important additions followed each other in rapid succession. Thus, in 1873, Obermeier discovered the Spirillum Obermeieri of relapsing fever. In 1879, Hansen announced the discovery of bacilli in the cells of leprous nodules. The same year Neisser discovered the gonococcus to be specific for gonorrhea. In 1880 the bacillus of typhoid fever was first observed by Eberth, and independently by Koch. In 1880, Pasteur published his work upon "chicken- cholera." In the same year Sternberg described the pneumococcus, calling it the Micrococcus Pasteuri. In 1882, Koch made himself immortal by his discov- ery of and work upon the tubercle bacillus. The same year Pasteur published a work upon Rouget du Pore, and Loffler and Shiitz reported the discovery of the bacillus of glanders. In 1884, Koch reported the discovery of the "comma bacillus," the cause of cholera, and in the same year Loffler discovered the diphtheria bacillus, and Nicolaier the tetanus bacillus. In 1892, Canon and Pfeiffer discovered the bacillus of influenza. In 1894, Yersin and Kitasato independently isolated the bacillus causing the. bubonic plague then prevalent at Hong-Kong. In 1894 Sanarelli discovered the bacillus icteroides, thought to be specific for yellow fever. A new era in bacteriology, and probably the most triumphant result of the modern scientific study of dis- ease, was inaugurated in 1890 by Behring, who presented to the world the " Blood-serum therapy," and showed as the result of prolonged, elaborate, and profound study of the subject of immunity that in the blood of animals with acquired immunity to certain diseases (diphtheria and tetanus) a substance was held in solution which was potent to save the lives of other animals suffering from the same diseases. CHAPTER I. BACTERIA. A bacterium is a minute vegetable organism consist- ing of a single cell principally composed of an albumin- ous substance, which Nencki has called mycoprotein. Nencki found the chemical analysis of bacteria in the active state to consist of 82.42 per cent, of water. In 100 parts of the dried constituents he found 84. 20 parts of mycoprotein; 6.04 of fat; 4.72 of ash; 5.04 of unde- termined substances. Mycoprotein, which has the composition C 52.32, H 7.55, N 14.75, *s a perfectly transparent, generally ho- mogeneous body, which probably varies somewhat ac- cording to the species from which it is obtained, the culture-medium in which it is grown, and the vital products which the organism produces by its growth. Sometimes the mycoprotein is granular, as in bacillus megatherium ; sometimes it contains fine granules of chlorophyl, sulphur, fat, or pigment. Bach cell is sur- rounded by a cell-wall, which in some species shows the cellulose reaction with iodin. When subjected to the influence of nuclear stains the bacteria not only take the stain faintly, but in such a manner as to show the existence of a large nucleus situ- ated in the centre of the cell and constituting its great bulk. The cell-wall generally is not stained, but when it does tinge, a delicate line of unstained material can sometimes be made out between the nucleus and the cell- wall, showing the existence of a protoplasm. The anilin dyes, which possess a great penetrating power, color the organisms so intensely as to preclude the differentiation of the cellular constituents. Under 29 30 PATHOGENIC BACTERIA. these conditions the bacteria appear as solidly-colored spheres, rods, or spirals, as the case may be. By careful staining of appropriate organisms a sug- gestion of internal structure beyond what has been men- tioned can be shown. Thus, in some of the bacilli dis- tinct "polar granules," rounded or oval, unstained bodies are observed at the ends of the cell. What their significance may be is unknown. A few bacilli may be observed which contain within them a row of deeply- stained granules, giving the organisms somewhat the appearance of a streptococcus chain. These are called metachromatic granules and are of unknown significance. The Bacillus megatherium is peculiar in having its pro- toplasm filled with small granules which stain more deeply than the bacilli themselves. The diphtheria bacillus and the cholera spirillum stain very irregularly in fresh cultures, as if the tingeable substance was not uniformly distributed throughout the protoplasm. Vacuo- lated bacteria and bacteria that will not stain or stain very irregularly may usually be regarded as degenerated organisms, which, because of piasmolysis or solution, can no longer stain homogeneously. The cell-walls of some of the bacteria seem at times to undergo a peculiar gelatinous change or to allow the ex- udation of gelatinous material from the protoplasm, so that the individuals appear surrounded by a distinct halo or capsule. Such capsules are seen to excellent advan- tage by the pneumococcus as found in blood or sputum, Friedlander's bacillus as seen in sputum, and by the Bacil- lus aerogenes capsulatus in blood or tissue. This is not only a peculiarity of certain individuals, but one which only takes place when they develop under certain condi- tions; thus, Friedlander points out that the capsule of his pneumonia bacillus, when it was found in the lung or in the "prune-juice" sputum, was very distinct, while it could not be demonstrated at all when the organisms grew in gelatin. From the cell-walls of many bacteria numerous deli- BACTERIA. 31 cate straight or wavy filaments project. These are called cilia or flagella, and seem to be organs of locomotion. Sometimes they are observed projecting only from the ends, or from one end; sometimes they are so numerous and so regular in their distribution as to give the organ- isms a woolly appearance. Messea ' has grouped the bacteria according to the ar- rangement of their flagella into monotricha, with a single terminal flagellum; amphitricha, with a flagellum at each end; lophotricha, with a bundle of flagella at one end; and peritricha, with a variable number of flagella round about the body. Many of the bacteria which are thus supplied with flagella are actively motile and swim about like mi- croscopic serpents. In all probability the locomotory powers of the bacteria are not entirely dependent upon the presence of the flagella, but may sometimes be due to contractility of the protoplasm within an elastic cell- wall. The micro-organisms most plentifully supplied with them are those of the rod and spiral shape. Only one of the spherical forms, Micrococcus agilis of Ali- Cohen, has been shown to have flagella. This and one other species are probably the only motile cocci. Ob- serving that the organisms known to be most active are those best supplied with flagella, it is reasonable to con- clude that the motility is dependent upon the flagella. The presence of flagella, however, does not necessarily imply motility, for some of the bacilli amply provided with these appendages are not motile. The flagella may not only serve as organs of locomotion, and be of use to the organism by conveying it from an area where the nutrition is less to one where it is greater, but, as Wood- head points out, may, in the non-motile species, serve to stimulate the passage of currents of nutrient material past the organism, so as to increase the food-supply. The flagellate bacteria have a greater number of repre- sentatives among those whose lives are spent in water 1 Rivista d'igiene e sanita publica, 1890, ii. 32 PATHOGENIC BACTERIA. and in fermenting and decaying materials than among those inhabiting the bodies of animals. This is an additional fact in favor of the view that locomotion and flagella are provisions favorable to the maintenance of the species by keeping the individuals supplied with food. It may be added that such parasitic disease-producing bacteria as do not habitually gain access to the tissues, but inhabit the intestine, as the bacillus of typhoid fever and the spirillum of cholera, are actively motile, like the saprophytes, while those habitually entering the tis- sues and multiplying there are motionless and without flagella. Of course this example is open to criticism, because the spirillum of relapsing fever, which has never been found elsewhere than in the blood and spleen of affected animals, is actively motile. One of the linear organisms, known as the Bacillus megatherium, has a distinct but limited ameboid move- ment. The commonly observed dancing movement of the spherical forms seems to be the well-known Brownian movement, which is simply a physical phenomenon. It is sometimes difficult to determine whether an organism is really motile or whether it is only vibrating. In the latter case it does not change its relative position to surrounding objects. When speaking of the movement of bacteria, we usually refer to individuals, but in some cases to the colonies, it being a peculiarity of organisms of the proteus group that their colonies upon 5 per cent, gelatin show definite migratory tendencies. The active movement of the bac- teria within the colony causes its shape to change con- stantly, so that it bears a faint resemblance to an ameba, and by the changing shape and active movement of its component individuals succeeds in moving about from place to place upon the surface of the gelatin. The bacteria are so minute that a special unit of meas- urement has been adopted by bacteriologists for their BACTERIA. 33 estimation. This is the micro-millimeter (/i), or one- thousandth part of a millimeter, and about equivalent to the one-twenty-five-thousaudth of an inch. As a rule, the spherical organisms are the smallest and the spiral organisms the longest, except the chains of bacilli called leptothrix. Their measurements vary from 0.15 fi (micrococcus of progressive abscess-formation in rabbits) to 2.8 fx (Diplococcus albicans amplus) for cocci, and from 1 X 0.2 ti (bacillus of mouse-septicemia) to 5 X 1.5 li (anthrax bacillus) for bacilli. Some of the spirilla are very long, that of relapsing fever measuring 40 it at times. This estimation of size almost prepares one for the estimation of weight given by Nageli, who found that an average bacterium under ordinary conditions weighed 10000000000 of a milligram. The bacteria multiply by binary division (fission). The bacterium which is about to divide appears a little larger than normal, and, if a spherical organism, more or less ovoid. No karyokinetic changes have been observed in the nuclei, though they may occur. When the conditions of nutrition are good, fission progresses with astonishing rapidity. Buchner and others have determined the length of a generation to be from fifteen to forty minutes. The results of binary division, if rapidly repeated, are almost appalling. " Cohn calculated that a single germ could produce by simple fission two of its kind in an hour ; in the second hour these would be multiplied to four ; and in three days they would, if their surroundings were ideally favorable, form a mass which can scarcely be reckoned in numbers, or, if reckoned, could scarcely be imagined — four thousand seven hundred and seventy-two billions. If we reduce this number to weight, we find that the mass arising from this single germ would in three days weigh no less than seventy-five hundred tons." "Fortunately for us," says Woodhead, "they can seldom get food enough to carry on this appalling rate of development, and a great number die both for 3 34 PA THOGENIC BA C TERIA . want of food and because of the presence of other con- ditions unfavorable to their existence." When the conditions for rapid multiplication are no longer good, the organism assumes a protective attitude and develops in its interior small oval eggs, seeds, or, as they are more correctly called, sfiores (Fig. i). Such a be d e f C^> cz 2) (o) O o CS=^ g=> Fig. i. — Diagram illustrating speculation : a, bacillus enclosing a small oval spore ; b, drumstick bacillus, with the spore at the end ; c, Clostridium ; d, free spores; e and/^ bacilli escaping from spores. spores developed within the bacteria are called endospores. When the formation of such a spore is about to com- mence, a small bright point appears in the protoplasm, and increases in size until its diameter is nearly or quite as great as that of the bacterium. As it nears perfection a dark, highly-refracting capsule is formed about it. As soon as the spore arrives at perfection the bacterium seems to die, as if its vitality were exhausted in the development of the permanent form. Endospores are generally formed in the elongate bac- teria— bacillus and spirillum — but Zopf has described similar bodies as occurring in micrococci. Escherich also claims to have found undoubted spores in a form of sarcina. The spores found in the bacilli are either round or oval. As a rule, each organism produces a single spore, which is situated either at its centre or at its end. When, as sometimes happens, the diameter of the spore is greater than the diameter of the bacillus, it causes a bulging of the organism, with a peculiar appearance described as Clostridium. When the distending spore is in the centre of the bacillus, it produces a barrel-shaped organism; when situated at the end, a " Trommelschlager, " or drum- stick-shaped one. The end-spores are almost character- istic of the anaerobic bacilli. As the degeneration of the BACTERIA. 35 protoplasm of the bacillus sets the spore free, it appears as a clear, highly-refracting sphere or ovoid situated in a little collection of granular matter. Spores differ from the bacteria in that their capsules seem to prevent evaporation and to enable them to with- stand drying and the application of a considerable amount of heat. Ordinarily, bacteria are unable to resist a tem- perature above 6o° C. for any considerable length of time, only a few resistant forms tolerating a temperature of yo° C. The spores, however, are uninjured by such temperatures, and can even successfully resist that of boiling water (ioo° C.) for a short time. The extreme desiccation caused by a protracted exposure to a tem- perature of 1500 C. will, however, destroy them. Not only can the spores resist a considerable degree of heat, but they are also unaffected by cold of almost any intensity. While the cell-wall of the bacterium is easily pene- trated by solutions of the anilin dyes, it is a matter of much difficulty to accomplish the staining of spores, so that we see they are probably more resistant to the action of chemical agents than the bacteria themselves. When a spore is accidentally dropped into some nu- trient medium a change is shortly observed. The proto- plasm, which has been clear, becomes somewhat granu- lar, the capsule a little less distinct; the body increases slightly in size, and in the course of time splits open to allow the escape of the young organism. The direction in which the escape of the young bacillus takes place is of interest, as varying in the different species. The Bacillus subtilis escapes from the side of the spore where a longitudinal fissure occurs, sometimes leaving the cap- sule of the spore in the shape of two small cups; the bacillus of anthrax escapes from the end. As soon as the young bacillus escapes it begins to in- crease in size, develops around its soft protoplasm a cha- racteristic capsule, and, having once established itself, presently begins the propagation of its species by fission. In addition to the endospores, of which we have just 36 PATHOGENIC BACTERIA. been speaking, there are arthrospores. The formation of these is much less clear. It seems to be the conver- sion of the entire microbe into a permanent form. This process is observed particularly in the micrococci, where in the chains or groups certain individuals become en- larged beyond the normal, and surrounded by a capsule. Hiippe, who has paid particular attention to the arthro- spores, believes that they have resisting powers far beyond those possessed by the bacteria themselves. Of the arthrospores little has, so far, been learned. It is not improbable that among the micrococci, and also among some of the smaller bacilli in whom no spores have been observed, the maintenance of the species when conditions of life become unfavorable is due to the as- sumption of a permanent form by some of the individ- uals, without the formation of any spore-like bodies. This is at present largely a matter of conjecture, but the indications pointing in that direction are numerous. It is believed by Frankel and others that sporulation in the bacteria is not a sign of the exhaustion of nutri- tion, but a sign of the vital perfection of the organism. These observers regard spore-formation as analogous to the flowering of higher plants, which takes place only when the conditions and development are best. Morphology. — The morphology of the bacteria is quite varied. Three principal forms, however, exist, from which the others seem to be but variations. The most simple appear as minute spheres, and from their fancied resemblance to little berries are called cocci or micrococci (Fig. 2, a). When the bacteria of this form multiply by fission the resulting two organisms not infrequently remain attached to each other, producing what is called a dipiococcus (Fig. 2, b). The diplococci sometimes consist of two perfect spheres, but more often show a flattening of the contiguous surfaces, which are not in absolute apposition (Fig. 2, g). In a few cases, as the gonococcus, the approximated surfaces are slightly concave, causing the organism to somewhat resemble the BACTERIA. 37 German biscuit called a "semmel," hence biscuit- or semmel-cocci (Fig. 2, //). Frequently a second binary di- vision occurs, causing four individuals to remain closely approximated, without disturbing the arrangement of the first two. When division of this kind produces a distinct tetrad, the organism is described as a tetracoccus, while to the entire class of cocci dividing so as to produce fours, eights, twelves, etc. on the same plane the name merismopedia is given (Fig. 2, e and f). 6990® / (l h CD © e © 3 Fig. 2. — Diagram illustrating the morphology of the cocci : a, coccus or micrococcus; b, diplococcus ; c, d, streptococci; e, f, tetragenococci or meris- mopedia; g, h, modes of division of cocci; i, sarcina; j, coccus with flagella; k, staphylococci. If, as sometimes happens, the divisions take place in three directions, so as to produce cubical masses or "pack- ages" of cocci, the resulting aggregation is described as a sarcina (Fig. 2, t). This form slightly resembles a dice or a bale of cotton in miniature. If the divisions always take place in the same direc- tion, so as to produce a chain or string of beads, the organism is described as streptococcus (Fig. 2, d). When there are diplococci joined in this manner a strepto-diplo- coccits is of course formed. More common than any of the forms already described is one in which, without any definite arrangement, the cocci occur in irregular groups having a fancied resem- blance to bunches of grapes. These are called staphylo- cocci, and, as it is very unusual to find cocci habitually occurring isolated, most cocci not classified under one of the above heads are called staphylococci. When cocci are associated in globular or lobulated 38 PATHOGENIC BACTERIA. clusters encased in a resisting glutinous, homogeneous mass, the name ascococcus has been used in describing them. A modified form of this, in which the cocci are in chains or solitary and are surrounded by an encase- ment almost cartilaginous in consistence, has been called laiconostoc. Certain bacteria, constituting a better-known if not more important group, are not spherical, but elongate or "rod-shaped," and bear the name bacillus (Fig. 3). <-~Lri Fig. 3. — Diagram illustrating the morphology of the bacilli : a, b, c, various forms of bacilli ; d, e, bacilli with flagella ; f, chain of bacilli, individuals dis- tinct; g, chain of bacilli, individuals not separated. I would remark that the absence of a standard by which to separate the cocci from the bacilli is the cause of much confusion. In my judgment, it would be well to place all individuals having one diameter greater than the other among the bacilli. This would prevent the error of describing one species as "oval cocci " and another as " nearly round bacilli," and by giving a definite standard would greatly aid in the formation of a differential table. The bacilli present a considerable variety of forms. Some are quite short, with rounded ends, so as to ap- pear elliptical ; some are long and delicate. Some have rounded ends, as subtilis ; others have square ends, as anthrax. Some are enormously large, some exceedingly small. Some are always isolated, never forming threads or chains ; others nearly always occur in these forms. The bacilli always divide by transverse fission, so that the only peculiarity of arrangement is the formation of threads or chains. In the older writings the short, stout bacilli were all BACTERIA. 39 described under the generic term bacterium. This genus, like some of the species it comprehended, has now passed out of use. Some of the flexile bacilli, whose movements are sinuous, much resembling the swimming of a snake or an eel, were described as vibrio ; but this name also has passed into disuse except in France, where the spirilla are all called vibrio. The long filaments formed by the division of bacilli without their distinct separation are sometimes called leptothrix, and when these long threads form distinct masses surrounded by a jelly-like material the name myconostoc is sometimes applied to them. Some of the elongate bacteria have a remarkably twisted form and bear some resemblance to a cork- screw. These are called spirilla (Fig. 4) (sometimes FlG. 4. — Diagram illustrating the morphology of the spirilla: a, b, c, spirilla; d, e, spirocheta. vibrio). A subdivision of them, whose individuals are not only twisted, but are also very flexible, is called spirocheta. The spirocheta of relapsing fever is the only described species of this genus. A spiral organism of a ribbon shape is called spiro- monas, while a similar organism of spindle shape is called a spiruliiia. One species of spiral bacteria in whose protoplasm sulphur-grounds have been detected has been called ophidiomonas. Some of the spirilla are exceedingly long and deli- cate, as the spirochseta of relapsing fever ; others which are stouter, like the spirillum of cholera, habitually occur 4o PATHOGENIC BACTERIA. in such short individuals as to be easily mistaken for slightly-bent bacilli. The bacteria thus far described are lower in their evo- lution than the following group, because even in their chains and clusters each individual is entirely inde- pendent. The higher forms of bacteria are characterized by a certain mutual dependence by which, for example, one end of a filament becoming attached, growth would take place at the other only. They also differ in having spe- cial elements developed for purposes of reproduction. The higher bacteria are all characterized by filament- ous forms and either real or apparent branchings. The filaments are usually regularly divided transversely, so as to appear as if composed of bacilli. The free ends only seem to be endowed with reproductive functions, and develop peculiar bodies known as conidia. Beggiatoa is a name given to free-swimming forms, motile by undulation of the protoplasm. The division of the filaments into segments is not apparent; they have no special sheath ; the protoplasm frequently contains sul- phur granules. Thiothrix differs in that an end of the filament is fixed so that the plant is not free to swim. In other points it much resembles beggiatoa. Leptothrix resembles thiothrix, but contains no sulphur granules. Cladothrix is characterized by false branchings. There are conidia formed for reproductive purposes. The parent may or may not be motile, but the conidium is flagellated and actively motile. Streptothrix is the highest form and the only one encountered in animal pathology. It is best illustrated by the Streptothrix actinomyces and Streptothrix Ma- dura, and may possibly include the tubercle bacillus and even the diphtheria bacillus if recent observations re- garding them are correct. The characteristic of the species is a true dichotomous BACTERIA. 41 branching. The filaments are not readily divisible into elements and form dense tangled masses. The organism is very pleomorphons, and sometimes forms streptococcns-like chains, the individuals of which are often spoken of as spores, though not possessed of spore-like qualities. Sometimes the filaments break up into bacilli-like fragments. The ends of the filaments often form enlarged club-shaped masses, which are looked upon by some as reproductive in function, but by the majority are thought to be degenerative in nature. They are well illustiated in the common ray fungus of actinomycosis. All of the ray fungi belong to the streptothrix group. Classification. — Leeuwenhoek, when he first saw the bacteria — and his successors even to so recent a date as to include Ehrenberg and Dujardin — did not doubt that they belonged to the infusoria. They are now known to belong to the vegetable kingdom. The extremely simple organization of bacteria naturally places them among the lowest members of the vegetable kingdom, in that class of the Cryptogamia known as Thallophytse, comprising the algae, lichens, and fungi. The algae are mostly water-plants, containing chloro- phyl and obtaining their nourishment from inorganic substances. The lichens are plants, some of which contain chloro- phyl. They live upon inorganic matter, which they generally absorb from the air. According to the new view of the subject, some, if not all, of these plants are regarded as fungi growing parasitically upon algae. The fungi, the lowest group of all, are minute or large plants, mostly devoid of chlorophyl, living upon organic matter, which they obtain as saprophytes from decom- posing animal and Vegetable matters, or as parasites upon the tissues or juices of living animals or plants. This lowest family, the fungi, are divisible into the — Hyphomycetes or Mucorini, or moulds; Saccharomycetes, or yeasts; and Schizomycetes, or bacteria. . 42 PATHOGENIC BACTERIA. Colin divided the bacteria, according to their mor- phology, into — Sphero-bacteria, or cocci ; Micro-bacteria — short rods ; Desmo-bacteria — bacilli ; Spiro-bacteria — spirilla. It has even been suggested to classify the bacteria by the size and number of their flagella, of which so little is known. The most convenient classification, though it cannot be purely scientific, seems to be the morphological one based upon that of Colin. I. Cocci, ] II. Bacilli, \ monomorphous forms of the lower order. III. Spirilla, i IV. Beggiatoa, V. Thiothrix, VI. Leptothrix, j- pleomorphous forms of the higher order. VII. Cladothrix, VIII. Streptothrix, J CHAPTER II. BIOLOGY OF BACTERIA. The distribution of bacteria is wellnigh universal. They and their spores float in the atmosphere we breathe, swim in the water we drink, grow upon the food we eat, and luxuriate in the soil beneath our feet. Nor is this all, for, entering the palpebral fissures, they develop upon the conjunctiva ; entering the nares, they establish them- selves in the nose ; the mouth is always replete with them ; and, as many are swallowed, the digestive appa- ratus always contains them. The surface of the body never escapes their establishment, and so deeply are some individuals situated beneath the epithelial cells that the most careful washing and scrubbing and the use of the most powerful germicides are required to rid the surgeon's hands of what may prove to be dangerous hindrances to the healing of wounds. The ear is not without its microscopic flora ; special varieties live be- neath the finger-nails, and especially the toe-nails, in the vagina, and beneath the prepuce. While so general, however, they are not ubiquitous. Tyndall succeeded in proving that the atmosphere of high Alpine altitudes was free from them, and likewise that the glacier ice contained none. Wherever man, ani- mals, or even plants, live, die, and decompose, bacteria are sure to be present. Notwithstanding their extreme familiarity with the animal body, there are certain parts of it into which bacteria do not enter, or, entering, remain vital for a very short time, for the body-juices and tissues of normal animals are free from them, and their occurrence there may almost ahvays be accepted as a sign of disease. The presence of bacteria in the air is generally de- 43 44 PATHOGENIC BACTERIA. pendent upon their previous existence in the soil, its pul- verization, and its distribution by currents of the atmo- sphere. Koch has shown that the upper stratum of the soil is exceedingly rich in bacteria, but that their num- bers decrease as the soil is penetrated, until below a depth of one meter there are very few. Remembering that bacteria live chiefly upon organic matter, this is readily understandable. Most of the organic matter is upon the surface of the soil. Where, as in the case of porous soil or the presence of cesspools and dung-heaps, the decomposing materials are allowed to penetrate to a considerable depth, the bacteria may occur much farther from the surface, yet they are rarely found at any great depth, because the majority of the known species require oxygen. The water of stagnant pools always teems with bacte- ria, but that of deep wells rarely contains many unless it is polluted from the surface of the earth. Being generally present in the soil, which the feet of men and animals grind to powder, the bacteria, together with the pulverized earth, are blown from place to place into every nook and cranny, until it is impossible to es- cape them. It has been suggested by Soyka that the currents of air passing over the surface of liquids might take up bacteria, but, although he seemed to show it ex- perimentally, it is not generally believed. Where bac- teria are growing in colonies they seem to remain un- disturbed by currents of air unless the surface becomes roughened or broken. Most of the bacteria which are carried about by the air are what are called saprophytes, and are perfectly harm- less to the human being ; but not all belong to this class, nor will they do so while tuberculous patients are al- lowed to expectorate upon the sidewalks, and typhoid patients' wash to dry upon the clothes-line, and their dejecta to be spread upon the ground. The growth of bacteria is profoundly influenced by environment, so that a consideration of the conditions BIOLOGY OF BACTERIA. 45 favorable or detrimental to their existence becomes a necessity. Conditions influencing the Growth of Bacteria. — (a) Oxygen. — The majority of bacteria grow best when exposed to the air. Some develop better when the air is withheld; some will not grow at all where the least amount of oxygen is present. Because of these pecu- liarities bacteria are divisible into the Aerobic bacteria, those growing in oxygen. Anaerobic bacteria, those not growing in the presence of oxygen. As, however, some of the aerobic forms will grow al- most as well without oxygen as with it, the term optional (facultative) anaeirobics has been applied to the special class made to include them. As examples of strictly aerobic bacteria the Bacillus subtilis and the Bacillus aerophilus may be given. These forms will not grow if oxygen is denied them. The staphylococci of suppuration and the bacilli of typhoid fever, pneumonia, and anthrax, as well as the spirillum of cholera, will grow almost equally well with or with- out oxygen, and hence belong to the optional anaerobics. The bacillus of tetanus and of malignant edema, and the non-pathogenic forms, the Bacillus butyricus, Bacillus muscoides, and Bacillus polypiformis, will not develop at all where any oxygen is present, and hence are strictly anaerobic. (b) Nutriment. — The bacteria do not seem able to derive their nourishment from purely inorganic matter. Pros- kauer and Beck, however, have succeeded in growing the tubercle bacillus in a mixture containing ammonium carbonate 0.35 per cent., potassium phosphate 0.15 per cent., magnesium sulphate 0.25 per cent., glycerin 1.5 per cent. They grow best where diffusible albumins are present. The ammonium salts are rather less fitted to support them than their organic compounds. The in- dividual bacterium varies very widely in the nutriment which it requires. Some of the water-microbes can live 46 PATHOGENIC BACTERIA. in distilled water to which the smallest amount of organic matter has been added; others require so concentrated a medium that only blood-serum can be used for their cultivation. Sometimes a species with a preference for a particular culture-medium can gradually be accustomed to another, though immediate transplantation causes the death of the transplanted organism. Sometimes the ad- dition of such substances as glucose and glycerin has a peculiarly favorable influence upon bacteria, causing, for example, the tubercle bacillus to grow upon agar-agar. (c) Moisture. — A certain amount of water is always necessary for the growth of bacteria. The amount can be exceedingly small, however, so that the Bacillus pro- digiosus is able to develop successfully upon crackers and dried bread. Materials used as culture-media should not be too concentrated; at least 80 per cent, of water should be present. Most bacteria grow best in liquid media; that is, they form the longest threads, and diffuse them- selves throughout the 4iquid so as to be present in far greater numbers than when on solid media. The statement that certain forms of bacteria can flour- ish in clean distilled water seems to be untrue. When transferred to such a medium the organisms soon die and undergo a granular degeneration of their substance. If, however, in their introduction a good-sized drop of cul- ture-material is carried with them, the distilled water ceases to be such, and becomes a dilute bouillon fitted to support life for a time. (d) Reaction. — Should the pabulum supplied to bacte- ria contain an excess of either alkali or acid, the growth of the organisms is inhibited. Most true bacteria grow best in a neutral or feebly alkaline medium. There are exceptions to this rule, for the Bacillus butyricus and the Sarcina ventriculi can grow well in strong acids, and the Micrococcus urea can tolerate excessive alkalinity. Acid media are excellent for the cultivation of moulds. (e) Light. — Most species of bacteria are not influenced in their growth by the presence or absence of light. The BIOLOGY OF BACTERIA. 47 direct rays of the sun, and to a less degree the intense rays of the electric arc-light, retard and in numerous in- stances kill bacteria. Some colors are distinctly inhibi- tory to their growth, blue being especially prejudicial. Some of the chromogenic forms will only produce their colors when exposed to the ordinary light of the room. The Bacillus mycoides roseus will not produce its red pigment except in the absence of light. The pathogenic bacteria have their virulence gradually attenuated if grown in the light. (/) Electricity. — Very little is known about the action of electric currents upon bacteria. Very powerful dis- charges of electricity through culture-media are said to kill the organisms, to change the reaction of the culture, and the rapidly reversed currents of high intensity to destroy the pathogenesis of the bacteria and change their toxic products into neutralizing protective (antitoxin ?) bodies. Much attention has recently been devoted to this subject by Smirnow, Arsonval and Charin, Bolton and Pease, Bonome and Viola, and others. (g) Movement. — When bacteria are growing in a liquid medium perfect rest seems to be the condition best adapted for their development. A slow-flowing move- ment does not have much inhibitory action, but violent agitation, as by shaking a culture in a machine, greatly hinders or prevents their growth. The practical appli- cation of this will show that rapidly flowing streams, whose currents are interrupted by falls and rapids, will, other things being equal, furnish a better drinking-water than a deep, still-flowing river. (//) Association. — It occasionally happens that bacteria grow better when associated with other species, or have their pathogenic powers augmented when grown in com- bination. Coley found the streptococcus toxin more active when combined with Bacillus prodigiosus. Occasionally the reverse is true, and Pawlowski found that mixtures of anthrax and bacillus prodigiosus were iess virulent than cultures of anthrax alone. 48 PATHOGENIC BACTERIA. Meunier1 found that when the influenza bacillus of Pfeiffer is inoculated upon blood agar in conjunction with the Staphylococcus aureus its growth is greatly favored. This does not depend upon any fertilizing effect of the staphylococci, but upon a change which they bring about in the hemoglobin, by which it is better adapted to the requirements of the influenza bacilli. A similar advantageous association is pointed out by Sauarelli, who found that the Bacillus icteroides grows well and retains its vitality long when grown in company with certain of the moulds. Rarely, the presence of one species of microorganism entirely eradicates another species. Hankin found that the Micrococcus Ghadialli destroyed the typhoid and colon bacilli, and suggested the use of this coccus to purify waters polluted with typhoid.2 (i) Temperature. — The question of temperature is of importance from its bearing upon sterilization. Accord- ing to Frankel, bacteria will scarcely grow at all below i6° and above 400 C. The researches of Fliigge show that the Bacillus sub- tilis will grow very slowly at 6° C, and as the tempera- ture is elevated it is said that until 12.50 C. is reached fission does not occur oftener than every four or five hours. When 250 C. is reached the fission occurs every three-quarters of an hour, and at 300 C. about every half hour. A few forms of bacteria which can grow at very high temperatures (6o°-70° C.) are described as thermophilic. They are found in manure piles and in hot springs. Tsiklinsky3 has described two varieties of actinomyces and a mould cultivated from earth that grow well at 48°-68° C, and not at all at the temperature of the room. 1 Societe de Biologie, Stance du n Juin, 1898; La Semaine midicale, June 15, 1898. 2 Brit. Med. Jour., Aug. 14, 1897, p. 418. 3 A'uss. Archiv.f. Path., etc., Bd. v., June, 1898. ^ BIOLOGY OF BACTERIA. 49 Most bacteria are killed by temperatures above 6o°-75° C. The spores can resist boiling water for some minutes, but are killed by dry heat if exposed to 1500 C. for an hour or to 1750 C. for five to ten minutes. The resistance of low forms of life to low temperatures is most astonishing. Cold inhibits the growth of all bac- teria, and immersion in freezing-mixtures will destroy a few. The spores seem capable of resisting almost any degree of cold. Ravenel1 performed some interesting experiments with liquid air. Anthrax spores were ex- posed to its action for three hours ; diphtheria bacilli for thirty minutes, typhoid bacilli for sixty minutes, and Bacillus prodigiosus for sixty minutes. The temperature to which the cultures were reduced was about — 31 2° F., yet in no case was the vegetative capability of the bacteria destroyed, and when planted in culture bouil- lon they all grew normally. His researches corroborate those of Pielet and Young and others. Bacteria usually grow best at the ordinary temperature of a comfortably heated room, and are not affected by its occasional slight changes. Some, chiefly the pathogenic forms, are not cultivable except at the temperature of the animal body (3J0 C); others, like the tubercle bacillus, grow best at a temperature a little above that of the body — 400 C. (/) x-Rays. — The action of the ^r-rays upon bacteria has been investigated by Bonome and Gros,2 Pott,3 and others. When the cultures are exposed to their action for prolonged periods their vitality and virulence seem to be slightly diminished. They are not killed by the .r-rays. (k) Antiseptics, etc. — The presence of certain substances — especially some of the mineral salts — in an otherwise perfectly suitable medium will prevent the development of bacteria, and when added to grown cultures of bac- 1 The Medical News, June 10, 1899. * Giornal. med. del Regis Esercito, an. 45, u. 6. s Lancet, vol. ii. No. 21, 1897. 5° PATHOGENIC BACTERIA. teria will destroy them. Carbolic acid and biclilorid of mercury are the best known examples. It is interesting to mention in this connection the results of the experiments of Trambusti, who found it possible to produce a tolerance to a certain amount of bichlorid of mercury by cultivating Friedlander's bacil- lus upon culture-media containing gradually increasing amounts of the salt, until from 1-15,000, which inhibited ordinary cultures, it could accommodate itself to 1-2000. Variations in the amount of oxygen, temperature, moist- ure, etc., beyond what have been described, are prej- udicial to the growth and development of bacteria, first inhibiting their growth, thus tending toward their de- struction. In the practical application of our knowledge of the biology of the bacteria we constantly make use of such precautions as removing from surgical dressings, sponges, etc., every substance that can possibly afford nutriment to bacteria, and heating such materials, as well as culture-media and a variety of other substances, to a temperature beyond that known to be the extreme limit of bacterial endurance. Some forms of the bacteria are never found except in the tissues of diseased animals. Such organisms are •called parasites. The parasitic group really is divisible into the purely parasitic and the occasionally parasitic bacteria. Of the first division the tubercle bacillus may be used as an illustration, for, so far as is known, it is never found in other places than the bodies and dejecta of diseased animals. The cholera spirillum illustrates the second group, for, while it produces the disease known as Asiatic cholera when admitted to the digestive tract, it is a constant inhabitant of certain waters, where it multiplies with luxuriance. Bacteria which do not enter the animal economy, or if accidentally admitted do no harm, but live upon decaying animal and vegetable materials, are called saprophytes. According to their products of metabolism, bacteria are often described as — BIOLOGY OF BACTERIA. 51 Zymogenic, or bacteria of fermentation. Saprogenic, or bacteria of putrefaction. Chromogenic, or color producers. Photogenic, or phosphorescent bacteria. Aerogenic, or gas producers. Pathogenic, or disease producers. The parasitic organisms alone possess much interest to the physician, but as in their growth the saprophytes ex- hibit many interesting vital manifestations, it is not well to exclude them or their products from the following consideration of the Results of Vital Activity in Bacteria. — 1. Fermenta- tion.— The alcoholic fermentation, which is a familiar phe- nomenon to the layman as well as to the brewer and the chemist, is not the work of a bacterium, but of a yeast- plant, one of the saccharomyces fungi. The acetic-acid, lactic-acid, and butyric-acid fermentations are, however, caused by bacilli. A considerable number of bacilli seem capable of converting milk-sugar into lactic acid, some- times associating this with coagulation of milk, some- times not. The production of coagulation in milk is not always associated with acid-production, but with the pro- duction of a curdling ferment similar to that belonging to the gastric juice. There seems to be no real specific micro-organism for the lactic-acid fermentation, although the Bacillus acidi lactici seems to be the most powerful generator of the acid. There may also be several bac- teria which produce the acetic fermentation, though it is generally attributed to a special common form, the Myco- derma aceti or Bacillus aceticus. The butyric fermenta- tion is generally due to the Bacillus butyricus, though it also may be caused by other bacilli, the one named sim- ply being the most common. (For an exact description of the chemistry of the fermentations reference must be made to text-books upon that subject, as their considera- tion here would occupy too much space.) 2. Putrefaction. — This process is in many respects sim- 52 PATHOGENIC BACTERIA. ilar to the preceding, except that instead of occurring in carbohydrates it takes place in nitrogenous bodies. The first step seems to be the transformation of the albumins into peptones, then the splitting up of the peptones into gases, acids, bases, and salts. In the process the innocu- ous albumins are frequently changed to toxalbumins, and sometimes to distinct animal alkaloids known as pto- maines. Vaughan and Novy declare the term "animal alkaloid" to be a misnomer, as ptomaines are sometimes produced from vegetable substances in the process of decomposition; they suggest the term "putrefactive al- kaloids" as preferable. Their definition of a ptomaine is "a chemical compound, basic in character, formed by the action of bacteria on organic matter." The chemis- try of these bodies is very complex, and for a satisfactory description of them Vaughan and Novy's book1 is excel- lent. Ptomaines probably play but a small part in patho- logical conditions. They are formed almost exclusively outside of the body, and only become a source of danger when ingested with the food. It is supposed that the cases of ice-cream and cheese-poisoning that sometimes occur are due to tyrotoxicon, a ptomaine produced by the putrefaction of the proteid substances of the milk before it is frozen into ice cream or made into cheese. The safe- guard is to freeze the milk only when perfectly fresh and avoid adding the sugar and flavoring substances, and al- lowing the whole to stand some time before it is frozen. Numerous others have been described, some toxic, some harmless. It is to compounds of this kind that the occasional cases of " Fleischvergiftung," "meat-poisoning," or " Botulismus," are due, the growth of various bacteria in stale meat bringing about in its proteid substances the development of toxic ptomaines. Kaensche2 carefully investigated the subject, and gives a synoptical table containing all the described bacteria of this class. His 1 Ptomaines and Leucomatnes. 2 Zeitschrift fur Hygiene, etc., Bd. xxii., Heft I, June 25, 1896. * BIOLOGY OF BACTERIA. 53 researches show that there are at least three different bacilli whose growth causes the development of poison- ous ptomaines in meat. 3. Chromogenesis. — Those bacteria which produce col- ored colonies or impart color to the medium in which they grow are called chromogenic ; those with which no color is associated, non-chromogenic. Most chromogenic bacteria are saprophytic and non-pathogenic. Some of the pathogenic forms, as the Staphylococcus pyogenes aureus, are, however, color-producers. It seems likely that the bacteria do not form the actual pigments, but certain chromogenetic substances, which, uniting with constitu- ents of the culture-medium, produce the colors. Galleotti l has described two kinds of pigment, one of which, being soluble, readily penetrates all neighboring portions of the culture-medium, like the colors of Bacillus pyocyaneus, and an insoluble pigment which does not tinge the solid culture-media at all, but is constantly found associated with the colonies, like the pigment of Bacillus prodigiosus. The pigments are found in their greatest intensity near the surface of the colony. The coloring matter never occupies the protoplasm of the bacteria (except the Bacillus prodigiosus, in whose cells occasional pigment-granules may be seen), but occurs in an intercellular excrementitious substance. The pigments are so varied as to give almost every known color. It sometimes happens that a bacterium will elaborate two or more colors. The Bacillus pyo- cyaneus thus produces pyocyanin and fluorescin, both being soluble pigments — one blue, the other green. Gessard has shown that when the Bacillus pyocyaneus is cultivated upon white of egg} it produces only the green fluorescent pigment, while in pure peptone solu- tion it grows with the production of blue pyocyanin alone. His experiments prove a very interesting fact, that for the production of fluorescin it is necessary that the culture-medium contain a definite amount of a 1 La Sperimentale, 1892, xlvi., Fasc. iii., p. 261. 54 PATHOGENIC BACTERIA. phosphatic salt. Sometimes one pigment is soluble, the other insoluble, so that the colony will appear one color, the medium upon which it grows another. Some organisms will only produce their colors in the light ; others, as the Bacillus mycoides roseus, only in the dark. Some produce them only at the room-temperature, but, though growing luxuriantly in the incubator, refuse to produce pigments at so high a temperature. Thus, Bacillus prodigiosus produces a brilliant red color when growing at the temperature of the room, but is colorless when grown in the incubator. The reaction of the cult- ure-medium is also of much importance in this connec- tion. Thus, the Bacillus prodigiosus produces an intense scarlet-red color upon alkaline and neutral media, but is colorless or pinkish upon slightly acid media. Colored lights seem to have no modifying influence upon the pig- ment-production. Even if for successive generations the bacterium be grown so as to be colorless, it speedily recovers its primitive color when restored to its old envi- ronment, no matter what the color of the light thrown upon it. 1 1 have found that bacteria which have been robbed of their color by incubation, when placed in the normal environment produce the original color, no mat- ter what color the light they receive. Some of the pig- ments— perhaps most of them — are formed only in the presence of oxygen. 4. Liquefaction of Gelatin. — When certain forms of bacteria are grown in gelatin the culture-medium is partly or entirely liquefied. This characteristic is en- tirely independent of any other property of the bacte- rium, and is one manifested alike by pathogenic and non-pathogenic individuals. Sternberg and Bitter have shown that if from a culture in which liquefaction has taken place the bacteria be removed by filtration, the filtrate will retain the power of liquefying gelatin, show- ing that the property is not resident in the bacteria, but in some substance in solution in their excreted products. 1 University Medical Magazine, July, 1894, vol. vi., No. 10, p. 675. BIOLOGY OF BACTERIA. 55 These products are described as " tryptic enzymes" by Fermi, who found that heat destroyed them. Mineral acids seem to check their power to act upon gelatin. Formalin renders the gelatin insoluble. As some of the bacteria not only liquefy the gelatin, but do so in a peculiar and constantly similar manner, the presence or absence of the change becomes extremely useful for the separation of different species. 5. Production of Acids and Alkalies. — Under the head of "Fermentation" the formation of acetic, lactic, and butyric acids has been discussed. These, however, are by no means all the acids resulting from microbic me- tabolism. Ziegler mentions formic, propionic, baldrianic, palmitic, and margaric as among those produced. As the acidity due to the microbic metabolism progresses, it im- pedes, and ultimately completely inhibits, the develop- ment of the bacteria. Milk, to which litmus is added, is particularly convenient for detecting the acids. Rosalie acid may also be used, the acid converting its red into an orange color. The same tests will also determine the alkali-production, which occurs rather less frequently than acid-formation and depends chiefly upon the salts of ammonium. The quantitative estimation of the acids can be best made by titration, and the fermentation-tube culture can be employed for the purpose. The contents of the bulb and branch should be shaken together, a meas- n ured quantity withdrawn, and titration with — sodium hvdroxid, or — hvdrochloric acid, performed. In rela- 20 ' tion to the acid, it is always important to know through the presence of what particular carbohydrate the acid was formed. The alkali most frequently formed by bacterial growth is ammonium, which is set free from its combinations, and either flies off as a gas or forms new combinations with acids simultaneously formed. Some bacteria pro- 56 PATHOGENIC BACTERIA. duce acids only, some alkalies only, others both acids and alkalies. Like the acids, the alkalies, when in excess, serve to check the further growth of the micro- organisms. 6. Production of Gases. — This seems, in reality, to be a part of the process of decomposition and fermentation. Among the gases due to bacterial action, C02, H2S, NH4, CH4, and others have been described. If the bacterium be anaerobic and develop at the lower part of a tube of gelatin, not infrequently a bubble of gas will be formed about the colonies. This is almost constant in tetanus and malignant edema. Ordinarily, the production or liberation of gases passes undetected, the vapors escaping from the surface of the culture-medium. To determine the gas production where it is suspected but not apparent, the ordinary fermentation-tubes can be employed. They are filled with glucose bouillon, steril- ized as usual, inoculated and allowed to grow. If gases are formed, the bubbles ascend and the gas accumulates at the top of the tube. In estimating quantitatively, one must be careful that the tube is not so con- structed as to allow the gas to escape as well as to ascend in the main reservoir. For the determination of the nature of the gases ordinarily produced, some of which are inflammable and some not, Fig. 5. — Smith's fer- ' mentation-tube. Theobald Smith has recommended the following methods: "The bulb is completely filled with a 2 per cent, so- lution of sodium hydroxid (NaOH) and tightly closed with the thumb. The fluid is shaken thoroughly with the gas and allowed to flow back and forth from the bulb to closed branch, and the reverse several times to insure intimate contact of the C02 with the alkali. Lastly, before removing the thumb all the gas is allowed to col- lect in the closed branch so that none may escape when the thumb is removed. If C02 be present, a partial BIOLOGY OF BACTERIA. 57 vacuum in the closed branch causes the fluid to rise sud- denly when the thumb is removed. After allowing the layer of foam to subside somewhat the space occupied by gas is again measured, and the difference between this amount and that measured before shaking with the sodium hydroxid solution gives the proportion of C02 absorbed. The explosive character of the residue is determined as follows: "The cotton plug is replaced and the gas from the closed branch is allowed to flow into the bulb and mix with the air there present. The plug is then removed and a lighted match inserted into the mouth of the bulb. The intensity of the explosion varies with the amount of air present in the bulb." 7. Production of Odors. — Of course, such gases as H2S and NH2 are sufficiently characteristic to be described as odors. There are, however, a considerable number of pungent odors which seem dependent purely upon odor- iferous principles dissociated from gases. Many of them are extremely unpleasant, as that of the tetanus bacillus. The odors seem to be peculiar individual characteristics of the organisms. 8. Production of Phosphorescence. — A Bacillus phos- phorescens and numerous other organisms have a dis- tinct phosphorescence associated with their growth. It is said that so much illumination is sometimes caused by a gelatin culture of some of these as to enable one to tell the time by a watch. Most of them are found in sea- water, and are best grown in sea-water gelatin. 9. Production of Aromatics. — The most important of these is indol, which was at one time thought to be pecu- liar to the cholera spirillum. For the method of deter- mining its presence, see " Dunham's Solution." At pres- ent we know that a variety of organisms produce it. Phenol, kresol, hydrochinon, hydroparacumaric acid, and paroxy-phenylic-acetic acid are by no means un- common products of bacteria. 10. Reduction of Nitrites. — A considerable number of bacteria are able to reduce nitrites present in the soil or 58 PATHOGENIC BACTERIA. in culture-media, prepared for them, into ammonia and nitrogen. To the horticulturist this is a matter of much interest. Wiuogradsky has found a specific nitrifying bacillus in soil, and asserts that the presence of ordinary bacteria in the soil causes the reduction of no nitrites so long as his special bacillus is withheld. Reduction of nitrites can be determined by the use of a nitrate broth made by dissolving in iooo c.c. of water i gram of peptone and 0.2 gram of potassium nitrate. The ingredients are dissolved, filtered, then deposited in tubes, and sterilized. As nitrites and ammonia are com- monly present in the air and are taken up by fluids, it is always well to control the test made by an uninoculated tube tested with the reagents in the same manner as the culture. Two test solutions are employed:1 !Boil, cool, filter, and add 156 c.c. of dilute (1-16) hydric acetate. II. Sulphanilic acid, 0.5 gram. Hydric acetate, diluted, 150.0 c.c. Keep the solutions in glass-stoppered bottles and mix equal parts for use at the time of employment. About 3 c.c. of the culture and an equal quantity of the uninoculated culture fluid are placed in test-tubes and about 2 c.c. of the test fluid slowly added to each. The development of a red color indicates the presence of nitrites, the intensity of the color being in proportion to the quantity of nitrites present. If a very slight pinkish or reddish color in the uninoculated culture fluid and a deeper red in the culture develop, it shows that there was a small amount of nitrites present already, but that more have been produced by the growth of the bacteria. The presence of ammonia in either fluid is easily deter- mined by the immediate development of a yellow color or precipitate when a few drops of Nessler's solution (see text-books upon Chemistry) are added. 1 Journal of the American Public Health Association, 1 888, p. 92. BIOLOGY OF BACTERIA. 59 Failure to determine either ammonia or nitrites may not mean that the nitrates were not reduced, but that they were reduced to N. It is, therefore, necessary to test the solutions for nitrates, which is done by the use of phenolsulphonic acid and sodium hydroxid, which in the presence of nitrates give a yellow color. 11. Combination of Nitrogen. — Not only do bacteria destroy or reduce nitrogen compounds, but some of them are also able to assimilate nitrogen from the air and com- bine it so as to be useful for the nourishment of vegetable and animal life. The most interesting organisms of this kind are found in association with the roots of the legu- minous plants, peas, clover, etc., and have been studied by Beyerinck. It seems to be by the entrance of these bac- teria into their roots that the plants are able to assimilate nitrogen from the atmosphere and enrich sterile ground. Every agriculturist knows how sterile soil is improved by turning under one or two crops of clover with the plough. 12. Peptonization of Milk. — Numerous bacteria possess the power of digesting — peptonizing — the casein of milk. The process diners with different bacteria, some digesting the casein without any apparent change in the milk, some producing coagulation, some gelatinization of the fluid. In some cases the digestion of the casein is so complete as to transform the milk into a transparent watery fluid. Milk usually contains bacteria, entering it from the dust of the dairy, possessing this power. In the process of peptonization the milk may become bitter, but need not change its original reaction. As the peptonization progresses the milk very often becomes poisonous, espe- cially to individuals under two years of age, and may bring about a fatal enterocolitis or "summer complaint." Vaughan and Novy believe the poisonous change to de- pend upon the formation of tyrotoxicon. Lubbert has shown,1 however, that the disease not only occurs in consequence of toxic substances formed from the split-up 1 Zeitschrift fur Hygiene, 1896, xxii., Heft 2, p 1. 60 PATHOGENIC BACTERIA. albumins, or from the presence of metabolic products of the bacteria, but also from the irritating bacteria them- selves. It is only in unusually warm weather that these bacteria are able to grow luxuriantly, hence enterocolitis is most common in summer. Sometimes the properties of coagulation and digestion of milk are valuable aids in the separation of different species of bacteria. Thus, the colon bacillus coagulates milk, but the typhoid bacillus does not. 13. Production of Disease. — Bacteria which produce diseases are known as pathogenic ; those which do not, as non-pathogenic. Between the two groups there is no sharp line of separation, for true pathogens may be culti- vated under such adverse conditions that their virulence will be entirely lost, while at times bacteria ordinarily harmless may be made virulent by certain manipulations or by introducing them into animals in certain combina- tions. In order to determine that a micro-organism is possessed of pathogenic powers the committee of bacte- riologists of the American Public Health Association recommended that: 1. When a given form grows only at or below i8°-20° C. inoculation of about 1 per cent of the body-weight with a liquid culture seven days old should be made into the dorsal lymph-sac of a frog. 2. When a species grows at 250 C. and upward an inocu- lation should be made into the peritoneal cavity of the most susceptible (in general) of warm-blooded animals — t. e., the mouse, either the white or the ordinary house mouse. The inoculation should consist of about 1 per cent, of the body-weight of the mouse of a 4-8-hour standard bouillon culture, or a broth or water suspension of one platinum loop from solid cultures. When such intraperitoneal injection fails, it is unlikely that other methods of inoculation will be successful in causing the death of the mouse. If the inoculations of the frog and mouse both prove negative, the committee think it un- necessary to insist upon any further tests of pathogenesis as being requisite for work in species differentiation. BIOLOGY OF BACTERIA. 61 Production of Enzymes by Bacteria. — Some of these have already been mentioned, as those which coagulate milk, dissolve gelatin, etc. There are, however, many others which have interesting actions upon animal and vegetable substances. Knowledge upon the subject is just becoming systema- tized, one of the best works being that of Emmerich and Low,1 who, observing that in old cultures of Bacillus pyocyaneus the bacteria became transformed into a gelat- inous mass, were led to experiment with concentrations of old cultures. For this purpose the well-grown and degenerating cultures were condensed to ^ volume in a vacuum apparatus, when their bacteriolytic powers were found to be much increased. Emmerich and Low were subsequently able to precipitate from the culture an enzyme, which they called Pyocyanase, and reached the rather hasty inference that the cessation of growth of bacteria in cultures depends upon the generative enzymes; that the enzymes destroy the dead bacteria; that enzymes will kill and dissolve living bacteria and destroy toxins, and, therefore, are useful for the treatment of infectious diseases. Further, that the antitoxins are simply the accumulated enzymes which the immunized animals have received during the progress of their treatment, and which, appearing in the serum, produce the effects so well known. It is very probable that many of the toxic effects of bacteria and their cultures depend upon the enzymic sub- stances present. 1 Zeitschrift fur Hygiene, 1899. CHAPTER III. INFECTION AND INTOXICATION. ZiEGLER defines infection as " the entrance of bacteria into the body and their increase there." In the majority of cases it means the entrance into and multiplication of bacteria in the tissues, though this cannot be given as a definition because of the possibility that certain bacteria, as, for instance, the cholera organisms, may begin their pathogenic effects by poisonous action upon the mucous membrane of the intestine before they succeed in enter- ing the tissues. While it may be true of a few infectious bacteria that entrance into the tissues is unessential for their pathogenesis, it is also true that some of the cavi- ties of the body contain bacteria which flourish in their fluids and cause no departure from normal conditions. When abnormal conditions arise, however, and enable them to leave their normal habitat, they may become the cause of serious ills. This is particularly the case with the intestine. Under normal conditions the colon bacil- lus and the streptococcus are pretty constant inhabitants of the entire tract, living a saprophytic existence upon the contained fecal matter. Should a portion of the intestinal wall become ulcerated or strangulated, these usually harmless bacteria penetrate into the tissues, some- times causing local, sometimes metastatic suppurative affections. The time at which infection takes place varies some- what according to the kind of bacterium with which we have to deal. Thus, in the case of some whose early operations are obscure, as typhoid and cholera germs, infection occurs at the moment at which the bacteria enter the alimentary canal. In the case of the colon 62 INFECTION AND INTOXICATION. 63 bacillus and streptococcus in the intestine and the staphy- lococcus in the nose and mouth, infection takes place when the bacteria invade the tissues. The mere entrance of bacteria into the tissues is not sufficient to constitute infection, because should the entering bacteria belong to varieties whose metabolic products are without effect upon the tissues no evidence of the invasion is apparent. Infection, therefore, implies (1) the invasion of the body or its tissues by bacteria and (2) injury of the body by the bacteria. Our knowledge has not yet progressed sufficiently to permit us to say that any known bacterium may not, under certain appropriate conditions, be a cause of disease, even though there are many species, such as the Bacillus subtilis and Bacillus prodigiosus, which have never been known to do so. In speaking of bacteria we constantly speak of pathogenic and. non-pathogenic species, as if there were some fixed difference by which we could separate them; yet a bacterium harmless for one animal is danger- ous for another, and one usually incapable of harming an animal may work havoc in its tissues should certain unusual and abnormal conditions present themselves. One of the best illustrations of disease produced by (usually) harmless bacteria is the condition known as sapremia. In it we find that the growth of saprophytic bacteria in the discharges from wounds, and in gan- grenous areas, leads to the formation of toxic ptomains, which, being absorbed by the lymphatics and into the blood, produce fever and other disagreeable symptoms. In this sapremic condition it is the activity of the bacteria in the dead or effete matter, not their invasion of the sound tissue, that causes the trouble, and the condition may depend upon the presence of bacteria that are entirely unable to live in the tissues of the body. The ability of bacteria to live in the tissues of a living animal varies very greatly. Thus, there are purely sapro- phytic bacteria that cannot live in the tissues at all; occa- sionally parasitic bacteria that usually enjoy a saprophytic 64 PATHOGENIC BACTERIA. >\ Va .A 6 ^ >>> 2 a J* d 'a w r<" og ajtiaj op-m'ci w n £ J§ fc •« .2 * 8 'S fp _« L'- J5 ? J g a £3 S &,S fi* .-B-s ■ -JB -g .a ■* 'I st-S ill '5i-§*i-s § £ ill! " S^l -ESS SUlS'gl * iHi ^ as as p _S2-°-Ho _ 5 2 £ a rt C "S "3 m e 3 '-p 'u 'J 5 -^ tc "o c fi +J •«■■ 'O t-i s a s a §c3a^&fr«g3i 2 * «> 1. c c: s 1 1 -a a «§ a § * § ? I 8 I I * dill-2 rt ,d >4 aj .Q d -a O oj ,d d .3 « _£! $ § 3 s .2 & § £ 3 .S ^ a, & 0 CJ -M JO o a . 01 .a w "3 -H-» »|J | >> K ,£j "S a el a M to •o a < .2 ft o w « H > a . S jQ 1h 9 &< -r; H-> O a Ctf « .2 P So CO cs > > a ^ a 2 - £ S a^1 >>il fih o St • '55 a g 2 i2^^^-2^ cu^ $£.a,art O Oh •d .j^ B PI 3 rt a CJ Si w CJ Sh ♦J 9 U 3; T-! -s! ^a pq *j o .5 •J5 a X on a M 03 < p c s y which to compare them. As soon as the micro- INFECTION AND INTOXICATION. 67 organism is isolated and grown experimentally the unnatural environment begins to tell upon it, and its biological characteristics begin to change. As it is transplanted again and again, artificial selection comes into play to modify the species. To illustrate this, I will mention the case of a streptococcus that is growing in the laboratory at the time of this writing. This micro- organism was isolated from the blood of a patient dead of puerperal sepsis. It at first grew very scantily upon glycerin agar-agar and blood-serum, and was fatal to rab- bits in intravenous injections of y1^ c.c. of a 24-hour-old bouillon culture. It has now been cultivated for a year or more, during which time its virulence has apparently entirely disappeared, so that at present 5 c.c. fail to pro- duce any symptoms when injected into the ear vein of a rabbit. It has also greatly altered its cultural appear- ances, so that it now grows luxuriously upon glycerin agar-agar and blood-serum. The attenuation of this streptococcus probably depends, in large measure, Upon the fact that but few of its individual cocci were capable of growing upon artificial media, and in transplanting it these vegetative cocci and their progeny have been gradually picked out, until they have entirely outlived the less vegetative but more pathogenic members of the colonies. If this coccus had been treated differently, it might have become as virulent as that with which Marmorek experi- mented. He kept his experiment organism constantly passing from rabbit to rabbit, using in the intervals a culture-medium consisting of bouillon containing asses' serum, human serum, ascitic fluid, etc. In this manner he was able to produce a coccus of very moderate vegeta- tive capacity, but so extraordinary in virulence that one one-hundred millionth of a cubic centimeter was suf- ficient to kill a rabbit. Many bacteria attenuate when kept in the laboratory. A few species, like the Bacillus anthracis, are so persist- ently virulent that when for experimental purposes it is 68 PATHOGENIC BACTERIA. desirable to secure an attenuated culture considerable pains must be taken to cultivate it at certain maximum temperatures or in media containing small amounts of antiseptic substances. When the virulence has been destroyed by experi- mental manipulations or by natural attenuation, it is sometimes difficult, sometimes impossible, to secure its return. The method usually adopted is that of rapid passage from animal to animal of the same kind, then to more resistant kinds, hoping to accustom it to life in the animal once more. It may be necessary to kill the animal to do this, inoculating beneath the skin or into the peritoneum, allowing some hours or a day for some of the bacteria to grow, killing the animal, and transfer- ring the secretions containing the survived bacteria to another animal, and so on. In this manner there is an artificial selection carried on by which there are secured from each animal those bacteria best qualified to live in that kind of animal. Another method frequently employed for exalting the virulence of bacteria is that of growing them in collo- dion sacs placed in the abdominal or other cavity of the body, or beneath the skin, where they receive by osmosis the fluids of the body and become accustomed to them. Muir and Ritchie state that attenuated diphtheria cult- ures may have their virulence raised by being injected into an animal together with the Streptococcus pyogenes; an attenuated culture of the bacillus of malignant edema by being injected with the Bacillus prodigiosus; an atten- uated streptococcus by being injected with the Bacillus coli communis, etc. A culture of typhoid fever bacillus may also be increased in virulence by being injected along with a dead culture of the Bacillus coli communis. These observations call attention to the results of mixed infections. While we are so trained by our laboratory ex- periments and our bacteriologic reading that we think of streptococcus, diphtheria, and tetanus infection as being caused by the streptococcus, diphtheria, and tetanus INFECTION AND INTOXICATION. 69 bacilli respectively, it should never be forgotten that under natural conditions infection of an unmixed charac- ter is made practically impossible by the prevalence of bacteria in general ; consequently, the streptococcus can scarcely enter the skin without being accompanied by the ever-present staphylococci ; the diphtheria bacillus finds bacteria already in the throat in which it begins to grow ; and the soil with which the tetanus bacillus enters the body carries with it quite a flora of its own. It is true that these accessory bacteria are in general unimportant ; yet that this is not entirely true is shown by the fact that the streptococcus which frequently grows in company with the diphtheria bacillus in the throat greatly modi- fies the course of the disease. Furthermore, it is the streptococcus that is usually responsible for the meta- static abscesses that develop. Again, because of its strictly anaerobic nature the tetanus bacillus may be entirely unable to grow in the tissues without the presence of aerobic bacteria to withdraw the oxygen from it. Roget found the combination of the bacillus of malig- nant edema and Bacillus prodigiosus greatly more viru- lent than the malignant edema bacillus by itself. Giarre found combinations of pneumococci and diphtheria ba- cilli caused a great increase in the effect of the pneumo- coccus. Ghadialli found a micrococcus which destroyed both the typhoid and colon bacilli. It is interesting to observe that the bacteria secured from cases of disease during epidemics are usually more virulent than those secured from sporadic cases, probably because of a natural selection by which the micro-organ- isms passing from patient to patient undergo an exaltation of virulence very similar to that carried out experimen- tally in the laboratory. 2. Number plays an important role in infection. It is true that in a few diseases (anthrax), when the viru- lence of the bacterium and the susceptibility of the ani- mal (guinea-pig) are alike great, the introduction of a single bacterium may be fatal. JO PATHOGENIC BACTERIA. Ordinarily, however, number plays a very important part in the process of infection. Park explains this by calling to mind what happens and can easily be de- monstrated in the transplantation of bacteria to fresh culture-media. When such a transplantation is made the greater number of the transplanted organisms quickly die. Those that live grow rapidly, so that while a short time after transplantation the number of living bacteria is less than the number actually put in, in a few hours these have increased amazingly. "With those bacteria whose virulence is great — i. e. those which are capable of growing with great ease in the body- fluids — a very few organisms will produce disease almost as quickly as a million, allowance only being made for the short time required for a few to become equal in num- ber to the million. At the other extreme of virulence, however, many millions may have to be introduced to permit of the development of any of the organisms in the body." It can be shown experimentally that a certain number of the pyogenic cocci can be injected into the perito- neum of a rabbit without provoking peritonitis, but that if this number be exceeded the animal will die. The effect of number must not be construed, however, to mean that the greater the quantity of culture injected the more rapid the outcome. After a fatal dose of any culture has been given to an animal a certain length of time must elapse for the development of pathogenesis, and doubling or trebling the dose will scarcely hasten the fatal outcome. This is well illustrated in the case of guinea-pigs and mice, which, when injected with anthrax, die in about twenty-four hours, whether the dose be ten times or a million times that which is fatal. 3. Avenue of Infection. — It makes a great difference in the subsequent events whether the infection takes place through usual or unusual channels. Thus, in the case of tuberculous infection of man, when the bacilli are inhaled or ingested and reach the internal organs, the INFECTION AND INTOXICATION. 71 usual affections are observed, and the case progresses, other things being equal, toward a fatal termination. If, however, the infection occur into the skin, a local tuber- culous disease, lupus, results, and in its usual course spreads slowly, lasts for many years, and does not tend toward a fatal issue. The injection of virulent cholera organisms into a vein or beneath the skin of a guinea-pig is followed by death from cholera-septicemia; but injection into the peritoneal cavity results, not in septicemia, but in choleraic peri- tonitis. When streptococci are injected beneath the skin they are apt to produce erysipelatous and suppurative inflam- mations, but when injected into the circulation they pro- duce septicemia. Pneumococci reaching the lung, pre- sumably by the respiratory passages, induce croupous pneumonia; but in the metastatic lesions of pneumonia we find abscess formation the usual outcome of their activity. Among the unusual pneumococcus infections cerebrospinal meningitis and conjunctivitis may be men- tioned. The fatality of the microbic diseases depends very largely upon the method of inoculation; thus, Klemperer found that dogs died more readily when pneumococci were injected beneath the skin than when they were injected into a vein. These illustrations are sufficient to suggest that certain bacteria are adapted for growth under certain conditions which exist in certain parts of the body, and that their maximum deleterious action is manifested only when they enter the body in such a manner as to be brought^ in contact with them. While not impossible, it therefore becomes improbable that the presence of typhoid bacilli upon the conjunctiva will be followed by successful severe infection, their natural sphere of pathogenesis being in the intestine. The same is true of the presence of the diphtheria bacilli in the intestine, they finding their best sphere of operation in the throat. 72 PATHOGENIC BACTERIA. II. The Subject of Infection. — i. Natural and Ac- quired Immunity. — The peculiar natural and acquired conditions of resistance to disease which constitute im- munity are so interesting and important that a separate chapter has been devoted to their discussion, wherein the student will fiud much that will merit thoughtful atten- tion. Immunity according to its degree will, of course, make infection impossible or difficult; and except in cases of extreme virulence of the bacteria immune animals do not become infected. In the rare cases in which infection does occur it is frequently so modified that the symptoms and lesions vary considerably from the usual type. 2. Vital Condition. — In susceptible animals the degree of susceptibility may vary greatly with conditions that arise. In some diseases the susceptibility is more marked in youth, in others in advanced age ; the existence of constitutional diseases, as diabetes, markedly predisposes to infection. Sex is sometimes a predisposition to infec- tion, in that organs possessed by one sex, and not by the other, readily become infected; thus, women and not men suffer from puerperal infections. 3. Injury. — Any damage or injury to the body favors the lodgement and activity of whatever bacteria may reach the damaged area. It seems to be a well-established, though not perfectly comprehensible, fact that tuberculosis of the bones and joints of children is determined by an injury of the part. The existence of a slightly damaged cardiac valve is frequently the nidus in which circulating bacteria find rest and produce the malignant or ulcerative endocarditis so frequent in the infectious diseases. Rosenbach was able to prove the predisposition of damaged tissue to bacterial invasion by injuring a cardiac valve by a sound passed into the aorta, and then inject- ing a culture of staphylococci. These micro-organisms operating upon the diseased valve produced malignant endocarditis. Sources of Infection. — The experiments of Nuttall INFECTION AND INTOXICATION. 73 and Schottelius have shown that the healthy animal is born free from bacterial life. In its entrance into inde- pendent existence, however, it is at once introduced to a world of bacteria that fall upon the skin, to be caught by hair or feathers, or to insinuate themselves between the epithelial cells of the epidermis, are inhaled into the re- spiratory passages, swallowed into the alimentary canal, and find their way into the various openings of the body, until each becomes a regular habitat for a number of species. There being no hereditary transmission of bacteria in health, the micro-organisms habitual to the body are but accidental intruders that chance has brought where moisture, heat, and nourishment have enabled them to colonize. Upon investigation we further find that the flora of each region is appropriate to the condi- tions there existing, so that the feebly alkaline saliva harbors quite a different flora from the acid secretions of the vagina, etc. The greater number of bacteria thus found inhabiting the body-surface and cavities are harm- less, but many of those familiar to us as disease-producers also occur. It therefore becomes quite evident that we constantly carry the source of many of our ills with us. Not all of these pathogenic parasites are attenuated, ex- periment as well as experience indicating that they are sufficiently virulent to await only the proper opportunity to bring about their familiar lesions. a. Skin. — It would be as useless as it would be tire- some to compile a list of the non-pathogenic bacteria that may be found upon the skin. The very fact that the skin surface is extensive, external, slightly moist, and constantly in contact with external objects ought to be sufficient guarantee that almost any bacterium may be found upon it. Of the pathogenic organisms, it is probable that the Staphylococcus epidermidis albus (Welch) is the most widely distributed. The Staphylococcus pyogenes albus and aureus are also quite common. Bordoni-Uffreduzzi found that some of the odors of the skin are peculiar to 74 PATHOGENIC BACTERIA. micro-organisms that inhabit it. Thus he found that cultures of his Bacillus graveolens, isolated from between the toes, gave off the disagreeable odor characteristic of " dirty feet." The Streptococcus pyogenes is also some- times present. Surgeons formerly dreaded the air as the carrier of infection to wounds ; they have now come to realize that it is the incised skin that infects itself, and that the most rigid disinfection is necessary to prevent it. b: Conjunctiva. — This is the most exposed moist sur- face of the body and gathers micro-organisms from the dust in large numbers. Its flora is almost unlimited, and a work that I did some years ago1 convinced me that there was no fixed flora. A variety of pathogenic organ- isms abound; thus it is quite a regular thing to find the pyogenic cocci in health. The Bacillus xerosis, whose relationship to xerosis is not determined, is common upon this membrane. The researches of Hildebrandt and Bernheim indicate that the conjunctival secretions have a germicidal power. The number of micro-organ- isms which I found would not suggest it. c. Nose and Respiratory Passages. — As bacteria are widely distributed in the dusts suspended in the air, they are necessarily taken into the nose with each re- spiratory movement. The moist chambers of the nose and the walls of the pharynx are splendidly adapted to the function of catching and retaining these bacteria, and probably very few of them succeed in penetrating as far as the lungs. Those that do so in health probably meet with very unfavorable conditions and are promptly dis- posed of. If, however, the lung is in any way diseased, we find that a variety of bacteria may present themselves and aid in damaging the tissues. Thompson and Hewlett2 estimate that 1500-14,000 bacteria are inspired every hour. As the expired air is nearly always sterile, they sought to determine what be- 1 Norris and Oliver's System of Diseases of the Eye, vol. ii., p. 489. 2 Brit. Med. Jour., Jan. 18, 1896, p. 137. INFECTION AND INTOXICA TION. 75 came of the organisms ; and agree with Lister and Hilde- brandt that the organisms are arrested and killed before they reach the air-cells. They found by killing a num- ber of animals and examining the tracheal surface that it was sterile, and concluded that the great majority of bac- teria are stopped in the nose against the moist surfaces of its vestibules, where they are found in great numbers in the crusts. An ingenious experiment performed con- sisted in placing some Bacilli prodigiosi upon the sep- tum naris and making a culture from the spot at intervals during two hours. Cultures made within five minutes showed confluent colonies of the bacteria, which became fewer and fewer in number until, after two hours, not a bacillus could be found. According to Wurtz and Ler- moyez, the nasal secretions exert a germicidal action, but this observation lacks confirmation. That micro-organisms do gain admission to the pul- monary tissue the same as other minute objects, is shown by the experiments of Buchner upon anthrax. Anthrax- spores and lycopodium powder were mixed together and distributed so that animals inhaled them. Tuberculosis, pneumonia, small-pox, measles, scarla- tina, and a variety of other infectious diseases probably result in many cases from inhalation of the specific micro-organisms. d. Mouth and Digestive Apparatus. — Probably the most important bacteriologic studies of the mouth are those of Miller of Berlin, who has isolated twenty or thirty different species. Of these a few, such as the Lepto- thrix innominata, Bacillus buccalis maximus, Leptothrix buccalis maxima, Iodococcus vaginatus, Spirillum sputi- genum, and Spirochaeta dentinum, are of invariable oc- currence, though all non-pathogenic. Of the pathogenic cocci, the Staphylococcus pyogenes aureus and albus, the pneumococcus, the Streptococcus pyogenes, Micro- coccus tetragenus, and a number of species pathogenic for animals only, have been found by different observers. Considerable difference seems to occur among different 76 PATHOGENIC BACTERIA. men concerning the presence of the pyogenic cocci which Miller rarely found in healthy mouths, and among which Vignal failed to find the streptococcus in his extensive researches. Block, on the other hand, found the strepto- coccus three times and the staphylococcus four times in the healthy human mouth. The small number of pathogens that flourish in the mouth may depend upon an antiseptic action of the saliva. Sanarelli endeavored to prove that such an action existed, and found that when Staphylococcus pyogenes, Streptococcus pyogenes, Micrococcus tetragenus, and the typhoid and cholera germs were added in very small amounts to filtered saliva, they did not grow, but in twenty-four hours had died. If more plentiful inocula- tions were made, enough of the germs survived to give an abundant development. The stomach, in spite of the fact that it constantly re- ceives myriads of bacteria from the ingested foods and drinks, and is always receiving saliva containing bacteria, is not a favorable environment for bacterial growth be- cause of the acidity of its secretion. In health very few bacteria live in the stomach, and in all probability a large majority of those ingested die in the presence of the acid secretions. The micro-organism most frequently observed in gastric contents is a yellow sarcina, probably identical with the Sarcina ventriculi of the older writers. It is not known to have any pathogenic powers, though such have been attributed to it, because in all cases in which the gastric secretions are disturbed and alkalinity or exces- sive acidity is present this sarcina is observed in increased numbers. In the diseased stomach the bacteria vary according to the existing conditions. Thus, if the secretions be alka- line, any of the bacteria that happen to be swallowed may thrive and the gastric flora become quite extensive. The most frequent bacteria observed in disease-conditions are those of lactic and butyric acid fermentation. The Oppler-Boas has attained to some reputation as an INFECTION AND INTOXICATION. 77 adjunct in the diagnosis of gastric carcinoma. Its oc- currence does not depend upon the carcinoma, but upon conditions more common in carcinoma than in other gastric diseases. The loss of natural acidity favors the growth of the bacillus, which in its turn evolves lactic acid. The intestine is the normal habitat of a number of species of bacteria, some of which are temporary, some permanent. The former are, for the most part, bacteria that have entered with the food and drink, escaped' de- struction in the stomach, and multiplied on their way down the intestine. The permanent residents enter shortly after birth, and, being particularly well adapted by nature for intestinal parasitism, establish them- selves permanently. The most common of the per- manent residents are the Bacillus coli communis, Ba- cillus lactis aerogenes (especially in milk-fed babies), and the Streptococcus coli gracilis (especially in meat- eaters). It seems to be true that carnivorous animals are inhabited by a greater number of intestinal parasitic bac- teria than herbivorous animals ; also that the colon is the home of a greater number than are found in any other part of the intestine. It has often been suggested that the bacteria of the di- gestive tract are essential to life in that they assist the function of converting proteid substances to peptone. To determine the truth of this, Nuttall and Thier- felder1 performed an interesting experiment. A preg- nant guinea-pig was delivered of its young by Cesarean section, and one of the offspring immediately transferred with sterile instruments to a sterile chamber, where it was kept and fed upon sterile milk. After a number of days, during which it lived comfortably, it was killed and its organs subjected to careful examination. The intes- tinal tract was found to be entirely free from bacteria. From this the experimenters conclude that bacteria are not essential to intestinal digestion. 1 Zeitschrift fur physiol. Chemie, 1896, Bd. xxii., Hefte 2 und 3. 78 PATHOGENIC BACTERIA. On the other hand, Schottelins ! hatched and kept some chickens under conditions of absolute sterility. The birds did fairly well in the beginning, but gradually pined and died on the seventeenth day. The control-chickens, whose digestive organs contained bacteria, thrived. e. The Sexual Apparatus. — The vagina has a flora of its own, consisting of a limited number of species that are able to endure its acid secretions. The uterus seems to be well guarded from bacterial in- vasion by the acid secretions of the vagina and by the alkaline cervical mucus. According to the studies of Gottschalk and Immerwahr,2 twenty-one out of sixty cases of endometritis which they studied bacteriologic- ally were characterized by sterile discharges. The penis and the vulva, in addition to the micro- organisms of the skin, contain in the smegma a peculiar bacillus — Bacillus smegmatis (q. v.) — which, while not known to be a pathogenic bacterium, is easily mistaken for the bacillus of tuberculosis. f. The external ear, being a canal of some depth, can- not fail to collect bacteria, and furnishes a certain non- pathogenic coccus, the Micrococcus cereus flavus, with great regularity. Whatever micro-organisms happen to enter may be found near the external meatus, but toward the tympanic membrane there are very few. With so large and varied a permanent flora upon and in our bodies we need not look far for the sources of the common infections. We carry them constantly with us, and are ever in danger from them. _ Fortunately it seems that something more than a breach in the continuity is necessary to enable them to harm us. Injuries of all parts of the body are common, but consequent infection is uncommon. For infection to occur it is necessary that bacteria (i) enter the tissues, (2) in a sufficient number, (3) when the usual resisting power is diminished. The element of numbers is an important one to consider in 1 Miinchener med. Wochenschrift, 1898, No. 36. 2 Archivf. Gynak., 1896, Bd. 50, H. 3. INFECTION AND INTOXICATION. 79 this relation, for in ordinary injuries probably very few bacteria are admitted to the tissues. Infection from external sources, while much less fre- quent than the autochthonous forms, are commonly much more severe, especially when the infecting bacterium has been operating in another animal of the same species. This increased severity probably depends upon increased virulence of the bacterium attained during its residence in the tissues. The infecting bacteria come to us through all conceiva- ble fomites. The dust in the air, the articles of food and drink, the soil we walk upon, the bodies and dis- charges of diseased animals, all contain infective micro- organisms. The avenues of infection are numerous. 1. The Surfaces of the Body. — These are all covered with epithelial protections which vary in character and thickness according to necessity. a. The Skin. — It is very improbable that bacteria, either resident upon the skin or accidentally brought in contact with it, can penetrate its uninjured structures. Only microscopic injuries are necessary, however, and infection can be produced by gently rubbing the bacteria into tissue, which is slightly abraded in the process. Any wound, a puncture, incision, laceration, abrasion, etc., that destroys the perfect continuity of the epithelium is a point of entrance for bacteria upon the skin, upon the instrument, or in the air. The bites of animals and insects may prove dangerous — as in rabies of dogs, and tse-tse fly-bites of herbivora — from bacteria in the saliva. There has been a tendency to pay a great deal of attention to the bites of insects since the study of plague has occupied us, and for a time it was supposed that the infection might readily be produced by the bites of parasites. Nuttall1 has, however, fully investigated the subject in relation to anthrax, chicken cholera, and mouse septicemia, and found that when bed- 1 Centralbl.f. Bakt. u. Parasitenk., April 12, 1898, xxiii., No. 14. 80 PATHOGENIC BACTERIA. bugs, fleas, etc., are allowed to prey upon infected mice and then upon healthy mice the latter did not become infected even when the biting insects were so pressed upon as to play the role of injecting syringes. The ba- cilli of the diseases mentioned occurred in vast numbers as long as ninety-six hours in the excrement of the in- sects, then disappeared, losing their virulence and vitality before this period had passed. b. The Mucous Membranes. — In the case of the moist, soft mucous membranes it does not seem necessary that a breach of continuity should occur for infection to take place. The lodgement of bacteria upon the surface, their multiplication, the effect of their toxin in producing super- ficial necrosis, probably precedes in many cases actual entrance of the bacteria into the tissues. Such a mode of operation suggests itself especially in such affections as diphtheria and gonorrhea, where exposure is sufficient guarantee of infection without a necessary injury. Doubtless in many cases of mucous membrane infections the entrance of the bacteria into the tissues is aided by phagocytes, especially where the relation of bacteria to the cells is intimate, as in gonorrhea. 2. The Respiratory Apparatus. — We are unable to say in how many of the specific infectious diseases the infec- tion takes place through the inspired air ; but it is in all probability frequent. Diseases such as small-pox, scarla- tina, measles, etc., in which infection occurs without actual contact with the patient, but simply from being near him, seem to depend upon the inspiration of the contagium in the air. Pneumonia, tuberculosis, influenza, and a number of diseases whose causes are known to us are also inhalation infections. It is probable that little difficulty is experienced by the pathogenic bacteria in penetrating the alveolar mucosa, which seems to be destructive to the non-pathogenic forms. Buchner's experiments upon the inhalation of anthrax spores are of interest as illustrating the co-opera- tion of factors in infection. He found that when rabbits INFECTION AND INTOXICATION. 8 1 were compelled to inhale anthrax spores in a pulverized liquid or in dry powder infection did not readily take place. If, however, the anthrax spores were mixed with lycopodium powder and inhaled, 50 out of 66 animals died of anthrax and 9 of pneumonia. Probably, for the an- thrax spores to germinate in the lung, it was necessary for them to reach the lymphatic spaces, to which they were carried by scavenging cells busy removing the lycopo- dium grains. 3. The Digestive Apparatus. — The colon bacillus and the streptococcus of the intestine are, as a rule, pure saprophytes, enjoying a harmless existence in the rich in- testinal contents. Should a lesion of the intestine occur, however, or should a marked depression of vitality take place, the former of these organisms seems particularly prone to pathogenic activity and in all cases of intestinal obstruction, strangulation, ulceration, perforation, etc., this bacillus may be looked for as the chief offender. There seems to be little reason for supposing that the colon bacillus can escape from the intestine into the blood during life without any apparent lesion. Beco,1 however, believes it possible, and showed that immediately after death the organisms could be found in small numbers in the spleen. When they were not found immediately after death, they were also not found after twenty-four hours. Achard 2 denies that bacteria pass out of the intestine during the death agony. In his studies of 49 cases, bac- teria were found in the blood and in the liver in 14 ; in 24 no bacteria were found during life, but after death ; in 1 1 no bacteria were found either during life or after death up to the time his autopsies were made. The micro- organisms that he usually found were streptococci and staphylococci during life, and the colon bacillus after death. Chrovstek and Egger,3 on the other hand, confirm the 1 Ann. de /' Inst. Pasteur, 1895, No. 3. 2 Archiv. de Mid. expir. et d'Anat. path., 1 895, No. I, p. 25. * Wiener klin. Wochenschrift, 1 897, No. 3. 82 PATHOGENIC BACTERIA. opinions of Wurtz and Bouchard that under certain con- ditions bacteria can pass from the intestine into the tissues and enter the blood while the heart is still beating in agony. The observations of White x that the human blood is normally germicidal for the colon bacillus, but loses this power in many cases shortly before death, may explain why the agonal invasion occurs in some cases and not in others. The occurrence of a lesion of the intestine is by no means sufficient to produce an infection, and in this particular the experiments of Neisser2 are very instructive. He fed mice, guinea pigs, and rabbits upon a variety of pathogenic and non-pathogenic bacteria both before and after injuries to the intestine caused by the ingestion of powdered glass, chemic agents, and irritating bacteria. He failed to show that, with the exception of those bac- teria whose particular tendency is to produce intestinal disease, any entered either the chyliferous vessels, the blood-vessels, or the organs. Adami 3 believes that colon bacilli and probably other micro-organisms are constantly being taken up from the intestine in small numbers in health, and are taken to the liver and other organs, where they are slowly de- stroyed. Usually no harm results from the absorption of the organisms, but as infection is always possible as the result of it he proposes the term "sub-infection" by which to describe it. The otherwise difficultly explainable cases in which the tubercle bacillus makes its first appearance deep in the bones, or staphylococci in osteomyelitis, etc., have led many to conclude that bacteria may pass through the uninjured intestinal wall and be carried to remote situa- tions by the blood. It should not be forgotten, however, 1 Boston Med. and Surg. Journal, cxl., No. 8. * Zeitschrift fur Hygiene, June 25, 1896, Bd. xxii., Heft 1. 1 Jour. Amer. Med. Assoc., Dec. 16 and 23, 1899, vol. xxxiii., Nos. 25 and 26. INFECTION AND INTOXICA TION 83 that in these cases there may have been a lesion long antedating the present trouble, and now healed, by which the passage of the bacteria into the blood may have been made easy. Intestinal infection by external bacteria, such as is observed in typhoid fever and cholera, probably takes place by the growth of the respective bacteria in the intestinal contents, the generation of their respective toxic products, their absorption, and a subsequent vital depression that permits of an action upon the tissues which could not have occurred primarily. . The anthrax bacillus seems capable of effecting an entrance into the tissues without opposition. Developing in the intestine in large numbers, it surrounds the villi with thick networks of bacillary threads, works its way into the lymphatics, and leads to a general infection. 4. The placenta is sometimes a source of infection, and in exanthematous diseases, such as variola and measles, and in anthrax, symptomatic anthrax, glanders, syphilis, relapsing fever, typhoid fever, and, in rare cases, tuber- culosis, it seems to be pretty clearly demonstrated that the cause of disease can transfer itself from the mother to the offspring, either without a lesion or by the pre- liminary formation of a lesion in the placenta. Forms of Bacterial Pathogenesis. — In general, it may be said that pathogenesis depends chiefly upon the ability of the micro-organisms to manufacture injurious substances. As, however, the clinical pictures resulting from infection and experimental intoxication produced by the injection of the separated bacterial poison into an animal are frequently very different, we must conclude that intoxication is but a part of infection, which in real- ity consists of the sum of all the vital phenomena mani- fested by the bacterium in its parasitic life. As infection is so markedly influenced by the viru- lence, number, avenue of entrance, and condition of the subject, it is far from being a process that takes place with regularity. For most infections there is a type 84 PATHOGENIC BACTERIA. symptom-complex, made up of the symptoms and con- ditions that usually present themselves; but wide devia- tion from this type is to be expected. The deviations are sometimes extraordinary and make the process at first unrecognizable. Thus, pneumococcus infection usually presents itself in the form of croupous, lobar pneumonia, but occasionally appears as otitis media, conjunctivitis, etc. Tuberculosis usually assumes the pulmonary form, and appears as a disease whose chief ravages affect that organ ; it is common as an affection of the bones and joints, and not uncommon as lupus on the skin. These three forms of the disease are so differ- ent from one another that for many years their identity was unsuspected. The Streptococcus pyogenes is usually associated with severe local suppurative affections. It may be the cause of erysipelas, of pseudomembranous sore throat, or of rapidly fatal septicemia. Typhoid infection usually oc- curs as clinical typhoid fever, but frequently makes its appearance as local suppuration or as general septicemia. Conditions present in the animal body go far to modify the course and appearances in disease; thus, should the operations of a micro-organism be chiefly upon the tissues iu their immediate vicinity, they usually operate at the point of original entrance. The staphylococci usually act locally at the seat of injury and infection, and bring about suppuration. Their accidental entrance into the lymphatic vessels with currents of lymph or enclosed in phagocytes leads to lymphangitis and then to lymph- adenitis. If they enter the circulation, the valves of the heart may become a nidus for their operation, and endo- carditis result; or metastatic abscesses may occur in con- sequence of their transportation to and deposition in remote tissues. Sometimes the micro-organism seems to find the con- ditions ill-adapted to excessive development, and con- sequently develops in a very limited — indeed, almost insignificant — manner; yet because of soluble toxic met- INFECTION AND INTOXICA TION 85 abolic products causes irreparable damage in remote organs. This is well illustrated in diphtheria, where a limited growth of the Klebs-Lofner bacillus upon a mucous membrane is attended with the absorp- tion of a powerful poisonous product that depresses the heart and brings about degenerations in the nervous and other tissues. Tetanus is another micro-organism of the same class, whose powers of growth in the animal body seem to be so feeble that in many cases (especially of experimental infection of the lower animals) no local lesion can be detected, yet by a very limited number of bacilli sufficient toxin may be manufactured to kill the animal by cramp asphyxia. Infection by other micro-organisms is characterized by ready growth in the lymphatics and capillaries, and at the time of death they are found in the blood and in all the tissues. Among these organisms we find many of the septicemias of the lower animals, such as anthrax, mouse septicemia, rabbit septicemia, swine plague; and a few of man, as anthrax, plague, relapsing fever, etc. Taking the typical characteristics of the micro-organ- ismal invasion, and willingly conceding that the opera- tions of the bacteria are not restricted to the method of infection suggested by the name of its class, I have pro- posed the division of bacteria into three groups: 1. Phlogistic — chiefly characterized by local irritation. 2. Toxic — characterized by local growth and toxin dis- semination. 3. Septic — whose chief field of activity is in the blood and lymphatic fluids. In all these forms the actual damage done seems to be dependent upon the formation of poisonous products. If these are insoluble or soluble with difficulty, their inju- rious effects must be manifested upon the cells with which they come in direct contact, and hence will be most obvious in the particular area in which the bacteria are actively growing. This form of irritation is well il- lustrated in the case of two familiar infections : first, the 86 PATHOGENIC BACTERIA. staphylococcus suppurations, in which the entire process is local and limited to the field of bacterial action; and second, by typhoid fever, in which the majority of the organisms remain in the intestinal and mesenteric lesions, where the local damage is marked, though the general histologic changes show that a mild intoxication prob- ably also takes place. In tetanus and diphtheria we find bacteria whose products are freely soluble, so that the damage done by the intoxication vastly outweighs that of the local lesions. In the Bacillus anthracis we have a micro-organ- ism whose toxin-producing ability is very uncertain. The damage done by its presence may be entirely de- pendent upon its tendency to blockade the capillaries and its ready affinity for oxygen. It is certainly largely dependent upon its great vegetative ability. Other bac- teria of the septic class seem to furnish more or less de- monstrable poison. Toxins. — Concerning the poisons generated by the bacteria we are at present in position to say very little. They are probably all proteid substances, most of them being properly classed among the toxalbumins. The toxins of diphtheria and tetanus, which have been studied more closely than any of the others, seem to belong to a separate class of poisons which do not give any of the albumin reactions. As a rule, the bacterial poisons are delicately organized, being destroyed by temperatures above 6o° C, by exposure to light and air, and by pro- longed keeping. An exception to this rule seems to occur in the semi-artificial toxic product of the tubercle bacillus known as tuberculin, which is not injured by heating to ioo° C. for hours at a time. The toxins seem to be soluble, leaving the protoplasm of the bacteria to appear in the filtered fluid in which they have grown, as in tetanus and diphtheria bacilli cultures ; and insoluble, present only in the bodies of the bacteria, as in the chol- era spirilla, typhoid fever bacillus, and pyogenic cocci. The physiologic action of the toxins varies with each INFECTION AND INTOXICATION. 87 species of bacteria, some having a definite selective affin- ity for certain cells, as tetanus toxin for the motor nerve cells. The action of certain toxins may be enzymic. lilimination of Bacteria from the Blood. — According to Kruse,1 the entrance of bacteria into the circulation is possible by — 1. Passive entrance of bacteria through the stomata of the vessels where the pressure of the inflammatory exu- date is greater than the intravascular pressure. 2. Entrance of the bacteria into a vessel in the bodies of leukocytes that have incorporated them. 3. Actual penetration of the vessel wall by the growth of the micro-organism. 4. Entrance into the vessels via the lymphatics either passively or in leukocytes. In all probability non-pathogenic bacteria, and path- ogenic bacteria which for any reason, such as the occur- rence of immunity from developed antitoxins, etc., have become non-pathogenic for the animal, are disposed of in much the same manner as inert minute particles injected into the circulation. Siebel has shown, regarding them, that they accumulate in the fine capillaries, especially in the lung, liver, spleen, and bone marrow, and are slowly transferred to the surrounding tissues, either to be col- lected in the connective tissues, carried to the lymphatic nodes, excreted with bile, succus entericus, etc., or dis- charged from the surface of the mucous membranes, pulmonary alveoli, tonsils, etc. They also escape from suppurating wounds to which they may be carried by leukocytes. No doubt many of the bacteria, being organ- ized particles, undergo dissolution in the body without excretion. Siebel did not find that the particles were excreted by the kidneys. Wyssokowitsch is in accord with Siebel, and has shown experimentally that the kid- ney rarely eliminates bacteria. Cavazzani found that unless the renal epithelium was injured no bacteria escaped from the blood. 1 Fliigge, Die Mikroorganismen, vol. i., p. 271. 88 PATHOGENIC BACTERIA. The observations of Adami1 indicate that the liver constantly excretes or destroys bacteria absorbed from the intestine. Weleminsky2 found that the mammary glands some- times participate in micro-organismal excretion, Bacillus pyocyaueus making its appearance in the milk in from five to eight hours after injection into the circulation. He concludes that only those bacteria that produce le- sions of the mammary gland are eliminated in the milk. The principal excretions with which bacteria are eliminated from the blood are those of the mucous mem- branes, the bile, and the sweat ; in diseased conditions in the urine when the renal epithelium is damaged and in the milk when the mammary gland is diseased. Bacteria are also discharged from the body in vast numbers in pathologic discharges, such as pus, sputum, and urine from the diseased animal. Special Phenomenon of Infection. — Agglutination. — The phenomenon of agglutination was first observed in connection with typhoid fever in 1896 by Widal and Griinbaum. The phenomenon soon became known in its typhoid fever relation as the "Widal reaction." It consists in the cessation of motion of the bacteria and the aggregation of the micro-organisms into clusters or groups — agglutination — when a small quantity of blood from an infected animal is added to a fresh active culture of the specific organism. In many cases the substance of the bacteria seems shrunken and the form distorted. The bacteria are not killed as a rule. As the subject is of chief interest in connection with typhoid fever, now being used every day for the clinical diagnosis of that disease, much space will be devoted to it in the chapter upon that subject. It is not by any means a typhoid fever reaction, how- ever, but a widespread phenomenon of infection, having 1 Jour. Amer. Med. Assoc, Dec, 1899. 2 LXIV Versamml. der deutschen Naturforscher und Aerzte, Braunschweig. Centralbl. f. Bakt. u. Parasitenk., April 16, 1898, xxiii., No. 15, p. 657. INFECTION AND INTOXICATION. 89 been observed in infection by the Bacillus coli commu- nis, Bacillus icteroides, bacillus of hog cholera, of teta- nus, of tuberculosis, etc. The reaction is, as a rule, specific; that is, it results from the action of serum from a certain infection upon the bacteria of that particular infection. In many cases serum may appear to cause agglutination with various related bacteria, but when the blood is properly diluted it is almost invariably the case that the bacteria of the particular infection under con- sideration are sensitive to the action of the serum in dilutions far greater than those that have ceased to act upon the related bacteria. In this way the phenomenon of agglutination more or less perfectly fulfils the double purpose of a differentiating test for similar bacteria and a method for recognizing different diseases. The cause of the phenomenon is not explained. It seems to have nothing to do with immunity. The ag- glutinating substance is present in all the normal and pathologic fluids of the infected animal, making its appearance some time after the inception of the process, though occasionally very promptly. It is present through- out the course of the disease, and may remain for many years afterward. The formation of agglutinations probably does not in- dicate any important cellular or vital reaction, as they may follow the addition of various chemic agents to cult- ures of the bacteria, and may depend upon metabolic products of the bacteria themselves, for they sometimes occur spontaneously in highly vegetative cultures. Mal- voz l found that the metabolic products contained in the cultures would produce the agglutinations. His experi- mental evidence consists in thoroughly mixing a fresh culture of the first vaccine of anthrax in \ c.c. of dis- tilled water, and adding to it a loopful of a six-day-old culture. A drop of the mixture allowed to stand for a few hours in a moist chamber will show typical agglu- tinations when examined under the microscope. 1 Ann. de V Inst. Pasteur, Aug. 25, 1899. CHAPTER IV. IMMUNITY AND SUSCEPTIBILITY. Immunity is resistance to disease. It is the ability of an animal to protect itself against the pathogenic action of bacteria. The absence or loss of this power character- izes the opposed condition known as susceptibility. The resistance may be an active process, endogenous and cytogenic in nature — active immunity ; or may be hematogenous, and result from exogenous active prin- ciples added to the blood — passive immunity. In the active form the cells of the body may be conceived as energetically engaged in destroying the bacteria, or in manufacturing destructive products to act upon them. In the passive form the cells take no part whatever, the phenomena resulting from the presence of the experi- mentally introduced active principle. To Infection . * f Active. Immunity, Natural and i Acquired. Cytogenic, Phagocytic. Hematogenic, Alexinic. Passive. h Antitoxic. [ To Intoxication. In discussing the products of bacterial energy it has been shown that the virulence of bacteria depends for the most part upon certain poisonous excretory products resulting from their metabolism, and that the essential ' difference between pathogenic and non-pathogenic bac- teria, except in a very few cases, is but a difference in their products. A few bacteria, like the anthrax bacillus, pro- »o IMMUNITY AND SUSCEPTIBILITY. 91 duce very little, if any, separable toxic substance, and seem to depend for pathogenesis upon vigor of growth in the channels of the body. Others produce non-specific irritating substances which react so similarly upon the organism that certain processes, as suppuration, fever, etc. , may result from the operation of any one of a con- siderable group of bacteria. Still others produce toxic substances whose operation is truly specific in that they act selectively upon the body cells, always affecting like cells in the same manner, producing similar symptoms. The best illustration of the micro-organisms of this group is the tetanus bacillus, whose toxin, acting upon the motor cells of the nervous system, produces the charac- teristic spasms of the disease. Immunity consists not only of overcoming the bacteria that cause disease, but in enduring and annulling their toxic effects. It is found that when the pathogenic bac- teria and their poisons are separated, the immune animal suffers no more effect from the one than the other. Thus, the rat is immune to diphtheria. Infection with living diphtheria bacilli will not kill it, and injection of power- ful toxin in considerable amounts does not injure it. The disease undoubtedly depends upon the diphtheria toxin to which the rat is immune, and for which reason the diph- theria bacillus is harmless to it. Fowls are immune to tetanus and will not succumb to infection. If, however, they are injected with large doses of strong tetanus toxin, they may die. In this case the fowls are able to annul the effects of as much toxin as the bacilli can form in their bodies, but are not able to dispose of unlimited quantities experimentally introduced. Immunity to disease, therefore, signifies immunity to the poisons causing disease, and can only be successfully studied in association with the correlated phenomena of intoxication. The reactions brought about in the body by the poisons of bacteria are similar to those caused by the toxalbumins of the higher plants, ricin, abrin, etc., and to some of the animal poisons, such as the venom of 92 PATHOGENIC BACTERIA. serpents and insects. For this reason considerable ref- erence must be made in the treatment of the subject to the phenomena associated with these poisons. Immunity is always a relative condition. Carl Fraen- kel expressed this admirably when he said, ua white rat is immune to anthrax in amounts sufficiently large to kill a rabbit, but it is perhaps not immune to a quantity sufficiently large to kill an elephant." The fowl can overcome as much toxin as the tetanus bacilli can pro- duce before their destruction in its body, but cannot overcome the effects of an unlimited quantity of the poison. The hedge-hog is immune to serpent's venom in the doses usually injected by the snakes, yet can be killed by the venom in larger doses. Many animals are immune to as many bacteria as reach them in the usual modes of infection, but will succumb to excessively large doses of the same bacteria artificially introduced. The standard of immunity may be expressed as the re- sistance manifested by the normal, healthy animal to the unmodified germs of disease. In attempting to establish that any animal is immune it is of the utmost importance to bear in mind that the virulence of bacteria is subject to the greatest variation under purely natural conditions. A few bacteria, as the anthrax bacillus, maintain a definite standard for long periods without observable attenuation, and may be manipulated artifically in the usual ways without exalta- tion of virulence. A greater number, of which the strep- tococcus and pneumococcus will serve as illustrations, are so variable that it is unusual for two organisms from different sources to have the same degree of virulence, or for any one organism to have the same degree of virulence for any considerable length of time. The meaning in- tended by the expression " unmodified germs of disease " is the natural virulence, without modification or manip- ulation in the laboratory. IMMUNITY AND SUSCEPTIBILITY. 93 Natural Immunity. Natural immunity is the natural, inherited resisting power peculiar to certain species of animals. A few diseases are so widely distributed as to take in all of the orders of the animal kingdom ; thus, tuberculosis has been observed in mammals, birds, reptiles, batrachi- ans, and fishes. Nearly all diseases are, however, more or less restricted, and are observed chiefly in animals with certain common peculiarities. An example of this is anthrax, which is a disease of warm-blooded animals, and may infect the great majority of mammals and a few birds, but will not infect the cold-blooded animals. Very commonly among closely related animals great dif- ferences in susceptibility exist, so that it can readily be determined that anthrax is far more infectious for her- bivorous than for carnivorous animals. Not infrequently remarkable variations are observed in animals of the same order; thus, among the rodents we find that while the mouse, guinea-pig, and rabbit readily succumb to an- thrax, the rat is immune. Rarely we find that differ- ences of susceptibility exist among families and genera, and even among species and varieties ; thus, the white mouse is immune to glanders, the house mouse is not very susceptible, but the field mouse is perhaps the most susceptible of all animals. These differences also exist between the susceptibility of man and the lower animals ; thus, while man, in com- mon with the lower animals, suffers from anthrax, glanders, actinomycosis, etc., he also suffers from cholera, typhoid fever, syphilis, lepra, scarlatina, and a variety of other diseases, never observed to occur spontaneously in the lower animals, which in turn are frequently afflicted with such diseases as symptomatic anthrax, hog cholera, swine plague, chicken cholera, mouse septicemia, rabbit septicemia, etc., which do not affect man. Racial differences of susceptibility also occur among men ; thus, negroes are said to be immune to yellow fever, and the Japanese are said to be immune to scarlatina, 94 PATHOGENIC BACTERIA. both of which diseases are highly infectious for the Caucasian. Explanation of Natural Immunity. — There are two agencies upon either or both of which natural immunity may depend ; first, the cells ; second, the humors of the body. I. The Activity of the Cells. — Phagocytosis. — The ameboid movements of the leukocytes and their ability to take inert particles into their protoplasm were observed by Virchow a half century ago while he was working upon the details of the cellular pathology. Carl Roser as early as 1881 observed that the leukocytes sometimes take up bacteria ; and a little later Sternberg, Koch, and others corroberated his observations. That this evidence of a phagocytic action by the cells might have any bearing upon immunity seems to have first occurred to the biolo- gist Metschnikoff, who, viewing the phenomena with a broad biological horizon, recognized in the activities of the leukocytes and related cells of the higher animals, processes universal among the unicellular animal organ- isms. From comparative studies of cellular processes in high and low forms of life, Metschnikoff concluded that their importance in preserving the health of the organ- ism by destroying the cause of disease could not be over- estimated, and elaborated the theory of phagocytosis, now inseparably associated with his name. Certain of his observations are familiar to every one who has studied biology and observed the ameba with its incorporated diatomes, desmids, etc., and the food vacuoles in which the digested products of similar organisms are contained; or observed the myriads of bacteria and other minute organisms flowing into the mouth opening of the Parame- cium, rotifier, vorticella, etc. All of these unicellular animals incorporate, destroy, and digest bacteria. Why, therefore, may not the phagocytic cells of the higher animals be endowed with similar powers? Indeed, the analogy is complete. The leukocytes — -es- pecially the polymorphonuclear and eosinophilic forms, to IMMUNITY AND SUSCEPTIBILITY. 95 which Metschnikoff gives the name ?nicrophages — and the endothelial and connective-tissue cells — which together with the large lymphocytes of the blood and occasional epithelial cells constitute what he describes as macro- phages, do take up bacteria as well as other particles, just as the free unicellular animals do, and appear to digest them. If an injection of anthrax bacteria be made into the lymph-sac of a frog, it will be found upon subse- quent examination that the bacteria have been consumed by the leukocytes, in whose protoplasm they appear. The bacteria contained in the leukocytes are shown by staining (vesuvin is said to be useful for the purpose) to undergo a gradual destruction, by which, though at first staining uniformly, they lose this property and appear pale and irregularly colored. Later, only the outline of the bacillus is visible, and finally it disappears. It cer- tainly seems as if the immunity of the frog depends upon the activity of its leukocytes in destroying the bacteria. The rabbit is susceptible to anthrax and succumbs to subcutaneous inoculations. If a little of the fluid from the gelatinous edema surrounding the seat of inoculation be examined microscopically after the death of the ani- mal, it will be found that, while this fluid contains abun- dant bacilli and large numbers of leukocytes, not a single bacillus is contained within a leukocyte. Contrasted with the other observations, one might readily conclude that the death of the rabbit depended upon the failure of its leukocytes to take up and destroy the bacteria. It appears to be the rule that when an animal is im- mune phagocytosis is active and the leukocytes readily take up the parasites ; when an animal is susceptible the leukocytes refuse to take up the parasites. Whether or not one is justified in concluding with Metschnikoff that the animal is immune because its leukocytes are active in operating upon the micro-organisms is questionable. "If One examines the exudate in erysipelas, it will be found, at the extending zone of the disease, that many of the streptococci are being taken up by the leukocytes, while 96 PATHOGENIC BACTERIA. in the older areas the streptococci are nearly all free." Is it possible that the extent of the disease is being con- tested by leukocytes waging an active warfare against the cocci, and that their success results in the demar- cation of the disease? As there is abundant evidence on all sides to show that the leukocytes do take up bacteria, the solution of the question of the relationship of the phenomenon to im- munity must depend upon the demonstration of certain involved questions. i. Do the leukocytes take up living bacteria? There are many reasons for thinking that in the illustrations cited, the bacteria may have been already dead when taken up by the leukocytes, having met their fate from other causes and become transformed into inert parti- cles upon which the leukocytes react as upon molecular substances in general. MetschnikofF,1 however, is pre- pared to demonstrate that the bacteria which tjie leuko- cytes attack are alive, for he successfully isolated leuko- cytes containing spores of anthrax, and upon transferring them to culture-media in which they died, observed the germination of the contained spores into bacilli. It must be remembered, however, that the resistance of the spore to deleterious influences is very great, so that it might be able to survive where adult bacilli would succumb. Also, that the spore, which as such is probably devoid of poi- sonous or irritative properties, might be seized upon when the adult bacillus would be carefully avoided by the leu- kocyte. However, whether in this case we are willing to accept the evidence as conclusive or not, there are abun- dant confirmations of the fact that the cells do take up living bacteria, for in various infectious diseases we find it not uncommon for the cells themselves to fall victims to the bacteria they have taken up. This is seen in such diseases as mouse septicemia, gonorrhea, tuberculosis. It is interesting to find that the phagocytes evince an 1 Vircko-u? s Archives, xcvi., p. 177 ; xcvii., p. 502. Annates de P Inst. Pas- teur, 1887, i., p. 321. See also Etudes sur r Inflammation, Paris, 1892. IMMUNITY AND SUSCEPTIBILITY. 97 actual selective tendency ; thus, the rabbit's leukocytes will not take up anthrax bacilli, but will take up diph- theria bacilli, both micro-organisms being fatally infec- tious for that animal. It has even been observed by Ruffer that in cases of mixed infection, the leukocytes may show a marked preference for one of the bacteria. Thus, in diphtheria, with combined streptococcus infection, the leukocytes seemed to take up the diphtheria bacilli with readiness, but did not touch the streptococci. The experiments of PfefFer,1 Massart and Bordet,2 Gabritschewsky,3 Buchner,4 and others have shown that the leukocytes are guided in the phagocytic mani- festations by the familiar force of chemotaxis, and that their migrations and operations are always dependent upon the existence of chemotactic products of the bacte- ria. Experiments in proof of this are easily performed by filling capillary tubes with cultures of various bacteria, sealing one end, and introducing the tubes beneath the skin of an animal. If the contents are chemotactic, leukocytes penetrate into the tube and a plug of leuko- cytes closes its open end. If no chemotactic force is exerted by the culture, no leukocytes enter it. The cultures that exert chemotactic powers in .the tube are those whose injection into the tissues is most likely to be followed by active phagocytosis. 2. Do the cells destroy the bacteria zvhich they incorpo- rate? This question has never been solved, and until we are able to answer it we will not be in a position to judge the true merits of the phagocytic theory. Metschnikoff 1 Ueber chemotaktische Bewegungen von Bacterien, Flagellaten und Vol- vociniin. Unlersuchungen aus d. Botan. Institut. zu Tubingen, II., 1888; also ibid., I., p. 363. 1 Recherches sur Virritabiliti des leukocytes et sur V intervention de cette irritabiliti dans la nutrition de cellules et dans V inflammation, Bruxelles, 1890. Also Annates de V Inst. Pasteur, 1891. Also Massart. Annates de V Inst. Pasteur, 1892, and Bordet, Communication faite a la Sociiti Royale des sciences mfdicales et naturelle de Bruxelles, seance p. 13, vi., 1892. 5 Ann. de I Inst. Pasteur, t. IV., p. 346. * Berliner klin. Wochenschrift, 1890, 30 and 47. 7 98 PATHOGENIC BACTERIA. has brought forth numerous evidences. For example, the varying action of staining reagents upon bacteria has been made use of to demonstrate retrogressive changes in the bacteria. In the frog's leukocyte Metschnikoff thought the loss of affinity of the bacteria for vesuvin might indicate its progressive dissolution, and in the giant cells, formed in the liver of the "Zieselmaus" he was able to show that the tubercle bacilli they contained were surrounded by a halo which he thought consisted of softening bacterial-cell protoplasm, but which Baum- garten thought might equally well be looked upon as softening cell protoplasm upon which the tubercle bacil- lus was operating destructively. The experiments that have been made with non-pathogenic bacteria seem to indicate that the destructive processes of the cells are not very rapid, for Wyssokowitsch found that spores of Bacil- lus subtilis remained alive in the spleen for three months. The observations that led to Metschnikoff' s theory of phagocytosis occurred during the period in which the bac- teria themselves, rather than their products, were looked upon as the cause of disease. As knowledge has accumu- lated, it has been found that animals which are immune to disease-producing bacteria are also immune to their filtered toxic products. This kind of immunity certainly cannot depend upon phagocytosis, but upon some more intricate process, and the theory as originally propounded becomes untenable. As Muir and Ritchie1 point out, "even if it were consistent with facts, it only removes the property of immunity a step further back — namely, to the phagocytes." " The phenomena of phagocytosis so admirably demonstrated by Metschnikoff may be re- garded as the result of immunity, but cannot be ac- cepted as its cause." With the development and progress of knowledge upon the subject Metschnikoff has never relinquished his orig- inal idea that the leukocytes are the essential agents, though the phenomena of immunization to toxins made 1 Manual of Bacteriology, Edinburgh and London, 1897. IMMUNITY AND SUSCEPTIBILITY. 99 it necessary for him to lay aside the idea of phagocytosis. He still believes these cells to be the essential agents, whether it be by incorporating and digesting the bacteria or by excreting products that annul their poisons. II. The Activity of the Humors. — The uncer- tainty regarding the ability of the phagocytes to destroy bacteria, and the probability that the bacteria are already dead when they incorporate them, oblige us to look else- where for the causes of bacterial death and immunity to their toxins. If the cells do not perform the function, may it be the juices of the body ? As early as 1884 Groh- man observed that fresh blood serum had the power of attenuatingthe anthrax bacillus ; and in 1887 von Fodor * found that by a more prolonged exposure to its influence the bacilli were killed. The matter was carefully studied by Nuttall2 in 1888, and he and Buchner3 found that bacteriolysis was a power common to many of the body juices. Nuttall investigated blood serum, aqueous humor, and serous fluids of the body, and found them all germi- cidal; while Buchner showed that in the blood the power resided exclusively in the serum, was not destroyed by dilution, and was not dialyzable. Nuttall found that the destruction of anthrax bacilli by rabbits' blood required from two to four hours, the temperature of 370 C. being maintained. Bacillus subtilis and Bacillus megatherium were also destroyed by the fresh serum, but Staphylococ- cus pyogenes aureus was not. Prudden found that hy- drocele fluid and abdominal effusions also possess germi- cidal powers. The germicidal power of the blood is not a permanent quality, but passes away in a day or two, and can be entirely set aside at any time by heating to 550 C. All bloods do not have the same degree of destroying 1 Cenlralbl.f. Bakt. u. Parasitenk., 1890, vii., p. 753. * Zeitschrift fur Hygiene, 1 888, iv., p. 353. * Centralbl. f. Bakt. u. Parasitenk, Bd. v., p. 817; Bd. vi., p. I, 561 ; xii ; No. 24, 1892. Berliner klin. Wochenschrift, 1892, p. 449. Munchener med. Wochemchrift, 189.2, Nos. 8 and 52 ; 1894, Nos. 24 and 25, pp. 717 and 744. I0O PATHOGENIC BACTERIA, power, it being the rule that the bloods of immune ani- mals act most destructively upon those bacteria against which the animal has the greatest resisting power. Thus, the rat which is immune to anthrax, has blood that is very destructive in its action upon the anthrax bacillus. However, the rule is one to which there are many puz- zling exceptions, for the dog is also quite resistant to an- thrax, though its blood is harmless to the bacilli, and the rabbit is susceptible to the disease although its blood is destructive to them. The power of the blood to destroy bacteria is not unlim- ited, for Nissen l found that when a few cholera spirilla are added to freshly-drawn rabbits' blood they are killed in about thirty minutes, but if the number exceeds about one million per cubic centimeter they increase in number. Behring2, endeavored to study the germicidal value of blood serums so as to measure their activity as compared with corrosive sublimate and carbolic acid, and found that u one part of fresh serum of the white rat added to eleven to fifteen parts of the serum of sheep (which is not anti- septic to anthrax) would prevent the growth of the bacilli in the latter; 3.5 c.c. of rats' serum mixed with an equal part of sheeps' serum would completely destroy, in twenty-four hours, the bacilli coming from the blood of a guinea-pig affected with anthrax. To obtain the same preventing and sterilizing action in sheeps' serum with corrosive sublimate and carbolic acid, it was necessary to use the first in the proportion of 1 : 1000, and the second of 2 : 100." In cases in which the activity of the serum does not kill the bacteria it frequently attenuates them ; and it may be that the immunity of animals whose serums are not germicidal depends upon attenuating substances which, robbing the organisms of their pathogenesis, enable the animal to dispose of them. The destructive and inhibiting powers of the serum 1 Zeitschrift fur Hygiene, 1889, vi., p. 487. * Die Bekampfung des Infektionskrankheiten, Leipzig, 1894, p. 493. IMMUNITY AND SUSCEPTIBILITY. IOI have been thought to depend upon various causes. Behring and Nissen1 supposed that the white rat was able to resist anthrax because of the extreme degree of alkalinity of its blood. They were supported in their views by Paul,2 who found that alkaline solutions (i : 3000 sodium carbonate) acted upon the anthrax bacilli like the blood of the rat ; and further, that if the rabbit's blood is neutralized, it loses its germicidal power. Von Foder also demonstrated that the resistance of the rabbit to anthrax is increased by the injection of alkali into the circulation, and that with this increased resistance the germicidal activity of the blood increases. He also found that when a rabbit is infected with anthrax there is a natural increase in the alkalinity of the blood during the first twenty-four hours, " when we may suppose that the powers of nature are brought to bear upon and resist the invading parasite;" and that with the further progress of the infection it rapidly diminishes. Hankin believed immunity to depend upon certain germicidal globulins which he isolated from the blood serum of rats. Vaughan,3 McClintock, and Novy attri- bute the germicidal action of the blood to nucleins which it contains in solution, and point out that the relationship of alkalinity of the serum to immunity probably depends upon the ready solubility of the nucleins in alkaline solutions. To the bactericidal substances present in the blood Buchner has applied the term Alexins. Hankin 4 calls them defensive proteids. They are destroyed by heating for one-half to one hour to 55°-6o° C, and are robbed of activity by dilution with eight to ten volumes of distilled water, though they can stand that dilution with physio- logic salt solution. They can be precipitated from the blood by the addition of forty per cent, of sodium sul- 1 Zeitschrift fiir Hygiene, 1890, Bd. vii. * Proceedings of the Royal Society of London, May 22, 1890. • Medical ATeivs, Dec, 1893. 4 Centralbl. f. Bakt. u. Parasitenk., xii., Nos. 22 and 23; xiv., No. 25. 102 PATHOGENIC BACTERIA. phate, but not with alcohol. The chemic composition of the bodies caused Buchner to class them with the pro- teids. Their composition seems to be very complex, and probably varies with different animals. The histogenesis of the germicidal substance has been given a great deal of attention. Those who hold that it is a nuclein or a cell globulin usually refer its origin to the leukocytes. Christmas-Dircking-Holmfeld l found that pus secured from animals immune to anthrax was fatal to the anthrax bacillus. Grawitz2 and Eichel3 have observed staphylococci and anthrax bacilli die in a few days when placed in pus obtained from turpentine abscesses. Deuys and Havet,4 and Buchner5 found that the bactericidal value of inflammatory exudates was much greater when it contained dead leukocytes than when they were filtered out. Alexin-like substances, therefore, seem to be liberated from the leukocytes, and in theory one may imagine suppuration to be the result of Nature's effort to concentrate germicidal substances by which to destroy bacteria, by aggregating large num- bers of leukocytes in the infected area. Hankin has ap- plied the term Alexocytes to certain of the leukocytes which he believes to contain the greatest quantity of bactericidal substance. Bordet 6 believes that the bactericidal substances es- cape from the leukocytes only when they are injured, and that their presence in blood serum depends upon the fact that in the process of coagulation many leukocytes have been destroyed. The appearance of the germicidal activity of the serum with the destruction of the leukocytes probably explains the curious discrepancy that, though the blood of an ani- mal, when withdrawn from its body, is capable of killing bacteria, the blood and juices of the same animal while in its body are unable so to do. Thus, when cultures of 1 Fortschritte der Med., 1887, 13. 4 La Cellule, x.. I. 2 Virchoiv's Archives, cxvi. 6 Mibichenermed. Wochensckrift, 1894, 25. 3 Ibid., cxxi. « Annales de I' Inst. Pasteur, 1895, 6. IMMUNITY AND SUSCEPTIBILITY. 103 pathogenic bacteria are inclosed in small collodion cap- sules and inserted into the abdominal or other cavity or beneath the skin, the contained bacteria are subject to the action of whatever fluids pass by osmosis through the collodion, but are protected from the phagocytes. In these capsules the bacteria usually grow luxuriantly, without infecting the animal. The phagocytists use this to prove that bacteria are not destroyed by the body juices. It may mean, however, that no inflammation of importance being set up and no leukocytes destroyed, the juices do not become germicidal ; or it may simply prove Buchner's observation that the germicidal substances do not dialyze. Laschtschenko ' has confirmed Van de Velde's obser- vation that heterogeneous serums dissolve the germicidal substances out of the leukocytes. He prepared an extract of rabbit's leukocytes with which he mixed various serums — from the calf, ox, hog, goat, sheep, horse, and dog — whose bactericidal energies had been destroyed by heating to 550 C. The serums appeared to dissolve the alexins out of the leukocytes, as the mixture became so germicidal that bacteria were quickly killed by ex- posure to them. The distribution of the bactericidal substances in the organs and tissues has been investigated by many, among whom may be mentioned Schottelius, Hennsen, Kotlar, Kopp, Wroblewski, Brieger, Kitasato, and Wassermann. The most recent researches are by Livingood and Wauters.2 Livingood3 investigated the subject thor- oughly, using portions of the organs themselves, and cooked infusions of them in performing his experiments. He concludes : 1. That there are substances in all the or- gans which exert an inhibitory influence on the growth of bacteria. 2. There are slight but inconsistent differences in the degree of inhibition exerted by the organs upon 1 Munchener med. Wochenschrift, 1899, No. 15. * Archiv. d. mid. Expir. et d'Atiat. Path., T. x., 1898, p. 751. * Centralb. f. Bakt. u. Parasitenk., 1898, Bd. xxxiii., p. 980. / 104 PATHOGENIC BACTERIA. organisms in general, and specific organisms. 3. There are no essential differences in the growth on the various media except in vegetation. 4. There are no differences in morphology shown by the test-organisms. Wauters comes to somewhat different results. He col- lected leukocytes by injecting staphylococci into the pleu- ral cavity of a rabbit. The exudate was collected, titrated, and mixed with blood serum previously heated to 6o° C. After an hour the mixture was centrifugated, the liquid part removed and replaced by an equal quantity of dis- tilled water. After an hour this also was centrifugated. He found that bacteria grew well in the plain heated serum and in the watery extract of the leukocytes, but not in the serous extract of the leukocytes. Of the lymphoid organs, Wauters found the extract of bone mar- row to be about twenty times as bactericidally powerful as a similar extract of lymphatic glands, and very much more powerful than extracts of the solitary follicles, vermi- form appendix, and spleen. Of the organs other than lymphoid, extracts from the brain, striped muscles, and thymus were capable of restraining bacterial growth for a time only; extracts of the liver, kidney, pancreas, adrenal, and testicle were found to possess bactericidal activities varying in wide limits according to the animal from which they are taken, while extracts of the lung and connective tissues were found to be very active. Of all the tissues, the bone marrow was most active. Wauters found that the erythrocytes and fatty tissue contained in the marrow were bactericidally inert, so that the vir- tue resided exclusively in the leukocytes. Inasmuch as the lymphocytes seem devoid of bactericidal powers, as is shown by the very feeble activity of extracts of the lymph-glands, the active substances must be present in the ameboid cells found in the bone marrow. The bac- tericidal power of the tissues may depend in large part upon leukocytes and similar cells, as is evinced by the observation that tissues most likely to contain consider- able numbers of leukocytes are most actively germicidal. IMMUNITY AND SUSCEPTIBILITY. 105 Thus through a long and somewhat wearying journey we are led back again to the leukocytes, and urged to view them not as phagocytes, but as alexocytes, whose powers lie not in their capacity for intracellular digestion and destruction of bacteria, but in the secretion and liberation of the germicidal substances. However, the occurrence of germicidal substances in the blood, although bacteria of disease may be destroyed by them, will not explain the phenomena of immunity. The rat is immune to diphtheria not only because its blood will destroy diphtheria bacilli, but also because the rat is able to endure without injury the toxin of the diph- theria bacillus. Enormous doses of strong diphtheria toxin produce only a slight local reaction in white rats — an endurance not to be explained either by phagocytosis or the germicidal action of the blood, so that the essence of immunity is not contained in either. III. The Presence of Antitoxin. — The term anti- toxin is used to express a peculiar protective energy which is manifested by the blood serum of animals that have been subjected to forced artificial immunization. Antitoxins rarely occur in the blood of normal animals. The facts and phenomena concerning antitoxin will be considered in a more appropriate place, but as it is possible that they have something to do with natural immunity a few words with regard to them must precede the chief consideration of the subject. Finding that neither the destruction of bacteria by phagocytes nor their attenuation or destruction by the body humors can explain immunity to toxins, we must next inquire whether the bacteria-destroying principles of the blood are also toxin-destroying substances. Ogata and Jasuhara1 found that injection of the blood of a frog or of a rat into a susceptible animal which had been inoculated with a virulent culture of the anthrax bacillus will restrain the development of the bacteria and prevent the death of the inoculated animal. Beh- 1 Centralbl.f. Bakt. u. Parasitenk., ix., p. 25. 106 PATHOGENIC BACTERIA. ring1 also immunized mice by injecting them with the blood of the rat, and found them proof against anthrax; and Hankin 2 not only protected mice in the same way, but also by injecting them with an albumose extracted from the spleen of the rat. Abel3 has found that the blood serum of healthy men sometimes affords protection against diphtheria toxin; Stern has found one normal serum capable of protecting against the germ of typhoid fever, and Metschnikoif, one against cholera. Fischel and Wunschheim4 found that new-born babies are immune to diphtheria, probably be- cause of a protective substance in the blood. Bolton5 has found a presumably normal horse whose blood was markedly antitoxic to diphtheria. These observations of antitoxin-like substances oc- curring in natural immunity, when considered by them- selves, are very suggestive. Unfortunately, however, they are outweighed by the negative observations, and it must be admitted that as a rule natural immunity is not accompanied by the occurrence of demonstrable an- titoxin in the blood. All protective and neutralizing energies may not of necessity be antitoxins in the accepted meaning of the term, and it is not impossible that the blood of an animal may contain toxin-neutralizing substances of some dif- ferent kind whose demonstration may be made difficult or impossible because of their failure to act when intro- duced into other animals. Hankin 6 has divided the protective proteids — alexins — into groups accordingly as they are found in animals with natural or acquired immunity. Those occurring in naturally immune animals he calls sozins ; those in acquired immunity, phylaxins. These proteids are sus- 1 Loc. cit. 2 Loc. cit. 3 Hueppe's Principles of Bacteriology, translated by E. O. Jordan, p. 374. 4 Zeitschrift fur Heilkunde, 1885, xvi., 429-482. 5 Journal of Experimental Medicine, July, 1896, vol. i., No. 3. 6 Loc. cit. IMMUNITY AND SUSCEPTIBILITY. 107 ceptible of further division into groups according to their mode of action; thus, if they act destructively upon the bacteria they are called micosozins and micophylaxins. If they neutralize bacterial toxins, they are called toxo- sozins and toxophylaxins. The existence of toxosozins and toxophylaxins is, how- ever, a matter of theory, not of demonstration; and until it can be shown that there are such bodies able to operate successfully against poisons in large doses, immunity will not be explained. At present, in the absence of any explanation of the ability of an animal to endure the bacterial intoxication, it is evident that immunity remains unexplained, and the conclusion of Sternberg \ that "natural immunity is due to a germicidal substance present in the blood serum which has its origin (chiefly at least) in the leukocytes and is soluble only in an alkaline medium," must be rejected as explaining only a part of the phenomena. Modification of Immunity. — Immunity is neither per- manent nor constant, but varies with many natural and artificial conditions. Thus, it is found that young ani- mals are, as a rule, much more susceptible to the in- fectious diseases than the adult animals. It is also found that newly-born animals are sometimes immune to diseases to which they will later become suscept- ible. I. Reduction of Immunity. — Any condition or combi- nation of conditions depressing the general vitality lessens the power of resisting infection a. Depressing hygienic conditions have long been asso- ciated with the occurrence of disease, and it is well estab- lished, both clinically and statistically, that infectious diseases are most common and severe where overcrowd- ln») Poor ventilation, improper diet, overwork, and insuf- ficient sleep exist. b. Noxious gases. — It has long been supposed that sewer-gas and other poisonous gases predispose "to disease. 1 Immunity and Serum- Therapy, 1895. 108 PATHOGENIC BACTERIA. Alessi,1 in investigating this subject, confined rats, rabbits, and guinea-pigs in cages, some of which were placed over the opening of a privy, while in others the excreta of the animals was allowed to accumulate in a receptacle below. The inhalation of the vapors from the excreta caused so marked a difference in the resisting powers of the animals that, while the control animals all resisted it successfully, the rats succumbed to an injec- tion of the typhoid fever bacillus in from twelve to thirty- six hours after from five to seventy-two days' exposure to the vapors ; guinea-pigs after seven to fifty-eight days ; and rabbits after three to eighteen days' exposure. Abbott,2 on the contrary, forced rabbits for as long as one hundred and twenty-nine days to breathe air which had been passed through sewage or through putrid meat infusions. He concludes that "the products of decom- position . . . play no part in either producing diseased conditions or in inducing susceptibility to infection." c. Fatigue has marked influence in reducing immunity, the fact being well recognized clinically. Charrin and Roger3 found that the white rat, which usually resists inoculation with anthrax, becomes infected and dies if compelled, before inoculation, to turn a revolving wheel until exhausted. d. Exposure to cold is viewed by clinicians as one of the most fruitful sources of reduction of immunity, and the occurrence of most of the infectious diseases not other- wise explained is referred to it. This is not without reason, for its influence upon pneumonia, bronchitis, etc. can scarcely be doubted. Experimentally, its influence has been shown in the classic experiment of Pasteur, who found that fowls that naturally resist anthrax become susceptible if given a cold bath before inoculation. e. Peculiarities of diet may reduce immunity. Hankin observed that the immunity of rats to anthrax was in 1 See abstract in the Centralbl. f. Bakt. u. Parasitenk., 1894, xv., 228. 1 Transactions of the Association of American Physicians, 1895. 8 Compte-rendu Soc. de Biol, de Paris, Jan. 24, 1890. IMMUNITY AND SUSCEPTIBILITY. 109 large measure destroyed by feeding the animals upon bread. The natural diet may have something to do with sus- ceptibility, for in anthrax we find that the herbivorous animals are all rather easily infected, while the carnivor- ous animals are infected with difficulty. f. Effect of Drugs, etc. — In the experiments of Platania, immune animals, such as frogs, pigeons, and dogs, were found to become susceptible to anthrax when under the influence of curare, chloral, or alcohol. Leo found that when white rats were fed upon phloridzin a glycosuria developed, during which they become susceptible to anthrax. Wagner1 found that pigeons become susceptible to anthrax when under the influence of chloral. It is a common observation that alcoholics are predis- posed to croupous pneumonia. The experimental demon- stration of the effect of alcohol in increasing susceptibility to disease is, however, difficult. Abbott's2 comprehen- sive work in which a large number of rabbits were daily intoxicated with alcohol (5-15 c.c.) introduced into the stomach through a rubber catheter, showed that the vital resistance to infection by the streptococcus pyogenes and bacillus coli communis was diminished. g. Operative manipulations may destroy immunity. The value of the spleen in preventing infectious diseases has been studied by a number of observers, but their results are conflicting. Thus, Bardach,3 Righi,4 and Montuori 5 found that the removal of the spleen increased susceptibility to infection ; Blumenreich and Jacoby 6 found that its removal was followed by a hyperleukocyto- sis, increase in the germicidal power of the blood, and corresponding increase in immunity; while Milkinow- 1 Wratsch, 1890, 39, 40. 1 Journal of Experimental Medicine, vol. i., No. 3, 1896. * Ann. de P Inst. Pasteur, 1889, No. 2, p. 577 ; 1891, No. I, p. 40. 4 La Riforma Medica, 1 893, pp. 170, 1 7 1. s Ibid., Feb., 1893, 17, 18. « Berliner klin. Wochenschrift, May 24, 1897. HO PATHOGENIC BACTERIA. Raswedenow ! found that the removal of the spleen was a weakening factor in the immunization of animals. Kurlow2 did not find the spleen more important than other organs in overcoming infections, and Kanthack found that its removal had practically no influence upon the natural immunity of animals to pyocyaneus infection. h. Diseased conditions of the animal are almost always associated with reduction of immunity because of the depressed vitality. Thus, in diabetes mellitus furuncles and carbuncles are very frequent, and local areas of gan- grene which may be dependent upon infection are fre- quent. In Flexner's3 studies of the terminal infections it was common to find that in cases of nephritis the organs contained pyogenic cocci. Pansini and Calabreuse found that the addition of uric acid to blood serum diminished its bactericidal activity. Glucose exerts a similar effect. Platania observed that the administration of phloridzin by exciting glycosuria destroyed immunity. i. Mixed Infections. — Clinically it is a familiar fact that in typhoid fever and in influenza pneumonia is apt to occur, both from the activity of the micro-organisms of the primary infection and from others frequently harm- less in health whose activities depend upon the general diminution of vitality. Probably for the same reason the occurrence of an acute infectious process like influenza commonly causes rapid spread of an existing chronic affection such as tuberculosis. Roger found that when animals immune to malignant edema were simultaneously inoculated with it and 1-2 c.c. of a bouillon culture of Bacillus prodigiosus, they would succumb to the malignant edema. Giarre observed that if an adult guinea-pig, which is refractory to infec- tion by the pneumococcus, were subsequently inoculated with diphtheria, it readily died of pneumococcus septi- 1 Zeitschrift fur Hygiene, 1896, xxi., 3. 4 Archiv filr Hygiene, 1889, lx., p. 450. * Journal of Experimental Medicine, 1896, vol. i., No. 3. IMMUNITY AND SUSCEPTIBILITY. Ill cemia. This observation may explain some of the pneu- monias occurring in consequence of or during the course of diphtheria. It is not understood what the two affections may have in common, but when yellow fever breaks out epidemic- ally it is apt to be associated with a simultaneous epi- demic of dengue fever. j. Traumatic injury seems to increase susceptibility, probably by providing a nidus in which the bacteria may develop free from the usual restraints. Attenuated cult- ures of the bacillus of symptomatic anthrax which would not kill a guinea-pig, may do so if simultaneously intro- duced into the tissues with a little lactic acid. Vaillard and Rouget1 found that if tetanus bacilli were introduced into the body, washed free from their toxin, they were readily taken up by the phagocytes and no signs of dis- ease followed. If, however, their toxin, some lactic acid or other damaging cheinic substance, were introduced with them and the cells of the tissue destroyed, they grew and caused tetanus. II. Exaltation of Immunity. — By means which rarely if ever arise under natural conditions it is pos- sible to intensify the natural immunity possessed by an animal. As, however, the process is identical with the development of immunity where none formerly existed, and is a purely artificial condition, it finds its best con- sideration below. Acquired Immunity. Acquired immunity is power to resist disease, depend- ing upon conditions arising during the life of the indi- vidual. It is a peculiarity of an individual, not of the species. In general, the condition resembles natural im- munity, but in a few particulars it is very different, and is characterized by certain peculiar phenomena. In the arrangement of the subject, as considered below, acciden- tally acquired immunity will be found to bear the greatest 1 La Bull, mid., 1891, p. 901. H2 PATHOGENIC BACTERIA. resemblance to natural immunity and to attain about the same degree of resisting power as is observed in natural immunity. Thus, after a horse recovers from traumatic tetanus it remains for a long time immune to the germs of tetanus and resistant to their toxin. It is, however, susceptible to intoxication with tetanus poison in moderate doses. Experimentally acquired immunity may differ from accidentally acquired immunity in that the resistance to both infection and intoxication may be increased to an amazing degree, the animal being gradually accustomed to the infection or intoxication until it can endure hun- dreds of times the fatal dose for a normal animal. This process of habituation is technically called im- munization or forced immunity, and is accompanied by certain phenomenal qualities of the blood serum, which becomes antimicrobic or antitoxic, or both. The greater number of the acquired immunities are active and cytogenic. Passive immunity makes its ap- pearance, however, among the acquired immunities only. That acquired immunity is not hereditary is amply illustrated by the fact that though nearly everyone has the diseases of childhood and becomes immune to them, children are still born susceptible to measles, mumps, chicken-pox, etc. I. Accidentally acquired immunity is the result of accidental conditions that arise in nature. a. Infectiojt is the most frequent cause of this form of immunity. Accidental infection commonly results in a more or less permanent immunity to disease. Thus, children born susceptible to measles, scarlatina, mumps, and the other infectious diseases of childhood are usually accidentally infected during early life, the sur- vivors usually remaining immune thereafter. Occasion- ally the immunity thus attained gives out and a rein- fection becomes possible. Such cases are, however, exceptions, not the rule. The immunity attained in this way is usually so active that the organism is not IMMUNITY AND SUSCEPTIBILITY. 1 13 only enabled to resist bacteria with virulence equal to those which caused the original infection, but also those of much greater virulence. b. Modified infection, by which infection with a mod- ified form of a disease, or with some closely related disease, may cause immunity. This is best exemplified in the vaccination against smallpox. The exact nature of vac- cinia and its true relation to variola are not yet settled, although the modern view, based upon a great amount of evidence, is that they are the same disease, variola a virulent form, vaccinia a modified, attenuated form. The observations leading to the early experiments and conclusions of Jenner were that milkmaids acci- dentally contracting vaccinia from the cow did not subsequently contract variola, having acquired immu- nity to the one affection by having suffered from the other. c. Aberrant diet may be a cause of immunity. The experiments of Hankin upon rats have already been quoted, and it will be remembered that when rats which are refractory to anthrax are fed upon a strictly vegetable diet their susceptibility is increased, while if they are fed upon meat their immunity is increased. In the natural condition it is not probable, though always possible, that an animal might select some un- usual food the ingestion of which would be followed by immunity to poisons. Ehrlich1 found that when mice were fed with food containing minute quantities of ricin they developed immunity to ricin. It may be possible that the immunity possessed by certain birds and mam- mals against serpent's venom depends upon the fact that they prey upon the snakes, and from ingested venom, liver, or blood of the reptile acquire the resisting power. Some authors assert that the snake-charmers of India, who seem to be immune to cobra poison, become so in consequence of making a habit of consuming some of the venom every day. As this is scarcely compatible with 1 Deutsche med. Wochenschrift, 1891, Nos. 32 and 44. * I 114 PA THOGENIC BA CTERIA. facts later to be discussed, it may not be the true ex- planation of the immunity. II. Experimentally acquired immunity differs from the above forms in that it depends upon conditions so purely artificial that they could not occur in nature. It is a subject of extreme interest and furnishes us with most remarkable biological puzzles. Some of the experimental immunities are active, some are passive, some of them consist in creating resisting powers where none normally existed, some in increasing those that were normally possessed by the animal. Active Immunity — a. Inoculation. — By this term I differentiate between the spontaneous infection that is the common cause of accidental immunity and the inten- tional experimental infection practised in the laboratory. Inoculation was practised centuries ago as a means of securing immunity to variola. The theory was good, but the practise had very decided drawbacks. In per- forming inoculation a mild case of the disease was selected, at a time when no epidemic was in progress, and from the variola pustule some of the matter was conveyed to an abraded surface of a healthy person. The result was a mild attack of smallpox, followed by perfect immunity. The disadvantages were that the case might assume serious aspects, and occasional deaths resulted from the treatment. Further, that the inoculated individual, having real variola, was a source of contagion and might excite an epidemic. In the laboratory, inoculation is practised to bring about immunity to many diseases, our knowledge of the phenomena of infection being drawn upon to prevent the death of the animal. It is the rule that in infecting an animal a certain number of bacteria are necessary. If less than this number are given, the animal shows no symptoms or recovers, afterward becoming immune to a much larger dose than was received. This increase of resisting power is made use of in the treatment known as immunization, to be described below. IMMUNITY AND SUSCEPTIBILITY. 115 b. I'accination. — This word, derived from vacca, a cow, had its origin in the use of matter from the pustules of cowpox, asjenner used it to prevent smallpox. In its etymologic sense it is not strictly applicable as now em- ployed, but it has become the convenient designation of all modified "viruses'''' or cultures of pathogenic bacteria. The vaccination against smallpox depends upon an attenuation of the variola germ as it passes through the cow, by which its energies in man are limited to the development of a local lesion and mild constitutional involvement, devoid of all contagiousness. The essence of the process is the attenuation of the germ in the cow. Laboratory experiments have enabled us to produce vac- cines to many diseases by manipulating their germs so as to destroy their pathogenic powers without limiting their immunizing powers. Pasteur1 found that if the Bacillus anthracis was grown at certain temperatures, it lost its virulence, so that it failed to kill animals larger than mice. He also observed that the inoculation of these attenuated germs into the cow was followed by no im- portant symptoms, though the cow became resistant to more virulent cultures. By a second inoculation of a vaccine or attenuated culture fatal for guinea pigs, and then a third, fatal to rabbits, the cow attained a perfect protection against infection with virulent anthrax. Vaccination against symptomatic anthrax has been sim- ilarly accomplished by Arloing, Cornevin, Thomas,2 and Kitt,3 who found that if the bacilli, dried in powdered muscle from affected animals, were exposed for some hours to a temperature of 850 C, they became attenuated and no longer pathogenic for cattle, though their inoculation into them was succeeded by perfect immunity. HafT- kine* has successfully used modified cultures of the chol- 1 Compte-rendu de la Soc. Biol, de Paris, 1881, xcii., pp. 662, 665. See, also, vol. xc. * Le Charbon Symptomatique du Bceuf., Paris, 1887. * Centralb. f. Bakt. u. Parasitenk. , i. p. 684. * Brit. Med. Jottrn., 1891, ii., p. 1278; 1895, ii., p. 1541. Ii6 PATHOGENIC BACTERIA. era spirillum and plague bacillus, and Wright,1 of typhoid fever bacilli, in vaccinating against the respec- tive diseases. In his experiments upon rabies, Pasteur2 found that drying the tissues in which they were enclosed was a satisfactory method of attenuating the germs of hydro- phobia, and that after a certain period of exposure to a dehydrating substance they ceased to be pathogenic, though their inoculation was succeeded by immunity. Thus, from the original observations in which the cow was the important factor, we now reach a time when vaccines, viruses, or attenuated cultures are prepared in the laboratory by a variety of methods. Some of the germs are dried, some heated, some grown upon media containing antiseptics, some are deprived of their spore- producing capacity, some washed free of their toxic prod- ucts, some are combined with bacteria of other species, some entirely killed in order that desired results shall fol- low from their inoculation, and sometimes the essential toxin is separated from the culture and used for immuniz- ing purposes, all these substances being known as vac- cines. It is particularly interesting to observe that dead cult- ures can be made use of for the production of immunity, probably because of the toxin which they contain. In some cases saprophytic bacteria may be made use of in producing immunity or increasing resistance to dis- ease. Hueppe and Wood claim that inoculation with saprophytic bacteria derived from water and the soil may protect animals against the pathogenic bacteria, and are said to have produced immunity to anthrax in this man- ner. Pawlowski has also found that the influence of one bacterium upon another, or the influence of one bacte- rium upon an animal infected with another, sometimes affords a protection similar to that of vaccination. Thus, he asserts that if rabbits are infected with anthrax, to 1 Brit. Med. Journ., Jan. 30, 1897, p. 256. 2 Compte-rendu de la Soc. Biol, de Paris, 1S89, cviii., p. 1228. IMMUNITY AND SUSCEPTIBILITY. 117 which they are susceptible, and then injected with a cult- ure of Bacillus prodigiosus, they will recover. c. Intoxication. — The phenomena of immunity are pe- culiar neither to infection by bacteria nor to the influence of their toxins, but are common to many forms of intoxi- cation. In consequence, with the advance of knowledge of the specific diseases, the study of the poisonous prod- ucts of bacteria, and the observation that the effects of many organic and some inorganic poisons bring about similar reactions in the organism, have greatly broad- ened the scope of immunity. The metabolic products of bacteria were early noticed and, indeed, as early as 1880, Toussaint and Chauveau \ taught that the protective effect resulting from the in- corporation into the body of attenuated disease germs depended upon the fact that such attenuated cultures contained the metabolic products of the bacteria and thus conferred immunity. This opinion was in direct opposi- tion to the view of Pasteur, who held that infection was essential. It was later discovered that although the bac- teria contained in a culture were killed, it might still confer immunity. Salmon and Smith,2 as early as 1886, found this true in the case of swine plague. Still later it was found that even if the dead bodies of the bacteria were removed by filtration, the metabolic products contained in the filtrate might confer immunity, this being shown by Foa and Bonome3 to be true of cultures of proteus, Charrin of cultures of Pyocyaneus, Roux and Chamberland4 of malignant edema, Roux5 of symptomatic anthrax, Gamaleia of the septicemia of pigeons and cholera, and Carl Fraenkel 6 of diphtheria. Hueppe7 is particular to caution us against supposing that immunity depends entirely upon accustoming the individual to the "specific" poisons of the disease germs, pointing out that in 1887 he had produced immunity by 1 Compte-rendu de la Soc. Biol, de Paris, 1890, 189 1. * Centralbl.f. Bakt. u. Parasitenk., ii., No. 18. s Zeitschrift fur Hygiene, v., 415. * Annates de V Inst. Pasteur, 1887, 12. 6 Ibid., 1888, 8. « Ibid., 1888, 2. * Loc. cit. ! Ii8 PATHOGENIC BACTERIA. the use of entirely attenuated bacteria that were purely saprophytic. His results were also confirmed by Chau- veau in 1889. The success of Behring1 and Roux2 in immunizing animals to the toxins of diphtheria and tetanus is well known, and close upon their researches with these toxins came Calmette's3 studies of serpents' venom, which showed that gradual progressive intoxication produced immunity, and Ehrlich's4 experiments with ricin and abrin and the production of immunity to these alkaloids. Still later Wassermann produced immunity to poisonous eel's blood, and Klemperer and Scheplewski to the " Botulism us-gift " (meat-poison). Immunity to a min- eral poison is seen among the arsenic eaters, and Bes- redka5 has recently found it possible to produce immu- nity to arsenic in rabbits, accompanied by the occurrence of an antitoxic substance (antiarsenine) in the blood. The immunity thus induced is active and probably cytogenic, and does not seem to differ perceptibly from that produced by the bacteria themselves. It is interesting to observe that the activity of the fil- tered culture depends upon the solubility of the metabolic products, and that in cases where these are with difficulty extracted from the germs the filtrates are very feebly active. Passive Immunity. — Passive immunities, not depend- ing upon the activity of the organism, can be developed experimentally. d. Antitoxins, derived from immunized animals, confer a perfect immunity upon the animal into whose blood they are introduced. As this subject is one of great im- portance to which much space must be devoted, it will be discussed in a subsequent part of the chapter. e. Tissue suspensions sometimes produce a protective reaction upon toxins when introduced into the body 1 Zeitschrift filr Hygiene, 1892, xii., I. 2 Annales de V Inst. Pasteur, 1888, ii., p. 629; 1898, p. 640 s Ibid., 1894, viii., p. 275. 4 Deutsche med. Wochenschrift, 1891, Nos. 32 and 44. 5 Ann. de V Inst. Pasteur, June, 1899, p. 30. IMMUNITY AND SUSCEPTIBILITY. 119 simultaneously with them. The first to observe this seems to have been Wassermann and Takaki,1 who found that when a portion of the spinal cord of a rabbit was crushed and suspended in a physiologic salt solution it would, when mixed with tetanus toxin, protect the rab- bit. This observation has been abundantly confirmed, and is all the more remarkable because the reaction does not seem to take place in vitro, but in corporo, for Wasser- mann found that if the nervous substance was injected twenty-four hours before the toxin or several hours after it, or into another part of the body, it still exerted the protection, acting like antitoxin. Marie2 denies this in part, and asserts that contact between the nervous sub- stance and toxin is essential, for if the nervous substance is introduced at one part of the body and the toxin injec- tion made at some remote part, as, for example, into a paw, the animals always die. Marie also observed that the gray matter of the cerebral cortex contained the greatest amount of toxin-destroying energy. MetschnikofT3 has also confirmed the protective effect of the comminuted brain substance, but does not agree with Wassermann in all particulars. Thu#, instead of looking upon it as an antitoxic effect, MetschnikofT views it as an inflammatory reaction by which the pulverized brain substance, being chemotactic, causes the accumulation of large numbers of leukocytes at the seat of injection, which are in all probability responsible for the toxin destruction. MetschnikofT observed that the brains of rabbits suffer- ing from tetanus exerted no protective effect. This observation will become more important when the subject of Wassermann's theory of immunity is reached later on. In order for the brain substance to exert its effect it must be removed from the animal and crushed. To inject tetanus toxin into the living brain is invariably to cause tetanus, but to remove the brain and crush it and mix it with the toxin is to destroy the toxin. MetschnikofT, however, 1 Berliner klin. Wochenschrift, Jan. 3, 1898. * Annates de F Inst. Pasteur, 1898, No. 2. 3 Ibid. 120 PATHOGENIC BACTERIA. assures us that the toxin is not destroyed by the contact, as mixtures of brain substance and toxin which were inactive for guinea pigs caused fatal tetanus in mice. Wassermann believes that the mixture of tetanus toxin and brain substance is neutral — i. e., inactive — because "every antitoxin producing toxin is specific in the sense that it produces its symptoms by chemic combination of its toxophoric atoms with some cell substance in the body of the susceptible animal." The tetanus toxin acting specifically upon the nervous system, unites with the nervous substance chemically in vitro, and is then unable to unite with that of the animal into which it is injected. Ingenious and suggestive as this hypothesis is, it may not be true, for Myers1 made a series of experiments that would seem to overthrow it. Taking cobra venom as the specific poison, and selecting the nervous system, upon which it undoubtedly acts in producing death by paralysis of the respiration, he found that there was no part of the nervous system that combined with it in vitro, or in any way changed it, the injections of the mixture invariably causing the death of the animal. Kanthack^ found that extracts of the liver protected against cobra venom, but Myers disproved this, and after investigating all of the major organs of the body dis- covered that there was but one organ in the body that had the power of annulling the effects of cobra poison, viz., the adrenal body. He found that the fresh organs in infusion and the dried organs pulverized were alike able to destroy the poison, and that the adrenals of all the animals studied exerted a similar effect. He further found that the degree of neutralization is very limited, and that o. i milligram of the venom being fatal to a guinea pig of 250-350 grams weight, the adrenal tissue when mixed with it was able to destroy somewhat more than this, although the law of multiples so characteristic of the antitoxins was not applicable here. 1 Lancet, July 2, 1898. 8 Report of the Local Government Board, 1895, vi., p. 212. IMMUNITY AND SUSCEPTIBILITY. 121 /. Inert particles are sometimes capable of producing very remarkable protective reactions when mixed with the toxins and injected into the cellular tissues. Staudensky1 has found that if ordinary commercial carmin is mixed with tetanus toxin in the proportion of 0.5 gram to 10 cubic centimeters, ten fatal doses can be administered to a guinea pig without resulting harm. Interestingly enough, if the solution of carmin is heated to 6o°-ioo° C, it loses its protective effect, though when dry carmin is heated in a sealed tube it is unchanged. When the mixture is kept for twenty-four hours it again becomes toxic, and if fresh mixtures are filtered free from the carmin its effect is lost. The toxin is, therefore, not destroyed by the carmin. Microscopic examination of the inflammatory exudate found at the seat of inoculation shows large numbers of leukocytes, which may be respon- sible for the toxin destruction. Immunization. — The process of rendering an animal immune is described as immunization, though at the pres- ent time this term is being more and more restricted to those cases in which the animal attains a high degree of immunity by a gradual process which I have already de- scribed as forced immunity. In the last few years this sub- ject has been very carefully studied in its most important relation to the therapeutic serums. These remedies are all produced by the same general management of the animal, though it may be that in one case some alkaloidal tox- albumin, in others some bacterial toxin, and in still others some sterilized culture, attenuated culture, or living virulent culture of bacteria is employed. An appropriate animal is selected and carefully examined to exclude diseased or other unfavorable conditions. It is then given a very small dose of the toxin (for convenience I shall refer to whatever preparation the animal receives as toxin), from the effect of which it is allowed to completely recover. If the effect has been too active, the toxin can be modified by adding some of the trichlorid of iodin 1 Annates de V Inst. Pasteur, Feb. 25, 1899, p. 126. 122 PATHOGENIC BACTERIA. solution recommended by Behring, or by diluting it, attenuating it by heat, or by using less virulent cultures, etc. The same dose is repeated, and after some days, if the reaction has been mild, a larger dose can be given. In a week, more or less, according to circum- stances, a still larger dose may be injected and so on until, by proper careful management through a sufficient length of time, the animal may be so accustomed to the toxin as to endure, without symptoms of inconvenience, hundreds of times the originally fatal dose for his kind. The condition is one of habituation or tolerance, and leads, of course, to a very unnatural degree of immunity. When we come to study this state of forced immunity we find ourselves encountered by a most interesting series of paradoxes and phenomena. No visible change is discerned during the process, the animal remaining well and strong throughout. The toxins injected at the regular periods are endured without inconvenience, and to all appearances the process of im- munization might go on indefinitely. Unexpectedly, however, the first obstacle and paradox appears. No matter how carefully the immunization process has been carried on, there may come a time when further increase will develop most unexpected symptoms, entirely charac- teristic of the disease, and, in my experience, invariably fatal. This condition was long ago pointed out by Behring, who showed that the injudicious or too rapid increase of the toxin doses in immunization threw the animal into a state of hypersensitivity in which it suc- cumbed to doses easily endured before. This hypersen- sitivity is not affected by the fact that the blood of the animal contains antitoxin, and is quite as likely to come on in highly antitoxic animals as in others. The duration of acquired immunity is very variable, according to the particular conditions by which it has been induced. Accidentally acquired immunity, as in the case of yellow fever, may be so perfect and perma- nent as to endure throughout the remainder of the indi- IMMUNITY AND SUSCEPTIBILITY. 123 vidual's life. In other cases it is only temporary, rarely continuing longer in diphtheria than thirty or sixty days. Passive immunity is probably never permanent, but continues only as long as the antitoxin is uneliminated. In cases in which diphtheria antitoxin is used for pro- phylactic purposes it seems to be effective for two or three months. Forced immunity is not permanent, but begins to decline as soon as the treatment of the animal is suspended, the return to the normal condition being slower than the production of the abnormal one. Two very important phenomenal manifestations of the blood serum of animals with forced immunity must be mentioned, namely, the antitoxic and the antimicrobic poivers. The Antitoxins. — By antitoxin is meant a peculiar protective energy manifested by the blood serum and other fluids of animals subjected to a high degree of forced im- munity. The first observation upon the protective power of immune blood was probably made in 1890 by Ogata and Jasuhara,1 who found that when animals were given a subcutaneous injection of blood from an animal im- munized to anthrax, they were able to resist the effects of inoculation with a virulent culture. In the same year Behring and Kitasato2 found that the blood serum of an animal immunized to diphtheria or tetanus when added to a culture of the respective bacilli neutralized its power to provoke disease, and that when added to the filtered culture it had the power of destroying its toxic effects. The next year, 1891, Kitasato3 discovered that when mice were inoculated with tetanus and symptoms of the disease appeared, they could be saved by the intra- abdominal injection of blood serum of an immunized mouse. About the same time Ehrlich * immunized ani- 1 Loc. cit. ' Deutsche med. Wochenscrift, 1890, No. 49. ■ Zeitschrift fiir Hygiene, 1892, xii., p. 256. * Deutsche med. IVochenschrift, 1891, Nos. 32 and 44. 124 PATHOGENIC BACTERIA. mals to ricin and abrin, and found that in the blood serum a protective substance appeared, which was potent to save animals into which it was injected, from fatal doses of the respective toxalbumins. Calmette,1 Phisalix, and Bertrand 2 also begun experimenting with the venom of serpents, and found that in the blood serum of animals immunized to this poison a protective substance made its appearance. Behring called the protective substances in the blood "antikorper" (anti-bodies). As the majority of them combated the activity of the toxins, it seemed natural to adopt the word antitoxin, and now scarcely any other term is applied to them. From the observations mentioned, Behring contrived to work out the details of the "Blood-serum Therapy," and after immense difficulty in overcoming the obstacles iu the way, proposed methods of preparing the necessary toxins, immunizing the animals, and preparing and using the serum. These methods were so satisfactory that the flood-tide of investigation which succeeded his publications has not made any essential changes. It has already been shown that the antitoxins are of questionable occurrence in ordinary forms of immunity. In some of the illustrations given the protective power may have depended upon entirely different entities, and surely after the experiments of Wassermann and Takaki with comminuted brain and spinal cord tissue, and those of Staudensky with carmin, we should be prepared to expect that there may be numerous other protective influences. The occurrence of antitoxin in the blood serum is to be considered as a phenomenon of forced immunization. During the immunization process it does not seem to develop in proportion to the toxic endurance of the ani- mal, but, as Roux has pointed out, is rather suddenly developed after the immunization has attained a high de- gree. During the continuance of the immunization it is 1 Annales de r Inst. Pasteur, 1894, viii., p. 275. * Compte-rendu Acad, des Sciences, cxviii., p. 556. IMMUNITY AND SUSCEPTIBILITY. 125 a variable, not a fixed quantity, and while the toxin en- durance of the animal is kept up without variation, the antitoxin may gradually diminish. This I have seen many times illustrated in horses producing diphtheria antitoxin, an excellent illustration being afforded by one particular horse that furnished at one time a serum con- taining 1400 units to each cubic centimeter of serum. The immunity was maintained by cautious toxin injec- tions for a long period subsequently, and the endurance of the horse remained unchanged for months, but the antitoxicity of its blood gradually declined, until from 1400 units it fell to 100 units. It was not worth while to keep the horse longer, and it was turned out to pasture, and later used to work about the farm. This rather sudden appearance of the antitoxin and its decline during the immunity of the animal prepare us for the information that the animal's immunity does not depend upon the antitoxin, but upon some other condi- tion. The probable proof of this is seen in the pecu- liar condition of hypersensitivity to which Behring and Wladimiroff called attention, and of which mention has been made. In these cases it makes no matter how much antitoxic strength is contained in the blood, the animal is just as sensitive to the toxin as if it had none, and as if it had not been immunized. Moreover, the hypersensitivity is not a cumulative action of the toxin that outweighs the antitoxin, as will be readily shown by a simple calculation. A horse weighing 1300 pounds, possessing about 100 pounds of blood, of which about one-third, or 30 pounds, is serum, has been immunized to diphtheria toxin according to the method described in the chapter upon Diphtheria (q. z>.); and while the serum contains 500 immunizing units of antitoxin in each cubic centimeter of blood serum, the horse falls into the hyper- sensitive condition and dies. What relation exists between the antitoxin in its blood and the toxin that produced death ? It is certain that there is none, and that the antitoxin 126 PATHOGENIC BACTERIA. that exerts a most remarkable protective influence upon other animals does not protect the animal by which it is formed. If the horse's blood furnishes a total of 30 pounds of serum, each pound being about equal to 500 c.c. of liquid, there is a total of 15,000 cubic centimeters of antitoxic serum in the horse. Suppose the minimum fatal dose of diphtheria toxin for a 250-gram guinea pig to be 0.0045. If the serum under consideration contain 500 units in each cubic cen- timeter (see the method of testing diphtheria antitoxic serum in the chapter upon Diphtheria), then ?0^00 c.c. will protect a guinea pig against 0.0045 c-c- °f the toxin ; g^oir c.c. against 0.045 c-c-5 5-00 c-c- against 0.45 c.c; ^q- c.c. against 4. 5 c.c. ; \ c.c. against 45 c.c. ; and 1 c.c. against 9 c.c. of the toxin. If each cubic centimeter of the serum of this horse is capable of destroying the toxic effect of 9 cubic centimeters of toxin, the total toxin- annulling capacity is 9x15,000 c.c. of serum in the horse's blood = 135,000 c.c. of toxin. We must next see how much toxin has been received by the horse during his immunization. The follow- ing doses, the figures referring to cubic centimeters of toxin, probably represent an average careful manipu- lation extending over a period of about three months : -nr, h h *> ** 2, 3, 5, 8, 10, 15, 20, 25, 50, 50, 100, 150, 200, 250, 300, 500, 500, 500, 500, making a total of about 4200 c.c. of diphtheria toxin. Now observe that the total quantity of toxin consumed by the horse is 4200 c. c. , but his protective energy is 135,000 c.c. of toxin, so that the blood of this horse, if drawn from his body, would furnish enough protection to save 327- horses from doses of toxin as large as the total amount administered to him during the entire course of his treatment. This illustration is not only extremely instructive in showing the paradoxic nature of the condition of hyper- sensitivity, but certainly proves that the antitoxicity of the blood is not the cause of immunity, but a phenome- IMMUNITY AND SUSCEPTIBILITY. 127 non of that state. It is also of great importance in con- sidering the origin of the antitoxin. Concerning the origin of the antitoxins, we must at once dismiss from our minds the thought that bacteria have anything to do with their formation other than through the toxins they generate. The immunization of animals to feebly toxic cultures, or to bacteria washed of their toxins, produces immunity, but immunity without antitoxic activity produces the antimicrobic power of the blood presenting itself in these cases. It is, therefore, the poison alone that is responsible for the phenomenon, and a moment's reflection upon the anti-bodies produced by immunization to ricin, abrin, venom, eel's blood, etc. will clearly establish this fact. How does the poison produce the antitoxin? A. Theory that the antitoxin is the toxin in a changed condition. — This thought presented itself early in the study of the subject, and doubtless suggested itself because in the forced immunity which is the foundation of anti- toxin formation the administration of large quantities of toxin was necessary, and it was only after large quan- tities had been administered that antitoxin was formed. Buchner is emphatic in his assertion that the antitoxin is "entgiftete" or changed products of the bacterial cells. The fact that the toxin met with a speedy elimi- nation seems not to have been taken into account, although evidences of the rapidity of this process are not wanting. I have seen horses covered with sweat a few minutes after toxin administration, and have observed diarrhea shortly after the injections. Various observers have found the unchanged toxin in the excretions. Smirnow,1 Kriiger,2 D' Arsonval and Charrin,3 Bolton and Pease,4 and others have, however, found that when diph- theria cultures are placed in a V-shaped tube and subjected 1 Berliner klin. IVochenschrift, 1895, Nos. 30 and 31, and Zeitschrift fur klin. Med , Bd. xxii., Nos. 1 and 2. * Deutsche med. Wochenschrift, 1895, No. 31. 3 La mid. Moderne, 1896, p. 71. ; Journal of Experimental Medicine. 128 PATHOGENIC BACTERIA. to electrolysis a peculiar change takes place by which the bacteria and toxin collect at one pole, while fluid containing a protective substance collects at the other. It was supposed that this was an evidence that the toxin had been changed into antitoxin, and that an analogous process to that which takes place in the body had been produced by the electrolysis. It may, however, have been the expression of another process. The calculation of antitoxin strength given above ought to be sufficient proof that the antitoxin is some- thing more than changed toxin, for in the blood of the horse given as an example we found 327 times as much antitoxin as it had received toxin, and in all of the electrolysis and other experiments that have been made there was but a small relative amount of protection re- sulting from the electrolysis of considerable culture. Vaillard opposes Buchner's view by showing that from an immunized rabbit ' ' a volume of blood equal to the entire amount that circulates in its body may be with- drawn without diminishing in an appreciable manner the antitoxic power of its serum." Roux also points out that the antitoxic power of the blood depends upon the method of immunization adopted rather than upon the quantity of toxin used, a few large doses producing a far less satisfactory result than many small ones. He found that the serum of an animal immunized by thirty-three small doses was capable of neutralizing in vitro 150 parts of toxin, while that of an animal which received the same amount in only nine doses neutralized only 25 parts of the same toxin. Several accidents that have occurred in the administra- tion of antitoxin have influenced some writers toward the conclusion that the antitoxin is altered toxin. Thus, Buschka,1 having accidentally inoculated himself with matter supposed to contain tetanus virus, gave himself a prophylactic injection of antitetanus serum and thereafter suffered from tetanus-like convulsions. -Marcuse2 ob- 1 Quoted by Hueppe in the Principles of Bacteriology. * Ibid. IMMUNITY AND SUSCEPTIBILITY. 1 29 served the appearance of paralysis just like that pro- duced by diphtheria toxin in a child injected with diph- theria antitoxin. B. Theory that the antitoxin is an enzyme produced in the culture. — This view of the subject is presented to us by Emmerich and Low,1 whose interesting observations upon cultures of Bacillus pyocyaneus showed that among the metabolic products of bacteria there are certain bac- teriolytic ferments or enzymes which check the further development of the culture after it has attained a certain age, and ultimately dissolve the contained dead bodies of its bacteria. By precipitating and concentrating they were able to separate a substance — pyocyonase — which quickly destroyed pyocyaneus and other bacilli, and when administered to animals exerted a protective power against both infection and intoxication. According to the view of these experimenters, antitoxin is nothing more than an enzyme similar in nature to the pyocyonase, whifch is introduced into the animal with the cultures used for immunization, and which, not being eliminated, accumu- lates in the blood, to which it subsequently confers the bacteria- and toxin-destroying functions. This theory is elaborated from most interesting and suggestive observations, but, unfortunately, will not stand the test of experimental investigation. In the first place, it would only apply to immunization by cultures of bacteria or filtered cultures of bacteria in which the cellular activities had generated the enzyme. Or, if we could persuade ourselves that the activity of the vegetable cells had been such that in ricin, abrin, venom, and eel's blood there might be some analogous enzymic product — a very doubtful matter — it could never be so modified as to explain the immunity which Besredka produced with arsenic and the anti-arsenine which de- veloped in the blood of his rabbits. Again, by reference to the calculations made above, it must be clear that the antitoxin cannot be such an 1 Zeitschrift fiir Hygiene, 1899. 9 130 PATHOGENIC BACTERIA. enzyme, for there was, as has been shown, 327 times as much energy in the horse's blood as had been introduced in the form of toxin or enzyme. C. Theory that the antitoxin is a product of cytic activity. — As the antitoxin cannot be a changed form of the toxin introduced into the immunized animal, and as it is not a power normal to the blood, its origin must be sought for elsewhere. All experiments directed toward finding any tissue storehouse from which the antitoxin is passed into the blood have failed. It is, however, present in the blood, in the tissue juices, and in the majority of the secretions, into which it enters from the blood. Probably the best explanation of the histogenesis of the antitoxin is contained in the theory of Ehrlich,1 which has become well known as the " Seiten-ketten Theorie" or "Lateral chain theory." According to Ehrlich' s view, the cells of the body are to be looked upon as of complex molecular structure, and as possessing numerous " lateral chains " which have varied combining powers. When toxin in increasing amounts is introduced into the body the cells are stimulated to regenerate that part of their structure with which it enters into combination, and to produce it in excess of the probable future needs. In this manner there is produced by the cells and poured into the blood a considerable quantity of toxin-combin- ing substance, which appears to us as antitoxin. The formation of this substance seems to be a new and per- sistent secretory property of the cells, according to the experiments of Thiele and Wolf,2 but experience has taught that it is by no means permanent, but begins to wane as soon as the stimulation of the toxin is withheld. Specific Action of the Antitoxins. — The protection afforded by diphtheria antitoxin seems to be exerted against diphtheria cultures and toxins only, so that the early experimenters easily fell into the error that each 1 Klinisches Jahrbuch, 1897. * Archiv fur Hygiene, 34, Heft I. IMMUNITY AND SUSCEPTIBILITY. 13 1 antitoxin was specific for its respective toxin and af- forded protection against no other. This, however, seems to be untrue, for while it is a fact that every anti- toxin is more potent in its action upon that toxin whose stimulation caused its formation, it is not infrequently the case that it will incidentally but to a less degree pro- tect against some others. Thus, quoting from Hueppe,1 "Antitoxins that are formed specifically in serum act in vitro upon poisons of a specifically different character in the same manner as upon poisons specifically similar, while the converse does not always obtain ; ' antivenin ' annuls the poisonous effect of abrin, but not of diph- theria toxin, tetanus toxin, or ricin ; antiabrin neutral- izes the toxic effect of snake venom, diphtheria toxin, and ricin, but not that of tetanus toxin ; tetanus anti- toxin is antagonistic to snake venom, but powerless against ricin and abrin ; rabies serum is potent against snake venom, but impotent against the diphtheria and tetanus toxins, and against ricin and abrin ; streptococ- cus serum is potent against snake venom, powerless against the others ; cholera serum is moderately effective against snake venom, but without effect against the others ; diphtheria antitoxin is powerless against snake venom, tetanus toxin, ricin, and abrin ; the antitoxic sera of swine erysipelas and typhoid are powerless against all these poisons." Hueppe, Gottstein, and Schleich advanced the theory that the specific action of the serums depended chiefly upon the fact that "those particular specific organs, tissues, cell territories, or cells which are involved in the disease in question are stimulated." Wassermann closely followed up this theory in the idea of toxin saturation which forms the basis of his theory of immunity yet to be described. Chemic Nature of Antitoxins. — The true nature of antitoxins is unknown. They are stable substances which are not destroyed by heat up to the point of coag- 1 Principles of Bacteriology, translation by E. O. Jordan, 1899, p. 385. 132 PA THOGENIC BACTERIA . illation of the serum containing them, are not injured by light, do not undergo any rapid spontaneous alteration when kept, sometimes not losing very much of their activity in a year or more. They are not destroyed by carbolic acid, trikresol, chloroform, formaldehyd, cam- phor, or other substances recommended for the preserva- tion of the serums. When the albumins are removed from the serums they lose most of the antitoxic strength, which seems to be thrown down with the albumins. Most of the virtue of the serum is precipitated with the globulins. All attempts to extract the antitoxin in a pure form have failed, the nearest approach to success having prob- ably resulted from the experiments of Brieger and Boer,1 who separated it as salts of heavy metals, especially zinc. Being in all probability of proteid nature, the anti- toxins are all destroyed in the alimentary canal. In the researches of Carriere2 the destruction was found to depend chiefly upon the pancreatic secretions, though in part upon the activity of intestinal juices, the contained bacteria, and the lining epithelium. Action of Antitoxin upon Bacteria. — Except when the immunization of the animal furnishing the protective serum is accomplished by the employment of living cultures or germ-containing cultures so as to be pos- sessed of the additional phenomenon of bacteriolysis, it cannot be said that the antitoxin has any direct action upon the bacteria. It is, indeed, said by Martini that the diphtheria bacillus, which usually grows well upon blood serum, will not grow upon antitoxic serum. This observation, however, lacks confirmation. Action of Antitoxin upon the Toxin. — This may be either a form of direct chemic action or an indirect action produced either by stimulation of the cells of the body or by bringing about combination between the toxin and certain substances in the blood or tissues. i. Theory of Chemic Action. — This is the original 1 Zeitschrift fiir Hygiene, 1896, Bd. xxi., p. 259. 2 Ann. de I , Inst. Pasteur, May 25, 1899, xiii. IMMUNITY AND SUSCEPTIBILITY. 133 theory of Behring, Ehrlich, Kanthack, and their fol- lowers, who, mixing toxin and antitoxin in vitro and in- jecting it into the body, see in the inertness of the mixture a chemic neutralization of the toxin, which is destroyed in the process. A few experiments made in the same line as those of Behring and Ehrlich seem very convincing. Thus if x toxin is mixed withjy antitoxin, x+y becomes an inert mixture. 10 x + 10 y and 100 x + 100 y are similarly inert. Of course, supposing that one of the other theories is correct, there is no real reason why definite proportions should not work out in the same way. There can be no doubt that the addition of antitoxin to toxin alters it chemically to a certain degree. Thus the experiments of Ehrlich with ricin are very instructive. If ricin is added to blood, the coagulation of which is prevented by citrate of sodium, the corpuscles agglu- tinate in masses and sediment. If, however, some anti- ricin — the serum of an animal immunized to ricin — be added to the blood before the ricin, the agglutination of the corpuscles does not take place. The reaction is a definite quantitative one. The action of antiricin upon ricin is viewed by Ehrlich as one akin to the formation of the double salts, one molecule of the antitoxin combining with a definite un- changeable quantity of toxin, the process being hastened by heat and retarded by cold and dilution. Kossel has shown, that the blood of the poisonous eel dissolves the blood corpuscles of animals into which it is injected. When eel serum is added to defibrinated blood from animals for which it is poisonous, the coloring matter is quickly dissolved from the corpuscles. If, however, some serum from an immunized rabbit is added to the blood beforehand, the eel's serum fails to dissolve out the hemoglobin. In this experiment the quantity of the serum of the animal immunized to the eel's blood must be directly proportional to the quantity of eel's blood used. 134 PATHOGENIC BACTERIA. Martin and Cherry found that the molecular structure of the toxin was changed by admixture with the anti- toxin. They found that under pressure toxin passes freely through a film of gelatin in a Chamberland filter, but that antitoxin does not. If a quantity of toxin equal to eight fatal doses per cubic centimeter is mixed with just enough antitoxin to neutralize it and the mixture allowed to stand for two hours, then filtered through gelatin, as much as 4 c.c, equalling thirty-two fatal doses, can be injected into guinea pigs without ill-effects. If the toxin had not been changed before being subjected to filtration, it should have passed through. An interesting paradox of immunity is made use of by Behring to aid in proving the directness of the chemic reaction. It is a fact that when rabbits are immunized to tetanus so as to be resistant to subcutaneous injections of the toxins they die of tetanus when the toxin is in- jected into the brain substance. Behring thinks this to be the result of the impenetrability of the blood-vessels to the antitoxin which does not dialyze. The toxin being injected into the brain substance acts directly upon the nervous system and produces death, although if it had to reach the same tissue by absorption through the lymphatics and circulation in the blood it must suffer neutralization in the latter. If in making the intracerebral injection the vessels are injured so that the blood flows out and comes in contact with the toxin, the animal will recover. Mixtures of toxin and antitoxin when injected into the brain provoke no tetanus. 2. Theory of Cytic Stimulation. — Buchner, Metschni- koff, Roux, Calmette, and others contend that that which we see in the so-called neutral mixture bears no definite relationship to what goes on in the body, and that the toxin is not neutralized by the antitoxin mixed with it, but destroyed by the cells of the body aroused to energetic action by the stimulating effect of the antitoxin. In support of this view Roux has shown that when so-called neutral mixture of tetanus toxin and antitoxin, incapable IMMUNITY AND SUSCEPTIBILITY. 135 of affecting mice, were injected into guinea pigs they died of tetanus ; therefore, the tetanus toxin was not destroyed. Roux and Vaillard have also found that similar " neutral mixtures," which failed to cause symp- toms in healthy animals, sometimes did so in diseased animals of the same species. In his investigations upon serpents' venom Calmette found the protective value of the serum was destroyed at temperatures causing coagulation (about 68° C), while the venom was able to endure temperatures of 700, 8o°, and even 900 C. When a mixture of venom and anti- veuene was proportioned so as to be harmless for rabbits, it was found that this mixture, if heated to the degree at which the antivenene was discharged, again became poisonous so that the heated mixture killed rabbits. Calmette used these observations to show that the action was not a chemic one, but it may be that certain im- portant considerations were neglected, for Martin and Cherry l found that this return to toxicity only took place when the venom and antivenene were mixed and imme- diately heated. If the mixture was allowed to remain for a short time in the incubating oven at the tempera- ture of 370 C, then heated to 68° C. and injected into rabbits, the animals did not die. These observers, there- fore, conclude that the reaction between the venom and antivenene is a chemic one which is stimulated by heat and requires time for completion. Concerning the effects of toxin-antitoxin mixtures upon the body, Mikanorow 2 reasoned that if toxin produced antitoxin by stimulating the cells to produce an anti- dote, neutral mixtures should not only be harmless for the animal, but, in case the toxin is changed by the antitoxin, should leave no immunity behind it. If, how- ever, the antitoxin stimulates the resisting power of the cells, the simultaneous introduction of toxin and anti- toxin should increase resistance. By frequent adminis- 1 British Med. Journal, 1898, II., p. 1 1 20. 1 Archiv fiir biol. IVissensckaft, 1897-1898, Bd. vi., p. 56. 136 PATHOGENIC BACTERIA. trations of antitoxin during a period of three months and ten days he was unable to find demonstrable antitoxin in the horse's blood. By a subsequent series of twelve injections, during a period of two months and five days, in which combined toxin and antitoxin were used, the antitoxin was formed and a value of 320 units per cubic centimeter was attained. These experiments furnish altogether too little data to be conclusive, but indicate that it is the toxin only that contributes to toxin forma- tion, and that in the mixtures of toxin and antitoxin, enough toxin remains unchanged to produce considerable antitoxin. It would seem natural that if there is a true chemic reaction between the toxin and antitoxin before injection into the animal body, in exactly neutral mixtures the antitoxin should be entirely destroyed by combining with the toxin, which in its turn should have no re- maining toxicity. Such a mixture should not confer passive immunity because it no longer contains antitoxin, nor yet stimulate active immunity because it contains no toxin. The fact is, however, that after the injection of a carefully made neutral mixture a high degree of im- munity remains for some time. I think the final proof that the reaction is not a purely chemic one is found in the calculations already given to illustrate the paradox of hypersensitivity. In this condi- tion we find animals with easily demonstrable amounts of antitoxin in the blood dying from ordinary toxin doses, no reaction between the toxin and antitoxin taking place. It might be urged that for the occurrence of this reaction some combining substance is necessary, but it must be remembered that in the test-tube experiments no such substance seemed to be necessary. 3. Theory of a Combining Ferment. — As in the coagu- lation of the blood and inflammatory exudates the union of the fibrin factors and ferments will not take place except in the presence of certain salts, so it may be true that the toxin is unable to combine with the antitoxin IMMUNITY AND SUSCEPTIBILITY. 137 unless some stimulating substance be present. Or the antitoxin itself may be the ferment which brings about a kind of union between toxin and cell — a harmless union — different from that which takes place when the toxin and cell come together. A/ode of Administration of Antitoxins. — Whether used experimentally or therapeutically the antitoxins must al- ways be injected subcutaneously or intravenously. Their administration by the mouth is followed by digestion in the intestine, as has been proved by the researches of Carri^re1 and Paltschikowski.2 It makes no difference where the injections are made, the flank usually being chosen as a part of the body where the skin is loose and little pressure brought to bear subsequently. There is no gain to be expected from administration in the neighbor- hood of the particular diseased or infected area, as the remedy does not act except through the blood. The suggestion of Roux that in the therapeutic ad- ministration of tetanus antitoxin the injection be made into the cerebral substance may have experimental foundation, but is a somewhat questionable procedure. Antimicrobic Phenomena. — The protective serums that develop in consequence of immunization to toxins of various kinds do not exert any destructive effect upon bacteria beyond that possessed by the normal serum of the animal used for the experiment. If, however, instead of using bacterial toxins, one produces the forced im- munization by the employment of cultures rich in viru- lent bacteria, the antitoxicity of the serum will vary in proportion to the amount of toxin contained in the culture, and a new phenomenon presents itself in the form of a great intensification of the bacteriolytic prop- erties of the serum. It does not seem to be necessary that virulence plays a very important role in the devel- opment of antimicrobic serums, for as Hueppe produced immunity with bacteria entirely devoid of virulence, it 1 Annates de 1 " Inst. Pasteur, May 25, 1899, xiii. * Both'n's Krankenhauszeitung, 1898, No. 42. 138 PATHOGENIC BACTERIA. seems possible to produce strongly germicidal serums with rich cultures of feeble virulence. In the treatment of the toxic diseases the antimicrobic serums are far less useful than the antitoxic serums, but in the invasive dis- eases their r61e is daily becoming more and more im- portant, and a place in the therapeusis of plague, suppu- rations, pneumococcus infections, typhoid fever, and cholera, is being assigned to them. The most striking illustration of the protective action and operation of an antimicrobic serum is probably observed in connection with the choleraic peritonitis of guinea pigs in what is known as "Pfeiffer's1 phenome- non." When virulent cholera organisms are injected into the peritoneal cavity of guinea pigs a peritonitis with some effusion develops and is fatal in about three days. The fluid within the peritoneum abounds with healthy, actively growing, motile cholera organisms. If a very small quantity (0.002 c.c.) of the serum of a guinea pig .immunized to cholera be injected into a guinea pig with well-developed choleraic peritonitis, it is found by the microscopic examination of occasional drops of fluid from the peritoneum that the energy of the micro-organism rapidly wanes, that they become less active, soon agglu- tinate in clumps, die, and undergo a granular degenera- tion, the animal recovering from the infection. The phe- nomenon was also observed when the serum of cholera convalescents was employed instead of that of immun- ized animals. The power of the serum to bring the changes about was destroyed by heating to 6o° C. Later the same phenomenon was observed in typhoid infection. Pfeiffer found that if some of the serum was injected into a healthy guinea pig, the fluid withdrawn from the body was capable of producing the same phenomenon in the test-tube. Metschnikoff also found that an identical reaction resulted when the serum was added to fresh peritoneal fluid outside the body, thus eliminating the 1 Zeiischrift fiir Hygiene, 1 894, Bd. 1 8 and 20. IMMUNITY AND SUSCEPTIBILITY. 139 probability that the endothelial or other cells had any- thing to do with it. He, however, concludes that the phenomenon depends upon the presence of substances derived from leukocytes, the only cells found in perito- neal fluid. Bordet modified Metschnikoff 's methods and used a suspension of cholera germs in bouillon. Two drops of this were added to one drop of anticholera serum and mixed. One drop of the mixture and one drop of normal guinea pig's serum were then brought in contact in a hanging-drop and examined microscopically. He found that in all cases in which the PfeifFer phenomenon would have taken place within the animal's body it took place in his artificial preparation. The explanation of Pfeiffer's phenomenon is not at all easy, but Kruse x has suggested a theory based upon the assumption that substances described as lysins are formed by the bacterial growth. These substances are of the nature of ferments, and attack and destroy the alexins, thus enabling the pathogenic bacteria to overcome the, bacteria-destroying mechanism of the tissues. Kruse sees in the process of immunization the formation of antilysin, by which the powers of the lysins are destroyed, and the alexins once more enabled to exert an active destructive influence upon the bacteria. That the germicidal substance is derived from the leukocytes is not improbable. Laschtschenko 2 found the action of heterogeneous serum very marked in liber- ating the alexins. This subject has, however, already been discussed {vide supra). Explanation of Acquired Immunity. — 1. The Ex- haustion Theory of Pasteur and Klebs. — This theory is of historic interest only. It was suggested in 1880 by Pasteur3 and Klebs,* who thought that when a bacterium developed in the body it used up some sub- 1 See Flugge's Die Mikroorganismen, 1892, vol. i., p. 414. 3 Munch, med. Wock., 1899, No. 15. 3 Compte-rendus de la Soc. Biol, de Paris, xci. 4 Archivfur Experimented Path. u. Phannakol.. xiii. 140 PATHOGENIC BACTERIA. stance essential to its life, then died out, leaving the soil unfit for future occupation. Such a view could only apply to immunity succeeding infection, not to passive immunity produced by the in- jection of antitoxins, or to active immunity produced by immunization to toxins. 2. The Retention Theory of Wernich l and Chau- VEAU,2 also of historic interest, supposes that by their growth in the body the bacteria leave behind them some metabolic product which interferes with their further and future development. As a theory this has much more to recommend it than its predecessor, being much more in accord with facts as we know them. Thus in our culture-media the bacteria die out long before the nutriment is exhausted, and will not grow when replanted even though the acidity or alka- linity be brought again to the most favorable point. The formation of antitoxin and antilysin is not in- compatible with this theory if one accepts that the phe- nomenal powers result from transformation of the toxin into antitoxin as held by Buchner. However, as immu- nity can be established to ricin, abrin, serpent's venom, arsenic, and numerous other non-micro-organismal poisons, it cannot be said to depend upon the presence in the blood of prejudicial metabolic products of bacteria, as was expressed in the original statement. Kruse's3 theory of lysins is a modification of Chau- veau's theory. Like the original retention theory, Kruse's theory applies solely to immunity to infection, not to immunity to poisons. III. Phagocytosis. — In acquired immunity the pha- gocytes are supposed by Metschnikoflf to acquire an appe- tite for the bacteria or to become educated to take up bac- teria of which they were originally fearful without injury. 1 Virchow's Archiv, 78. 8 Compte-rendus de la Soc. de Biol, de Paris, xc. and xci. 8 See Flugge's Die Mikroorganismen, vol. i. IMMUNITY AND SUSCEPTIBILITY. 141 The phenomena of phagocytosis as seen in acquired immunity are very interesting. Every one that has studied anthrax infection and examined the blood and juices of the dead animal, is familiar with the fact that the leukocytes never touch the bacteria. If, however, the animal be vaccinated to anthrax, the phagocytes are said to take up the virulent bacilli regularly. In the researches of Werigo1 upon the immunity of the rabbit to symptomatic anthrax it was found that in the normal animal the phagocytosis is irregular, many bacteria being taken up by the leukocytes, but enough allowed to remain to carry on the diseased process. In the vaccinated animal the phagocytosis is prompt and efficient. He thinks that in the process of immunization the leukocytes become so sensitive to the bacterial in- fluences that the least particle of their toxic products is sufficient to attract them to find and destroy the bacteria. In infection with vibrio Metschnikovi it has been shown by Metschnikoff that the phagocytes of unpro- tected animals do not take up the bacteria, but that in vaccinated animals the phagocytes are loaded with them. It is thus apparent to the reader that according to the degree of immunity the phagocytes are active. Whether or not they are active because the animal is immune and the bacteria harmless for them, or whether the animal is immune because they are hurtful to the bacteria, remains an important question as yet unsolved. IV. Germicidal Action of the Blood. — This phenomenon, like that of phagocytosis, is apt to develop in proportion to the degree of immunity attained. It does not always follow, however, for in immunization to the specific toxins of the micro-organismal affections the blood does not become germicidal to the respective germs. The so-called antimicrobic serums character- izing forced immunity have already been discussed in a 1 Archiv de Med. experimentalle et d' Anatotnie pathologique, t. x., p. 725. 142 PATHOGENIC BACTERIA. preceding paragraph, and their activities pointed out. Like the antitoxic serums, however, the antimicrobic serums seem to be formed only in high degrees of forced immunity. V. Antitoxins. — A few years ago it might have been unhesitatingly declared that acquired immunity de- pended upon the presence of antitoxins in the blood. We have of late accumulated much experimental evidence which, when sifted, seems to indicate that the antitoxins present in the blood of an animal may have very little to do with its immunity. These observations consist of numerous immunities in which no antitoxins can be demonstrated in the blood, as in the various vaccinations against anthrax, typhoid, cholera, symptomatic anthrax, etc., and some very different conditions in which, with a great deal of antitoxin in the blood, animals succumb to specific infection or intoxication. The most forcible illustration of this is seen in the hypersensitivity of horses furnishing diphtheria and tetanus antitoxin, a full discussion of which has been given. In the face of such illustrations we must conclude that antitoxin is one of the phenomena or reactions of im- munity, but that the condition does not depend upon it. VI. Ehrlich-Wasserman Lateral Chain Theory. — Ehrlich,1 in his careful studies of the toxins and anti- toxins, finds it convenient to look upon the protoplasm of the body cells as having a complex molecular structure, with lateral chains of different combining powers and affinities for certain specific toxins. He says: "Every antitoxin-producing toxin is specific in the sense that it produces its symptoms by chemical combination of its toxophoric atoms with some cell substance in the body of a susceptible animal." Upon this hypothesis, Wasserman 2 has concluded that when a toxin and its specific cell affinity are mixed in a test-tube the mixture should be neutral. He found that when tetanus toxin was mixed in vitro with an 1 Klinisches Jahrbuch, 1897. * Loc. cit. IMMUNITY AND SUSCEPTIBILITY. 143 emulsion of spinal cord, for which it was known to have a strong affinity (tetanus acts chiefly upon the spiual cord) that the mixture became harmless for mice and even for guinea pigs. It not only was true of mixtures made without the body, but was also operative when the two components of the mixture were injected in different parts of the body and at different times, the nervous tissue being in- jected always before the toxin up to periods as long as twenty-four hours. It was supposed that in these cases the protection af- forded by the nervous matter depended upon the satura- tion of the toxophoric atoms in the toxin by the cell sub- stances, and it was deduced that acquired immunity to poisons depends upon the ability of the cells to saturate the toxophoric atoms of the respective toxins by their combining lateral chains. As in these combinations the combining substance is used up, more of it is produced, the production being in excess of the requirements. It is supposed by the promulgators of these views that anti- toxin is the excess of such combining substance as has escaped into the blood. From this survey of the subject it must be evident that in investigating both natural and acquired immunity we have unravelled a great mass of most interesting and im- portant facts; and out of them have constructed a very fair knowledge of the reactions and phenomena of a process whose essence has so far escaped us. At present we only know natural immunity as a remarkable spontaneous power of resistance or endurance, and acquired immunity as a similar power developing in animals not naturally so endowed. The ability of the animal to endure the toxins, which is the key to the whole situation, depends upon some tolerance the 7iature of which has not yet been satisfactorily determined. CHAPTER V. METHODS OF OBSERVING BACTERIA. Bacteria may be examined either stained or un- stained. The former condition would always be prefer- able if the process of coloring the organisms did not injure them. Unfortunately, it is generally the case that the dry- ing, heating, boiling, macerating, and acidulating to which we expose the organisms in the process of staining alter their shape, make their outlines less distinct, break up their arrangement, and disturb them in a variety of other ways. Because of the possible errors of appearance re- sulting from these causes, as well as because it must be determined whether or not the individual is motile, in making a careful study of a bacterium it must always be examined in the living, unstained condition. The simplest method of making such an examination would be to take a drop of the liquid, place it upon a slide, put on a cover, and examine. While this method is simple, it cannot be recommended, for if the specimen should need to be kept for a time much evaporation takes place at the edges of the cover- glass, and in the course of an hour or two has changed it too much for further use. The immediate occurrence of evaporation at the edges also causes currents of liquid to flow to and fro beneath the cover, carrying the bacteria with them and making it almost impossible to determine whether the organisms under examination are motile or not. The best way to examine living micro-organisms is in what is called the hanging drop (Fig. 6). A hollow- 144 METHODS OF OBSERVING BACTERIA. 145 ground slide is used, and with the aid of a small camel' s- hair pencil a ring of vaselin is drawn on the slide about, not in, the concavity at its centre. A drop of the mate- rial to be examined is placed in the centre of a large clean cover-glass, and then placed upon the slide so that the drop hangs in, but does not touch, the concavity. The micro-organisms are now hermetically sealed in an air-chamber, and appear under almost the same condi- tions as in the culture. Such a specimen may be kept from day to day and examined, the bacteria continuing to live until the oxygen or nutriment is exhausted. By means of a special apparatus (Fig. 7), in which the mi- croscope is stood, the growing bacteria may be watched at any temperature, and very exact observations made. FlG. 6. — The " hanging drop " seen from above and in profile. The hanging drop should always be examined at the edge, as the centre is too thick. In such a specimen it is possible to determine the shape, size, grouping, division, sporulation, and motility of the organism under observation. Care should be exercised to use a rather small drop, especially for the detection of motility, as a large one vibrates very readily and masks the motility of the sluggish forms. When the bacteria to be observed are in solid or semi- solid culture, a small quantity of the culture should be mixed up in a drop of sterile bouillon or water and ex- amined. 10 146 PATHOGENIC BACTERIA. In the early days of study efforts were made to facili- tate the observation of bacteria by the use of carmin and hematoxylon. Both of these reagents tinge the proto- plasm of the organisms a little, but so unsatisfactorily that since Weigert introduced the anilin dyes for the purpose both of these tissue-stains have been rejected. The affinity between the bacteria and the anilin dyes is peculiar, and many times is so certain a reaction as to become an essential factor in the differentiation of species. For the study of bac- teria in the stained con- dition we now employ the anilin dyes only. The best anilin dyes made at the present time, and those which have become the stand- ard for all bacteriologi- cal work, are made in Germany by Dr. Griib- ler. In ordering the stain the name of this Fig. 7.— Apparatus for keeping objects under manufacturer should microscopic examination at constant tempera- 1 , . ~ , tures r always be specified. A whole volume could easily be devoted to scientific staining. Indeed, the tech- nical difficulties encountered are so great that no explana- tions can be too thorough to be useful. The special meth- ods essential for such bacteria as have peculiar staining re- actions will be given with the description of the organism. General methods only will be discussed in this chapter. METHODS OF OBSERVING BACTERIA. 147 Cover-glass Preparations for General Examination. — For bacteriological purposes thin covers (No. 1) are generally required, because thick glasses interfere with the focussing of the oil-immersion lenses. The cover- glasses can never be too clean. It is best to immerse them first in a strong mineral acid, then to wash them in water, then in alcohol, then in ether, and keep them in ether until they are to be used. Except that it some- times cracks, bends, or fuses the edges of the glasses, a better and more convenient method of cleaning them is to wipe them as clean as possible, seize them in fine-pointed forceps, pass them repeatedly through a small Bunsen flame until it becomes greenish yellow, then slowly ele- vate the glasses above the flame, so as to allow them to anneal. This maneuvre removes the organic matter by combustion. It is not expedient to use covers twice for bacteriological work, though if well cleaned they may subsequently be employed for ordinary microscopic ob- jects. The material to be examined must be spread in the thinnest possible layer upon the surface of a perfectly clean cover-glass and dried. It must next be fixed to the glass by immersion for twenty-four hours in equal parts of absolute alcohol and ether, or, as is much easier and more rapid, be passed three times through aflame, experience having shown that when drawn through the flame three times the desired effect seems best accom- plished. The Germans recommend that a Bunsen burner or a large alcohol lamp be used, that the arm holding the forceps containing the cover-glass describe a circle a foot in diameter, and that, as each revolution occupies a second of time, the glass be made to pass through the flame from apex to base three times. This is supposed to be exactly the requisite amount of heating. The rule is a good one for the inexperienced. Inequality in the size of various flames may make it desirable to have a more accurate rule. Novy l suggests 1 Laboratory Work in Bacteriology, 1 899. 1 48 PA THOGENIC BA CTERIA . that as soon as it is found that the glass is so hot that it can no longer be held against the finger it is sufficiently heated for fixing. After fixing, the material is ready for the stain. Every laboratory should be provided with several stock-solutions of the more ordinary dyes. These stock-solutions are saturated alcoholic solutions made by adding 25 grams of the dye to 100 c.cm. of alcohol. Of these it is well to have fuchsin, gentian violet, and methylene blue always made up. The stock-solutions will not stain, but are the standards for the manufacture of the working stains. For ordinary staining an aqueous solution made in a simple manner is employed. A small bottle is nearly filled with distilled water, and the stock-solution is added, drop by drop, until the color becomes just sufficiently in- tense to prevent the ready recognition of objects through it. For exact work it is probably best to give these stains a standard composition, using 5 c.c. of the satu- rated alcoholic solution to 95 c.c. of water. Such a watery solution possesses the power of readily penetrat- ing the dried protoplasm of the bacterium, taking the stain with it. Alcohol does not have this power. As in the process of staining the cover is apt to slip from the fingers and spill the stain, it is well to be pro- vided with cover-glass forceps (Fig. 8), which hold the Fig. 8. — Stewart's cover-glass forceps. glass in a firm grip and allow of all manipulations with- out danger to the fingers or clothes. The ordinary in- struments are entirely unfitted for the purpose, as capil- lary attraction draws the stain between the blades and makes certain the soiling of the fingers. Sufficient stain is allowed to run from a pipette upon the smeared side METHODS OF OBSERVING BACTERIA. 149 of the cover-glass to flood it, but not overflow, and is allowed to remain for a moment or two, after which it is thoroughly washed off with water. If the specimen is one prepared for temporary use, it can be examined at once, mounted in a drop of water, but under these con- ditions will not appear as advantageously as if dried and then mounted in Canada balsam. Sometimes the material to be examined is too solid to spread upon the glass conveniently. Under such circum- stances a drop of distilled water can be added and a minute portion of the material be mixed in it upon the glass. The entire process is, in brief : 1. Spread the material upon the cover ; 2. Dry — do not heat ; 3. Pass three times through the flame ; 4. Stain two to three minutes; 5. Wash thoroughly in water; 6. Dry; 7. Mount in Canada balsam. This simple process suffices to stain most bacteria. Ohlmacher1 deserves credit for his observation that when the " fixed " preparation is immersed for a moment or two in a 2-4 per cent, solution of formalin, the brill- iancy of the stain is considerably increased. Staining Bacteria in Sections of Tissue. — It not infrequently happens that the bacteria to be examined are scattered among or enclosed in the cells of tissues. Their demonstration is then a matter of some difficulty, and the method employed is one which must be modified according to the kind of organism to be stained. Very much, too, depends upon the preservation of the tissue to be studied. As bacteria disintegrate rapidly in dead tissue, the specimen for examination should be secured as fresh as possible, cut into small fragments, and im- mersed in absolute alcohol from six to twenty-four hours to kill the cells and bacteria. Afterward they are re- moved from the absolute alcohol and kept in 80-90 per cent., which does not shrink the tissue. Bichlorid of mercury may also be used, but absolute alcohol seems to answer every purpose. 1 Medical News, Feb. 16, 1896. 150 PATHOGENIC BACTERIA. The ordinary methods of imbedding suffice. The sim- pler of these are probably as follows: I. Celloidin. — From the hardening reagent (if other than absolute alcohol) — 12-24 hours in 95 per cent, alcohol, 6-12 " " absolute alcohol, 12-24 " " thin celloidin (consistence of oil), 6-12 " " thick celloidin (consistence of molasses). The solutions of celloidin are made in equal parts of absolute alcohol and ether. Place upon a block of dry wood, allow to evaporate until the block can be overturned without dislodging the specimen ; then place in 70-80 per cent, alcohol until ready to cut. The knife must be kept flooded with alco- hol while cutting. II. Paraffin — 12-24 hours in 95 per cent, alcohol, 6-12 " " absolute alcohol, 4 u " chloroform, benzole, or xylol, 4-8 " "a saturated solution of paraffin in one of the above reagents. Place in melted paraffin in an oven or paraffin water- bath, at 50°-6o° C, until the volatile reagent is all evap- orated, and the tissue impregnated with paraffin. Im- bed in freshly melted paraffin in any convenient mould. In cutting, the knife must be perfectly dry.- When it is necessary, subsequently, to remove the im- bedding material, dissolve the paraffin in chloroform, benzole, xylol, oil of turpentine, etc., which in turn can be removed with 95 per cent, alcohol. Celloidin is soluble in absolute alcohol, ether, and oil of cloves. It is very convenient to fasten the cut sections upon the slide — paraffin sections by Myer's glycerin-albumin mixture, oil of cloves and collodion or gum arabic solu- tion; celloidin sections by firmly pressing filter paper upon them and rubbing hard, then allowing a little vapor of ether to run upon them. ME THODS OF OBSER VING BA CTERIA. 1 5 1 III. Glycerin-Gelatin. — As the penetration of the tissue by celloidin is attended with lessened staining-qualities of the tubercle bacillus, it has been recommended by Kolle ' that the tissue be saturated with a mixture of glycerin, i part; gelatin, 2 parts; and water, 3 parts; cemented to a cork or block of wood, hardened in absolute alcohol and cut as usual for celloidin with a knife wet with alcohol. For staining bacteria (other than the tubercle and lepra bacilli) in tissue, two universal methods can be recommended: Loffler's Method. — The cut sections of tissue are stained for a few minutes in Loffler's alkaline methylene- blue solution (q. v.\ and then differentiated in a 1 per cent, solution of hydrochloric acid for a few seconds. The section is subsequently dehydrated in alcohol, cleared up in xylol, and mounted in balsam. Pfeiffer's Method. — The sections are stained for one- half hour in diluted Ziehl's carbol-fuchsin (q. v.), then transferred to absolute alcohol made feebly acid with acetic acid. The sections must be carefully watched, and as soon as the original, almost black-red color gives place to a red violet color the section is removed to xylol, where it is cleared preparatory to mounting in balsam. For ordinary work the following simple method is recommended: After the sections are cut the paraffin must be, and the celloidin had better be, removed. From water the sections are placed in the same watery stain used for cover-glasses and allowed to remain five to eight minutes. They are next washed in water for several minutes, then decolorized in 0.5-1 per cent, acetic-acid solution. The acid removes the stain from the tissues, and ultimately from the bacteria as well, so that one must watch carefully, and as soon as the color almost disappears from the sections remove them to absolute alcohol. At this point the process may be interrupted to allow the tissue-elements to be counter- 1 Fliigge's Mikroorganismen. 152 PATHOGENIC BACTERIA. stained with alum carmin or any stain not requiring acid for differentiation, after which the sections are dehydrated in absolute alcohol, cleared in xylol, and mounted in Canada balsam. As will be mentioned hereafter, certain of the bacteria which occur in tissue do not allow of the ready penetra- tion of the color. For such forms a more intense stain must be employed. One of the best of these stains, which can be employed by the given method both for cover-glasses and tissues, is Loftier' s alkaline methylene blue : Saturated alcoholic solution of methylene blue, 30 ; 1 : 10,000 aqueous solution of caustic potash, 100. Some bacteria, as the typhoid-fever bacillus, decolorize so rapidly as to contraindicate the use of acid for the dif- ferentiation, washing in water or alcohol being sufficient. Gram's Method of Staining Bacteria in Tissue. — Gram was the fortunate discoverer of a method of stain- ing bacteria in such a maimer as to saturate them with an insoluble color. It will be seen at a glance what a marked improvement this is on the method given above, for now the stained tissue can be washed thoroughly in either water or alcohol until its cells are colorless, with- out fear that the bacteria will be decolorized. Its prose- cution is as follows : The section is stained from five to ten minutes in a solution of a basic anilin dye, pure anilin (anilin oil) and water. This solution, first devised by Ehrlich, is known as Ehrlich's solution. The ordinary method of preparing it is to mix the following : Pure anilin, 4 ; Saturated alcoholic solution of gentian violet, 11 ; Water, 100. Instead of gentian violet, methyl violet, Victoria blue, or any pararosanilin dye will answer. The rosanilin dyes, such as fuchsin, methylen blue, vesuvin, etc., will not react with iodin. The mixture does not keep well — in METHODS OF OBSERVING BACTERIA. 153 fact, seldom longer than six to eight weeks, sometimes not more than two or three ; therefore it is best to prepare it in very small quantity by pouring about 1 c.cm. of pure anilin into a test-tube, rilling the tube about one-half with distilled water, shaking the mixture well, then fil- tering as much as is desired into a small dish. To this the saturated alcoholic solution of the dye is added until the surface becomes distinctly metallic in appearance. Friedlander recommends that the section remain from fifteen to thirty minutes in warm stain, and in many cases the prolonged process gives better results. From the stain the section is given a rather hasty wash- ing in water, and then immersed from two to three min- utes in Grain's solution (a dilute Lugol's solution) : Iodin crystals, 1 ; Potassium iodid, 2 ; Water, 300. While the specimen is in the Gram's solution it appears to turn a dark blackish-brown color. When removed from the solution it is carefully washed in 95 per cent, alcohol until no more color is given off and the tissue assumes a grayish color. If it is simply desired to find the bacteria, the section is dehydrated in absolute alcohol for a moment, cleared up in xylol, and mounted in Canada balsam. If it is necessary to study the relation between the bacteria and the tissue- elements, a nuclear stain, such as alum carmin or Bis- marck brown, may be subsequently used. Should a nuclear stain requiring acid for its differentiation be desirable, the process of staining must precede the Gram method altogether, so that the acid shall not act upon the stained bacteria. The success of Gram's method rests upon the fact that the combination of mycoprotein, anilin dye, and the iodids forms a compound insoluble in alcohol. The process described may be summed up as follows : 154 PATHOGENIC BACTERIA. Stain in Ehrlich's anilin-water gentian violet five to thirty minutes ; Wash momentarily in water ; Immerse two to three minutes in Gram's solution ; Wash in 95 per cent, alcohol until no more color comes out ; Dehydrate in absolute alcohol ; Clear up in xylol ; Mount in Canada balsam. This method stains a large variety of bacteria very beautifully, but, unfortunately, does not stain them all, and as some of those which do not stain are important it seems well to mention the — Spirillum of cholera and of chicken-cholera ; Bacillus mallei (of glanders) ; Bacillus of malignant edema ; Bacillus pneumoniae of Friedlander ; Micrococcus gonorrhoeae of Neisser ; Spirochaete Obermeieri of relapsing fever ; Bacillus of typhoid fever ; Bacillus of rabbit-septicemia ; Bacillus of symptomatic anthrax ; Bacillus of hog-cholera ; Bacillus coli communis ; Bacillus icteroides ; Bacillus of influenza ; Bacillus pestis bubonica ; Bacillus rhinoscleromatis ; Spirillum of Denecke ; Spirillum of Finkler and Prior ; Spirillum of Metschnikoff. No matter how carefully the method is performed an unsightly precipitate is sometimes deposited upon the tissue, obscuring both its cells and contained bacteria. Muir and Ritchie obviate this (1) by making the staining solution with 1 : 20 aqueous solution of carbolic acid instead of the saturated anilin solution, and (2) by clear- ing the tissue with oil of cloves after dehydration with alcohol. The oil of cloves, however, is itself a powerful METHODS OF OBSERVING BACTERIA. 155 decolorant and must be washed out in xylol before the section is mounted in Canada balsam. Gram's method is chiefly employed for staining bac- teria in tissues, but the fact that not all bacteria are colored by it is of considerable differential import- ance, as the difficulty of separating the species of bacteria is so great that use must be made of every aid. Gram's method for cover-glass preparations is employed for differentiating between different species of bacteria. A thin layer of the bacteria to be exam- ined is spread upon the cover-glass, dried, and fixed. The cover, held in the grip of a cover-glass forceps, is flooded with Ehrlich's solution. The solution is kept warm by holding the cover flooded with the stain over a small flame. The process of staining is continued from two to five minutes. If the heating causes the stain to evaporate, more of it must be dropped upon the glass, so that it does not dry up and incrust. The stain is poured off, and the cover placed in a small dish of Gram's solution and allowed to remain one-half to two minutes, the solution being agitated. It is pos- sible to apply the Gram solution in the same manner in which the stain is used, but as a relatively larger quantity should be employed, the dish seems preferable. The cover is next washed in 95 per cent, alcohol until the blue color is wholly or almost lost, after which it can be counter-stained with eosin, Bismarck brown, vesuvin, etc., washed, dried, and mounted in Canada balsam. Given briefly, the method is : Stain with Ehrlich's solution two to five minutes ; Gram's solution for one-half to two minutes ; Wash in 95 per cent, alcohol until decolorized ; Counter-stain if desired ; wash the counter-stain off with water ; Dry; Mount in Canada balsam. 156 PATHOGENIC BACTERIA. Method of Staining Spores. — It has already been remarked that the peculiar quality of the spore-capsules protects them from the influence of stains and disinfect- ants to a certain extent. On this account they are much more difficult to color than the adult bacteria. Several methods are recommended, the one generally employed being as follows : Spread the thinnest possible layer of material upon a cover-glass, dry, and fix. Have ready a watch-crystalful of Ehrlich's solution, preferably made of fuchsin, and drop the cover-glass, prepared side down, upon the surface, where it should float. Heat the stain until it begins to steam,, and allow the specimen to remain in the hot stain for five to fifteen minutes. The cover is now transferred to a 3 per cent, solution of hydro- chloric acid in absolute alcohol for about one minute. Abbott recommends that the cover-glass be submerged, prepared side up, in a dish of this solution and gently agitated for exactly one minute, then removed, washed in water, and counter-stained with an aqueous solution of methyl or methylene blue. In such a specimen the spores should appear red, the bacilli blue. I have not generally found that spores color so easily, and for many species the best method seems to be to place the prepared cover-glass in a test-tube half full of carbol-fuchsin : Fuchsin, 1 ; Alcohol, 10 ; 5 per cent, aqueous solution of phenol crystals, 100, and boil it for at least fifteen minutes, after which it is decolorized, either with 3 per cent, hydrochloric or 2-5 per cent, acetic acid, washed in water, and counter- stained blue. Muir and Ritchie1 recommend that cover-films be pre- pared and stained as for tubercle bacilli, then decolorized with a 1 per cent, sulphuric acid solution in water or 1 Manual of Bacteriology, London, 1897. METHODS OF OBSERVING BACTERIA. 157 methylated spirit, then washed in water and counter- stained with saturated aqueous methylen blue for half a minute, washed with water, dried, and mounted in Canada balsam. Moller ' finds it decidedly advantageous to prepare the films, before staining, by immersion in chloroform for two minutes, following this by immersion in 5 per cent, chromic acid solution for one-half to two minutes. Anjeszky2 recommends the following method of stain- ing spores which is said to always give good results even with anthrax bacilli : A cover-glass is thinly spread with the spore-containing fluid and dried. While it is drying some y2 per cent, hydrochloric acid is warmed in a por- celain dish over a Bunsen flame until it steams well and bubbles begin to form. When the solution is hot and the smear dry, the cover-glass is dropped upon the fluid, which is allowed to act upon the unfixed smear for three to four minutes. The cover is now removed, washed with water, dried, and fixed for the first time, then stained with Ziehl's carbol-fuchsin solution, which is warmed twice until fumes arise. The stain is allowed to cool, decolorized with 4-5 per cent, sulphuric acid solution, and counterstained for a minute or two with malachite green or methylen blue. The whole pro- cedure should not take longer than eight to ten minutes. Fiocca3 suggests the following rapid method : ''About 20 c.cm. of a 10 per cent solution of ammonium are poured into a watch-glass, and 10-20 drops of a saturated solution of gentian violet, fuchsin, methyl blue, or saf- ranin added. The solution is warmed until vapor begins to rise, then is ready for use. A very thinly-spread cover- glass, carefully dried and fixed, is immersed for three to five minutes (sometimes ten to twenty minutes), washed in water, washed momentarily in a 20 per cent, solution of nitric or sulphuric acid, washed again in water, then 1 Centralbl. f. Bakt. u. Parasitenk., Bd. x., p. 273. 2 Ibid., Feb. 27, 1898, xxiii., No. 8, p. 329. s Ibid., July I, 1893, x'v-> No. '• 158 PATHOGENIC BACTERIA. counter-stained with a watery solution of vesuvin, chrys- oidin, methyl blue, malachite green, or safranin, according to the color of the preceding stain. This whole process is said to take only from eight to ten minutes, and to give remarkably clear and beautiful pictures." Method of Staining Flagella. — This is much more difficult than the staining of either the bacteria or their spores, because each species seems to behave differently in its relation to the stain, so that the chemistry of the micro-organismal products must be taken into considera- tion. The best method introduced is that of Loffler.1 In it three solutions are used : A. A 20 per cent, solution of tannic acid, 10 ; Cold saturated aqueous solution of ferrous sulphate, 5 ; Alcoholic solution of fuchsin or methyl violet, 1. B. A 1 per cent, solution of caustic soda. C. An aqueous solution of sulphuric acid of such strength that 1 c.cm. will exactly neutralize an equal quan- tity of Solution B. Some of the bacteria to be stained are mixed upon a cover-glass with a drop of distilled water. This is the first dilution, but is too rich in bacteria to allow the flagella to show well, so that it is recommended to prepare a second dilution by placing a small drop of distilled water upon a cover and taking a small portion from the first cover to the second, spreading it over the entire sur- face. The material is allowed to dry, and is then fixed by passing it three times through the flame. When this is done with forceps there is some danger of the prepara- tion becoming too hot, so Loffler recommends that the glass be held in the fingers while the passes through the flame are made. The cover-glass is now held in forceps, and the mordant, Solution A, is dropped upon it until it is well covered. 1 Centralbl.f. Bakt. u. Parasitenk., 1890, Bd. vii., p. 625. METHODS OF OBSERVING BACTERIA. 159 The cover is warmed until it begins to steam, and the stain replaced as it evaporates. It must not be heated too strongly ; above all things, must not boil. This solution is allowed to act from one-half to one minute, is then washed in distilled water, then in absolute alcohol until all traces of the solution have been removed. The real stain — Loftier recommends an anilin-water fuchsin (Ehrlich's solution) — which should have a neutral reaction, is now dropped on so as to cover the specimen, and heated for a minute until vapor begins to arise; it is then washed off carefully, dried, and mounted in Canada balsam. To obtain this neutral reaction enough of the 1 per cent, sodium-hydrate solution is added to an amount of the anilin-water-fuchsin solution having a thickness of sev- eral centimeters to begin to change the transparent into an opaque solution. Such a specimen may or may not show the flagella. If not, before proceeding farther it is necessary to study the products of the bacterium in cul- ture-media. If by its growth the organism elaborates alkalies, Solution C, in proportion from 1 drop to 1 c.cm. in 16 c.cm. of the mordant A, must be added, and the process repeated again and again until the proper amount is determined. On the other hand, if the organism by its growth produces acid, Solution B must be added, drop by drop, until 1 in 16 cm. have been attained, and numerous experiments made to see when the flagella will appear. LofBer has fortunately worked out the amounts required for some of the species, and of the more important ones the following amounts of Solutions B and C must be added to 16 c.cm. of Solution A to attain the desired effect : Cholera spirillum, %-i drop of Solution C ; Typhoid fever, 1 c.cm. of Solution B ; Bacillus subtilis, 28-30 drops of Solution B ; Bacillus of malignant edema, 36-37 drops of Solution B. Part of the success of the staining depends upon 160 PATHOGENIC BACTERIA. having the bacteria thinly spread upon the glass, and as free from albuminous and gelatinous materials as possi- ble. The cover-glass must be cleaned most painstakingly : too much heating in fixing must be avoided. After using and washing off the mordant, the preparation should be dried before the application of the anilin-water-fuchsin solution. Pitfield1 has devised a simple and good method of staining flagella. A single solution at once mordant and stain is employed. It is made in two parts, which are filtered and mixed. A. Saturated aqueous solution of alum, 10 c.cm. ; Saturated alcoholic solution of gentian-violet, i c. cm. B. Tannic acid, i gr. ; Distilled water, 10 c.cm. The solutions should be made with cold water, and immediately after mixing the stain is ready for use. The cover-slip is carefully cleaned, the grease being burned off in a flame. After it has cooled the bacteria are spread upon it, well diluted with water. After drying thoroughly in the air, the stain is gradually poured on and by gentle heating brought almost to a boil ; the slip covered with the hot stain is laid aside for a minute, then washed in water and mounted. In such preparations I have always been able to see the flagella well, but usually find that while the flagella are very distinct, the bodies of the bacteria are scarcely visible. Bunge suggests a mordant consisting of a concentrated aqueous tannin solution and a i : 20 solution of liq. ferri sesquichloridi in water. The best mixture seems to be 3 parts of the tannin solution to 1 part of the diluted iron solution. To 10 c.cm. of this mixture 1 c.cm. of a concentrated aqueous fuchsin solution is added. It is not necessary to prepare this mordant fresh for each 1 Med. News, Sept. 7, 1895. METHODS OF OBSERVING BACTERIA. 161 staining, as it seems to improve with age. The use of acid and alkaline solutions added to the mordant is dis- pensed with. The bacteria are carefully fixed to the glass, stained with the mordant for five minutes, warming a little to- ward the end, washed, dried, and subsequently colored with carbol-fuchsin warmed a little. Van Ermengem ' has devised a somewhat complicated method of staining flagella which has given great satis- faction to many who have used it. Three solutions, which he describes as the Bain fixateitr, Bain sensibili- sateur, and Bain redncteur et reinforcateur, are to be used as follows : i. Bain Jixateur : 2 per cent, solution of osmic acid, i part ; 10-25 Per cent, solution of tannin, 2 parts. The cover-glasses, which are very thinly spread, dried, and fixed, are placed in this bath for one hour at the room temperature, warmed until steam arises, and then kept hot for five minutes. They are next washed with distilled water, next with absolute alcohol, then again with distilled water. All three washings must be very thorough. 2. Bain sensibilisateur : 5 per cent, solution of nitrate of silver in distilled water. The films are allowed to remain in this for a few seconds, and are then immediately transferred to the third bath. 3. Bain reducteur et reinforqateur : Gallic acid, 5 grams Tannin, 3 " Fused potassium acetate, 10 " Distilled water, 350 c.cm. 1 Travaux du Lab. d'hygiene et des bad. de Gand., t. i., p. 3. Abstracted in the Centralbl. f. Bakt. u. Parasitenk., 1894, Bd. xv., p. 969. 11 l62 PATHOGENIC BACTERIA. The preparations are kept in this solution for a few seconds, then returned to the nitrate of silver solution until they begin to turn black. They are then washed, dried, and mounted. Mervyn Gorden recommends that the method be modi- fied by allowing the preparations to remain in the second bath for two minutes, then transferring to the third bath for one and a half to two minutes, and then washing, drying, and mounting without returning to the second bath. Muir and Ritchie find it advantageous to use a fresh supply of the third solution for each specimen. Bacteria can best be measured by an eye-piece microm- eter. As these instruments vary somewhat in con- struction, the unit of measurement for each objective magnification or the method of manipulating the adjusta- ble instruments must be learned from dealers' catalogues. Photographing bacteria requires special apparatus and methods, which are fully described in text-books upon the subject. CHAPTER VI. STERILIZATION AND DISINFECTION. Before considering the cultivation of bacteria and the preparation of media for that purpose it is necessary to discuss methods of destroying bacteria whose acci- dental presence might ruin our experiments. The dust of the atmosphere, as has already been shown, is almost constant in its micro-organismal contamination, so that the spores of moulds and bacilli, together with yeasts and micrococci, constantly settle from it upon our glassware, enter our pots, kettles, funnels, etc., and would ruin every culture-medium with which we operate did we not take measures for their destruction. Micro-organisms may be killed by heat or by the action of chemicals, the processes being generically termed sterilization. The term sterilization is usually employed to denote the destruction of bacteria by heat, in contra- distinction to disinfection, which usually means the destruction of the bacteria by the use of chemical agents. A chemical agent causing the death of bacteria is called a germicide. An object which is entirely free from bacteria and their spores is described as sterile. Certain substances whose action is detrimental to the vitality of bacteria and prevents their growth amid other- wise suitable surroundings are termed antiseptics. The study of sterilization, disinfection, and antisepsis will naturally lead us through the following subdivisions : I. The sterilization and protection of instruments and glassware used in experimentation. II. The sterilization and protection of culture-media. III. The disinfection of the instruments, ligatures, etc. and the hands of the surgeon, and the use of antiseptics. IV. The disinfection of sick-chambers and their con- tents, as well as the dejecta and discharges of patients suffering from contagious and infectious diseases. 163 1 64 PA THOGENIC BA CTERIA . The Sterilization and Protection of Instruments and Glassware Used in Experimentation. — Steriliza- tion may be accomplished by either moist or dry heat. For the perfect sterilization of objects capable of with- standing it dry heat is preferable, because more certain in its action. If we knew just what organisms we had to deal with, we might be able in many cases to save time and gas, but while some simple non-spore-producing forms are killed at a temperature of 6o° C, others can withstand boiling for an hour ; it is therefore best to employ a temperature high enough to kill all with cer- tainty. Platinum wires used for inoculation are held in the direct flame until they become incandescent. In sterilizing such wires attention must be bestowed upon the glass handle, which should be held in the flame for at least half its length for a few moments when used for the first time each day. Carelessness in this respect may cause the loss of much time by contaminating cultures. Knives, scissors, and forceps may be exposed for a very brief time to the direct flame, but this affects the temper of the steel when continued too long. They may also be boiled, steamed, or carbolized. All glassware is sterilized by exposure to a sufficiently high temperature, 1500 C. or 3020 F., for one hour in the well-known hot-air closet (Fig. 9). A temperature of 1500 C. is sufficient to kill all known bacteria and their spores if continued for an hour. Rubber stoppers, corks, wooden apparatus, and other objects which are warped, cracked, charred, or melted by so high a temperature must be sterilized by moist heat in the steam apparatus for at least an hour before they can be pronounced sterile. It must always be borne in mind that after sterilization has been accomplished the same sources of contamination that originally existed are still present, and begin to operate as soon as the objects are removed from the sterilizing apparatus. To Schroder and Van Dusch belong the credit of STERILIZATION AND DISINFECTION 165 having first shown that when the mouths of flasks and tubes are closed with plugs of sterile cotton no germs can filter through. This observation has been of ines- timable value to every bacteriologist. Before sterilizing Fig. 9. — Hot-air sterilizer. flasks and tubes we plug them with ordinary raw cotton, and are sure that afterward their interiors will remain free from the access of germs until opened. Instruments may be sterilized wrapped in cotton, to be opened only when ready for use ; or instruments and rubber goods sterilized by steam can subsequently be wrapped in sterile cotton and kept for use. It is of the utmost importance to carefully protect every sterilized object, and to allow as little dust to collect upon it as possible, in order that the object of the sterilization be not de- feated. As the spores of moulds falling upon cotton sometimes grow and allow their mycelia to work their way through and drop into a culture-medium, Roux i66 PATHOGENIC BACTERIA. has introduced little paper caps with which the cotton stoppers are protected from the dust. These are easily made by curling a small square of paper into a ' ' cornu- copia, ' ' fastening by turning up the edge or putting in a pin. The paper is placed over the stopper before the sterilization, after which no contamination of the cotton can occur. Sterilization of Culture-media. — As almost all of the culture-media contain about 80 per cent, of water, which would be evaporated in the hot-air closet, so that the material would be destroyed, hot-air sterilization is not appropriate for them. Sterilization by streaming steam is the best and surest method. The prepared media are placed in flasks or tubes care- fully plugged with cotton and previously sterilized with dry heat, and then sterilized in what is known as Koch's steam appa- ratus (Fig. 10) or in Arnold's Fig. 10. — Koch's steam sterilizer. Fig. 11. — Arnold's steam sterilizer. steam sterilizer (Fig. 11), which is more convenient and more generally useful. The temperature of boiling water, ioo° C, does not STERILIZATION AND DISINFECTION. 167 kill many spores, so that the exposure of culture-media to streaming steam is of little use unless applied in a systematic manner — intermittent sterilization — based upon a knowledge of sporulation. In carrying out the intermittent sterilization the cul- ture-medium is exposed for fifteen minutes to the passage of streaming steam in the apparatus or to some tem- perature judged to be sufficiently high, so that the bac- teria contained in it are killed. As the spores remain uninjured, the medium is stood aside in a cool place for twenty-four hours, and the spores allowed to develop into perfect bacteria. When the twenty-four hours have passed the culture- medium is again placed in the apparatus and exposed to Fig. 12. — Autoclave for rapid sterilization FlG. 13. — Kny-Sprague steam sterilizer, by superheated steam under pressure. the same temperature, until these newly-developed bac- teria are also killed. Eventually, the process is repeated 1 68 PATHOGENIC BACTERIA. a third time, lest a few spores remain alive and capable of spoiling the material. When properly sterilized in this way, culture-media will remain free from contamina- tion until time and evaporation cause them to dry up. Fig. 14. — Pasteur-Chamberland filter arranged to filter under pressure. If hermetically sealed, a sterile medium will keep indef- initely. If it should be necessary to sterilize culture-media at once, not waiting the three days required by the inter- mittent method, it may be done by superheated steam in STERILIZATION AND DISINFECTION. 169 an autoclave (Fig. 12). Here under a pressure of two or three atmospheres sufficient heat is generated to destrpy the spores. For the sake of convenience many laboratory workers use the autoclave habitually for the sterilization of all media not injured by the high temperature. The steril- ization to be complete requires that the exposure shall be for fifteen minutes at no° C. (six pounds pressure). The media to be sterilized should be placed in the autoclave, the top firmly screwed up, but the escape-valve allowed to be open until steam is freely generated within and replaces the hot air. The valve is then closed, and the temperature maintained for fifteen minutes or longer if the media is in bulk in flasks. The cooling is allowed to take place gradually, for if the pressure is suddenly re- lieved the fluids boil rapidly and the cotton plugs may be forced into the tubes or flasks by the air pressure. The chief objection to the use of the autoclave is that the high temperature brings about certain chemical changes in the culture-media by which its reaction is altered. Liquids may also be sterilized by filtration — i. e. by passing them through unglazed porcelain or some other material whose interstices are sufficiently fine to resist the passage of the bacteria. This method is largely employed for the sterilization of the unstable toxins and anti- toxins, which are destroyed by heat. Various substances have been used for filtration, as stone, sand, powdered glass, etc., but experimentation has shown porcelain to be the only reliable filter against bacteria. Even this material, whose interstices are so small as to allow the liquid to pass through with great slowness, is only cer- tain in its action for a time, for after it has been used considerably the bacteria seem able to work their way through. To be certain of the efficacy of such a filter the fluid first passed through must be tested by cultiva- tion methods. The complicated Pasteur-Chamberland and the simple Kitasato and Reichel filters are shown in Figures 14, 15, and 16. 170 PATHOGENIC BACTERIA. After having been used a porcelain filter must be dis- infected, scrubbed, dried thoroughly, and then heated in a Bunsen burner or blowpipe flame until all the organic matter is consumed. In this firing process the filter first turns black as the organic matter chars, then becomes white as it is consumed. The greatest care must be exercised in cleansing, and especially must care be taken that the porcelain is dry before entering the fire, as it will certainly crack if moist. Before using a new filter it should be sterilized by dry Fig. 15. — Kitasato's filter: a, por- celain bougie ; b; attachment for suc- tion-pump; c, reservoir; d, sterile receiver. Fig. 16. — Reichel's bacteriologic filter of unglazed porcelain : A, sterile re- ceiver ; B, porcelain filter ; c, d, attach- ments for pump. heat, then connected with receivers and tubes, also care- fully sterilized. It should not be forgotten that the fil- tered material is still a good culture-medium and must be handled with the greatest care. While the filtration of water, peptone solution, and bouillon is comparatively easy, gelatin and blood-serum pass through with great difficulty, and speedily gum the filter, so that it is useless until fired. A convenient apparatus used by the author for the rapid STERILIZATION AND DISINFECTION. 171 filtration of large quantities is shown in the accompany- ing illustration (Fig. 17). Fig. 17. — Apparatus for the rapid filtration of toxins, etc. The Disinfection of Instruments, Ligatures, Sutures, the Hands, etc. — There are certain objects used by the surgeon which cannot well be rendered incandescent, exposed to dry heat at 1500 C, steamed, or intermittently heated without injury. For these objects disinfection must be practised. Ever since Sir Joseph Lister intro- duced antisepsis, or disinfection, into surgery there has been a struggle for the supremacy of this or that highly- recommended germicidal substance, with two results — viz. that a great number of feeble germicides have been discovered, and that belief in the efficacy of all germi- cides has been somewhat shaken; hence the origin of the successful aseptic surgery of the present day, which strives to prevent the entrance of germs into, rather than their destruction after admission to, the wound. For a complete discussion of the subject of antiseptics in relation to surgery the reader must be referred to the large text-books of surgery, where much space is thus occupied. A short list of useful germicides of which the respective values are given, and a brief discussion of some of the more important measures, can alone find space in these pages. The antiseptic value of some of the principal substances used may be expressed as fol- lows, the figures indicating the strength of the solution necessary to prevent the development of bacteria : 172 PATHOGENIC BACTERIA. Pyoktanin (methyl violet) . i : 2,000,000 — 1 : 5000. Formalin 1 : 25,000 — 1 : 5000. Bichlorid of mercury . . . 1 : 14,300. Hydrogen peroxid 1 : 20,000. Nitrate of silver 1:12,500. Creolin 1 : 5000 to 1 : 200. Chromic acid 1 : 5000. Thymol 1 : 1340. Salicylic acid 1 : 1000. Potassium bichromate . . . 1 : 909. Trikresol 1 : 1000 — 1 : 500. Zinc chlorid 1 : 526. Potassium permanganate . 1 : 285. Nitrate of lead 1 : 277. Izal 1 : 200. Boracic acid 1 : 143. Chloral hydrate 1 : 107. Ferrous sulphate 1 : 90 — 1 : 200. Calcium chlorid 1 : 25. Creosote 1 : 20. Carbolic acid 1 : 20 : : 1 : 50. Alcohol 1 : 10. Ether. Pure ether will not kill anthrax spores immersed in it for eight days. The value of antiseptics, like that of disinfectants, is always relative, the destructive as well as the inhibitory power of the solution varying with the micro-organism upon which it acts. The following table, from Boer, will illustrate this : Methyl Violet {Pyoktanin). Restrains. Kills. Anthrax bacillus 1 : 70,000 1 : 5000 Diphtheria 1 : 10,000 1 : 2000 Glanders 1 : 2500 1 : 150 Typhoid 1 : 2500 1 : 150 Cholera spirillum 1 : 30,000 1 : 1000 Large numbers of both strongly and feebly antiseptic substances have purposely been omitted from the above lists, compiled from Sternberg and Micquel, as either in- appropriate for ordinary use or as having been replaced by better agents. STERILIZATION AND DISINFECTION. 173 The newest, and one of the best germicides for all pur- poses is formaldehyde. Its use as a vapor for the sterili- zation of infected rooms was first suggested by Trillat in 1895, but it did not make much stir in the medical world until a year or more had passed and a 40 per cent, solu- tion of the gas, under the name of "Formalin," had been placed upon the market The original method con- sisted of the evolution of the gas from methyl alcohol by volatilizing it in a steam apparatus, and passing the vapor over a heated metal plate. At present the original auto- clave has been replaced by the apparatus shown in Fig. 19, in which a solution of formochloral is volatilized by heating under a pressure of three atmospheres. The gas is very penetrating, easily diffusing itself, and is said to have enormous bactericidal powers. In experi- ments conducted by Prof. Robinson, of Bowdoin Col- lege, the gas penetrated mat- tresses and killed cultures in tubes wrapped up in them. There seems to be little doubt of the ability of the Fig. 18.— The Trillat autoclave. Fig. 19. — Sanitary formaldehyde re- generator. formaldehyde gas to disinfect, but there are few apparatus upon the market at present that seem capable of discharg- ing a sufficient volume of the gas with sufficient rapidity to do the work. The physician, therefore, who desires to disinfect with confidence should choose an apparatus 174 PATHOGENIC BACTERIA. that has been shown by competent experiments to fill the requirements. It is not always necessary to use an expensive appa- ratus in order to disinfect with formaldehyde; one can, under forced conditions, hang up in the room a number of sheets saturated with the 40 per cent, formaldehyde solution. Of course, the number of sheets must be in- creased for a large room. Care must also be exercised that the hands do not become wet with the concentrated formaldehyde solution. The "formalin," or 40 per cent, solution of the gas, when fresh and tightly corked, is fatal to most bacteria in dilutions of from 1 : 5000 to 1 : 25,000. It can be employed with great advantage to spray the walls and floors of rooms. It cannot be employed upon the skin or mucous membranes, because of its marked irritating effect. The disinfection of the skin, both the hands of the surgeon and the part about to be incised, is a matter of importance. It is almost impossible to secure absolute sterility of the hands, so deeply do the skin-cocci pene- trate between the layers of the scarf-skin. The method at present generally employed, and recommended by Welch and Hunter Robb, is as follows : The nails must be trimmed short and perfectly cleansed. The hands are washed thoroughly for ten minutes in water of as high a temperature as can comfortably be borne, soap and a brush previously sterilized being freely used, and afterward the excess of soap washed off in clean hot water. The hands are then immersed for from one to two minutes in a warm saturated solution of permanganate of potassium, then in a warm saturated solution of oxalic acid, until complete decolorization of the permanganate occurs, after which they are washed free from the acid in clean warm water or salt-solution. Finally, they are soaked for two minutes in a 1 : 500 solution of bichlorid of mercury, after which they are ready for use. Lockwood,1 of St. Bartholomew's Hospital, recommends 1 Brit. Med. Jour., July II, 1896. STERILIZATION AND DISINFECTION. 175 after the use of the scissors and penknife, scrubbing the hands and arms for three minutes in hot water and soap to remove all grease and dirt. The scrubbing brush ought to be steamed or boiled before use, and kept in 1 : 1000 biniodid of mercury solution. When the soap- suds have been thoroughly washed away with plenty of clean water, the hands and arms are thoroughly washed and soaked for not less than two minutes in a solution of biniodid of mercury in methylated spirit; 1 part of the biniodid in 500 of the spirit. Hands that cannot bear 1 : 1000 bichlorid and 5 per cent, carbolic solutions, bear frequent treatment with the biniodid. After the spirit and biniodid have been used for not less than two min- utes, the solution is washed off in 1 : 2000 or 1 : 4000 biniodid of mercury solution. Catgut cannot be sterilized by boiling without deterio- ration. The present method of preparing it is to dry it in a hot-air chamber and then boil it in cumol, which is afterward evaporated and the skeins preserved in sterile test-tubes or special receptacles plugged with sterile cot- ton. Cumol was first introduced for this purpose by Kronig, as its boiling-point is i68°-i78° C, and thus sufficiently high to kill spores. The use of cumol for the sterilization of catgut has been carefully investigated by Clarke and Miller.1 Ligatures of silk and silkworm-gut are boiled in water immediately before using, or are steamed with the dressings, or placed in test-tubes plugged with cotton and steamed in the steam sterilizer. At present, in most hospitals, instruments are boiled before using in a 1-2 per cent, soda solution. Plain water has the disadvantage of rusting the instruments, and during the operation they are either kept in the boiled water or in carbolic solution. Andrews makes special mention of the fact that the instruments must be com- pletely immersed to prevent rusting. During the operation the wound is frequently washed 1 Bull, of the Johns Hopkins Hospital, Feb. and March, 1896. 176 PATHOGENIC BACTERIA. with normal salt solution, applied by sterile marine or gauze sponges. The water and the salt solution used for surgical pur- poses are to be sterilized before using, either by steaming for a prolonged period, or by the intermittent method. Large hospitals are generally furnished with special appa- ratus for supplying sterile distilled water in large quantity. To La Place belongs the credit of observing that the efficacy of bichlorid of mercury is greatly increased by the addition of a small amount of acid, by which the penetration is increased and the formation of insoluble albuminates lessened. The knowledge that the action of germicides is chem- ical, and that the destruction of the bacteria is due to the combination of the germicide with the mycoprotein, is apt to lessen our confidence in the permanence of their action. Geppert has shown of bichlorid of mercury that in the reaction between it and anthrax spores the vitality of the latter seems lost, but that the precipitation of the bichlorid from this combination by the action of ammo- nium sulphid restores the vitality of the spore. Again, the fact that some of the antiseptics, as nitrate of silver and bichlorid of mercury, are at once precipi- tated by albumins, and thus lose their germicidal and antiseptic powers, limits the scope of their employment. I think it may be safely said that carbolic acid is the most reliable and most generally useful of all the germi- cides and antiseptics. The Disinfection of Sick-chambers, Dejecta, etc. — What has just been remarked concerning the unreliability of many of the germicidal substances is eminently a propos of the disinfection of dejecta. It is useless to mix bichlorid of mercury with typhoid stools or tubercu- lar sputum rich in albumin, and imagine these substances rendered harmless in consequence. It should not be for- gotten that the sick patient is less the means of convey- ing the contagium than the objects with which he is in contact, which can be carried to other rooms or houses STERILIZATION AND DISINFECTION. 177 during or after the progress of the disease. A careful consideration of the condition of the sick-room will lead us to a clear understanding of its bacteriological condition. The Air of the Sick-room. — It is impossible to sterilize or disinfect the atmosphere of a room during its occu- pancy by the patient. The disinfecting capacity of the solutions given above must make obvious the concentra- tion of their useful solutions, and show the foolishness of placing beneath the bed or in the corners of a room small receptacles filled with carbolic acid or chlorinated lime. These can serve no purpose for good, and may be potent for harm by obscuring the disagreeable odors emanating from materials which should be removed from the room by the still more disagreeable odors of the dis- infectants. The practice of such a custom is only com- parable to the old faith in the virtue of asafetida tied in a corner of the handkerchief as a preventive of cholera and smallpox. During the period of illness a chamber in which the patient is confined should be freely ventilated, so that its atmosphere is constantly changing and replacing the closeness so universally an accompaniment of fever by fresh, pure air — a comfort to the patient and a protection to the doctors and nurses. After recovery or death one should rely less upon fu- migation than upon the disinfection of the walls and floor, the similar disinfection of the wooden part of the furniture, and the sterilization of all else. The fumes of sulphur may do some good — when combined with steam, much good — but are greatly overestimated, and the sulphurous vapors are rapidly giving way to the more penetrating and germicidal formaldehyde vapor. To apply this, the room to be sterilized is carefully closed, the carefully selected apparatus set in action, and the discharged vapor allowed to act undisturbed for some hours, after which the windows and doors are all thrown open to fresh air and sunlight. 12 i78 PATHOGENIC BACTERIA. Instead of the gas, a 40 per cent, solution, which can be sprayed upon the ceiling, walls, floor, and contents of the room from a large atomizer, is sometimes used. Ex- perience has not shown, however, that this possesses any distinct advantages. So far as is at present known, the disinfection by form- aldehyde is complete and leaves nothing to be desired. One important point must always be considered — that is, the apparatus — which should be capable of discharging enough of the gas in a short time. The Dejecta.— A little thought will direct attention to those of the dejections which are dangerous to the com- munity and promote efforts for their complete steriliza- tion. In cases of diphtheria the vomit, expectorations, and nasal discharges are most important. They should be received in old rags or in Japanese paper napkins — not handkerchiefs or towels — and should be burned. The sputum of tuberculous patients should either be collected in a glazed earthen vessel which can be subjected to boil- ing and disinfection, or, as is an excellent plan, should be received in Japanese rice-paper napkins, which can at once be burned. These napkins are not quite as good as the small pasteboard boxes (Fig. 20) recommended by Fig. 20. — Pasteboard cup for receiving infectious sputum. When used the pasteboard can be removed from the iron frame and burned. some city boards of health, because, being highly absorb- ent, the sputum is apt to soak through and soil the fin- gers, etc. Tuberculous patients should be provided with rice-paper instead of handkerchiefs, and should have their towels, knives, forks, spoons, plates, etc. kept strictly STERILIZATION AND DISINFECTION. 1 79 apart from the others of the household (though the pa- tients, whose mental acuity makes their sensibilities very pronounced, need never be told of their isolation), and frequently boiled for considerable lengths of time. The excreta from typhoid-fever and cholera cases re- quire particular attention. These, and indeed all alvine matter possibly the source of infection or contagion, should be received in glazed earthen vessels and imme- diately intimately mixed with a 5 per cent, solution of chlorinated lime (containing 25 per cent, of chlorin) if semi-solid, or with the powder if liquid, and allowed to stand for an hour before being thrown into the drain. The Clothing, etc. — All bed-clothing which has been used in the sick-room, all towels, napkins, handkerchiefs, night-robes, underclothes, etc. which have been used by the sick, and all towels, napkins, handkerchiefs, caps, aprons, and outside dresses worn by the nurse, should be regarded as infected and subjected to sterilization. The only satisfactory method of doing this is by prolonged subjection to steam in a special apparatus ; but, as this is only possible in hospitals, the next best thing is boiling for some time in the ordinary wash-boiler. When possi- ble, the clothes should be soaked in 1 : 2000 bichlorid solu- tion before or after boiling, and in drying should hang in the sun and wind. Woollen underwear can be treated exactly as if of cotton. The woollen outer clothing of the patient, if infected, requires special treatment. For- tunately, the infection of the outer woollen garments is unusual. The only reliable method for their purification is prolonged exposure to hot air at no°C. In private practice it becomes a grave question what shall be done with these articles. Prolonged exposure to fresh air and sunlight will aid in rendering them harmless ; when it is certain that articles of wool are infected, they may be sent to the city hospital or to certain of the moth-destroy- ing and fumigating establishments which can be found in all large cities, and be baked. 180 PATHOGENIC BACTERIA. The Furniture, etc. — The wholesale destruction of fur- niture practised in earlier times has at present become unnecessary. The doctor, if he properly performs his functions, will save much trouble and money for his patient by ordering the immediate isolation of his charge in an uncarpeted, scantily- and cheaply-furnished room the moment an infectious disease is suspected, before much infection can have occurred. However, if before his removal the patient has occupied another bed, its clothing should be promptly handled in the above- described manner. After the illness the walls of the rooms, including the ceiling should be sprayed with formalin, or, where it can- not be obtained, may be rubbed with fresh bread, which Loffler has shown to be efficacious, though scarcely prac- ticable, in collecting the bacteria, or, if possible, should be whitewashed. If the walls are hung with paper, they may be dampened with i : iooo bichlorid-of-mercury so- lution before new paper is hung. Aronson1 says: "For the disinfection of living-rooms there is no method that can compare in the remotest degree, as regards certainty and simplicity, with that by means of formaldehyde gas. For example, any one who has seen the process of cleansing walls by rubbing them down with bread, as carried out by the disinfecting corps, will agree with me that, however effective it may be from a theoretical point of view, it is absolutely inefficient in practice. The possibility of disinfecting rooms and all their contents with certainty, by means of a simple, cheap, harmless, and easily managed method must be hailed as a great advance." The floor should be scoured with 5 per cent, carbolic- acid solution or 1 : 1000 bichlorid of mercury, and all the wooden articles wiped off two or three times with the same solution employed for the floor. In this scouring no soap can be used, as it destroys the virtue of the germicide. If a straw mattress was used, it should be 1Ve rein fiir 0(fentliche Gesundheitspflege, Berlin, April 26, 1897. STERILIZATION AND DISINFECTION. 181 burned and the cover boiled. If a hair mattress was used, it can be steamed or baked by the manufacturers, who generally have ovens for the purpose. Curtains, shades, etc., should receive proper attention; but, of course, the greater the precautions exercised in the beginning, the fewer the articles which will need attention in the end. They should be removed before the case has developed. Strehl has succeeded in demonstrating that when 10 per cent, formalin solution is sponged upon artificially infected curtains, etc., the bacteria are killed by the action of the disinfectant. This knowledge will be an important ad- junct to our means for disinfecting the furniture of the sick-chamber. The patient, whether he lives or dies, may also be a means of spreading the disease unless specially cared for. After convalescence the body should be bathed with a weak bichlorid-of-mercury solution or with a 2 per cent, carbolic-acid solution, or with 25-50 per cent, alco- hol, before the patient is allowed to mingle with society, and the hair should either be cut off or carefully washed with the above solution. In desquamative diseases it seems best to have the entire body anointed with cos- molin once daily, the unguent being well rubbed in, in order to prevent the particles of epidermis being distrib- uted through the atmosphere. Carbolated cosmolin may be better than the plain, not because of the very slight antiseptic value it possesses, but because it helps to allay the itching which may be part of the desquamative process. After the patient is about the room again, common sense will prevent the admission of strangers until all the disinfective measures have been adopted, and after this, touching, and especially kissing him, should be omitted for some time. The dead who die of infectious diseases should be washed in a strong disinfectant solution, and should be given a private funeral in which the body, if exposed, 1 82 PATHOGENIC BACTERIA. should not be touched. In my judgment, the body is best disposed of by cremation. It seems, however, to be an error to suppose that a dead body can remain for an indefinite period a source of infection. Esmarch ! has made a series of laboratory ex- periments to determine what the fate of pathogenic bac- teria in the dead body really is. From his results it seems clear that in septicemia, cholera, anthrax, malignant edema, tuberculosis, tetanus, and typhoid the pathogenic bacteria all die sooner or later, generally more rapidly in conditions of decomposition than in good preservation of the tissues. Lack of oxygen may be a cause of their disappearance. 1 Zeitschrift fur Hygiene, 1893. CHAPTER VII. CULTIVATION OF BACTERIA; CULTURE-MEDIA. Accuracy of observation requires that the bacteria be separated from their natural surroundings and artificially grown upon certain prepared media of standard compo- sition, in such a manner that only organisms of the same kind are together. One after another various organic and inorganic mix- tures have been suggested, but, although almost any compound containing organic matter, even in small amounts, will suffice for the nourishment of bacteria, a certain few have met with particular favor as being most valuable. Rather than give a complete review of the work which has already been done, in the following pages the most useful preparations only will be considered. Our knowledge of the biology of the bacteria has shown that they grow best in a mixture containing at least 80 per cent, of water, of a neutral or feebly alka- line reaction, and of a composition which, for the patho- genic forms at least, should approximate the juices of the animal body. It might be added that transparency is a very desirable quality, and that the most gener- ally useful culture-media are those that can be readily liquefied and solidified. All accurate bacteriologic culture experiments re- quire that an exact knowledge of the reaction of the media used shall be at hand. This matter is so important that I give the following excerpt from the Report of the Committee of Bacteriologists of the American Public Health Association : ' 1 Journal of the American Public Health Association, Jan., 1898, p. 72. 183 184 PA THOGENIC BACTERIA. " The first thing to obtain is a standard ' indicator' which will give uniform results. These requirements are best fulfilled by phenolphthalein. This indicator was first suggested by Schultze in combination with the titration method for obtaining the desired reaction for culture-media,1 but its general adoption seems to have been retarded by Dahmen,2 who claimed that it was not feasible owing to the complications that might arise from the presence of carbonates and ammonium salts in the solution to be tested. These objections to the use of phenolphthalein do exist, but may be readily overcome. The amount of free and combined ammonia present in the culture-media at the time the reaction is determined has been found not to exceed 0.003 Per cent., which is less than one-tenth the amount which interferes with the accuracy of the indicator, while the production of carbon dioxid is obviated to a very great degree by neutralizing with sodium hydroxid instead of sodium carbonate, and any of this gas which may be absorbed from the atmosphere is practically all driven off by the heat during the preparation of the media." The great advantage of the use of phenolphthalien over other indicators lies in the fact that it takes into account the reaction of weak organic acids and of organic compounds which have an amphoteric reaction, but in which the acid character predominates. Turmeric possesses the same properties, but the change of color from yellow to brown is less satisfactory than the development of purple-red color. Furthermore, turmeric paper changes rather slowly, while with phenolphthalein the color appears almost instantly. Another advantage to be gained from the use of this latter indicator is its behavior toward the phosphates. Petri and Moussen 3 and Timpe 4 have shown that the amphoteric reaction of media is associated with the presence of phosphates, and that there are present in peptone and gelatin proteid bodies which possess both an acid and a basic nature, but in which the acid character predominates. These observers agree that to deter- mine accurately the reaction of such amphoteric compounds phenolphthalein (or turmeric paper) should be used as an indicator. It is known that at the neutral point of phenolphthalein any free phosphoric acid present enters into combination, and the monobasic and tribasic salts of this acid are changed to the dibasic form (Na2HPOi). Now disodium hydrogen phosphate reacts alka- 1 Centralbl. f. Bakt. u. Parasitenk. , 1891, Bd. x., p. 53. 2 Ibid., 1892, Bd. xii., p. 620. 3 Arbeiten aus dem k. Gesundheitsamte, 1893, Bd. viii., p. 311. * Centralbl. f. Bakt. u. Parasitenk., 1893, Bd. xiv., p. 845; 1 894, Bd. xv., PP- 394-644; 1893, Bd. xvii., p. 416. CUL TT VA TION OF BA CTERIA . 1 85 line to litmus, lacmoid, rosolic acid, and methyl orange, but neu- tral to phenolphthalein and turmeric. " Studies made at the Lawrence Experiment Station show that this acid salt may be added to culture-media in amounts greatly exceeding those naturally present in the media without producing any apparent influence upon bacterial development. From these facts it is clear that the use of any of the above-mentioned indi- cators, other than phenolphthalien and turmeric, in the presence of this dibasic phosphate, prevents the addition of a sufficient amount of free alkali to effect neutralization, and as the amount of phosphates in media varies considerably, the reaction passes be>-ond accurate control when litmus and other substances of its class are used as indicators." " The question of the proper reaction of media for the cultiva- tion of bacteria and the method of obtaining this reaction have been discussed in a valuable paper by Mr. George W. Tuller, pub- lished in the Journal of the American Public Health Association, Oct., 1895, vol. xx., p. 321." ' ' Method of determining the degree of reaction of culture- media : For this most important part in the preparation of culture- media, burettes graduated into one-tenth c.cm. and three solutions are required — 1. A 0.5 per cent, solution of commercial phenolphthalein 50 per cent, alcohol. 2. A — solution of sodium hydroxid. 20 x. A — solution of h\-dric chlorid. 0 20 J Solutions 2 and 3 must be accurately made and must correspond with the normal solutions soon to be referred to. " Solutions of sodium hydroxid are prone to deterioration from the absorption of carbon dioxid, and the consequent formation of sodium carbonate. To prevent as much as possible this change, it is well to place in the bottle containing the stock solution a small amount of calcium hydroxid, while the air entering the burettes or the supply bottles should be made to pass through a U-tube containing caustic soda, to extract from it the carbon dioxid." The medium to be tested, all ingredients being dissolved, is brought to the prescribed volume by the addition of distilled water to replace that lost by boiling, and after being thoroughly stirred, 5 c.cm. are transferred to a 6-inch porcelain evaporating- dish. To this 45 c.cm. of distilled water are added and the 50 c.cm. of fluid are boiled for three minutes over a flame. One cubic centimeter of the solution of phenolphthalein (No. 1) is then 1 86 PA THOGENIC BA CTERIA . added, and by titration with the required reagent (No. 2 or No. 3) the reaction is determined. In the majority of instances the re- action will be found to be acid, so that the — sodium hydroxid is the reagent most frequently required. This determination should be made not less than three times and the average of the results obtained taken as the degree of the reaction. One of the most difficult things to determine in this process is exactly when the neutral point is reached as shown by the color developed, and to be able in every instance to obtain the same shade of color. To aid in this regard, it may be remarked that in bright daylight the first change that can be seen on the addition of alkali is a very faint darkening of the fluid which, on the addi- tion of more alkali, becomes a more evident color and develops into what might be described as an Italian pink. A still further addition of alkali suddenly develops a clear and bright pink color, and this is the reaction always to be obtained. All titrations should be made quickly and in the hot solutions to avoid compli- cations arising from the presence of carbon dioxid." The next step in the process is to add to the bulk of the medium the calculated amount of the reagent, either alkali or acid, as may be determined. For the purpose of neutralization a normal solu- tion of sodium hydroxid or of hydric chlorid is used, and after being thoroughly stirred the fluid thus neutralized is again tested in the same manner as at first, to insure the proper reaction of the medium being attained. When neutralization is to be effected by the addition of an alkali, it not infrequently happens that after the calculated amount of normal solution of sodium hydroxid has been added, the second test will show that the medium is acid to phenolphthalein, to the extent sometimes of 0.5 to 1 per cent. This discrepancy is perhaps due to side reactions which are not understood. The reaction of the medium, however, must be brought to the desired point by the further addition of sodium hydroxid, and the titrations and additions of alkali must be re- peated until the medium has the desired reaction (z. e., 0.0 per cent. — 0.005 Per cent., see below). After the prescribed period of heating, it is frequently found that the medium is again slightly acid, usually about 0.5 per cent. Without correcting this the fluid is to be filtered and the calculated amount of acid or alkali is to be added to change the reaction to the one desired. A still further change in reaction is not infrequently to be observed after sterilization, the degree of acidity varying apparently with the composition of the media and the degree and continuance of the heat." " Manner of expressing the reaction : Since at the time the re- CULTIVATION OF BACTERIA. 187 action is first determined enlture-niedia are more often acid than alkaline, it is proposed that acid media be designated by the plus sign and alkaline media by the minus sign, and that the degree of acidity or alkalinity be noted in parts per hundred. Thus, a medium marked + 1.5 would indicate that the medium was acid, and that 1.5 per cent, of - sodium hydroxid is required to make it neutral to phenolphthalein ; while — 1.5 would indicate that the medium was alkaline and that 1.5 per cent, of - acid must be added to make it neutral to the indicator." " Standard reaction of media ( provisional) : " Experience seems to vary somewhat as to the optimum degree of reaction which shall be uniformly adopted in the preparation of standard culture-media. To what extent this is due to varia- tion in natural conditions as compared with variations of labora- tory procedure, it seems impossible to state. Somewhat different degrees of reaction for optimum growth are required, not only in or upon the media of different composition and by bacteria of different species, but also by bacteria of the same species when in different stages of vitality. The bulk of available evidence from both Europe and America points to a reaction of + 1.5 as the opti- mum degree of reaction for bacterial development in inoculated culture-media. While this experience is at variance with that in several of our own laboratories, it has been deemed wisest to adopt •+ 1.5 as the provisional standard reaction of media, but with the recommendation that the optimum growth reaction be always recorded with the species." Bouillon is one of the most useful and most simple of the media. Its preparation is as follows : To 500 grams of finely-chopped lean, boneless beef, 1000 c.cm. of clean water are added and allowed to stand for about twelve hours on ice. At the end of this time the liquor is de- canted, that remaining on the meat expressed through a cloth, and then, as the entire quantity is seldom regained, enough water added to bring the total amount up to 1000 c.cm. This liquid is called the meat-infusion. To it 10 grams of Witte's or Fairchild's dried beef-peptone and 5 grams of sodium chlorid are added, and the whole boiled until the albumins coagulate. Smith * says that when the peptones are added before boiling most of them are lost, and therefore recommends that the meat-infusion be 1 Trans. Assoc. Amer. Phys., 1896. 1 88 PATHOGENIC BACTERIA. boiled and filtered and the solid ingredients added and dis- solved subsequently. This observation referred especially to bouillon intended for the culture of diphtheria bacilli for toxin. The reaction is then carefully titrated accord- ing to the directions already given. For rough work in students' classes litmus-paper is commonly used as an indicator, the alkaline solution being added drop by drop until a faint blue appears on the red paper. The method of using phenolphthalein suggested by Timpe is to con- tinue the addition of the alkaline solution until a drop of the bouillon produces a red spot upon phenolphthalein- paper. Such a paper can easily be made by using a solu- tion of 5 grams of phenolphthalein to i liter of 50 per cent, alcohol. The bibulous paper is cut into strips, moistened with the solution, and then hung up to dry. It keeps quite well. Acids do not change the appearance of the paper, but small traces of alkali turn it red. The bouillon thus prepared is a clear fluid of a straw color, much resembling normal urine in appearance. It is dispensed in previously sterilized tubes with cotton plugs — about 10 c.cm. to each — and is then sterilized by steam three successive days for fifteen to twenty minutes each, according to the directions already given for frac- tional sterilization. (See p. 167.) When it is desirable to prepare the bouillon from beef- extract, the method is very simple. To 1000 c.cm. of clean water 10 grams of Witte's dried beef-peptone, 5 grams of sodium chlorid, and about 2 grams of beef- extract are added. The solution is boiled until the con- stituents are dissolved, titrated, and filtered when cold. If it is filtered while hot, there is always a subsequent precipitation of meat-salts, which clouds it. Bouillon and other liquid culture-media are best dis- pensed and kept in small receptacles — test-tubes or flasks — in order that a single contaminating organism, should it enter, may not spoil the entire bulk. A very con- venient simple apparatus used by bacteriologists for fill- ing tubes with liquid media is shown in Figure 21. It CULTIVATION OF BACTERIA. 189 need not be sterilized before using, as the culture-medium will be sterilized by the intermittent method after the tubes are rilled. The test-tubes and flasks into which the culture-medium is filled must, however, be previously sterilized by dry heat. The dry-heat sterilization is done, of course, after the cotton plugs are in place. Bouillon is the basis of most of the culture-media. The addition of 10 per cent, of gelatin makes it "gela- tin ;" that of 1 per cent, of agar-agar makes it "agar- Fig. 21. — Funnel for filling tubes with culture-media (Warren): a, funnel containing the culture-media in liquid condition ; b, pinch-cock by which the flow of fluid into the test-tube is regulated ; c, rubber tubing. agar." The preparation of these media, however, varies somewhat from that of plain bouillon. Sugar Bouillon is bouillon containing in solution known percentages of such sugars as glucose, lactose, 190 PATHOGENIC BACTERIA. saccharose, etc. Smith1 points out that the bouillon as usually prepared is apt to contain considerable muscle sugar; this should be destroyed before the new sugar is added, else confusion of results must be expected. To exclude the muscle sugars and secure dextrose-free bouillon he inoculates the beef-infusion in the evening with the colon bacillus and stands it in the incubator. Next morning, the growth of the colon bacillus having destroyed the sugars, the bouillon is prepared from the sugar-free meat-infusion as already described, and the requisite percentage of sugar added. The sugar bouillons should not be sterilized in the autoclave, as the high temperatures alter the sugars. Geiatin. — The culture-medium known as gelatin has de- cided advantages over the bouillon, not only because it is an excellent food for bacteria, and, like the bouillon, trans- parent, but because it is also solid. Nor is this all : it is a transparent solid which can be made liquid or solid at will. It is prepared as follows: To 1000 c.cm. of meat- infusion or to 1000 c.cm. of water containing 2 grams of beef-extract in solution, 10 grams of peptone, 5 grams of salt, and 100 grams of gelatin ("Gold label " is the best commercial article) are added, and heated until the ingre- dients are all dissolved. It is then titrated or alkalinized to litmus by adding sodium hydroxid solution as described. Double boilers are very slow, and if proper care is exer- cised there is little danger of the gelatin burning. It must be stirred occasionally, and the flame should be so distributed by wire gauze as not to act upon a single point of the bottom of the kettle. The preparation is now re- turned to the fire and boiled for about an hour. At the end of the hour the albumins of the meat-infusion will be coagulated and the gelatin thoroughly dissolved. Giinther has shown that the gelatin congeals better if allowed to dissolve slowly in warm water before boiling. As much water as has been lost by vaporization during the process of boiling should be replaced. It is well to 1 Journal of Experimental Medicine, ii., No. 5, p. 546. CUL TI VA TION OF BA CTERrA . 1 9 1 cool the liquid to about 6o° C, then add the water mixed with the white of an egg, and then boil again for half an hour, and filter. If a double boiler is preferred, it is well to fill the outer boiler with a saturated solution of calcium chlorid, as suggested by Wilson. The boiling-point of this solu- tion is so high that the gelatin or agar-agar in the inner receptacle will boil vigorously. The solution of calcium chlorid can be used again and again, water being added to replace that which evaporates. If the filter-paper is of good quality, properly folded (pharmaceutical filter), wet with boiling water, and if the gelatin is properly dissolved, the whole quantity should pass through before cooling too much. Should only half go through before cooling, the remainder must be re- turned to the pot, heated to boiling once more, and then passed through a new filter-paper. As a matter of fact, gelatin generally filters readily. A wise precaution is to catch the first few centimeters in a test-tube and boil them, so that if a cloudiness shows the presence of un- coagulated albumin, the whole mass can be boiled again. The finished gelatin is at once distributed into sterilized tubes and then sterilized like the bouillon by the frac- tional method. The sterilization can also be satisfactorily performed by the use of the autoclave at iio°-ii5° C. for 15 minutes. Of course, the gelatin or any other culture-medium can be kept en masse indefinitely, but should a contaminating micro-organism accidentally enter, the whole quantity will be spoiled ; if, on the other hand, it is kept in tubes, several of them may be lost without much inconvenience. Under proper precautions of sterilization and protection it should all keep well. Agar-agar. — Agar-agar is the commercial name of a Ceylonese sea-weed which dissolves .in boiling water with resulting thick jelly when cold. The jelly, which solidi- fies between 400 and 500 C, cannot again be melted ex- cept by the elevation of its temperature to the boiling- 193 PATHOGENIC BACTERIA. point, so that this culture-medium, which is nearly trans- parent, is almost as useful as gelatin. In addition to its readiness to liquefy and solidify, it is sufficiently firm to allow of the incubation-temperature — i. e. 37 ° C. — at which gelatin is always liquid, and no better than bouillon. The preparation of this medium is generally described in the text-books as one " requiring considerable patience and much waste of filter-paper. " In reality, it is not dif- ficult if a good heavy filter-paper be obtained and no attempt be made to filter the solution until the agar-agar is perfectly dissolved. It is prepared as follows : To 1000 c.cm. of bouillon made as described above, preferably of meat instead of beef-extract, 10 to 15 grams of agar-agar are added. The mixture is boiled vigorously for an hour in an open pot over the direct gas flame or in the double boiler with saturated calcium chloride solution in the outside pot. After being cooled to about 6o° C. and after titration an egg beaten up in water is added, and the liquid is boiled again until the egg is entirely coagulated. After the boiling the agar-agar is filtered, just as the gelatin was, through a carefully-folded pharmaceutical filter wet with boiling water. It may expedite matters to pour in about one-half of the solution, keep the re- mainder hot, and subsequently add it when necessary. The formerly much-employed hot-water and gas-jet filters are unnecessary. If properly prepared, the whole quantity will filter in from fifteen to thirty minutes. Ravenel l prepares his agar-agar by making two solu- tions, one representing the meat-infusion, but twice the usual strength, the other the agar-agar dissolved in one- half the usual quantity of water. The agar-agar is dis- solved by exposure to superheated steam in the autoclave, after which the two solutions are poured together and boiled until all of the albumins are precipitated. The coagulation of the albumins of the meat-infusion serves to clarify the agar-agar. x Journal of Applied Microscopy, June, 1898, vol. i., No. 6, p. 106. CUL TI VA TION OF BA CTERIA . 1 93 Allegar informs me that he finds the use of powdered agar-agar very satisfactory because of the readiness with which it dissolves and the ease with which it niters. If agar-agar is to be made with beef-extract, the bouil- lon should be made first and filtered when cofd} after which the agar-agar is added and dissolved. If this is not done, it is sure to contain a precipitate of crystalline urates from the meat-extract, which makes it unsightly though it does not interfere with its nutrient qual- ities. Agar-agar is dispensed in tubes like the gelatin and bouillon, sterilized by steam by the intermittent process, or in the autoclave, and after the last sterilization, before cooling, each tube is inclined against a slight elevation, so as to offer an extensive flat surface for the culture. After the agar-agar jelly solidifies its contraction causes some water to collect at the lower part of the tube. This should not be removed, as it keeps the material moist, and also because it has a distinct influence upon the cha- racter of the growth of the bacteria. Glycerin Agar-agar. — For an unknown reason certain of the bacteria which will not grow upon the agar-agar as prepared above will do so if 3-7 per cent, of glycerin be added. Among these is the tubercle bacillus, which, not growing at all upon plain agar-agar, will grow well when glycerin is added — a fact discovered by Roux and Nocard. The glycerin may also be added to bouillon or any other medium. Blood Agar-agar was recommended by R. Pfeiffer for the cultivation of the influenza bacillus. It is ordinary agar-agar whose surface is coated with a little blood secured under antiseptic precautions from the finger-tip, ear-lobule, etc., of man, or the veins of one of the tower animals. Some bacteriologists prepare a hemoglobin agar-agar by spreading a little powdered hemoglobin upon the surface of the agar-agar. This has the disad- vantage that powdered hemoglobin is not sterile, and the medium must be sterilized after its addition. 13 194 PATHOGENIC BACTERIA. The blood agar-agar should be kept in the incubator a day or two before use so as to insure perfect sterility. Blood-serum. — The great advantage possessed by this medium is that it is itself a constituent of the body, and hence offers opportunities for the development of the parasitic forms of bacteria under the most natural con- ditions possible. It is the most difficult of all the media to prepare. The blood must be obtained from a slaughter- house in an appropriate receptacle, the best things for the purpose being tall narrow jars of about i liter capacity, with a tightly-fitting lid. The jars are sterilized by heat or by washing with alcohol and ether, are carefully dried, closed, and carried to the slaughter-house where the blood is to be obtained. As the blood flows from the severed vessels of the animal the jars are filled one by one. It seems advisable to allow the first blood to escape, as it is likely to become contaminated from the hair. By waiting until a coagulum forms upon the hair the danger of con- tamination is obviated. The jars when full are allowed to stand undisturbed until quite firm coagula form within them. If these have any tendency to cling to the glass, each one should be given a few violent twists, so as to break away the fibrinous attachments. After this the jars are carried to the laboratory and stood upon ice for forty-eight hours, by which time the clots will have re- tracted considerably, and a moderate amount of clear serum can be removed by sterile pipettes and placed in sterile tubes. If the serum obtained is red and clouded from the presence of corpuscles, it may be pipetted into sterile cylinders and allowed to sediment for twelve hours, then repipetted into tubes. It is evident that such com- plicated maneuvring will offer many possible chances of infection ; hence the sterilization of the serum is of the greatest importance. If it is desirable to use the serum as a liquid medium, it is exposed to a temperature of 6o° C. for one hour upon each of five consecutive days. If it is thought best to coagulate the serum and make a solid culture-medium, CULTIVATION OF BACTERIA. 195 it may be exposed twice, for an hour each time — or three times if there is distinct reason to think it contam- inated— to a temperature just short of the boiling-point. During the process of coagulation the tubes should be inclined, so as to offer a large surface for the growth of the culture. The serum thus prepared may be white, or have a reddish-gray color if many corpuscles are pres- ent, and is opaque. It cannot be melted, but once solid remains so. Koch devised a special apparatus (Fig. 22) for coag- ulating- blood-serum. The bottom should be covered Fig. 22. — Koch's apparatus for coagulating and sterilizing blood-serum. with cotton, a single layer of tubes placed upon it, and the temperature elevated until coagulation occurs. The repeated sterilizations may be conducted in this appa- ratus, or may be done equally well in the steam appa- ratus, the cover of which is not completely closed, for if the temperature of the serum is raised too rapidly it is certain to bubble. Like other culture-media, blood-serum and its combi- nations may be sterilized in the autoclave, thus saving much time. The serum should be coagulated first, else bubbling is apt to occur. The autoclave temperature is apt to make the preparation very firm and hard, consider- able fluid being pressed out of it. 196 PATHOGENIC BACTERIA. It is said that considerable convenience in the blood- serum manipulations is secured by the addition of neu- trose, which prevents it coagulating when heated. It can then be sterilized like bouillon and when used can be solidified by the addition of agar-agar. Fresh blood-serum can be kept on hand in the labora- tory, in sterile bottles, by adding an excess of chloro- form. In the process of coagulation and sterilization the chloroform is evaporated and the serum is unchanged by its presence. Loffler's blood-serum mixture, which seems rather better for the cultivation of some species than the blood- serum itself, consists of 1 part of a beef-infusion bouillon containing 1 per cent, of glucose and 3 parts of liquid blood-serum. After being well mixed this is distributed in tubes, and sterilized and coagulated like the blood- serum itself. Most organisms grow more luxuriantly upon it than upon either plain blood-serum or other culture-media. Its special usefulness is for the Bacillus diphtheriae, which grows upon it with rapidity and with quite a characteristic appearance. Alkaline Blood-serum. — According to Lorrain Smith, a very useful culture-medium can be prepared asibllows: To each 100 c.cm. of blood-serum add i-r.5 c.cm. of a 10 per cent, solution of sodium hydrate and shake it gently. Put sufficient of the mixture into each of a series of test- tubes, and, laying them upon their sides, sterilize like blood-serum, taking care that their contents are not heated too quickly, as then bubbles are apt to form. The result should be a clear, solid medium consisting chiefly of alkali-albumins. It is especially useful for the bacillus diphtheriae. Deycke's Alkali-albuminate.— 1000 grams of meat are macerated twenty-four hours with 1200 c.cm. of a 3 per cent, solution of potassium hydrate. The clear brown fluid is filtered off and pure hydrochloric acid carefully added while a precipitate forms. The precipitated albuminate is collected upon a cloth filter, mixed with a small quan- CULTIVATION OF BACTERIA. 197 tity of liquid, and made distinctly alkaline. To make solutions of it of definite strength it can be dried, pul- verized, and redissolved. The most useful formula used by Deycke was a 2^ per cent, solution of the alkali-albuminate with 1 per cent, of peptone, 1 per cent, of NaCl, and gelatin or agar-agar enough to make it solid. Potatoes. — Without taking time to review the old method of boiling potatoes, opening them with sterile knives, and protecting them in the moist chamber, or the much more easily conducted method of Esmarch in which the slices of potato are sterilized in the small dishes in which they are afterward kept and used, we will at once pass to what seems the most simple and satisfactory method of using this valuable medium — that of Bolton and Globig :l With the aid of a cork-borer a little smaller in diam- eter than the test-tube ordinarily used a number of cyl- inders are cut from potatoes. Rather large potatoes should be used, the cylinders being cut transversely, so that a number, each about an inch and a half in length, can be cut from one potato. The skin is removed from the cylinders by cutting off the ends, after which each cylinder is cut in two by an oblique incision, so as to leave a broad, flat surface. The half-cylinders are placed each in a test-tube previously sterilized, and then are exposed three times, for half an hour each, to the pass- ing steam of the sterilizer. This steaming cooks the potato and also sterilizes it. Such cultures are apt to deteriorate rapidly, first by turning very dark ; second, by drying so as to be useless. Abbott has shown that if the cut cylinders be allowed to stand for twelve hours in running water before being dispensed in the tubes, they do not turn dark. Drying may be prevented by adding a few drops of clean water to each tube before sterilizing. It is not necessary to have a special small chamber blown in the tube to contain this water; only 1 The Medical News, vol. L, 1887, p. 138. 198 PATHOGENIC BACTERIA. a small quantity need be added, and this will not touch the potato, which does not reach the bottom of the rounded tube. Potatoes differ considerably in reaction, and so give us very variable results. If the work done is to be accu- rate, it may be necessary to correct this reaction if the acids have not been sufficiently removed by the washing in running water already described. To do this the cut cylinders are placed in a measured quantity of distilled water and steamed for about an hour. The reaction of the water is then determined by titration and the desired amount of sodium hydroxid added, after which the potatoes are again steamed in the corrected solution for about thirty minutes, and then placed in tubes. A potato-juice has also been suggested, and is of some value. It is made thus : To 300 c.cm. of water 100 grams of grated potato are added, and allowed to stand on ice over night. Of the pulp 300 c.cm. are expressed through a cloth and cooked for an hour on a water-bath. After cooking, the liquid is filtered, titrated if desired, and receives 4 per cent, of glycerin. Upon this medium the tubercle bacillus grows well, especially when the reaction of the medium is acid. Milk. — Milk is useful as a culture-medium. When the milk stands the cream which rises to the top is a source of inconvenience, so that it is best to secure from a dairy fresh milk from which the cream has been removed by a centrifugal machine. It is given the desired degree of alkalinity, placed in sterile tubes, and sterilized by steam by the intermittent method. The opaque nature of this culture-medium often permits the undetected develop- ment of contaminating organisms. A careful watch should therefore be kept upon it lest it spoil. Litmus Milk. — This is milk to which just enough of a saturated watery solution of pulverized litmus is added to give a distinct blue color. Cow's milk is inclined to be acid in reaction, and a small amount of sodium car- CUL TI VA TION OF BA CTERIA . 1 99 bonate may be necessary to give it a distinct bine. The use of litmus is probably the best method of determining whether bacteria by their growth produce acids or alka- lies. The watery solution of litmus, being a vegetable in- fusion, is likely to spoil; hence it should always be treated like the culture-media and sterilized by steam every time the receptacle in which it is kept is opened. Petruschky's Whey. — In order to differentiate be- tween acid and alkaline producers among the bacteria, Petruschky has recommended a neutral whey colored with litmus. It is made as follows: To a liter of fresh skimmed milk 1 liter of water is added. The mixture is violently shaken. About 10 c.cm. are now taken out as a sample to determine how much hydrochloric acid must be added to produce coagulation of the milk, and, having determined the least quantity required for the whole bulk, it is added. After coagulation the whey is filtered off", exactly neutralized and boiled. After boiling it is generally found clouded and acid in reaction. It is therefore filtered again, and again neu- tralized. Litmus is finally added to the neutral liquid, so that it has a violet color, which can readily be changed to blue or red by alkalies or acids. The medium is a very useful aid in differentiating the typhoid and colon bacilli, showing well the alkaline formation of the typhoid bacillus. Peptone Solution, or Dunham's solution, is very use- ful for the detection of certain faint colors. It is a per- fectly clear, colorless solution, made as follows: Sodium chlorid, 0.5^ Boil until the ingredients Witte's dried peptone, 1. V dissolve; then filter, fill Water, 100. J into tubes, and sterilize. It is one of the best media for the detection of indol. In it the bacillus pyocyaneus produces its blue color. A very important fact in regard to peptone has been pointed 200 PATHOGENIC BACTERIA. out by Garini,1 who found that many of the peptones upon the market were impure, and on this account failed to show the indol reaction for bacteria known to produce indol. He recommends the use of the biuret reaction for testing the peptone to be employed. The reagent used is Fehling's copper solution, with which pure pep- tone strikes a violet color not destroyed upon boiling, while impure peptone gives a red or reddish-yellow pre- cipitate. Both the peptone and copper solution should be in a dilute form to make successful tests. The addition of 4 c.cm. of the following solution — Rosalie acid, 0.5, 80 per cent, alcohol, 100. makes it become an excellent reagent for the detection of acids and alkalies. The solution is pale rose in color. If the bacterium produces acids, the color fades; if alka- lies, it intensifies. As the color of rosalic acid is destroyed by glucose, it cannot be used in culture-media contain- ing it. Theobald Smith2 calls attention to the fact that Dun- ham's solution is unsuited to the growth of many bac- teria, some failing altogether to grow in it, and recom- mends that, instead, bouillon free of dextrose shall be used. All bacteria grow well in it, and the indol-reaction is pronounced in sixteen-hour-old cultures. His method of preparation is as follows: beef-infusion, prepared either by extracting in the cold or at 6o° C, is inoculated in the evening with a rich fluid culture of some acid-pro- ducing bacterium (Bacillus coli), and placed in the ther- mostat. • Early next morning the infusion, covered with a thin layer of froth, is boiled, filtered, peptone and salt added and the neutralization and sterilization carried on as usual. To test for the presence of indol, the bacterium is 1 Centralbl. f. Bakt. u. Parasitenk., xiii., p. 790. 2 Journal of Exp. Medicine, Sept. 5, 1897, vi., p. 546. CULTIVATION OF BACTERIA. 201 planted in the culture-medium, allowed to grow for upward of twelve hours, and then subjected to the com- bined action of a nitrite and chemically pure sulphuric acid. In making the test, Smith adds to each tube i c.cm. of a o.oi per cent, solution of KN02, freshly prepared, and 10 drops of chemically pure H2SO<. The presence of indol is characterized by the production of a red color. It is not intended that the student shall infer that there are no culture-media other than these, which have been selected because of their usefulness and popularity. Many other compounds and as many simple substances are employed ; for example, eggs, white of egg} urine, bread, sputum, sugar solutions, hydrocele fluid, aqueous humor, etc. CHAPTER VIII. CULTURES, AND THEIR STUDY. The objects which we have had before us in the prep- aration of the culture-media were numerous. We have prepared them so as to allow us to separate — or, rather, to isolate — bacteria, to keep them in healthy growth for considerable lengths of time, to enable us to observe their biologic peculiarities, and to introduce them without dif- ficulty into the bodies of animals. The isolation of bacteria was impossible until the fluid culture-media of the early observers were replaced by the solid media, and was exceedingly crude until Koch gave us the solid, transparent media and the well-known "plate-cultures." A growth of artificially-planted micro-organisms in which an immense number are massed together is called a culture. If such a growth contains but one kind of organism, it is known as a pure culture. It has become the habit at present to use the term "cul- ture" rather loosely, so that it does not always signify a growth of micro-organisms artificially planted, but may signify a growth taking place under natural conditions j thus, typhoid bacilli are said to exist in the spleens of patients dead of that disease "in pure culture," because no other bacteria are there ; and sometimes, when in ex- pectorated fragments of cheesy matter from tuberculosis pulmonalis the tubercle bacilli are very numerous and unmixed with other bacteria, the term "pure culture" is again used to describe the condition. Three principal methods are at present employed to enable us to secure pure cultures of bacteria, but before beginning a description of them it is well to observe that 202 CULTURES, AND THEIR STUDY. 203 the peculiarities of certain pathogenic forms enable us to use special means, taking advantage of their eccentrici- ties, for their isolation, and that the general methods are in reality more useful for the non-pathogenic than for the pathogenic forms. All three methods depend upon the observation of Koch, that when germs are equally distributed through- out some liquefied nutrient medium which can be solidi- fied in a thin layer, the growth of the germs takes place in little scattered groups or families, called colonies, dis- tinctly separated from each other and capable of trans- plantation to tubes of culture-media. Plate-cultures. — The plate-cultures, originally made by Koch, require considerable apparatus, and of late years have given place to the more ready methods of Petri and Von Esmarch. So great, however, is the historic interest attached to the plates that it would be a great omission not to describe Koch's method in full. Apparatus. — Half a dozen glass plates, about 6 by 4 inches in size, free from bubbles and scratches and ground at the edges, are carefully cleaned, placed in a sheet-iron box made to receive them, and then put in the hot-air closet, where they are sterilized. The box, which is tightly closed, al- lows the sterilized plates to be kept on hand indefinitely before using. A moist chamber, or double dish, about 10 inches in di- ameter and 3 inches deep, the upper half being just enough larger than the lower to allow FlG- 23— Complete levelling appa- , . . ,, ratus for pouring plate-cultures, as it to close over it, is carefully taught by Koch washed. A sheet of bibulous paper is placed in the bottom, so that some moisture can be retained, and a 1 : 1000 bichlorid solution is poured in and brought in contact with the side*, top, and bottom 204 PATHOGENIC BACTERIA. by turning the dish in all directions. The solution is emptied out, and the dish, which is always kept closed, is ready for use. A levelling apparatus is required (Fig. 23). This con- sists of a wooden tripod with adjustable screws, and a glass dish covered by a flat plate of glass upon which a low bell-jar stands. The glass dish is filled with broken ice and water, covered with the glass plate, and then exactly levelled by adjusting the screws under the legs of the tripod. When level the cover is placed upon it, and it is ready for use. Method (Fig. 24). — A sterile platinum loop is dipped into the material to be examined, a small quantity se- FlG. 24. — Method of holding tubes during inoculation. cured, and stirred about so as to distribute it evenly through a tube of the melted gelatin. If the material under examination is very rich in bacteria, one loopful may contain a million individuals, which, if spread out in a thin layer, would develop so many colonies that it would be impossible to see any one clearly ; hence the necessity for a dilution. From the first tube a loopful of gelatin is carried to a second tube of melted gelatin and stirred well, so as to distribute the organisms evenly through it. In this tube we may have no more than ten thousand organisms, and if the same method of dilution be used again, the third tube may have only a few hun- dreds, and a fourth only a few dozen colonies. After the tubes are prepared, one of the sterile glass plates is caught by its edges, removed from the iron box, and placed beneath the bell-glass upon the cold plate CULTURES, AND THEIR STUDY. 205 covering the ice-water of the levelling apparatus. The plug of cotton closing the mouth of tube No. 1 is re- moved, and to prevent contamination during the outflow of the gelatin the mouth of the tube is held in the flame of a Bunsen burner for a moment or two. The gelatin is then cautiously poured out upon the plate, the mouth of the tube, as well as the plate, being covered by the bell-glass to prevent contamination by germs in the air. The apparatus being level, the gelatin spreads out in an even, thin layer, and, the plate being cold from the ice beneath, it immediately solidi- fies, and in a few moments can be removed to the moist cham- ber prepared to receive it. As x x . Fig. 25. — Glass bench. soon as plate No. 1 is prepared, the contents of tube No. 2 are poured upon plate No. 2, allowed to spread out and solidify, and then superimposed on plate No. 1 in the moist chamber, being separated from the plate already in the chamber by small glass benches (Fig. 25) made for the purpose and sterilized. After the contents of all the tubes are thus distributed, the moist chamber and its contents are allowed to stand for some hours, to permit the bacteria to grow. Where each or- ganism falls a colony develops, and the success of the whole method depends upon the isolation of a colony and its transfer to a tube of culture-medium where it can grow unmixed and undisturbed. The description must have made evident the fact that only such culture-media can be used for plate-cultures as can be melted and solidified at will — viz. gelatin, agar- agar, and glycerin agar-agar. Blood -serum and Loffler's mixture are entirely inappropriate. The great drawback to this excellent method is the cumbersome apparatus required and the comparative im- possibility of making plate-cultures, as is often desirable, in the clinic, at the bedside, or elsewhere than in the laboratory. The method therefore soon underwent mod- ifications, the most important being 2o6 PA THOGENIC BACTERIA. Petri's Dishes. — These small dishes (Fig. 26), about 4 inches in diameter and ]/2 inch deep, with accurately fitting lids, are about as convenient as anything that has been devised in bacteriological technique. They dis- FlG. 26. — Petri dish for making plate-cultures. pense with plates and plate-boxes, with moist chambers and benches, and usually with the levelling apparatus, though this is still employed in connection with the Petri dishes in some laboratories. The method of the employment of Petri dishes is very simple. The dishes are carefully cleaned, polished, and sterilized by hot air, care being taken that they are placed in the hot-air closet right side up, and after sterilization are kept covered and in that position. The dilution of the material under examination is made with gelatin or agar-agar tubes in the manner described above, the plugs are removed, the mouth of the tube is cautiously held for a moment in the flame, then the contents of each tube are poured into one of the sterile dishes, whose top is elevated just sufficiently to allow the mouth of the tube to enter. The gelatin is spread over the bottom of the dish in an even layer, is allowed to solidify, labelled, and then stood away for the colonies to develop. -Esmarch Tubes. — This method, devised by Esmarch, converts the walls of the test-tube into the plate and dis- penses with all other apparatus. The tubes, which are inoculated and in which the dilutions are made, should contain less than half the usual amount of gelatin or agar-agar. After inoculation the cotton plugs are pushed into the tubes until even with their mouths, and then covered with a rubber cap, which protects them from wetting. A groove is next cut in a block of ice, and CULTURES, AND THEIR STUDY. 207 the tube, held almost horizontally, is rolled in this until the entire surface of the glass is covered with a thin layer of the solid medium (Fig. 27). Thus the tube becomes the plate upon which the colonies develop. Fig. 27. — Esmarch tube on block of ice (redrawn after Abbott). Several little points need to be observed in carrying out Esmarch' s method. The tube must not contain too much culture-medium, or it cannot be rolled into an even layer. In rolling the contents should not touch the cotton plug, lest it be glued to the glass and its subsequent use- fulness be injured. No water must be admitted from the melted ice. The offspring of each bacterium growing upon the film of gelatin constituting a plate-culture form a mass which has already been pointed out as a colony. These small bacterial families may be seen through a micro- scope when still much too small for detection by the naked eye, and because of their minuteness should always be studied with the microscope. The original plates of Koch are very inconvenient for such examination, because it is impossible to remove them from the moist chamber and lay them upon the stage of the microscope without exposing them to the danger of contamination by the atmosphere, so that the advantages of Petri dishes and Esmarch tubes, where the examination may be made through the glass tube or 208 PATHOGENIC BACTERIA. through the bottom of the inverted dish, will be more than ever apparent. The colonies should be viewed from time to time in their growth, drawings being made of the appearances, so as to form a series showing the developmental cycle. Most colonies will be found to originate as spherical, cir- cumscribed, slightly granular, yellowish, greenish, or brownish dots, and later to send out offshoots or filaments or to develop concentric rings or characteristic liquefac- tions. A few appear from the very first as woolly clumps of entangled threads. Some of the most diverse forms of colonies are repre- sented in the accompanying illustrations (Figs. 28-32). Figs. 28, 29, 30. — The various appearances of colonies of bacteria under the microscope : a, colony of Bacillus liquefaciens parvus (Luderitz) ; b, colony of Bacillus polypiformis (Liborius); c, colony of Bacillus radiatus (Luderitz). A pure culture, when obtained from colonies growing upon a plate, must always be made from a single colony, the transplantation being accomplished under a low power of the microscope. The naked eye can rarely be depended upon to recognize the purity of a colony or its isolation. Selecting as isolated, large, and characteristic a colony as possible, it is brought to the centre of the field. A platinum wire, securely fused into a glass handle about 8 inches long, is sterilized by being made incandescent in a Bunsen flame, cooled, and then cautiously manipu- lated until, while it is watched through the microscope, CULTURES, AND THEIR STUDY. 209 it is seen to touch the colony and take part of its con- tents away. In this maneuvre the wire must not touch the objective, the glass, or anything except the colony. Having secured the adhesion of a few bacteria to the sterile wire, the pure culture is made by introducing them into a sterile culture-medium. If the pure culture is to be made in bouillon, the tube is held obliquely, so that when the cotton plug is cau- tiously removed no germs can fall in from the air. The plug is removed by a twisting movement. The wire, with- out being allowed to touch the mouth or sides of the tube, is plunged into its contents and stirred about until the bacteria are de- tached, and is then re- FlGS. 31, 32. — The various appearances of colonies of bacteria under the microscope : a, colony of Bacillus muscoides (Liborius) ; b, colony of Bacillus anthracis (Fliigge). moved and the plug replaced. The wire should be im- mediately sterilized by heating to incandescence, lest the bacteria be pathogenic and capable of doing subsequent harm. If the culture is to be made in gelatin, a different method is employed. The tube is either held horizon- tally, or, as is perhaps better, inverted ; the cotton plug 14 2IO PATHOGENIC BACTERIA. is removed cautiously ; the wire bearing the bacteria from the colony is introduced until its point enters the centre of the gelatin, and is then carefully pushed on until a vertical puncture from the surface to the bottom of the gelatin is made. This is the puncture-culture — " stichcultur " of the Germans. If the bacteria are only to be planted upon the surface of the culture-medium, the wire is drawn over the surface of a tube of obliquely solidified gelatin, agar-agar, blood- serum, etc. with a steady, slow movement, so as to scatter the germs along its path and cause the development of the bacteria in an enormous colony or mass of colonies in a line following the longest diameter of the exposed surface from end to end. This is the stroke-culture — "strichcultur." The method of holding the tubes, cotton plugs, and platinum wire during the process of inoculation is shown in Figure 24. Sometimes it is desirable to preserve an entire colored colony as a microscopic specimen. To do this a perfectly clean cover-glass, not .too large in size, is momentarily warmed, then carefully laid upon the surface of the gelatin or agar-agar containing the colonies. Sufficient pressure is applied to the surface of the glass to exclude bubbles underneath, but the pressure must not be too great, as it may destroy the integrity of the colony. The cover is gently raised by one edge, and if successful the whole colony or a number of colonies, as the case may be, will be found adhering to it. It is treated exactly as any other cover-glass preparation, is dried, fixed, stained, and mounted, and kept as a permanent specimen. It is called an adhesion preparation — u klatsch praparat. ' ' Very often, when one is in a hurry, pure cultures from single colonies may be secured by a very simple manipu- lation suggested by Banti.1 The inoculation is made into the water of condensation at the bottom of an agar- 1 Centralbl. f. Bakt. und Parasitenk., 1895, xvii., No. 16. CULTURES, AND THEIR STUDY. 21 1 agar tube, without touching the surface. The tube is then inclined so that the water flows over the agar, after which it is stood away in the vertical position. Colonies will grow where bacteria have been floated upon the agar-agar, and may be picked up later in the same manner as from a plate. In other cases pure cultures may best be secured by animal inoculation. For example, when the tubercle bacillus is to be isolated from milk or urine which con- tains rapidly growing bacteria that would outgrow the slow-developing tubercle bacillus, it is better to inject some of the fluid into the abdominal cavity of a guinea- pig and await the development of tuberculosis, and then seek to secure the bacillus from the unmixed material in the softened lymphatic glands. Anthrax bacilli are also more easily secured in pure culture by inoculating a mouse and recovering the bacilli from a spleen or the heart's blood after death, than by going to the trouble of making plates and picking out the colonies. In many cases when it is desired to isolate the micro- coccus tetragenus, the pneumococcus, and others, it is easier to inoculate the most susceptible animal and recover the germ from the organs than to plate it out and search for the colony among many others which may be similar to it. The development of bacteria in liquids is of less in- terest than that upon solid media. The growth generally manifests itself by a diffused turbidity. Sometimes flocculi float in the otherwise clear medium. Some forms grow most rapidly at the surface of the liquid, and produce a distinct membranous pellicle called a mycoderma. In such a growth multitudes of degenerated bacteria and large numbers of spores are to be observed. On the other hand, it occasionally happens that the growth occurs chiefly below the surface, and may produce gelat- inous masses which are known as zooglea. In gelatin the bacteria exhibit a great variety of ap- pearances, many of which are beautiful and interesting. 212 PATHOGENIC BACTERIA. Certain bacteria, as the tubercle bacillus, will not grow at all upon gelatin. Some forms which are rigidly ae- robic will only grow upon or near the surface ; others, anaerobic, only in the deeper parts. The majority, how- ever, grow both upon the surface and in the puncture made by the wire. Sometimes the consistence of the gelatin is unaltered ; sometimes it is liquefied throughout, sometimes only at the surface. Sometimes offshoots ex- tend from the colonies into the gelatin, giving the culture Fig. 33. — Various forms of gelatin puncture-cultures : a, Bacillus typhi ab- dominalis ; b, B. anthracis ; c, B. mycoides ; d, B. mesentericus vulgatus ; e, B. of malignant edema ; f, B. radiatis. a bristling appearance. Figure 33 will serve to illustrate different varieties of gelatin growth. The growth in gelatin is generally so far removed from the walls of the tube (a central puncture nearly always being made in the culture-medium, in order that the growth be symmetrical) that it is next to impossible to make a microscopical examination of it with any power beyond that given by a hand-lens. Much attention has been given of late to the preparation of microtome sections of the gelatin growth. To accom- plish this the glass is warmed sufficiently to allow the gelatin to be removed and placed in Miiller's fluid (bi- CULTURES, AND THEIR STUDY. 213 chromate of potassium 2.-2.5, sulphate of sodium 1, water 100), where it is hardened. When quite firm it is washed in water, passed through alcohols ascending in strength from 50 to 100 per cent., imbedded in cel- loidin, cut wet, and stained like a section of tissue. A ready method of doing this has been suggested by Winkler,1 who bores a hole in a block of paraffin with the smallest-size cork-borer, soaks the block in bichlorid solution for an hour, pours liquid gelatin into the cavity, allows it to solidify, inoculates it by the customary punc- ture of the platinum wire, allows it to develop sufficiently, and when ready cuts the sections under alcohol, subse- quently staining them with much-diluted carbol-fuchsin. Very pretty museum specimens of plate- and puncture- cultures in gelatin can be made by simultaneously killing the micro-organisms and permanently fixing the gelatin with formalin, which can either be sprayed upon the gelatin or applied in dilute solution. As gelatin fixed in formalin cannot subsequently be liquefied, such prep- arations will last indefinitely. The growths which occur upon agar-agar are in many ways less characteristic than those in gelatin, but as this medium does not liquefy except at a high temperature (ioo° C), it has that great advantage over gelatin. The colorless or almost colorless condition of the preparation also aids in the detection of such chromogenesis as may be the result of the micro-organismal growth. Sometimes the growth is colored, sometimes not ; some- times the production of a soluble pigment colors the agar-agar as well as the growth ; sometimes the growth is one color and the agar-agar another. Sometimes the growth is filamentous, sometimes a smooth, shining band. Occasionally the bacterium does not grow upon agar-agar unless glycerin be added (tubercle bacillus) ; sometimes it will not grow even then (gonococcus). Still less characteristic are the growths upon potato. Most bacteria produce rather smooth, shining, irregu- 1 Fortschritte der ATedicin, Bd. xi., 1893, No. 22. 214 PATHOGENIC BACTERIA. larly-extending growths, which often show very beautiful colors. Fig. 34. — New model incubating-oven with electro-regulator. In milk and litmus milk one must observe the presence or absence of acid-production, the coagulation which may CULTURES, AND THEIR STUDY. 215 or may not accompany it, and the subsequent gelatiniza- tion or digestion of the coagulum. Blood-serum is liquefied by some bacteria. The ma- jority of organisms are not very characteristic in their development upon it. Others, as the bacillus of diph- theria, are, however, characterized by their shape, color, and rapidity of development at given temperatures. While most of the saprophytic bacteria will grow well at the ordinary temperature of a well-warmed room, the important pathogenic forms require to be kept at the temperature of the body. To do this accurately an in- cubating oven becomes a necessity. Various forms, of wood and metal, are in the market, the one shown in the illustration (Fig. 34) being one of the newest and best. It scarcely need be pointed out that gelatin cultures cannot be grown in the incubating oven, as the medium will not remain solid at temperatures above 20-220 C. CHAPTER IX. THE CULTIVATION OF ANAEROBIC BACTERIA. The cultivation of micro-organisms which will not grow where the least amount of oxygen is present is always attended with much difficulty, and can seldom be accomplished with certainty. Many methods have been suggested, but not one can be described as satisfactory. Koch originally cultivated anaerobic bacteria upon plates by covering the surface of the soft gelatin with a thin film of mica previously sterilized by incandescence. Some anaerobic forms will grow quite well by such a simple exclusion of the air, but the strictly anaerobic forms will not develop at all. Hesse originated the plan, still sometimes followed, of making a deep puncture in recently boiled and rapidly sterilized gelatin or agar-agar, then covering the surface with sterilized oil, through which no oxygen was sup- posed to penetrate (Fig. 35). The fermentation-tube devised by Smith is an excel- lent ready means of determining that bacteria will de- velop under anaerobic conditions. The obligatory aerobs grow only in the bulb, the obligatory anaerobs only in the closed tube, the facultative or optional organisms in both. In order that the anaerobs can grow, Smith points out that it is essential that the bouillon contains some sugar. Iyiborius suggested the plan of having a tube nearly full of gelatin or agar-agar, boiling it just before inocu- lation, so as to expand and drive out whatever air it might contain, making the inoculation while the culture- medium was still fluid, cooling rapidly in ice-water, and sealing up the tube in a blowpipe as near the surface of the gelatin as possible. 216 CULTIVATION OF ANAEROBIC BACTERIA. 217 Esmarch used a regular "Esmarch tube," into the central cavity of which melted sterile gelatin was poured to exclude the air. Buchner invented a method by which, by the use of pyrogallic acid, the oxygen was absorbed from the atmo- sphere in which the culture was kept, and the growth allowed to continue in the nitrogen and carbonic acid which remained (Fig. 36). His method was to place the tube which had been inoculated in a much larger outer test-tube containing alkaline pyrogallic acid. The large Fig. 35. — Hesse's method of making anaerobic cultures. Fig. 36. — Buchner's method of making an- aerobic cultures. Fig. 37. — Frankel's meth- od of making anaerobic cul- tures. tube was closed with a rubber cap, and the absorption of the oxygen allowed to progress. Gruber, instead of absorbing the oxygen as Buchner does, prefers to use an air-pump and exhaust the contents of the tube. He uses a tube having a slender neck and a perforated rubber stopper. After the inoculation is made the air is pumped out and the slender neck sealed in the blowpipe. After this the tube can be warmed and 2l8 PATHOGENIC BACTERIA. the melted gelatin or agar-agar rolled on its sides, as sug- gested by Esmarch, if desired. Better than any of the preceding is the method of Frankel, which removes the air and replaces it by hy- drogen. Frankel prepares an ordinary Esmarch tube, removes the cotton stopper, and replaces it by a carefully sterilized rubber cork containing two tubes (Fig. 37). The tubes are connected with a hydrogen generator, and the gas is allowed to pass through until all the oxygen is forced out and replaced by the hydrogen, after which the ends of the tubes are sealed in the flame (Fig. 36). Liborius has designed a special tube for accomplish- ing the same thing. Kitasato and Weil found the addition of 0.3-0.5 per cent, of sodium formate to be of use in aiding the rapid- ity of the development of anaerobic cultures. Liborius found that 2 per cent, of glucose added to the culture- medium also increased the rapidity of the process. The methods now generally employed by bacteri- ologists for the anaerobic cultivations embrace all the essentials of the foregoing methods. One of the best arrangements for the purpose is that devised by Ravenel. His inoculations are deeply made in culture-media as free from air as possible. The tubes are loosely plugged, and are placed in an air-tight chamber the bottom of which contains pyrogallic acid — pyrogallic acid 1, solu- tion of caustic potash 1, water 10. The apparatus is connected by two tubes with an exhaust-pump on one side, and with a hydrogen apparatus on the other, by which means the atmosphere is exhausted and replaced by hydrogen until only pure hydrogen remains, after which the chamber is permanently sealed and the germs allowed to grow. Such a chamber can be constructed to hold a number of tubes or Petri dishes, yet not be too large to be stood in an incubator. Whatever oxygen may have escaped the exhaustion or have entered by the process of leakage is at once absorbed by the pyrogallic acid in the lower chamber of the apparatus. CULTIVATION OF ANAEROBIC BACTERIA. 219 Apparatus for plating out strictly anaerobic bacteria that have met with great favor are those invented by Botkin (Fig. 38) and Novy (Fig. 39). The first mentioned combines the replacement of the air by hydrogen and the absorption of the oxygen possibly remaining by alkaline pyrogallic acid; the other simply replaces the oxygen by hydrogen. In using Botkin' s apparatus the uncovered Petri dishes are placed one above the other in the rack c, and covered with the bell-glass A. Liquid paraffin is poured in the dish b until it is about half full. From a Kipp's apparatus hydrogen gas enters the little rubber tube a, subsequently escap- ing by the tube b. When only pure hydrogen escapes the rubber tubes a and b are withdrawn, and the appa- ratus remains filled with hy- drogen. Lest a little oxygen should remain, it is best to have the dishes at the top and bottom of the rack filled with alkaline pyrogallic acid. Tetanus can be cul- tivated in this apparatus. The jars recently intro- duced by Novy are similar in principle, depending upon the replacement of the air by hydrogen. They are so constructed that when the stopper occupies a certain relative position to the neck the gas can enter and exit, but when the stopper is turned a little the jar is hermet- ically sealed. Alkaline pyrogallic acid in a test-tube, or in the bottom of the jar, will serve to absorb any remain- ing oxygen. The larger jar (Fig. 39, a) is intended for Petri dishes, the smaller one (b) for test-tube cultures. Roux has suggested the simplest method of cultivating anaerobic. bacteria. The germs are distributed through Fig. 38. — Botkin's apparatus for mak- ing anaerobic cultures. 220 PATHOGENIC BACTERIA. freshly boiled, still liquid, gelatin or agar-agar, as in making the dilutions for plate-cultures, then drawn into a long, slender sterile piece of glass tubing of small calibre. When the tube is full the ends, which should have been narrowed, are closed in a flame, and the cul- Fig. 39. — Novy's jars for anaerobic cultures. ture is hermetically sealed in an air-tight chamber. The chief difficulty is in transplanting the growing colony. To do this the tube must be opened with a file or a dia- mond at the point where the colony desired is observed. CHAPTER X. EXPERIMENTATION UPON ANIMALS. Bacteriology has to-day become a science whose principal objects are to discover the cause, explain the symptoms, and prepare the cure of diseases. We can- not hope to achieve these objects except by the intro- duction of bacteria into animals, where their effects and the effects of their products can be studied. No one should more heartily condemn wanton cruelty to animals than the physician and the naturalist. In- deed, it is hard to imagine a class of men so much of whose lives is spent in relieving pain, and who know so much about pain, being guilty of the wholesale butchery and torture accredited to them by a few of the laity, whose eyes, but not whose brains, have looked over the pages of physiological text-books. Experimentation upon animals has given us almost all our knowledge of physiology, most of our valuable therapeutics, and the only scientific methods of treating tetanus and diphtheria. Experiments upon animals we must make, and, as animals differ in their susceptibility to diseases, large numbers and different kinds must be employed. The bacteriological methods are not cruel. Two prin- cipal modes of introducing bacteria are employed : the subcutaneous injection and the intravenous injection. Subcutaneous injections into animals are made exactly as hypodermic injections are given to man. Any hypodermic syringe that can be conveniently cleaned and disinfected may be employed for the purpose. Those expressly designed for bacteriological work and most frequently employed are shown in Fig. 40. Those 221 222 PATHOGENIC BACTERIA. of Meyer and Roux resemble ordinary hypodermic syringes; that of Koch is supposed to possess the decided advantage of not having a piston to come into contact with the fluid to be injected. This is, however, some- what disadvantageous inasmuch as the cushion of com- pressed air that drives out the contents is elastic, and un- less carefully watched will follow the injection into the body of the animal. In making subcutaneous injections there is no disadvantage or danger from the entrance of Fig. 40. — 1, Roux's bacteriological syringe; 2, Koch's syringe; 3, Meyer's bacteriological syringe. air beneath the skin, but in intravenous injections it is commonly supposed to be dangerous. All syringes should be disinfected with carbolic acid solutions before and after using, the carbolic acid being allowed to act for some time and then washed out with sterile water. Syringes should not be boiled, as it ruins the packings, whether of asbestos, leather, or rubber. The intravenous injections differ only in that the needle of the syringe is introduced into a vein. This is easy in a large animal like a horse, but is very difficult in a small animal, and wellnigh impossible in anything smaller than a rabbit. Such injections when given to rabbits are gen- erally made into the ear-veins, as those most conspicuous and accessible (Fig. 41). A peculiar and important fact to remember is, that the less conspicuous posterior vein EXPERIMENTATION UPON ANIMALS. 223 is much better adapted to the purpose than the anterior. The introduction of the needle should be made from the hairy surface of the ear. If the ear is manip- ulated for a moment or two before the injec- tion is begun, vaso- motor dilatation occurs and the blood- vessels all become larger and more con- spicuous. The vein should be compressed at the root of the ear until the needle is in- troduced, and the in- jection made as near *IG- 41- — Method of making an intravenous the root as possible. inJectio" into a rabblt\ ?bserv1e tbat the n!edle . , . - enters the posterior vein from the hairy surface. The introduction of bacteria into the lymphatics is only possible by injecting liquid preparations of them into some organ with com- paratively few blood-vessels and large numbers of lym- phatics. The testicle is best adapted to this purpose, the needle being introduced deeply into the organ. Sometimes the inoculation can be made by the platinum wire, a very small opening made in the skin by a snip of the scissors being sufficient. Sometimes intra-abdominal and intra-pleural injections are made, and in cases where it becomes necessary to determine the presence or absence of tuberculosis or glanders in tissues it may be necessary to introduce small pieces of the suspected tissue under the skin or into the abdominal cavities. To do this is not difficult. The hair is carefully, closely cut over the point of election, which is generally on the abdomen near the groin, the skin picked up with forceps, a snip made through it, and the points of the scissors introduced for half an inch 224 PATHOGENIC BACTERIA. or so and then separated. By this maneuver a subcuta- neous pocket is formed, into which the tissue is easily forced. The opening should not be large enough to re- quire subsequent stitching. Small animals, like rabbits and guinea-pigs, can be held in the hand, as a rule. Rabbit-holders of various forms can be obtained from dealers. Dogs, cats, sheep, and goats can be tied and held in troughs. A convenient form of mouse-holder, invented by Kitasato, is shown in Fig. 42. In all these experiments one must remember that the amount of material introduced into the animal must be in proportion to its size, and that injection-experiments upon mice generally are so crude and destructive as to warrant the comparison drawn by Frankel, that to inject a few minims of liquid into the pleural cavity of a mouse is " much the same as if one would inject through a fire- hose three or four quarts of some liquid into the respira- tory organs of a man. ' ' The blood of animals, when it is necessary to experi- ment with it, is best secured from a large vein, generally the jugu- lar. From small animals, such as guinea-pigs, it may be secured by introducing a small cannula into the carotid artery. Our observations of animals by no means cease with their death. Indeed, he cannot be a bacteriol- ogist who is not already a good pathologist and expert in the recog- nition of diseased organs. When an autopsy is to be made upon a small animal, it is best to wash it for a few moments in a disinfecting solution, to kill the germs present upon the hair and the skin, as well as to moisten the hair arid enable it to be kept out of the incision. Fig. 42. — Mouse-holder. EXPERIMENTATION UPON ANIMALS. 225 The animal should be tacked to a board if small, or tied, by cords fastened to the legs, to the corners of a table if large, and should be dissected with sterile knives and scissors. When a culture is to be made from the interior of an organ — say the spleen — it should be incised deeply with a sterile knife and the culture made from its centre. Fragments intended for subsequent microscopical ex- amination should be cut very small (cubes of 1 can,), placed in absolute alcohol for a few hours, then trans- ferred to weaker alcohol, 80-90 per cent., for preserva- tion. The technique of imbedding and staining the tis- sues can be found in almost any reliable text-book on pathology or on the special subject of microscopical technique. Collodion capsules are quite frequently employed for the purpose of growing bacteria in a confined position in the body of an animal, where they can freely receive and utilize the body-juices without being subjected to the action of the phagocytes. In such capsules the bacteria usually grow plentifully, and not rarely their virulence is greatly increased. The capsules can be made of any size, though they are probably most easily handled when of about 5-10 c.cm. capacity. Their size is always an objection to their use, because of the disturbance they cause in the animal's abdominal cavity when introduced. The capsules are made by carefully coating the outside of the lower part of a test-tube with collodion until a suf- ficiently thick homogeneous layer is formed. During the coating process the tube must be twirled alternately within and without the collodion, so that it is equally distributed upon its surface. When the desired thick- ness is attained, and the collodion is sufficiently firm, the tube is plunged under water and the hardening process checked. A cut is next made around the upper edge of the col- lodion film, and it is removed by carefully turning it 15 226 PATHOGENIC BACTERIA. inside out In this manner a mould of the test-tube is formed. The test-tube is next constricted to a degree that will not interfere with the future introduction of culture- media in a fine pipette or inoculation with a platinum loop, and that will permit of ready sealing in a flame when necessary ; the rounded end is then cut off, and the edges are smoothed in a flame. The collodion bag is now carefully fitted over the end of the tube, shrunken on by a gentle heating, and cemented fast with a little fresh collodion ap- plied to the line of union. Novy rec- ommends that a thread of silk be wound around the point of union, to hold the collodion in place and to aid in handling the finished sac. It now appears as in Fig. 43, b. The sac is next filled with distilled water up to the thread, the tube is plugged with cotton, and the whole placed in a larger test-tube containing distilled water, the cotton-plug being packed tightly around the smaller tube, so that the collodion sac does not reach the bottom of the large tube, but hangs suspended in the water it con- tains. The whole is now carefully sterilized by steam. When ready for use, a tube of bouillon is inoculated with the culture intended to be placed in the animal ; the water in the capsule is then pipetted out, and is re- placed by the inoculated bouillon carefully introduced with a pipette. The constricted portion is then sealed in a flame, and the capsule picked up with forceps and introduced into the peritoneum by an aseptic operation. Fig. 43. — Prepara- tion of collodion sacs : a, test-tube constricted and cut ; b, sac attached to the tube. CHAPTER XL THE RECOGNITION OF BACTERIA. The most difficult thing in bacteriology is to be able to recognize the bacteria which come under observation. A certain few micro-organisms are so characteristic in shape and grouping as to be separated by a microscopic examination. Some, as the tubercle bacillus, are charac- teristic in their reaction to the anilin dyes, and can be differentiated at once by this peculiarity. Some, as the Bacillus mycoides, are so characteristic in their agar-agar growth as to eliminate others. The red color of Bacillus prodigiosus and the blue of Bacillus janthinus will speak almost positively for them. The potato culture of the Bacillus mesentericus fuscus and its close relative the vul- gatus is quite sufficient to enable us to pronounce upon them. Unfortunately, however, there are several hun- dreds of described species which lack any one distinct character that may be used for differential purposes, and require that for their diagnosis we shall wellnigh ex- haust the bacteriological technique in an almost fruitless effort to recognize them. A series of useful tables has been compiled by Eisen- berg, and is now almost indispensable to the worker. Unfortunately, in tabulating bacteria we constantly meet species described so insufficiently as to make them worse than useless on account of the confusion caused. The only way to recognize a species is to study it thoroughly and compare it, step by step, with the descrip- tions and tables of known species compiled by Eisenberg and others. 227 CHAPTER XII. THE BACTERIOLOGIC EXAMINATION OF THE AIR. It has been repeatedly emphasized — and indeed at the present time almost every one knows — that micro-organ- isms float almost everywhere in the air, and that their presence there is a constant source of danger, not only of contamination in our bacteriologic researches, but also a menace to our health. Such micro-organisms are neither ubiquitous nor equally disseminated, but are much more numerous where the air is dusty than where it is pure — much more so where men and animals are accustomed to live, than upon the ocean or upon high mountain-tops. The purity of the atmo- sphere bears a distinct relation to the purity of the soil over which its currents blow. The micro-organisms that occur in the air are for the most part harmless saprophytes which have been sepa- rated from their nutrient birthplace and carried about by the wind. They are almost always taken up from dried materials, experiment having shown that they arise from the surfaces of liquids in which they grow with much dif- ficulty. They are by no means all bacteria, and a plate of sterile gelatin exposed for a brief time to the air will generally grow moulds and yeasts as well as bacteria. The bacteria present are occasionally pathogenic, espe- cially in localities where the discharges of diseased animals have been allowed to collect and dry. For this reason the atmosphere of the wards of hospitals and of rooms in which infectious cases are being treated is much more apt to contain them than the air of the street. However, the dried expectoration of cases of tuberculosis, of in- 228 BACTERIOLOGIC EXAMINATION OF AIR. 229 fluenza, and sometimes of pneumonia, causes the specific bacteria of these diseases to be far from uncommon in street-dust. Gunther points out that the majority of the bacteria which occur in the air are cocci, sarcina being very abundant. Most of them are chromogenic and do not liquefy gelatin. It is unusual to find a considerable variety of bacteria at a time ; generally not more than two or three species are found. It is an easy matter to determine whether bacteria are present in the air or not, all that is necessary being to expose sterile plates or Petri dishes of gelatin to the air for a while, close them, and observe whether or not bac- teria grow upon them. To make a quantitative estimation is, however, much Fig. 44. — Hesse's apparatus for collecting bacteria from the air. more difficult. Several methods have been suggested, of which the most important may be considered. The method suggested by Hesse is simple and good. It consists in making a measured quantity of the air to 230 PATHOGENIC BACTERIA. be examined pass through a horizontal sterile tube about 70 cm. long and 3.5 cm. wide (Fig. 44), the interior of which is coated with gelatin in the same manner as an Ksmarch tube. The tube, having been prepared, is closed at both ends with sterile corks carrying smaller glass tubes closed with cotton. When ready for use the tube at one end is attached to a hand-pump, the cotton is removed from the other end, and the air passed through very slowly, the bacteria having time to precipitate upon the gelatin as they pass. When the required amount has passed the tubes are again plugged, the apparatus stood away for a time, and subsequently, when they have grown, the colonies are counted. The number of colo- nies in the tube will represent pretty accurately the number of bacteria in the amount of air which passed through the tube. In such a cylindrical culture it will be noted that if the air is passed through with the proper slowness, the colonies will be much more numerous near the end of entrance than that of exit. The first to fall will probably be those of heaviest specific gravity — i, e. the moulds and yeasts. A still more exact method is that of Petri, who uses small filters of sand held in place in a wide glass tube by small wire nets (Fig. 45). The sand used is made to pass through a sieve whose openings are of known size, is heated to incandescence, then arranged in the tube so that two of the little filters, held in place by their wire-gauze coverings, are superimposed. One or both ends of the tube are closed with corks having a narrow glass tube. The apparatus is heated and sterilized in a hot-air sterilizer, and is then ready for The method of employment is very simple. By :'';:.'? Fig. 45 — Petri's sand filter for air- examination. use. means of a hand-pump 100 liters of air are made to pass through in from ten to twenty minutes. The sand from BACTERIOLOGIC EXAMINATION OF AIR. 231 L the upper filter is then carefully mixed with sterile melted gelatin and poured into sterile Petri dishes, where the colonies develop and can be counted. Sternberg re- marks that the chief objection to the method is the pres- ence in the gelatin of the slightly opaque sand, which interferes with the recognition and count- ing of the colonies. This objection has, (x^% however, been removed by Sedgwick and Miquel, who use a soluble material — granu- lated or pulverized sugar — instead of the sand. The apparatus used for the sugar- experiments differs a little from the original of Petri, but the principle is the same, and can be modified to suit the experimenter. Petri points out in relation to his method that the filter catches a relatively greater number of bacteria in proportion to moulds than the Hesse apparatus, which depends upon sedimentation. A particularly useful form of apparatus is a granulated sugar-filter suggested by Sedgwick and Tucker, which has an ex- pansion above the filter, so that as soon as the sugar is dissolved in the melted gela- tin it can be rolled out into a lining like that of an Esmarch tube. This cylindrical expansion is divided into squares which make the counting of the colonies very easy (Fig. 46). The number of germs in the atmosphere Fig. 46.— Sedg- will naturally be very variable. Roughly, ™e SfJxpa™ex- the number may be estimated at from 100 amination. to 1000 per cubic meter. In reality, the bacteriologic examination of air is of very little value, as so many possibilities of error may occur. Thus, when the air of a room is quiescent there may be very few bacteria in it ; let some one walk across the floor and dust at once rises, and the number 232 PATHOGENIC BACTERIA. of bacteria is considerably increased : if the person be a woman with skirts, more bacteria will probably be raised from the floor than would be disturbed by a man ; if the room be swept, the increase is enormous. From these and similar contingencies it becomes very difficult to know just when and how the air is to be examined, and the value of the results is correspondingly lessened. The most valuable examinations are those which aim at the discovery of some definite organism or organisms regardless of the number per cubic meter. CHAPTER XIII. BACTERIOLOGIC EXAMINATION OF WATER. Unless water has been specially sterilized or distilled and received and kept in sterile vessels, it always con- tains some bacteria. The number will bear a very dis- tinct relation to the amount of organic matter in the water, though experiment has shown that certain patho- genic and non-pathogenic bacteria can remain vital in perfectly pure distilled water for a considerable length of time. Ultimately, owing to the lack of nutriment, they undergo a granular degeneration. The majority of the water-bacteria are bacilli, and as a Fig. 47. — WolfhugePs apparatus for counting colonies of bacteria upon plates. rule they are non-pathogenic. Wright,1 in' his examina- tion of the bacteria of the water from the Schuylkill River, found two species of micrococci, two species of cladothrices, and forty-six species and two varieties of bacilli. Of course, at times the most virulent forms of pathogenic bacteria — those of cholera and typhoid fever — occur in polluted water, but this is the exception, not the rule. The method of determining quantitatively the number 1 Memoirs of the National Academy of Sciences, vol. vii., Third Memoir. 233 234 PATHOGENIC BACTERIA. of bacteria in water is very simple, and can generally be prosecuted without much apparatus. The principle de- pends upon the equal distribution of a given quantity of the water to be examined through a sterile liquid medium, and the subsequent solidification of this medium in a Fig. 48. — Heyroth's instrument fur counting colonies of bacteria in Petri dishes. thin layer, so that all the colonies which develop may be counted. The method, which originated with Koch, may be per- formed with the Koch plates or with Petri dishes or with Esmarch rolls. It is always best to make a num- ber of these plate-cultures with different amounts of the water to be examined, using, for example, 0.01, 0.1, 0.5, and 1.0 c.cm. added to a tube of gelatin, agar-agar, or glycerin agar-agar. The exact method must depend somewhat upon the quality of the water to be examined. If the number of bacteria per cubic centimeter is small, large quantities may be used, but if there are millions of bacteria in every cubic centimeter, it may be necessary to dilute the BACTER/OLOGIC EXAMINATION OF WATER. 235 water to be examined in the proportion of 1 : 10 or 1 : 100 with sterile water, mixing well, and making the plate- cnltnres from the dilutions. It is best to count all the colonies if possible, but when there are hundreds or thousands scattered over the plate, an average estimation of a number of squares ruled upon a glass background (Fig. 47), as suggested by Wolf hiigel, is most convenient. In his apparatus a large plate of glass is divided into small square di- visions, the diagonals being spe- cially indicated by color. The plate or Petri dish is stood upon the glass, and the number of colonies in a number of small squares is easily counted, and the total number of colonies es- timated. In counting the colo- nies a lens is indispensable. Special apparatuses have been devised for counting the Colo- re . • ,• 1 /T-%. 0\ Fig. 49. — Esmarch's instrument nies in Petri dishes (Fig. 48) , . . . , . v #° ^ ' for counting colonies of bacteria and in Esmarch tubes (Fig. 49). in tubes- The majority of the water- bacteria are rapid liquefiers of gelatin, for which reason it seems better to employ agar-agar than gelatin for making the cultures. In ordinary hydrant-water the bacteria number from 2-50 per cubic centimeter ; in good pump-water, 100-500 ; in filtered water from rivers, according to Giinther, 50-200 are present ; in unfiltered river-water, 6000-20,000. Ac- cording to the pollution of the water the number may reach as many as 50,000,000. The waters of wells and springs are dependent for their purity upon the character of the earth or rock through which they filter, and the waters of deep wells are much more pure than those of shallow wells, unless contamina- tion takes place from the surface of the ground. Ice always contains bacteria if the water contained 236 PATHOGENIC BACTERIA. them before it was frozen. In Hndson-River ice Prud- den found an average of 398 colonies in a cubic centi- meter. A sample of water when collected for examination should be placed in a clean sterile bottle or in a her- metically-sealed pre-sterilized glass bulb, and must be examined as soon as possible, as the bacteria multiply rapidly in water which is allowed to stand for a short time. In determining the species of bacteria found in the water reference must be made to the numerous mono- graphs upon the subject, and to tables such as those compiled by Eisenberg. The discovery of certain important pathogenic bacteria, as those of cholera and typhoid, will be considered under the specific headings. Unfortunately, the bacteriologic examination of waters does not throw satisfactory light upon their exact hygi- enic usefulness. Of course, if cholera or typhoid-fever bacteria are present, the water is harmful, but the quality of the water cannot be gauged by the number of bacteria it contains. The drinking-water furnished large cities is not infre- quently contaminated with sewage, and contains intes- tinal bacteria — Bacillus coli communis. For the ready determination of this organism, which is an important one as an indicator that the water is polluted, Smith1 has made use of the fermentation-tube in addition to the plate. His method is to add to each of the fermentation- tubes containing 1 per cent, dextrose-bouillon a certain quantity of water. The evolution of 50-60 per cent, of gas by the third day is a strong indication that the colon bacillus is present. Plates may be used to confirm the presence of the bacillus, but are hardly necessary, as there is scarcely another bacterium met with in water that is capable of producing so much gas. Filtration with sand, etc. diminishes the number of bacteria for a time, but, as the organisms multiply in 1 American "Journal of the Aledical Sciences, 1895, IIO, p. 301. BACTERIOLOGIC EXAMINATION OF WATER. 237 the filter, the benefit is not permanent. The filters must frequently be renewed. Porcelain filters seem to be the only positive safeguard, and even these, the best of which seems to be the Pasteur-Chamberland, allow the bacteria to pass through if userl too long without renewal or with- out firing. CHAPTER XIV. BACTERIOLOGIC EXAMINATION OF SOIL. Almost all soil contains bacteria in its upper layers. Their number and character, however, depend some- what upon the surrounding conditions. Near the hab- itations of men, where the soil is cultivated, the ex- crement of animals, largely made up of bacteria, is spread upon it to increase its fertility, this being a treat- ment which not only adds new bacteria to those already present, but also enables those present to grow very much more luxuriantly because of the increased amount of organic matter they receive. The researches of Fliigge, C. Frankel, and others show that the bacteria of the soil do not penetrate very deeply — that they gradually decrease in number until the depth of a meter is reached, then rapidly diminish until at a meter and a quarter they rather abruptly cease to be found. Many of the soil-bacteria are anaerobic, and for a careful consideration of them the reader must be referred to monographs upon the subject. The estimation of their number seems to be devoid of any dis- tinct practical importance. C. Frankel has, however, originated a very accurate method of determining it. By means of a special boring apparatus (Fig. 50) earth can be secured from any depth without digging and without danger of mixing that secured with that of the superficial strata. With sterile liquefied gelatin a definite 238 Fig. 50. — Fran- kel's instrument for obtaining earth from various depths for bacteriologic study. BACTERIOLOGIC EXAMINATION OF SOIL. 239 amount of this soil is mixed thoroughly and the mixture solidified upon the walls of an Esmarch tube. The col- onies are counted with the aid of a lens. Fliigge found in virgin earth about 100,000 colonies in a cubic centi- meter. Samples of earth, like samples of water, should be examined as soon as possible after being secured, for, as Gunther points out, the number of bacteria changes because of the unusual environment, exposure to increased amounts of oxygen, etc. The most important bacteria of the soil are those of tetanus and malignant edema, in addition to which, how- ever, there are a great variety which are pathogenic for rabbits, guinea-pigs, and mice. In the "Bacteriological Examination of the Soil of Philadelphia," Ravenel1 came to the conclusion that — 1. Made soils, as commonly found, are rich in organic matter and excessively damp through poor drainage. 2. They furnish conditions more suited to the multi- plication of bacteria than do virgin soils, unless the latter are contaminated by sewage or offal. 3. Made soils contain large numbers of bacteria per gram of many different species, the deeper layers being as rich in the number and variety of organisms as the upper ones. After some years the number in the deeper layers probably becomes proportionally less. Made soils are more likely than others to contain pathogenic bacteria. In 71 cultures that were isolated and carefully studied by Ravenel, there were two cocci, one sarcina, and five cladothrices; all the others were bacilli. 1 Memoirs of the National Academy of Sciences, First Memoir, 1896. CHAPTER XV. TO DETERMINE THE THERMAL DEATH-POINT. Several, methods may be employed for this purpose. Roughly, it may be done by keeping a bouillon-culture of the micro-organism to be studied in a water-bath whose temperature is gradually increased from that of the body to 750 C. Into a fresh bouillon-culture thus exposed to heat, the experimenter cautiously, and at given intervals, intro- duces a platinum loop or a capillary pipette, and with- draws a drop of the culture which he inoculates into fresh bouillon and stands aside to grow. It is economy to make the transplantations rather infrequently at first and frequently later on in the experiment, when the tem- perature is ascending. In an ordinary determination it would be well to make a transfer at 400 C, one at 450 C, another at 500, still another at 550, and then beginning at 6o° make one for every additional degree up to 750 C. or above. The day following the experiment it will be observed that all the cultures grow except those heated beyond a certain point, as 6o° C. and upward, when it can properly be concluded that 6o° C. is the thermal death-point. If all the transplantations grow, of course the maximum temperature that the bacteria can endure was not reached, and the experiment must be performed again with higher temperatures. When more accurate information is desired, and one wishes to know how long the micro-organism can endure some such temperature as 6o° C. without losing its vital- ity, a dozen or more bouillon-tubes may be inoculated with the germ to be studied, and stood in the water-bath at the temperature to be investigated. The first can be 240 TO DETERMINE THERMAL DEATH-POINT. 241 removed as soon as it is certainly heated through, another in five minutes, another in ten minutes, or at whatever intervals the thought and experience of the experimenter shall suggest. In both of the described procedures one must be care- ful that the temperature in the test-tube is identical with that of the water in the bath. There is no reason why a sterile thermometer should not be placed in the heated tube in the first case, and in the second experiment one of the test-tubes exposed under conditions similar to the others might contain a thermometer which would show the temperature of the contents of the tube containing it, and so be an index of the rest. Another method of accomplishing the same end is to use Sternberg's bulbs. These are small glass bulbs blown on one end of a piece of glass tubing, which is subsequently drawn out to capillarity at the opposite end. If such a bulb be heated, and its capillary tube dipped into inoculated bouillon, in cooling, the fluid is drawn in so as to fill it one-third or one-half. A number of these tubes are filled in this manner with freshly inoculated culture-medium and then floated, tube upward, upon a water-bath whose temperature is gradually elevated, the bulbs being removed from time to time as the required temperatures are reached. Of course, as the bulbs are already inoculated, all that is necessary is to stand them aside for a day or two, and observe whether or not the bacteria grow, judging the death-point exactly as in the other case. To Determine the Antiseptic and Germicidal Value of Reagents. — There are various methods whose modi- fications can be elaborated according to the extent and thoroughness of the investigation to be made. I. The Antiseptic Value. — Remembering that an anti- septic is a substance that inhibits bacterial growth, the method that will at once suggest itself is that of adding varying quantities of the antiseptic to be investigated to culture-media in which the bacteria are subsequently 16 242 PATHOGENIC BACTERIA. planted. It is always well to use a considerable number of tubes. Bouillon is generally employed. If the anti- septic is non-volatile, it may be added before sterilization, which is to be preferred; but if it is volatile, it must be added by means of a sterile pipette, with the greatest precaution as regards asepsis, immediately before the test is to be made. Control-experiments — i. e. without the addition of the antiseptic — should always be made. The results of antiseptic action are two: retardation of growth and complete inhibition of growth. As the tubes used for the study of the antiseptic are watched in their development, it will usually be noticed that those con- taining very small quantities develop almost as rapidly as the control-tubes; those containing more, a little more slowly; those containing still more, very slowly, until at last there comes at time when the growth is not deferred, but prevented. Sternberg points out that certain circumstances may modify the results obtained. They are: i. The composition of the nutrient media, with which the antiseptic may be incompatible. 2. The nature of the test-organism, no two organisms being exactly alike in their susceptibility. 3. The temperature at which the experiment is con- ducted, a relatively greater amount of the antiseptic being necessary at temperatures favorable to the organ- ism than at temperatures unfavorable. 4. The presence of spores which are always more resistant than the asporogenous forms. II. The Germicidal Value. — Koch's original method of doing this was to dry the micro-organisms upon sterile shreds of linen or silk, and then soak them for varying lengths of time in the germicidal solution. After the bath in the reagent the threads were washed in clean, sterile water and then transferred to fresh culture- media, and their growth or failure to grow observed. It will be observed that this method is aimed at the deter- mination of the time in which a certain solution will kill. TO DETERMINE THERMAL DEATH-POINT. 243 Sternberg suggested a method in which the time should remain constant (two hours' exposure), and the object be the determination of the exact dilution of the reagent required to destroy the bacteria. " Instead of subjecting a few of the test-organisms attached to a silk thread to the action of the disinfecting agent, a certain quantity of the recent culture — usually 5 c.cm. — has been mixed with an equal quantity of a standard solution of the germicidal agent, . . . and after two hours' contact one or two ose-fuls would be introduced into a suitable nutri- ent medium to test the question of disinfection." A very simple and popular method of determining the germicidal value is to make a series of dilutions of the reagent to be tested ; add to each a couple of loopfuls of a fresh liquid culture, and at varying intervals of time transfer a loopful to fresh culture-media. By a little ingenuity this method may be made to yield information as to both time and strength. When it is desired to secure information concerning the progress of the germicidal action of reagents, body- fluids, etc., especially in the unusual and interesting cases in which the material subjected to the test may exert a restraining action for a time only, or bring about destruction of some or many, but not all of the germs, the use of the Petri dish can be introduced. For example, it is desired to determine whether a blood-serum is germicidal or not. Into about 5 c.cm. of the serum contained in a test-tube, two or three loopfuls of any desired bacterium, in liquid culture, are added. The tube is well agitated and immediately one loopful is transferred to a tube of melted gelatin, distributed through it, and poured into a Petri dish. After one minute the operation is repeated, in five minutes again, and so on as often as is desired. The dishes are stood away until the bacteria develop into colonies, which are then counted with a Wolfhiigel apparatus. On the first dish there may be 100 colonies; on the second, 80; on the third, 50; on the fourth, 20; on 244 PATHOGENIC BACTERIA. the fifth, 30; on the sixth, 150; on the seventh, 1000, etc. ; indicating that the serum exerted a destructive action upon some but not all of the bacteria, and that this power disappeared after the lapse of a certain time, allowing the bacteria to develop ad libitum. When the germicide to be studied is a gas, as in the case of sulphurous acid or formaldehyd, a different method must, of course, be adopted. It may be sufficient simply to place a few test-tube cul- tures of various bacteria, some with plugs in, some with plugs out, in a closed room in which the gas is afterward evolved. The germicidal action is shown by the failure of the cultures to grow upon transplantation to fresh cul- ture-media. This crude method may be supplemented by an examination of the dust of the room. Pledgets of sterile cotton are rubbed upon the floor, washboard, or any dust-collecting surface present, and subsequently dropped into culture-media. Failure of growth under such circumstances is very certain evidence of good dis- infection. These tests are, however, very severe, for in the cultures there are immense numbers of bacteria in the deeper portions of the bacterial mass upon which the gas has no opportunity to act, and in the dust there are many sporogenous organisms of extreme resisting power. Failure to kill all the germs exposed in such manner is no indication that the vapor cannot destroy all the ordi- nary pathogenic organisms. More refined is the method of saturating sterile sand or fragments of blotting-paper or absorbent cotton with cultures and exposing them, moist or dry, to the action of the gas. Such materials are best made ready in Petri dishes, which are opened immediately before and closed immediately after the experiment. A piece of cotton or blotting-paper or a little sand transferred to fresh culture- media will not give any growth where the disinfection has been thorough. By transplanting from different depths, the sand may be used incidently to show to what depth the gas is capable of penetrating. TO DETERMINE THERMAL DEATH-POINT. 245 Easier of execution, but rather more severe, is a method in which cover-glasses are employed. A num- ber of them are spread with cultures of various bacteria, allowed to dry, and then exposed to the gas as long a's required. The cover-glasses are afterward dropped into culture-media to permit the growth of the germs not destroyed. Animal-experiments may also be employed to deter- mine whether or not a germ that has survived exposure to the action of reagents has its pathogenic power destroyed. An excellent example of this is seen in the case of the anthrax bacillus, a virulent form of which will kill rab- bits, but after being grown in media containing an insufficient amount of a germicide to kill it will often lose its rabbit-killing power, though still able to fatally infect guinea-pigs, or may lose its virulence for both rabbits and guinea-pigs, though still able to kill white mice. PART II. SPECIFIC DISEASES AND THEIR BACTERIA. A. THE PHLOGISTIC DISEASES. I. THE ACUTE INFLAMMATORY DISEASES. CHAPTER I. SUPPURATION. Suppuration was at one time supposed to be an inevitable outcome of the majority of wounds, and, although bacteria were observed in the discharges, the old habit of thought and insufficiency of information caused most surgeons to believe that they were sponta- neously developed there. The development of antiseptic surgery, and the ex- tremes to which it was carried by specialists, almost attain to the ridiculous, for not only were the hands of the operator, his instruments, sponges, sutures, ligatures, and dressings kept constantly saturated with irritating germicidal solutions, but at one time the air over the wound was carefully saturated with pulverized antiseptic lotions during the whole operation by means of a steam atomizer. This rather monstrous outcome of the appli- cation of Lister's system to surgery was the very natural result of the erroneous idea that the germs which cause the suppurative changes in wounds entered the exposed tissues principally from the atmosphere, and that the hands and instruments of the operator, while certainly means of infection, were secondary in importance to it. The researches of more recent date, however, have shown not only that the atmosphere cannot be disin- fected, but also that the air of ordinarily quiet rooms, 246 SUPPURA TION. 247 while containing numerous saprophytic organisms, very rarely contains many pathogenic bacteria. We now also know that a direct stream of air, such as is generated by an atomizer, causes more bacteria to be conveyed into a wound than would ordinarily fall upon it, thereby in- creasing instead of lessening the danger of infection. It may therefore be stated, with a reasonable amount of certainty, that the atmosphere is rarely an important factor in the process of suppuration. Suppuration, while in most cases the result of micro- organismal operation, is not a specific infectious process, but a form of reaction that may result from a variety of injurious agents. The pustules of croton oil, the ex- perimental pleurisies produced by turpentine, etc., are examples of the non-specific form of the process. Being, therefore, only the expression of violent irrita- tion of the tissue, it is to be expected that as many bac- teria as are capable of producing marked local damage of the tissues together with chemotactic influences, may be found associated with it. Very naturally some bac- teria with these powers are more usual in occurrence than others and will appear as the common causes of the process, while every now and then some familiar organ- ism that has not previously manifested phlogistic powers, unexpectedly makes its appearance in pus. We have already called attention to the fact that vari- ous micro-organisms are so intimate in their relation to the skin that it is almost impossible to get rid of them, and have cited in this relation the experiments of Welch,1 Robb, and Ghriskey, whose method of disinfecting the hands has been recommended. The investigations of these observers have shown that, no matter how rigid the disinfection of the patient's skin, the cleansing of the operator's hands, the sterilization of the instruments, and the precautions exercised, a certain number of wounds in which sutures are employed will always sup- purate. The cause of the suppuration is a matter of vast 1 Amer. Jour. Med. Sciences, 1891, p. 439 248 PATHOGENIC BACTERIA. importance in surgery and in surgical bacteriology, yet it is one which it is impossible to remove. We carry it constantly with us upon our skins. Staphylococcus Epidermidis Albus. Welch has described, under the name Staphylococcus epidermidis albus, a micrococcus which seems to be habit- ually present upon the skin, not only upon the surface, but also deep down in the Malpighian layer. He is of the opinion that it is the same organism which is familiar to us under the name of Staphylococcus pyogenes albus, but in an attenuated condition. If his opinion be correct, and we have seated deeply in our derm a coccus which can at times cause abscess-formation, the conclusions of Robb and Ghriskey, that sutures of catgut when tightly drawn may be a cause of skin-abscesses by predisposing to the development of this organism, are certainly justi- fiable. Not only does the coccus occur in the attenuated form described, but we have very commonly present upon the skin, generally as a harmless saprophyte, the important Staphylococcus pyogenes albus, which is a common cause of suppuration. Staphylococcus Pyogenes Albus. Although, as stated, the Staphylococcus pyogenes albus is a common cause of suppuration, it rarely occurs alone, the studies of Passet showing that in but 4 out of 33 cases which he investigated was this coccus found by itself. When pure cultures of the coccus are injected subcu- taneously into rabbits and guinea-pigs, abscesses some- times result ; sometimes there is no result. Injected into the circulation of these animals, the staphylococci sometimes cause septicemia, and after death can be found in the capillaries, especially of the kidneys. From these illustrations it will be seen that the organism is feebly pathogenic. In its vegetative characteristics the Staphylococcus SUPPURA TION. 249 albus is almost identical with the species next to be de- scribed, but differs from it in that there is no golden color produced. Upon the culture-media it grows white. Staphylococcus Pyogenes Aureus. Generally present upon the skin, though in smaller numbers, is the dangerous and highly virulent Staphylo- coccus pyogenes aureus (Fig. 51), or "golden staphylococ- cus ism identical with those of the preceding species, it seems convenient to describe them together, pointing out such " of Rosenbach. As the morphology of this organ- , and indeed the generality of its characters, are Fig. 51. — Staphylococcus pyogenes aureus, from an agar-agar culture; x iooo (GUnther). differences as occur step by step. In doing this, how- ever, it must not be forgotten that, although the Staphy- lococcus albus has been described first, the Staphylococcus aureus is the more common organism of the suppurative diseases. Although they had been seen earlier by several ob- servers, the staphylococci were not isolated and care- fully described until 1884, when Rosenbach worked upon them. The results of his study, followed by Passet and a host of others, have now given us pretty accurate information about them. 250 PATHOGENIC BACTERIA. The cocci are distributed rather sparingly in nature, seeming not to find a purely saprophytic existence a suitable one. They occur, however, wherever man and animals have been, and can be found in the dust of houses, hospitals, and especially surgical wards where proper precautions are not exercised. They are common upon the skin, they live in the nose, mouth, eyes, and ears of man, they are nearly always beneath the finger- nails, and they sometimes occur in the feces, especially in children. The cocci are rather small, measuring about o. 7 ft in diameter. When examined in a delicately-stained con- dition the organisms may be seen to consist of hemi- spheres separated from each other by a narrow interval. The contiguous surfaces are flat, thus differing from the gonococcus, whose contiguous surfaces are concave. The grouping is not very characteristic. In both liquid and solid culture-media the organisms either occur in solid masses or are evenly distributed. It is only in the organs or tissues of a diseased animal that it is possible to say that a true staphylococcus grouping is present. The organism stains brilliantly with aqueous solu- tions of the anilin dyes. In tissues it can be beautifully stained by Gram's method. The staphylococci grow well either in the presence or absence of oxygen at a temperature above 180 C, the most rapid development being at about 370 C. Upon the surface of gelatin plates small whitish points can be observed in forty-eight hours (Fig. 52). These rapidly extend to the surface and cause extensive liquefaction. Hand in hand with the liquefaction is the formation of an orange color, which is best observed at the centre of the colony. Under the microscope the colonies appear as round disks with circumscribed, smooth edges. They are distinctly granular and dark-brown. When the col- onies are grown upon agar-agar plates the formation of the pigment is much more distinct. In gelatin punctures the growth occurs along the whole length of the needle-track, and causes an extensive lique- SUPPURA TION. 251 faction in the form of a long, narrow, blunt-pointed, in- verted cone (Fig. 53) full of clouded liquid, at the apex of which a collection of golden or orange-yellow precipitate is always present. It is this precipitate in particular that gives the organism its name, "golden staphylococcus." The growth of the golden staphylococcus upon agar- agar is subject to considerable variations in the color produced. Sometimes, perhaps rarely, it is golden ; more commonly it is yellow, often cream color. Aloug the whole line of inoculation a moist, shining, usually well-circumscribed growth occurs. When the growth takes place rapidly, as in the incubator, it exceeds the rapidity of color-production, so that the centre of the growth is distinctly colored, the edges remaining white. Fig. 52. — Staphylococcus pyogenes aureus: colony two days old, seen upon an agar-agar plate; X40 (Heim). Upon potato the growth is luxuriant, producing an orange-yellow coating over a large part of the surface. The potato-cultures give off a sour odor. When grown in bouillon the organism causes a diffuse cloudiness. In milk coagulation takes place, and is followed by gradual digestion of the casein. The Staphylococcus albus is exactly the same as the aureus, with the exception that in all media it is con- stantly colorless. 252 PATHOGENIC BACTERIA. Experiments have shown that the Staphylococcus aureus, like its congener, the albus, exists in an atten- uated form, and there is every reason to believe that in the majority of instances it inhabits the surface of the body in that condition. When virulent the golden staphylococcus is a danger- ous and often deadly organism. Its pathogeny among animals is decided. When introduced subcutaneously, abscesses almost invariably follow, except in a certain Fig. 53. — Staphylococcus pyogenes aureus : puncture-culture three days old in gelatin (Frankel and Pfeiffer). few comparatively immune species, and not infrequently lead to a fatal termination. In such cases the organisms may be cultivated from the blood of the large vessels, though by far the greater number collect in, and fre- quently obstruct, the capillaries. In the lungs and spleen, and still more frequently in the kidneys, infarcts are formed by the bacterial emboli. The Malpighian SUPPURA TION. 253 tufts of the kidneys sometimes are full of cocci, and become the centres of small abscesses. The coccus is almost equally pathogenic for man, though the fatal outcome is much more rare. It enters the system through scratches, punctures, or abrasions, and when virulent generally causes an abscess, as various experimenters who inoculated themselves have discov- ered to their cost. Garre applied the organism in pure culture to the uninjured skin of his arm, and in four days developed a large carbuncle with a surrounding zone of furuncles. Bockhart suspended a small portion of an agar-agar culture in salt-solution, and scratched it gently into the deeper layers of the skin with his finger- nail ; a furuncle developed. Bumm injected the coccus suspended in salt-solution beneath his skin and that of sev- eral other persons, and produced an abscess in every case. The Staphylococcus aureus is not only found in the great majority of furuncles, carbuncles, abscesses, and other inflammatory diseases of the surface of the body, but also plays an important role in a number of deeply- seated diseases of the internal organs. Becker and others obtained it from the pus of osteomyelitis, demonstrating that if, after fracturing or crushing a bone, the staphylo- coccus was injected into the circulation, osteomyelitis would result. Numerous bacteriologists have demon- strated its presence in ulcerative endocarditis. Rodet has been able to produce osteomyelitis without previ- ous injury to the bones ; Rosenbach was able to produce ulcerative endocarditis by injecting some of the staphy- lococci into the circulation in animals whose cardiac valves had been injured by a sound passed into the carotid artery ; and Ribbert has shown that the injection of cultures of the organism may cause the valvular lesion without the preceding injury. The Staphylococcus aureus is an easy organism to ob- tain, and can be secured by plating out a drop of pus in gelatin or in agar-agar. Such a preparation, however, generally does not contain the Staphylococcus aureus alone, but shows colonies of the Staphylococcus albus as 254 PATHOGENIC BACTERIA. well. In addition to these two principal forms, one sometimes discovers an organism identical with the pre- ceding, except that its growth on agar-agar and potato is of a brilliant lemon-yellow color, and its pathogeny for animals much less. This is the Staphylococcus citreus of Passet. It is not quite so common, and not so patho- genic as the others, and consequently much less im- portant. Streptococcus Pyogenes. Another organism whose colonies are frequently ob- tained from the pus containing the staphylococci is the Streptococcus pyogenes of Rosenbach (Fig. 54). It was found by him in 18 of 33 cases of suppurative lesions studied, fifteen times alone and five times with the Staphylococcus aureus. It is a spherical organism of variable size (0.4-1 ft in diameter), constantly Fig. 54. — Streptococcus pyogenes, from the pus taken from an abscess; x iooo (Frankel and Pfeiffer). Fig. 55. — Streptococ- cus pyogenes : culture upon agar-agar two days old (Frankel and Pfeif- fer). associated in pairs and chains of from four to twenty in- dividuals. A special variety of it, known as Streptococ- SUPPURA TION. 255 cus longus, sometimes forms chains of more than one hundred members. The organism stains well with ordinary aqueous solu- tions of the anilin dyes, and also by Gram's method. Like the coccus already described, it is not motile and does not seem to form spores, though sometimes a large individual — much larger than the others in its chain — may be ob- served, and may suggest the thought of arthro-sporulation. Upon gelatin plates very small colonies of translucent appearance are observed. When superficial, they spread out to form flat disks about 0.5 mm. in diameter. The microscope shows them to be irregular and granular, to have a slightly yellowish color, and to have numerous irregularities around the edges, due to projecting chains of the cocci. No liquefaction occurs. In gelatin puncture-cultures no liquefaction is observed. The minute spherical colonies grow along the whole needle-track and form a slightly opaque granular line. Upon agar-agar an exceedingly delicate transparent growth develops slowly along the line of inoculation. It consists of almost transparent, colorless small colonies which do not become confluent. The growth upon blood-serum much resembles that upon agar-agar. The streptococcus does not seem to grow upon potato, or produces an invisible growth only. In bouillon the cocci develop rather slowly, seeming to prefer a neutral or feebly acid reaction. The culture- medium remains clear, while numerous small flocculi are suspended in it. When the flocculi-formation is very distinct the name Streptococcus conglotneratus is used to describe the organism. These masses sometimes ad- here to the sides of the tube; sometimes they form a sedi- ment. Rarely, there is general clouding of the medium {Streptococcus diffusus). In mixtures of bouillon and blood-serum or ascitic fluid the streptococcus grows much better, especially at incubation temperatures, and in such mixtures the lux- uriant development causes the liquid to appear clouded. 256 PATHOGENIC BACTERIA. The organism seems to grow well in milk, which is coagulated and digested. The streptococcus is not very sensitive to acids, and can be grown quite well in media with a slightly acid reaction. Sternberg found that the streptococci succumb to a temperature of 52°-54° C. continued for ten minutes. Their vitality in culture is not great. Unless fre- quently transplanted they die. In bouillon they are said to die in five to ten days. On solid media they seem to retain their vegetative and pathogenic powers much longer. They resist drying well. Their growth in arti- ficial media is accompanied by the production of an acid which probably acts destructively upon the bacteria themselves. The Streptococcus pyogenes is generally not very patho- genic for animals. Subcutaneous injections into mice and rabbits are, as a rule, without either general or local manifestations of importance. If, however, an ear of a rabbit is carefully inoculated with a small amount of a pure culture, a small patch resembling erysipelas usually results. The disturbance passes away in a few days and the animal recovers. If, however, the streptococcus is highly virulent, the rabbit dies in from twenty-four hours to six days from a general septicemia. The cocci may be found in large numbers in the heart's blood and in the organs. In less virulent cases minute disseminated abscesses are some- times found. According to Marmorek,1 the virulence can be increased to a remarkable degree by rapid passage through rabbits, and maintained by the use of a culture-medium consist- ing of three parts of human blood-serum and one of bouillon. The blood of the ass, and ascitic and chest fluids may also be used. By these means Marmorek suc- ceeded in intensifying the virulence of his culture to such a degree that one hundred millionth of a c.cm. injected into the ear vein was fatal to a rabbit. 1 Ann. de P Inst. Pasteur, Tome ix., No. 7, July 25, 1895, p. 593. SUPPURA TION. 257 Petruschky l found the virulence of the culture to be well retained if the culture was planted in gelatin, trans- planted every five days, and when grown kept on ice. Hoist2 succeeded in keeping an exceedingly virulent Streptococcus brevis on artificial culture-media for eight years without any particular precautions and found its virulence unchanged. Probably the virulence and attenuation are peculiarities of the organism itself. Dried streptococci are said by Frosch and Kolle to re- tain their energies longer than those growing on culture- media.3 Like the staphylococci, the streptococcus is frequently associated with internal diseases, and has been found ill erysipelas, ulcerative endocarditis, periostitis, otitis, men- ingitis, emphysema, pneumonia, lymphangitis, phleg- mons, sepsis, and in the uterus in cases of infective puer- peral endometritis. In man the streptococci occur in the most active forms of suppuration. Its relation to diph- theria is of interest, for, while, in all probability, the great majority of cases of pseudomembranous angina are caused by the Klebs-Loffler bacillus, yet an undoubted number of cases are met with in which, as in Prudden's 24 cases, no diphtheria bacilli can be found, but which seem to be caused by a streptococcus exactly resembling that under consideration. There is no clinical difference in the picture of the throat-lesion produced by the two organisms, and the only positive method of diagnosticating the one from the other is by means of a careful bacteriologic examina- tion. Such an examination should always be made, as it has much weight in connection with the treatment. Of course, in streptococcus angina no benefit could be ex- pected from the diphtheria antitoxic serum. 1 Centralbl. filr Bakt. utid Parasitenk., Bd. xviii., No. 1 6, May 4, 1 895, P- 55«. 2 Ibid., Bd. xix., No. II, Mar. 21, 1896. 3 Fiugge's Die Mikroorganismen. 17 258 PATHOGENIC BACTERIA. Hirsli x has shown that under pathological conditions streptococci are by no means rare organisms in the in- testinal canal of infants, and may cause a streptococcic enteritis. In these cases the organisms are found in large numbers in the stomach and in the stools, and later in the course of the disease in the blood and urine of the living child and in the internal organs of the cadaver. Liebman2 reports two cases of streptococcic enteritis that were cerefully studied bacteriologically. Flexner,3 in a series of autopsies upon cases of death from various diseases, found the bodies invaded by num- erous micro-organisms, causing what he has called " term- inal infections," and hastening the fatal issue. Of 793 autopsies at Johns Hopkins Hospital, 255 from chronic heart or kidney diseases, or both, were sufficiently well studied bacteriologically to meet the needs of a statis- tical inquiry. Tubercular infection was not included. Of the 255 cases, 213 gave positive bacteriological results. "The micro-organisms causing the infections, 38 in all, were the Streptococcus pyogenes, 16 cases; Staphylococcus pyogenes aureus, 4 cases; Micrococcus lanceolatus, 6 cases; gas bacillus (B. Aerogenes capsulatus), three times alone and twice combined with the Bacillus coli communis; the gonococcus, anthrax bacillus, Bacillus proteus, the last combined with the Bacillus coli, the Bacillus coli alone, a peculiar capsulated bacillus, and an unidentified coc- cus. ' ' It is interesting to observe how many cases were accompanied by the streptococcus. All the streptococci may not have been streptococcus pyogenes, but for con- venience in his statistics they were regarded by Flexner as identical. The presence of streptococci in the blood in scarlatina has been observed in 30 cases by Crooke, by Frankel and Trendenburg, Raskin, Leubarth, Kurth, and Babes. 1 Centralbl. fur Bakt. und Parasitenk., Bd. xxii., Nos. 14 and 15, p. 369. 2 Ibid., Bd. xxii., Nos. 14 and 15, p. 376. 3 Journal of Experimental Medicine, vol. i., No. 3, 1896. SUPPURA TION. 259 In 11 cases studied by Wright1 a general streptococcus infection occurred in 4, a pneumococcic infection in 1, and a mixed infection of pyogenic cocci in 1. Lemoine2 found streptococci in the blood during life in 2 out of 33 cases studied. Pearce s studied 17 cases and found streptococci in the heart's blood and liver in 4 cases, in the spleen in 2 cases, in the kidney in 5 cases. In 2 of the cases it was mixed with the Staphylococcus pyogenes aureus. The streptococcus is the most common bacterium found in the suppurative sequelae of scarlatina, fre- quently occurring alone, sometimes with the staphylo- cocci, sometimes with the pneumococci. As found asso- ciated with scarlatina the organisms have no peculiarities by which it is possible to differentiate them from the Staphylococcus pyogenes. The streptococcus of Rosenbach is thought to be iden- tical with a streptococcus described by Fehleisen as the Streptococcus erysipelatis (Fig. 56). It may seem unwise to omit the Streptococcus erysipelatis as a major topic for discussion, but the similarity of the organism to that just described has caused me to consider them in the same chapter. The streptococci of erysipelas can be obtained in almost pure culture from the serum which oozes from a puncture made in the margin of an erysipelatous patch. They are small cocci, forming long chains — generally from six to ten individuals, but sometimes reaching a hundred in number. Occasionally the chains can be found collected in tangled masses. They can be cultivated at the room- temperature, but grow much better at 30-370 C. They are not particularly sensitive to the absence of oxygen, but develop a little more rapidly in its presence. The erysipelas cocci, like the Streptococcus pyogenes, are not motile, form no spores, and are destroyed by a 1 Boston Med. and Surg. Journal, March 21, 1895. 2 Bull, et mini. Soc. d'hdp. de Paris, 1896, 3 sM xiii. 8 Journal of the Boston Society of the Medical Sciences, March, 1898. 260 PATHOGENIC BACTERIA. low degree of heat. They stain well with aqueous solu- tions of anilin dyes and also by Gram's method. The colonies upon gelatin and the development in gelatin tubes, upon agar-agar, and upon blood-serum are identical with the descriptions of the Streptococcus pyogenes. No growth occurs on potato. The growth in bouillon is generally luxuriant, and in a short time causes the medium to be filled with chains of the cocci. As the growth progresses these chains gather in clusters and fall to the bottom as a whitish Fig. 56. — Streptococcus erysipelatis, seen in a section through human skin ; x 500 (Frankel and Pfeiffer). granular precipitate, above which the liquid remains clear. When injected into animals Fehleisen's coccus behaves exactly like the Streptococcus pyogenes. Observation has shown that dire results may follow the entrance of this organism into exposed wounds, and that it causes not only local suppuration, but sometimes a general infection. The empiric experience that the occasional accidental SUPPURA T/OJV. 261 infection of malignant tumors with erysipelas cocci was followed by sloughing and subsequent disappearance of the tumor, suggested inoculation with the Streptococcus erysipelatis as a therapeutic measure. The dangerous character of the remedy, however, caused many to re- frain from its use, for when one inoculated the living erysipelas germs into the tissues he never could estimate the exact amount of disturbance that would follow. The difficulty seems to have been overcome by Coley, who recommends the toxin instead of the living coccus for injection. A virulent culture is obtained, inoculated into small flasks of slightly acid bouillon, allowed to grow for three weeks, then reinoculated with Bacillus prodigiosus, allowed to grow for ten or twelve days at the room-temperature, well shaken up, poured into bottles of about f 3ss capacity, and rendered perfectly sterile by an exposure to from 50-600 C. for an hour. It is claimed that the combined toxins of erysipelas and prodigiosus are much stronger than the simple erysipelas toxin. The best effects are found in cases of sarcoma, where the toxin causes a rapid necrosis of the tumor tissue, which can be scraped out with an appropriate instrument. Numerous cases are on record in which this treatment has been most efficacious ; but, although Coley recom- mends it and Czerny still upholds it, the majority of sur- geons have failed to secure the desired results. Recently (1895) considerable attention has been be- stowed upon the anti-streptococcus serum of Marmorek, which is said to act specifically upon cases of strepto- coccus-infection, both general and local. Numerous cases are upon record in which the serum seemed to exert a beneficial action. It would seem as if an antiphlogistic serum should occupy an important place in the future of medicine. The serum is prepared upon the same plan as that of Behring, except that living virulent streptococci instead of the sterile toxin are injected into the horse. 262 PATHOGENIC BACTERIA. Bacillus Pyocyaneus. In some cases the pus evacuated from wounds exhibits a peculiar bluish or greenish color, from the presence of wi • US**.* « J v T , *«v Fig. 57. — Bacillus pyocyaneus, from an agar-agar culture; x 1000 (Itzerott and Niemann). the Bacillus Pyocyaneus (Figs. 57, 58). This is a short, delicate bacillus of small size, measuring 0.3 : 1-2 /•«, ac- cording to Fliigge, frequently united in chains of four or six. It has round ends, is actively motile, has one terminal flagellum, does not form spores, and can exist with or without oxygen, though it is an almost purely aerobic organism. It closely resembles a harmless bacillus found in water, the Bacillus fluorescens liquefaciens, from which Ruzicka1 thinks it probably descended. It stains well with the ordinary solutions, but does not retain the color by Gram's method. The superficial colonies upon gelatin plates form small, irregular, ill-defined collections, which produce a fluores- cence of the neighboring gelatin. The gelatin softens gradually, and about five days elapse before liquefaction is complete. 1 Centralbl.f. Bakt. u. Parasitenk., July 15, 1898, p. XI. SUPPURA TION. 263 The microscope shows the colonies to be round, coarsely-granulated masses with notched or filamentous borders. They have a yellow-green color. Upon the surface they form a deli- _ cate clump with a smooth r ' surface, finely granular, dis- .T^\ tinctly green in the middle &h. and pale at the edges. The colonies sink into the gel- atin as the liquefaction \ progresses. £<> •> -y-j Fig. 58. — Bacillus pyocyaneus: colonies upon gelatin (Abbott). In gelatin puncture-cultures most of the development occurs at the upper part of the tube, where a deep saucer of liquefaction forms. The growth slowly descends into the medium, and is the point of origin of a beautiful fluorescence. The bacterial growth sinks to the bottom as it ages. At times a delicate mycoderma forms on the surface. Upon agar- agar the growth is at first bright green, developing all along the line of inoculation. The green pigment (fluorescin) is soluble, and soon saturates the cul- ture-medium and makes it very characteristic. As the culture ages, or if the medium upon which it grows contains much peptone, a second pigment (pyocyanin) is developed, and the bright green fades to a deep blue- green, dark-blue, or in some few cases to a deep reddish- brown. A well-known feature of the growth upon fresh agar- agar, upon which much stress has recently been laid by Martin is the formation of crystals in fresh cultures. Crystal-formation in cultures of other bacteria usually takes place in old, partially dried agar-agar cultures. The bacillus pyocyaneus, however, produces crystals in a few 264 PATHOGENIC BACTERIA. days upon fresh media. In my experience freshly iso- lated bacilli manifest this capability more markedly than those which have been for some time part of the labo- ratory stock of cultures, and subject to frequent trans- plantation.1 Upon potato a luxuriant greenish or brownish, smeary layer is produced. Milk is coagulated and peptonized. This bacillus is highly pathogenic for laboratory ani- mals. About 1 c.cm. of a fresh bouillon culture, if in- jected into the subcutaneous tissue of a guinea-pig or a rabbit, causes a rapid edema, a suppurative inflammation, and death in a short time (twenty-four hours). Some- times the animal lives for a week or more, then dies. There is a marked hemorrhagic subcutaneous edema at the seat of inoculation. The bacilli can be found in the blood and in most of the tissues. When the dose is too small to prove fatal, suppuration occurs in many cases. When sterilized cultures are injected, the same results follow, a relatively larger quantity, of course, being re- quired. Intraperitoneal injections cause suppurative peritonitis. Blum reports a case of pyocyaneus infection with pyo- cyaneus-endocarditis in a child.2 Lartigau,3in his study of "The Bacillus Pyocyaneus as a Factor in Human Pathology," speaks as follows: " The Bacillus pyocyaneus, like many pathogenic micro- organisms, is occasionally found in a purely saprophytic role in various situations in the human economy. It has been found in the saliva by Pausini, in sputum by Frisch, and in the sweat by Eberth and Audanard. Abelous demonstrated its presence in the stomach as a saprophyte. Its existence in suppurating wounds has long been known, and Koch early detected its presence in tuberculous cavities, regarding it as an organism in- 1 See Centralbl.f. Bakt., xxi., April 6, 1897, p. 473. 2 Centralbl.f. Bakt. u. Parasitenk., Feb. 10, 1899, xxv., No. 4. 8 Phila. Med. Journal, Sept. 17, 1898. SUPPURA TION. 265 capable of playing any pathologic role. The etiologic relation of the organism to certain cases of purulent otitis media in children was pointed out by Martha, Maggiora and Gradenigo, Babes, Kossel, and others. H. C. Ernst obtained it from a pericardial exudate during life. G. Blumer demonstrated its presence in practically pure cultures in a case of acute angina simulating diphtheria ; Jadkewitsch, B. Motz, and Le Noir obtained the bacil- lus in cases of urinary infection. The cases of Triboulet, Karlinski, Oettinger, Ehlers, and Barker are interesting instances of its role in cutaneous lesions. u In addition to these lesions, other morbid processes have been associated in some cases with the bacillus of blue pus, such as meningitis and bronchopneumonia by Monnier ; diarrhea of infants by Neumann, Williams, Theircelin and Lesage, and other observers ; dysen- tery by Calmette and by Lartigau ; and general in- fection by Ehlers, Neumann, Ottinger, Karlinski, Mon- nier, Krannhals, Calmette, Finkelstein, and L. F. Barker." It is interesting to observe, in passing, that this path- ogeny can be set aside by the immunity which develops after a few inoculations with sterilized cultures. These are easily prepared, as the thermal death-point deter- mined by Sternberg is 560 C. The bacillus appears to be rather common as a sapro- phyte, and, as it has been found in the perspiration, probably is not uncommon upon the skin. Before leaving the subject of suppuration attention must be called to several rather common bacteria which may at times be the cause of troublesome suppuration. Among these are the pneumococcus of Frankel and Weichselbaum, the typhoid bacillus, and the Bacillus coli communis {q. v.). The fineumococcus has not infrequently been discov- ered most unexpectedly in abscesses of the brain and other deep-seated organs, and seems to have powerful chemotactic powers. For a careful consideration of it 266 PATHOGENIC BACTERIA. the reader must be referred to the chapter upon Pneumo- nia, where it is considered in full. The Bacillus coli communis, which is always present in the intestine, seems at times to enter the blood- or lymph- channels and stimulate suppuration, and numerous cases are on record showing this. The points most frequently attacked seem to be the bile-ducts and the vermiform ap- pendix, though the significance of the organism in appen- dicitis has no doubt been overrated. It has also been found in the kidney in scarlatinal nephritis, and is thought to be the exciting cause of some cases. It was originally described by Passet as the Bacillus pyogenes fcetidus. For a more particular study of this organism the reader is referred to the chapter devoted to its consideration. The Bacillus typhosus is probably less frequently a cause of suppuration than either of the others, yet it seems to be the occasional cause of the purulent sequelae of typhoid fever. A case has recently been reported by Flexner in which metastatic abscesses were found to be caused by it. The Micrococcus tetragenus has also been found in the pus of acute abscesses: it is quite common in the cavities of pulmonary tuberculosis, and may aid in the destructive processes involved in the general phthisical infection. Micrococcus Gonorrhoeae. All authorities now accept the "gonococcus" to be the cause of gonorrhea. It was first observed in the urethral and conjunctival secretions of gonorrhea and purulent ophthalmia by Neisser in 1879. The organisms are of hemispherical shape, arranged in pairs, so that the inner surfaces are separated from each other by a narrow interval. Sometimes, instead of pairs of cocci, fours are seen, the group no doubt resulting from the division of a pair. The described hemispherical shape is not exactly cor- rect, for a good lens generally shows the approximated surfaces to be somewhat concave rather than flat. The SUPPURA TION. 267 Germans see in the organism a resemblance to their pop- ular biscuit called a "semmel." The gonococcus is small, is not motile, like other cocci, is not provided with flagella, and does not have spores. It stains readily with all the aqueous anilin dyes — best with rather weak solutions — but not by Gram's method. It can be found in the urethral discharges of gonorrhea from the beginning until the end of the disease, though in the later days its numbers may be outweighed by other 0m $?*•■■ ffSJM*:,.. ,\ *jfe % V Fig. 59. — Gonococcus in urethral pus ; x 1000 (Fr'ankel and Pfeiffer). organisms. Wertheim cultivated the gonococcus from a case of chronic urethritis of two years' standing, and proved its virulence by producing with it gonorrhea in a human being. The organisms are generally found within the pus-cells (Fig. 59) or attached to the surface of epithelial cells, and should always be sought for as diagnostic of gonorrhea, especially as urethritis some- times is caused by other organisms, as the Bacillus coli communis1 and the Staphylococcus pyogenes. The cultivation of the gonococcus is not an easy task, but one which requires considerable bacteriologic skill. 1 Van der Pluyn and Loag : Centralbl. f. Bakt. u. Parasilenk., Bd. xvii., Nos. 7, 8, Feb. 28, 1895, p. 233. 268 PATHOGENIC BACTERIA. Wertheim accomplished it by diluting a drop of the pus in a little liquid human blood-serum, then mixing this with an equal part of melted 2 per cent, agar-agar at 400 C. , and pouring into Petri dishes. As soon as the media became firm the dishes were stood in the incubator at 3J° C, and in twenty-four hours the colonies could be observed. Those upon the surface showed a dark centre, around which a delicate granular zone could be made out. When one of these colonies is transferred to a tube of human blood-serum or the above mixture obliquely co- agulated, isolated little gray colonies occur ; later these become confluent and produce a delicate smeary layer upon the medium. The main growth is surrounded by a thin, veil-like extension which gradually fades away into the medium. A slight growth occurs upon the water of condensation. Turro says that the gonococci may also be cultivated upon acid gelatin, upon gelatin containing acid urine, and also in acid urine itself, in which the gonococci grow near the surface, while the pus-cocci which may be mixed with them sink deeper into the medium. His work has not been confirmed by other investigators. Heiman,1 who made an extensive series of culture-ex- periments, find that the gonococcus grows best in a mixt- ure of 1 part of pleuritic fluid and 2 parts of 2 per cent, agar. Wright2 prefers a mixture of urine, blood-serum, peptone, and agar-agar. Laitinen3 found agar-agar mixed with one-third to one-half its volume of cyst or ascites fluid, and bouillon containing 1 per cent, of peptone and 0.5 per cent, of sodium chlorid, mixed with one-third to one-half its volume of cyst or ascites fluid, very useful. The gono- coccus could be maintained alive upon these media for two months. L,aitinen found that the gonococcus pro- 1 Med. Record, Dec. 19, 1886. 2 Amer. Jour. Med. Sciences, Feb., 1895. 3 Centralbl. f. Bakt. u. Parasitenk., June I, 1898, vol. xii., No. 20, p. 874. SUPPURA TION. 269 duces acids in the early days of its development, and alkalies subsequently. He was unable to isolate any toxin from the cultures. It is ordinarily presumed that gonorrhea cannot be communicated to animals, but Turro asserts that the gonococci when grown upon acid gelatin readily com- municate urethritis to dogs, and that no Icesio continui is necessary, the simple introduction of the organisms into the meatus sufficing to produce the disease. The injection of gonococci into the subcutaneous tissue does not produce abscess. There is no doubt that the gonococcus causes gonor- rhea, as it has on several occasions been intentionally inoculated into the human urethra with resulting typical gonorrhea. It is constantly present in the disease, and very frequently also in the sequelse — endometritis, salpin- gitis, oophoritis, cystitis, peritonitis, arthritis, conjuncti- vitis, endocarditis, etc. — and, so far as can at present be determined, is never found under normal conditions. In the beginning of their activities the cocci grow in the superficial epithelial cells, but soon penetrate between the cells to the deeper layers, where they continue their irritation as the superficial cells desquamate. Authorities differ as to whether the gonococci can penetrate squamous and columnar epithelium with equal facility. The periurethral abscesses that occur in the course of gonorrhea are generally due to the Staphylococci aureus and albus, not directly to the gonococcus. In certain of the remote secondary inflammations the gonococci disappear after a time, and either the inflam- mation subsides or is maintained by other bacteria. In synovitis this does not seem to be true, and the inflam- mation excited may last for months. As long as the gonococci persist the patient may spread contagion. It must be pointed out that after apparent recovery from the disease the cocci sometimes remain latent in the urethra, and cause a relapse if the patient partake of some substance, as alcohol, irritating to the 270 PATHOGENIC BACTERIA. mucous membranes. Bearing this in mind, patients should not too soon be discharged as cured. The gonococci are not easily killed, but withstand dry- ing very well. Kratter was able to demonstrate their presence upon washed clothing six months after the orig- inal soiling, and also found that they still stained well. Bumm found cocci similar to the gonococcus in the urethra, and points out that neither the shape nor the position in the cells is positively characteristic, but that, in addition, there must be refusal to stain by Gram's method before we can say with certainty that cocci found in urethral pus are gonococci. All of the urethral inflammations do not depend upon the gonococcus, and in true gonorrhea all of the inflam- matory symptoms do not depend upon the gonococcus, as the epithelial denudation following the disease permits the entrance of the common pus cocci of the urethra into the peri-urethral tissues. The peri-urethral abscesses and salpingitis, etc., not infrequently depend upon the ordi- nary pus cocci, and I have seen a case of gonorrhea with double orchitis and general septic infection, with endo- carditis, in which the gonococci had no role in the sep- sis, which was caused by a large dumbbell-coccus that stained beautifully by Gram's method. Mumps, or Epidemic Parotitis. This epidemic, infectious disease of childhood, charac- terized by enlargement of the parotid and submaxillary glands, and rarely of the testicles, ovaries, and mammae, has not been proved to have a specific micro-organism. Pasteur thought the disease due to bacilli which he found in the blood. Capitan and Charrin1 and Olivier found in the blood, urine, and saliva both cocci and ba- cilli, but their studies are too early, and hence too crude to be of any value. Bouchard, Boisnet, and Bordas also found micro-organ- isms in the blood and saliva. 1 Comptes Rendu Soc. de Bioc. de Paris, May 28, 1 88 1. SUPPURA TION. 2 7 1 Netter, Laveran,1 Catrin, Mecray, and Walsh2 have all studied cases and isolated a diplococcus thought to be specific. The organism is described as occurring in pairs and in fours, sometimes in zooglea. It grows slowly in the ordinary media, clouding bouillin in twenty-four hours, and appearing on gelatin after forty-eight hours as small white punctiform colonies which develop very slowly and liquefy some considerable time after coalescence. It grows on potato, and has a whitish appearance not easy to detect. Laveran and Catrin found the organism in 67 out of 72 cases examined. In their method a few drops of exudate are withdrawn from the inflamed gland with a hypodermic needle, some of the negative results being due to the fact that the needle withdrew no exudate. The blood gave pure cultures in 10 out of 15 trials. Mecray and Walsh report that by disinfecting the mouths of patients, suffering from mumps, with a satu- rated boric acid solution, and cleansing Stensen's duct, by careful massage expressing its secretion, and then allowing a piece of cotton saturated with a boric acid solution to remain for five minutes between the orifice of the duct and the jaw, they were able to secure from the interior of the duct upon a bougie of sterile catgut a micrococcus identical with that Laveran had found. Of tubes inoculated with the contents of Stensen's duct 6 gave a mixed growth. All, however, showed the diplo- coccus. Out of 8 carefully made blood examinations, 3 gave pure cultures of the coccus and 3 mixed cultures; 2 were negative. From Stensen's duct in healthy children they obtained the various oral bacteria, but not the diplococcus found in the cases of mumps. The experimenters do not think it possible that this diplococcus is the Staphylococcus epidermidis albus, as its growth is slower and the lique- faction of gelatin is accomplished only after a longer 1 La Semaine Medicale, 1894 or I £95, No. 7. 2 Medical Record, Sept. 25, 1896. 272 PATHOGENIC BACTERIA. time than is required by the staphylococcus. They did not succeed in producing mumps in animals. In their experience a dog was encountered which suffered from swelling of the parotids, malaise, etc., after playing with a child suffering from mumps. Concerning the diplococctis, it appeared in twos and fours ; rarely in larger groups. Each was regularly rounded and about the size of the pus cocci. The colonies are small, white, glistening, distinctly defined, regularly circular spots, at first discrete and of slow growth, gradually coa- lescing. The slow growth is characteristic. In study- ing pure cultures, some gelatin tubes three days after in- oculation were set aside, no growth being noted; three days later the small white colonies became distinctly vis- ible. At ordinary temperatures gelatin is not liquefied until ten or twelve days, and the liquefaction proceeds slowly. A faint white streak appears on potato on the third day, and spreads as a delicate whitish film. The growth upon blood-serum is more rapid than on other media, but the colony is not so distinctly white in color. Litmus milk is changed to pink on the third day and is coagulated. Milk is thought to be an excellent nutrient medium, and a possible ready means of spreading con- tagion. In the paper of Mecray and Walsh no mention is made of the relation of the cocci to pus cells or other organized constituents of the secretion from which they were obtained; no animal inoculations were done and nothing is said about the reaction to Gram's method of staining or possible motility the cocci might possess. Michaelis and Bein,1 of Leyden's clinic, found a diplo- coccus (previously observed by Leyden in the sputum), which occurred chiefly in the pus cells. In severe cases of the disease, which they studied by culture and micro- scopic section, the organism was not only secured from Stensen's duct, but in 2 cases from the pus of an abscess (parotid ?) and in 1 case from the blood. 1 Deutsche med. Wochenschrift, May 13, 1897. SUPPURA TION. 273 In spite of the small number of cases studied, they were of the opinion that their coccus is the specific one. It is about 1 fi in size and resembles the gonococcus, though it is smaller. The cocci generally lie in the cells, sometimes 8 or 10 in one pus cell, and are occasion- ally distributed throughout the pus in long chains or strings. They stain readily with the usual anilin dyes, especially with Loftier' s methylene-blue, and can be decolorized by the Gram method. They grow slowly upon the ordinary media, forming living, transparent, dew-like points on agar-agar. These little drops do not coalesce. In peptone-bouillon they form white, rather granular than flocculent deposit, the bouillon itself re- maining clear. The growth is said to be more rapid in strongly than feebly alkaline media. The cocci are said to grow upon ascites-fluid and upon milk, the latter coag- ulating in the course of forty-eight hours. They are capable of slight movement. Numerous inoculation ex- periments were made, only one animal, a white mouse, succumbing. Control-experiments failed to disclose the same organisms in the healthy human parotid or its se- cretion. All the observers agree in finding in the secretions of the gland and in the blood diplococci that grow slowly, produce small colonies, and coagulate milk. No one has shown their specificity by inoculation, evidence of course necessary before the claim of real importance can be accepted. 18 CHAPTER II. CEREBROSPINAL MENINGITIS. Diplococcus Intracellulars Meningitidis. The acute sero-purulent form of inflammation of the cerebral and spinal meninges not infrequently presents itself as a complication of certain well-known infectious processes, as croupous pneumonia, more rarely as a primary sporadic or epidemic affection. The disease is usually associated with one or the other of three micro- organisms, the pneumococcus, the streptococcus, and the Diplococcus intracellulars meningitidis of Weichsel- baum. In more rare cases the staphylococci, the typhoid bacillus, and other bacteria may present themselves. As early as 1887 Weichselbaum 1 found in six cases which he studied a diplococcus that had not been suc- cessfully cultivated, although it may have been identical with one found by Leichtenstern 2 in the purulent exu- date of a case of meningitis. Weichselbaum' s studies and description of the coccus seem to have attracted but little attention at first, and references to them are but brief in most of the text-books. The common opinion seemed to prevail that as its presence did not appear to be essential to the occurrence of cerebrospinal menin- gitis, as its inoculation into animals showed its path- ogenic power to be limited, its importance was but trivial. The careful studies of Joger,3 Scherer,4 Council- man, and Mallory and Wright,5 embracing a large num- ber of cases, have shown the presence of the diplococcus of Weichselbaum in so large a number that its import- ance has become correspondingly great. 1 Fortschritte der Med., v., 1 8 and 19. * Deutsche med. Wochensckrift, 1885. 3 Zeitschrift fur Hygiene, xix., 2, 351. 4 Centralbt. f. Bakt. u. Parasitenk., xvii., 13 and 14. 5 American Jour. Med. Sci., March, 1898, vol. cxv., No. 3. 274 CEREBROSPINAL MENINGITIS. 275 The micro-organism is a diplococcus of biscuit shape, bearing the greatest resemblance in form and arrange- ment to the gonococcus. This resemblance is further increased by the fact that the cocci usually occur en- closed in the protoplasm of the leukocytes. Weichsel- baum, by whom this arrangement was observed, found it constant in sections of the brain and its membranes, though in the exudate of the disease a good many free cocci also occur. It was this peculiarity that led him to call the organism the Diplococcus intracellularis. Many of the cocci thus enclosed within the cells are apparently dead and degenerated, as evinced by the fact that they stain badly and refuse to grow when the pus is trans- ferred to culture-media. Tlie bacterium is easily stained by the usual methods with aqueous solutions of the anilin dyes. Its relation to Gram's method has not yet with certainty been deter- mined. According to Weichselbaum, it does not stain by Gram's method. The organism was successfully cultivated by Weichsel- baum, but does not readily adapt itself to artificial media. It develops upon agar-agar and glycerin agar-agar, upon Loffler's blood-serum mixture, and, according to Gold- schmidt,1 upon potato. Weichselbaum was, however, unable to find that it developed upon potato. It does not grow in bouillon or gelatin. There is nothing char- acteristic about the cultures. The cocci grow only at the temperature of the body, attain only a sparse devel- opment, and form a more or less confluent line of minute, rounded, grayish colonies which are easily overlooked upon opaque media like blood-serum. The general characteristics of the growth are not unlike those of the pneumococcus, streptococcus, etc. When sown upon agar-agar plates the deep colonies scarcely develop at all, appearing under the low-power lens as minute, irregularly rounded granular masses. The surface colonies are larger, and consist of an opaque 1 Centralbl.f. Bakt. u. Parasitenk., ii., 22, 23. 276 PATHOGENIC BACTERIA. yellowish-brown central nucleus about which a flat, rounded disk spreads out. The edges may be dentate ; the color is grayish or yellowish near the centre, becom- ing less intense as the thin edges are reached ; the for- mation is finely granular. The vitality of the culture is low, and when cultivated the cocci tend to die out readily, ceasing to grow when transplanted after eight or ten days. It becomes neces- sary, therefore, when studying the organism to trans- plant it frequently — Park says every two days.1 The organism can be secured for cultivation either from the purulent matter of the exudate found at autopsy, or from the fluid obtained by lumbar puncture. To obtain this fluid Park gives the following directions : " The patient should lie on the right side with the knees drawn up and the left shoulder depressed. The skin of the patient's back, the hands of the operator, and the large antitoxin syringe should be sterile. The needle should be 4 c.cm. in length, with a diameter of 1 mm. for children, and larger for adults. The puncture is gener- ally made between the third and fourth lumber vertebrae. The thumb of the left hand is pressed between the spinous processes, and the point of the needle is entered about 1 cm. to the right of the median line and on a level with the thumb-nail, and directed slightly upward and inward toward the median line. At a depth of 3 or 4 c.cm. in children and 7 or 8 c.cm. in adults the needle enters the subarachnoid space, and the fluid flows out in drops or in a stream. If the needle meets a bony ob- struction, withdraw and thrust again rather than make lateral movements. Any blood obscures microscopic examination. The fluid is allowed to drop into abso- lutely sterile test-tubes or vials with sterile stoppers. From 5 to 15 c.cm. should be withdrawn. No ill-effects have been observed from the operation." In making a culture from this fluid Park points out that as many of its contained cocci are dead, a consider- 1 Bacteriology in Medicine and Surgery, 1899, p. 518. CEREBROSPINAL MENINGITIS. 277 able quantity of the fluid (say about 1 c.ctn.) must be used. In the 55 cases studied by Councilman, Mallory, and Wright the cocci were found by culture or by micro- scopic examination in 38. The cocci have also been cultivated from the nasal discharges in the 6 cases studied by Weichselbauin and in 18 studied by Scherer. The results of animal inoculations made with the Diplococcus intracellularis meningitidis are on the whole disappointing. Subcutaneous inoculations into the lower animals are continually without effect. Intrapleural and intraperitoneal injections of cultures of the organism into mice and guinea pigs are sometimes fatal, the dead animals showing a serofibrinous inflammation with the presence of the cocci. The intravenous injection of the coccus into rab- bits is followed by death without important or conclusive symptoms, and usually without the cocci in the blood. Weichselbauin endeavored to reproduce the original cerebrospinal meningitis in animals by trephining and injecting the cocci beneath the dura. In this manner he inoculated three rabbits and three dogs. Two of the rabbit injections failed, probably because the injected material escaped at once from the wound. The third rabbit died, and showed marked congestion of the mem- branes of the brain and a minute softened and hemor- rhagic area. In these the cocci were found by culture to be abundant. The three dogs all died with congestion and pus formation in the membranes and areas of soften- ing in the brain substance. The cocci were recovered from two of the dogs, but the lesions of the third animal, which lived twelve days, contained none. It is not known by what channels the infection by the Diplococcus intracellularis meningitidis takes place. Weichselbaum supposed it might enter by the nasal, auditory, or other passages, especially the nose, where he constantly found it. In this connection it is interesting to note that the only two of the fifty supposedly healthy 278 PATHOGENIC BACTERIA. persons studied by Scherer in which this coccus was found suffered from 'coryza, which is an almost constant symptom of cerebrospinal meningitis. The distribution of the Diplococcus intracellularis in nature is as yet unknown. It has been found in cerebrospinal meningitis by those who have looked for it, has been found in the nose in coryza twice by Scherer, has been found in the conjunctiva by Carl Frankel1 and Axenfeld,2 and in rhinitis and otitis by Joger.3 It occurs in above 50 per cent, of the cases of cerebrospinal meningitis, but fails satisfactorily to fulfil the requirements of the laws of specificity. For staining the meningococcus the method of Pick and Jacobsohn4 is highly praised by Carl Frankel, who modifies it by adding three times as much carbol-fuchsin as is recommended in the original method, which is as follows : Mix 20 c.cm. of water with 8 drops of saturated methylene-blue solution; then add 45-50 drops of carbol- fuchsin. Allow the fluid to act upon the cover-glass for five minutes. The cocci alone are blue, all else red. Carl Frankel, in discussing the micro-organism, points out that its morphologic peculiarities have much in common with the pneuinococcus, so that the most re- fined methods of differentiation should always precede a positive diagnosis. Its resemblance to the gonococcus should also be kept in mind. Steel5 describes what may be a variety of the menin- gococcus that occurs in the simple posterior basic menin- gitis of infants. The organism differs from that of Weichselbaum in having a more permanent saprophytic existence upon culture-media, where it often lives as long as thirty days. It is easily stained by methylene blue, but not by Gram's method. 1 Zeitschrift fiir Hygiene, June 1 4, 1899. 2 Lubarsch and Oestertag, Ergebnisse der allg. Path. u. path. Anat., iii., S- 573- s Deutsche med. Wochenschrift, 1894, S. 407- * Berlin, klin. Wochenschrift, 1896, S. 811. 5 Pediatrics, Nov. 15, 1898. CHAPTER III. PNEUMONIA. Diplococcus Lanceolatus. The term "pneumonia," while generally understood to refer to the lobar disease particularly designated as croupous pneumonia, is a vague one, really comprehend- ing a variety of inflammatory conditions of the lung quite dissimilar in character. This being true, no one should be surprised to find that a single organism cannot be described as "specific" for all. Indeed, pneumonia must be considered as a group of diseases, and the various microbes found associated with it must be described suc- cessively in connection with the peculiar phase of the disease in which they occur. I. Lobar or Croupous Pneumonia. — The bacterium, which can be demonstrated in at least 75 per cent, of the cases of lobar pneumonia, which is now almost uni- versally accepted as the cause of the disease, and about whose specificity very few doubts can be raised, is the Diplococcus lanceolatus, or pneumococcus of Frankel and Weichselbaum. Priority of discovery in the case of the pneumococcus seems to be in favor of Sternberg,1 who as early as 1880 described an identical organism which he secured from his saliva. Curiously enough, Pasteur2 seems to have captured the same organism, also from saliva, in the same year. The researches of the observers whose names are attached to the organism were not completed until five years later. It is to Frankel,3 Telamon,4 and particularly 1 National Board of Health Bulletin, 18S1, vol. ii. * Comptes rendus Acad, des Set., 1881, xcii., p. 159. s Deutsche med. Wochenschrift, 1 885, 31. * Communication a la Sociili anatom. de Paris, Nov. 30, 1883. 279 280 PATHOGENIC BACTERIA. to Weichselbaum,1 however, that we are indebted for the discovery of the relation which the organism bears to pneumonia. The organism (Fig. 60) is variable in its morphology. When grown in bouillon it is oval, has a pronounced dis- position to occur in pairs, and not infrequently forms chains of five or six members, so that some have been disposed to look upon it as a streptococcus (Gamaleia). In the fibrinous exudate from croupous pneumonia, in Fig. 60. — Diplococcus pneumoniae, from the heart's blood of a rabbit; x 1000 (Frankel and Pfeiffer). the rusty sputum, and in the blood of rabbits and mice containing them the organisms are arranged in pairs, exhibit a distinct lanceolate shape, the pointed ends generally approximated, and are usually surrounded by a distinct halo or capsule of clear, colorless, homogeneous material, thought by some to be a swollen cell-wall, by others a mucus-like secretion given off by the cells. When 1 Wiener vied. Jahrbuch, 1886, p. 483. PNEUMONIA. 281 grown ordinarily in culture-media, and especially upon solid media, the capsules are absent. The organism is without motility, has 110 spores, and does not seem to be able to resist any unfavorable con- ditions when grown artificially. It stains well with the ordinary solutions of the anilin dyes, and gives most beautiful pictures in blood and tissues when stained by Gram's method. The capsule does not stain. To demonstrate the capsule, the glacial acetic acid Fig. 61. — Capsulated pneumococci in blood from the heart of a rabbit; carbol- fuchsin, partly decolorized ; x iooo. method of Welch ' may be used. The cover-glass is spread with a thin film of the material to be examined, which is dried and fixed as usual. Glacial acetic acid is dropped upon it for an instant, poured (not washed) off, and at once followed by anilin-water gentian-violet, in which the staining continues several minutes, the stain being poured off and replaced several times until the acid 1 Bulletin of the Johns Hopkins Hospital, Dec, 1892, p. 128. 282 PATHOGENIC BACTERIA. has all been replaced. Finally, the preparation is washed in water containing i or 2 per cent, of sodium chlorid, and may be examined at once in the salt solution, or mounted in balsam after drying. The capsules are prob- ably more distinct when the examination is made in water. The pneumococcus is no stranger to us; it may some- times be found in the saliva of healthy individuals, and the inoculation of human saliva into rabbits frequently causes a septicemia in which the bacillus is found abun- dantly in the blood and tissues. Because of its frequent presence in the saliva it was described by Fliigge as the Bacillus septicus sputigenus. When desired for purposes of study, it may be obtained by inoculating rabbits with pneumonic sputum and re- covering the organisms from their heart's blood, or it may be secured from the rusty sputum of pneumonia by the method employed by Kitasato for securing tubercle ba- cilli from sputum. A single mouthful of fresh sputum is secured, washed in several changes of sterile water to free it from bacteria of the mouth and pharynx, carefully separated, and a central portion transferred to an appro- priate culture-medium. The organism grows upon all the culture-media except potato, but only between the temperature-extremes of 240 and 420 C. ; the best development is at 370 C. The growth is always limited, probably because the formic acid produced serves to check it. The addition of an unusual amount of alkali to the culture-medium favors the growth. The organisms readily lose their virulence in culture- media, and cease to be pathogenic after a few days. In his experiments with antipneumococcic serum Washbourn found, however, that a pneumococcus isolated from pneu- monia sputum and passed through one mouse and nine rabbits developed a permanent virulence when kept on agar-agar made carefully, so that it was not heated beyond ioo° CM and alkalinized 4 c.cm. of normal caustic soda PNEUMONIA. 283 solution beyond the neutral point determined with rosalic acid, to each liter. The agar-agar is first streaked with sterile rabbit's blood, then inoculated. The cultures are \'\c. 62. — Diplococcus pneumoniae : colony twenty-four hours old upon gelatin ; x 100 (Frankel and Pfeiffer). kept at 37. 50 C. Not only is this true, but ordinarily they seem to be unable to accommodate themselves to a purely saprophytic life, and unless continually trans- planted to new media die in a week or two, sometimes sooner. Kinyoun recommended to the writer that virulence could be retained for a considerable time by keeping blood from an infected rabbit, in a hermetically sealed glass tube, on ice. This plan seems to work admirably if the blood is not kept too long. The colonies which develop at 240 C. upon 15 per cent, gelatin plates are described as small, round, cir- cumscribed, finely granular white points which grow slowly, never attain any considerable size, and do not liquefy the gelatin (Fig. 62). If, instead of gelatin, agar-agar be used and the plates kept at the temperature of the body, the colonies which 284 PATHOGENIC BACTERIA. develop upon the plates appear as transparent, delicate, drop-like accumulations, scarcely visible to the naked eye, but under the microscope distinctly granular, the central darker portion being frequently surrounded by a paler marginal zone. In gelatin puncture-cultures, made with 15 instead of the usual 10 per cent, of gelatin, the growth takes place along the entire path of the wire in the form of little whitish granules distinctly separated from each other. The growth in gelatin is always very limited. Upon agar-agar and blood-serum the growth consists of minute, transparent, semi-confluent, colorless, dew- drop-like colonies, which die before attaining a size which permits of their being seen without careful in- spection. Upon glycerin agar-agar the growth is more luxuriant. In bouillon the organisms grow well, clouding the medium very slightly. Milk is quite well adapted as a culture-medium, its casein being coagulated. It does not grow upon potato.1 When it is desired to maintain or increase the virulence of a culture it must be very frequently passed through the body of a rabbit. The degree to which the virulence can be raised in this way is remarkable. C. W. Lincoln has succeeded in reducing the fatal dose for rabbits to ToTTOlfFoTFo °* a c-cm- If a small quantity of a pure culture of the virulent organism is introduced into a mouse, rabbit, or guinea- pig, the animal dies in one or two days. Exactly the same result can be obtained by the introduction of a piece of the lung-tissue from croupous pneumonia, by the introduction of some of the rusty sputum, and gener- ally by the introduction of saliva. The post-mortem shows that an inflammatory change has taken place at the point of inoculation, with a fibrin- 1 Ortmann asserts that the pneumococcus can be grown on potato at 370 C, but this is not generally confirmed. The usual acid reaction of the potato would indicate that it was a very unsuitable culture-medium. PNEUMONIA. 285 011s exudate resembling somewhat that in diphtheria. At times, and especially in dogs, there may be a little pus formed. The other appearances are those of a general disturbance. The spleen is much enlarged, is firm and red brown. The blood in all the organs contains large numbers of the bacteria, most of which exhibit a distinct lanceolate form and have their capsules very distinct. The disease is a pure septicemia unassociated with pronounced tissue-changes. In cases of the kind described the lungs show no pneu- monic changes. Likewise, if the hypodermic needle used for injection be plunged through the breast-wall into the pulmonary tissue, no pneumonia results. Mon- ti, however, claims to have found that a true character- istic pneumonia results from the injection of cultures into the trachea of susceptible animals. This observa- tion lacks confirmation. Not all animals are susceptible. Guinea-pigs, mice, and rabbits are highly sensitive to the operations of the organism ; dogs are comparatively immune. From this brief review of the peculiarities of the pneu- mococcus it must be obvious that its reputation in pneu- monia depends more upon the regularity with which it is found in that disease than upon its capacity to produce a similar affection in the lower animals. As in numerous other diseases, we are unable to furnish an absolute proof of specificity according to the postu- lates of Koch. The disease is peculiar in that recovery from it is fol- lowed either by no immunity or by one of such brief dura- tion as to allow of frequent relapses ; and it is well known that many cases show a subsequent predisposition to fresh attacks of the disease. This brevity of immunity lessens the probability that in the future we shall dis- cover an antitoxin that shall be powerful in its influ- ence upon the course and termination of the disease. The experiments of G. and F. Klemperer,1 a few years 1 Berliner klin. Wochenschrift, 1891, No>. 34 and 35. 286 PATHOGENIC BACTERIA. ago, showed that the serum of immunized rabbits pro- tected animals inoculated with the pneumococcus. The principle failed, however, when applied to human medi- cine. The treatment of pneumonia by the injection of blood-serum from convalescents, as tried by Hughes and Carter,1 has also been abandoned as useless and dangerous. A more modern and refined antipneumococcic serum has been investigated experimentally by Washbourne,2 De Renzi,3 and Paul.4 Washbourne 5 prepared an antipneumococcic serum effi- cacious in protecting rabbits against ten times the fatal dose of live pneumococci in doses of o. 3 c. cm. In general, the lines upon which he operated were those of Behring, Marmorek's work with the streptococcus furnishing most of the details. A pony was subjected to immunization for a period of five months, allowed to rest three or four months until the live pneumococci introduced were all destroyed, and then bled. Two cases of human pneu- monia seem to have received some benefit from the injec- tion of large doses of this serum. The serums of Pane and De Reuzi were not so powerful as those of Wash- bourne, requiring about 1 c.cm. to protect a rabbit. McFarland and Lincoln 6 have succeeded in confirming the experimental work of Pane, De Renzi, and Wash- bourne, and by immunizing a horse to large doses of highly virulent cultures of the pneumococcus have ob- tained from it a serum, of which \-\ c.cm. protected rab- bits from doses of the live bacilli many times as large as necessary to produce death. The antipneumococcic serums have not been given a sufficient clinical application for us to judge what merits they may have. 1 Therapeutic Gazette, Oct. 15, 1892. 2 Brit. Med. Journal, Feb. 27, 1897, p. 510. 8 II Policlinico, Oct. 31, 1896, Supplement. * Centralbl. f. Bakt. u. Parasitenk., May 29, 1897, xxi., 17 and 18, p. 664. 5 Brit. Med. Journal, Feb. 27, 1897, p. 510. 6 Journal of the American Medical Association, Dec. 16, 1899, p. 1534. PNEUMONIA. 287 The pneuinococcus causes other lesions than croupous pneumonia; thus, Foa, Bordoni-Uffreduzzi, and others have found it in cerebrospinal meningitis; Frankel, in pleuritis; Weichselbaum, in peritonitis; Banti, in peri- carditis; numerous observers have found it in acute ab- scesses; Gabbi has isolated it from a case of suppurative tonsillitis; Axenfeld has observed an epidemic of con- junctivitis caused by it; and Zaufal, Levy, and Schrader and Netter have been able to demonstrate its presence in the pus of otitis media. It has also been reported as oc- curring in the joints in arthritis following pneumonia. The pneuinococcus is often present in the mouths of healthy persons. The conditions under which it enters the lung to produce pneumonia are not known. In the opinion of most authorities, something more than the simple entrance of the bacterium into the lung is required for the production of the disease, but what that something is, is still a matter of doubt. It would seem to be some systemic depravity, and in support of this view we may point out that pneumonia is very frequent, and almost universally fatal, among drunkards. Whether, however, any vital depression or systemic depravity will predispose to the disease, or whether it depends for its origin upon the presence of a certain leucomai'ne, time and further study will be required to tell. Bacillus Pneumonia of Friedlander — Bacillus Capsu- latus Mucosus (Fig. 63). — An unfortunate accident has ap- plied the name "pneuinococcus" to an organism very dif- ferent from the one just described. It was discovered by Friedlander ' in 1883 in the exudate from the lung in croupous pneumonia, and, being thought by its discov- erer to be the cause of the disease, very naturally was called the pneuinococcus, or, more correctly, the pncu- mobacillus. The grounds upon which the pathogeny of the organism was supposed to depend were very insuffi- cient, and the bacillus of Friedlander — or, as Fliigge prefers to call it, the Bacillus pneumoniae — has ceased to 1 Fortschritte der Medizitt, 1883, 22. 288 PATHOGENIC BACTERIA. be regarded as specific, and is now looked upon as an accidental organism whose presence in the lung is, in most cases, unimportant. As the two organisms are similar in more respects than their names, Friedlander's bacillus requires at least a brief description. It is distinctly a bacillus, but sometimes, when occur- ring in pairs, has a close resemblance to the pneumo- coccus of Frankel and Weichselbaum. Very frequently it forms chains of four or more elements. It is also com- monly surrounded by a transparent capsule. It is non- Fig. 63. — Bacillus pneumonias of Friedlander, from the expectoration of a pneumonia patient; x 1000 (Frankel and Pfeiffer). motile, has no spores and no flagella. It stains well with the ordinary anilin dyes, but does not retain the color when stained by Gram's method. Frankel points out that Friedlander's error in suppos- ing this bacillus to be the chief parasite in pneumonia depended upon the fact that his studies were made by the plate method. If some of the pneumonic exudate be mixed with gelatin and poured upon plates, the bacilli grow into colonies at the end of twentv-four hours, and appear as small white spheres which spread upon the PNEUMONIA. 289 gelatin to form white masses of a considerable size. Under the microscope these colonies are rather irregular in outline and somewhat granular. The bacillus grows at as low a temperature as 160 C, and, according to Sternberg, has a thermal death-point of 560 C. When a colony is transferred to a gelatin puncture-cul- ture, quite a massive growth occurs. Upon the surface a somewhat elevated, rounded white mass is formed, and in the track of the wire innumerable little colonies spring up and become confluent, so that a " nail-growth " results. No liquefaction occurs. When old the cultures sometimes become brown in color. Upon the surface of agar-agar at ordinary temperatures quite a luxuriant white or brownish-yellow, smeary, cir- cumscribed growth occurs. The growth upon blood- serum is the same. Upon potato the growth is abundant, quickly covering the entire surface with a thick yellowish-white layer, which sometimes contains bubbles of gas. Gas is also sometimes developed in gelatin cultures. A most superficial comparison will suffice to show the great difference in vegetation between these two so-called pneumococci. Friedlander had considerable difficulty in causing any pathogenic changes by the injection of his bacillus into animals. Rabbits and guinea-pigs were immune, and the only actual pathogenic results which Friedlander ob- tained were in mice, into whose lungs and pleura he injected the cultures. In the status prcesens of bacteriologic knowledge the bacillus of Friedlander is regarded as an organism, generally a harmless saprophyte, but at times capable of producing inflammations. Curry1 has found Friedlander' s bacillus in combina- tion with the pneumococcus in acute lobar pneumonia, 1 Journal of the Boston Society of Medical Sciences, March, 1898, vol. ii., No. 8, p. 137. 19 290 PATHOGENIC BACTERIA. in conjunction with the diphtheria bacillus in otitis media associated with croupous pneumonia, and in the throat in diphtheria. In pure culture it was obtained from the vegetations upon the valves of the heart in a case of acute endocarditis with gangrene of the lung ; in the middle ear, in a case of fracture of the skull with otitis media ; and from the throat, in a case of tonsillitis. Occasionally Friedlander's bacillus becomes the cause of pneumonia of lobular or catarrhal type, an interesting case of this kind having been studied by Smith.1 The histology of the lung was remarkable in that the "alve- olar spaces in the consolidated areas were dilated and filled for the most part with the capsule bacilli." In some alveoli there seemed to be pure cultures of the bacilli, in others there were a few red and white blood- corpuscles, and in some there was a little fibrin. The bacillus obtained from this case when injected into the peritoneal cavity of guinea-pigs produced death in eleven hours. The peritoneal cavity after death contained a large amount of thick, slimy fluid ; the intestines were injected and showed a thin fibrinous exudate upon the surface ; the spleen was enlarged and softened, and the adrenals much reddened. Cover-glass preparations from the heart, blood, spleen, and peritoneal cavity showed large numbers of the capsule bacilli. Howard2 has also called attention to the importance of this bacillus in connection with numerous acute and chronic infectious processes, among which may be men- tioned croupous pneumonia, suppuration of the antrum of Highmore and frontal sinuses, endometritis, perirenal abscesses, and peritonitis. 2. Catarrhal Pneumonia. — This form of pulmonary inflammation occurs in local areas, generally situated about the distribution of a bronchiole. It cannot be said to have a specific micro-organism, as almost any 1 Journal of the Boston Society of Medical Sciences, May, 1 898, vol. ii., No. 10, p. 174. 2 Phila. Med. Journal, Feb. 19, 1898, vol. i., No. 8, p. 336. PNEUMONIA. 291 irritant foreign materials accidentally inhaled can cause it. The majority of the cases, however — and especially those which are distinctly peribronchial — are caused by the presence of the staphylococcus and streptococcus of suppuration. Friedlander's bacillus may also aid in pro- ducing local inflammations. 3. Tubercular Pneumonia. — At times the process of pulmonary tuberculosis is so rapid, and associated with the production of so much semi-liquid, semi-necrotic material, that the auto-infection of the lung is greatly favored ; the tubercle bacilli are distributed to the entire lung or to large parts of it, and a distinct inflammation occurs. Such a pneumonia may be caused by the tubercle bacillus alone, but more often it is aided by accompany- ing staphylococci, streptococci, tetragenococci, pneumo- cocci, pneumobacilli, and other organisms apt to be pres- ent in a lung in which tuberculosis is in progress and ulceration and cavity-formation are advanced. 4. Mixed Pneumonias. — It frequently happens that pneumonia occurs in the course of, or shortly after the convalescence from, influenza. In these cases a mixed infection is present, and there is no difficulty in deter- mining that both the influenza bacillus and the pneumo- coccus are present. Again, sometimes the pneumococci and staphylococci operate simultaneously, and produce a purulent pneumonia with abscesses as the conspicuous feature. As almost any combination of the described bacteria is possible in the lungs, and as these combi- nations will all produce varying inflammatory conditions, it must be left for the student to imagine what the par- ticular characters of each may be. Among these mixed pneumonias may be mentioned those called by Klemperer and Levy "complicating pneumonias," occurring in the course of typhoid, etc. II. THE CHRONIC INFLAMMATORY DISEASES. CHAPTER I. TUBERCULOSIS. The Bacillus Tuberculosis (Koch).1 Tuberculosis is one of the most dreadful and, un- fortunately, most common diseases of mankind. It affects alike the young and the old, the rich and the poor, the male and the female, the enlightened and the savage. Nor do its ravages cease with human beings, for it is common among animals, occurring with great frequency among cattle, less frequently among goats and hogs, and sometimes, though rarely, among sheep, horses, dogs, and cats. Wild animals under natural conditions seem to escape the disease, but when caged and kept in zoological gar- dens even the most resistant of them — lions, tigers, etc. — are said at times to succumb to it, while it is the most common cause of death among captive monkeys. The disease is not even limited to mammals, but occurs in a somewhat modified form in birds, and, it is said, even affects reptiles at times. It is not a disease of modern times, but one which has persisted through centuries ; and though, before the ad- vent of the microscope, not always clearly separated from cancer, it has not only left unmistakable signs of its existence in the early literature of medicine, but has also imprinted itself upon the statute-books of some countries, as Naples, where its ravages were great and the means taken for its prevention radical. 1 Berliner klin. Wochenschrift, 1882, 15. 292 TUBERCULOSIS. *93 While the great men of the early days of pathology clearly saw that the time must come when the parasitic nature of this disease would be proved, and some, as Klebs, Villemin, and Cohnheim, were "within an ace " of the discovery, it remained for Robert Koch to succeed in demonstrating and isolating the specific bacillus, now so well known, and to write so accurate a description of the organism and the lesions it produces as to render it almost unparalleled in medical literature. The tubercle bacillus (Fig. 64) is a rod-shaped organ- Fig. 64. — Section of a peritoneal tubercle from a cow, showing the tubercle bacilli; x 500 (Frankel and Pfeiffer). ism with rounded ends and a slight curve, measuring from 1.5-3.5 t1 *n length and from 0.2-0.5 // m breadth. It very commonly occurs in pairs, which may be asso- ciated end to end, but generally overlap somewhat and are not attached to each other. In organisms found in pus and sputum a peculiar beaded appearance is very common (Fig. 65). By some these fragmentations are thought to be bacilli in the stage of sporulation (see Fig. 66). Koch originally held this view himself, but re- searches have not been able to substantiate the opinion. 294 PATHOGENIC BACTERIA. The appearance is no doubt one of involution or degene- ration. The fragments do not resemble spores as seen in other organisms. When spores occur in the continuity of Fig. 65. — Tubercle bacillus in sputum (Frankel and Pfeiffer). bacilli, they usually occur as easily recognized discrete oval refracting bodies. The fragments seen in the tubercle bacillus are irregular in shape instead of oval, have ragged '< (r '* Fig. 66. — Tubercle bacilli : I, forms suggesting speculation, because of the presence of large chromophylic granules ; 2, forms described as beaded ; the open spaces in the fragmented rods are sometimes mistaken for spores; 3, branched forms of the tubercle bacillus sometimes seen in sputum. surfaces, and are without the highly refracting index peculiar to ordinary spores. The space between the bacillary fragments cannot be TUBERCULOSIS. 295 made to stain like the spores of other species. Finally, all known spores resist heat more strongly than the fully- developed bacilli, but experiments have shown that these degenerative forms are no more capable of resisting heat than the tubercle bacilli themselves. As sh6wn in Fig. 66, 3, it is occasionally observed that the bacilli present projecting processes or branches. The frequency of this observation has changed our views of the systematic classification of the organism, which proba- bly is erroneously placed among the bacilli, belonging more properly to the higher bacteria and being related to the actinomyces. It is probably a streptothrix. The organism is not motile, and does not possess flagella. While this statement is dogmatically made, there is some evidence accumulating to show that the tubercle bacillus is sometimes motile. Ferran ' and Schumowski 2 have observed a slow swimming movement. Ferran even claims to have observed the flagella upon which the movements depend. I have watched a pure culture of the tubercle bacillus under the microscope, in the hang- ing drop, and have seen a bacillus separate itself from its fellows and sail off slowly and steadily. I am not sure, however, that such occasional observations are con- vincing, and prefer to consider the bacillus non-motile. The tubercle bacillus is peculiar in its reaction to the anilin dyes. It is rather difficult to stain, requiring that the dye used shall contain a mordant (Koch); but it is also very tenacious of the color once assumed, re- sisting the decolorizing power of strong mineral acids (Ehrlich). This peculiarity delayed the discovery of the bacillus for a considerable time, but, now that we are familiar with it, gives us a most valuable diagnostic character, for, with the exception of the bacillus of lepra and the Bacillus smegmatis, none reacts in the same way. 1 Sent, mid., 1897, No. 48. 2 Centralbl f. Bakt. u. Parasitenk., May 20, 1898, xxiii., No. 19, p. 838. 296 PATHOGENIC BACTERIA. Koch first stained the bacillus with an aqueous solu- tion of a basic anilin dye to which some potassium hydrate was added, subsequently washing with water and counter-staining with vesuvin. Ehrlich subsequently modified Koch's method, showing that pure anilin was a better mordant than potassium hydrate, and that the use of a strong mineral acid would remove the color from everything but the tubercle bacillus. This modi- fication of Koch's method given us by Ehrlich is at the present time acknowledged to be the best method of staining the bacillus. Many other methods have been suggested, all of them, perhaps, more convenient than Ehrlich's, but none so good. As being that most frequently performed by the physician, we will first describe the method of seeking the bacillus in sputum, for the purpose of making a diag- nosis of the disease. If the material to be examined is sputum, and one desires to be very exact in his examination, it is well to have the patient cleanse the mouth thoroughly upon waking in the morning, and after the first fit of coughing expectorate into a clean wide-mouthed bottle. The object of this is to avoid the presence of fragments of food in the sputum. A better result will be secured if the examination is made on the same day, because if the bacilli are few they occur most plentifully in the small caseous flakes to be described farther on, which are easily found at first, but which break up and become part of a granular sediment that always forms in decomposed sputum. The fresh sputum when held over a black surface generally shows a number of grayish-yellow, irregular, translucent granules somewhat smaller than the head of a pin. These consist principally of the caseous material from tuberculous tissue, and are the most valuable part of the sputum for examination. One of the granules is picked up with a pointed match-stick and spread over the surface of a perfectly clean cover-glass. If no such TU13ERCUL0SIS. 297 fragment can be found, the purulent part is next best for examination. In cases in which this ordinary procedure fails to reveal bacilli whose presence is strongly indicated by the clin- ical signs, the exact method of searching for them is to partially digest the sputum with caustic potash, and then collect the solid matter with a centrifugal apparatus. If a very few bacilli are present in the sputum, this method will often secure them. The material spread upon the cover-glasses should not be too small in amount. Of course a massive, thick layer will become opaque in staining, but should the layer spread be, as is often advised, u as thin as possible," there may be too few bacilli upon the glass to enable one to make a satisfactory diagnosis. As usual, the material is allowed to dry thoroughly, and is then passed three times through the flame for purposes of fixation. EhrlicJC s Method, or the Koch-Ehrlich Method. — The cover-glasses thus prepared are floated, smeared side down, upon, or immersed, smeared side up, in, a small dish of Ehrlich's anilin-water gentian-violet solution : Anilin, 4, Saturated alcoholic solution of gentian violet, 11, Water, 100, and placed in an incubator or a paraffin oven, and kept for twenty-four hours at about the temperature of the body. When removed from the stain they are washed momentarily in water, and then alternately in 25-33 per cent, nitric acid and 60 per cent, alcohol, until the blue color of the gentian violet is almost entirely lost. It must be remembered that the action of the strong acid is a powerful one, and that too long a time must not be allowed for its application. A total immersion of thirty seconds is quite enough in most cases. After final thor- ough washing in 60 per cent, alcohol the specimen is 298 PATHOGENIC BACTERIA. counter-stained in a dilute aqueous solution of Bismarck brown or vesuvin. The excess of stain is then washed off in water, and the specimen dried and mounted in bal- sam. The tubercle bacilli will appear of a fine dark blue, while the pus-corpuscles, epithelial cells, and other bacteria, having been decolorized by the acid, will be colored brown by the counter-stain. This method, requiring twenty-four hours for its com- pletion, is naturally one which has fallen into disuse for practitioners who desire in the briefest possible time to know simply whether bacilli are present in the sputum or not. Among clinicians Ziehl's method with carbol-fuchsin has met with great favor. After having been spread, dried, and fired, the cover-glass is held in the bite of an appropriate forceps (cover-glass forceps), and the stain1 dropped upon it from a pipette. As soon as the entire cover-glass is covered with stain it is held over the flame of a spirit-lamp or a Bunsen burner until the stain begins to volatilize a little, as indicated by a white vapor. When this is observed, the heating is sufficient, and the temper- ature can be subsequently maintained by intermittent heating. If evaporation is allowed to take place, a ring of in- crustation occurs at the edge of the area covered by the stain and prevents the proper action of the acid. To prevent this more stain should now and then be added. The staining is complete in from three to five minutes, after which the specimen is washed off with water, the excess of water absorbed with paper, and 3 per cent, hydrochloric acid in 70 per cent, alcohol, 25 per cent, aqueous sulphuric, or 33 per cent, aqueous nitric acid solution dropped upon it for thirty seconds, or until the red color is just extinguished. The acid is washed off 1 Carbol-fuchsin: Fuchsin, 1 ; Alcohol, 10; 5 per cent, phenol in water. 100. TUBERCULOSIS. 299 with water, and the specimen is dried and mounted in Canada balsam. Nothing will be colored except the tu- bercle bacilli, which will appear red. Gabbett modified the staining by adding methylene blue to the acid solution, which he makes according to this formula: Methylene blue, 2 Sulphuric acid, 25 Water, 75' In Gabbett's method, after staining with carbol-fuch- sin the specimen is washed with water, acted upon by the methylene-blue solution for exactly thirty seconds, washed with water until only a very faint blue remains, dried, and finally mounted in Canada balsam. By this method the tubercle bacilli are colored red, and the pus- corpuscles, epithelial cells, and the unimportant bacteria blue. Taking into consideration that the tubercle bacil- lus contains a large amount of fat (40 per cent, of ether- soluble constituents, de Schweinitz and Dorset), Dorset1 suggests the use of Soudan III. as the best stain for the differential staining of tubercle bacillus. The method which he recommends is as follows : Cover-glass prepa- rations are made and fixed in the ordinary way and then immersed for ten minutes in a cold, saturated, 80 per cent, alcoholic solution of Soudan III. The excess is then re- moved by washing in several changes of 70 per cent, alcohol for five minutes, or in 95 per cent, alcohol. The bacilli appear bright red, and the beaded appearance is said to be very distinct. Tissues are stained and decolorized in the same manner and subsequently counter-stained with methylene blue, dehydrated with absolute alcohol, cleared in clove oil, and mounted in Canada balsam. Dorset found that Soudan III. is selective for the tubercle bacillus, and does not stain other micro- 1 New York Medical Journal, Feb. 4, 1899, p. 148. 300 PATHOGENIC BACTERIA. organisms upon which he tried it. He deems it posi- tively differential for the separation of the tubercle bacillus and the smegma bacillus, as it does not color the latter. It is said that the staining can be accomplished with Merck's Soudan III., but not with Griibler's. Inmost hands the method has not been a success. The possible relation that the number of bacilli in the expectoration of consumptives might bear to the progress or treatment of the case has been elaborately investigated by Nuttall.1 The total quantity of sputum expectorated in twenty-four hours was caught in covered, scrupulously clean conical glasses and measured therein. The pro- portion of muco-purulent to fluid matter was noted. Depending upon the degree of viscidity and number of bacilli present in the sputum, a varying amount of 5 per cent, caustic potash solution was added to it (from one- sixth to an equal volume), and after the caustic potash had rendered the sputum perfectly fluid more or less water was added to dilute the mixture. The sputum, having been measured, was poured into a perfectly clean wide- mouthed bottle containing fine sterilized gravel or broken glass. Rinsings of a measured amount of the caustic pot- ash solution were used to free the conical glass from what matter might remain and were added to the sputum. The contents of the bottle were agitated in a shaking machine for five minutes, and allowed to stand until the caustic potash solution had had time to act. As soon as the sputum had become homogeneous an equal volume of water was added, and the whole shaken again. The sputum thus treated was of a pale-green or yellowish- brown color, and contained only small fragments of elas- tic tissue. It was allowed to stand two to four hours, and then shaken again for five to ten minutes. By means of a burette of original design drops of ex- actly equal ' size were secured and caught upon clean sterile cover-glasses. The drops were subsequently 1 Bull, of the Johns Hopkins Hospital, May and June, 1891, ii., 13. TUBERCULOSIS. 301 spread into an even film by a very fine platinum wire, while the cover-glass was rotated upon a "turn-table." After spreading, the cover-glasses were laid upon a level brass plate slightly warmed to facilitate drying. After drying, the cover-glasses were coated with a serum film by spraying, and the temperature raised to 8o°-90° C. to coagulate the serum and retain the bacteria in place, after which they were carefully stained with carbol- fuchsin and decolorized with a solution of 150 parts of water, 50 parts of alcohol, and 20-30 drops of pure sul- phuric acid. Prior to this the cover-glass was washed in three alcohols and subsequently in water, and if necessary in acid and alcohol again. A special arrangement of the microscope was devised for the purpose, and the number of bacilli' in each drop estimated with extreme care. The number varied from 472 to 240,000. To estimate the number of bacilli in a given quantity the number of drops to a cubic centimeter is multiplied by the number of bacilli in the drop, and then by the number of cubic centimeters to be estimated. The method is an ingenious one, but a glance down the columns of figures in the original article will be sufficient to show that the counting of the bacilli is devoid of any practical value. This is only to be expected when one considers the pathology of the disease and remembers that accidents, such as unusually violent cough one day, modified by the use of sedatives the next, may cause wide variations in the quality if not in the quantity of the sputum. The detection of tubercle bacilli in the urine is some- times easy, sometimes difficult. The centrifuge should be used and the collected sediment spread upon the glass. If there is no albumin in the urine, it is well to add a little white of egg in securing a good fixation of the urinary sediment to the glass. The staining is identical with that of sputum, but it must be remembered that the smegma bacillus is apt to be present in the urine and, therefore, the precaution should be taken of washing the 302 PATHOGENIC BACTERIA. specimen with strong alcohol, so that this bacillus may- be decolorized. It is very difficult to find tubercle bacilli in the feces because of the small number present. When the tubercle bacilli are to be sought for in sections of tissue, considerable difficulty is at once encountered, partly because of the thickness of the section and partly because of the presence of nuclei which color intensely. Again, Ehrlich's method must be recommended as the most certain and best method of staining a large number of bacilli. The sections of tissue, if imbedded in celloidin or par- affin, should be freed from the foreign substances. Like the cover-glasses, they are placed in the stain for twelve to twenty- four hours at a temperature of 37 ° C. Upon removal they are allowed to lie in water for about ten minutes to wash away the excess of stain and to soften the tissue, which often shrinks and becomes brittle. The washing in nitric acid (20 per cent.) which follows may have to be continued for as long as two minutes. Thor- ough washing in 60 per cent, alcohol follows, after which the sections can be counter-stained, washed, dehydrated in 95 per cent, and absolute alcohol, cleared in xylol, and mounted in Canada balsam. A method which has attained great and deserved praise is Unna's. It is as follows: The sections are placed in a dish of twenty- four-hours-old, newly-filtered Ehrlich's solution, and allowed to remain twelve to twenty-four hours at the room-temperature or one to two hours in the incubator. From the stain they are placed in water, where they remain for about ten minutes to wash. They are next immersed in acid (20 per cent, nitric acid) for about two minutes, and become greenish-black. From the acid they are placed in absolute alcohol, and are gently moved to and fro until the pale-blue color returns. They are then washed in three or four changes of clean water until they become almost colorless, and are then removed to the slide by means of a section-lifter. The TUBERCULOSIS. 303 water is absorbed with filter-paper, and then the slide is heated over a Bnnsen burner until the section becomes shining, when it receives a drop of xylol balsam and a cover-glass. It is said that sections stained in this manner do not fade as quickly as those stained by Ehrlich's method. The tubercle bacillus also stains well by Gram's method, but as this is a general method by which many different bacteria are colored, it is ill adapted for purposes of differ- entiation, especially when the prosecution of the charac- teristic methods is not more difficult. So far as is known, the tubercle bacillus is a purely parasitic organism. It has never been found except in the bodies and excretions of animals affected with tuber- culosis, and in dusts of which these are component parts. This purely parasitic nature greatly interferes with the isolation of the organism, which cannot be grown upon the ordinary culture-media. Koch first achieved its arti- ficial cultivation by the use of blood-serum. When planted upon this medium the bacilli are first apparent to the naked eye in about two weeks, and occur in the form of small dry, whitish flakes, not unlike fragments of chalk. These slowly increase at the edges, and grad- ually form scale-like masses of small size, which under the microscope are seen to consist of tangled masses of bacilli, many of which are in a condition of involution. The best method of obtaining a culture is to inoculate a guinea-pig with tuberculous material, allow an artificial tuberculosis to develop, kill the animal after a couple of months, and make the cultures from the centre of one of the tuberculous glands. Of course many technical difficulties must be over- come. The sputum or other tuberculous material used for inoculation may be injected beneath the skin by a hypodermic syringe, or placed in a little subcutaneous pocket made by snipping the skin of the thigh with scis- sors and dissecting it loose so that the fragment is easily introduced. The animal is allowed to live for a month 304 PA THOGENIC BA CTERIA . or six weeks, and then killed. The autopsy is performed according to directions already given. A large lymphatic gland with softened contents or a nodule in the spleen being selected for the culture, an incision is made into it with a sterile knife, or a rigid sterile platinum wire is introduced ; some of the contents are removed and planted upon glycerin agar-agar, or, as Nocard and others recommend, upon glycerinized potato. After receiving the inoculated material the tubes are closed, either by a rubber cap placed over the cotton stopper, which is cut off and pushed in, or by a rubber cork above the cotton, the purpose of this corking being to prevent evaporation. The tubes must be kept in an incubator at the tempera- ture of 37°-38° C. A very successful method of isolating the tubercle bacillus has recently been published by Smith.1 For making the cultures dogs' serum is used. A dog is bled from the femoral artery, the blood being caught in a sterile flask, where it is allowed to coagulate. The serum is removed by a sterile pipette, placed in sterile tubes, and coagulated at 75°-76° C. Smith prefers to use a test- tube with a ground cap, having a small tubular aperture at the end, instead of the ordinary test-tube with the cotton-plug. The object of this is to prevent the contents from drying during the prolonged period of incubation that is necessary. To the same end the ventilators of the incubator are closed, and a large evaporating dish of water stood inside, so that the atmosphere may be constantly saturated with moisture. The tubes are inoculated with bits of tissue the size of a small pea, torn from the tuberculous foci. The fragments of tissue are not crushed or comminuted, but are simply laid upon the undisturbed surface of the blood-serum and then incubated for several weeks. If no growth is apparent after this period, the bit of tissue is stirred about a little and the tube returned to the 1 Transactions of the Association of American Physicians, 1898, vol. xiii., p. 417. TUBERCULOSIS. 305 incubator, where growth almost immediately begins from the bacilli, which have been scattered over the surface as the bit of tissue was moved about. Smith secures the tubercle bacillus from sputum by intraperitoneal inoculation of a guinea-pig, and prefers metastatic tuberculous foci to local foci of disease from which to secure material for inoculation. The guinea-pig should not be allowed to die, but should be chloroformed at the end of the third week. The special tubes recommended by Smith are not essential, as Ravenel, working in the laboratory of the Pennsylvania State Live Stock Sanatory Board, has successfully achieved the cultivation of the bacilli in cotton-stoppered tubes sealed with paraffin. Kitasato has published a method by which Koch has been able to secure the tubercle bacillus in pure culture from sputum. After carefully cleansing the mouth the patient is allowed to expectorate into a sterile Petri dish. By this method the contaminating bacteria from the mouth and the receptacle are excluded, and the expecto- rated material is made to contain only such bacteria as were present in the lungs. The material is carefully washed a great many times in renewed distilled sterile water until all bacteria not enclosed in the muco-purulent material are removed ; it is then carefully opened with sterile instruments, and the culture-medium — glycerin agar-agar or blood-serum — is inoculated from the centre. Kitasato has been able by this method to demonstrate that many of the bacilli ordinarily present in tuber- cular sputum are dead, although they continue to stain well. Kitasato' s method of washing the sputum has been modified and simplified by Czaplewski and Hensel.1 In their studies of whooping-cough, instead of washing the flakes in water in dishes, they shook them in peptone water in test-tubes. The shaking in the test-tube being so much more thorough than the washing in dishes, fewer 1 Centralbl. f, Bakt. u. Parasitenk., xxii., Nos. 22 and 23, p. 643. 20 3°6 PATHOGENIC BACTERIA. changes are necessary, three or four washings being sufficient. In 1887, Nocard and Roux gave a great impetus to investigations upon tuberculosis by their discovery that the addition of 4-8 per cent, of glycerin to bouillon and agar-agar made them suitable for the development of the bacillus, and that a much more luxuriant development could be obtained upon these media than upon blood-se- rum. The growth upon such "glycerin agar-agar" (Fig. 67) very much resembles that upon blood-serum. The growth upon bouillon with 4 per cent, of glycerin is also luxuriant. A critical study of the relationship of massive development and glycerin was made by Kimla, Poupe, and Vesely,1 who found that the most luxuriant growth occurred when the culture-media con- tained from 5 to 7 per cent, of glycerin. As tubercle bacilli require considerable oxygen for their proper de- velopment, they grow only upon the surface of the bouil- lon, where a thick wrinkled surface growth forms. This growth is rather brittle, and after a time subsides. The tubercle bacillus can be grown in gelatin to which glycerin has been added, but as its development takes place only at 37°-38° C, a temperature at which gelatin is always liquid, its use for the purpose is disadvantageous. Pawlowski was able to cultivate the bacillus upon potato, but Sander, who found that it could be readily grown upon various vegetable compounds, especially 1 Revue de la Tuberculose, 1898, vi., p. 25. Fig. 67. — Bacillus tuberculosis on "glycerin agar-agar." TUBERCULOSIS. 307 upon acid potato mixed with glycerin, also found that upon such compounds its virulence was constantly lost. According to French writers, the virulence of the bacillus is not diminished when it grows upon potato. It has also been said that the continued cultivation of the tubercle bacillus upon culture-media lessens its parasitic nature, so that in the course of time it can be induced to grow feebly upon the ordinary agar-agar, and that pro- longed cultivation destroys its virulence. - In a letter recently received from Ravenel, I learn that &{Xsx jive years' continuous cultivation upon artificial Fig. 68. — Bacillus tuberculosis : adhesive cover-glass preparation from a fourteen- day-old blood-serum culture; x 100 (Frankel and Pfeiffer). media the tubercle bacillus which he uses for making tuberculin still kills guinea-pigs in three weeks after intraperitoneal inoculation. It is really surprising to note the extremely simple compounds in which the tubercle bacillus can be grown. Instead of requiring the most concentrated albuminous media, as was once supposed, Proskauer and Beck1 have shown that the organism can grow in non-albuminous media containing asparagin, and that it can even be in- 1 Zeitschrift fur Hygiene, August io, 1894, xviii., No. I. 308 PATHOGENIC BACTERIA. duced to grow upon a mixture of commercial ammonium carbonate, o. 35 per cent. ; primary potassium phosphate, 0.15 per cent; magnesium sulphate, 0.25 per cent.; glycerin, 1.5 percent. It was even found that tuberculin was produced in this inorganic mixture. The tubercle bacillus seems to require a considerable amount of oxygen for its development. It is also pecu- liarly sensitive to temperatures, not growing at a tem- perature below 290 C. or above 420 C. Temperatures above 750 C. kill it after a short exposure. It does not develop well in the light, and when its virulence is to be maintained should always be kept in the dark. Sunlight kills it in from a few minutes to several hours, according to the thickness of the mass exposed to its influence. At one time the widespread character of tuberculosis suggested the idea that tubercle bacilli were ubiquitous in the atmosphere, that we all inhaled them, and that it was only our vital resistance that prevented us all from becoming its victims. Cornet1 has shown that this is untrue, and that tubercle bacilli exist only in the atmospheres contaminated by consumptives. His experiments were made by collecting dusts from numerous places — streets, sidewalks, houses, rooms, walls, etc. Injecting them into guinea-pigs, whose constant susceptibility to the disease makes them a very delicate reagent for its detection, Cornet showed the bacilli to be present only in the dust with which pul- verized sputum was mixed, and found such infectious dust to be most common where the greatest carelessness in respect to cleanliness prevailed. Our present knowledge of the life-history of the tubercle bacillus, by showing its indisposition to multiply outside the bodies of animals, the deleterious influence of sun- light upon it, the absence of positive permanent forms, and its sensitivity to temperatures beyond a certain range, confirms all that Cornet has pointed out, and shows us 1 Zeilschrifi fiir Hygiene, v. TUBERCULOSIS. 309 why the expectoration of consumptives has not rendered our atmospheres pestilential. As long as tuberculosis exists among men or cattle, it shows that the existing hygienic precautions are insuf- ficient. While not so radical as to suggest the unreason- able isolation of patients and destruction of property once practised in the kingdom of Naples, I favor the registra- tion of all tuberculous cases as a means of collecting accurate data concerning their origin, insist upon domestic sterilization and disinfection, and would have special hospitals for as man}-, especially of the poorer classes, among whom hygienic measures are almost always opposed, as could be persuaded to occupy them. It has already been declared the duty of the physician to use every means in his power to prevent the spread of infection in the households in his care, and no disease is more deserving of attention than this neglected one. Patients should cease to kiss the members of their fam- ily and friends; their individual knives, forks, spoons, cups, etc. should be carefully kept apart — secretly if the patient be sensitive upon the subject — from those of the family, and scalded after each meal ; the napkins and handkerchiefs, as well as whatever clothing or bed-cloth- ing is soiled by the discharges, should be kept apart from the common wash, and boiled ; and of course the expec- toration should be carefully attended to, received in a suitable receptacle, sterilized or disinfected, and never allowed to dry, for it has been shown that the tubercle bacillus can remain vital in dried sputum for as long as nine months. A very neat arrangement for collecting and disposing of the expectoration is recommended by some boards of health. It consists of a metal case into which a pasteboard box is fitted. When the box is to be emptied the whole of the pasteboard portion is removed, and, together with the expectoration, burned. The metal part is disinfected, provided with a new pasteboard box, and is again ready for use. (See Fig. 20, page 178.) The physician should also give directions for disinfecting the 3 1 0 PA THOGENIC BA CTERIA . bedroom occupied by a consumptive before it becomes the chamber of a healthy person. Boards of health are now becoming more and more in- terested in tuberculosis, and, though exceedingly slow and conservative in their movements, are disseminating literature among doctors for distribution to their patients, with the hope of achieving by volition that which they would otherwise regard as cruel compulsion. The channels by which the tubercle bacillus enters the organism are varied. A few cases are on record where the micro-organisms have passed through the place7ita, so that a tuberculous mother was able to infect her unborn child. It is not impossible that the passage of bacilli in this manner through the placenta causes the development of tuberculosis in infants after birth, the disease having remained latent during fetal life, for Birch-Hirschfeld has shown that fragments of a fetus, itself showing no tubercular lesions, but coming from a tuberculous woman, caused the death from tuberculosis of guinea-pigs into which they were inoculated. The most frequent channel of infection is the respira- tory tract, into which the finely-pulverized pulmonary dis- charges of consumptives and dust of infected rooms and streets enter. Fliigge, Laschtschenko, Heyman-Sticher, and Beninde x find that the greatest danger of infection is in the atomized secretions of the tuberculous respira- tory apparatus, discharged during cough. Nearly everyone discharges finely comminuted secretions during cough- ing and sneezing, as can easily be determined by holding a mirror before the face at the time. Even though dis- charged by consumptives these atoms of moisture are not infectious, except when tubercle bacilli are present in the sputum. Experiment showed that they usually do not pass further than 0.5 meter from the patient, though occasionally they may be driven 1.5 meters. The impor- tance of a knowledge of these facts in dealing with tuber- culous individuals is very great, and teach us that visits 1 Zeitschrift fur Hygiene, etc., Bd. xxx., pp. 107, 125, 139, 163, 193. Plate II. Tuberculosis of the lung : the upper lobe shows advanced cheesy consoli- dation with cavity-formation, bronchiectasis, and fibroid changes; the lower lobe retains its spongy texture, but is occupied by numerous miliary tubercles. TUBERCULOSIS. 3 1 1 to consumptives should not be prolonged; that no one should remain continually in their presence, nor habitu- ally sit within two meters of them, and that the patients should always hold a handkerchief before the face during cough. The rooms also should frequently be washed up with a disinfecting solution. Probably all of us at some time in our lives inhale living virulent tubercle bacilli, yet not all suffer from tuberculosis. Personal predispo- sition seems of great importance, for it has been shown that without the formation of tubercles virulent bacilli may be present for considerable lengths of time in the bronchial lymphatic glands — the dumping-ground of the pulmonary phagocytes. In order that infection shall occur it does not seem necessary that the least abrasion or laceration shall exist in the mucous lining of the respiratory tract. The tubercle bacillus is a foreign body of irritating prop- erties, and, lodging upon a cell, is soon engulfed in its protoplasm, or, arrested by a leucocyte, is dragged off to some other region in whose narrow passages a most hos- tile strife doubtless takes place. Infection also commonly takes place through the gas- trointestinal tract by infected food. At present an over- whelming weight of evidence points to the presence of bacilli in the milk of cattle affected with tuberculosis. It does not seem necessary that tuberculous ulcers shall be present in the udders ; indeed, the bacilli have been demonstrated in considerable numbers in milk from udders without tubercular lesions discoverable to the naked eye. The meat from tuberculous animals is less dangerous than the milk, because the meat is nearly always cooked before being eaten, while the milk is generally taken uncooked. The bacilli enter the intestinal lymphatics, sometimes produce lesions immediately beneath the mu- cous membrane, and lead later on to the formation of ulcers ; but generally they first involve the mesenteric lymphatic glands. The thoracic duct is sometimes af- 312 PATHOGENIC BACTERIA. fected, and from such a lesion it is easy to understand the development of a general miliary tuberculosis. The oc- casional absorption of tubercle bacilli by the lacteals, and their entrance into the systemic circulation and subse- quent deposition in the brain, bones, joints, etc., are sup- posed to explain primary lesions of these tissues. Infection is said also to take place occasionally through the sexual apparatus. In sexual intercourse tubercle bacilli from tuberculous testicles may be discharged into the female organs, with resulting tuberculous lesions. The infection in this way generally is from the male to the female, primary tuberculosis of the testicle being much more common than primary tuberculosis of the uterus or ovaries. While most probably rare, in comparison with the preceding, wounds also are avenues of entrance for the tubercle bacilli. Anatomical tubercles are not uncom- mon upon the hands of anatomists and pathologists, most of these growths being tuberculous in character. An interesting fact concerning these dermal lesions is the exceedingly small number of bacilli which they contain. The macroscopic lesions of tuberculosis are too familiar to require a description of any considerable length. They consist in nodes, nodules, or collections of agminated nodules, called tubercles, scattered irregularly through the tissues, which are devitalized or disorganized by their presence. When tubercle bacilli are introduced beneath the skin of a guinea-pig, the animal shows no sign of disease for a week or two ; it then begins to lose appetite and gradually to diminish in flesh and weight. Examination at this time will show a nodule at the point of injection and enlargement of the neighboring lymphatic glands. The atrophy increases, the animal shows a febrile reaction, and dies at the end of a period of time vary- ing from four to twelve weeks. Post-mortem exam- ination shows a cluster of tubercles at the point of inoculation, enlargement of lymphatic glands both near TUBERCULOSIS. 313 and remote from the primary lesion (due to the presence of tubercles), and a widespread invasion of the lungs, liver, kidneys, peritoneum, and other organs and tissues, with tuberculous tissue in a more or less advanced con- dition of necrosis. Sometimes there are no tubercles discoverable at the point of inoculation. There is no regularity in the distribution of the disease. Tubercle bacilli are demonstrable in immense numbers in all the diseased tissues. The disease as seen in the guinea-pig is more extended than in other animals because of its greater susceptibility, and the death of the animal is more rapid than in other species for the same reason. Intraperi- toneal injection of tubercle bacilli in guinea-pigs causes a much more rapid disease than that following sub- cutaneous inoculation, and is accompanied with wide- spread lesions of the abdominal organs. The animals die in from three to six weeks. In rabbits the lesion runs a longer course with similar lesions. In bovine animals and sheep the infection is usually first seen in the alimentary apparatus and associated organs, and may be limited to them. Pulmonary disease sometimes oc- curs. In man the disease is chiefly pulmonary, though gastro-intestinal and general miliary forms are common. The development of the lesions in whatever tissue or animal always depends upon the distribution of the bacilli by the lymph or the blood. The experiments of Koch, Prudden and Hodenphyl, and others have shown that when dead tubercle bacilli are injected into the subcutaneous tissues of rabbits small local abscesses develop in the course of a couple of weeks, showing that the tubercle bacilli are chemotac- tically potent. While it is extremely interesting to observe that this chemotactic property exists, it seems to be by some other irritant that most of the lesions of tuberculosis are caused. When the dead tubercle bacilli, instead of being injected en masse into the areolar tissue, are so introduced into the body — as by intravenous injection — as to disseminate 314 PATHOGENIC BACTERIA. themselves or remain in small groups, the result is quite different, and much more closely resembles that of the action of the living organism. Baumgarten, whose researches were made upon minute tubercles of the iris, has shown that the first manifesta- tion of the irritation caused by the bacillus is not the attraction of leucocytes, but the stimulation of the fixed connective-tissue cells of the part affected. These cells increase in number by karyokinesis, and form about the irritating bacterium a minute cellular collection which forms the primitive tubercle. The leucocytes are of secondary advent, and are no doubt attracted both by the substance shown by Prudden and Hodenphyl to exist in the bodies of the dead bacilli and by the necrotic changes which already affect the primary cells. For reasons not understood, the amount of chemotaxis varies greatly in different cases. Some- times the tubercles will be sufficiently purulent in type to justify the name "tuberculous abscess;" sometimes there will be a marked absence of leucocytes. The important toxic substance produced by the bacillus does not cause the chemotaxis, for when the leucocytes are absent from the tubercles the coagulation-necrosis which is so characteristic persists. The group of epithelioid cells and leucocytes constitut- ing the primitive tubercle scarcely reaches visible pro- portions before coagulation-necrosis begins. The proto- plasm of the affected epithelioid cells takes on a hyaline character, and seems abnormally viscid, so that contigu- ous cells have a tendency to unite. The chromatin of their nuclei becomes dissolved in the nuclear juice and gives stained nuclei a pale but homogeneous appearance. Sometimes this nuclear change is only observed very late. As the necrosis advances some of the cells flow together and form large protoplasmic masses — giant-cells — which contain as many nuclei as there were component cells. It may be that these nuclei multiply by karyokinesis after the protoplasmic coa- TUBERCULOSIS. 315 lescence, but only one observer, Baumgarten, has found signs of this process in giant-cells. While these changes are in progress in the epithelioid cells leucocytes may collect in such numbers as to become the chief components of the tubercle. In the further progress of coagulation-necrosis, the most deli- cate cells (the leucocytes) die first ; and not infrequently a tubercle rich in leucocytes shows extensive degenera- tion of these cells, with recurring prominence of the original epithelioid cells. It has been taught by some that the giant-cells are produced by the union of the leucocytes, but after a care- ful study of these cells I am convinced that such an origin for these monstrous cells must be very rare, and that they result solely from the epithelioid cells.1 Giant-cells are not always formed, for the- necrotic changes are sometimes so violent and widespread as to convert the whole cellular mass into a granular detritus of unrecognizable fragments. Tubercles are constantly avascular, the avascularity being an important factor in the necrosis of the larger tuberculous masses, but probably playing no important part in the degeneration of the small tubercles, which is purely toxic. Tubercles may be developed in any tissue and in any organ. In whatever situation they occur, space is occu- pied at the expense of the tissue, whose component cells are pushed aside or else included in the nodule. In mil- iary tuberculosis of the kidney it is not unusual to find a tubercle including a whole glomerule, and resolving its component thrombosed capillaries and epithelium into necrotic fragments. As almost all tissues contain a supporting tissue-frame- work of connective-tissue fragments, some of these must be embodied in the new growth. The fibres which pos- sess little vitality are more resistant than cells, and, after all the cells of a tubercle have been destroyed, will be 1 International Medical Magazine, vol. i., No. io, 1892; vol. iii., No. 2, 1894. 3i6 PATHOGENIC BACTERIA. distinctly visible among the granules, giving the tubercle a reticulated appearance. As a rule, tubercles steadily increase in size by the in vasion of fresh tissue. The tubercle bacillus does not seem to find the necrotic centres of the tubercles adapted to its growth, and completes its life-cycle with the tissue-cells. It is unusual to find healthy-looking bacilli in the necrotic Fig. 69. — Miliary tubercle of the testicle : a, zone of epithelioid cells and leucocytes ; b, area of coagulation-necrosis ; c, giant-cell with its processes : peripherally arranged nuclei and necrotic center ; d, seminiferous tubule (Cam- eron, in International Text- Book of Surgery). areas, most of them being observed at the edges of the tubercle, where the nutrition is good. From such edges the bacilli are occasionally picked up by leucocytes and transported through the lymph-spaces, until the phago- cyte falls a prey to its prisoner, dies, and sows the seed of a new tubercle. However, for the spread of tubercle TUBERCULOSIS. 317 bacilli from place to place phagocytes are not always necessary, for the bacilli seem capable of transportation by streams of lymph alone. Notwithstanding the steady advance which takes place in most observed cases of tuberculosis, and the thoroughly comprehensible microscopic explanation of it, many cases of tuberculosis recover. The periphery of every tubercle is a zone of reac- tion, with a tendency to granulation and organization. If the vital condition is such that through inappro- priate nutriment or through unusually active phago- cytosis the activity of the bacilli is checked or their death brought about, this tendency to cicatrization is allowed to progress unmolested, and the necrosed mass becomes surrounded by a zone of newly-formed contracting fibrillar tissue, by which it is perfectly isolated. In such isolated masses lime-salts are commonly deposited. Some- times this process is perfected without the destruction of the bacilli, but with their incarceration and inhibition. Such a condition is called latent tuberculosis, and may at any time be the starting-point of a new infection and lead to a fatal termination. In 1890, Koch ! announced some observations upon the toxic products of the tubercle bacillus and their relation to the diagnosis and treatment of tuberculosis, which at once aroused an enormous, but, unfortunately, a transitory enthusiasm. These observations, however, are of capital importance. Koch observed that when guinea-pigs are inoculated with a mixture containing tubercle bacilli the wound ordinarily heals readily, and soon all signs of local dis- turbance other than enlargement of the lymphatic glands of the neighborhood disappear. In about two weeks there occurs at the point of inoculation a slight induration which develops into a hard nodule, then ulcerates, and remains until the death of the animal. If, however, in the course of a short time the animals are reinoculated, the course 1 Deutsche med. IVochemchrift, 1891, No. 343. 318 PATHOGENIC BACTERIA. of the process is altogether changed, for, instead of heal- ing, the wound and the tissue surrounding it assume a dark color and become obviously necrotic, and ulti- mately slough away, leaving an ulcer which rapidly and permanently heals without enlargement of the lymph- glands. Having made this observation with injected cultures of the living bacillus, Koch next observed that the same change occurred when the secondary inoculation was made with pure cultures of the dead bacilli. It was also observed that if the material used for the secondary injection was not too concentrated and not too often repeated (only every six to forty-eight hours), the animals thus treated improved in condition, and, instead of dying of the tuberculosis induced by the primary injection in from six to ten weeks, continued to live, sometimes (Pfuhl) as long as nineteen weeks. Koch also discovered that a 50 per cent, glycerin extract of cultures of the tubercle bacillus produced the same effect as the dead cultures originally used, and gave this substance, tuberculin, to the scientific world for experimental purposes, in the hope that the prolon- gation of life observed in the guinea-pig might be true in the case of man. The active substance of the " tuberculin" seems to be an albuminous derivative insoluble in absolute alcohol. It is a proteid substance and gives all the albumin reac- tions. It differs from the toxalbumins in its resistance to heat, being able to resist exposure to 1200 C. for hours without change. Tuberculin is almost harmless for healthy animals, but extremely poisonous for tuberculous animals, its injection into them being not infrequently followed by death. The method of preparation of tuberculin is rather simple. Flasks exposing a considerable surface of liquid are filled with bouillon containing 4-6 per cent, of glyc- erin. The bouillon is preferably made with veal instead of beef. The surfaces are inoculated with pure cultures TUBERCULOSIS. 319 of the tubercle bacillus and are stood in an incubator. In the course of two weeks a slight surface growth is appar- ent, which in the course of time develops into a pretty firm pellicle and gradually subsides. At the end of some weeks development ceases and the pellicle sinks. Fig. 70. — Massive culture of the tubercle bacillus upon the surface of glycerin- bouillon, used in the manufacture of tuberculin. The contents of a number of flasks are then collected in an appropriate vessel and evaporated over a water-bath to one-tenth their volume, then filtered through a Pasteur- Chamberland filter. This is crude tuberculin. When such a product is injected in doses of a fraction 320 PATHOGENIC BACTERIA. of a cubic centimeter an inflammatory and febrile reac- tion occurs. The inflammation sometimes causes super- ficial tuberculous lesions (lupus) to ulcerate and slough away, and for this reason is of some value in therapeutics, although attended with the dangers mentioned above. The fever is sufficiently characteristic to be of diagnostic value, though the tuberculin can only be used as a diag- nostic agent in practice upon animals. The tuberculin test of cows and other animals suspected of having tuberculosis is easily carried out. The tuber- culin as Koch prepared it is now known as "concentrated " or " Koch's tuberculin," to differentiate it from the "di- luted tuberculin," which is the same thing so diluted with i per cent, carbolic acid solution that i cubic centi- meter equals a dose. The dose of the concentrated tuber- culin is 0.4-0. 5 c. cm., that of the diluted tuberculin ic.cm. For the test to be a satisfactory diagnostic one, the temperature of the animal should be taken every few hours for a day or two before the tuberculin is to be used, in order that the normal diurnal and nocturnal variations of temperature shall be known. The tuberculin is then administered by hypodermic injection into the shoulder or flank, and the temperature taken every two hours for the next twenty-four hours. A reaction of two degrees beyond that normal to the individual animal is positive of tuberculosis. After one reaction of this kind the ani- mal will not again react to a like dose of tuberculin for weeks or months. It is not impossible that the serum-agglutination test may, in the future, be applied to the diagnosis of tuber- culosis. Arloing found that when to homogeneous cult- ures of the tubercle bacillus the serum of a normal goat is added, no change occurs. If, however, the serum is from a goat that has received injections of strong tuber- culin or of tubercle bacilli, typical agglutinations like those of Widal's typhoid test occur. The action of tuberculin upon the animal organism is peculiar, but readily understandable. // does not exert TUBERCULOSIS. 321 the slightest influence upon the tubercle bacillus, but acts upon the tuberculous tissue. In the description of the tissue-changes already given it has been shown that the tubercle bacillus effects the coagulation-necrosis of the cells, but does not derive its nutriment from the dead tissue. As the cells die and are incorporated in the necrotic mass, the bacilli find the conditions of life un- favorable, and likewise die. The active bacilli, therefore, are found at the margins of the tuberculous tissues, where the cells are fairly active. The necrosis is due to bacillary poisons. When tuberculin is injected into the organism the result is to augment greatly the amount of poisonous influence upon the cells surrounding the bacilli, to destroy their vitality, to remove the favorable conditions of growth from the organism, and to leave it for a time checkmated. This action of tuberculin is accompanied by a marked hyperemia of the peri-tuberculous tissue, with transuda- tion of serum, softening of the tuberculous mass, and absorption of poisonous material into the blood. The process is always followed by a marked febrile reaction. Virchow, who well understood the action of the tuber- culin, soon showed that as a diagnostic and therapeutic agent in man its use was attended with great danger. The destroyed tissue was absorbed, and with it the bacilli, which were transported to new areas, in which a rapid invasion occurred. Old tuberculous lesions which had been encapsulated were softened, broken down, and be- came sources of dangerous infection to the individual, so that, a short time after its enthusiastic reception as a "gift of the gods," tuberculin was placed upon its proper footing as a diagnostic agent valuable in veterinary prac- tice, but dangerous in human medicine, except in cases of lupus and other external forms of the disease where the destroyed tissue could be discharged from the surface of the body. A recent important work upon tuberculin done by Koch1 has resulted in a new preparation, TR or tuberculin-R. 1 Deutsche med. Wochenschrift, 1897, No. 14. 21 322 PATHOGENIC BACTERIA. In his experience the attempts made to produce im- munity to the tubercle bacillus by the injection into animals of attenuated cultures proved failures, because abscesses invariably followed their introduction, whether dead or alive, and nodular growths in the lungs were constant sequelae of their injection into the circulation. In such nodules the bacilli could be found unabsorbed and unaltered. It seemed as if the fluids of the body could not effect solution of the bacteria. The ineffectual attempts at immunization, with the results given, probably depend upon the inability of the tissues to take up from the bacilli whatever immunizing substances they might contain, first, because of the impossibility of dissolving them, and, second, because the irritating powers they possess interfere with the direct action of normal fluids and uninjured body-cells, and always subject the bacteria to semi-pathological conditions. From these data, which he carefully studied out, Koch concluded that it would be necessary to bring about some artificial condition advantageous to the absorption of the bacilli, and for the purpose tried the action of diluted mineral acids and alkalies. The chemical change brought about in this manner facilitated absorption, but the ab- sorption of bacilli in this altered condition was not fol- lowed by immunity, probably because the chemical com- position of tubercle- toxin (or whatever one may name the poisonous products of the bacillus) was changed by the reagents used. Tuberculin, with which Koch performed many experi- ments, was found to produce immunity only to tubercu- lin, not to bacillary infection. Pursuing the idea of fragmenting the bacilli, or in some way treating them chemically in order to increase their solubility, Koch found that a 10 per cent, sodium hydrate solution yielded an alkaline extract of the bacillus, which, when injected into animals, produced effects similar to those following the administration of tuberculin, except that they were briefer in duration and more constant in TUBERCUL OSIS. 323 result. The marked disadvantage of abscess-formation following the injections, however, remained. This fluid, when filtered, possessed the properties of tuberculin. The mechanical fragmentation of the bacilli had been used by Klebs in the studies of antiphthisin and tubercu- locidin. Koch now used it with advantage in his studies, and pulverized living, fresh, virulent, but perfectly dry bacteria in an agate mortar, in order to liberate the ba- cillary substance from its protecting envelope of fatty acid. In the trituration only a very small quantity of the bacteria could be handled at a time, and Koch seemed thoroughly aware of the risk incurred from inhalation of the finely pulverized bacillary mass. Having reduced the bacilli to fragments, they were removed from the mortar in distilled water, and collected by centrifugation, in a small glass tube, as a muddy re- siduum at the bottom of an opalescent, clear fluid. For convenience he named the clear fluid TO, the sediment TR. TO was found to contain tuberculin. In order to separate the essential poison of the bacteria as perfectly as possible from the irritating tuberculin, the TR or fragments were dried perfectly, triturated once more, re-collected in fresh distilled water and re-centrifugated. After the second centrifugation microscopic examination showed that the bacillary fragments had not been resolved into a uniform mass, for when TO was subjected to stain- ing with carbol-fuchsin and methylene-blue it was found to exhibit a blue reaction, while in TR a cloudy violet reaction was obtained. The addition of 50 per cent, of glycerin had no effect upon TO, but caused a cloudy white deposit to be thrown down from TR. This last reaction showed that TR con- tained fragments of the bacilli which are insoluble in glycerin. Experiment showed that TR had decided immunizing powers. Injected into tuberculous animals in too large dose it produced a reaction, but its effects were entirely independent of the reaction. Koch's aim in using this 324 PATHOGENIC BACTERIA. substance in therapeutics was to produce immunity in the patient without reactions, by gradual but rapid in- crease of the dose. In so large a number of cases did Koch produce immunity to tuberculosis by the adminis- tration of TR, that he thinks it proved beyond a doubt that the observations are correct. In making the TR preparation Koch advises the use of a fresh, highly virulent culture not too old. It must be perfectly dried in a vacuum exsiccator, and the tritu- ration, in order to be thorough, should not be done upon more than ioo mg. of the bacilli at a time. A satisfac- tory separation of the TR from TO is said only to occur when the perfectly clear TO takes up at least 50 per cent, of the solid substance, as otherwise the quantity of TO in the final preparation is so great as to produce undesirable reactions. The fluid is best preserved by the addition of 20 per cent, of glycerin, which does not injure and prevents decomposition of the TR. The finished fluid contains 10 mg. of solid constituents to the c.cm., and before administration should be diluted with physiological salt solution (not solutions of carbolic acid). When administering the remedy to man the in- jections are made with a hypodermic syringe into the tissues of the back. The beginning dose is ^^ of a mg., rapidly increased to 20 mg., the injections being made daily. In speaking of the results of experiments upon guinea- pigs, Koch says: "I have, in general, got the impression in these ex- periments that full immunization sets in two or three weeks after the use of large doses. A cure in tubercu- lous guinea-pigs, animals in which the disease runs, as is well known, a very rapid course, may, therefore, take place only when the treatment is introduced early — as early as one or two weeks after the infection with tuber- culosis. "This rule avails also for tuberculous human beings, TUBERCULOSIS. 325 whose treatment must not be begun too late. ... A patient who has but a few months to live cannot ex- pect any value fronj the use of the remedy, and it will be of little value to treat patients who suffer chiefly from secondary infection, especially with the streptococcus, and in whom the septic process has put the tuberculosis entirely in the background." By proper administration of the TR Koch was able to render guinea-pigs so completely immune that they were able to withstand inoculations of virulent bacilli. The point of inoculation presents no changes when the remedy is administered, and the neighboring lymph- glands are generally normal, or when slightly swollen contain no bacilli. One very important objection found by Trudeau and Baldwin against commercially prepared TR is that it is possible for it to contain unpulverized, and hence live, virulent tubercle bacilli. In the preparation of the rem- edy it will be remembered that no antiseptic or germicide was added to the solutions, by which the effects of acci- dental failure to crush every bacillus could be overcome, Koch having specially deprecated such additions as pro- ducing destructive changes in the TR. Until this objec- tion can be removed, and our confidence that our attempts to cure patients will not cause their death be restored, it becomes a question whether TR can find a place in human medicine at all, or must remain an interesting scientific laboratory demonstration. Baumgarten and Walz ' find that the administration of tuberculin-R to guinea-pigs is without curative effect. They emphasize that the results obtained are like those of the old tuberculin; that "small doses are of no ad- vantage, while the larger the doses one employs, the greater are the disadvantages that result from their em- ployment." Probably the most interesting use to which the tuber- culin-R has thus far been put is found in the experi- 1 Centralbl.f. Bakt. und Parasitenk., April 12, 1898, xxiii., No. 14, p. 593. 326 PATHOGENIC BACTERIA. merits of Fisch,1 who immunized a horse with it, hoping to produce an antitoxin that might be useful in treating tuberculosis. His experiment seems to have met with remarkable success, for the serum thus secured, which he calls "Antiphthisic Serum, TR," is found to thor- oughly immunize guinea-pigs to tuberculosis, to cure tuberculous guinea-pigs in the early stages of the dis- ease, and to neutralize the effects of tuberculin upon tuberculous animals. Upon human beings it is too early to make a positive report, but Fisch' s cases have shown remarkable improve- ment. The subject is pregnant with interest and deserves attention. Hirshfelder2 claims to have cured a large number of cases of tuberculosis by the use of a preparation known as oxytuberculin. It consists of a 4 per cent, glycerin- bouillon culture of very virulent tubercle bacilli, which after being sterilized for one hour, and filtered, receives the addition of 8-10 volumes of hydrogen peroxid, and is then sterilized for ninety-six hours in a steam apparatus. During the sterilization the fluid is kept in a glass vessel, plugged with cotton wool. The peroxid of hydrogen is renewed every twelve hours. From the fluid obtained in this way the excess of the peroxid is removed by alkalinization. Before being em- ployed in human medicine the remedy is tested upon guinea-pigs. The dose may gradually be increased to 20 c.cm. The theory of action is based upon a claimed destruction of the toxic property of the tuberculin by the oxidation of the peroxid of hydrogen, which leaves a harmless but potent immunizing substance in the fluid. Paterson3 has suggested, for the production of immun- ity to tuberculosis, the use of gradually increasing doses of the serum of a fowl immunized to avian tuberculosis ' x Jour, of the Amer. Med. Assoc, Oct. 30, 1897. J Deutsche med. Wochenschrift, 1897, No. 19, and Jour, of the Amer. Med. Assoc, 1897. 3 Amer. Medico-Surg. Bull., Jan. 25, 1898. TUBERCULOSIS. 327 by gradually increased doses of sterilized, attenuated, and virulent cultures of the bacillus of avian tuberculosis. Curative results were observed in fowls thus treated, and in mammals similarly treated, and the inference drawn is that men treated in the same manner can be similarly benefited. The dose recommended is 2 c.cin. The theory depends upon the supposed identity or near relationship of the bacilli of avian and mammalian tu- berculosis. Klebs has claimed much advantage from the treatment of tuberculosis by antiphthisin. According to the ex- perimental studies of Trudeau and Baldwin, however, antiphthisin is only much diluted tuberculin, and exerts no demonstrable influence upon the tubercle bacillus in vitro, does not cure tuberculosis in guinea-pigs, and probably inhibits the growth of the tubercle bacillus upon culture-media to which it has been added, only by its acid reaction. On the other hand, Ambler has used antiphthisin with excellent results in the treatment of human tubercu- losis. Numerous experimenters, prominent among whom are Tizzoni, Cattani, Bernheim, and Paquin, have experi- mented with the tubercle bacillus and tuberculin, hoping that the principles of serum-therapy might be applicable to the disease. Nothing positive has, however, been achieved. The first-named observers claim to have im- munized guinea-pigs, in whose blood an antitoxin formed; the last-named thinks the serum of immunized horses a specific for tuberculosis. The field of experimentation is an inviting one, though the chronic course of the dis- ease lessens the certainty with which the results can be estimated. Maragliano's antitubercle serum is prepared in an uncertain manner, the tubercle-toxin with which the animals are immunized not being clearly described. It has been used in a very large number of cases in human medicine, and many cures as well as improvements are 328 PATHOGENIC BACTERIA. reported. Behring comments upon it by saying that " Maragliano's tubercle-antitoxin contains no antitoxin." Babes and Proca, in an experimental research upon the action of the antituberculous serum, claim for it a decided specific action, and demonstrate experimentally that ani- mals inoculated with tubercle bacilli and injected with the serum are protected from the spread of the disease. Mafucci and diVestra found that by injecting guinea- pigs with serum from sheep immunized by injections first of dead, then of living cultures of tubercli bacilli, although no cures were brought about, the vitality of the animals was maintained longer. Unprotected animals died in fifty to fifty-three days. Those injected after in- fection, seventy-four days, and those injected before infec- tion, ninety-one days. The author1 made an elaborate study of so-called Anti- tiiberculin. For a long period donkeys were injected with increasing doses of tuberculin, in order that an antitoxin — antituberculin — might be generated in their blood. Experiments upon guinea-pigs showed that the serum was powerless to immunize against the tubercle bacillus, or to cure established tuberculosis. The serum, however, had the power of annulling the effects of tuber- culin upon tuberculous animals. While a failure experi- mentally, certain clinicians claim that in practice it ex- erts a beneficial action upon patients. Indeed, presuming that an antituberculin is formed, it is but natural that it should do good in all cases in which it is probable that the patient is poisoned by tuberculin or a similar product. Rather nearer the desideratum are the experiments of De Schweinitz,2 who injected cows and horses with increas- ing quantities of bouillon cultures of a greatly attenuated tubercle bacillus, and subsequently found that the serum possessed the property of rendering guinea-pigs immune to the virulent bacilli. It is said that this serum has pro- duced beneficial results in human medicine. 1 Jour, of the Atner. Med. Assoc, Aug. 21, 1897. 2 Centralbl. f. Bakt. und Parasitenk., Sept. 15, 1897, Bd. xxii., Nos. 8 and 9. TUBERCULOSIS. 329 Fowl-tuberculosis {Bacillus tuberculosis avium). — The cases of tuberculosis which occasionally occur sponta- neously in chickens, parrots, ducks, and other birds were originally attributed to the Bacillus tuberculosis hominis, but the recent work of Rivolta, Mafucci, Cadio, Gilbert, Roger, and others have shown that, while very similar in many respects to the Bacillus tuberculosis, the organism found in the avian disease has distinct peculiarities which stamp it a different variety, though not a separate species. Cadio, Gilbert, and Roger succeeded in infect- ing fowls by feeding them upon food containing tubercle bacilli, and keeping them in cages in which dust con- taining tubercle bacilli was placed. The infection was aided by lowering the temperature with antipyrin and lessening vitality by starvation. Morphologically, the organisms are similar, the bacillus of fowl-tuberculosis being a little longer and more slender than that of mam- malian tuberculosis, and showing a more marked tend- ency to assume club and branched forms. The frag- mented or beaded forms occur as in human tubercle bacilli. Bovine Tuberculosis {Bacillus tuberculosis bovis). — In his monograph upon tuberculosis Koch called attention to certain differences that exist between the bacilli of human and animal tuberculosis, but very little attention was devoted to the subject. The well-known tubercu- lous diseases of cattle were found to have lesions resem- bling those of human tuberculosis, containing bacilli that in a general way resemble those found in human tuber- culosis and stain similarly. The conclusion that the bacilli and processes were identical was inevitable. It is not determined that there is any other difference between the two bacilli than can be "accounted for upon biological grounds, each organism being slightly modified to accommodate itself to its environment. There are, however, a few quite well-marked differences which have been subjected to investigation, and which may have some important bearings upon public sanitation. 33° • PATHOGENIC BACTERIA. While occasional desultory experiments have been made, and from time to time investigators have per- formed inoculation experiments, the subject seems to have met its most thorough study at the hands of Smith.1 He carefully compared a series of bacilli obtained from human sputum with a series obtained from the tuberculous lesions of cattle, horses, hogs, cats, dogs, and other animals. Briefly summarized, his observations lead us to the following conclusions : 1. Vegetation. — The human tubercle bacillus grows upon dog's serum much more luxuriantly and rapidly than the bovine bacillus. 2. Morphology. — The size of the bovine forms is very constant, the individuals being quite short (1-2 //). They are straight, not very regular in outline, and sometimes of a spindle, sometimes a barrel, and sometimes an oval shape. The bacilli of human tuberculosis, on the other hand, are prone to take an elongate form under artificial cultivation. 3. Staining. — The bacillus of bovine tuberculosis usually takes an even color with readiness ; that of hu- man tuberculosis differs in presenting more irregularity of penetration, by which the so-called beaded appearance is produced. They are also more apt to contain rounded, deeply-staining bodies suggestive of spores, at or near the ends. When grown upon very moist surfaces many of the bacilli of human tuberculosis are stained with great difficulty. 4. Pathogenesis. — a. Guinea-pigs. — The bacilli of bovine tuberculosis are much more virulent than those of human tuberculosis, intraperitoneal inoculation of the former producing death in adult animals in from seven to sixteen days; of the latter, in from ten to thirty-eight days. Sub- cutaneous inoculation of the bovine bacillus causes death in less than fifty days; of the human bacillus, in from fifty to one hundred days. 1 Transactions of the Association of American Physicians, 1896, xi., p. 75. "and 1898, xiii., p. 417. TUBERCUL OSIS. 33 1 b. Rabbits. — All of the rabbits which were inoculated into the ear vein with the bovine bacillus died in from seventeen to twenty-one days. Those receiving human bacilli sometimes lived several months. c. Cattle. — Cows and heifers receiving intrapleural and intra-abdominal injections of the human bacilli usually gained in weight and showed no symptoms. When ex- amined post mortem, circumscribed chronic lesions were found. Those inoculated with the bovine bacillus lost weight, suffered from constitutional symptoms, and showed at the necropsy extensive lesions. Two-thirds of the cattle inoculated experimentally with the bovine bacillus died. 5. Lesions. — In general the lesions produced by the bovine bacillus were rapid, extensive, and necrotic. Many bacilli were present. Those produced by the human bacillus were more apt to be productive, chronic, and un- accompanied by large numbers of bacilli. The bacilli of human tuberculosis produced lesions with many giant cells; those of bovine tuberculosis, lesions with rapid coagulation-necrosis. The lesions resulting from the intravenous injection of human bacilli into rabbits re- sembled those observed by Prudden and Hodenphyle1 after the intravenous injection of boiled, washed tubercle bacilli. From these data it is evident that the bovine bacillus is by far the more virulent and dangerous organism. While the human bacillus infects cattle with difficulty, the bovine bacillus infects animals, and probably man, with great readiness. The urgent need of securing milk free from all possible tuberculous infection will be evi- dent to all who reflect upon the subject. Upon culture-media a distinct rapidity of growth is observable, and we find that, instead of growing only when glycerin is present, the Bacillus tuberculosis galli- narum will grow upon blood-serum, agar-agar, and bouil- lon as ordinarily prepared. It will not grow upon potato. 1 N. Y, Med. Jour., June 6-20, 1891. 332 PATHOGENIC BACTERIA. The bacillus will grow at 42°-45° C. quite as well as at 370 C, while the growth of the mammalian tubercle bacillus ceases at 420 C. Moreover, the temperature of 430 C. does not attenuate its virulence. The thermal death-point is 700 C. Upon culture-media it can retain its virulence for two years. The growth upon artificial culture-media is luxuriant, and lacks the dry quality characteristic of ordinary tubercle-bacillus cultures. As it becomes old a culture of fowl-tuberculosis turns slightly yellow. Birds are the most susceptible animals for experimental inoculation, the embryos and young more so than the adults ; guinea-pigs are quite immune, or after inocula- tion develop cheesy nodes, but do not die. Artificial inoculation can be made in the subcutaneous tissue, in the trachea, and in the veins, never through the intestine. After inoculation the birds die in from one to seven months. The chief seat of the disease is the liver, where cellular nodes, lacking the central coagula- tion and the giant cells of mammalian tuberculosis, and enormously rich in bacilli, are found. The disease never begins in the lungs, and the fowls which are diseased never show bacilli in the sputum or the dung. Rabbits are-easily infected, an abscess forming at the seat of inoculation, and later nodules forming in the lung, so that the distribution is quite different from that seen in birds. It is very probable that the bacillus occa- sionally infects man. The bacillus stains like the tubercle bacillus, but takes the stain rather more easily. The resistance to acids is about the same. The possibility that this bacillus is de- rived from the same stock as the tubercle bacillus is strengthened by the experiments of Farmi and Saleano, who succeeded in increasing its virulence by combining it with glucose and lactic acid until it became fatal to guinea-pigs. Bacillus Resembling the Tubercle Bacillus. — While there can be little doubt that the bacilli of human, TUBERCULOSIS. 333 bovine, and avian tuberculosis are closely related to each other and may have had a common ancestry, there are a few other micro-organisms whose morphology and stain- ing peculiarities suggest a closer relationship than other considerations would warrant. The most important of these is the Bacillus leprae of leprosy (q. v.), which is separately discussed. Another very similar organ- ism is the Bacillus smegmatis, or smegma bacillus. Al- varez and Tavel, Matterstock, Klemperer and Bittu,1 and others have found a peculiar bacillus in the smegma taken from beneath the prepuce in man and from between the labia minora in women, as well as in the folds of the groin and about the anus, in urine, and occasionally in the saliva and sputum. The organism stains with carbol-fuchsin as does the tubercle bacillus, and resists the decolorant action of acids. It is, however, very readily decolored by the use of absolute alcohol. The bacillus is about the same size and shape as the tubercle bacillus, and is very readily mistaken for it. Its presence is to be expected in all cases of suspected tuberculosis of the genito-urinary ap- paratus, and in staining specimens of urine or other secre- tions washing with strong alcohol is a precaution against error. The final differentiation may have to rest upon animal inoculation. So far as is known, the smegma bacillus is a harmless saprophyte. It has been confounded with Lustgarten's bacillus of syphilis, but must be a separate species, as Lustgarten's bacillus was found in lesions of the internal organs. It is harmless to the lower animals when inocu- lated into them. Its cultivation is very difficult. Doutrel- epont and Matterstock, however, have achieved its culti- vation upon coagulated hydrocele fluid, which was browned by their growth. They were unable to transplant the growth successfully. Novy2 recommends the cultivation of the smegma bacillus by inoculating a tube of melted agar-agar 1 Virchow's Archives, v., 103. 5 Laboratory Work in Bacteriology, 1899. 334 PATHOGENIC BACTERIA. cooled to 500 C. with the appropriate material, and mix- ing with it about 2 c.cm. of blood withdrawn from a vein of the arm with a sterile hypodermic syringe. The blood- agar mixture is poured into a sterile Petri dish and set aside for a day or two at 37 ° C. The colonies that form are to be examined for bacilli that resist decolorization with acids. Novy mentions bacilli which are occasionally found in milk, butter, timothy hay, cow-dung, etc., which stain like the tubercle bacillus and may be mistaken for it. Guinea-pig inoculations must be resorted to in cases of doubt, but as some of these organisms sometimes kill the guinea-pigs after a month or two, and as small nodules or tubercles may be present in the animals in the mesen- tery, peritoneum, liver, lung, etc., the diagnosis may have to be subjected to the further confirmation of a histolog- ical examination of the lesions in order to exclude tuber- culosis. In cases of this kind it should not be forgotten that the tubercle bacillus can be present in the substances mentioned, so that the exact differentiation becomes a very fine one. Acid-resisting bacilli are also said by Novy to occur upon the conjunctiva. Pseudo-tuberculosis. — Eberth, Chantemesse, Charrin, and Roger have reported certain cases of so-called pseudo- tuberculosis. The disease occurred spontaneously in guinea-pigs, and was characterized by the formation of cellular nodules in the liver and kidneys much resembling miliary tubercles. Cultures made from them showed the presence of a small motile bacillus which could easily be stained by ordinary methods (Fig. 71). When introduced subcutaneously into guinea-pigs the original disease was produced. The Bacillus pseudo-tuberculosis is characterized by Pfeiffer as follows : The organism is rod-shaped, the rods varying in length. Upon gelatin and agar-agar circular colonies with a dark nucleus surrounded by a transparent zone are found. In gelatin punctures the bacilli grow all TUBERCULOSIS. 335 along the line of puncture and form a surface growth with consecutive markings. The bacilli grow readily upon agar, but not so readily on potato. The bacillus is fatal to mice, guinea-pigs, rabbits, and hares about twenty days after inoculation. At the seat of inoculation an abscess develops, the neighboring lym- Fig. 71. — Bacillus pseudo-tuberculosis from agar-agar; x 1000 (Itzerott and Niemann). phatic glands enlarge and caseate, and nodules resembling tubercles form in the internal organs. The bacilli studied by Pfeiffer were isolated from a horse supposed to have glanders. CHAPTER II. LEPROSY. Bacillus Leprae (Hansen).1 Leprosy is a disease of great antiquity, and very early received much attention and study. In giving the laws to Israel, Moses included a large number of rules for its recognition, the isolation of the sufferers, the determina- tion of recovery, and observances to be fulfilled before the convalescent could once more mingle with his people. The Bible is replete with accounts of miracles wrought upon lepers, and during the times of biblical tradition it must have been an exceedingly common and malignant disease. At the present time, although we in the Northern United States hear very little about it, leprosy is still a widespread disease. It exists in much the same form as two thousand years ago in Palestine, Syria, Egypt, and the adjacent countries. It is exceedingly common in China, Siam, and parts of India. Cape Colony has many cases. In Europe, Norway, Sweden, and parts of the Mediterranean coast furnish a considerable number of cases. Certain islands, especially the Sandwich Islands, are regular hot-beds for its maintenance. The United States is not exempt, the Gulf coast being chiefly af- fected. At one time the view was prevalent that the disease was spread only by contagion, at another that it was miasmatic. At present the tendency is to view it as contagious to a degree rather less than tuberculosis. Sometimes it is hereditary. The cause of leprosy is now pretty certainly deter- mined to be the lepra bacillus (Fig. 72), which was dis- 1 Virchow's Archives, 1879. 336 LEPROSY. 337 covered by Hansen, and subsequently clearly described by Neisser. The bacillus is almost the same size as the tubercle bacillus — perhaps a little shorter — but lacks the curve which is so constant in the latter. It stains in very much the same way as the tubercle bacillus, but permits of a rather more rapid penetration of the stain, so that Fig. 72. — Bacillus leprae, seen in a section through a subcutaneous node ; x 500 (Frankel and Pfeiffer). the ordinary aqueous solutions of the anilin dyes color it quite readily. It stains well by Gram's method, by which beautiful tissue specimens can be prepared. The peculiar property of retaining the color in the presence of the mineral acids which characterizes the tubercle bacillus also characterizes the lepra bacillus, and the methods of Ehrlich, Gabbett, and Unna can be used for its detection. Like that of the tubercle bacillus, its protoplasm often presents open spaces or fractures, which have been re- 22 , 33§ PA THOGENIC BACTERIA. garded by some as spores, but which are even less likely to be spores than the similar appearances in the tubercle bacillus. The organism almost always occurs singly or in irreg- ular groups, filaments being unknown. It is not motile. Many experimenters have endeavored to grow this ba- cillus upon artificially prepared substances, but in spite of modern methods, improved apparatus, and refined media, few claim to have met with success. Bordoni-Uffredozzi was able to grow upon a blood-serum- glycerin mixture a bacillus which partook of the staining peculiarities of the lepra bacillus as it appears in the tissues, but differed very much from it in its morphology. After numerous generations this bacillus was induced to grow upon ordinary culture-media. It commonly pre- sented a club-like form, which was regarded by Baum- garten as an involution appearance. Frankel points out that the bacillus of Bordoni is possessed of none of the essential characters of the lepra bacillus except its stain- ing. Czaplewski ! offers a confirmation of the work of Bor- doni-Uffredozzi, together with a description of a bacillus supposed to be the lepra bacillus, which he succeeded in cultivating from the nasal secretions of a leper. The bacillus was first isolated upon a culture-medium consisting of glyceriuized serum without the addition of salt, pepton, or sugar. The mixture was placed in flat dishes, coagulated by heat, and sterilized by the inter- mittent method. The secretion, rich in lepra bacilli, was taken up with a platinum wire and inoculated upon the culture-medium by a series of linear strokes. The dishes (Petri dishes were used for the experiment) were securely closed with paraffin and stood in the incubating-oven at 370 C. Upon the surface of the medium there grew numerous colonies of staphylococcus aureus, the bacillus of Fried- 1 Cenlralbl. f. Bakt. und Parasitenk., Jan. 31, 1898, vol. xxiii., Nos. 3 and 4, p. 97. LEPROSY. 339 lander and a number of colonies consisting of fine, slender, often somewhat nodose bacilli about the size and form of the lepra bacillus. These colonies were grayish-yellow, humped in the middle, 1-2 mm. in diameter, irregularly rounded, and irregular at the edges. They could be inverted entire with the platinum wire and were excavated on the under side. The consistence was crumbly. When a transfer was made from one of these colonies to fresh media, in a few days the growth became apparent and assumed a band-like form, with a plateau-like eleva- tion in the center. The bacillus thus isolated grew with moderate rapidity upon all the ordinary culture-media except potato. Upon blood-serum the growth was more luxuriant and fluid than upon the solid media. Upon coagulated serum the growth was rather dry and elevated, and was frequently so loosely attached to the surface of the medium as to be readily lifted up by the platinum wire. The growth was especially good upon sheep's blood- serum with the addition of 5 per cent, of glycerin. The growth upon the LofBer-mixture was excellent. Upon agar-agar the growth is not so good as upon blood-serum ; it is more luxuriant upon glycerin agar- agar than upon plain agar-agar; it is grayish and flatter upon agar-agar than upon blood-serum. The growth never extends to the water of condensation to form a floating layer, as does that of the tubercle bacillus. The colonies that form upon agar-agar are much like those described by Bordoni-Uffredozzi, and appear as iso- lated, grayish, rounded flakes, thicker in the center than at the edges, and characterized by an irregular serrated border from which a fine irregular network extends upon the medium. These projections consist of bundles of the bacilli. Upon gelatin the bacillus develops well after it has grown artificially for a number of generations. Upon the surface of gelatin the growth is, in general, similar 34° PATHOGENIC BACTERIA. to that upon agar-agar. In puncture-cultures most of the growth is on the surface in the form of a whitish, or grayish, or yellowish folded layer. In the depths of the gelatin the development occurs as a granular rather thick column. The medium is hot liquefied. Bouillon is not* clouded; no superficial growth occurs. The vegetation occurs only at the bottom of the tube in the form of a powdery sediment. Czaplewski found that the bacillus stained well with Loffler's methylen-blue, and with the aqueous solutions of the anilin dyes. It also stains by Gram's method, and has the same resisting power to the decolorizing action of mineral acids and alcohol as the lepra bacillus as seen in tissue. The young bacilli color homogeneously, but older ones are invariably granular. They are usually pointed at the ends when young, but may be rounded or knobbed when older. The more rapidly the bacillus grows, the longer and more slender it appears. All attempts to infect the lower animals with leprosy, either by the purulent matter or solid tissue from lepers, or by inoculating them with the supposed specific bacilli that have been isolated, have failed. Ducrey seems to have cultivated the lepra bacillus in grape-sugar, agar, and in bouillon "m vacuo." His results need confirmation. Very few instances are re- corded in which actual inoculation has produced leprosy in either men or animals. Arning was able to secure permission to experiment upon a condemned criminal in the Sandwich Islands. The man was of a family entirely free from disease. Arning introduced beneath his skin fragments of tissue freshly excised from a lepra nodule, and kept the man under observation. In the course of some months typical lesions began to develop at the points of inoculation and spread gradually, ending in general lepra in the course of about five years. Melcher and Artmann introduced fragments of lepra nodules into the anterior chambers of the eyes of rabbits, and observed the death of the animals after some months LEPROSY. 341 with typical lepra lesions of all the viscera, especially the cecum. While the lepra bacillus has much in common with the tubercle bacillus, there is not the slightest evidence of any real identity. It has already been shown that lepra bacilli do not grow upon artificial media, and that they cannot be readily transmitted by inoculation. The fol- lowing description will show that the relation of the bacilli to the lesions is entirely different from that of the tubercle bacilli to the tubercles. Like the Bacillus tuberculosis, the Bacillus leprae proba- bly only occurs in places frequented by persons suffering from the disease. That individuals are infected by the latter less readily than by the former bacilli probably depends upon the fact that leprotis infection seems to take place most commonly by the entrance of the organ- isms into the individual through cracks or fissures in the skin, while the tuberculous infection occurs through the more accessible respiratory and digestive apparatus. The lepra nodes are usually superficial, affecting the skin and subcutaneous tissues, but may occur in the organs. Virchow has seen a case in which lepra bacilli could be found only in the spleen.1 Sticker 2 (Lepra Konferenz) is of the opinion that the primary infection in lepra takes place in the nose, sup- porting his opinion by observations upon 153 accurately- studied cases, in which 1. The nasal lesion is the only constant one in both the nodular and anaesthetic forms. 2. The nasal lesion is peculiar — i. e. characteristic — and entirely different from all other lepra lesions. 3. The clinical symptoms of lepra begin in the nose. 4. The relapses in the disease always begin with nasal symptoms, such as epistaxis, congestion of the nasal mucous membrane, a sensation of heat, etc. 1 Mittheilungen und Verkandlungen der internationalen wissenschaftlischen Lepra- Konferenz zu Berlin, Oct., 1897, ii. Thiel. 1 Ibid. 342 PATHOGENIC BACTERIA. 5. In incipient cases the lepra bacilli were first found in the nose. Once established in the body, the bacillus by its growth produces chronic inflammatory nodes — the analogues of tubercles. The nodes consist of lymphoid and epithelioid cells and fibres, and, unlike tubercles, the lepra nodes are vascu- lar, so that much of the embryonal tissue completes its transformation to fibres. The bacilli are not distributed through the nodes like tubercle bacilli, but are found in groups enclosed within the protoplasm of certain large cells — the "lepra cells." These cells seem to be over- grown and partly degenerated lymphoid cells. Some- times they are anuclear, sometimes they contain several nuclei (giant-cells). Bacilli also occur in the lymph- spaces and in the nerve-sheaths. Iyepra nodules do not degenerate like tubercles, and the formation of ulcers, which constitutes a large part of the disease, seems largely due to the action of external agencies upon the feebly vital pathological tissue, which is unable to recover itself when injured. According to the recent studies of Johnston and Jamie- son,1 the bacteriological diagnosis of nodular leprosy can be made by spreading the serum obtained by scraping a leprous nodule upon a cover-glass, drying, fixing, and staining with carbol-fuchsin and Gabbet's solution as for the tubercle bacillus. In such preparations the bacilli are present in enormous numbers, thus forming a marked contrast to the tubercular skin diseases, in which very few can be found. In that form known as anesthetic leprosy, nodules form upon the peripheral nerves, and by connective-tissue formation, as well as the entrance of the bacilli into the nerve-sheaths, cause irritation, then degeneration, of the nerves. The anesthesia which follows these peripheral nervous lesions is one of the conditions predisposing to the formation of ulcers, etc. by allowing injuries to occur 1 Montreal Med. Journal, Jan., 1897. LEPROS Y. 343 without detection and to progress without observation. The ulcerations and occasional loss of phalanges that follow these lesions occur, probably, in the same manner as in syringomyelia. The disease advances, having first manifested itself upon the face, extensor surfaces, elbows, and knees, to the lymphatics and the internal viscera. Death ultimately Fig. 73. — Tubercular leprosy in a negro (Corlett). occurs from exhaustion, if not from the frequent inter- current affections to which the conditions predispose. While not so contagious as many other diseases, it has been abundantly proved that leprosy is transmissible, and it may be regarded as an essential sanitary precaution that lepers should be strictly segregated. CHAPTER III. GLANDERS. Bacillus Mallei (Loffler and Schiitz).1 Glanders is an infectious mycotic disease which, very fortunately, is almost confined to the lower animals. Only occasionally does it secure a victim from hostlers, drovers, soldiers, and bacteriologists, whose frequent association with and experimentation upon animals bring them in frequent contact with those which are diseased. Of all the infectious diseases studied by scientists, none has caused the havoc which glanders has wrought. Several men of prominence have succumbed to accidental in- fection. Glanders was first known to us as a disease of the horse and ass characterized by the occurrence of discrete, clean- ly-cut ulcers upon the mucous membrane of the nose. These ulcers are formed by the breaking down of nodules which can be detected upon the diseased membranes, and show no tendency to recover, but slowly spread and dis- charge a virulent pus. The edges of the ulcers are in- durated and elevated, the surfaces often smooth. The disease does not progress to any great extent before the submaxillary lymphatic glands begin to enlarge. Later on these glands form large lobulated masses, which may soften, open, and become discharging ulcers. The lungs may also become infected by inspiration of the infectious material, and contain small foci not unlike tubercles in appearance. The animals ultimately die of exhaustion. In 1882, shortly after the discovery of the tubercle bacillus, Loffler and Schiitz discovered in the discharges and tissues of this disease the specific micro-organism, the glanders bacillus {Bacillus mallei. Fig. 74), which is its cause. 1 Deutsche med. Wochenschrift, 1882, 52. 344 GLANDERS. 345 The glanders bacillus is somewhat shorter and dis- tinctly thicker than the tubercle bacillus. It has rounded ends, and it generally occurs singly, though upon blood- Fig. 74. — Bacillus mallei, from a culture upon glycerin agar-agar; x 1000 (Frankel and Pfeiffer). serum, and especially upon potato, several joined indi- viduals may be found. Long threads are never formed. The bacillus is non-motile. Various observers have claimed the discovery of spores, but although in the interior of the bacilli there have been observed irregular spaces like the similar spaces in the continuity of the tubercle bacillus not colored by the stains, they have not yet been definitely proven to be spores. The ob- servation of Loffler that the bacilli can be cultivated after being kept in a dry state for three months makes it appear as if some permanent form (spore) occurs. No flagella have been demonstrated upon the bacillus. Like the tubercle bacillus, the glanders bacillus does not seem to find conditions outside the animal body suit- able for its existence, and probably does not occur except as a parasite. The organism only grows between 250 and 42 ° C, and generally grows very slowly, so that attempts at its isola- 346 PATHOGENIC BACTERIA. tion and cultivation by the usual plate method are apt to fail, because the numerous other organisms in the material grow much more rapidly. The best method of isolation seems to be the use of an animal reagent. It has been said that glanders princi- pally affects horses and asses. Recent observations, how- ever, have shown the goat, cat, hog (slightly), field-mouse, wood-mouse, marmot, rabbit, guinea-pig, and hedgehog all to be susceptible animals. Cattle, house-mice, white mice, and rats are immune. The guinea-pig, being a highly susceptible as well as a readily procurable animal, naturally becomes the reagent for the detection and isolation of the bacillus. When a subcutaneous inoculation of some glanders pus is made, the disease can be observed in guinea-pigs by a tumefaction in from four to five days. Somewhat later this tumefaction changes to a caseous nodule, which ruptures and leaves a chronic ulcer with irregular mar- gins. The lymph-glands speedily become involved, and in a month to five weeks signs of general infection are present. The lymph-glands suppurate, the testicles un- dergo the same process, and still later the joints exhibit a suppurative arthritis containing the bacilli. The ani- mal finally dies of exhaustion. In guinea-pigs no nasal ulcers form. In field-mice, which are even more suscepti- ble, the disease is much more rapid. No local lesions are visible. In two or three days the animal seems un- well, the breathing is hurried, it sits still with closed eyes, and without any other preliminaries tumbles over on its side, dead. From the tissues of the inoculated animals the pure cultures are most easily made. Perhaps the best places to secure the culture are from softened nodes which have not ruptured or from the suppurating joints. Strauss has, however, given us a method which is of great use, because of the short time required. The material sus- pected to contain the glanders bacillus is injected into the peritoneal cavity of a male guinea-pig. In three or GLANDERS. 347 four days the disease becomes established. The testicles enlarge a little ; the skin over them becomes red and shining. The testicles themselves begin to suppurate, and often discharge through the skin. The animal dies in about two weeks. If such an animal be killed and its testicles examined, the tunica vaginalis testis will be found to contain pus, and sometimes to be partially ob- literated by inflammatory exudation. The bacilli are pres- ent in this pus, and can be secured from it in pure cultures. The value of Strauss' s method has, however, been less- ened by the discovery by Kutcher,1 that a new bacillus, which he has classified among the pseudo-tubercle ba- cilli, produces a similiar testicular swelling when injected into the abdominal cavity. Also by Levy and Stein- metz,2 who found that Staphylococcus pyogenes aureus was also capable of provoking suppurative orchitis. However, the result is easily controlled by the culture of the glanders bacillus from the pus in cases of glanders. The purulent discharges from the noses of horses and from other lesions of large animals generally contain very few bacilli, so that their detection by the use of the guinea-pig inoculation is made much more simple. The bacillus is an aerobic organism, and can be grown in bouillon, upon agar-agar, better upon glycerin agar- agar, very well upon blood-serum, and quite character- istically upon potato. It grows in gelatin, but this is not an appropriate medium, because the bacillus develops best at temperatures at which the gelatin is liquid. Upon 4 per cent, glycerin agar-agar plates the colonies appear upon the second day as pale-yellow or whitish, shining round dots. Under the microscope they appear as brownish-yellow, thick granular masses with sharp borders. The culture upon agar-agar and glycerin agar-agar occurs as a moist, shining layer not possessed of distinct peculiarities. Upon blood-serum the growth is rather 1 Zeitschrift fur Hygiene, Bd. xxi., Heft i., Dec. 6, 1895. 1 Berliner klin. Wochenschrift, March 18, 1895, No. II. 348 PATHOGENIC BACTERIA. characteristic. The colonies along the line of inoculation first develop as circumscribed, clear, transparent drops, which later become confluent and form a transparent layer unaccompanied by liquefaction. The most characteristic growth is upon potato. It first appears in about forty-eight hours as a transparent, honey-like, yellowish layer, developing only at incuba- tion-temperature and soon becoming reddish-brown. As this brown color of the colony develops, the potato for a considerable distance around it becomes greenish- brown. (See Frontispiece.} No other known organism produces the same appearance upon potato. In litmus milk the growth of the glanders bacillus is associated with the production of an acid that reddens the reagent, with the formation of a firm coagulum and the subsequent separation from it of a clear reddish whey. The organism loses its virulence if cultivated for many generations upon artificial media. The bacillus is killed in five minutes by exposure to 55° C. That this bacillus is the cause of glanders there is no room to doubt. Loftier and Schiitz have succeeded by the inoculation of horses and asses in producing the well-known disease. The organisms when in cultures can be stained with the watery anilin-dye solutions, but are difficult to stain in tissues. They do not stain by Gram's method. The chief difficulty in staining the bacillus in tissues is the readiness with which it gives up the stain in the presence of decolorizing agents. LofHer at first accom- plished the staining by allowing the sections to lie for some time (five minutes) in the alkaline methylene-blue solution, then transferring them to a solution of sulphuric and oxalic acids — Concentrated sulphuric acid, 2 drops ; 5 per cent, oxalic-acid solution, 1 drop ; Distilled water, 10 c.cm. GLANDERS. 349 for five seconds, then transferring to absolute alcohol, xylol, etc. The bacilli appear dark blue upon a paler ground. This method gives very good results, but has been largely superseded by the use of Kiihne's carbol- methylene blue : Methylene blue, 1.5 Alcohol, 10. 5 per cent, aqueous phenol solution, 100. Kiihne's method of staiuing is to place the section in the stain for about half an hour, wash in water, decolorize carefully in hydrochloric acid (10 drops to 500 c.cm. of water), immerse at once in a solution of lithium carbonate (8 drops of a saturated solution of lithium carbonate in 10 c.cm. of water), place in a bath of distilled water for a few minutes, dip into absolute alcohol colored with a little methylene blue, dehydrate in anilin oil containing a little methylene blue in solution, wash in pure anilin oil, not colored, then in a light ethereal oil, clear in xylol, and mount in balsam. When stained in sections of tissue the bacilli are found to occupy the interior of small inflammatory zones not unlike tubercles in appearance. These nodules can be seen with the naked eye scattered through the livers, kidneys, and spleens of animals dead of experimental glanders. The nodules consist principally of leucocytes, but also contain numerous epithelioid cells. As is the case with tubercles, the centres of the nodules are prone to degenerate, soften, and also to suppurate. The retro- gressive processes upon exposed surfaces, where the break- ing down of the nodules allows their contents to escape, are the sources of the typical ulcerations. At times the process is progressive, and some of the lesions heal by the formation of a stellate scar. Baumgarten regarded the origin and course of the his- tological lesions of glanders to be much like those of the tubercle. In his studies epithelioid cells first accumulated, and were followed by leucocytes. Tedeschi was not able 35° PATHOGENIC BACTERIA. to confirm the results of Baumgarten's work, but found the primary change to be due to a necrosis of the affected tissue followed by an invasion of leucocytes. The recent researches of J. H. Wright l are in accord with those of Tedeschi rather than with those of Baumgarten, for Wright observed first a marked degenerative effect upon the tissue, and then an inflammatory exudation amount- ing in some cases to actual suppuration. As has been mentioned, cultures of the bacillus lose their virulence more or less after four or five generations in artificial media. While this is true, attempts to atten- uate fresh cultures by heat, etc. have so far failed. Leo has pointed out that white rats, which are immune to the disease, may be made susceptible by feeding with phloridzin and causing a glycosuria. Kalning, Preusse, Pearson, and others have pre- pared a substance, "mallein," from cultures of the bacillus, and suggested its employment for diagnostic purposes. It seems to be quite useful in veterinary medicine, the reaction occasioned by its injection being similar to that caused by the injection of tuberculin in tuberculous patients. The manufacture of mallein is not attended with great difficulty. The bacilli are grown in glycerin bouillon for several weeks, killed by heat, the culture filtered through porcelain and evaporated to one- tenth of its volume. It has also been prepared from potato cultures, which are said to produce a stronger toxin. A febrile reaction of more than 1.50 C. following the injection is said to be specific of the disease. Babes has asserted that the injection of this toxic product into susceptible animals will protect them from the disease. Various experiments have been made with curative objects in view. Certain observers claim to have seen good results follow the injection of mallein in repeated small doses. Others, as Chenot and Picq, find the blood- serum from immune animals like the ox to be curative when injected into infected guinea-pigs. 1 your, of Exp. Med., vol. i.5 No. 4, p. 577. CHAPTER IV. SYPHILIS. Bacilli of Lustgarten1 and Van Niessen.1 Although syphilis is almost as well known as it is widespread, we have not yet discovered for it a definite specific cause. Whether it is due to a protozoan par- asite, or whether it is due to a bacterium, the future must decide. Numerous claims have been made by those whose studies have revealed organisms of one kind or another in syphilitic tissues, but no one has yet suc- ceeded either in isolating, cultivating, or successfully in- oculating them. In 1884 and 1885, Lustgarten published a method for the staining of bacilli which he had found in syphilitic tissues and assumed to be the cause of the disease. The staining, which is very complicated, requires that the sections of tissue be stained in Ehrlich's anilin-water gentian-violet solution for twelve to twenty-four hours at the temperature of the room, or for two hours at 400 C. ; washed for a few minutes in absolute alcohol ; then im- mersed for about ten seconds inai^ per cent, perman- ganate-of-potassium solution, after which they are placed in an aqueous solution of sulphurous acid for one to two seconds, thoroughly washed in water, run through alco- hol and oil of cloves, and finally mounted in Canada balsam dissolved in xylol. If the bacilli are supposed to be present in pus or dis- charges from syphilitic lesions, the cover-glasses spread with the material are stained in the same manner, except that for the first washing distilled water instead of abso- lute alcohol is used. 1 Wiener med. Wochenschrift, 1884,47. * Centralbl. f. Bakt. u. Parasitenk., 1 898, xxiii., No. 2. 351 352 PA THOGENIC BA CTERTA . This method undergoes a modification in the hands of De Giacomi, who prefers to stain the cover-glasses in hot anilin-water-fuchsin solution for a few moments, sections in the same solution cold for twenty-four hours ; then immerse them first in a weak, then in a strong, solution of chlorid of iron. The cover-glasses are washed in water, sections in alcohol, and subsequently passed through the usual reagents for dehydration and clearing. Fig. 75. — Bacillus of syphilis (Lustgarten), from a condyloma; x 1000 (Itzerott and Niemann). In some syphilitic tissues these methods suffice to de- fine distinct bacilli with a remarkable similarity to the tubercle bacillus. The organism is about the same size as the tubercle bacillus, and even more frequently curved, but often presents a club-like enlargement of one end (involution-form?). The bacilli very frequently occur singly, though more often in groups, and never lie free, but are always enclosed in cells. These bacilli are not always found in syphilitic lesions, nor is their demonstra- tion easy under the most favorable circumstances. Laist- garten emphasizes particularly that they are only demon- strable after the most painstaking technical procedures. The probability of the specificity of this organism was considerably lessened by the observation by Matterstock, Travel, and Alvarez that in preputial smegma, and also SYPHILIS. 353 in vulvar smegma from healthy individuals, a similar organism, identical both in morphology and staining peculiarities, could be demonstrated. Of course the oc- currence of Lustgarten' s bacillus in the internal organs could not but argue against the probability of its identity with the smegma bacillus ; but Lustgarten himself pointed out that the bacilli of both tuberculosis and leprosy stain by his method, and thus gave Baumgarten the right to suggest that the few cases well adapted for the demon- stration of the Lustgarten bacilli might be cases of mixed infection of tuberculosis and syphilis. The most recent research upon the bacteriology of syphilis is that of van Niessen,1 who claims to have cul- tivated a syphilis bacillus from the blood of a few cases. Blood secured from a deep puncture at the end of a thoroughly disinfected finger is caught in a sterile glass, diluted with an equal quantity of distilled water and kept for from ten to fourteen days at a temperature of io°-20° R. (i3°-i5° C). Very often the blood of syphi- litics is found subject to accidental contamination by various well-known bacteria. When this is not the case, however, the serum remains almost perfectly clear and contains a large number of bacilli — syphilis bacilli. The bacillus can be transplanted to bouillon, in which it grows with the production of grayish- white shreds and floating flocculi, some of which are suspended in the liquid, while others form a membrane upon the surface. When transplanted to obliquely solidified gelatin and kept at room temperature, in the course of forty-eight hours a very fine, grayish-white, thready mass like cloudy streaks, and having a peculiar reflecting surface, can be seen. Under a lens this is seen to consist of lines of threads which sometimes seem to penetrate into the depths of the gelatin. After a time a layer is formed upon the surface of the medium. Some liquefaction of the medium occurs and causes the growth to slide down upon 1 Centralbl. f. Bakt. und Parasitenk., Bd. xxiii., No. 2, Jan. 19, 1898, p. 49; No. 344, Jan. 31, 1898, p. 97; and No. 546, Feb. 11, 1898, p. 177. 23 354 PATHOGENIC BACTERIA. itself so as to assume the form of a fragment of a tape- worm. Upon agar-agar after the lapse of two days the growth consists of a central pellicle along the line of in- oculation, with little sprouts projecting in all directions from the edges. The growth is grayish, with an occa- sional yellowish tinge. Punctures in agar-agar were unsuccessful, but in gela- tin the appearance of the growth is similar to that of the cholera spirillum. The bacillus also grows upon potato in the form of an elevated layer of exactly the same color as the potato. In the course of time the entire potato becomes colored a dark gray. It also grows in milk, urine, serum, and water. The colonies of this bacillus are quite characteristic, but so varied in appearance as to make one suspect that the plate upon which which they grow is contaminated with various other species of bacteria. In general, the colonies may be said to appear slowly as transparent whitish drops, which become grayish and later yellow- ish, and finally brownish in color. The gelatin about them presents concentric, wave-like rings, depending upon the liquefaction of the medicine. When the growth is more rapid and occurs at higher temperatures bundles of threads, somewhat resembling the early stages of a mould, are observed. Examining microscopically, one finds in the slowly growing colonies a surrounding zone of small centrifugally arranged fine threads or hairs extending in all directions, with one or two exceptionally long bundles extending beyond the others and beyond the limits of the colony. The long threads are never found to divide. Many of the colonies are highly suggestive of those of anthrax. The bacillus is motile in very slight degree. It forms spores. It is, in general, about the size of the tubercle bacillus. . The vegetation of the organism is said to be peculiar in that the bacillary stage is of short duration and soon SYPHILIS. 355 gives place to the formation of septate, V-shaped, and branched forms. It seems to be normally a strepto-ba- cillns in its early stages, but eventually becomes very pleomorphous, varying in appearance from a chain of oval cocci to the hypha of the moulds. There seems to be nothing peculiar about the staining-capacity of the bacillus. It stains with the ordinary solutions of the aniliu dyes, retains the stain of Gram's method, and is decolorized by mineral acids. Dohle ' succeeded in staining certain protoplasmic bodies in the tissues in syphilis, which resembled the actively motile protoplasmic bodies which he had pre- viously encountered in the discharges. They were for the most part round or oval, sometimes with irregular outlines, and were provided with flagella. The staining took place in a mixture of hematoxylon and carbol-fuch- sin, subsequently treated with iodin or chromatin, and washed in alcohol. Convinced that these bodies were the cause of syphilis, he excised small fragments from gummata and other syphilitic tissues, and placed them beneath the skin of guinea-pigs, which subsequently fell ill with a chronic marasums which ultimately caused death. In the inoculation experiments of van Niessen there were observed as evidences of the specificity of the organism discovered by him: (i) abortion in pregnant female rabbits; (2) extra-genital primary lesions on the ears of inoculated rabbits in the form of nodes; (3) sec- ondary ulcer and tumor formations, and irregular lesions, such as occasional thrombosis and pneumonia. The researches of others have, up to the present time, entirely failed to confirm the results of Van Niessen' s work. 1 Miinch. med. Wochenschrift, 1897, No. 43. CHAPTER V. ACTINOMYCOSIS. Streptothrix Actinomyces (Rossi-Doria). In 1845, Langenbeck discovered that the specific dis- ease of cattle known as actinomycosis could be com- municated to man. His observations, however, were not given to the world until 1878, one year after Bollinger1 had discovered the cause of the disease in animals. Fig. 76. — Actinomyces bovis, from the tongue of a calf; x 500 (Frankel and Pfeiffer). J. Israel 2 first observed the disease in man and studied its pathogenesis. The best paper on the subject is prob- 1 Deutsche Zeitschrift fur Thiermedizin, 1 877. 2 Virchow's Archives, 1874-1878. 356 ACTINOMYCOSIS. 357 ably that by Bostron,1 who carefully studied the micro- scopical lesions of the disease. Actinomycosis is a disease almost peculiar to the bovine animals, though sometimes occurring in hogs, horses, men, and other animals. The first manifestations of the disease are usually found either about the jaw or in the tongue, in either of which localities there are produced considerable enlargements which are sometimes dense and fibrous (wooden tongue) and sometimes suppurative. In sections of these nodular formations small yellowish granules surrounded by some pus can be found. These granules when viewed beneath the microscope exhibit a peculiar rosette-like body — the ray-fungus or actinomyces. The fungus is of sufficient size to be detected in pus, etc., by the naked eye. It can be colored, in sections of tissue, by the use of Gram's method, or better by Weigert's fibrin-stain. Tissues pre-stained with carmin, then stained by Weigert's method, give beautiful pict- ures. The entire fungus-mass consists of several distinct zones embracing entirely different elements. At the centre of the mass there is found a granular substance containing numerous bodies resembling chains of micro- cocci or spores. Extending from this centre into the neighboring tissue is a radiating, branched, thickly- tangled mass of mycelial threads. These threads seem to terminate in a zone of conspicuous club-shaped radiat- ing forms which give the colonies the rosette-like appear- ance. The clubs are inconspicuous in the fungus as seen in the lesion of the human form of the disease. The pleomorphism and branched network formed by the growth of the micro-organism class it among the higher bacteria in the genus streptothrix. When the artificial cultivations are properly crushed, spread out and stained, it is found that the mycelial threads are from 0.3-0.5// in thickness, are quite long, and frequently 1 Zeitschrift fur Hygiene, 1 889. 358 PATHOGENIC BACTERIA. show at their ends a flask- or bottle-like expansion — the club— which probably depends upon a gelatinization of the cell-membrane. It is the club that is the chief char- acteristic of the organism. As the fungus occurs in the tissues, the radiations are very distinct, the clubs all being directed outward, closely packed together, forming a rounded mass. When stained by carmin and Gram's stain the threads appear blue-black, the clubs red. The cells of the tissues affected and a larger or smaller collec- tion of leucocytes form the surrounding resisting tissue- zone. The degree of chemotactic influence exerted bv the organism seems to depend partly upon the tissue affected and partly upon the individuality of the animal. When the animal is but slightly susceptible, and when the tongue is the part affected, the disease is characterized by the production of enlargement due to the formation of cicatricial tissue. If, on the other hand, the animal is highly susceptible or the jaw is affected, the chief symptom is suppuration, with the formation of cavities communicating by sinuses. Before the nature of the affection was understood it was confounded with various diseases of the bones, prin- cipally with osteosarcoma. From the tissues primarily affected the disease spreads to the lymphatic glands, and not infrequently to the lungs. Israel has pointed out certain cases of human actinomycosis beginning in the peribronchial tissues, probably from inhalation of the fungi. Jones is of the opinion that the disease, if not iden- tical with, is closely allied to, tuberculosis, and that the occasional branched forms of tubercle bacilli prove the tendency of the individual bacillus to form a reticulum. The organism may be grown upon all the artificial culture-media, as has been shown by O. Israel,1 Wolff, and others. To secure it in pure culture the material known to 1 Virchow's Archives, cxv. A CTINOM YCOSIS. 359 contain the actinomyces granules, secured so as to be as free from other micro-organisms as possible, is crushed between glass plates or in a mortar, and then transferred to plates or tubes as desired. The colonies make their appearance as small gray dots, which consist of a trans- lucent, radiating network of filaments. If kept at the temperature of incubation for a few days, they become opaque nodules with radiating processes about the periph- ery. The growth occurs best in free access of oxygen, though the organism is a facultative anaerobe. Upon blood-serum the nodular growths present a yel- lowish or sometimes brick or rust-red color, and may sur- round themselves with a whitish down of fine threads. The colonies cling closely to the culture-media and are firm, so that they are crushed with difficulty. If the sur- face is scraped, spores and fine threads are secured. If the mass is crushed, the branched filaments of the strep- tothrix may be secured. The colonies become confluent as time goes on, and a thick wrinkled membrane is produced. The growth liquefies the blood-serum. In gelatin puncture cultures an arborescent growth is produced and the gelatin liquefies. Upon agar-agar and glycerin agar-agar the picture is similar to that of the blood-serum growth, but has less and sometimes only a grayish color. The agar-agar turns brown as the culture ages. In bouillon the growth takes the form of large granules if allowed to stand quietly; of numerous small granules if frequently shaken up. The granules are similar in structure to those formed upon the dense media. The bouillon does not become clouded. Upon potato the growth much resembles that upon blood-serum, but is slower. The color is reddish-yellow and the white down early makes its appearance. The growth also takes place in eggs, when long branched filaments are observed. When the actinomyces are grown upon artificial media their virulence is retained for a considerable length of 360 PATHOGENIC BACTERIA. time. The disease cannot usually be successfully inocu- lated into the laboratory experiment animals, as the intro- duced fungus elements seem either to become absorbed or encapsulated by connective tissue and do not grow. If successfully introduced into the abdominal cavities of rabbits, there are produced in the peritoneum, mesentery, and omentum typical nodules containing the actinomyces rays. The organism can also be grown in raw eggs, into which it is carefully introduced through a small opening made under aseptic precautions. In the eggs the organism forms peculiar, long, branched mycelial threads quite un- like the short forms developing upon agar-agar. The characteristic rosettes which are constantly found in the tissues are never seen in artificial cultures. The exact manner by which the organism enters the body is unknown. In some cases it may be by direct inoculation with pus, but there is reason to believe that the organism occurs in nature as a saprophyte, or as an epiphyte upon the hulls of certain grains, especially barley. Woodhead records a case where a primary me- diastinal actinomycosis in the human subject was sup- posed to be traced to perforation of the posterior pharyn- geal wall by a barley spikelet swallowed by the patient. Cases of actinomycosis are fortunately of rather rare occurrence in human medicine, and do not always occur in those brought in contact with the lower animals. The fungi may enter the organism through the mouth and pharynx, through the respiratory tract, through the di- gestive tract, or through wounds. The invasion has been known to take place at the roots of carious teeth, and is more liable to occur in the lower than in the upper jaw. Israel reported a case in which the primary lesion seemed to occur external to the bone of the lower jaw, as a tumor about the size of a cherry, with an external opening. In two cases of the disease observed by Murphy of Chicago both began with tooth- ache and swelling of the jaw. A few cases of dermal ACTINOMYCOSIS. 361 infection are recorded. Elsching1 has seen a case in which calcified actinomyces grains were observed in the tear duct. When inhaled, the organisms gain entrance to the deeper portions of the lung, and bring about a suppura- Fig. 77. — Section of liver from a case of actinomycosis in man (Crookshank). tive bronchopneumonia with adhesive inflammation of the contiguous pleura. After the formation of the pleu- ritic adhesions the disease may penetrate the newly- 1 Centralbl.f. Bakt. u. Parasitenk., xviii., p. 7. i . 362 PATHOGENIC BACTERIA. formed tissue, extend to the chest-wall, and form external sinuses. Or it may penetrate the diaphragm and invade the abdominal organs, causing an interesting and charac- teristic lesion in the liver and other large viscera (see Fig. 77)- Microscopically the lesion consists chiefly of a round- cell infiltration with circumscribing granulation-tissue leading to the formation of cicatricial bands. In the form known as "wooden tongue" the disease runs an essentially chronic course, with the production of consid- erable amounts of connective tissue. But few cases recover, the disease terminating by death from exhaustion or from complicating pneumonia or other organic lesions. CHAPTER VI. MYCETOMA, OR MADURA-FOOT. Streptothrix Madur>e (Vincent1). A CURIOUS disease of not infrequent occurrence in the Indian province of Scinde is one known as mycetoma, Madura-foot, or pied de Madura. It almost invariably affects natives of the agriculturist class, and in most cases begins in or is referred by the patient to the prick of a thorn. It generally affects the foot, more rarely the hand, and in one instance was seen by Boyce in the shoulder and hip. It is more common in men than in women, individuals between twenty and forty years of age suffering most frequently, but persons of any age or sex may suffer from the disease. It is insidious in its onset, as has been said, generally following a slight injury, such as the prick of a thorn. No symptoms are observed in what might be called an incubation stage of a couple of weeks' duration, but after this time elapses a nodular growth gradually forms, attaining in the course of time the size of a marble. Its deep attachments are indistinct and diffuse. The skin becomes purplish, thickened, indurated, and adherent. The points most frequently invaded at the onset are the ball of the great toe and the pads under the bases of the fingers and toes. In the course of months, although progressing slowly, the lesions attain very perceptible size, distinct tumors being present. Later, sometimes not until after a year or two, the nodes begin to soften, break down, discharge their purulent contents, and originate ulcers and com- municating sinuses. The discharge at this stage is a thin sero-pus, and is always mixed with a number of 1 Annates de V Inst. Pasteur, 94, 3. 363 364 PATHOGENIC BACTERIA. fine round black or pink bodies, described, when black, as resembling gunpowder ; when pink, as resembling fish-roe. It is the detection of these particles upon which the diagnosis rests, and upon which the divis- ion of the disease into the melanoid and pale varieties depends. The progress of the disease causes an enormous size and a peculiar deformity of the affected part. The mal- ady is generally painless. The micro-organismal nature of the disease was early suspected. In spite of the confusion caused by some who confounded the disease with and described it as "Guinea-worm," Carter held that it was due to some indigenous fungus as early as 1874. Boyce and Surveyor believe that the black particles of the melanoid variety represent a curious metamorphosis of a large branching septate fungus, and that the white particles of the other variety are the remains of a lowly-organized fungus and of caseous particles. Kanthack tried to prove the identity of the fungus with the well-known actinomyces, but there seems to be considerable doubt about the correctness of his view. Vincent succeeded in isolating the micro-organism by puncturing one of the nodes with a sterile pipette, and has cultivated it upon artificial media. Acid vege- table infusions seem suitable to its growth. It develops scantily in bouillon at the room-temperature, better at 370 C. — in from four to five days. In twenty to thirty days the colony attains the size of a little pea. In the liquid media the colonies which cling to the glass, and thus remain near the surface of the medium, develop a rose- or bright-red color. Cultures in gelatin are not very abundant, are colorless, and are unaccompanied by liquefaction. Upon the surface of agar-agar strikingly beautiful rounded, glazed colonies are formed. They are at first colorless, but later become rose-colored or bright red. The majority of the clusters remain isolated, some of them Plate III. Mycktoma. — I. Section showing black granules and general features of the lesions as they appear under a low magnifying power. Zeiss aY 2. Showing structure and appearances of the hyphae of the mycelium obtained from the granules. Zeiss apochromat., 4 mm. 3. Two bouillon cultures, showing the powder-puff-ball appearance. In one the black granule is seen in the center of the growth. 4. Potato culture of the hyphomycete obtained from the gran- ules. The black globules are composed of a dark-brown fluid. (James H. Wright.) MYCETOMA, OR MADURA-FOOT. 365 attaining the size of a small pea. They are generally umbilicated like a variola pustule, and present a curious appearance when the central part is pale and the periphery red. As the colony ages the red color is lost and it be- comes dull white. The colonies are very adherent to the surface of the medium, and are said to be of cartilaginous consistence. The organism also grows in milk without coagulation. Upon potato the development is meagre, slow, and with very little tendency to chromogenesis. The color- production is more marked if the potato be acid in reac- tion. Some of the colonies upon agar-agar and potato have a powdery surface, no doubt from the occurrence of spores. It is, of course, an aerobic organism. Under the microscope the organism is found by Vin- cent to be a streptothrix — a true branched fungus con- sisting of long bacillary branching threads in a tangled mass. In many of the threads spores could be made out. Vincent was unable to communicate the disease to animals by inoculation. Microscopic study of the diseased tissues in cases of mycetoma is not without interest. The healthy tissue is said to be sharply separated from the diseased masses. The latter appear as large degenerated tubercles, except that they are extremely vascular. The mycelial or filamentous fungous mass occupies the centre of the degeneration, where its long filaments can be beautifully demonstrated by the use of appropriate stains, Gram's method being excellent for the purpose. The tissue sur- rounding the disease-nodes is infiltrated with small round cells. The youngest nodules are seen to consist of granu- lation-tissue, which in its organization is checked by the coagulation-necrosis which is sure to overtake it. Giant- cells are few. Not infrequently small hemorrhages occur from the ulcers and sinuses of the diseased tissues ; the hemor- rhages can be explained from the abundance of small blood-vessels in the diseased tissue. 366 PATHOGENIC BACTERIA. Although the disease has been described as occurring in Scinde, it is not limited to that province, having been met with in Madura, Hissar, Bicanir, Delhi, Bombay, Baratpur, Morocco, Algeria, and one case by Bastini and Campana in Italy. In America the disease is almost unknown, a total of five cases being on record. CHAPTER VII. FARCIN DU BCEUF. Streitothrix Farcinica (Rossi-Doria1). The peculiar disease which sometimes affects numbers of cattle in Guadeloupe, and which was described by the older writers as farcin du bceuf, has been carefully studied by Nocard. It is a disease of cattle character- ized by a superficial lymphangitis and lymphadenitis, affecting the tracheal, axillary, prescapular, and other glands. The affected glands enlarge, suppurate, and discharge a creamy, sometimes a grumous, pus. The internal organs are often affected with a pseudo-tubercu- losis whose central areas undergo a purulent or caseous degeneration. In the researches of Nocard it was discovered, by staining by Gram's and by Kuhne's methods, that in the centres of the tubercles micro-organisms could be defined. They resembled long delicate filaments rather intricately woven, characterized by distinct ramifications which made clear the proper classification of the organ- ism as a Streptothrix (Streptothrix farcinica). The organism was successfully cultivated by Nocard upon various culture-media at the temperature of the body. It is aerobic. In bouillon the organism develops in the form of color- less masses irregular in size and shape, some of which float upon the surface, others of which sink to the bottom of the liquid. Sometimes the surface is covered by an irregular fenestrated pellicle of a gray color. Upon agar-agar the growth develops in small, rather 1 Annali de I' Institute d'igiene sperimrniah di Roma, 91. 367 368 PATHOGENIC BACTERIA. discrete, irregularly rounded, opaque masses of a yellow- ish-white color. The surfaces of the colonies are tuber- ciliated, and an appearance somewhat like a lichen is observed (see Fig. 78). Upon potato very dry scales of a pale-yellow color rapidly develop. The growth upon blood-serum is less luxuriant, but similar to that upon agar-agar. In milk the organism produces no coagulation by its growth, and does not alter the reaction. Microscopic study always reveals the organism as the same tangled mass of filaments seen in the tissues. The old cultures are rich in spores, which are very small and 1 H j • Fig. 78. — Streptothrix of farcin du bceuf growing on glycerin agar-agar. develop upon the most superficial portions of the growth. These spores resist the penetration of stains to a rather unusual extent. Cultures retain their virulence for a long time : Nocard found one virulent after it had been kept for four months in an incubating oven at 400 C. FARCIN DU BCEUF. 369 The streptothrix of farcin du bceuf \s pathogenic for guinea-pigs, cattle, and sheep ; dogs, rabbits, horses, and asses are immune. When the culture or some pus containing the micro- organism is injected subcutaneously into a guinea-pig, a voluminous abscess results. Not long afterward the lym- phatic vessels and glands of the region are the seat of swell- ing and induration, and extensive phlegmons form, which rupture externally and discharge considerable pus. The animal, of course, becomes extremely ill and seems about to die ; instead, it slowly recovers its normal condition. In other animals, as the cow and the sheep, the subcu- taneous inoculation results in an abscess relatively less extensive. This ulcerates, then indurates, and seems to disappear, but after the lapse of several weeks or months opens again in the form of a new abscess. In animals which are immune or nearly immune, like the horse, the ass, the dog, and the rabbit, the subcuta- neous inoculation is followed by the formation of a small abscess which speedily cicatrizes. Intraperitoneal inoculation in the guinea-pig gives rise to an appearance resembling tuberculosis. The omentum may be extensively involved and full of softened nodes. The liver, spleen, and kidneys appear full of tubercles, but careful examination will satisfy the observer that the tubercles are only upon the peritoneal surfaces, not in the organs. Intravenous introduction of the cultures produces a condition much resembling general miliary tuberculosis. All the organs contain the pseudo-tubercles in consider- able numbers. 24 CHAPTER VIII. RHINOSCLEROMA. Bacillus Rhinosci.eromatis (von Frisch1). In Austria, Hungary, Italy, and some parts of Ger- many there sometimes occurs a peculiar disease of the anterior nares, characterized by the occurrence of circum- scribed tumors, known as rhinoscleroma. The tumor- masses are somewhat flattened, isolated or coalescent, grow with great slowness, and recur if excised. The dis- ease commences in the mucous membrane and the adjoin- ing skin, and spreads to the skin in the neighborhood by a slow invasion, involving the upper lip, jaw, hard palate, and sometimes the pharynx. The growths are without evidences of inflammation, do not ulcerate, and consist microscopically of infiltration of the papilla and corium of the skin, with round cells which change in part to fibrillar tissue. The tumors possess a well-developed lymph- vascular system. Sometimes the cells undergo hyaline degeneration. In these little tumors the researches of Von Frisch dis- covered little bacilli much resembling both in morphol- ogy and vegetation the pneumo-bacilli of Friedlander, and, like them, surrounded by capsules. The only marked difference between the so-called bacillus of rhi- noscleroma and the Bacillus pneumoniae of Friedlander is that the former stains well by Gram's method, while the latter does not, and that the former is rather more distinctly rod-shaped than the latter, and more often shows its capsule in culture-media. The bacillus can easily be cultivated, and in all media resembles the bacillus of Friedlander too closely to be distinguished from it. Even when inoculated into animals the bacillus behaves much like Friedlander's bacillus. Inoculation has, so far, failed to produce the disease either in men or in the lower animals. 1 Wiener med. Wochenscrift, 1882, 32. 370 B. THE TOXEMIAS. CHAPTER I. TETANUS. Bacillus Tetani (Fliigge). One of the most exquisitely toxic bacteria of which we have any knowledge is the bacillus discovered in 1884 by Nicolaier,1 obtained in pure culture by Kitasato* in 1889, and now universally recognized as the cause of tetanus. It is a peculiar organism, whose striking feature is a considerable enlargement of one end, in which a bright round spore is seen (Fig. 79). The bacilli which Fig. 79. — Bacillus tetani; x 1000 (Frankel and Pfeiffer). are not sporiferous, are long, rather slender, have rounded ends, seldom unite in chains or pairs, are motile, and have no flagella. The bacilli stain readily with ordi- 1 Deutsche med. Wochenschrift, 1884, 42. " Ibid , 1S89, No. 31. 371 372 PATHOGENIC BACTERIA. nary aqueous solution of the anilin dyes, and also very readily by Gram's method. The tetanus bacillus is a common saprophytic organ- ism which can be found in most garden-earth, in dust, in manure, and sometimes in the intestinal discharges of animals. It is extremely difficult to isolate and cultivate, because it will not grow where the smallest Fig. 80. — Bacillus tetani : six-days- Fig. 81. — Bacillus tetani : culture old puncture-culture in glucose-gelatin four days old in glucose-gelatin (Fran- (Frankel and Pfeiffer). kel and Pfeiffer). amount of oxygen is present It is a typical obligatory anaerobic micro-organism. Curiously enough, in spite of the difficulty that attends the cultivation of the tetanus bacillus, Farran1 and Grixoni believe it to be 1 Centralbl.f. Bakt. u. Parisitenk., July 15, 1898, p. 28. TETANUS. 373 an optional anaerobe, originally an aerobic organism. When trained to grow in the presence of oxygen it loses its virulence. The method now generally employed for the isolation of this bacillus is that originated by Kitasato, and based upon his observation that its spores can resist high temper- atures. After finding that the typical bacilli are present in earth or pus, or whatever the material to be investi- gated was, Kitasato exposed a portion of it for an hour to a temperature of 8o° C. By this heating all the fully- developed bacteria, tetanus as well as the others, and the Fig. 82. — Bacillus tetani : five-days-old colony upon gelatin containing glucose ; x 1000 (Fiankel and Pfeiffer). great majority of the spores except those of tetanus, were destroyed, and, as little other than tetanus spores re- mained, their cultivation was made comparatively easy. The resistance which the tetanus bacilli manifest toward heat is only part of a great general resisting power of which they are possessed. It is said that they can retain their vitality in the dried condition for months. Stern- berg says they can resist 5 per cent, carbolic solutions 374 PATHOGENIC BACTERIA. for ten hours, but will not grow after fifteen hours' im- mersion. 5 per cent, carbolic acid, to which 0.5 per cent, of hydrochloric acid has been added, destroys them in two hours. They are also destroyed in three hours by 1 : 1000 bichlorid-of-mercury solution ; but when to such a solution 0.5 per cent, of hydrochloric acid is added, its activity is so increased that the spores are destroyed in thirty minutes. The resistance to heat is only within certain limits, for exposure to passing steam for from five to eight minutes is certain to kill the spores. The colonies of the tetanus bacillus, when grown in an atmosphere of hydrogen upon gelatin plates, somewhat resemble those of the well-known hay bacillus. There is a dense rather opaque central mass from which a more transparent zone is readily separable. The margins of this outer zone are made up of a radiating fringe of pro- jecting bacilli (Fig. 82). The liquefaction that occurs is much slower than that caused by bacillus subtilis. When grown in gelatin puncture-cultures the develop- ment occurs deep in the puncture, and consists of mul- titudes of short-pointed processes radiating from the puncture, somewhat resembling a fir tree (Fig. 80). Liquefaction begins in the second week and causes the disappearance of the radiating processes. The liquefac- tion spreads slowly, but may involve the entire mass of gelatin and resolve it into a grayish-white syrupy liquid, at the bottom of which the, bacilli accumulate. The growth in gelatin containing glucose is much more rapid ; that in agar-agar punctures is much slower, but similar to the gelatin cultures except for the absence of liquefac- tion. The organism can also be grown in bouillon, and attains its maximum development at a temperature of 370 C. Much gas is given off from the cultures. Cultures of the tetanus bacillus in all media give off a peculiar, very disagreeable, characteristic odor. The methods for excluding the oxygen from the cul- tures and replacing it by hydrogen, as well as other methods suggested for the cultivation of the strictly TETANUS. 375 anaerobic organisms, are given under the appropriate heading (Anaerobic Cultures), and need not be repeated here. A very simple method of cultivating the bacillus in bouillon for the purpose of securing a large amount of toxin has been suggested by the author.1 An ordinary bottle is filled with bouillon to the mouth, and closed by a perforated rubber stopper containing a glass tube a couple of inches long. Connected with this glass tube, by means of a short piece of rubber tubing (a), is the bulb of a broken pipette ^ (b), the other end of which is plugged with cotton (Fig. 83). When the steam sterilization takes place the expanding fluid ascends to the reservoir repre- sented by the pipette bulb, de- scending again as the fluid cools. When the sterilization is com- pleted the reservoir is detached, the inoculation made by passing a very fine pipette into the bottle, the projecting glass tube drawn out to a fine tube, and the bottle stood in hot water until the ex- panding fluid ascends to the apex Of the pointed glass tube. The durinS sterilization; 2, after in- . 1 1 j • n oculation and sealing. tube is now sealed in a name & and the bottle and its contents allowed to cool. In cool- ing the retracting fluid leaves a vacuum which at once draws up any minute bubbles of air remaining, and allows the tetanus bacillus to grow in a condition of very fair anaerobiosis. Tetanus bacilli exist in nature as widely distributed saprophytes. They are quite common in the soil, and the fact that they are most plentiful in manured ground 1 Centralbl. f. Bake. u. Parasitenk., xix., Nos. 14 and 15, April 25, 1896, p. 550. Fig. 83. — Tetanus bottle : t, 376 PATHOGENIC BACTERIA. has suggested that they originate in the intestines of horses and reach the earth from their excrement. The relation of the bacillus to manure is very interest- ing, inasmuch as it is most common in manured ground and about stables. It is most probable that the relation is simply dependent upon the fact that in manured ground, where there is much nutriment, the bacilli flourish better than in sterile ground. The occasional occurrence of the bacilli in the excrement of herbivorous animals is to be expected because of the earth they commonly swallow with the food cropped from the ground. Verneuil has observed that tetanus rarely occurs at sea except upon cattle transports. Upon such vessels, how- ever, there is likely to be considerable earth and earthy dust from hay, straw, etc., which may carry the bacilli. Le Dantec1 has shown that the tetanus bacillus is a common organism in New Hebrides, where there are no horses. In these islands the natives poison their arrows by dipping them into a clay rich in tetanus bacteria. The work of Kitasato has given us a very exact knowledge of the tetanus bacillus and completely estab- lishes its specific nature. The organisms generally enter the animal body through a wound caused by some implement which has been in contact with the soil, or enter abrasions from the soil directly. Doubtless many of the wounds are so small that their existence is overlooked, and this, together with the fact that the period of incubation of the dis- ease, especially in man, is of considerable duration (three to ten days), and at times permits the wound to heal before any symptoms of intoxication occur, serves to explain at least some of the reported cases in which no wound is said to have existed. It would seem that in some rare cases tetanus can occur without the previous existence of a wound. Such a case has been reported by Kamen, who found that the intes- tine of a person dead of the disease was rich in the 1 See abstracts in the Centralbl. f. Bakt. u. Parasitenk., ix., 286; xiii., 351. TETANUS. 377 Bacillus tetani. Kamen is of the opinion that the bacilli can grow in the intestine and be absorbed, espe- cially where there are imperfections in the mucosa. It is not impossible, though he does not think it probable, that the bacteria growing in the intestine could elaborate enough toxin to produce the disease by absorption. All animals are not alike susceptible to the disease. Men, horses, mice, rabbits, and guinea-pigs are all sus- ceptible ; dogs are much less so. Most birds are scarcely at all susceptible either to the bacilli or to the poison. Amphibians are immune, though it is said that frogs can be made susceptible by elevation of their body- temperature. When a white mouse is inoculated with an almost infinitesimal amount of bouillon or solid culture, or is inoculated with garden-earth containing the tetanus bacillus, the disease is almost certain to follow, the first symptoms coming on in from one to two days. The mouse develops typical tetanic convulsions, which begin first in the neighborhood of the inoculation, but soon become general. Death follows sometimes in a very few hours. In rabbits the period of incubation is nearly two weeks, and in man may be three weeks. The conditions in the animal body are not favorable for the development of the bacilli, because of the free supply of oxygen contained in the blood, and we find that they grow with great slowness, remain localized at the seat of inoculation, and never enter the blood- or lymph-circulation. Doubtless most cases of tetanus are cases of mixed infection in which the bacillus enters with bacteria, which greatly aid its growth by using up the oxygen in their neighborhood. The amount of poison produced must be exceedingly small and its power tre- mendous, else so few bacilli growing under adverse con- ditions could not produce fatal toxemia. The poison is produced rapidly, for Kitasato found that if mice were inoculated at the root of the tail, and afterward the skin and the subcutaneous tissues around the inoculation were 378 PATHOGENIC BACTERIA. either excised or burned out, this treatment would not save the animal unless the operation were performed within an hour after the inoculation. Some incline to the view that the toxin is a ferment, and the experiments of Nocard, quoted before the Acad- emie de Medecine, October 22, 1895, might be adduced in support of the theory. He says: "Take three sheep with normal tails, and insert under the skin at the end of each tail a splinter of wood covered with the dried spores of the tetanus bacillus; watch these animals care- fully for the first symptoms of tetanus, then amputate the tails of two of them 20 cm. above the point of inocula- tion, . . . the three animals succumb to the disease with- out showing any sensible difference. ' ' The circulating blood of diseased animals is fatal to susceptible animals because of the toxin which it con- tains; and the fact that the urine is also toxic to mice proves that the toxin is excreted by the kidneys. From pure cultures of tetanus bacilli grown in various media, and from the blood and tissues of animals affected with the disease, Brieger succeeded in separating two alkaloidal substances — " tetanin " and " tetano- toxin," both very poisonous and productive of tonic convulsions; and Brieger and Frankel later isolated an extremely poi- sonous toxalbumin. The pathology of the disease is of much interest be- cause of its purely toxic nature. There is generally a small wound with a slight amount of suppuration. At the autopsy the organs of the body are normal in appear- ance, except the nervous system, which bears the great- est insult. It, however, shows little else than congestion either macroscopically or microscopically. An interesting fact contributed to our knowledge of the disease has been presented by Vaillard and Rouget, who found that if the tetanus bacilli were introduced into the body freed from their poison, they were unable to produce any signs of disease because of the prompt- ness with which the phagocytes took them up. If, how- TETANUS. 379 ever, their poison was not removed, or if the body-cells were injured by the simultaneous introduction of lactic acid or other chemical agents, the bacilli would imme- diately begin to manufacture the toxin and produce symptoms of the disease. The toxin is easily prepared, being readily soluble in water. The most ready method of preparation is to grow the bacilli in bouillon, keeping the culture-medium at a temperature of 370 C, and allowing it to remain un- disturbed for from two to four weeks, by which time it will have attained a toxicity so great that 0.000005 c.cm. will cause the death of a mouse. The toxin is very rapidly destroyed by heat, and cannot bear any temperature above 6o°-65° C. It is also decomposed by light. The best method of keeping it is to add 0.5 per cent, of phenol, and then store it in a cool, dark place. It will nott keep its strength very long under the best conditions. The purified toxin of Brieger and Cohn was surely fatal to mice in doses of 0.00000005 gram. Lambert,1 in his comprehensive review of the use of tetanus antitoxin, points out that this is the most poisonous substance that has ever been discovered. Like most of the bacterial toxins, the tetanus poison is- only effective when produced in or injected into the tissues and absorbed directly into the circulation. It is harmless when given by the digestive tract, Ramon 2 having ad- ministered by the mouth 300,000 times the fatal hypo- dermic dose without producing any symptoms. The toxin seemed to pass out with the feces. The toxin has a very painful local action associated with spasm of the muscular fibres with which it comes in contact. Pitfield 3 injected it into the calf of his leg and experienced the local effects of the poison for twelve hours. By the gradual introduction of such a toxin into ani- mals Behring and Kitasato have been able to produce in 1 New York Med. Jour., June 5, 1897. * Deutsche med. Wochenschrift, Feb. 24, 1898. * Therapeutic Gazette, Mar. 15, 1897. 380 PA THOGENIC BA CTERIA . their blood a distinctly potent and valuable antitoxic substance. The method for the production of this tetanus anti- toxic serum is very much like that for the diphtheria antitoxic serum {q. v.), except that a much longer time is required for its production, that the doses of toxin are of necessity smaller because its toxicity is greater, and that trichlorid of iodin or Gram's solution will probably need to be added to the toxin to prevent too powerful a local reaction. Horses, dogs, and goats may be used. As tetanus cases are not very common, and the anti- toxic serum when produced is not very stable in its prop- erties, Tizzoni and Cattani have successfully prepared it in a solid form, in which, it is claimed, it can be kept indefinitely, shipped any distance, and used after simple solution in water. Their method is to precipitate the antitoxin from the blood of immunized dogs with alcohol. Numerous cases of the beneficial action of this antitoxin are on record. The strength of the serum is generally expressed 1 : 1,000,000, 1 : 10,000,000, etc., which indicates that 1 c.cm. of the serum is capable of protecting 1,000,000 or 10,000,000 grams of guinea-pig from infection. The experiments of Alexander Lambert show that a protective power of 1 : 800,000,000 can be attained. As Welch has pointed out, the antitoxin of tetanus has proved to be rather a disappointment in human medicine, and also for the treatment of large animals, such as the horse. The results following its injection, in combination with the sterile toxin, into mice, guinea-pigs, and rabbits are highly satisfactory, but the amount needed, in pro- portion to the body-weight, to save the animal from the toxin being manufactured in its body by bacilli increases so enormously with the day or hour of the disease as to make the dosage, which increases millions of times where that of diphtheria antitoxin increases but tenfold, a matter of difficulty and uncertainty. Nocard also calls atten- tion to the fact that the existence of tetanus is unknown TETANUS. 381 until there is sufficient toxemia to produce spasms, and that therefore it is impossible to attack the disease in its inception ; we are obliged to meet it upon the same grounds as diphtheria in the later days of the disease — a time when it is well known that the chances of im- provement are greatly lessened. Of course, as there is no other remedy that combats the disease at all, the antitoxin is one which, when ob- tainable, should always be employed. While tetanus antitoxin is extremely disappointing in practice for the cure of the disease, even when it is admin- istered, as Roux has suggested, by injection beneath the dura mater, it is most satisfactory for its prevention. In the biological laboratory of the H. K. Mulford Co., where a large number of horses are constantly being injected with toxins of various kinds, and having antitoxic blood with- drawn day after day, the number of horses that died of tetanus was alarming, on several occasions reaching as high as 10 per cent. It occurred to me that these horses should be kept immunized to tetanus by periodical injec- tions of tetanus antitoxin — say every three months. This was tried with the greatest success, and the results given in a paper by Mc£arland and Ranck1 show that teta- nus immediately disappeared from these stables, the deaths falling to less than 1 per cent. The inference that is to be drawn is that " an ounce of prevention is better than a pound of cure, " and if the surgeon would admin- ister a prophylactic injection of the antitoxin in every case in which the occurrence of tetanus was at all likely, the disease would rarely develop. An interesting observation has been recently made by Wasserman,2 who, assuming that the destruction of nerve-cells in the cerebrum and cord during tetanus tox- emia might have something to do with immunity, be- lieved it possible to obtain from these cells an immuniz- ing substance. Investigating the subject, he found that 1 Proceedings of the American Veterinary Medical Association , 1 899, p. 258. 2 Berlin, klin. Wochenschrift, 1898, No. I. 382 PATHOGENIC BACTERIA. when fresh brain or spinal cord was rubbed up in a mor- tar with physiological salt solution, and injected into ani- mals, the mixture had the power not only to confer upon them an immunity lasting for twenty-four hours, but also was potent enough to neutralize the effects of an injec- tion of tetanus toxin ten times as large as that necessary to kill the animal in doses of 1 c.cm. These observations may offer a possible solution of the difficult problem laid before us by Montesano and Mon- tesson/ who unexpectedly found the tetanus bacillus in pure culture in the cerebro-spinal fluid of a case of para- lytic dementia that died without a tetanic symptom. A peculiar method of administering tetanus antitoxin was suggested by Chauffard and Ouenu,2 who injected it into the cerebral substance and found such administra- tion to bring about apparent cure in one case. Bacilli Resembling the Tetanus Bacillus. — Tavel 3 has called attention to a bacillus commonly found in the intestine, sometimes in large numbers in the ap- pendix in cases of appendicitis, and looked upon by one of his colleagues, Fraulein Dr. von Mayer, as the probable common cause of appendicitis. He calls it the " Pseudo- tetanusbacillus. " The bacillus is slender and measures 0.5 X 5-7 //. It is rather more slender than the tetanus bacillus. Its spores are oval, situated at the end of the rod, and cause a slight bulging rather pointed at the end. The bacillus is provided with not more than a dozen flagella, usually only four to eight, thus differing markedly from the tetanus bacillus, which has many cilia. The flagella are easily stained by Loffler's method without the addition of acid or alkali. The organism does not stain as well by Gram's method as the true tetanus bacillus. The growth in bouillon is rather more rapid than that of the tetanus bacillus. It will not grow in gelatin. The 1 Centralbl.f. Bakt., u. Parasitenk., Dec, 1897, Bd. xxii., Nos. 22, 23, p. 663. 2 Presse mid., No. 5, 1898. 3 Centralbl.f. Bakt. etc., Mar. 31, 1898, xxiii., No. 13, p. 538. TETANUS. 383 growth in agar-agar is very luxuriant and is accompanied by the evolution of much gas. Upon obliquely solidified agar-agar the colonies are round, circumscribed, and often encompassed by a narrow clear zone, which is often notched. The organism grows in serum only in vacuum. The spores are killed at 8o° C. The organism produced no symptoms in mice, guinea- pigs, and rabbits even when 2-5 c.cm. were subcutane- ously introduced. Sanfelice1 and Lubinski2 have also observed in earth and meat infusions a bacillus which is morphologically and culturally like the tetanus bacillus, but differs from it in not producing any toxin. Kruse3 has also described a tetanus-bacillus-like micro- organism that grows aerobically. It is not pathogenic. 1 Zeitschrift filr Hygiene, vol. 14. 1 Centralbl. f. Bakt. u. Parasitenk., xvi., 19. * Haggis, Die Mikroorganismen, vol. ii., p. 267. CHAPTER II. HYDROPHOBIA, OR RABIES. No micro-organism of hydrophobia has as yet been discovered, yet the peculiarities of the disease are such as to leave no doubt in the mind of a bacteriologist that one must exist. To find it is now the desideratum. Although many men have labored upon hydrophobia, no name is so well known or so justly honored as that of the great pioneer in bacteriology, Pasteur. The profes- sion and laity are alike familiar with his name and work, and although at times the newspapers of our country and certain members of the profession have opposed the methods of treatment which he has suggested as the re- sult of his experimentation, we cannot but feel that this skepticism and opposition are due either to ignorance of the principles upon which Pasteur reasoned or to a culpable conservatism. The most vehement opponent that Pasteur has in America seems to disbelieve the existence of rabies. It is impossible to argue with him. Hydrophobia, or rabies, is a specific toxemia to which dogs, wolves, skunks, and cats are highly susceptible, and which can, through their saliva, be communicated to men, horses, cows, and other animals. The means of communication is almost invariably a bite, hence the inference that the specific organism is present in the saliva. The animals that are infected manifest no symptoms during a varying incubation-period in which the wound generally heals kindly. This period may last for as long a time as twelve months, but in rare cases may be only some days. An average duration of the period of incu- bation might be stated as about six weeks. 384 HYDROPHOBIA, OR RABIES. 385 As the incubation-period comes to an end there is an observable alteration in the wound, which becomes red- dened, sometimes may suppurate a little, and is painful. The victim, if a man, is much alarmed and has a sensa- tion of horrible dread. The period of dread passes into one of excitement, with paralysis of the pharyngeal muscles and inability to swallow, which in many cases amounts to a wild delirium and ends in a final stage of convulsion and palsy. The convulsions are tonic, rarely clonic, and subsequently cause death by interfering with the respiration, as do those of tetanus and strychnia. During the convulsive period much difficulty is experi- enced in swallowing liquids, and it is supposed that the popular term "hydrophobia" arose from the reluctance of the diseased to take water because of the inconveni- ence and occasional spasms which the attempt causes. This description, brief and imperfect as it is, will illustrate the parallelism existing between hydrophobia and tetanus. In the latter we observe the entrance of infectious material through a wound, which, like the bite in hydrophobia, sometimes heals, but often suppu- rates a little. We see in both affections an incubation- period of varying duration, though in hydrophobia it is much longer than in tetanus, and convulsions of tonic character causing death by asphyxia. It is maintained by some that the stage of excitement argues against the specific nature of the disease, but these subjective symptoms are like the mental con- dition of tuberculosis, which leads the patient to make a hopeful prognosis of his case, and the mental condition of anthrax, in which it is stated that no matter how dan- gerous his condition the patient is seldom much alarmed about it. Pasteur1 and his co-workers, Chamberland and Roux,2 found that in animals that die of rabies the salivary glands, the pancreas, and the nervous system contain the 1 Compte rendu, Acad, des Sciences, Paris, 1889, cviii., p. 1228. * Ibid., Oct. 26, 1885. 25 386 PATHOGENIC BACTERIA. infection, and are more appropriate for experimental pur- poses than the saliva, which is invariably contaminated with accidental pathogenic bacteria. The introduction of a fragment of the medulla ob- longata of a dog dead of rabies beneath the dura mater of a rabbit causes the development of rabies in the rabbit in about six days. The medulla of this rabbit introduced beneath the dura mater of a second rabbit produced a more violent form of the disease in a shorter time, and by frequently repeated implantations Pasteur found that an extremely virulent material could be ob- tained. Inasmuch as the toxins of diphtheria and tetanus circulate in the blood, and not infrequently saturate the nervous systems of animals affected, it might be concluded that the material with which Pasteur worked was a toxin. This is readily disproven, however, not only by the fact that the toxin would weaken instead of strengthen by the method of transfer from animal to animal, it not being a vital entity, but also by the dis- covery that when an emulsion of the nervous system of an affected animal is filtered through porcelain, or when it is heated for a few moments to ioo° C, or exposed for a considerable time to a temperature of 750 or 8o° C, its virulence is entirely lost. This would seem to prove that that which is in the nervous system and communi- cates the disease is a living, active body — a parasite, and in all probability a bacterium. However, all endeavors to discover, isolate, or cultivate this organism have failed. Pasteur noted that the virulence of the poison was less in animals that had been dead for some time than in the nervous systems of those just killed, and by experi- mentation showed that when the nervous system was dried in a sterile atmosphere the virulence was attenu- ated in proportion to the length of time it had been dry. This attenuation of virulence of course suggested to Pasteur the idea of a protective vaccination, and by in- oculating a dog with much attenuated, then with less HYDROPHOBIA, OR RABIES. 387 attenuated, then with moderately strong, and finally with, strong, virus, the dog developed an immunity which enabled it to resist the infection of an amount of viru- lent material that would certainly kill an unprotected animal. It is remarkable that this thought, which was a theory based upon a broad knowledge, but experience with comparatively few bacteria, should every day find more and more grounds for confirmation as our knowledge of immunity, of toxins, and of antitoxins progresses. What Pasteur did with rabies is what we now do in producing the antitoxin of diphtheria — i. e. gradually accommodate the animal to the poison until its body-cells are able to neutralize or resist it. As the poison cannot be secured outside of the body because the bacilli, micro- cocci, or whatever they may be cannot be secured outside of the body, he does what Behring originally did in diphr theria — introduces attenuated poison-producers — bacilli crippled by heat or drying, and capable of producing only a little poison — accustoms the animal to these, and then to stronger and stronger ones until immunity is established. The genius of Pasteur did not cease with the produc- tion of immunity, but, we rejoice to add, extended to the kindred subject of therapy, and has now given us a cure for hydrophobia. For the production of a cure in infected cases very much the same treatment is followed as has been der scribed for the production of immunity. The patient must come under observation early. The treatment con- sists of the subcutaneous injection of about 2 grams of an emulsion of a rabbit's spinal cord which had been dried over caustic potash for from seven to ten days. This beginning dose is not increased in size, but each day the emulsion used is from a cord which has not been dried so long, until, when the twenty-fifth day of treat- ment is reached, the patient receives 2 grams of emulsion of spinal cord dried only three days, and is considered immune or cured. 388 PATHOGENIC BACTERIA. It will be observed that this treatment is really no more than the immunization of the individual during the incubation stadium, and the generation of a vital force — shall we call it an antitoxin ? — in the blood of the animal in advance of the time when the organism is expected to saturate the body with its toxic products. This, in brief, is the theory and practice of Pasteur's system of treating hydrophobia. It is exactly in keeping with the ideas of the present, and is most extraordinary in its reasonings and details when we remember that the first application of the method to human medicine was made October 26, 1885, nearly ten years before the time we began to understand the production and use of anti- toxins. Frantzius ' has studied the bile of animals immunized to rabies, and found that it possessed a marked neutral- izing effect upon the rabies poison, so that when 0.2 gram of bile and 0.2 gram of comminuted rabid rabbit's medulla are simultaneously introduced beneath the dura of a healthy rabbit, no disease occurs. The bile of healthy oxen, sheep, hogs, etc., was also studied, but found to be without effect. He concludes that the bile is the most powerful rabies antitoxin (?) yet discovered. The action of the bile in this combination is probably identical with that discovered by Koch, who found that the bile of cattle suffering from the "Rinderpest" ex- erted an immunizing power by which healthy animals could be protected from the disease. Hogyes of Budapest2 believes that Pasteur was mis- taken in supposing that the drying Was of importance, and thinks the dilution is of chief importance. He, therefore, makes an emulsion of rabbits' medulla (1 gram of medulla to 10 c.cm. of sterile broth). Of this stock solution, prepared freshly every day, the first dilution used is 1 : 10,000 ; then on succeeding days 1 : 8000, 1 Cmtralbl. f. Bakt. u. Parasitenk., May 13, 1898, xxii., No. 18. 2 Acad, des Sciences de Buda-Pest, Oct. 17, 1897; Centralbl. f. Bakl. u. Parasitenk., 1887, ii., 579. HYDROPHOBIA, OR RABIES. 389 1 : 6000, 1 : 5000, 1 : 2000, 1 : 1000, 1 : 500, 1 : 250, 1 : 200, 1 : 100, and finally the full strength, 1 : 10. Cabot1 prepared a stock solution of 8 parts of rabbit's brain and 80 parts of glycerin and water. The quantity of glycerin added comprised one-fifth of the total bulk. After the emulsion was made, it was filtered through sterile cheese-cloth. This emulsion containing the glyc- erin, if kept in the ice-chest, will be of standard viru- lence during the entire period of immunization. As the result of his experimentation, Cabot finds that the dilu- tion-method is attended with some danger to the animal immunized, which is not present in the dried-cord method of Pasteur. The latter method, therefore, is the one to be preferred. 1 Journal of Experimental Medicine, 1899, vol. iv., No. 2. CHAPTER It I. DIPHTHERIA. Bacillus Diphtheria (Klebs1-L6ffler4), In 1883, Klebs pointed out the existence of a bacillus in the pseudo-membranes upon the fauces of patients suffering from diphtheria, but it was not until 1884 that Loffler succeeded in isolating and cultivating the organ- ism, which is now known by both their names — the Klebs-Lomer bacillus. The bacillus as described by Loffler is about the length * of the tubercle bacillus, about twice its diameter, has a Fig. 84.— Bacillus diphtheriae, from a culture upon blood-serum ; x 1000 (Frankel and Pfeiffer). curve similar to that which characterizes the tubercle bacillus, and has rounded ends (Fig. 84). It does not 1 Congress fiir innere med. Verhandlungen, 1883. 1 Mittheilungen aus der Kaiserliche "Gesimtfheitsamt, 2; 390 DIPHTHERIA. 391 form chains, though two, three, and rarely four individ- uals may be found joined ; generally the individuals are all separate from one another. The morphology of the bacillus is peculiar in its considerable irregularity, for among the well-formed individuals which abound in fresh cultures a large number of peculiar organisms are to be found, some much larger than normal, some with one end enlarged to a club-shape, some greatly elongated, with both ends expanded into club-shaped enlargements. These bizarre forms seem to represent an involution-form of the organism, for, while present in perfectly fresh cul- tures, they are so abundant in old cultures that scarcely a single well-formed bacillus can be found. It not infre- quently happens that in unstained bacilli distinct gran- ules can be defined at the ends — polar granules — thus giving the organism somewhat the appearance of a diplococcus. Occasionally branched forms are observed, and it is not impossible that the diphtheria bacillus may belong to the higher bacteria, finding its real place among the streptothrices. The bacillus can be readily stained by aqueous solu- tions of the anilin colors, but more beautifully and characteristically with L,6ffler's alkaline methylene blue : Saturated alcoholic solution of methylene blue, 30 ; 1 : 10,000 aqueous solution of caustic potash, 100 ; and an aqueous solution of dahlia, as recommended by Roux. The Neisser method of staining the diphtheria bacillus is as follows : The prepared cover-glass is immersed for from two to three seconds in Alcohol (96 per cent.), 20 parts. Methylene blue, 1 part. Distilled water, 950 parts. Acetic acid (glacial), 50 " Then for three to five seconds in Bismarck brown, 1 part. Boiling distilled water, 500 parts. 392 PATHOGENIC BACTERIA. The true diphtheria bacilli appear brown with a dark-blue body at one or both ends ; the pseudo-diphtheria bacilli usually exhibit no polar bodies. Park ' in his large experience found that neither the Neisser nor the Roux stains gave any more information concerning the virulence of the bacilli than the Loffler alkaline-methylene-blue. When cover-glass preparations are stained with these solutions, the bizarre forms already mentioned are much more obvious than in the unstained individuals, and the contrast between the polar granules, which color in- tensely, and the remainder of the bacillus, which tinges slightly, is marked. Through good lenses the organisms are always distinct bacilli, notwithstanding the fact that the ends stain more deeply than the centres, and it is only through poor lenses that the organisms can be mis- taken for diplococci. The bacilli stain well by Gram's method, this being a good method to employ for their definition in sections of tissue, though Welch and Abbott assert that Weigert's fibrin method and picro-carmin give the most beautiful results. The diphtheria bacillus does not form spores, and is delicate in its thermal range. Loffler found that it could not endure a temperature. of 6o° C, and Abbott has shown that a temperature of 580 C. for ten minutes is fatal to it. Notwithstanding this susceptibility, the organism can be kept alive for several weeks after being dried upon shreds of silk or when surrounded by dried diphtheria membrane. No flagella have been demonstrated upon the bacillus. It is non-motile. Fernbach thinks that when the organisms are grown in a medium exposed to a passing current of air, the lux- uriance of their development is increased, though their life-cycle is shorter. The growth can also take place when the air is excluded, so that the bacillus must be classed among the optional anaerobic organisms. 1 Bacteriology in Medicine and Surgery, 1900. DIPHTHERIA. 393 The diphtheria bacillus grows readily upon all the ordinary media, and is a very easy organism to obtain in pure culture. Loffler has shown that the addition of a small amount of glucose to the culture-medium increases the rapidity of the growth, and suggests a special medium which bears his name — Loffler's blood- serum mixture : Blood-serum, 3 ; Ordinary bouillon + i per cent, of glucose, i. This mixture is filled into tubes, coagulated, and steril- ized like blood-serum, and is one of the best-known media in connection with the study of diphtheria. The studies of Michel ! have shown that the develop- ment of the culture is much more luxuriant and rapid when horse serum instead of beef or calves' blood is used. Horse's blood can easily be secured by the introduction of a trocar into the jugular vein ; 5 liters of it can be with- drawn without causing the animal any inconvenience or producing symptoms. The impossibility of clinically making an accurate di- agnosis of diphtheria without a bacteriologic examination has caused many private physicians and many medical societies and boards of health to equip laboratories where accurate examinations can be made. The method re- quires some apparatus, though a competent bacteriologist can often make shift with a bake-oven, a wash-boiler, and other household furniture instead of the regular sterilizers and incubators, which are expensive. When it is desired to make a bacteriologic diagnosis of a suspected case of diphtheria or to secure the bacillus in pure culture, a sterile platinum wire having a small loop at the end, or a swab made by wrapping a little cotton around the end of a piece of wire and carefully sterilizing in a test-tube, is introduced into the throat and touched to the false membrane, after which it is smeared carefully ove* the surface of at least three of 1 Centralbl.f. Bakt. u. Parasilenk., Sept. 24, 1897, Bd. xxii., Nos. 10 and II. 394 PATHOGENIC BACTERIA. the blood-serum-mixture tubes, without either again touching the throat or being sterilized. The tubes thus inoculated are stood away in an incubating oven at the temperature of 370 C. for twelve hours, then examined. If the diphtheria bacillus is present upon the first and second tubes, there will be a smeary yellowish-white layer, with outlying colonies on the second tube, while the third tube will show rather large isolated whitish or slightly yellowish colonies, smooth in appearance, but rather ir- regular in outline. Very often the colonies are china- white in appearance. These colonies, if found by micro- scopic examination to be made up of diphtheria bacilli, will confirm the diagnosis of diphtheria, and will at the same time give pure cultures when transplanted. There are very few other bacilli which grow so rapidly upon Loffler's mixture, and scarcely one other which is found in the throat. Ohlmacher recommends the microscopic examination of the still invisible growth in five hours. A platinum loop is rubbed over the inoculated surface ; the material secured is then mixed with distilled water, dried on a cover-glass, stained with methylene blue, and examined. This method, if reliable, will be very valuable in making an early diagnosis preparatory to the use of the antitoxin. The presence of diphtheria bacilli in material taken from the throat does not necessarily prove the patient to be diseased. Virulent bacilli can often be discovered in the throats of healthy persons who have knowingly or unknowingly come in contact with the disease. The bacteriologic examination is only an adjunct to the clinical diagnosis, and must never be taken as positive in itself. The bacillus grows similarly upon blood-serum and Loffler's mixture. Upon glycerin agar- agar and agar-agar the colonies are much larger, more translucent, always without the yellowish-white or china-white color of the blood-serum cultures, and generally are distinctly divided into a small elevated centre and a flatter surrounding zone DIPHTHERIA. 395 with indented edges, sometimes with a distinctly radiated appearance. It must be remarked that when sudden transplantations are made from blood-serum to agar- agar the growth resulting is meagre, but the oftener this growth is transplanted to fresh agar-agar the more luxuriant it becomes. The growth in gelatin puncture-cultures is character- ized by small spherical colonies which develop along the a b c Fig. 85. — Diphtheria bacilli (from photographs taken by Prof. E. K. Dun- ham, Carnegie Laboratory, New York): a, pseudo-bacillus; b, true bacillus; c, pseudo-bacillus. entire length of the needle-track. The gelatin is not liquefied. Upon the surface of gelatin plates the colonies that develop do not attain anything like the size of the colo- nies upon Loffler's mixture. They appear to the naked eye as whitish points with smooth contents and regular 396 PATHOGENIC BACTERIA. thoueh sometimes indented borders. Under the micro- scope they appear as granular, yellowish-brown colonies with irregular borders (Fig. 86). When planted in bouillon the organism sometimes causes a diffuse cloudiness at first, but, not being motile, soon settles to the bottom in the form of a rather floccu- lent precipitate which has a tendency to cling to the sides of the glass, but leaves the bouillon clear. Sometimes a delicate pellicle of granular appearance forms upon Fig. 86. — Bacillus diphtheria?, colony twenty-four hours old upon agar-agar; x ioo (Frankel and Pfeiffer). the surface, especially when the cultivation is made by the method of Fernbach with a passing current of air. This mycoderma, which may appear quite regular when the flask is undisturbed, is so brittle that it at once falls to pieces if the flask be moved, the minute fragments forming a miniature snow-storm in the tube. Spronck1 has determined that the characteristics of the growth of the diphtheria bacillus in bouillon, as well as the amount of toxin-production, vary according to the amount of gducose in the bouillon. He divides the cult- ures into three types : 1 Annales de f Inst. Pasteur, October 25, 1895, vol. ix., No. 10, p. 758. DIPHTHERIA. 397 . Type A. The reaction of the bouillon becomes acid and remains acid, the acidity increasing. The bacilli accumulate at the bottom of the clear liquid. The toxin-production is meagre. Type B. There is no change from alkalinity to acidity, but the original alkalinity of the bouillon steadily in- creases. The culture is very rich, the bottom of the flask shows a considerable sediment, the liquid is cloudy, and a delicate growth occupies the surface. The toxicity is very great. Type C. In a few days the reaction of the culture becomes acid, and then later on changes to alkaline. During the acid period the liquid is clear, with a white surface-growth. When the alkalinity returns the bouillon clouds and the surface-growth increases in thickness. Sediment accumulates at the bottom of the flask. The toxicity of the culture is much less than in Type B. Spronck regards the varying reaction as due to the fermentation of the glucose, and asserts that the most luxuriant and toxic cultures are those in which no glucose is present. To exclude as much of the undesir- able sugar as possible, he makes the bouillon from the stalest meat obtainable, preferring it when just about to putrefy. Of the meats experimented with, beef was found to be the best. In large cities meat is ordinarily kept sufficiently long before being offered for sale to meet Spronck's require- ment. Upon potato the bacillus develops only when the reac- tion is alkaline. The potato-growth is not characteristic. Welch and Abbott always secured a growth of the organ- ism when planted upon potato, but do not mention the reaction of the medium they employed. Milk is an excellent medium for the cultivation of the Bacillus diphtherial, and is possibly at times a means of infection. Litmus milk is an excellent medium for ob- serving the changes of reaction brought about by the growth of the bacillus. At first the alkalinity, which 398 PATHOGENIC BACTERIA. is always favorable to the development of the bacillus, is destroyed by the production of an acid. When the culture is old the acid is replaced by a strong alkaline reaction. Palmirski and Orlowski ' assert that the bacillus pro- duces indol, but only after the third week. Smith, how- ever, came to a contrary result, and found that when diphtheria bacillus grew in the dextrose-free bouillon that he recommends no indol was produced.2 The entrance of the diphtheria bacillus into ths internal organs can scarcely be regarded as a common occurrence, though in the severe cases it is by no means rare, as will be shown by the synopsis of Pearce's work given below. It must be remembered, however, that his cases were all fatal, and that the conditions found may not parallel the usual course of affairs. Diphtheria bacilli were first found in the heart's blood, liver, spleen, and kidney, by Frosch.3 Kolisko and Paltany 4 had already found it in the spleen, and other observers in various lesions of the deeper tissues and occasionally in the organs. In the blood and organs it is very commonly associated with the Streptococcus pyogenes and sometimes with other bacteria. While present in nearly all of the inflammatory sequelae of diphtheria, the Klebs-Loffler bacillus may have very little influence in producing them, as in such conditions it is almost invariably associated with the pyogenic cocci, either the streptococci or staphylococci. Howard5 has observed a case of ulcerative endocarditis depending upon the diphtheria bacillus, and Pearce6 has observed it in i case of malignant endocarditis, in 19 out of 24 cases of broncho-pneumonia, in 1 case of empyema, in 16 cases of middle-ear disease, eight times 1 Central/)!, f. Bakt. u. Parasitenk., Mar., 1895. ''■Jour, of Exp. Med., Sept., 1807, vol. ii., No. 5, p. 546. 8 Zeilschrift fiir Hygiene, etc., 1893. xiii> Heft I. 4 Wiener klin. Wochenschrift, 1889. 5 American Journal of Medical Sciences, Dec, 1894. 6 Journal of the Boston Society of Medical Sciences, Mar., 1898. DIPHTHERIA. 399 in inflammation of the antrum of Highmore, 1 case of inflammation of the sphenoidal sinuses, 1 case of throm- bosis of the lateral sinuses, in 2 cases of abscesses of the cervical glands, and in oesophagitis, gastritis, vulvo- vaginitis, dermatitis, and conjunctivitis following or associated with diphtheria. Diphtheria as it occurs in man is generally a disease characterized by the formation of a pseudomembrane upon the fauces. In unusual cases the diphtheria pseudo- membrane may occur elsewhere than upon the fauces. It is not infrequent as a form of rhinitis; it may occur in the mouth, upon the genital organs, or upon wounds. Williams ' has reported a case of diphtheria of the vulva. Nisot and Bumm have also reported cases of puerperal diphtheria in which the bacilli were cultivated from the membranes. It is a local infection, due to the presence and development of the bacilli in the pseudomembrane, but is accompanied by a general toxemia resulting from the absorption of a violently poisonous substance produced by the bacilli. The bacilli are found only in the membranous exudation, and most plentifully in its older portions. As a rule, they do not distribute themselves through the circulation of the animal, though at times they may be found in the heart's blood and internal organs. The disease pursues a course of rather variable length, and in favorable cases the patient recovers gradually, the pseudomembrane first disappearing, leaving an inflamed mucous membrane behind it, upon which the virulent diphtheria bacilli persist, always for several weeks, sometimes for months. Smith2 describes the bacterio- logical condition as follows: "The microscope informs us that during the earliest local manifestations, the usual scant miscellaneous bacterial flora of the mucosa is quite suddenly replaced by a rich vegetation of the easily-dis- 1 American Journal of Obstetrics and Diseases of Women and Children, Aug., 1898. * Boston Medical and Surgical Journal, 1898, i., p. 157. 400 PATHOGENIC BACTERIA. tinguishable diphtheria bacillus. Frequently no other bacteria are found in the culture-tube. This vegetation continues for a few days, then gradually gives way to another flora of cocci and bacilli, and finally the normal condition is reestablished." The Streptococcus pyogenes and Staphylococcus pyogenes aureus and albus are, in many cases, found in association with it, especially when there are very severe lesions of the throat, attended with invasion of the internal organs. In a series of 234 cases carefully and statistically studied by Blasi and Russo-Travali,1 it was found that in 26 cases of pseudomembranous angina due to streptococci, staphy- lococci, colon bacilli, and pneumococci, 2 patients died, the mortality being 3.84 per cent. In 102 cases of pure diphtheria 28 died, a mortality of 27.45 per cent. Seventy- six cases showed diphtheria bacilli and staphylococci; of these, 25, or 32.89 per cent., died. Twenty cases showed the diphtheria bacilli and Streptococcus pyogenes, with 6 deaths — 30 per cent. In 7 cases, of which 3, or 43 per cent., were fatal, the diphtheria bacillus was in com- bination with streptococci and pneumococci. The most dangerous forms met were 3 cases, all fatal, in which the diphtheria bacillus was found in combination with the Bacillus coli. In 157 cases of diphtheria and scarlatina studied at the Boston City Hospital by Pearce,2 there were 94 cases of diphtheria, 46 cases of complicated diphtheria (29 with scarlet fever, 11 with measles, and 5 with measles and scarlet fever), and 17 cases of scarlet fever (in 3 of which measles was also present). Of the 94 cases of uncomplicated diphtheria, the Klebs-Loffler bacilli were present in the heart's blood in 4, twice alone and twice with streptococci. In 9 cases the streptococcus occurred alone ; in 1 case the pneumo- coccus occurred alone. In the liver the bacillus was 1 Annales de T Inst. Pasteur, 1896, p. 387. ' Journal of the Boston Society of Medical Sciences, Mar., 1898. DIPHTHERIA. 4°l found in 24 cases, alone in 12 and together with the streptococcus in 12 ; the streptococcus occurred in 27 cases, alone in 14, with the Klebs-Loffler bacillus in 12, and with Staphylococcus pyogenes aureus in 1. The Staphylococcus pyogenes aureus occurred in 4 cases, alone in 3 and associated with the streptococcus in 1. The pueumococcus occurred alone in 1 case. In the spleen the Klebs-Loffler bacillus occurred eigh- teen times, fifteen times alone and three times associated with the streptococcus. The streptococcus occurred in 24 cases, alone in 21, associated with the Klebs-Loffler bacillus twice, and with the Staphylococcus pyogenes aureus once. The Staphylococcus pyogenes aureus occurred twice, once alone and once with the strepto- coccus. The pneumococcus occurred twice alone. In the kidney the Klebs-Loffler bacillus occurred in 23 cases, in 15 alone, in 5 associated with the streptococcus, and in 2 with the Staphylococcus pyogenes aureus. The streptococcus occurred in 26 cases, in 19 of which it was the only organism present. The Staphylococcus pyogenes aureus occurred in 8 cases, in 4 of which it was in pure culture. The pneumococcus occurred four times, three times in pure culture and once with the Klebs-Loffler bacillus. In the 46 cases of complicated diphtheria, the hearfs blood showed pure cultures of the streptococcus nine times and the streptococcus associated with the Klebs- Loffler bacillus once. The diphtheria bacillus occurred alone once. In the liver in 10 cases the Streptococcus occurred alone, in 7 cases associated with the Klebs-Loffler bacillus and in 3 cases with the Staphylococcus pyogenes aureus. The diphtheria bacillus occurred in pure culture in 5 cases. The spleen contained streptococci only thirteen times and mixed with the diphtheria bacillus twice. The diphtheria bacillus was found in pure culture in 5 cases. The kidney contained pure cultures of streptococci in 26 402 PATHOGENIC BACTERIA. 10 cases, streptococci associated with diphtheria bacilli five times and with Staphylococcus pyogenes aureus three times. The diphtheria bacillus occurred alone in 7 cases. The Staphylococcus pyogenes aureus and the pneumo- coccus each alone once and both together once. "The clinical significance of this general infection with the Klebs-LofHer bacillus is not apparent. It occurred generally, but not always, in the gravest cases, or those known as ' septic ' cases. It is probable that it may be due to a diminished resistance to the tissue- cells, or of the germicidal power of the blood. In this series of fatal cases the number of infections with the streptococcus and with the Klebs-L,6ffler bacillus was about even, though slightly in favor of the strepto- coccus." The mixed infections are simply diphtheria plus the pathogenic effects of the combined bacteria. The diph- theria bacillus probably begins the process, growing upon the mucous membrane, devitalizing it, and produc- ing the coagulation-necrosis. Whatever pyogenic germs happen through accident to be present are thus afforded an opportunity to enter ; and added to the diphtheria there may be ulceration, suppuration, gangrene, etc. , at the seat of local disease, and metastatic abscesses from lymphogenic and hematogenic distribution of the pyogenic bacteria. It may be well to remark that all pseudomembranous diseases of the throat are not diphtheria, but that some of them, exactly similar in clinical picture, result from the activity of the pyogenic organisms alone, and are neither diphtheria nor contagious. Diphtheritic inflammations of the throat are not always accompanied by the formation of the usual pseudomem- brane. It occasionally happens that in the larynx a rapid inflammatory edema without a fibrinous surface-coating causes a fatal suffocation. Only a bacteriological exam- ination will reveal the nature of the disease in such cases. The pseudomembrane which characterizes diphtheria DIPHTHERIA. 4°3 consists of a combined necrosis of the tissues acted upon by the toxin and coagulation of an inflammatory exudate. When examined histologically it is found that the mucous membrane is chiefly affected in its superficial elements. The superficial layers of cells are embedded in coagulated exudate — fibrin — and show a peculiar hyaline degeneration. Sometimes the membrane seems to consist exclusively of hyaline cells ; sometimes the fibrin formation is secondary to or subsequent to the hyaline degeneration. Leucocytes caught in the fibrin also become hyaline. From the superficial layer the process descends often to the deepest layers, all of the cells being incorporated in the coagulated fibrin and showing the hyaline degeneration. The neighboring capillaries also become hyaline and the membrane becomes a coagulation-necrotic mass. The occasional laminated appearance of the membrane probably depends upon different depths having been affected at different periods, or to a difference in the process by which it has been formed. The pseudomembrane is continuous with the subjacent tissues by a fibrinous reticulum, and is removed with difficulty, leaving an abraded surface. The coagulation-necrosis of diphtheria seems to depend upon the local effect of the toxin. In the horses which receive large quantities of it in the course of immunization for antitoxin production it is common for a large subcutaneous inoculation to be succeeded by a fluctuating necrosis, which commonly becomes in- fected from the skin and suppurates. Morax and Elmas- sian1 found that when very strong diphtheria toxin is applied to the conjunctiva of rabbits every three minutes for eight or ten hours, typical diphtheritic changes are produced. Herman Biggs,2 in an interesting discussion of the occurrence of the diphtheria bacillus and its relation to diphtheria, comes to the following conclusions : 1 Annales de P Inst. Pasteur, 1898, p. 210. * Am. Jour, of the Med. Sciences, Oct., 1896, vol. xxii., No. 4, p. 411. 404 PATHOGENIC BACTERIA. 1. "When the diphtheria bacillus is found in healthy throats investigation almost always shows that the indi- viduals have been in contact with cases of diphtheria. The presence of the bacillus in the throat, without any lesion, does not, of course, indicate the existence of the disease. 2. "The simple anginas in which virulent diphtheria bacilli are found are to be regarded from a sanitary stand- point in exactly the same way as the cases of true diph- theria. 3. " Cases of diphtheria present the ordinary clinical feat- ures of diphtheria, and show the Klebs-Loffler bacilli. 4. "Cases of angina associated with the production of membrane in which no diphtheria bacilli are found might be regarded from a clinical standpoint as diph- theria, but bacteriological examination shows that some other organism than the Klebs-Loffler bacillus is the cause of the process." No more convincing proof of the existence of a power- ful poison in diphtheria could be desired than the evi- dences of general toxemia resulting from the absorption of material from a comparatively small number of bacilli situated upon a little patch of mucous membrane. In animals artificially inoculated the lesions produced are not identical with those seen in the human subject, yet they have the same general features of local infection with general toxemia. Guinea-pigs, kittens, and young pups are susceptible animals. When half a cubic centimeter of a twenty-four- hour-old bouillon culture is injected beneath the skin of such an animal, the bacilli multiply at the point of in- oculation, with the production of a patch of inflamma- tion associated with a distinct fibrinous exudation and the presence of an extensive edema. The animal dies in twenty-four to thirty-six hours. The liver is enlarged, and sometimes shows minute whitish points, which in microscopic sections prove to be necrotic areas in which the cells are completely degenerated and the chromatin of DIPHTHERIA. 405 their nuclei is scattered about in granular form. Similar necrotic foci, to which attention was first called by Oertel, are present in nearly all the organs in cases of death from the toxin. The bacilli are constantly absent from these lesions. Welch and Flexner ' have shown these foci to be common to numerous irritant poisonings, and not peculiar to diphtheria. The lymphatic glands are usually enlarged ; the adrenals are also enlarged, and, in cases into which the live bacilli have been injected, are hemorrhagic. It might be argued, from the different clinical pictures presented by the disease as it occurs in man and in animals, that they were not expressions of the same thing. A careful study, however, together with the evi- dences adduced by Roux and Yersin, who found that when the bacilli were introduced into the trachea of animals opened by operation a typical false membrane was produced, and that diphtheritic palsy often followed, and of hundreds of investigators, who find the bacilli constantly present in the disease as it occurs in man, must satisfy us that the doubt of the etiological role of the bacillus rests on a very slight foundation. All possible skepticism of the specificity of the bacillus on my part was dispelled by an accidental infection that kept me housed for three weeks during the busiest season of the year. Without having been exposed to any known diphtheria contagion, while experimenting in the labora- tory, a living virulent culture of the diphtheria bacillus was drawn into a pipette and accidentally entered my mouth. Through carelessness no precautions were taken to prevent serious consequences, and as a result of the accident, two days later, my throat was full of typical pseudomembrane which private and Health Board bac- teriological examinations showed contained pure cultures of the Klebs-Loffler bacilli. One reason for skepticism in this particular is the supposed existence of a pseudodiphtheria bacillus, which 1 Bull, of the Johns Hopkins Hospital, Aug., 1891. 406 PATHOGENIC BACTERIA. has so many points in common with the real diphtheria bacillus that it is difficult to distinguish between them. The chief points of difference between these bacilli are that the pseudodiphtheria bacillus seems to be shorter than the diphtheria bacillus when grown upon blood- serum; that the cultures in bouillon seem to progress much more rapidly at a temperature of from 20°-22° C. than those of the true bacillus ; and that the pseudobacil- lus is not pathogenic for animals. These slight distinc- tions are, however, exactly what should be expected of an organism whose virulence had been lost, and whose vegetative powers had been altered, by persistent manip- ulation or by unfavorable surroundings. The diphtheria bacilli are always present in the throats of patients suffering from diphtheria, and constitute the element of contagion by being accidentally discharged by the nose or mouth by coughing, sneezing, vomiting, etc. Whoever comes in contact with such materials is in danger of infection. It is of great interest to notice the remarkable results obtained by Biggs, Park, and Beebe in New York, where the bacteriological examinations conducted in connection with diphtheria show that the virulent bacilli may be found in the throats of convalescents as long as five weeks after the discharge of the membrane and the commence- ment of recovery, and that they exist not only in the throats of the patients themselves, but also in the throats of their care-takers, who, while not themselves infected, may be the means of conveying the disease from the sick-room to the outer world. Even more extraordinary are the observations of Hewlett and Nolen,1 who found the bacilli in the throats of patients seven, nine, and in one case twenty-three weeks after convalescence. The importance of this observation must be apparent to all readers, and serves as further evidence why most thor- ough isolation should be practised in connection with this dreadful disease. 1 Brit. Med. Jour., Feb. I, 1896. DIPHTHERIA. 4°7 Park ' found virulent diphtheria bacilli in about i per cent, of the healthy throats examined in New York City. Diphtheria was, however, prevalent in the city at the time. Most of the persons in whose throats they existed had been in direct contact with cases of diphtheria. Very many of those whose throats contained the virulent bacilli did not develop diphtheria. He concludes that the mem- bers of a household in which a case of diphtheria exists should be regarded as sources of danger, unless cultures from their throats show the absence of diphtheria bacilli. In connection with the contagiousness of diphtheria the recent experiments of Reyes are interesting. He has demonstrated that in absolutely dried air distributed diphtheria bacilli die in a few hours. Under ordinary conditions their vitality, when dried on paper, silk, etc., continues for a few days. In air that is moist the dura- tion of vitality is prolonged to about a week. In sand exposed to a dry atmosphere they die in five days in the light; in sixteen to eighteen days in the dark. When the sand is exposed to a moist atmosphere the duration of vitality is doubled. In fine earth they remained alive seventy-five to one hundred and five days in dry air, and one hundred and twenty days in moist air. From time to time reference has been made to the toxin elaborated by the diphtheria bacillus. Roux and Yersin2 first demonstrated the existence of this substance in cultures passed through a Pasteur porcelain filter. The toxin is intensely poisonous; it is not an albumin- ous substance, and can be elaborated by the bacilli when grown in non-albuminous urine, or, as suggested by Uschinsky. in non-albuminous solutions whose prin- cipal ingredient is asparagin. The toxic value of the cultures is greatest in the second or third week. In addition to the toxin, a toxalbumin has been isolated by Brieger and Frankel. 1 Report on Bacteriological Investigations and Diagnosis of Diphtheria, from May 4, 1893, to May 4, 1894, Scientific Bulletin No. 1, Health Department, City of New York. 2 Ann. de I'Inst. Pasteur, 1888, 1889, 1890, and 1894. 408 PATHOGENIC BACTERIA. Behring1 discovered that the blood of animals rendered immune to diphtheria by inoculation, first with attenu- ated and then with virulent organisms, contained a neu- tralizing substance which was capable of annulling the effects of the bacilli or the toxin when simultaneously or subsequently inoculated into non-protected animals. This substance, in solution in the blood-serum of the immu- nized animals, is the diphtheria antitoxin. The preparation of the antitoxin for therapeutic pur- poses received a further elaboration in the hands of Roux. The subject is one of great interest, but must be consid- ered briefly in a work of this kind. The antitoxin is manufactured commercially at present, the method being the immunization of large animals to great quantities of the toxin, and the withdrawal of their antitoxic blood when the proper degree of immunity has been attained. The details are as follows : The Preparation of the Toxin. — The method employed at the present time consists in growing the most virulent bacilli obtainable in alkaline bouillon for from five to seven days at a temperature of 370 C. After the given time has passed, it will be found that the acidity prima- rily produced by the bacillus gives place to a much more intense alkalinity than originally existed. The acme of the toxin-production seems to keep pace with this alka- line production. When ' ' ripe, " o. 4 per cent, of trikresol is added to the cultures, which are then filtered through porcelain or paper, or even simply allowed to sediment. As the dead bacilli are not irritating, their presence is harmless. If the bacillus employed is virulent and the conditions of culture favorable, the filtered culture should be so toxic that 0.0025-0.005 would be fatal to a 250-gram guinea-pig within four days. Park and Williams2 did an elaborate work upon the production of diptheria toxin. They found that "toxin of sufficient strength to kill a 400-gram guinea-pig 1 Die Blutserumtherapie. 2 Jour, of Exper. Med., vol. i., No. I, Jan., 1896, p. 164. DIPHTHERIA. 409 in three days and a half in a dose of 0.025 c.cm., developed in suitable bouillon, contained in ordinary Erlenmeyer flasks, within a period of twenty-four hours. In such bouillon the toxin reached its greatest strength in four to seven days (0.005 c.cm. killing a 500-gram guinea-pig in three days). This period of time covered that of the greatest growth of the bacilli, as shown both by the appearance of the culture and by the number of colonies developing on agar plates." "The bodies of the diphtheria bacilli did not at any time contain toxin in considerable amounts." "The type of growth of the bacilli and the rapidity and extent of the production of toxin depended more on the reaction of the bouillon than upon any other single factor." " The best results were obtained in bouillon which, after being neutralized to litmus, had about 7 c.cm. of normal soda solution added to each liter. An excessive amount of either acid or alkali prevented the development of toxin." "Strong toxin was produced in bouillon con- taining peptone ranging from 1 to 10 per cent." "The strength of toxin averaged greater in the 2 and 4 per cent, peptone solution than in the 1 per cent." "When the stage of acid reaction was brief and the degree of acidity probably slight, strong toxin developed while the culture bouillon was still acid; but when the stage of acid reaction was prolonged little if any toxin was produced until just before the fluid became alka- line." "Glucose is deleterious to the growth of the diphtheria bacillus and to the production of toxin when it is present in sufficient amounts to cause by its disintegration too great a degree of acidity in the culture-fluid. When the acid resulting from the decomposition of glucose is neu- tralized by the addition of an alkali the diphtheria bacillus again grows abundantly." Smith ' differs from Park and Williams in regard to the presence of dextrose in the culture-media, and claims that 1 Journal of Experimental Medicine, May and July, 1899, p. 373. 410 PATHOGENIC BACTERIA. when it is present in quantities not exceeding 0.2 per cent, in peptone bouillon freed from fermentable acid- producing substances (muscle-sugar) it leads to the maxi- mum accumulation of toxin by utilizing the available peptones to the best advantage. Martin 1 believes that it is essential to provide a stand- ard peptone for use in cultures intended to be highly toxic, and has recommended for this purpose what he calls a "bouillon de panse," which is prepared by adding to 200 grams of finely chopped hogs' stomachs, 10 c.cm. of pure hydrochloric acid and 1000 c. cm. of water. The mixt- ure is kept at 500 C. for from twelve to twenty-four hours, during which time the proteids of the stomach are con- verted into peptones. The mixture is now heated to ioo° C, to destroy the excess of pepsin, and passed through a cloth. The liquid is warmed again to 8o° C. and alka- linized, then filtered through paper. After this it is to be elevated to 1200 C, filtered again through paper, dis- pensed in flasks, and sterilized in the autoclave. The dipththeria bacillus grows abundantly in the medium, without the production of any acid, and produces toxin of which T^¥ c.cm. killed a 500-gram guinea-pig. The mixture can be used as thus prepared, or can be mixed with an equal volume of veal infusion. I have tried this method, but have not found it any better, if as good, as the ordinary bouillon made accord- ing to the suggestions of Park and Williams. The Immunisation of the Animal. — The animals chosen to furnish the antitoxic serum should be animals which present a distinct natural immunity to ordinary doses of the toxin, and should be sufficiently large to furnish large quantities of the finished serum. Behring originally employed dogs and sheep ; Arouson at first preferred the goat ; but Roux introduced the horse, which is more easi- ly immunized than the other animals mentioned, and, being large enough to furnish a considerable quantity of serum, recommends itself strongly for the purpose. 1 Ann. de P Inst. Pasteur, Jan. 25, 1898, vol. xii., No. 1. DIPHTHERIA. 41 1 The animal chosen should be free from tuberculosis and glanders, as tested by tuberculin and mallein, but need not be expensive. A horse with a disabled foot will answer well. Rheumatic horses should be rejected. In the beginning a small dose of the toxin — about -^ ccin. — should be given hypodermically to detect indi- vidual susceptibility. Horses vary much in this particu- lar, as Roux has pointed out. I have found light-colored horses to be distinctly more susceptible than dark-colored ones, a fact which has some substantiation in the clinical observation that blonde children suffer more severely from diphtheria than dark-complexioned ones. If well borne, the preliminary injection is followed in about six days by a larger dose, in six days more by a still larger one, and the increase is continued every six days or so, according to the condition of the animal, until enormous quantities — 500 or even 1000 c.cm. — are introduced at a time. As the expression of quantity alone is very misleading, and to know exactly what toxin-strength the horse is receiving, I use a special term, factor, by which to express it. Instead of stating that the animal received 10, 50, or 100 c.cm. of toxin, I record that it receives 10, 50, or 100 factors, the term factor being used to express 100 times the least certainly fatal dose of toxin per 100 grams of guinea-pig. The number of factors in a given quantity of toxin naturally varies with its strength, and it will at once be seen that it is advanta- geous to express the strength regardless of the quantity. The toxin causes some local reaction — at first a dis- tinct inflammation, later a painful edema and a febrile reaction. The amount of local irritation is much less marked when the injections are made slowly ; and a gravity apparatus, which is filled with the amount of serum to be injected, suspeuded from the ceiling of the stable so that the toxin is allowed to take its own time to enter the tissues, can be recommended (Fig. 87). Sometimes it takes several hours to inject 500 c.cm. in this manner. 412 PATHOGENIC BACTERIA. Reservoir for toxin. Rubber tube. The amount of local reaction, edema, etc., the appetite and general condition, the temperature-curve, and the stability of the body-weight, must all be taken into con- sideration, so that the administration shall not be too rapid and the animal thrown into a condition of cachexia. One of the principal things to be avoided is haste. Too frequent or too large dosage is almost certain to kill the animal or bring about a con- dition of hypersensitivity to the toxin. Behring found that mixing the toxin with trichlorid of iodin lessened the irritant effect upon susceptible ani- mals. I prefer not to use susceptible horses. As the antitoxin protects the horse perfectly against the toxin, it is said that a pre- liminary dose, as suggested by Pawlowski, will enable one to omit all the small preliminary doses of toxin, and render the horse im- mune at once. Thus, I have frequently administered ioo c.cm. of antitoxin of about ioo units strength to a horse one day and 500 c.cm. of strong toxin (500 factors) the next. This is just 500 times as much toxin as has twice killed a horse in the laboratory. After the lapse of a few days the same quantity can be administered again, and in a week a third time. In this rapid way antitoxin can often be secured at short notice. I have not found this method of any particular advantage. The possibility of producing serum rapidly may depend upon the method, but the production of strong serums de- pends chiefly upon the horse, and not upon its treatment. Pinch cock. s Hypodermic needle. Fig. 87. — Apparatus used by the author for injecting toxins into horses by gravity. DIPHTHERIA. 4T3 The Preparation of the Serum for Therapeutic Pur- poses.— When, because of the tolerance to large quanti- ties of toxin, the horse seems to possess antitoxic blood, a small incision is made through the skin of the neck, a trocar thrust into the jugular vein, and the blood allowed to flow through a cannulated tube into sterile bottles. It is allowed to coagulate, and kept upon ice for two days or so, that the clear serum may be pipetted off. This serum is the antitoxic serum. It does not always mate- rialize according to the desires of the experimenter, sometimes proving surprisingly strong in a short time, sometimes very weak after months of patient prepara- tion. The serums are preserved by Roux with camphor, by Behring with carbolic acid (0.5 per cent.), and by Aron- son with trikresol (0.4 per cent.). I prefer to use tri- kresol, as it is not poisonous, is a reliable antiseptic, and has a very pronounced local anesthetic action. Formalin has been tried, but it gelatinizes the serum and causes much local pain when injected beneath the skin. Dried antitoxic serum has also been placed upon the market under the impression that it will keep longer and bear shipment better than any other. This is not, how- ever, shown to be the case, and as the dried serum dis- solves with difficulty it is much less convenient than the usual preparations. It is also less likely to be sterile than the liquid forms. The strength of the serum is expressed in what are known as immunising units. This denomination origin- ated with Behring and Ehrlich, whose normal serum was of such strength that o. 1 c.cm. of it would protect against ten times the least certainly fatal dose of toxin when simultaneously injected into guinea-pigs. Each cubic centimeter of this normal serum they called an immunizing unit. Later it was shown that the strength of the serum could easily be increased tenfold, so that 0.01 c.cm. of the serum would protect the guinea-pig against the ten-times fatal dose. Each cubic centimeter 414 PATHOGENIC BACTERIA. of this stronger serum was described as an antitoxic unit, and, of course, contained ten immunizing units. Still later it was shown that the limits of strength were by- no means reached, and he succeeded in making serums three hundred times the normal strength, each cubic centimeter of which contained 300 immunizing units, or 30 antitoxic units. In the course of the development of strength in the serum the exact meaning of " immunizing unit " grad- ually became obscured, until it is at present an expres- sion of strength rather than one of quantity. While it is difficult to define an immunizing unit, it is not at all difficult for one skilled in laboratory technique to determine the number present in a sample of serum. There are three rules of practice: 1. Determine accurately the least certainly fatal dose of a sterile diphtheria toxin for a standard guinea-pig. 2. Determine accurately the least quantity of the serum that will protect a guinea-pig against ten times the determined least certainly fatal dose of toxin. 3. Express the required dose of antitoxic serum as a fraction of a cubic centimeter and multiply it by ten. The result is one unit. Example : It is found that 0.01 c.cm. of toxin kills at least 9 out of 10 guinea-pigs. It is then regarded as the least certainly fatal dose. Guinea-pigs receive ten times this dose (0.1 c.cm.) and varying quantities of the serum, measured by dilution, say, t^ c.cm.,^0 c.cm., ^0 c.cm. The first two live. The frac- tion ^(j is now multiplied by 10 ; 2^V(j X 10 = ^ = 1 unit, and we find that each cubic centimeter of the serum contains 250 Units. The most accurate definition of an immunizing unit is: ten times the least amount of antitoxic serum that will protect a standard (300-gram) guinea-pig against ten times the least certainly fatal dose of diphtheria toxin. The strongest serum I have ever obtained contained 1700 units per cubic centimeter. The correctness of the mode of testing just described depends upon the ability of one unit of antitoxin exactly to neutralize one unit, of toxin. Ehrlich ! points out, 1 Klinisches JaJu'buch, 1897. DIPHTHERIA. 415 however, that while the majority of properly made toxins have about the same combining power, they do not necessarily correspond in this particular, because when the cultures are allowed to remain too long in the incubat- ing-oven, a change, that takes place very slowly in the cold, transforms the toxins formed by the bacilli into certain other bodies, which he calls toxoids. This would make no particular difference, the substances all being poisonous, except that the toxoids have different combin- ing powers from the toxins and may, therefore, cause confusion. The toxoids consist of three groups, which he describes as pro-toxoids, because they have a greater affinity for the antitoxin union than the toxins ; syn- toxoids, which have an equal affinity for the antitoxin ; and epi-toxoids, which have less affinity for the antitoxin than the toxin. The existence of these bodies can be determined by finding the exact limits of toxin-antitoxin neutralization and toxin-antitoxin fatality. The point at which a mixture of toxin and antitoxin is inactive he describes as L0 ; that at which such a mixture becomes fatal by the addition of a little more toxin as L+. The difference between L0 and L,+ should exactly equal one minimum fatal dose of toxin, but only does so when no excess of epitoxoid is present. When epitoxoids are present and have to be displaced by the added toxins, the difference between L0 and L+ becomes enormous. Thus, Ehrlich investigated one fresh active toxin and found L0 = 50 doses of toxin, L+ = 100 doses of toxin, the differ- ence between L0 and h+ not being one single minimum fatal dose, but fifty of them. From this it will be seen that all calculations based upon L0 or upon the exact neutralization of the toxin by the antitoxin must be erroneous, because the combining powers of the antitoxin are by no means exhausted in such a mixture. L 1 should, therefore, always be determined and made use of as the test-dose. The determination of L+ must depend, however, upon 416 PATHOGENIC BACTERIA. a standard unit of antitoxin by which it may be deter- mined, and to this end Ehrlich has suggested the follow- ing alterations in the directions for testing the diphtheria- antitoxin. These alterations have been confirmed in Germany by a decree of March 29, 1897. l I. As a standard for the estimation of the antitoxin an anti- toxin-powder of accurately determined strength, protected against the influence of oxygen and water, is employed. This is con- tained in carefully measured quantities in especially prepared vacuum-tubes. The apparatus at the time present in the labora- tory are filled each with 2 grams of a dry antitoxin 1700 times the normal strength. II. To secure the greatest possible degree of permanence the antitoxin should be dissolved in a mixture of equal parts of 10 per cent, solution of sodium chlorid and glycerin. A tube is to be opened every three months and a new solution prepared. Of the dry antitoxin at the time preserved in the laboratory, the con- tents of a tube are dissolved in 200 c.cm. of the mixture described, and thus a test antitoxin-solution 17 times the normal strength is prepared. III. The present test-dose of toxin is determined with the aid of an immunity-unit, such as is contained, for instance, in 1 c.cm. of a T'T dilution of the test-antitoxin 17 times the normal strength. To this amount of antitoxin increasing amounts of toxin are added, and by means of most careful experimental observations the limit is determined at which just that excess of toxin becomes manifest which causes death of the animal in the first four days. The amount of toxin thus obtained represents the immediate test-dose. By means of the same dose of serum, for the more exact characterization of the toxin, the determination of a second limit is made, for the purpose of learning the dose of toxin that is just neutralized by admixture with the amount of serum named. IV. The determination of the strength of a diphtheria-antitoxin is made by means of the test-dose of toxin as follows : The test- dose of toxin in question — for instance, 0.355 c.cm. of tested toxin at the time present in the laboratory — is mixed with 4 c.cm. of antitoxin corresponding to the test figures given. As the test-dose of toxin is estimated for 1 c.cm. of antitoxin of normal strength, or for 4 c.cm. of antitoxin yi the normal strength, an antitoxin of x strength will have to be diluted % x, and in test1 ing an antitoxin 100 times the normal strength, ¥^. 1 See Levy and Klemperer's Clinical Bacteriology, translated by A. A. Eshner, Philadelphia, 1900. DIPHTHERIA. 417 As the quantity to be injected at each dose diminishes according to the number of units per cubic centimeter the serum contains, it is of the highest importance that the serums be as strong as possible. Various methods of concentration have been suggested, such as the partial evaporation of the serum in vacuo, but none have proved satisfactory. Bujwid1 and H. C. Ernst2 find that when an antitoxic serum is frozen and then thawed, it separates into two layers, an upper watery stratum and a lower yellowish one ; the antitoxic value of the yellowish layer is about three times that of the original serum, the sepa- rated upper layer being chiefly water. Ehrlich asserts that 500 units are valueless for treat- ment: 2000 units are probably an average dose, but, as the remedy seems harmless, it is better to err on the side of too much than on that of too little. Forty thousand units have been administered with beneficial results. It is a common clinical experience that paralysis is more frequent after the use of antitoxin than in cases treated without it. In a paper upon this subject3 I have shown that this is to be expected, as the palsies usually occur after the bad cases of the disease, of which a far greater number recover when antitoxin is used for treat- ment. The largest collection of statistics upon the results of antitoxic treatment in diphtheria in the hospitals of the world are probably those collected by Prof. Welch,4 who, excluding every possible error in the calculations, "shows an apparent reduction of case-mortality of 55.8 per cent." One of the most important things in the treatment is' to begin it early enough. Welch's statistics show that 1 1 15 cases of diphtheria treated in the first three days of the disease yielded a fatality of 8.5 per cent., whereas 1 Centralbl. f. Bakt. u. Parasitenk., Sept., 1897, Bd. xxii., Nos. 10 and 11, p. 287. * Journal of the Boston Society of the Medical Sciences, May, 1898, vol. ii., No. 8, p. 137. 3 Medical Record, New York. 1897. * Bulletin of the Johns Hopkins Hospital. 27 418 PATHOGENIC BACTERIA. 546 cases in which the antitoxin was first injected after the third day of the disease yielded a fatality of 27.8 per cent. After the toxin has set up destructive organic lesions in various organs and tissues of the body, no amount of neutralization will restore the integrity of the parts, so that the antitoxin must fail in these cases. The urticaria which sometimes follows the injection of antitoxic serum seems to depend upon the globulins it contains. I have found that the "keeping" qualities of the se- rums, when properly preserved, are of long duration. Samples examined two years after having been exposed for sale in the markets have been found unchanged. The serums most prone to deteriorate seem to be those of highest potency, but even here the good qualities are unchanged for months. Serums, however, are by no means regular in their deterioration, and no very old serum should be used for the treatment of diphtheria. Freezing is without effect and ordinary temperature- changes are harmless to the serum. The antitoxic power is destroyed at 6o° C, the point at which the serum coagulates. The antitoxin is precipitated with the glob- ulins.1 The erythemata are probably in some way associated with the globulocidal action of the blood. The serums from different horses probably vary much in both their irritant and globulocidal properties, so that antitoxins prepared by mixing the serums from a number of horses are probably preferable to those from single horses. The transitory nature of the immunity afforded by prophylactic injections of the antitoxin is probably de- pendent upon the fact that the antitoxin is slowly ex- creted through the kidneys. Bacilli Resembling the Diphtheria Bacillus. — The Pseudodiphtheria bacillus — Bacillus pseudodiphthericus — 1 See paper by J. P. Atkinson, Journal of Experimental Medicine, Sept. .and Nov., 1899, vol. iv., Nos. 5 and 6. DIPHTHERIA. 4X9 was first described by Loffler1 as occurring in diphthe- ria pseudomembraues and in the healthy mouth and pharynx. It is also found upon the conjunctiva, espe- cially in xerosis conjunctivae, and corresponds to the previously described Bacillus xerosis conjunctivae. By some authors this bacillus is thought to cause chronic ulcerative keratitis and chalazion. The pseudodiphtheria bacillus is also found in the nose and upon the skin, where it usually associates itself with the Staphylococcus aureus. It has been found in impetigo, acne, and vari- ola pustules. It has also at times been isolated from the internal organs, as in the cases of Egyptian dysentery studied by Kruse and Pasquale.2 Ohlmacher has also found it with other bacteria in pneumonia, Babes in gangrene of the lung, and Howard 3 in a case of ulcera- tive endocarditis not succeeding diphtheria. While various authors have endeavored to point out morphological and cultural differences by which the diphtheria and pseudodiphtheria bacilli can be differen- tiated, it must be admitted that the variations of the latter organism are so numerous that all rules fail. The only criterion for specific differentiation is the ability of the true diphtheria bacillus to form toxin, in which capacity the pseudodiphtheria bacillus is entirely lack- ing. The introduction of the diphtheria bacillus into animals is characterized by an indurated serofibrous in- flammatory area, that of the pseudodiphtheria bacillus by no pathological changes. Park4 carefully studied this subject, and found that all bacilli with the exact morphology of the diphtheria bacillus, found in the human throat, are virulent Klebs- Loffler bacilli, while forms found in the throat closely resembling them, but more uniform in size and shape, shorter in length, and of more equal staining properties 1 Centralbl. f. Bakt. u. Parasitenk , ii., 105. 1 Zeitschrift fur Hygiene, xvi., I. 3 Bull. Johns Hopkins Hospital, 93, 30. 4 Scientific Bulletin No. 1, Health Department, City of New York, 1895. 420 PATHOGENIC BACTERIA. with Loffler's alkaline methylene blue solution, can be, with reasonable safety, regarded as pseudodiphtheria bacilli, especially if it be found that they produce an alkaline rather than an acid reaction by their growth in bouillon. The pseudodiphtheria bacilli were found in about i per cent, of throats examined in New York; they seem to have no relationship to diphtheria. They are never virulent. The observation of Martini,1 that the diphtheria ba- cillus will not grow in fluid antitoxic serum in which the pseudodiphtheria bacillus thrives I have not been able to confirm. Both the real and the pseudobacilli flourish upon coagulated antitoxic serum. Having practically the same cultural and staining re- actions as the diphtheria bacillus, the question presents itself, Is the pseudodiphtheria bacillus the diphtheria ba- cillus in an attenuated condition ? This question we are, as yet, unable to answer. Every attempt to bring back virulence to the pseudobacilli has failed, and we know it only as a saprophyte, except upon the conjunctivae, when, if it is identical with the Bacillus xerosis conjunctivae, it seems able to take up parasitic existence very successfully and lead to recognized lesions. It seems to me that inas- much as it is, as a rule, almost impossible to increase the virulence of any diphtheria bacillus, its persistent attenu- ation should not stand in the way of regarding it as the extreme decree of attenuation to which a bacterium can descend, and accepting it as a non-virulent and non- infectious form of diphtheria bacillus. 1 Centralbl.f. Bakt. u. Parasitenk., Jan. 30, 1897, Bd. xxi., No. 3. CHAPTER IV. CHOLERA AND SPIRILLA RESEMBLING THE CHOLERA SPIRILLUM. Spirillum Cholera Asiatics (Koch1). Cholera is a disease from which certain parts of India are never free. The areas in which it is endemic are the foci from which the great epidemics of the world, as well as the constant smaller epidemics of India, probably spread. No one knows when cholera was first introduced into India, and the probabilities are that it is indigenous to that country, as yellow fever is to Cuba. Very early mention of it is made in the letters of travellers, in books and papers on medicine of a century ago, and in the governmental statistics, yet we find that little is said about the disease except in a general way, most attention being directed to the effect upon the armies, native and European, of India and adjacent countries. The opening up of India by Great Britain in the last half century has made possible much accurate scientific observation of the disease and the relation which its epi- demics bear to the manners and customs of the people. The filthy habits of the people of India, their poverty, their crowded condition, and their religious customs, all serve to aid in the distribution of the disease. We are told that the city of Benares drains into the Ganges River by a most imperfect system, which distributes the greater part of the sewage immediately below the banks upon which the city is built. It is a matter of religious ob- servance for every zealot who makes a pilgrimage to the "sacred city " to take a bath in and drink a large quan- 1 Deutsche med. Wochenschrift, 1884-1885, Nos. 19, 20, 37, 38, and 39. 421 422 PATHOGENIC BACTERIA. tity of this sacred but polluted water, and, as may be imagined, the number of pious Hindoos who leave Benares with comma bacilli in their intestines or upon their clothes is great, for there are few months in the year when there are not at least some cases of cholera in the city. The frequent pilgrimages and great festivals of the Hindoos and Moslems, by bringing together an enormous number of people who crowd in close quarters where filth and bad diet are common, cause a rapid increase in the number of cases during these periods and the dispersion of the disease when the festivals break up. The disease extends readily along the regular lines of travel, visiting town after town, until from Asia it has frequently ex- tended into Europe, and by the steamships plying on foreign waters has been several times carried to our own continent and to the islands of the seas. Many cases are on record which show conclusively how a single ship, having a few cholera cases on board, may be the cause of an outbreak of the disease in the port at which it arrives. The discovery of the organism which seems to be the specific cause of cholera was made by Koch, who was appointed one of a German cholera-commission to study the disease in Egypt and India in 1883-84. Since his discovery, but a decade ago, the works upon cholera and the published investigations to which the spirillum has been subjected have produced an immense literature, a -large part of which was stimulated by the Hamburg epidemic of a few years ago. The micro-organism described by Koch, and now gen- erally accepted to be the cause of cholera, is a short individual about half the length of a' tubercle bacillus, considerably stouter, and distinctly curved, so that the original name by which it was known was the ' ' comma bacillus" (Figs. 88, 89). A study of the growth of the organism and the forms which it assumes upon different culture-media soon con- CHOLERA. 423 vinces lis that we have to do with an organism in no way related to the bacilli. If the conditions of nutrition are i>>; in? Fig. 88. — Spirillum of Asiatic cholera, showing the flagella; x 1000 (Gunther). diminished so that the multiplication of the bacteria by simple division does not progress with the usual rapidity, we find a distinct tendency toward — and in some cases, as upon potato, a luxuriant development of — long spiral threads with numerous windings — unmistakable spirilla. Frankel has found that the exposure of cultures to unusu- ally high temperatures, the addition of small amounts of alcohol to the culture-media, etc., will so vary the growth of the organism as to favor the production of spirals instead of commas. One of the most common of the numerous forms observed is that in which two short curved individuals are so joined as to produce an S-shaped curve. The cholera spirilla are exceedingly active in their movements, and in hanging-drop cultures can be seen to swim about with great rapidity. Not only do the comma-shaped organisms move, but when distinct spirals exist, they, too, move with the rapid rotary motion so common among the spirilla. The presence of flagella upon the cholera spirillum can be demonstrated without difficulty by LofBer's 424 PATHOGENIC BACTERIA. method {q. v.). Each spirillum possesses a single flagel- lum attached to one end. Inoculation-forms of most bizarre appearance are very common in old cultures of the spirillum, and very often * K VS r% ■ « 1 ■ - >v* Fig. 89. — Spirillum of Asiatic cholera, from a bouillon culture three weeks old, showing numbers of long spirals; x 1000 (Frankel and Pfeiffer). there can be found in fresh cultures many individuals which show by granular protoplasm and irregular outline that they are partly degenerated. Cholera spirilla from various sources seem to differ in this particular, some of the forms being as pronounced in their involution as the diphtheria bacilli. In partially degenerated cultures in which long spirals are numerous Hiippe observed, by examination in the "hanging drop," in the continuity of the elongate mem- bers, certain large spherical bodies which he described as spores. These bodies were not enclosed in the organisms like the spores of anthrax, but seemed to exemplify the form of sporulation in which an entire individual trans- forms itself into a spore (arthrospore). Koch, and indeed all other observers, failed to find signs of fructification in the cholera organism, and the true nature of the bodies described by Hiippe must be regarded as doubtful. CHOLERA. 425 The cholera spirillum stains well with the ordinary aqueous solutions of the anilin dyes ; fuchsin seems par- ticularly appropriate. At times the staining must be con- tinued for from five to ten minutes to secure homogeneity. The cholera spirillum does not stain by Gram's method. It may be colored and examined while alive ; thus Cornil and Babes, in demonstrating it in the rice-water dis- charges, "spread out one of the white mucous fragments upon a glass slide and allow it to dry partially ; a small quantity of an exceedingly weak solution of methyl violet in distilled water is then flowed over it, and it is flattened out by pressing down on it a cover-glass, over which is placed a fragment of filter-paper, which absorbs any excess of fluid at the margin of the cover-glass. Comma bacilli so prepared and examined with an oil-immersion lens (x 700-800) may then be seen : their characters are the more readily made out because of the slight stain which they take up, and because they still retain their power of vigorous movement, which would be entirely lost if the specimen were dried, stained, and mounted in the ordinary fashion." The colonies of the spirillum when grown upon gel- atin plates are highly characteristic. They appear in the lower strata of the gelatin as small white dots, grad- ually grow out to the surface, effect a gradual liquefaction of the medium, and then appear to be situated in little pits with sloping sides (Fig. 90). This peculiar appear- ance, which gives one the suggestion that the plate is full of little holes or air-bubbles, is due to the evapora- tion of the liquefied gelatin. One of the best methods of securing pure cultures of the cholera spirillum, and also of making a diagnosis of the disease in a suspected case, is probably that of Schottelius. The method is very simple : A small quan- tity of the fecal matter is mixed with bouillon and stood in an incubating oven for twenty-four hours. If the cholera spirilla are present they will grow most rapidly at the surface of the liquid when the supply of air is 426 PATHOGENIC BACTERIA. good. A pellicle will be formed, a drop from which, diluted in melted gelatin and poured upon plates, will show typical colonies. Under the microscope the principal characteristics can be made out. The. colony of the cholera spirillum scarcely resembles that of any other organism. The little colonies which have not yet reached the surface of the gelatin begin very soon to show a pale-yellow color and FlG. 90. — Spirillum of Asiatic cholera: colonies two days old upon a gelatin plate ; x 35 (Heim). to exhibit irregularities of contour, so that they are almost never smooth and round. They are coarsely granular, and have the largest granules in the centre. As the colony increases in size the granules also increase in size, and attain a peculiar transparent character which is suggestive of powdered glass. The commencement of liquefaction causes the colony to be surrounded with a transparent halo. When this occurs the colony begins to sink, from the digestion and evaporation of the medium, and also to take on a peculiar rosy color. CHOLERA. 427 In puncture-cultures in gelatin the growth is again so characteristic that it is quite diagnostic (Fig. 91). The ■ JNMBU Y\G. 91. — Spirillum cholera Asiatica ; gelatin puncture-cultures aged forty- eight and sixty hours (Shakespeare). growth takes place along the entire puncture, but devel- ops best at the surface, where it is in contact with the atmosphere. An almost immediate liquefaction of the medium begins, and, keeping pace with the rapidity of the growth, is more marked at the surface than lower down. The result of this is the occurrence of a short, rather wide funnel at the top of the puncture. As the growth continues evaporation of the medium takes place slowly, so that the liquefied gelatin is lower than the solid surrounding portions, and appears to be surmounted by an air-bubble. The luxuriant development of the spirilla in gelatin produces considerable solid material to sediment and fill up the lower third or lower half of the liquefied area. This solid material consists of masses of spirilla which have probably completed their life-cycle and become inactive. Under the microscope they exhibit the most 428 PATHOGENIC BACTERIA. varied involution-forms. The liquefaction reaches the sides of the tube in from five to seven days. Liquefac- tion of the medium is not complete for several weeks. According to Frankel, in eight weeks the organisms in the liquefied culture all die, and cannot be transplanted. Kitasato, however, has found them living and active on agar-agar after ten to thirty days, and Koch was able to demonstrate their vitality after two years. When planted upon the surface of agar-agar the spi- rilla produce a grayish-white, shining, translucent growth along the entire line of inoculation. It is in no way peculiar. The vitality of the organism is retained much better upon agar-agar than upon gelatin, and, according to Frankel, the organism can be transplanted and grown when nine months old. The growth upon blood-serum likewise is without dis- tinct peculiarities, and causes gradual liquefaction of the medium. Upon potato the spirilla grow well, even when the reaction of the potato is acid. In the incubator at a temperature of ^7° C. a transparent, slightly brownish or yellowish-brown growth, somewhat resembling the growth of glanders, is produced. It contains large numbers of long spirals. In bouillon and in peptone solution the cholera organ- isms grow well, especially upon the surface, where a folded, wrinkled mycoderma is formed. Below the my- coderma the culture fluid generally remains clear. If the glass be shaken and the mycoderma broken up, fragments of it sink to the bottom. In milk the development is also luxuriant, but takes place in such a manner as not visibly to alter its appear- ance. The existence of cholera organisms in milk is, however, rather short-lived, for the occurrence of any acidity at once destroys them. Wolffhugel and Riedel have shown that if the spirilla are planted in sterilized water they grow with great ra- pidity after a short time, and can be found alive after CHOLERA. 429 months have passed. Frankel points ont that this ability to grow and remain vital for long periods in sterilized water does not guarantee the same power in unsterilized water, for in the latter the simultaneous growth of other bacteria in a few days serves to extinguish the cholera germs. One of the characteristics of the cholera spirillum is the metabolic production of indol. The detection of this substance is easy if the spirilla are grown in a transparent colorless solution. As the cholera organisms also produce nitrites, all that is necessary is to add a drop or two of chemically pure sulphuric acid to the culture-medium for the production of the well-known reddish color. Several toxic products of the metabolism of the spirilla have been isolated. Brieger, Frankel, Roux and Yersin have isolated toxalbumins; Villiers, a toxic alkaloid fatal to guinea-pigs; and Gamal£ia, two substances about equally toxic. The cholera spirilla can be found with great constancy in the intestinal evacuations of all cholera cases, and can often be found in the drinking-water, milk, and upon vegetables, etc. in cholera-infected districts. There can be little doubt that they find their way into the body through the food and drink. Many cases are reported in the literature upon cholera that show how the disease- germs enter the drinking-water, and are thus distributed ; how they are sometimes thoughtlessly sprinkled over veg- etables, offered for sale in the streets, with water from polluted gutters ; how they enter milk with water used to dilute it ; how they are carried about in clothing and upon foodstuffs ; how they can be brought to articles of food upon the table by flies which have preyed upon cholera excrement; and how many other interesting in- fections are made possible. The literature upon these subjects is so vast that in a sketch of this kind it is scarcely possible to mention even the most instructive examples. One physician is reported to have been in- 43° PATHOGENIC BACTERIA. fected with cholera while experimenting with the spirilla in Koch's laboratory. The evidence of the specificity of the cholera spirillum when collected shows that it is present in the choleraic dejections with great regularity, and that it is as con- stantly absent from the dejecta of healthy individuals and those suffering from other diseases ; but these facts do not admit of satisfactory proof by experimentation upon animals. Animals are never affected by any dis- ease similar to cholera during the epidemics, nor do foods mixed with cholera discharges or with pure cultures of the cholera spirillum affect them. This being true, we are prepared to receive the further information that sub- cutaneous injections of the spirilla a're often without serious consequences, though cultures differ very much in this respect, some always causing a fatal septicemia in guinea-pigs, others being as constantly harmless. Intraperitoneal injection of the virulent cultures pro- duces a fatal peritonitis in guinea-pigs. One reason that animals and certain men are immune to the disease seems to be found in the distinct acidity of the normal gastric juice, and the destruction of the spi- rilla by it. Supposing that this might be the case, Nicati and Rietsch, Von Ermengen and Koch, have suggested methods by which the micro-organisms can be introduced directly into the intestine. The first-named investigators ligated the common bile-duct of guinea-pigs, and then in- jected the spirilla into the duodenum with a hypodermic needle. The result was that the animals usually died, some- times with choleraic symptoms ; but the excessively grave nature of the operation upon such a small and delicately constituted animal as a guinea-pig greatly lessens the value of the experiment. Koch's method is much more satisfac- tory. By injecting laudanum into the abdominal cavity of guinea-pigs the peristaltic movements are checked. The amount given for the purpose is very large, about i gram for each 200 grams of body-weight. It generally narcotizes the animals for a short time, but they recover CHOLERA. 431 without injury. After administering the opium the con- tents of the stomach are neutralized by introducing through a pharyngeal catheter 5 c.cm. of a 5 per cent, aqueous solution of sodium carbonate. With the gastric contents thus alkalinized and the peristalsis paralyzed a bouillon culture of the spirilla is introduced. The ani- mal recovers from the manipulation, but shows an indis- position to eat, is soon observed to be weak in the pos- terior extremities, subsequently is paralyzed, and dies within forty-eight hours. The autopsy shows the intes- tine congested and filled with a watery fluid rich in spi- rilla— an appearance which Frankel declares to be exactly that of cholera. In man, as well as in these artificially injected animals, the spirilla are never found in the blood or the tissues, but only in the intestine, where they fre- quently enter between the basement membrane and the epithelial cells, and aid in the detachment of the latter. Issaeff and Kolle found that when virulent cholera spirilla are injected into the ear-veins of young rabbits the animals die on the following day with symptoms re- sembling the algid stage of human cholera. The autopsy in these cases showed local lesions of the small intestine very similar to those observed in cholera in man. Guinea-pigs are also susceptible to intraperitoneal in- jections of the spirillum, and speedily succumb. The symptoms are — rapid fall of temperature, tenderness over the abdomen, and collapse. The autopsy shows an abundant fluid exudate containing the micro-organism, and injection and redness of the peritoneum and viscera. Although in reading upon cholera at the present time we find very little skepticism in relation to Koch's "comma bacillus," we do find occasional doubters who believe with Von Pettenkoffer that the disease is mias- matic. Petteukoffer's theory is that the disease has much to do with the ground-water and its drying zone. He regards as the principal cause of the disease the de- velopment of germs in the subsoil moisture during the warm months, and their impregnation of the atmosphere 432 PATHOGENIC BACTERIA. as a miasm to be inhaled, instead of ingested with food and drink. This idea of Pettenkoffer's, combined with his other idea that individual predisposition must pre- cede the inception of the disease, is scarcely compatible with what has gone before, and cannot possibly be made to explain the march of the disease from place to place with caravans, or its distribution over extended areas when fairs and religious gatherings among the Hindoos break up, the people from an infected centre carrying cholera with them to their homes. While it is an organism that multiplies with great rapidity under proper conditions, the cholera spirillum is not possessed of much resisting power. Sternberg found that it was killed by exposure to a temperature of 520 C. for four minutes. Kitasato, however, found that ten or fifteen minutes' exposure to a temperature of 550 C. was not always fatal. In the moist con- dition the organism may retain its vitality for months, but it is very quickly destroyed by desiccation, as was found by Koch, who observed that when dried in a thin film its power to grow was destroyed in a few hours. Kitasato found that upon silk threads the vitality might be retained longer. Abel and Claussen l have shown that it does not live longer than twenty to thirty days in fecal matter, and often disappears in one to three days. The organism is very susceptible to the influence of carbolic acid, bichlorid of mercury, and other germicides. The organism is also destroyed by acids. Hashimoto2 found that it could not live longer than fifteen minutes in vinegar containing 2.2-3.2 per cent, of acetic acid. This low vital resistance of the microbe is very fortu- nate, for it enables us to establish safeguards for the pre- vention of the spread of the disease. Excreta, soiled clothing, etc. are readily rendered harmless by the proper use of disinfectants. Water and food are rendered in- nocuous by boiling or cooking. Vessels may be disin- 1 Centralbl. f. Bakt. u. Parasitenk., Jan. 31, 1895, v°l- xvii., No. 4. 2 Kwai Med. Jour., Tokyo, 1893. CHOLERA. 433 fected by thorough washings with jets of boiling water thrown upon them through hose. Baggage can be steril- ized by superheated steam. It often becomes a matter of importance to detect the presence of cholera in drinking-water, and, as the dilu- tion in which the bacteria exist in such a liquid may be very great, much difficulty is experienced in finding them by ordinary methods. One of the most expeditious meth- ods that have been recommended is that of Loffler, who adds 200 c.cm. of the water to be examined to 10 c.cm. of bouillon, allows the mixture to stand in an incubator for twelve to twenty-four hours, and then makes plate- cultures from the superficial layer of the liquid, where, if present, the development of the spirilla will be most rapid because of the presence of air. A similar method can be used to detect the spirilla when their presence is suspected in feces. Gruber and Wiener, Haff kine, Pawlowsky, and Pfeiffer have all succeeded in immunizing animals against the toxic substances removed from cholera cultures or against living cultures properly injected. There seems, accord- ing to the researches of Pfeiffer, to be no doubt that in the blood of the protected animals a protective substance is present. In the peritoneal infection of guinea-pigs the spirilla grow vigorously in the peritoneal cavity, and can be found in immense numbers after twelve to twenty- four hours. If, however, together with the culture used for inoculation, a few drops of the protective serum be in- troduced, Pfeiffer found that instead of multiplying the organisms underwent a peculiar granular degeneration and disappeared, the unprotected animal dying, the pro- tected animal remaining well. Pfeiffer and Vogedes l have suggested the application of this " immunity-reaction " for the positive differentia- tion of cholera spirilla in cultures. A hanging-drop of a 1 : 50 mixture of powerful anti-cholera serum and a particle of cholera culture is made and examined under 1 Centralbl. fur Bakt. und Parasitenk., March 21, 1896, Bd. xix., No. II. 23 434 PATHOGENIC BACTERIA. the microscope. The cholera spirilla at once become in- active, and are in a short time converted into little rolled- up masses. If the culture added be a spirillum other than the true spirillum of cholera, instead of destruc- tion of the micro-organisms following exposure to the serum, they multiply and thrive in the mixture of serum and bouillon. The specific immunity-reaction of the cholera serum has been carefully studied by I/oburnheim,1 and is specific against cholera alone. The protection is not due to the strongly bactericidal property of the serum, but to its stimulating effect upon the body-cells. If the serum be heated to 6o°-70° C, and its bactericidal power thus destroyed, it is still capable of producing immunity. The immunity produced by the injection of the spirilla into guinea-pigs continues in some cases as long as four and a half months, but the power of the serum to con- fer immunity is lost much sooner. Of the numerous attempts which have from time to time been made, and are still being made, to produce immunity against cholera in man or to cure cholera when once established in the human organism, nothing very favorable can at the present time be said. Experi- ments in this field are not new : we find Dr. Ferran ad- ministering hypodermic injections of pure virulent cul- tures of the cholera spirillum in Spain as early as 1885, in the hope of bringing about immunity. The more mod- ern work of Haffkine2 seems to be followed by a distinct diminution of mortality in protected individuals. Ac- cording to the work of this investigator, two vaccines are used, one of which, being mild, prepares the animal (or man) for a powerful vaccine, which, were it not preceded by the weaker form, would bring about extensive tissue- necrosis and perhaps death. Protection certainly seems to follow the operation of these vaccines. 1 Zeitschrift fur Hygiene, xx., p. 438. 2 Le Bull, mid., 1892, p. 11 13; Indian Med. Gazette, 1893, p. 97; Brit. Med. Jour., 1893, p. 278. CHOLERA. 435 Haffkine's studies embrace more than 40,000 inocula- tions performed in India. From his latest paper (Dec, 1895) the following extract will show the results : "1. In all those instances where cholera has made a large number of victims, that is to say, where it has spread sufficiently to make it probable that the whole population, inoculated and uninoculated, were equally exposed to the infection, — in all these places the results appeared favorable to inoculation. u2. The treatment applied after an epidemic actually breaks out tends to reduce the mortality even during the time which is claimed for producing the full effect of the operation. In the Goya Garl, where weak doses of a relatively weak vaccine had been applied, this reduction was to half the number of deaths ; in the coolies of the Assam-Burmah survey-party, where, as far as I can gather from my preliminary information, strong doses have been applied, the number of deaths was reduced to one-seventh. This fact would justify the application of the method in- dependently of the question as to the exact length of time during which the effect of this vaccination lasts. "3. In Lucknow, where the experiment was made on small doses of weak vaccines, a difference in cases and deaths was still uoticeable in favor of the inoculated fourteen to fifteen months after vaccination in an epidemic of exceptional virulence. This makes it probable that a protective effect could be obtained even for long periods of time if larger doses of a stronger vaccine were used. "4. The best results seem to be obtained from applica- tion of middle doses of both anticholera vaccines, the second one being kept at the highest possible degree of virulence obtainable. "5. The most prolonged observations on the effect of middle doses were made in Calcutta, where the mortality from the eleventh up to the four hundred and fifty-ninth day after vaccination was, among the inoculated, 17.24 times smaller, and the number of cases 19.27 times smaller than among the not inoculated." 436 PATHOGENIC BACTERIA. Pawlowsky and others have found that the dog is sus- ceptible to cholera, and have utilized the observation to prepare an antitoxic serum in considerable quantities. The dogs were first immunized with attenuated cultures, then with more and more virulent cultures, until a serum was obtained whose value was estimated at 1 : 130,000 upon experimental animals. Freymuth^nd others have endeavored to secure favor- able results from the injection of blood-serum from con- valescent patients into the diseased. One recovery out of three cases treated is recorded — not a very glittering result. In all these preliminaries the foreshadowing of a future therapeusis must be evident, but as yet nothing really satisfactory has been achieved. Spirilla resembling the Cholera Spirillum. The Finkler and Prior2 Spirillum (Vibrio Proteus). — Somewhat similar to the spirillum of cholera, and in some respects closely related to it, is the spirillum ob- tained from the feces of a case of cholera nostras by Finkler and Prior in 1884. It is a shorter, stouter or- ganism, with a more pronounced curve than the cholera spirillum, and rarely forms the long spirals which char- acterize the latter. The central portion is also somewhat thinner than the ends, which are a little pointed and give the organism a less uniform appearance than that of cholera (Fig. 92). Involution-forms are very common in cultures, and occur as spheres, spindles, clubs, etc. Like the cholera spirillum, each organism is provided with a single flagellum situated at its end, and is actively motile. Although at first thought to be a variety of the cholera germ, marked differences of growth were soon observed, and showed the organism to be a separate species. The growth upon gelatin plates is quite rapid, and leads to such extensive liquefaction that four or five dilutions 1 Deutsche med. Wochenschrift, 1893, No. 43. 2 Ibid., 1884. SPIRILLA RESEMBLING CHOLERA. 437 must frequently be made before the growth of a single colony can be observed. To the naked eye the colonies * Fig. 92. — Spirillum of Finkler and Prior, from an agar-agar culture ; x 1000 (Itzerott and Niemann). appear as small white points in the depths of the gelatin (Fig. 93). They, however, rapidly reach the surface, Fig. 93. — Spirillum of Finkler and Prior: colony twenty-four hours old, as seen upon a gelatin plate; x 100 (Frankel and Pfeiffer). begin liquefaction of the gelatin, and by the second 438 PATHOGENIC BACTERIA. day appear about the size of lentils, and are situated in little depressions. Under the microscope they are of a yellowish-brown color, are finely granular, and are sur- rounded by a zone of sharply circumscribed liquefied gelatin. Careful examination with a high power of the microscope shows a rapid movement of the granules of the colony. In gelatin punctures the growth takes place rapidly along the whole puncture, forming a stocking-shaped liquefaction filled with cloudy fluid which does not pre- cipitate rapidly ; a rather smeary, whitish mycoderma is generally formed upon the surface. The much more ex- tensive and more rapid liquefaction of the medium, the wider top to the funnel-shaped liquefaction at the surface, Fig. 94. — Spirillum of Finkler and Prior : gelatin puncture-cultures aged forty-eight and sixty hours (Shakespeare). the absence of the air-bubble, and the clouded nature of the liquefied material, all serve to differentiate it from the cholera spirillum. Upon agar-agar the growth is also very rapid, and in a short time the whole surface of the culture-medium is SPIRILLA RESEMBLING CHOLERA. 439 covered with a moist, thick, slimy coating, which may have a slightly yellowish tinge. The cultures upon potato are also very different from those of cholera, for instead of a temperature of 370 C. being required for a rapid development, the Kinkier and Prior spirilla grow rapidly at the room-temperature, and produce a grayish-yellow, slimy, shining layer, which may cover the whole of the culture-medium. Blood-serum is rapidly liquefied by the growth of the organism. Buchner has shown that in media containing some glucose an acid reaction is produced. The spirillum does not grow well, if at all, in milk, and speedily dies in water. The organism does not produce indol. The spirillum can be stained well by the ordinary dyes, and seems, like the cholera spirillum, to have a special affinity for the aqueous solution of fuchsin. In connection with this bacillus the question of patho- genesis is a very important one. At first it was sus- pected that it was, if not the spirillum of cholera itself, a very closely allied organism. Later it was regarded as the cause of cholera nostras. At present its exact pathological significance is a question. It was in one case secured by Knisl from the feces of a suicide, and has been found in carious teeth by Miiller. When injected into the stomach of guinea-pigs treated according to the method of Koch, about 30 per cent, of the animals die, but the intestinal lesions produced are not the same as those produced by the cholera spirillum. The intestines in such cases are pale and filled with watery material having a strong putrefactive odor. This fluid teems with the spirilla. It seems very unlikely, from the collected evidence, that the Finkler and Prior spirillum is associated with pathogenesis in the human species. As Frankel points out, it is probably a frequent and harmless inhabitant of the human intestine. 44o PATHOGENIC BACTERIA. The Spirillum of Denecke1 (Vibrio Tyrogenum). — Another organism with a distinct resemblance to the cholera spirillum is one described by Denecke as occur- ring in old cheese (Fig. 95). Its form is much the same as that of the spirillum of cholera, the shorter indi- viduals being of equal diameter throughout. The spirals which are produced are longer than those of the Finkler and Prior spirillum, and are more tightly coiled than those of the cholera spirillum. Like its related species, this micro-organism is actively motile. It grows at the room-temperature, as well as at 370 C, in this respect, as in its reaction to stains, much resembling the other two. Upon gelatin plates the growth of the colonies is much more rapid than that of the cholera spirillum, but slower than that of the Finkler and Prior spirillum. The col- FiG. 95. — Spirillum Denecke, from an agar-agar culture; x 1000 (Itzerott and Niemann). onies appear as small whitish, round points, which soon reach the surface of the gelatin and commence liquefac- tion. By the second day they are about the size of a pin's head, have a yellow color, and occupy the bottom of a conical depression. The appearance is much like that of a plate of cholera spirilla. 1 Deutsche med. Wochenschrift, 1 885. SPIRILLA RESEMBLING CHOLERA. 441 The microscope shows the colonies to be of irregular shape and coarsely granular. The color is yellow, and is pale at the edges, gradually becoming intense toward the centre. The colonies are surrounded at first by distinct lines of circumscription, later by clear zones, which, ac- cording to the illumination, are pale or dark. From this description it will be seen that the colonies differ from those of cholera in the prompt liquefaction of the gelatin, their rapid growth, yellow color, irregular form, and dis- tinct lines of circumscription. In gelatin punctures the growth takes place all along the track of the wire, and forms a cloudy liquid which precipitates at the apex in the form of a coiled mass. Upon the surface a delicate imperfect yellowish myco- 1 fl vMB lEM H i Fig. 96. — Spirillum Denecke : gelatin puncture-cultures aged forty-eight and sixty hours (Shakespeare). derma forms. Liquefaction of the entire gelatin gen- erally requires about two weeks. Upon agar-agar this spirillum grows as a thin yellow- ish layer which does not seem inclined to spread widely. The culture upon potato is luxuriant if grown in the incubating oven. It appears as a distinct yellowish moist 442 PATHOGENIC BACTERIA. film, and when examined microscopically is seen to con- tain long beautiful spirals. The organism sometimes produces indol, but is irreg- ular in its action in this respect. The spirillum of Denecke is mentioned only because of its morphological relation to the cholera spirillum, not because of any pathogenesis which it possesses. It probably is not associated with any human disease. Ex- periments, however, have shown that when the spirilla are introduced into the intestines of guinea-pigs whose gastric contents are alkalinized and whose peristalsis is Fig. 97. — Spirillum Metschnikowi, from an agar-agar culture; x IOOO (Itzerott and Niemann). paralyzed with opium, about 20 per cent, of the animals die from intestinal disease. The Spirillum of Gamaleia1 (Vibrio Metschnikowi). — Very closely related to the cholera spirillum in its morphology and vegetation and possibly, as has been suggested, a descendant of the same original stock, is the spirillum which Gamaleia cultivated from the intestines of chickens affected with a disease similar to chicken- cholera. This spirillum is a curved organism, a trifle shorter and thicker than the cholera spirillum, a little 1 Annates de I Inst. Pasteur, 1888. SPIRILLA RESEMBLING CHOLERA. 443 more curved, and with similar rounded ends (Fig. 97). It forms long spirals in appropriate media, and is actively motile. Each spirillum is provided with a terminal flagel- lum. No spores have been positively demonstrated. The organism, like the cholera vibrio, is very suscep- tible to the influence of acids, high temperatures, and drying, so that spores are probably not formed. It grows well both at the temperature of the room and at that of incubation. The thermal death-point is 500 C, continued for five minutes. The bacterium stains easily, the ends more deeply than the center. It is not stained by Gram's method. Upon gelatin plates a remarkable similarity to the FlG. 98. — Spirillum Metschnikowi ; puncture-culture in gelatin forty-eight hours old (Frankel and Pfeiffer). colonies of the cholera spirillum' is developed, yet there is a difference, and Pfeiffer points out that "it is com- paratively easy to differentiate between a plate of pure cholera spirillum and a plate of pure Spirillum Metsch- nikowi, yet it is almost impossible to pick out a few colo- nies of the latter if mixed upon a plate with the former." Frankel regards this bacterium as a kind of interme- 444 PATHOGENIC BACTERIA. diate species between the cholera and the Fiukler-Prior spirilla. The colonies upon gelatin plates appear in about twelve hours as small whitish points, and rapidly develop, so that by the end of the third day large saucer-shaped areas of liquefaction resembling colonies of the Finkler-Prior spirilla occur. The liquefaction of the gelatin is quite rapid, the resulting fluid being turbid. Generally there will be upon a plate of Vibrio Metschnikowi some colo- nies which closely resemble cholera by occupying small conical depressions in the gelatin. Under a high power of the microscope the contents of the colonies, which ap- pear to be of a brownish color, are observed to be in rapid motion. The edges of the bacterial mass are fringed with radiating organisms (Fig. 98). In gelatin tubes the culture is very much like that of cholera, but develops more slowly. Upon the surface of agar-agar a yellowish-brown growth develops along the whole line of inoculation. On potato at the room-temperature no growth occurs, but at the temperature of the incubator a luxuriant yellowish-brown growth takes place. Sometimes the color is quite dark, and chocolate-colored potato cultures are not uncommon. In bouillon the growth which occurs at the tempera- ture of the incubator is quite characteristic, and very different from that of the cholera spirillum. The entire medium becomes clouded, of a grayish-white color, and opaque. A folded and wrinkled mycoderma forms upon the surface. When glucose is added to the bouillon no fermentation or gas-production results. When grown in litmus milk the original blue color is changed to pink in a day, and at the end of another day the color is all destroyed and the milk coagulated. Ulti- mately the clots of casein sediment in irregular masses, and clear colorless whey is supernatant. The addition of sulphuric acid to a culture grown in a SPIRILLA RESEMBLING CHOLERA. 445 medium rich in peptone produces the same rose color observed in cholera cultivations. The organism is pathogenic for animals, but not for man. Pfeiffer has shown that chickens, pigeons, and guinea-pigs are highly susceptible animals. The birds when inoculated under the skin generally die — pigeons always. W. Rindfleish has pointed out that this positive fatal outcome of the introduction of the spirillum into pigeons makes it a valuable diagnostic point for the differentiation of this spirillum from that of cholera. According to his researches, the simple subcutaneous in- jection of the most virulent cholera cultures is never fatal to pigeons. The birds only die when the injections are made into the muscles in such a manner that the muscular tissue is injured and becomes a locus 7tiinoris resistentice. When guinea-pigs are treated according to the method of Koch for the inoculation of cholera, the temperature of the animal rises for a short time, then abruptly falls to 330 C. or less. Death follows in twenty to twenty-four hours. A distinct inflammation of the intestine, with exudate and numerous spirilla, may be found. The spirilla can also be found in the heart's blood and in the organs of such guinea-pigs. When the bacilli are introduced by subcutaneous inoculation, the autopsy shows a bloody edema and a superficial necrosis of the tissues. In the blood and all the organs of pigeons and young chickens the organisms can be found in such large num- bers that Pfeiffer has suggested the term ' ' vibrionensep- ticsemia" for the condition. In the intestines very few al- terations are noticeable, and very few spirilla can be found. Gainal£ia has shown that pigeons and guinea-pigs can be made immune by inoculating them with cultures ster- ilized for a time at a temperature of ioo° C. Mice and rabbits are immune except to very large doses. Spirillum Berolinensis. — This organism (Fig. 99), which was discovered by Neisser1 in the summer of 1893, 1 Hygienische Rundschau, 1 893. 446 PATHOGENIC BACTERIA. is of great interest in comparison with the spirillum of cholera and its related forms. Its morphology is in every particular exactly like that of the cholera spirillum, but its growth is a little more rapid. It grows upon the same culture-media and at the same temperature. The colonies are, however, quite different. Upon the second day, when grown upon gelatin plates, the colonies of the Spirillum Berolinensis appear finely granular and paler than those of cholera. The borders are generally smooth and circular. As it be- comes older the colony takes on a slightly brownish color, and may be nodulated or radiately lobulated. The gelatin is very slowly liquefied. *<'*&. Fig. 99. — Spirillum Berolinensis, from an agar-agar culture; x 1 000 (Itzerott and Niemann). In puncture-cultures the development takes place along the entire puncture, and causes a gradual liquefaction of the gelatin. Upon agar-agar the growth is generally similar to that of the cholera spirillum, but at times is copious, dry, and ragged, and suggests leather by its appearance. When introduced intraperitoneally into guinea-pigs the animals die in from one to two days. The indol reaction is exactly like that given by cul- SPIRILLA RESEMBLING CHOLERA. 447 tares of the cholera spirillum. The spirillum does not stain by Gram's method. Spirillum Dunbar. — This organism (Fig. 100) was de- ■a 'jr.-* '«V „ -*• Fig. 100. — Spirillum Dunbar, from agar-agar; x 1 000 (Itzerott and Niemann). scribed in 1893 by Dunbar and Oergel, who secured it from the water of the Elbe River. It much resembles the cholera spirillum, but it never exhibits sigmoid forms. It stains poorly, the ends taking the color much better than the central portion. Gelatin is liquefied by the growth of this organism more quickly than by the cholera spirillum. The colo- nies upon gelatin and the puncture-cultures in gelatin are identical with those of the cholera spirillum. On agar-agar a luxuriant whitish-yellow layer is pro- duced. In bouillon and peptone solution the addition of dilute sulphuric acid produces the red color of nitro-indol. It is said that cultures grown at a temperature of 22° C. phosphoresce in the dark. The spirillum seems to be pathogenic for guinea-pigs when introduced into the stomach according to Koch's method for cholera. Spirillum Danubicus. — This organism (Fig. 101) also 448 PATHOGENIC BACTERIA. much resembles cholera. It was first isolated by Heider l in 1892. In appearance it is rather delicate and decidedly curved. It is often united in sigmoid and semicircular forms, and exhibits long spirals in old cultures. It is actively motile, each organism presenting a terminal flagellum. The growth upon gelatin plates is rapid. Small light- gray colonies, resembling those of cholera, but exhibit- ing a dentate margin, are observed. The growth in gelatin punctures also much resembles cholera, and the agar-agar growth can scarcely be distinguished from it. The potato growth has a distinct yellowish-brown color. Milk is coagulated in three or four days. A\> XT' f i ^- ) . .. " Fig. ioi. — Spirillum Danubicus, from an agar-agar culture; x 1000 (Itzerott and Niemann). This spirillum does not produce indol. Heider found the spirillum pathogenic for guinea-pigs. Spirillum I. of Wernicke. — This organism is about twice as large as the cholera spirillum, liquefies gelatin more rapidly, produces indol, and is feebly pathogenic for guinea-pigs. Spirillum II. of Wernicke. — This spirillum is smaller than the cholera spirillum, liquefies gelatin more slowly, 1 Centralbl. f. Bakt. u. Parasitenk., xiv., 341. SPIRILLA RESEMBLING CHOLERA. 449 produces indol, and is highly pathogenic for rabbits, guinea-pigs, pigeons, and mice. Spirillum Bonhoffi. — This organism (Fig. 102) was found in water by Bonhoff. It has a decided resem- FlG. 102. — Spirillum Bonhoffi, from a culture upon agar-agar ; x IOOO (Itzerott and Niemann). blance to the cholera spirillum, but is rather stouter and less curved. Curved forms — i. e. semicircles, sig- moids, and spirals — occur in old cultures especially. These organisms are colored badly with ordinary stains, dahlia seeming to be the most appropriate color, and ac- complishing the process better if warmed. The organ- ism is motile, and has a long flagellum attached to one end. The colonies develop slowly upon gelatin plates, first appearing in forty-eight hours as little grayish points. The margin of the colony is sharply circumscribed ; the interior is broken up. The gelatin is not liquefied. In gelatin punctures there is no liquefaction observable. Upon agar-agar the development at the temperature of the incubator, which is more rapid than that at the temperature of the room, results in the production of a bluish-gray layer. The growth upon potato has a brownish color. The 29 45° PATHOGENIC BACTERIA. growth in bouillon and in peptone solutions is accompa- nied by the production of indol. The spirillum is pathogenic for mice, guinea-pigs, and canary birds. Spirillum Weibeli. — This spirillum (Fig. 103) was found in 1892 by Weibel in spring-water which had a long time Fig. 103. — Spirillum Weibeli, from agar-agar; x iooo (Itzerott and Niemann). before been infected by cholera. It is short, rather thick, and distinctly bent, often forming S-shaped figures. The colonies before liquefaction sets in are described as pale-brown, transparent, circular, and homogeneous. Liquefaction is much more rapid than in cholera, and causes the borders of the colonies to become irregular. In the centre of each colony a little depression is ob- served. In gelatin puncture-cultures the growth is rapid, be- ginning first upon the surface, where a large flat, saucer- shaped liquefaction, extending to the sides of the tube, forms. Scarcely any growth takes place in the puncture, but the superficial liquefaction, separated by a horizontal line from the normal gelatin, descends slowly. Upon agar-agar a grayish-white layer is formed. No growth has been obtained upon potato. SPIRILLA RESEMBLING CHOLERA. 451 In alkaline peptone solution a slow but luxuriant growth takes place. Spirillum Milleri. — This spirillum (Fig. 104) was found in the mouth by Miller in 1885. It resembles the cholera 1 *% / FlC 104. — Spirillum Milleri, from an agar-agar culture; x 1000 (Itzerott and Niemann). spirillum somewhat, but is much more like the spirillum of Finkler and Prior, with which many bacteriologists think it identical. Upon gelatin the colonies are small, finely granular, have a narrow border-zone and a pale-brown color. The gelatin is rapidly liquefied. Upon agar-agar a thick yellowish layer is produced. The organism seems not to be pathogenic. Spirillum Aquatilis. — Giinther in 1892 found this or- ganism (Fig. 105) in the water of the river Spree. It is similar to the cholera spirillum in shape, has a loug terminal flagellum, and is motile. The colonies which form upon gelatin are circular, have smooth borders, and look very much as if bored out with a tool. They have a brown color and are finely granular. In gelatin puncture-cultures the growth occurs almost exclusively at the surface. The agar-agar cultures are similar to those of cholera. Scarcely any development occurs in bouillon. By the 452 PATHOGENIC BACTERIA. growth of the organism sulphuretted hydrogen gas is produced. The spirillum does not grow at all upon potato. Giinther did not find the organism to be pathogenic. Spirillum Terrigenus. — This species, also discovered by Giinther, was secured from earth. It generally occurs in a slightly curved form, but sometimes is spiral. It is actively motile and has a terminal flagellum. The colonies, which appear in twenty.-four hours, are small, structureless, and transparent, and later take on a ' ' fat-drop ' ' appearance. Upon agar-agar a thin white coating is formed. Milk is coagulated by the growth of the organism. No indol is produced. The organism does not stain by Gram's method, and is said not to be pathogenic for guinea-pigs or for mice. Fig. 105.— Spirillum aquatilis, from an agar-agar culture ; x 1000 (Itzerott and Niemann). Vibrio Schuylkiliensis. — This form, closely resembling the cholera spirillum, was found by Abbott1 in sewage- polluted water from the Schuylkill River at Philadelphia. The colonies upon gelatin plates resemble very closely those of Spirillum Metschnikovi. In gelatin puncture- cultures the appearance is exactly like the true cholera spir- 1 Jour, of Exper. Med., vol. i., No. 3, July, 1896, p. 419. SPIRILLA RESEMBLING CHOLERA. 453 ilium. At times the growth may be a little more rapid. The growth on agar is very luxuriant, and gives off a pronounced odor of indol. Loffler's blood-serum is ap- parently not a perfectly adapted medium, but upon it the organisms grow, with resulting liquefaction. Upon po- tato at the point of inoculation there is a thin, glazed, more or less dirty yellow, shading to brownish deposit that is sometimes surrounded by a flat, dry, lusterless zone. In litmus milk a slightly reddish tinge is found after twenty-four hours at body temperature. After forty-eight hours this is increased and the milk is coagulated. In peptone solutions indol is produced. No gas is pro- duced in glucose-containing culture-media. The organ- ism is a facultative anaerobic spirillum. The thermal death-point is 500 C. for five minutes. The organism is pathogenic for pigeons, guinea-pigs, and mice. The pathogenesis is much like that of the Spirillum Metschnikovi. No Pfeiffer's phenomenon was observed with the use of the serum of immunized ani- mals. Immunity was produced in pigeons, and it was found that their serum was protective against both the Vibrio Schuylkiliensis and Spirillum Metschnikovi, the immun- ity thus produced being of about ten days' duration. In a second paper by Abbott and Bergy x it was shown that the vibrios were found in river water during all four seasons of the year, and in all parts of the river within the city, both at low and at high tide. They were also found in the sewage emptying into the river. The spirilla were also found in the water of the Delaware River as frequently as in that from the Schuylkill. One hundred and ten pure cultures of spirilla were iso- lated from the sources mentioned and subjected to routine tests. It was found that few or none of them were iden- tical in all points. There seems, therefore, to be a family of river spirilla related to each other like the different colon bacilli are related. 1 Journal of Experimental Medicine, vol. ii., No. 5, p. 535. 454 PATHOGENIC BACTERIA. The opinion of the writers is that "the only trust- worthy difference between many of these varieties and the true cholera spirillum is the specific reaction with serum from animals immune from cholera, or by Pfeiffer's method of intraperitoneal testing in such animals." In discussing these spirilla of the Philadelphia waters Bergy l says: "The most important point with regard to the occur- rence of these organisms in the river water around Phil- adelphia, is the fact that similar organisms have been found in the surface-waters of the European cities in which there had recently been an epidemic of Asiatic cholera, notably at Hamburg and Altona. . . . The fore- most bacteriologists of Europe have been inclined to the opinion that the organisms which they found in the sur- face-waters of the European cities were the remains of the true cholera organism, and that the deviations in the morphologic and biologic characters from those of the cholera organism were brought about by their prolonged existence in water. No such explanation of the occur- rence of the organisms in Philadelphia waters can be given." 1 Jour, of the Amer. Med. Assoc, Oct. 23, 1897. C. THE BACTEREMIAS. CHAPTER I. ANTHRAX. Bacillus Anthracis. The disease of cattle known as anthrax or "splenic fever" is of infrequent occurrence in this country and in England. In France, Germany, Hungary, Russia, Persia, and the East Indian countries it is a dreaded and common malady which robs herdsmen of many of their valuable stock. Siberia perhaps suffers most, the disease being so exceedingly common and malignant as to deserve the name "Siberian pest." Certain local areas, such as the Tyrol and Auvergne, in which it seems to be constantly present, serve as distributing foci from which the disease spreads rapidly in summer, afflicting many animals, and ceasing its depredations only with the advent of winter. It seems to be distinctly a disease of the summer season. The animals most frequently affected are cows and sheep. Among our laboratory animals white mice, house-mice, guinea-pigs, and rabbits are highly suscepti- ble ; dogs, cats, most birds, and amphibians are almost perfectly immune. White rats are infected with difficulty. Man is only slightly susceptible, the manifestation of the disease as seen in the human species being different from the same disease in the lower animals in that it is usually a local affection — malignant carbuncle — and only at times gives rise to a general infection. Anthrax was one of the first of the specific diseases proven to be caused by a definite micro-organism. As early as 1849, Pollender discovered small rod-shaped bodies in the blood of animals suffering from anthrax, but the exact relation which they bore to the disease was 455 456 PATHOGENIC BACTERIA. not pointed out until 1863, when Davaine,1 by a series of interesting experiments, proved to most unbiased minds their etiological significance. The further confirmation of Davaine' s conclusions and actual proof of the matter rested with Pasteur and Koch, who, observing that the bacilli bore spores, cultivated them successfully outside the body, and then produced the disease by the inocula- tion of pure cultures. The anthrax bacilli (Fig. 106) are large rods with a Fig. 106. — Bacillus anthracis : colony three days old upon a gelatin plate; ad- hesive preparation ; x 1000 (Frankel and Pfeiffer). rectangular form, caused by the very slight rounding of the corners. They measure 5-20 // in length and are from 1 n to 1.25 /i in breadth. The pronounced tendency is toward the formation of long threads, in which, how- ever, the individuals can generally be made out ; at times isolated rods occur. In the threads the bacilli seem en- larged a little at the ends, and give somewhat the appear- ance of a bamboo cane. The formation of spores is pro- lific : each spore has a distinct oval shape, is transparent, and does not alter the contour of the bacillus in which it occurs. Spores are generally formed in the presence of 1 Compt. rendu, 57, 59-61, 77. ANTHRAX. 457 oxygen upon the surfaces of the culture-media. When a spore is placed under favorable conditions for its devel- opment and is carefully watched, it may be observed to increase in length a trifle, then to undergo a rupture at one end, from which the new bacillus projects. The spores of anthrax (Fig. 107), being large and easily ob- FlG. 107. — Bacillus anthracis, stained to show the spores; x icoo (Tr'ankel and Pfeiffer). tainable, are excellent subjects for the study of sporula- tion, for the action of germicides and antiseptics, and for demonstration by stains. When dried upon threads of silk they will retain their vitality for several years, and are highly resistant to heat and disinfectants. Spores of anthrax are killed by five minutes' exposure to a temperature of ioo° C. It is said by some that spores subjected to 5 per cent, carbolic acid can germinate when introduced into susceptible animals, their resist- ance to this strength carbolic solution being so remarkable that they are not usually destroyed by it under twenty- four hours. Spores are also killed in a short time by- exposure to 1 : 1000 bichlorid-of-mercury solution. The bacilli are not motile and are not provided with flagella. They stain well with ordinary solutions of the 458 PATHOGENIC BACTERIA. anilin dyes, and can be beautifully demonstrated in the tissues by Gram's method and by Weigert's fibrin method. Picro-carmin, followed by Grain's method, gives a beauti- ful, clear picture. The spores can be stained with carbol- fuchsin, the bacilli decolorized with a very weak acid and then counter-stained with a watery solution of methyl blue. Upon the surface of gelatin plate-cultures the bacillus forms beautiful and highly characteristic colonies (Fig. 108). To the naked eye they appear first as minute Fig. 108. — Bacillus anthracis: colony upon a gelatin plate ; x ioo (Frankel and Pfeiffer). round whitish dots occurring upon the surface, and caus- ing liquefaction of the gelatin as they increase in size. Under the microscope they can be seen in the gelatin as egg-shaped, slightly brownish granular bodies, not attain- ing their full development except upon the surface, where they spread out into flat, irregular, transparent growths bearing a partial resemblance to tufts of curled wool. From a tangled centre large numbers of curls extend, each made up of parallel threads of bacilli. As soon as the colony attains any considerable size liquefaction be- gins. These colonies make beautiful adhesive prepara- tions. If a perfectly clean cover-glass be passed once ANTHRAX. 459 through a flame and laid carefully upon the gelatin, the colonies can generally be picked up entire when the glass is removed. Such a specimen can be dried, fixed, and stained in the same manner as an ordinary cover-glass preparation. In gelatin puncture-cultures the growth is even more characteristic than are the colonies. The bacilli begin to grow along the entire track of the wire, most luxuri- antly at the surface, where oxygen is plentiful. As the growth progresses fine filaments like bristles, extend from the puncture into the neighboring gelatin giving the growth somewhat the appearance of an evergreen tree inverted (Fig. 109). Fig. 109. — Bacillus anthracis : gelatin puncture-culture seven days old (Gunther). The more superficial of these threads reach about half- way to the sides of the tube, while the deeper ones are shorter and shorter, until near the apex branches cease. When the projections are pretty well developed a distinct surface-growth will be discerned, and if the tube be tilted, one can observe that the gelatin beneath it has liquefied. As the growth becomes older the liquefaction increases, until ultimately the entire gelatin is fluid and the growth is precipitated. Upon agar-agar the characteristics are few. The growth takes place all along the line of inoculation as a slightly translucent, slightly wrinkled layer with irreg- 46° PATHOGENIC BACTERIA. ular edges, from which sufficient bacillary threads pro- ject to give it a ciliated appearance to the naked eye. When the culture is old the agar-agar turns a distinct brown. Spore-formation is luxuriant upon agar-agar. On potato the growth is white, creamy, sometimes rather dry in appearance. Sporulation is marked. Blood-serum cultures lack peculiarities ; the culture- medium is slowly liquefied. The bacillus only grows between the extremes of 200 and 45° C, best at 2,7° C. The exposure of the organ- ism to the temperature of 42-430 C. for twenty-four hours is sufficient to destroy its virulence. The culture-media should always be faintly alkaline, as anthrax bacilli will not grow in the presence of free acid. The micro-organism under consideration is a parasitic microbe, yet is one which, because of its spores, can, in a latent form, exist without the animal organism until appropriate conditions for its natural development are presented. Ordinarily, the infection takes place either through the respiratory tract or through the alimentary canal. Buchner has shown that when animals are allowed to inhale anthrax spores they die of typical anthrax. The spores establish themselves in the alveoli of the lung, penetrate the epithelium, enter the vascular sys- tem, and soon give rise to typical lesions. Strange to say, the appearance caused by the inhalation of the bacilli in their perfect form is entirely different, for a rapid multiplication occurs without sporulation, and causes a violent irritative pneumonia with serous or sero- fibrinous exudate in which large numbers of the bacilli occur. In these cases there may be no general infection. When the bacilli are taken into the stomach in food they meet with a rapid death because of the acidity of the gastric juice. Should spores, however, be ingested, they are able to endure the gastric juice, to pass into the intestine, and, as soon as proper conditions of alkalinity are encountered, to develop into bacilli. They develop ANTHRAX. 461 rather rapidly, surround the villi with thick networks of bacillary threads, separate the epithelial cells, enter the lymphatics, and thus find the appropriate environ- ment for the production of a general infection. Sometimes the bacillus enters the body through a wound, cut, scratch, or fly-bite. This is especially the case with men who come in contact with diseased cattle. As has already been pointed out, a malignant pustule is apt to follow, and may cause death. Men whose occupations bring them in contact with skins and hair from animals dead of anthrax are not only liable to wound-infection, but are sometimes the subjects of a pul- monary form of the disease — "wool-sorter's disease" — caused by inspiration of the spores attached to the wool. The disease as we see it in the laboratory is accom- panied by few but marked lesions. The ordinary method of inoculation is to cut away a little of the hair from the abdomen of a guinea-pig or rabbit or the root of a mouse's tail, make a little subcutaneous pocket with a snip of a pair of sterile scissors, and introduce the spores or bacilli from a pure culture upon a rather heavy platinum wire, the end of which is flattened, pointed, and perforated. An animal inoculated in this way gen- erally dies, according to the species, in from twenty-four hours to three days. The symptoms are weakness, fever, loss of appetite, and sometimes a bloody discharge from nose and bowels. There is much subcutaneous edema. At the autopsy very little change is observed at the seat of inoculation. The subcutaneous tissue beneath it for a considerable distance around is occupied by a peculiar colorless gelatinous edema which contains the bacilli. The abdominal cavity shows injection and congestion of its viscera. The spleen is considerably enlarged, is dark in color, and of mushy consistence. The liver is somewhat enlarged. When the thorax is opened, the lungs may be slightly congested, but otherwise no changes are to be found. When the various organs, which present no appreciable 462 PATHOGENIC BACTERIA. changes to the naked eye, are subjected to a microscopic examination, the appropriate staining methods bring out a most remarkable and beautiful change. The capil- lary system is almost universally occupied by bacilli, which extend throughout its meshworks in long threads. Most beautiful bundles of these bacillary threads can, at times, be found in the glomeruli of the kidney and in the minute capillaries of the intestinal villi. In the larger vessels, where the blood-stream is rapid, the bac- teria are relatively few, so that the burden of bacillary obstruction is borne by the minute vessels. The con- dition is thus one of pure septicemia, and bacilli can be secured in pure cultures from the blood and tissues. Death from anthrax seems to depend essentially upon the obstruction to the circulation caused by the multi- tudes of bacilli in the capillaries, and upon the appro- priation of the oxygen by the bacilli, leaving the tissues to be poisoned by the carbon dioxid. It is very questionable whether the anthrax bacillus produces a toxic substance or not. Hoffa l isolated a basic substance from anthrax cultures and called it anthracin. Haukin and Westbrook2 found an albumose fatal in laro-e doses, and immunizing in small ones. Brieger and Frankel3 isolated a toxalbumin from the tissues of animals dead of anthrax ; Martin4 separated protalbu- mose, deuteroalbumose, peptone, an alkaloid, leucin, and tyrosin. The albumoses were not very poisonous, but the alkaloid was capable of producing death after the development of somnolence. The animals were edema- tous. Marmier5 thought he had isolated a toxin of non- albuminous nature and immunizing power. The most recent thorough work upon the subject is that of Con- radi,6 who in an elaborate research failed to find that the 1 Ueber die Natur. des Milzbrandgifts, Wiesbaden, 1886. 2 Ann. de T Inst. Past., 1892. No. 9. s Ueber Ptomaine, Berlin, 1885-1886. 4 Proceedings of the Royal Society, May 22, 1890. 5 Ann. de I Inst. Past., 1895, p. 533. 6 Zeitschrift fur Hyg., June 14, 1899. ANTHRAX. 463 anthrax bacillus produces any soluble extracellular or intracellular poison capable of affecting susceptible ani- mals, and concludes that it is highly improbable that the anthrax bacillus produces any toxic substance at all. The susceptibility of the anthrax bacillus to the influ- ence of heat, cold, antiseptics, etc. not only permitted Buchner, Behring, and others to produce biological curi- osities in the form of bacilli unable to bear spores and robbed of their pathogenic powers, but also suggested to Pasteur the important practical measure of protective vaccination. Pasteur found that the inoculation of non- virulent bacilli into cows and sheep, and their reinocula- tion with slightly virulent bacilli, gave them the ability to withstand the action of highly virulent organisms. Loffler, Koch, and Gaffky, however, found that these immunized animals were not absolutely protected from intestinal anthrax. The methods of diminishing the virulence of the anthrax bacilli are numerous. Toussaint, who was cer- tainly the first to produce immunity in animals by inject- ing them with sterile cultures of the bacillus, found that the addition of 1 per cent, of carbolic acid to blood of animals dead of anthrax destroyed the virulence of the bacilli ; Chamberland and Roux found it removed when 0.1-0.2 per cent, of bichromate of potassium was added to the culture-medium ; Chauveau used atmospheric pressure to the extent of six to eight atmospheres and found the virulence diminished ; Arloing found that direct sunlight operated similarly ; Lubarsch found that the inoculation of the bacilli into immune animals, such as the froe, and their subsequent recovery from its blood, diminishes the virulence markedly. Protection can be afforded in still other ways. The simultaneous inoculation of bacteria not at all related to anthrax will sometimes recover the animal, as Hiippe found. Hankin found in the cultures chemical sub- stances, especially an albuminose, which exerted a pro- tective influence. Chamberland has shown that pro- 464 PATHOGENIC BACTERIA. tective inoculation by Pasteur's method has diminished the death-rate from 10 per cent, for sheep and 5 per cent, for cattle to about 0.94 per cent, for sheep and 0.34 per cent, for cattle, so that the utility of the method is scarcely questionable. In 1890, Ogata and Jasuhara showed that in the convalescents from anthrax among their experimental animals an antitoxic substance was present in the blood in such quantities that 1 : 800 parts per body-weight of dog's serum containing the antitoxin would protect a mouse. Similar results have been at- tained by Marchoux. Experiments of interest have been performed to show that the natural immunity enjoyed by many animals can be destroyed. Behring found that if the alkalinity of the blood of rats was diminished, they could become affected with anthrax, and numerous observers have shown that when anthrax bacilli and unrelated organisms, such as the erysipelas cocci, Bacillus prodigiosus, and Bacillus pyocyaneus, are simultaneously introduced into immune animals, the immunity is destroyed and the animals succumb to the disease. Frogs have been made to suc- cumb to the disease by exposure to a temperature of 370 C. after inoculation. Pasteur destroyed the immunity of fowls by a cold bath after inoculation. In the natural order of events anthrax in cattle is probably the result of the inhalation or ingestion of the spores of the bacilli from the pasture. At one time much discussion arose concerning the infection of the pasture. It was argued that, the bacilli being enclosed in the tissues of the diseased animals, the infection of the pasture must be due to the distribution of the germs from the buried cadaver to all parts of the field, either through the activity of earth-worms, which ate of the earth surrounding the corpse and then deposited the spores in their excrement at remote areas (Pasteur), or to currents of moisture in the soil. Koch seems, however, to have demonstrated the fallacy of the theories by show- ing that the conditions under which the bacilli find them- ANTHRAX. 465 selves in buried cadavers are exactly opposed to those favorable to fructification or sporulation, and that in all probability the majority of bacteria suffer the same fate as the animal cells, and disintegrate, especially if the ani- mal be buried at a depth of two or three meters. Frankel points out particularly that no infection of the soil by the dead animal could be worse than the pollution of its surface by the bloody stools and urine, rich in bacilli, discharged upon it by the animal before death, and that in all probability it is the live, and not the dead, animals that are to be blamed as sources of infection. As every animal affected with anthrax is a source of danger to the community in which it lives, to the men who handle it as well as the animals who browse beside it, such animals, as soon as the diagnosis is made, should be killed, and, together with the hair and skin, be burned. When this is impracticable, Frankel recommends that they be buried to a depth of at least 1^2-2 meters, so that the sporulation of the bacilli is impossible. The dejecta should also be carefully disinfected with 5 per cent, carbolic-acid solution. Of course, animals can be infected through wounds. This mode of infection is, however, more common among men, who suffer from the local disease mani- fested as the malignant carbuncle, than among animals. Bacilli Resembling the Anthrax Bacillus. — Oc- casionally bacilli are encountered presenting all the mor- phological and cultural characteristics of the anthrax bacillus, but devoid of any disease-producing power — Bacillus anthracoides of Hiippe and Wood,1 Bacillus anthracis similis of McFarland,2 and Bacillus pseudo- anthracis,3 etc. Exactly what relation they may bear to the anthrax bacillus is uncertain. They may be entirely different organisms, or they may be individuals whose pathogeny has been lost through unfavorable environment. 1 Berliner klin. Wochenschrift, 1889, 16. * Centralbl.f. Bakt., vol. xxiv., No. 26, p. 556. 3 Hygienische Rundschau, 1894, No. 8. 30 CHAPTER II. TYPHOID FEVER. Bacillus Typhosus (Eberth^Gaffky2). The bacillus of typhoid fever {Bacillus typhi abdomi- nalis [Fig. no] or Bacillus typhosus) was discovered by Eberth and Koch 3 in 1880, and was first secured in Pig. iio. — Bacillus typhi abdominalis, from a twenty-four-hours-old agar-agar culture; x 650 (Heim). pure culture from the spleen and affected lymphatic glands by Gaffky four years later. The organism is a small, short bacillus about 1-3 a (2-4//- Chantemesse, Widal) in length and 0.5-0.8/* broad (Sternberg). The ends are rounded, and it is rather ex- ceptional for the bacilli to be united in chains, though this arrangement is common in potato cultures. The size and morphology vary distinctly with the nature of the culture-medium and the age of the culture. Thoinot and Masselin 4 in describing these morphological peculi- 1 Virchow's Archiv, 1881 and 1883. ' Mittheilungen aus dem Kaiserl. Gesundheitsamts, 2. 3 Ibid., I, 45. * Prtcis de Microbie, Paris, 1893. 466 TYPHOID FEVER. 467 arities mention that when grown in bouillon it is a very slender bacillus ; in milk it is a large bacillus ; upon agar-agar and potato it is very thick and short ; and in old gelatin cultures it forms very long filaments. The organisms are actively motile, the motility prob- ably being caused by the numerous flagella with which the bacilli are provided. The flagella stain well by Loffler's method, and, as they are numerous (ten to twenty) and readily demonstrable, the typhoid bacillus is the favorite subject for their study. The movements of Fig. hi. — Bacillus typhi abdominahs, from an agar-agar culture six hours old, showing the flagella stained by Loffler's method; x 1000 (Krankel and Pfeiffer). the short bacilli are oscillating, those of the longer indi- viduals serpentine. The organism stains quite well by the ordinary meth- ods, but loses the color entirely when stained by Gram's method. A peculiarity of the bacillus is the readiness with which it gives up its color in the presence of solvents, so that it is particularly difficult to stain it in tissue. When sections are to be stained the best method is to allow the tissue to remain in Loffler's alkaline methylene- 468 pa THOGENIC BACTERIA. blue for from fifteen minutes to twenty-four hours, then wash in water, dehydrate rapidly in alcohol, clear up in xylol, and mount in Canada balsam. Ziehl's method also gives good results. The sections are stained for fif- teen minutes in a solution of distilled water ioo, fuch- sin i, and phenol 5. After staining they are washed in distilled water containing 1 per cent, of acetic acid, dehydrated in alcohol, cleared, and mounted. In such preparations the bacilli may be found in little groups, which are easily discovered, under a low power of the microscope, as reddish specks, and readily resolved into bacilli with the high power of the oil-immersion lens. In bacilli stained by this alkaline methylene-blue solu- tion dark-colored dots may sometimes be observed near the ends of the rods. These dots were at first regarded as spores, but are now denominated polar granules, and are thought to be of no importance. The typhoid bacillus is both saprophytic and parasitic. It finds abundant conditions in nature for its growth and development, and, enjoying strong resisting powers, can accommodate itself to environment much better than the majority of pathogenic bacteria, and can be found in water, air, soiled clothing, dust, sewage, milk, etc. con- taminated directly or indirectly by the intestinal dis- charges of diseased persons. The bacillus is also occasionally present upon green vegetables sprinkled with water containing it, and epi- demics are reported in which the infection was traced to oysters from a certain place where the water was infected through sewage. Newsholme1 found that in 56 cases of typhoid fever about one-third was attributable to the eating of raw shell-fish. In such cases the evidence accumulated serves to show that the shell-fish were from sewage-polluted beds. The bacillus probably enters milk occasionally in water used to dilute it or to wash the cans. 1 Brit. Med. Jour., Jan., 1895. TYPHOID FEVER. 469 The resisting powers of the organism have already been described as great. It can grow well at the room- temperatnre. The thermal death-point is given by Stern- berg as 6o° C. The bacilli can, according to Klemperer and Levy,1 remain vital for three months in distilled water, though in ordinary water the commoner and more vigorous saprophytes outgrow them and cause their dis- appearance in a few days. When buried in the upper layers of the soil the bacilli retain their vitality for nearly six months. Robertson 2 found that when planted in soil and occasionally fed by pouring bouillon upon the surface, the typhoid bacillus maintained its vitality for twelve months. He suggests that it may do the same in connection with leaky drains. Cold has no effect upon typhoid bacilli, for freezing and thawing several times are without injury to them. They have been found to remain alive upon linen for from sixty to seventy-two days, and upon buckskin for from eighty to eighty-five days. Sternberg has succeeded in keeping hermetically sealed bouillon cultures alive for more than a year. In the presence of chemical agents the bacillus is also able to retain its vitality, o. 1 to 0.2, per cent, of carbolic acid added to the culture-media being without effect upon its growth. At one time the tolerance to carbolic acid was thought to be character- istic, but it is now known to be shared by other bacteria. The bacilli seem to be killed in a short time by thorough drying. The bacillus is best secured in pure culture, either from an enlarged lymphatic gland or from the splenic pulp of a case of typhoid. To secure the bacillus in this way the autopsy should be made as soon after death as possible, lest the bacillus coli invade the tissue. Cultures of the typhoid bacillus may be obtained, but with difficulty, from the alvine discharges of typhoid patients. In examining this material, however, it must 1 Clinical Bacteriology. * Brit. Med. Jour., Jan. 8, 1898. 470 PATHOGENIC BACTERIA. be remembered that the bacilli are certain to be present only in the second and third weeks. As numerous saprophytic bacteria are present in the feces, the resistance which the typhoid bacillus exhibits to carbolic acid can be made use of in obtaining the pure culture. To each of several tubes of melted gelatin 0.05 per cent, of carbolic acid is added. This addition is most easily calculated by supposing the average amount of gelatin contained in a tube to be 10 c.cm. To the aver- age tube y1^- c.cm. of a 5 per cent, solution of carbolic acid is added, and gives very nearly the desired quantity. A minute portion of the feces is broken up with a platinum loop and stirred in the tube of melted gelatin ; a drop from this dilution is transferred to the second tube, a drop from it to a third, and then the contents of each tube are poured upon a sterile plate or into a Petri dish, Fig. 112. — Bacillus typhi abdominalis : superficial colony two days old, as seen upon the surface of a gelatin plate; x 20 (Heim). or rolled, according to Esmarch's plan, in the manner already described. The carbolic acid present in these cases prevents the great mass of saprophytes from de- veloping, but allows the perfect development of the typhoid bacillus (Fig. 112) and its near congener, the Bacillus coli communis (Fig. 115). The deep colonies that develop upon such gelatin plate- TYPHOID FEVER. 471 cultures are seen under the microscope to be brownish- yellow in color, spindle-shaped, and sharply circum- scribed. When superficial they are larger and form a bluish iridescent layer with notched edges. These colo- nies are often described as resembling grape-vine leaves. The center of the superficial colonies is the only portion which shows the yellowish-brown color. The margins of the colony appear somewhat reticulated. The gelatin is not liquefied. Unfortunately, the appearances of the colonies of the typhoid bacillus and the colon bacillus are identical, and make it impossible to select a single colony of either with certainty. The only solution of the problem is to transfer a large number of colonies to some culture- medium in which a characteristic of one or the other species is manifested, and then study the growth ; or to grow the colonies upon some special medium in which differences, such as rapidity of growth or acid-produc- tion, etc., cause the colonies of the different species to assume characteristic appearances. A method recently suggested by Eisner1 has materially aided the separation of these allied bacteria by using a culture-medium upon which the two bacilli develop dif- ferently. The Eisner medium can be made by allowing 1 kgm. of grated potatoes (the small red German potato is best) to macerate in 1 liter of water over night. The juice is carefully pressed out, and filtered cold to get rid of as much starch as possible. The filtrate is now boiled and filtered again. The next step is a neutralization, in which Eisner used litmus as an indicator, and added 2.5- 3 c.cm. of a ^ normal solution of sodium hydrate to each 10 c.cm. of the juice. Abbott prefers to use phenol- phthalein as an indicator. The final reaction should be slightly acid. Ten per cent, of gelatin (no peptone or sodium chlorid) is now dissolved in the solution, which is boiled for the purpose, and must then be again neu- 1 Zeitschrift fiir Hygiene, xxii., Heft 1, 1895; Dec. 6, 1896. 472 PA THOGENIC BA CTERIA . tralized to the same point as before. After filtration, the medium receives the addition of i per cent, of potassium iodid. It is filled into tubes and sterilized. When water or feces suspected of containing the ty- phoid bacillus are mixed in this medium and poured upon plates, no bacteria develop well except the colon and typhoid bacilli. These two bacteria, however, differ very markedly in their appearance upon the medium, for the colon bacillus appears as usual in twenty-four hours, while at that time the typhoid bacillus, if present, will have produced no colonies discoverable by the microscope. It is only after forty-eight hours — long after the colon colonies have attained considerable size and are conspic- uous— that the little colonies of the typhoid bacillus appear as small, round, shining, dew-like points, which are finely granular and in marked contrast to their coarsely granular predecessors. Unfortunately, many of the small colonies that develop in Eisner's medium sub- sequently prove to be those of the colon bacillus. Kashida x prefers to make the differential diagnosis by observing the marked acid production of the Bacillus coli upon a medium consisting of bouillon containing i^ per cent, of agar, 2 per cent, of milk-sugar, 1.0 per cent, of urea, and 30.0 per cent, of tincture of litmus. The cul- ture-medium should be blue. When liquefied and inocu- lated with the colon bacillus, poured into Petri dishes, and stood for sixteen to eighteen hours in the incubator, the blue color passes off and the culture-medium becomes red. If a glass rod dipped in hydrochloric acid be held over the dish, vapor of ammonium chlorid is given off. The typhoid bacillus produces no acid in this medium, and there is consequently no change in its color. For the differentiation of the typhoid bacillus from the allied bacillary forms, Hiss 2 recommends the use of two special media. The first consists of 5 grams of agar-agar, 1 Centralbl. f. Bakt. u. Paristenk., Bd. xxi., Nos. 20 and 21, June 24, 1897. 2 "Journal of Experimental Medicine, Nov., 1897, vol. ii., No. 6. TYPHOID FEVER. 473 80 grams of gelatin, 5 grains of Liebig's beef-extract, 5 grams of sodium chlorid, and 10 grams of glucose to the liter. The agar is dissolved in the 1000 c.cm. of water, to which have been added the beef-extract and sodium chlorid. When the agar is completely melted the gelatin is added and thoroughly dissolved by a few minutes' boiling. The medium is then titrated to determine its reaction, phenol- phthalein being used as the indicator, and enough HC1 or NaOH added to bring it to the desired reaction — i. e. a re- action indicating 1.5 per cent, of normal acid. To the clear medium add one or two eggs, well beaten in 25 c.cm. of water; boil for forty-five minutes, and filter through a thin filter of absorbent cotton. Add the glucose after cleaning. The medium is used in tubes, in which it is planted by the ordinary puncture of the many allied forms studied. The typhoid bacillus alone has the power of cloud- ing this medium uniformly without showing streaks or gas-bubbles. The second medium is used for plating. It contains 10 grams of agar, 25 grams of gelatin, 5 grams of beef- extract, 5 grams of sodium chlorid, and 10 grams of glu- cose. The method of preparation is the same as for the tube-medium, care always being taken to add the gela- tin after the agar is thoroughly melted, so as not to alter this ingredient by prolonged exposure to high tempera- ture. This preparation should never contain less than 2 per cent, of normal acid. Of all the organisms with which Hiss experimented, the Bacillus typhosus alone displayed the power of producing thread-forming colonies upon this medium. The colonies of the typhoid bacillus when deep in the medium appear small, generally spherical, with a rough, irregular outline, and by transmitted light are of a vitreous greenish or yellowish-green color. The most character- istic feature consists of well-defined filamentous out- growths, ranging from a single thread to a complete fringe about the colony. The young colonies are, at 474 PATHOGENIC BACTERIA. times, composed solely of threads. The fringing threads generally grow out nearly at right angles to the periphery of the colony. The colonies of the colon bacillus are, on the average, larger than those of the typhoid bacillus; they are spher- ical or of a whetstone form, and by transmitted light are darker, more opaque, and less refractive than the typhoid colonies. By reflected light, to the unaided eye they are pale yellow. The surface-colonies are large, round, irreg- ularly spreading, and are brown or yellowish-brown in color. Hiss claims that by the use of these reagents the typhoid bacillus can be readily detected in typhoid stools. Piorkowski1 recommends a culture-medium composed of urine, two days old, to which 0.5 per cent, of peptone and 3.3 per cent, of gelatin have been added. When grown upon this medium, the colonies of the typhoid bacillus appear radiated and filamentous, those of the colon bacillus round, yellowish, and sharply defined at the edges. The cultures should be kept at 22° C, and the colonies should appear in twenty-four hours. When transferred to gelatin puncture-cultures the ba- cilli develop along the entire track of the wire, with the formation of minute confluent spherical colonies. A small thin whitish layer develops upon the surface near the center. The gelatin is not liquefied, but sometimes is slightly clouded in the neighborhood of the growth. The growth upon the surface of obliquely solidified gela- tin, agar-agar, or blood-serum is not very luxuriant. It forms a thin, moist, shining, translucent band with smooth edges. When a potato is inoculated and stood in the incubat- ing-oven, no growth can be detected at the end of the second day, unless the observer be skilled and the exam- ination thorough. If, however, the medium be touched with a platinum wire, it is discovered that its entire sur- face is covered with a rather thick, invisible layer of a sticky vegetation which the microscope shows to be made 1 Berliner klin. Wochenschrift, Feb. 13, 1899. TYPHOID FEVER. 475 up of bacilli. This is described as the "invisible growth." No other bacillus gives the same kind of growth upon potato. Unfortunately, it is not constant, for occasionally there will be encountered a typhoid bacillus which will show a distinct yellowish or brown- ish color. The typical growth seems to take place only when the reaction of the potato is acid. In bouillon the only change produced by the growth of the bacillus is a diffuse cloudiness. In milk a slight and slow acidity is produced. The growth in milk is not accompanied by coagulation. The typhoid bacillus does not produce indol. The chief hindrance to the ready isolation of the ty- phoid bacillus is the closely-allied Bacillus coli com- munis. This organism, being habitually present in the intestine, exists there in typhoid fever, and adds no little complication to the bacteriological diagnosis by respond- ing in exactly the same manner as the typhoid bacillus to the action of carbolic acid, by having colonies almost exactly like those of typhoid, by growing in exactly the same manner upon gelatin, agar-agar, and blood-serum, by clouding bouillon in the same way, by being of almost exactly the same shape and size, by having flagella, by being motile, and, in fact, by so many pronounced simi- larities as almost to warrant the assertion of some that it and the typhoid bacillus are identical. Not the least significant fact about the colon bacillus is that it is also pathogenic and capable of exciting acute inflammatory processes which are not infrequent, and which sometimes serve to increase the seriousness of typhoid fever. At the present time we are in more or less of a quan- dary about this extraordinary resemblance, but base our differentiation of the species upon certain constant, slight, but distinct differences (see table on p. 513). The open lymphatics and vessels of the intestinal ulcers of typhoid favor the absorption of the bacteria in the di- gestive tract, and the colon bacillus enters the blood no 476 PATHOGENIC BACTERIA. longer to be a saprophyte, but now to be a virulent pus- producer, and in many cases of typhoid we find suppura- tions and other milder inflammations due to this microbe. This is also a stumbling-block, for the typhoid bacillus when distributed through the blood may act in exactly the same manner. The typhoid bacillus may enter the body, at times, through dust (Klemperer and Levy), but no doubt, in the great majority of cases, enters the digestive tract at once through the mouth. It may possibly enter through the rectum at times. Eichhorst mentions the infection of soldiers in military barracks through the wearing of drawers previously worn by comrades who had suffered from typhoid. When ingested the resisting power of the bacillus per- mits it to pass uninjured through the acid secretions of the stomach and to enter the intestine, where the chief local disturbances are set up. The bacilli enter the solitary glands and Peyer's patches, and multiply slowly during the one to three weeks of the incubation of the disease. The immediate result of their residence in these lymphatic structures is increase in the number of cells, and ultimately the necrosis and slough- ing which cause the typical post-mortem lesion (Fig. 113). From the intestinal lymphatics the bacilli pass, in all probability, to the mesenteric glands, which become en- larged and softened, and finally extend to the spleen and liver, and sometimes to the kidneys. The growth of the bacilli in the kidneys causes the albuminuria of the dis- ease, and the bacilli are found in the urine in about 25 per cent, of the cases. Smith l found the bacilli in the urine in three out of seven cases which he investigated, Richardson 2 in nine out of thirty-eight cases. They did not occur before the third week, and remained in one case twenty-two days after cessation of the fever. Some- times they were present in immense numbers, the urine 1 Brit. Med. Jour., Feb. 13, 1897. 2 Journal of Experimental Medicine, May, 1 898. TYPHOID FEVER. 477 being actually clouded by their presence. Petruschy ' found that albuminuria sometimes occurred without the presence of typhoid bacilli, that the presence of bacilli in the urine is infrequent rather than otherwise, that the bacilli never appear in the urine in the early part of the disease, and hence are of little importance for diagnostic purposes. GwynMias found as many as 50,000,000 per can. of urine, and mentions a case of Cushing's in which the bacilli persisted in the urine for six years after the Fig. 113. — Intestinal perforation in typhoid fever. Observe the threads of tissue obstructing the opening. (Museum of the Pennsylvania Hospital.) (Keen, Surgical Complications and Sequels of Typhoid Fever.) primary attack of typhoid fever. Their occurrence, no doubt, depends primarily upon a typhoid bacteremia, by which they are brought to the kidney. After recovery from typhoid fever, their persistence in the urine prob- ably depends upon growth in the bladder. It is of im- 1 Centralbl.f. Bakt. u. Parasitenk., May 13, 1898, No. 13, p. 578. 1 Phila. Med. Jour., Mar. 3, 1900. 47^ PATHOGENIC BACTERIA. portance from a sanitary point of view to remember that the urine as well as the feces is infectious. Occasionally the bacilli succeed in entering the general circulation, and, finding a lodgement at some remote part of the body, set up local inflammatory processes sometimes terminating in suppuration. The pyogenic power of the typhoid bacillus was first pointed out by A. Frankel, who observed it in a suppu- ration that occurred four months after convalescence. Low1 found virulent typhoid bacilli in the pus of ab- scesses occurring from one to six years after convales- cence. Weichselbaum has seen general peritonitis from rupt- ure of the spleen in typhoid fever with escape of the bacilli. Otitis media, ostitis, periostitis, and osteomye- litis are very common results of the lodgement of the bacilli in bony tissue ; and Ohlmacher has found the ba- cilli in suppurations of the membranes of the brain. The bacilli are also encountered in other local suppurations occurring in or following typhoid fever. Flexner and Harris2 have seen a case in which the distribution of the bacilli was sufficiently widespread to constitute a real septicemia, the bacillus being isolated from various or- gans of the body. The bacilli are commonly found in the bile, where they sometimes persist for a long time, as in the case studied by Miller,3 when they were found in this viscus seven years after recovery from typhoid fever, dishing4 invariably found the bacilli in the bile in clumps looking like the agglutinations of theWidal reaction. He thinks it probable that these clumps form nuclei upon which bile-salts can be precipitated and calculus-formation begin. The presence of gall-stones, together with the long-lived infective agents, may at any subsequent time 1 Sitz. der K. K. Gesellschaft d. Aerzt. in Wien. Aerztl. Central- Attz., 1898, No. 3. 2 Bull. Johns Hopkins Hospital, Dec, 1897. s Ibid., May, 1898. 4 Ibid., ix., No. 86. TYPHOID FEVER. 479 provoke a cholecystitis. Cushing has collected six cases of operation for cholecystitis with calculi in which ty- phoid bacilli were present, and five in which the Bacillus coli communis was present in the gall-bladder. The bacilli can be found in the intestinal lesions, in the mesenteric glands, in the spleen, in the liver, in the kidneys, and in any local lesions which may be present. Their scattered distribution and their occurrence in minute clumps have already been alluded to. They should always be sought for at first with a low power of the microscope. Ordinarily no bacilli can be found in the blood, but it has been shown that the blood in the roseolae some- times contains them, so that the eruption may be re- garded as one of the local irritative manifestations of the bacillus. A particularly careful work upon this subject has been done by Richardson,1 who found that by carefully disin- fecting the skin, freezing it with chlorid of ethyl, making a crucial incision, and cultivating from the blood thus obtained, he was able to secure the typhoid bacillus in thirteen out of fourteen cases thus examined. It was, however, necessary to examine a number of spots in each case. As a means of diagnosis the matter is of some impor- tance, as the occurrence of rose spots and the cultivation from them of the bacilli often preceded the occurrence of the Widal reaction by a number of days. The amount of local disturbance, in proportion to the constitutional disturbance, is, in the majority of cases, slight, and almost always partakes of a necrotic charac- ter, which suggests that in typhoid we have to do with a toxic bacterium whose disease-producing capacity resides in the elaboration of a toxic substance. This, indeed, is true, for Brieger and Frankel have separated from bouillon cultures a toxalbumin which they thought to be the specific poison. Klemperer and Levy also point out 1 Phila. Med. Jour., Mar. 3, 1900. 480 PA THOGENIC BA CTERIA . further clinical proof in certain exceptional cases dying with the typical picture of typhoid, yet without char- acteristic post-mortem lesions, the only confirmation of the diagnosis being the discovery of the bacilli in the spleen. PfeifFer and Kolle1 found that the toxic substance resided only in the bodies of the bacilli, and could not, like the toxins of diphtheria and tetanus, be dissolved in the cul- ture-medium. This was an obstacle to their immuniza- tion-experiments as well as those of Loffler and Abel,2 later to be described, for the only method of immuniz- ing animals to large quantities of the bacilli was to make massive agar-agar cultures, scrape the bacilli from the surface, and distribute them through nutrient bouillon. When injected into guinea-pigs the typhotoxin of Brieger is productive of increased secretion of saliva, in- creased rapidity of respiration, diarrhea, and mydriasis, and usually causes a fatal termination in from twenty- four to forty-eight hours. As the discovery of the bacilli in the spleen, and espe- cially the securing of a pure culture of the bacilli from the spleen, are sometimes attended with considerable dif- ficulty because of the dissemination of the colonies throughout the organ, E. Frankel recommends that as soon as the orgau is removed from the body it be wrapped in cloths wet with a solution of bichlorid of mercury and kept for three days in a warm room, in order that a con- siderable and massive development of the bacilli may take place. Typhoid fever is a disease which is communicable to animals with difficulty. They are not affected by bacilli in fecal matter or in pure culture mixed with the food, and are not diseased by the injection into them of blood from typhoid patients. Gaffky failed completely to pro- duce any symptoms suggestive of typhoid fever in rab- bits, guinea-pigs, white rats, mice, pigeons, chickens, 1 Deutsche med. Wochenschrift, Nov. 12, 1896. J Centralbl.f. Bakt. u. Parasitenk., Jan. 23, 1 896, Bd. xix., No. 23, p. 51. TYPHOID FEVER. 481 and calves, and found that Java apes could feed daily upon food polluted with typhoid germs for a considerable time, yet without symptoms. The introduction of viru- lent cultures into the abdominal cavity of animals is fol- lowed by peritonitis. Germano and Maurea ' found that mice succumbed in from one to three days after intraperitoneal injection of 1-2 c. cm. of a twenty-four-hour-old bouillon culture. Sub- cutaneous injections in rabbits and dogs caused abscesses. Losener found the introduction of 3 mgr. of an agar- agar culture into the abdominal cavity of guinea-pigs to be fatal. When animals are treated in the manner described in the chapter upon Cholera — i. e. the gastric contents ren- dered alkaline, a large quantity of laudanum injected into the peritoneal cavity, and the bacilli introduced through an esophageal catheter — Klemperer, Levy, and others found that there was produced an intestinal con- dition which very much resembled typhoid as it occurs in man. The virulence of the bacillus can be very greatly increased by rapid passage from guinea-pig to guinea-pig. In the experiments of Chantemesse and Widal the symptoms following the injection of virulent culture into guinea-pigs were briefly as follows : ' ' Very shortly after the inoculation there is a rise of temperature, which continues from one to four hours, and is succeeded by a depression of the temperature, which continues to the fatal issue. Meteorism and great tenderness of the abdo- men are observed. At the autopsy a sero-fibrinous or sero-purulent peritonitis is observed — sometimes hemor- rhagic. There is also generally a pleurisy, either serous or hemorrhagic. All the abdominal viscera are con- gested. The intestine is congested — contains an abun- dant mucous secretion. The Peyer patches are enlarged. The spleen is enlarged, blackish, and often hemorrhagic. In cases which are prolonged the liver is discolored. The kidneys are congested, the adrenals filled with blood. 1 Zeigler's Beilrage, lid. xii , Heft, 3, p. 494. 31 482 PATHOGENIC BACTERIA. " In such cases the bacillus can be found upon the in- flamed serous membranes, in the inflammatory exudates, in the spleen in large numbers, in the adrenals, the liver, the kidneys, and sometimes in the lungs. The blood is also infected, but to a rather less degree. ' ' In cases described as chronic, the bacillus disappears completely in from five to twenty-four hours, and pro- duces but one lesion, a small abscess at the point of inoc- ulation. " Sanarelli has observed that if some of the poisonous products of the colon bacillus or the Proteus vulgaris be injected into the abdominal cavity of an animal recover- ing from a chronic case, it speedily succumbs to typical typhoid fever." Petruschky * found that mice that recovered from sub- cutaneous injections of typhoid cultures frequently suf- fered from a more or less widespread necrosis of the skin at the point of injection. I experienced great difficulty in immunizing a horse to the disease, because every injection of virulent living organisms was followed by a necrosis equalling in size the distended area of subcutaneous tissue. Large quantities of filtered cultures produce symptoms similar to those resulting from inoculation with the bacilli. Animals can easily be immunized to this bacillus, and then, according to Chantemesse and Widal, develop in their blood an antitoxic substance capable of protecting other animals. Stern 2 has also found that in the blood of human convalescents a substance exists which has a protective effect upon guinea-pigs. His observation is in accordance with a previous one by Chantemesse and Widal, and has recently been abundantly confirmed. The immunization of dogs and goats by the introduc- tion of increasing doses of virulent cultures has been achieved by Pfeiffer and Kolle3 and by Loftier and Abel.4 1 Zeitschrift fur Hygiene, 1892, Bd. xn., p. 261. » Ibid., 1894, xvi., p. 458. 3 Ibid., 1896. * Centralbl.f. Bakt. u. Parasitenk., Jan. 23, 1896, Bd. xix., No. 23, p. 51. TYPHOID FEVER. 483 From these animals serums were secured not exactly an- titoxic, but anti-infectious or auti-microbic in operation, and possessed of marked specific germicidal action upon the typhoid bacilli when simultaneously introduced into the peritoneal cavity of guinea-pigs. The action of the typhoid serum is specific, and exerts exactly the same action upon the typhoid bacilli as the cholera serum exerts upon the cholera spirilla, killing and dissolving them (Pfeiffer's phenomenon). So far, no serum has been produced that is efficacious in human medicine. The specific reactions of the serums artificially pre- pared can be used to differentiate cultures of the colon and typhoid bacilli, the typhoid bacilli alone exhibiting the specific effect of the typhoid serum. Richardson ' has found it very convenient to keep on hand in the laboratory filter-paper saturated with typhoid serum and dried, then cut into }4 c.cm. squares. To make a differential test of the typhoid bacillus one of these little squares is dropped in ]/2 c.cm. of a 24-hour- old bouillon culture of the suspected bacillus and al- lowed to stand for five minutes. Then a drop of the fluid placed upon a slide and covered with a cover- glass will show typical agglutinations if the culture be one of typhoid-fever bacilli. In a second mention of this method2 he has found its use satisfactory in practice and the paper serviceable after fourteen months' keeping. Christophers3 found that the serum from typhoid patients occasionally caused agglutinations in cultures of the colon bacillus, but concludes that this does not lessen the specificity of the reaction, as there may be two combined specific actions of these serums. Experi- ments on rabbits established that typhoid and colon serums could be produced, each specific in its agglutin- 1 Centralbl f. liakt. u. Parasitenk., 1897, p. 445. 1 Journal 0/ Experimental Medicine, May, 1898, p 353, note. 3 Brit. Med. Jour., Jan. 8, 1898. 484 PATHOGENIC BACTERIA. ating power upon bouillon cultures of its respective organism. Loffler and Abel also prepared a colon serum which exerted a like specific action upon the colon bacillus, but was without effect upon the typhoid bacillus. In 1896, Widal and Griinbaum,1 working indepen- dently, discovered that when blood-serum from typhoid- fever patients is added to cultures of the typhoid bacillus a definite reactive phenomenon occurs. This phenome- non, which is now familiarly known as the "Widal re- action," consists in complete loss of the motion so char- acteristic of the typhoid bacillus, and the collection of the micro-organisms into clusters or groups — agglutina- tion. There seems, also, to be a change in the bacillary substance, so that the bacteria appear shrunken and distorted. Gruber and Durham 2 made the first thorough and sys- tematic studies of the agglutinating and immobilizing property of serum, beginning their publications in Janu- ary, 1896. The work of Gruber and his associates is of great importance, and merits careful reading and study. So long as the matter was in Gruber' s hands, it was an interesting scientific observation. In Widal's hands it developed into a reliable means of diagnosing typhoid fever and other diseases. For convenience, the subject will be considered under separate divisions. The Time at ivhich the Reaction Develops. — The reac- tion usually comes on about the seventh day, some- times earlier ; and Johnston and McTaggart usually found it quite well marked by the fifth day. They also observed it in three cases at the end of forty-eight hours from the beginning of the fever. Widal and Sicard state that the reaction usually occurs on the first day of infec- tion. Not infrequently the reaction fails to develop 1 La Semaine medicate, 1 896, p. 295. 2 Munch, tned. Woche7isci-ift, 1896, No. 13. TYPHOID FEVER. 485 until later. Most cases, however, react well in the second week, and nearly all show reaction in the third week. Rarely the reaction is delayed still later. In rare cases of typhoid the reaction may never occur, even though the case prove fatal. Together with Prof. James M. Anders, of the Medico- Chirurgical College, I1 made more than a thousand blood-examinations to determine the value of the serum- test in typhoid fever and as an aid in the diagnosis of febrile diseases. The Widal test was properly and continuously applied to 230 cases of typhoid fever among soldiers who had enlisted in the Spanish-American War and who were treated in the Medico-Chirurgical Hospital. Of these, 219 reacted positively, or 95.64 per cent, of the total number examined. One of the fatal cases, which was otherwise typical typhoid fever, failed to show a reaction so late as the seventeenth day of the illness ; this was excluded, for the obvious reason that had the patient survived for a longer period a positive reaction might have been obtained. Thus in 10 of the cases no Widal reaction was present ; these were tested from time to time so late as the end of the first week of convalescence, or return of normal temperature. The percentage of cases, therefore, showing absence of the reaction was 4.36 per cent, as against 4.5 per cent, indicated by the table of Drs. Stengel and Kneass. Of the 219 cases giving a positive result, 128 showed the reaction prior to the appearance of the rose-spots, or before the eighth day ; 36 showed the reaction during the second week ; 45 between the seventeeth and twenty-first day of the disease ; 8 not until the twenty-fifth day, and 2 as late as the twenty-eighth day of the illness.2 1 Phi/a. Med. /our., April 8-1 5, 1899. 1 These results may be misleading, as they might seem to indicate that the blood of these cases was tested every day and the reaction first noted on the day given. In reality the blood was sometimes not examined until the day mentioned. 486 PATHOGENIC BACTERIA. In addition to the 230 cases considered above, in which the clinical symptoms and course were typical, we noted the presence of the Widal reaction in 10 cases that were atypical, and, apart from the presence of the sero-reaction, so lacking in the characteristic typhoid features as to be placed in the category of non-typhoid diseases. In a series of 300 cases taken from the records of the Episcopal, Presbyterian, and St. Agnes' Hospitals, Dr. W. H. Bell l found that the Widal reaction was unob- tainable until the disease was well advanced, the average in this series being the ninth day, and the earliest the fifth day of the disease. Delepine2 points out that during the first week the reaction is often slow and not quite clear, and that to establish an assured diagnosis a re-examination is often necessary. The results obtained in Osier's wards in the Johns Hopkins Hospital by Block and Gwyn up to Novem- ber, 1898, show that in 151 cases the reaction was pres- ent in 144. "In 4 of the negative cases the clinical course was not certain. In only 46 of the last 108 cases was the reaction obtained; in only 26 cases was the reac- tion present before the seventh day of the disease ;" in 4 cases it developed on the twenty-second, twenty-sixth, thirty-fifth, and forty-second days respectively. In a very few cases it is absent altogether. In this connection it may be noted that there are cases in which the reaction is missed during the primary attack, or until the period of convalescence (Achard, Blumenthal), and, in still others, it first makes its appearance in the relapse (Biggs, Park, Stahl, Bell, et a/). In the American Year-Book of Medicine and Surgery for 1898, Drs. Stengel and Kneass have collected and tabulated, from the previous statistics of different writers, 2392 cases of typhoid, in which the reaction was positive in 2283 and negative in 109. Also 1387 non-typhoid 1 University Med. Mag., June, 1898. - Allbutt's System of Medicine, vol iii , p. 1147. TYPHOID FEVER. 487 cases, of which 22 reacted positively and 1365 negatively. The results in these cases show — Reactions in typhoid cases 95.5 per cent. No reactions in non -typhoid cases 98.4 " Correct results in . 96.5 '* Incorrect results in 3.5 " Surely, this table indicates a small proportion of nega- tive responses. II. The duration of the reaction is by no means con- stant. In the greater number of cases the agglutinating property of the blood begins to diminish in the first weeks of convalescence, and marked reduction in its activity is noticed in a few months. Few cases continue to show it longer than one year. Widal and Sicard1 found that it sometimes disappears as early as the eight- eenth day after convalescence. Brenner saw it disap- pear on the seventeenth day in one case and on the twenty-fifth day in another case. E. Fraukel found it absent after the twenty-eighth day in one case. In all the cases observed by Widal and Sicard the reaction dis- appeared within a year. Sometimes, however, the reac- tion has been observed to persist much longer, and in a series of cases which had had typhoid fever a varying number of years before the examination, Widal and Sicard found it still present in one case 3 years after recovery, in one case 7 years after recovery, and in another 9 years after convalescence. Widal tested 80 cases of typical or suspected typhoid fever at periods ranging from 1^ to 9 years after convalescence, obtain- ing 6 positive and 16 negative results. Musser and Swan2 found that in their series of cases the reaction disappeared in one case on the thirty-eighth day, while in one other case it persisted for ten years. I was much interested in the following series of 30 cases which show the persistence of the reaction and 1 Compt. rendu de la Soc. de Biol., Dec. 1 9, 1896, No. 33. * Journal American Med. Assoc, Aug. 14, 1897. 488 PATHOGENIC BACTERIA. confirm the observations already quoted, were students, nurses, and physicians. Most of these Studies of the Blood of 30 Cases Convalescent from Typhoid Fever for Varying Lengths of Time, Illustrating the Dura- tion of the Widal Reaction : Time since convalescence. I month G 6 months D 18 « Dr. R 1 year F. S. C 2 years H 9 9 9 13 14 15 15 16 16 17 18 20 S. . . . c G L. C. S. . Miss P. . S B R. W. . . R. . . . Mrs. McB. J. R. . • M. . . . A. D. . . Miss W. . I W. J. R. . Stern. . . Miss F. . Miss J. . . D O. B. R. . B M. . . . P. . . S. P. H. . Reaction. + + 0 + + O + o + o + + O + O + o + ? O O + ? O o o O O o o O o One person, not included in the table, suffered from two attacks of typhoid fever, one eight, the other thirteen years ago. At present the blood does not possess any reactive power. When once established the reaction is usually constant; but occasionally cases are observed in which it is absent for short intervals, usually during the period of its estab- lishment or disappearance. TYPHOID FEVER. 489 The occasional persistence of the reaction for some years may lead to errors of diagnosis, but is not apt to do so if the previous history of the case can be properly studied. It is said that the reaction sometimes disappears shortly before death. III. The Distribution of the Agglutinating Substance. — Being a substance dissolved in the blood, the aggluti- nating substance is present in the various secretions ex- tracted from it. In Widal's paper, reviewing the whole subject,1 he says the agglutinating substance has been found in the blood, urine, the serous fluid from blisters, the pleural pericardial, and peritoneal fluids, milk, bile, seminal fluid, aqueous humor, tears, pleural exudates, and to a less extent in the spleen, liver, and mesenteric glands. Catrin2 found it in the pus of a phlegmonous inflam- mation occurring during typhoid fever, and Block asserts that it is present in the typhoid stools. Thiercelin3 found it absent from the spontaneous sweat when present in the blood and milk. In a case with two relapses whose blood was strongly agglutinative I failed to find any agglutinative principle in the urine. The fact that the typhoid bacilli are found scattered through the body in little groups would suggest that agglutination took place in the circulating blood of the infected individual. Salimbeni, however, asserts that such agglutinations do not occur. IV. The relation zvhich the reaction bears to the germi- cidal activity of the blood has been pretty carefully studied, but nothing definite established. In general it is found that the fresher the blood, the more readily bacteriolysis is observed. Johnston and McTaggart4 state that " with 1 Ann. de f Inst. Pasteur, May, 1897, No. 5. J Gas. de Med. de Paris, Oct. 15, 1896. 3 Compt. rendu de la Soc. de Biol., Dec. 19, 1896, No. 33. * Montreal Med. Journ., Mar., 1897. 490 PA THOGENIC BA CTERIA . blood-solutions this phenomenon is frequently witnessed. The clumped bacteria, if watched for an hour or so, may be seen to break up in granules which gradually become indistinct and vanish while under observation, until practically no trace remains of the clumps which shortly before studded the entire field of the microscope. The change is more liable to occur in culture some days old than in young cultures, and more likely with attenuated than virulent cultures." It does not occur with all the samples of typhoid blood, and is not well marked in very dilute blood-solutions. The agglutinative substance is different from the bac- tericidal substance, and the agglutination of the bacteria is not to be looked upon as the beginning of their destruc- tion. It was found by Widal and Sicard that many of the typhoid serums with a high degree of agglutinating power were entirely devoid of bactericidal powers. ' ' The bactericidal, immunizing, and agglutinative properties of the sera, although generally present at the same time, are, however, mutually independent." Jemmal l found that the phenomenon of agglutination was most marked during the period of most intense infec- tion and when the bactericidal activity was greatest. Widal and Sicard2 also trace a relationship between the two. They were able to keep typhoid cultures alive for two months in strongly agglutinative serums without destroying their vitality; and indeed, one of the original methods that Widal suggested for studying the reaction required that the typhoid bacillus should grow in diluted, but of course agglutinative, serum. V. Transmission of the Agglutinating Power from Parent to Child. — Mosse and Dannie3 report the case of a woman who had typhoid in the eighth month of pregnancy. At the time of delivery the blood and mammary secretion of the mother gave a positive reaction, and the blood of the 1 Ceniralbl. f. inner e Med., Jan. 23, 1897. '. Compt. rendu de la Soc. de Biol., Mar., 1897, No. 8. s Philadelphia Pediatric Society, 1897. TYPHOID FEVER. 491 child reacted positively for thirty-three days after birth. J. P. C. Griffith1 has seen a similar case. Barber2 reports a case of a woman who on the second day of typhoid fever gave birth to a child whose blood, two days later, gave the serum-reaction; subsequently the infant had a slight fever and an attack of diarrhea. Pepper and Stengel3 mention a case in which the blood of an infant gave positive agglutinative reactions. Charrier and Apert4 examined the tissues of an embryo from a mother in the third week of typhoid fever. There was a total absence of any agglutinating property in the fetal organism, though it existed in the blood of the placenta. VI. The Chemic Nature of the Agglutin. — Widal and Sicard found that filtration through porcelain lessens the quantity of agglutinative substance found in the body fluids. The addition of 15 per cent, of sodium chlorid precipitates fibrinogen, which carries down much of the agglutinating substance with it. Further precipitation with magnesium sulphate throws down the globulins and more of the agglutinating substance. Dieulafoy5 states that in milk the casein is the active principle. Widal and Sicard conclude that it is not in the serum- albumin, but in the globulins and fibrinogen that most of the agglutin resides. In dialyzed liquids they found the agglutinating substance absent. Achard and Ben- saud, on the other hand, found it dialyzable. The agglutin has a marked resisting power, as is shown bv the fact that it can be dried without change, and is active after being sealed in tubes for months. It resists decomposition for a time. Temperatures of 6o° C. diminish its power, but it is only destroyed at 8o° C. Sunlight has no effect upon it. VII. The relation of the agglutinating substance to the 1 See Amer. Jour. Med. Sci., N. S., Jan. -June, 1897, vol. 113, p. 621. 1 New York Med. Jour., April 16, 1898, vol. lxvii., No. 16. 5 Year- Book of Medicine, 1897. * Compt. rendu de la Soc. de Biol, de Paris, Jan. I, 1897. 6 Bull. deTAcad. de Mid., 1896, 346. 492 PATHOGENIC BACTERIA. leukocytes is a matter of interest. Achard and Bensaud ' have decided that there is no relation between the two. They experimentally secured plasma devoid of leuko- cytes by using leeches and centrifugation, and found it active, and also secured leukocytes by filtering the fluid through cotton. The living active leukocytes did not cause any phenomenon on the part of the bacilli when brought into contact with them. VIII. The Relation of Agglutinating Power to Im- munity. There are numerous reasons for regarding the reaction as one of immunity, and many able men, among whom are Maragliano and Pfeiffer, adhere to this view. It is well known that the reaction usually develops some days after the inception of the disease, is most marked during the third week, and remains as a post-typhoid con- dition for about the length of time that immunity to the disease is supposed to exist. This looks significant, and is at least interesting, if of no especial importance. The reaction is also observed to be less intense in mild than in severe cases, though there are numerous excep- tions to this. Achard2 noticed that after relapses the agglutinative power of the serum was increased. In some of their writings, Widal and Sicard look upon the reaction as one of defence on the part of the organism, especially during the period of infection. Charrin also shares this opinion. In the so-called vaccination against typhoid fever prac- tised by Wright and Semple, the prophylactic injections of sterilized cultures of virulent typhoid bacilli beneath the skin impart to the blood of the individual a more or less marked immunity, the occurrence of which it is sup- posed can be gauged by observing when the agglutinative power of the blood occurs. Welch points out that in experimental immunity to typhoid, in which there is an increase of immunity to great heights, there may be a corresponding rise of the 1 Semaine med., 1896, p. 393. 1 Bull, de la Soc. mid. des Hop., Oct. 16, 1896. TYPHOID FEVER. 493 agglutinative power, whereby degrees of this power may be obtained which are entirely unknown during natural infection. There is, however, no necessary correspond- ence between the height of immunity and that of aggluti- nation; especially may the latter lessen or disappear when the former is present. On the other hand, the power of agglutination often occurs almost simultaneously with the onset of the disease, when no immunity can have been established; many cases with a high degree of agglutinating power are without immunity and suffer relapses; and the phenomenon is most marked during the acme of the disease, and it sub- sequently declines. The facts that the phenomenon is more marked in bad cases than in mild ones, and that the blood possesses a higher degree of agglutinating power after relapses, are given little weight in the argu- ment, for, supposing the most recent view of Widal to be correct, and the reaction to be simply a phenomenon of infection, it is only to be expected that the results of severe infection should be greater than those of mild infection, and of prolonged infection than of brief ill- nesses. Relapses have also occurred in cases in which the blood showed an agglutinating strength operative in dilution of i : 2000. Unable to prove that a definite relationship between immunity and the agglutinative power of the blood exists, most thinkers have now adopted Widal's view, and look upon the reaction simply as a u phenomenon of infection." Malvoz1 looks upon the phenomenon as depending upon metabolic products of the bacteria which are accumulated in the body during the disease. His proof is found in the effect of certain chemic substances which agglutinate, and in the effects of old upon fresh cultures. He says: "If we take an emulsion of the first vaccine of anthrax, made by thoroughly mixing some of the culture with 1 Ann. de T Inst. Pasteur, August 25, 1889. 494 PATHOGENIC BACTERIA. 0.5 c.cm. of distilled water, and add to it a drop or two of a six- day-old culture of the same bacillus, a drop of it allowed to stand for a few hours in a moist chamber, when examined under the microscope, will show typical agglutinations. The addition of a little distilled water will have the same effect ; hence the infer- ence is drawn that the reaction is a passive, not an active, histo- genic one." IX. The specific nature of the reaction is now univer- sally accepted. There are, however, certain cases of irregular phenomena which diminish the absolute reli- ability of the test. These may or may not be dependent upon conditions which it is within the province of expe- rience to explain. Normal human serum, when concentrated, occasion- ally exerts a slight agglutinating effect upon the typhoid bacillus. This seldom occurs except with concentrations exceeding those employed for diagnostic purposes. • The blood of certain animals normally possesses an agglutinating property for the typhoid and other bacilli. Some individuals are peculiar in that their blood acts more strongly in its agglutinating and bacteriolytic action than others. A few diseases, which may or may not be related to typhoid, occasionally produce in the blood of their vic- tims a reactive effect upon the typhoid bacillus. The number of cases in which such errors can occur is shown, by reference to the statistics of Pepper, Stengel, and Kneass (given above), to be very small, and the validity of the test is very slightly influenced by them. It will be shown elsewhere that for a final opinion upon the specificity of any reaction attempted for purposes of scientific diagnosis certain definite criteria are given which allow of little or no deviation, and to which non-conformity by those reporting irregularities must be submitted before their weight is established. Villiez and Battle ' found that in three cases of malaria from Madagascar a positive reactive phenomenon oc- curred in one. 1 Presse ?neJ., 1896, No. 84. TYPHOID FEVER. 495 The reactive phenomena are very slightly interchange- able for species of bacteria closely allied to the typhoid bacillus, so that a serum with strong agglutinative properties may slightly affect the colon bacillus, and a serum from an animal immunized to the colon bacillus may be able to affect slightly the typhoid bacillus. Far from lessening the value of the test, this, as Welch points out, only argues for the close relationship of the species acted upon. The typical reaction does not occur with the other closely allied members of the typhoid group of bacilli. The attempts to make the colon bacillus agglutinate by application of typhoid serum to their culture usually fails. In cases of suspected typhoid, in which the reac- tion upon the colon bacillus takes place, it is impos- sible to eliminate the possibility of combined typhoid and colon-bacillus infection, as suggested by Johnston and McTaggart. Widal and Courmont found that all human sera, whether normal or typhoid, have a slight action upon the colon bacillus in dilutions of 1 : 10, whereas normal serum, as a rule, has no effect upon the typhoid bacillus in this dilution. The agglutinative power of the specific serum upon the bacteria is so delicate that bacteriologists now make use of the phenomena for the differentiation of similar species. Richardson's serum-paper, which will be fully described elsewhere, is a most important adjunct to our means of separating the colon and typhoid bacilli. In a number of cases that I studied, serums which reacted strongly upon the typhoid bacillus failed to pro- duce any alteration in cultures of Bacillus coli communis, Bacillus suispestifer, and Bacillus icteroides. X. The technic is of the utmost importance. Widal ' suggested that a small quantity of blood be withdrawn with a sterile syringe from the median cephalic vein, and a few drops of it added to a fresh bouillon culture of the 1 Semaine mid., 1896, p. 259. 496 PA THOGENIC BA CTERIA . typhoid bacillus in the proportion of i : 10 or i : 15. Twenty-four hours afterward the cloudiness characteristic of the growth of the typhoid bacillus in bouillon had entirely disappeared, because the bacilli, massed together in agglutins, had all sedimented, and were found as a flocculent mass at the bottom of the tube. This he called the "rapid method," in contradistinction to a more rarely employed "slow or culture method," in which the serum in the given proportion was added to the sterile bouillon, which was inoculated with the typhoid bacillus and then stood in an incubating oven for about fifteen hours, or until the growth could be observed in a flocculent mass at the bottom of the tube. Pugliesi * used serum obtained from blisters. Both of these methods were so disturbing to the patient because of the large amount of blood required, that, had no improvement in the technic been devised, it is prob- able that the reaction would have attained little import- ance as a method of diagnosis. Widal himself made the first improvement, and suggested that instead of a syringe- ful of blood a few drops secured from the finger-tip would suffice. He also recommended observing the reaction through the microscope instead of awaiting the results of the slow sedimentation that succeeds the addition of the blood to a culture. Widal found that it was not nec- essary to use fresh serum, but that serum kept for some time produced all the phenomena in quite as typical a manner. He further found that when the blood was dried and subsequently redissolved, it was capable of causing the reaction. Wyatt Johnston, entirely independent of Widal's work, found that successful reactions could be secured from blood dried upon paper, and immediately proceeded to make practical use of his observation by requesting that specimens of blood dried upon paper be sent to the labor- atory at Montreal, where they would be studied and re- ported upon. A ready and certain means of diagnosis of 1 Riforma Medica, Oct., 1896. TYPHOID FEVER. 497 doubtful cases of typhoid fever having been desired for a long time, this offer met with ready acceptance, and it was not long before a large number of cases had been studied and the success of the measure shown to be complete. Indeed, the outcome of Johnston's work was the establishment, at the laboratory of the Board of Health of the Province of Quebec, and later at most public laboratories in this country, of a system of free public examinations, by which the physicians of the larger cities and towns can have their diagnoses con- firmed. The paper upon which the blood is dried is moistened with germ-free water, and a drop of the solution placed upon a cover-glass which has just been passed through a flame. A drop of a bouillon culture, or of a watery sus- pension of an agar-agar culture of the typhoid bacillus, is mixed with it, the cover placed upon a concave slide prepared with vaselin so as to make a hanging drop, and is then examined under a dry lens of moderate power (one-fourth or one-fifth inch). This was Johnston's tech- nic. Wesbrook, of Minneapolis, endeavored to improve it by having the blood drops dried upon weighed pieces of tin-foil, prepared for the purpose in the laboratory. The advantage of this is that the exact weight of the blood secured can be determined, and accurate dilution made for the occasional cases requiring them. Cabot has suggested the use of the medicine-dropper: he secures blood from the finger and drops it into a recep- tacle, and afterward follows it by a given number of drops of bouillon-culture from the same medicine-dropper. In our work we have not found it easy to do this, and it is disadvantageous in that it must be performed at the bedside. Some men have successfully employed the pipet used for counting leukocytes. This seems excellent for bed- side work. Delepine has suggested a method that can be employed both in the laboratory and at the bedside. A drop of blood 32 498 PATHOGENIC BACTERIA. or serum is picked up with the bacteriologist's platinum- loop, placed upon a glassslide, and mixed with nine similar drops of the culture to be used. The mixture forms a dilution of i : 10. I have recommended1 the employment of capillary glass tubes of equal size, which, when allowed to draw in blood from a prick on the finger-tip, will contain a quantity of blood easily estimated from the length of the column in the capillary tube. Knowing the quantity of contained blood, it is easy to estimate how much fluid- culture one must add to make a definite dilution. The tube is crushed and stirred in the diluting culture, so that none of the blood is lost. Hewlett and Sydney 2 recommend a similar method which is probably more exact. I prefer fresh agar-agar cultures, distributed through- out sterile clean water, rather than bouillon cultures, be- cause of the larger number of bacteria they contain, the consequently greater number of agglutinations formed, and the readiness with which they are found upon micro- scopic examination. It is necessary, however, to make a microscopic examination of the diluted culture before adding the serum or blood, in order to be sure that there are no natural clumps of bacteria present to simulate the specific agglutination. This is of great importance. The natural clumps of bacilli are more apt to occur in cultures grown upon fresh, moist agar-agar than upon that kept for a short time until the surface has become partially dried. Research by numerous authors has shown that errors are apt to occur when concentrated dilutions of the typhoid blood or serum are used, and that it is only in cases in which sufficient dilutions of the serum produce agglutina- tion that a positive diagnosis of typhoid can be made. Stern3 concluded that the dilution should be i : ioo, 1 New York Med. Jour., Sept. 25, 1897. 2 British Med. Jour., April 28, 1900. s Centralbl. f. innere Med., Dec. 5, 1896. TYPHOID FEVER. 499 or even i : 200. Widal, however, found 1 : 60 sufficient. Dilutions of 1 : 10 are satisfactory for routine work, and it is only necessary to use the high dilutions in doubtful cases. Welch says : " From the observations thus far reported, although they are insufficient in number for definite conclusions, there would seem to be only a small liability of failure to recognize genuine typhoid cases by resorting to dilutions of 1 : 40 or 1 : 50, but unquestionably a few would escape recognition, and for this reason lower dilutions should also be used, and if those between 1 : 10 and 1 : 50 give decided reaction there should be at least suspicion of typhoid. It is not, therefore, to be recommended that one make the test with only high dilutions, such as 1 : 50. The negative result of the preliminary test with equal parts of serum and culture suffices to exclude typhoid reaction. The examination, if positive, may then be made with a low dilution of the serum, and for this Widal's recommendation of 1 : 10 or 1:15 may well be adopted. If with this dilution the microscopic reaction is complete and almost immediate, as is often the case, there is practically no risk in making a positive diagnosis. But for absolute certainty, and above all, in cases in which the result of the reaction is not prompt, complete, and unmistakable, higher dilutions should be employed ; if the amount of serum permits only one such, it maybe 1 : 50, but preferably intermediate dilutions should also be made, and it is desirable, if not absolute^ neces- sary, to try dilutions higher than 1 : 50." A time limit is also to be fixed, and when, with the ordinary dilution of 1 : 10, a reaction does not come on within fifteen minutes, any subsequent agglutinative phenomena should be looked upon with suspicion. We have, however, seen reaction delayed an hour and longer in 1 : 10 dilutions in cases known to be typhoid. With the weak dilutions, 1 : 50, etc., the time limit must be ex- tended to at least an hour. Usually the reaction occurs so promptly that no difficulty is experienced. XI. The condition of the culture is also to be carefully considered from several standpoints, else the validity of the test may be called into question. 1. Age. — Young cultures of the typhoid bacillus are actively motile, and are apt to contain rather elongate individuals. Both these features are essential to success. 500 PATHOGENIC BACTERIA. The more actively motile the bacilli, the greater the con- trast when this motility ceases ; the longer the bacilli, the more typical are the agglutinations. Cultures less than twenty-four hours old are best. At a pinch, how- ever, they can be employed so long as they remain motile (forty-eight hours). 2. Activity. — Johnston first pointed out that virulent bacilli frequently transplanted from culture-medium event- ually developed an abnormal condition in which reactions were apt to occur with normal blood. The best method of avoiding this error seems to be to keep a stock culture in the laboratory, and transplant it from agar to agar only as often as is necessary to keep it in good condition — say, once in three or four weeks. A transplantation from this to whatever culture-medium is preferred is made about twenty-four hours before testing. 3. Virulence. — Johnston is emphatic upon the use of attenuated rather than virulent, freshly-isolated cultures, stating1 that "with virulent cultures the presence of agglutinative substances in non-typhoid bloods may lead to pseudo-reactions. " These reactions are characterized by a rapid clumping without the characteristic loss of motion. Kuhnan2 seems to differ with Johnston, how- ever, and finds that the reaction with non-virulent cult- ures is nearly double that obtained with virulent ones. Foerster3 did not observe that the difference between the reaction obtained with virulent and attenuated bacilli was great. He experimented with nine different bacilli, and expresses the difference observed as 5 : 8. 4. Reaction of the Culttire-medinm. — Johnston also in- vestigated this subject, and found that when the bacilli are cultivated upon acid media they may entirely lose their ability to agglutinate. Care should be taken to insure that the culture-medium employed always has the same degree of alkalinity. On the other hand, the media 1 Montreal Med. Jour., Mar., 1897. 1 Berliner klinische Wochenschrift, May 10, 1897, No. 19. 3 Zeitschrift f. Hygiene, 1897, vol. 24, p. 500. TYPHOID FEVER. 501 must not be too alkaline, as this condition will also cause erroneous results. 5. Vitality of the Culture. — Except for the fact that dead bacilli are not motile, and hence cannot show a loss of motility as a part of the reactive phenomenon, they are useful for making the test. In fact, the absence of danger of infection, and the convenience with which the sterilized cultures can be sent from place to place to be used by physicians who are unacquainted with bac- teriologic technic, have made Wright and Semple l rec- ommend their use. The reaction is probably physical rather than vital, as it occurs in dead as well as in living organisms. 6. Peculiarities of Reaction. — It is interesting to note the different forms of reaction, which may be classified as follows : a. Cessation of motion (not regarded as typical). b. Formation of small aggregations without loss of motion of the free bacilli (not regarded as typical). c. Complete cessation of motion and formation of a reticulated agglutinated mass of bacilli covering the whole field. d. Complete cessation of motion with the formation of small, fairly uniform aggregations. This usually occurs quickly and is accompanied by distortion of the bacilli. e. Complete cessation of motion and the formation of large-sized aggregations, some of which are enormous. The bacilli are shrunken and twisted. (This form of re- action was usually almost instantaneous in its occurrence, and probably indicated that the highest degree of the blood-alteration had taken place. I saw it most marked in a case with two relapses. It is sometimes accompanied by bacteriolysis.) f. Rapid agglutination and loss of motion followed by prompt and complete solution of the bacteria. The bac- teriolysis is probably entirely independent of the other phenomena. It may occur in normal blood, but few of 1 British Med. Jour., May 15, 1897. 502 PATHOGENIC BACTERIA. these were examined, and the one case in which I en- countered it was typical typhoid. XII. The Clinical Value of the Serum Diagnosis. — Granting the precision of method of serum diagnosis, it is doubtless of great diagnostic importance. As shown by the statistics given, the reaction failed to develop in only 4.5 per cent, out of a total of 2393 cases of typhoid fever. Again, it is highly probable that this small per- centage of failures would be farther reduced if the test (when negative at the first examination) were repeated every day or two until convalescence is fully established. Rumpf1 and Kraus and Buswell2 report a number of cases of typhoid which were favorably influenced by the hypodermic injection of small quantities of sterilized cult- ures of Bacillus pyocyaneus. Following the lines of experimentation suggested by Haffkine's researches upon preventive vaccination against cholera Asiatica, Pfeiffer and Kolle, and Wright and Sem- ple have used the subcutaneous injection of sterilized cult- ures as a prophylactic measure. One cubic centimeter of a bouillon culture sterilized by heat is thought to be suffi- cient Wright and Semple 3 report 18 cases in which it was used, and by experiment showed the blood to be changed similarly to that of typhoid patients and convalescents. This change consisted in the destruction of motility and agglutination of the bacilli, as seen in Widal's reaction. Walger 4 reports four cases treated successfully with a serum obtained from convalescent patients. Ten c.cm. were given at a dose, and the injection was repeated in one case with relapse. Jez5 believes that the antitoxic principle in typhoid fever is contained in some of the internal organs instead of the blood, and claims to have obtained remarkable results in eighteen cases treated with extracts of the bone- 1 Deutsche med. Wochenschrift, 1893, No. 41. 2 Wien. klin. Wochenschrift, July 12, 1894. 3 Brit. Med. Jour., 1897,' i., p. 256. 4 Munch, med. Wochenschrift, Sept. 27, 1898. ^ 5 Med. moderne, Mar. 25, 1899. TYPHOID FEVER. 503 marrow, spleen, and thymus of rabbits previously in- jected with the typhoid bacillus. Chantemesse,1 Pope,2 and Steele,3 have all used serums from animals immunized to typhoid cultures for the treatment of typhoid fever, with more or less success. An analysis of the results will, however, show them to be very inconclusive. One of the most important and practical points for the physician to grasp in relation to the subject of typhoid fever is the highly virulent character of the discharges, both feces and urine. In every case the greatest care should be taken for their proper disinfection, a rigid attention paid to all the details of cleanliness in the sick- room, and the careful sterilization of all articles which are soiled by the patient. If country practitioners were as careful in this particular as they should be, the disease would be much less frequent in regions remote from the filth and squalor of large cities with their unmanageable slums, and the distribution of the bacilli to villages and towns, by watercourses polluted in their infancy, might be checked. 1 Gaz. des Hopitaux, 1898, lxxi., p. 397. 3 Brit. Med. Jour., 1897, i., 259. s Ibid., Apr. 17, 1897. CHAPTER III BACILLI RESEMBLING THE TYPHOID BACILLUS. Bacillus Coli Communis. The Bacillus coli was first isolated from human feces in 1885 by Emmerich,1 who thought that it was the spe- cific cause of Asiatic cholera. Many investigators have since studied its peculiarities, until at the present time it is one of the best-known bacteria. It is habitually present in the fecal matter of most ani- mals except the horse, and in water and soil contaminated « \ Fig. 114. — Bacillus coli communis, from an agar-agar culture; x 1000 (Itzerott and Niemann). with it. With water or dust it gains entrance into the mouth, where it can frequently be found, and occurs accidentally in foods and drinks. During life the organ- ism sometimes enters wounds externally from the surface of the body or internally from the intestine, and is a cause of suppuration — or at least occurs in the pus. The Bacillus pyogenes foetidus of Passet is almost certainly identical with it. 1 Deutsche mid. Wochenschrift, 1885, No. 2. 504 BACILLI RESEMBLING TYPHOID BACILLUS. 505 The bacillus is rather variable culturally, and is some- what polymorphic. Probably both size aud form depend to a certain extent upon the culture-medium on which it grows. On the average, it measures 1-3 X 0.4-0.7/*. It usually occurs in the form of short rods, but very short coccus-like elements and quite elongate forms are often found in the same culture. The individual bacilli are frequently isolated or in pairs. Chains are the ex- ception. They are provided with flagella, which are very variable in number, generally from four to a dozen, though there may be more. It forms no spores. The bacillus stains well with the ordinary aqueous solutions of the anilin dyes, but does not retain the stain after immersion in Grain's solution. The bacillus is motile, though in this particular it is subject to irregularity, the organisms from some cultures Fig. 115. — Bacillus coli communis: superficial colony two days old upon a gelatin plate; x 21 (Heim). always swimming actively, even when the culture is some days old, others being exceedingly sluggish even when young and actively growing, and a few cultures seem to consist of bacilli that do not move at all. Fresh cultures which, when grown at incubation temperature, consist of entirely non-motile bacteria are probably Bacil- lus coli immobilis, not Bacillus coli communis. The bacillus is readily cultivated upon the ordinary 506 PATHOGENIC BACTERIA. media. Upon gelatin plates the colonies develop in twenty-four hours. Those situated below the surface appear round, yellow-brown, and homogeneous. As they grow older they increase in size and become opaque. The superficial colonies are larger and spread out upon the surface. Their edges are dentate and resemble grape- leaves, often showing radiating ridges suggestive of the veins of a leaf. They may have a slightly concentric appearance. The colonies rapidly increase in size and become more and more opaque. The gelatin is not liquefied. In gelatin punctures the culture, developing rapidly upon the surface, and also in the needle's track, causes the formation of a nail-like growth. The head of the nail may reach the walls of the test-tube. Not infre- quently gas is formed in ordinary gelatin, and when i per cent, of glucose is dissolved in the medium the gas- production is often so copious and rapid as to form large bubbles, which by their distention subsequently break it up into irregular pieces. Sometimes the gelatin becomes slightly clouded as the bacilli grow. Upon agar-agar along the line of the inoculation a grayish-white, translucent, smeary growth takes place. It is devoid of any characteristics. The entire t surface of the culture-medium is never covered, the. growth re- maining confined to the inoculation-line, except where the moisture of the condensation-fluid allows it to spread out at the bottom. Kruse says that in old cultures crys- tals may form. Bouillon is soon evenly clouded by the development of the bacteria. Sometimes a delicate pellicle forms upon the surface. There is rarely much sediment in the cul- ture. Wiirtz found that the bacillus produced ammonia in culture-media free from sugar, and thus caused an intense alkaline reaction in the culture-media. The cultures usually give off an odor that varies somewhat, but is, as a rule, unpleasant. BACILLI RESEMBLING TYPHOID BACILLUS. 507 Indol is formed in both bouillon and pepton solu- tions. Phenol is not produced. Litmus added to the culture-media is ultimately decolorized by the bacilli. The presence of indol is probably best determined by Salkowski's method. To the culture 1 c.cm. of a 0.02 per cent, aqueous solution of potassium nitrate aud a few drops of concentrated sulphuric acid are added. If. a rose color develops, indol is present. Nitrates are reduced to nitrites by the growth of the bacillus. Upon potato the growth is luxuriant. The bacillus forms a yellowish-brown, glistening layer spreading from the line of inoculation over about one-half to two-thirds of the potato. The color shown by the potato-cultures varies considerably, sometimes being very pale, some- times quite brown. It cannot, therefore, be taken as a characteristic of much importance. Sometimes the po- tato becomes greenish in color. Sometimes the growth on potato is almost invisible. In milk there are rapid coagulation and acidulation, with the evolution of much gas. . The bacillus seems to require very little nutriment. It grows in Uschinsky's asparagin solution, and is frequently found living in river and well waters. It is quite resistant to antiseptics and germicides, and grows in culture-media containing from 0.1-0.2 per cent. of carbolic acid. It lives for months upon artificial media. The bacillus begins to penetrate the intestinal tissues almost immediately after death, and is the most frequent contaminating micro-organism met with in cultures made at autopsy. Exactly how it penetrates the tissues is not known. It may spread by direct continuity of tissue, or via the blood-vessels. While under normal conditions a saprophytic bacte- rium, the colon bacillus is far from harmless. It not infrequently is found in the pus of abscesses remote from the intestine, and is almost always found in suppura- 508 PATHOGENIC BACTERIA. tions connected with the intestines, as, for example, appendicitis. It is a question whether the colon bacillus is always virulent, or whether it becomes virulent under abnormal conditions. Klencki1 found that it was very virulent in the ileum, and less so in the colon and jejunum, espe- cially in dogs. He also found that the virulence was greatly increased in a strangulated portion of intestine. Other observers, as Dreyfuss, found that the colon bacil- lus as it occurs in normal feces is non-pathogenic. Most experimenters, however, believe that pathological con- ditions, such as disease of the intestine, ligation of the intestine, etc., cause increased virulence. Adelaide Ward Peckham, in an elaborate study of the " Influence of Environment on the Colon Bacillus,"2 con- cludes that while the conditions of nutrition and develop- ment in the intestine seem to be most favorable, the colon bacillus is ordinarily not virulent, because " its first force is spent upon the process of fermentation, and as long as opportunities exist for the exercise of this function the affinities of this organism appear to be strongest in this direction. "Moreover, the contents of the intestine remain acid until they reach the neighborhood of the colon, and by that time the tryptic peptons have been formed and absorbed to a great extent. "During the process of inflammation in the digestive tract a very different condition may exist. The peptic and tryptic enzymes may be partially suppressed. Fermenta- tion of carbohydrates and proteid foods then begins in the stomach, and continues after the mass of food is passed on into the intestine. The colon bacillus cannot, therefore, spend its force upon fermentation of sugars, because they are already broken up and an alkaline fer- mentation of the proteids is in progress. It also cannot form peptons from the original proteids, for it does not 1 A tin. de I 'Inst. Pasteur, 1895, No. 9. 2 Journal of Experimental Medicine, Sept., 1897, vol. ii., No. 4, p. 549. />'. ICILLI RESEMBLING TYPHOID BACILLUS. 509 possess this property, and unless trypsin is present it must be dependent upon the proteolytic activity of other bac- teria for a suitable form of proteid food. Perhaps these bacteria form an albuminate molecule, which like leucin and tyrosin cannot be broken up into indol, and thus there might be caused an important modification of the metabolism of the colon bacillus, which might have either an immediate or remote influence upon its acquisi- tion of disease-producing properties, for our own experi- ments indicate that the power to form indol, and the actual forming of it, are to some extent an indication of the possession of pathogenesis." To the laboratory animals the colon bacillus is patho- genic in varying degree. Intraperitoneal injections into mice cause their death in from one to eight days if the culture is virulent. Guinea-pigs and rabbits also suc- cumb to intraperitoneal and intravenous injection. Sub- cutaneous injections are of less effect, and in rabbits seem to produce abscesses only. When the bacilli are injected into the abdominal cavity a sero-fibrinous or purulent peritonitis occurs, the bacilli being very numerous in the abdominal fluids. The pathogeny of the colon bacillus is due to irritating, chemotactic substances in its protoplasm. The experi- ments of PfeifFer and Kolle and Loffler and Abel have proved very conclusively that the poisonous principle is in, and cannot by any means be separated from the bodies of the bacteria. Frequent transplantation lessens the virulence, passage through animals increases it. Numerous observers have found that cultures of the bacillus obtained from cholera, cholera nostras, and other intestinal diseases are much more pathogenic than those obtained from normal feces or from pus. Cumston,1 from a careful study of thirteen cases of sum- mer infantile diarrheas, comes to the following conclu- sions: 1 International Medical Magazine, Feb., 1 897. "5 io PA THOGENIC BA CTERIA . The bacterium coli seems to be the pathogenic agent of the greater number of summer infantile diarrheas. This organism is the more often associated with the streptococcus pyogenes. The virulence, more considerable than in the intestine of a healthy child, is almost always in direct relation to the condition of the child at the time the culture is taken, and does not appear to be proportional to the ulterior gravity of the case. The mobility of the Bacterium coli is in general pro- portional to its virulence. The jumping movement, nevertheless, does not correspond to an exalted virulence in comparison with the cases in which the mobility was very considerable, without presenting these jumping movements. The virulence of the Bacterium coli found in the blood and other organs is identical with that of the Bac- terium coli taken from the intestine of the same indi- vidual. Lesage,1 in studying the enteritis of infants, found that in 40 out of 50 cases depending upon the Bacillus coli the blood of the patient agglutinated the cultures ob- tained, not only from his own stools, but from those of all the other cases. From this uniformity of action Le- sage very properly suggests that the colon bacilli in these cases are all of the same species. The agglutinating reaction occurs only in the early stages and acute forms of the disease. It is not difficult to immunize an animal against the colon bacillus. Loffler and Abel immunized dogs by progressively increased subcutaneous dosage of live bac- teria, grown in solid culture and distributed through water. The injections at first produced hard swellings. The blood of the immunized animals possessed an active bactericidal influence upon the colon bacteria. It was not in the correct sense antitoxic. In intestinal diseases, such as typhoid, cholera, and 1 Semaine Medicale, Oct. 20, 1897. BACILLI RESEMBLING TYPHOID BACILLUS. 51 1 dysentery, the bacillus not only seems to acquire an un- usual degree of virulence, but because of the existing denudation of mucous surfaces, etc., finds it easy to enter the general system, with the result of secondary remote suppurative lesions in which it is the essential factor. When absorbed from the intestine it frequently enters the kidney and is excreted with the urine, causing, inci- dentally, local inflammatory areas in' the kidney, and occasionally cystitis. A case of urethritis is reported to have been caused by it. In infants cholera infantum may not infrequently be caused by the colon bacillus, though probably in this disease other bacteria play a very important role. The bile-ducts are very often invaded by the bacillus, which may cause inflammation, obstruction, suppuration, or calculous formation. The bacillus has also been met in puerperal fever, Winckel's disease of the new-born, endocarditis, menin- gitis, liver-abscess, bronchopneumonia, pleuritis, chronic tonsillitis, and urethritis. For the determination of the colon bacillus the im- portant points are the motility, the indol reaction, the milk-coagulation, and the active gas-production. As, however, all of these features are shared by other bac- teria to a greater or less degree, the only positive differ- ential point upon which very great reliance can be placed is the immunity-reaction of the serum of an immunized animal, which not only protects susceptible animals from the effects of inoculation, but produces with fresh cul- tures of the bacillus exactly the same reaction as that observed in connection with the blood and serum of typhoid patients, and convalescents and immunized ani- mals. This reaction has been considered at length in speaking of typhoid fever. For the few who are convinced that the colon and typhoid bacilli are identical, the fact that the typhoid serum is specific for the typhoid bacillus, and the colon serum for the colon bacillus, with rare exceptions. 512 PA THOGENIC BA C TERIA . should be important evidence of their separate individ- uality. I have no doubt that the Bacillus coli communis is not a single species of bacteria, but is a name ap- plied to a group whose individual differences are too similar to enable us to differentiate them. This opin- ion seems to be shared by other bacteriologists, some of whom have attempted a separation into groups, types, or families. In order to establish a type species of the Bacillus coli communis, Smith1 says: " I would suggest that those forms be regarded as true to this species which grow on gelatin in the form of deli- cate, bluish, or more opaque, whitish expansions with irregular margin, which are actively motile when exam- ined in the hanging drop from young surface-colonies taken from gelatin plates which coagulate milk within a few days; grow upon potato, either as a rich-pale or brownish-yellow deposit, or merely as a glistening, barely recognizable layer, and which give a distinct indol reac- tion. Their behavior in the fermentation-tube must conform to the following scheme: ' ' Variety a : "One per cent, dextrose-bouillon (at 370 C). Total gas approximately }4\ HC02 approximately yi\ reaction strongly acid. " One per cent, lactose-bouillon: as in dextrose-bouil- lon (with slight variations). "One per cent, saccharose-bouillon; gas-production slower than the preceding, lasting from seven to four- teen days. Total gas about 2/3; HC02 nearly %. The final reaction in the bulb may be slightly acid or alkaline, according to the rate of gas-production. "Variety ft: " The same in all respects, excepting as to its behavior in saccharose-bouillon; neither gas nor acids are formed in it." 1 American "Journal of the Medical Sciences, 1895, IIO, p. 287. BACILLI RESEMBLING TYPHOID BACILLUS. 513 Characteristics for Differentiation. Typhoid Bacillus. Bacilli usually slender. Flagella numerous (10-20), long, and wavy. Growth not very rapid, not particularly luxuriant. Upon Eisner's culture-medium de- velops slowly, the colonies remain- ing small. Upon fresh acid potato the so-called " invisible growth " formerly thought to be differential. Acid-production in whey not exceed- ing 3 per cent. Sometimes slight in ordinary media, and sometimes succeeded by alkaline production. Grows in media containing sugars without producing any gases. Produces no indol. Growth in milk unaccompanied by coagulation. In Maassen's asparagin-glycerin solu- tion the bacillus does not grow. Gives the Widal reaction with the serum of typhoid blood. Colon Bacillus. Bacilli inclined to be a little thicker. Flagella fewer (8-10). Growth rapid and luxuriant. This character is by no means constant. Upon Eisner's medium develops more rapidly, the colonies being larger. (Sometimes the colonies are small and remain so.) Upon potato a brownish-yellow, dis- tinct pellicle. Acid-production well marked. Gas-production well marked. Indol-production marked. Milk coagulated. Grows in Maassen's solution. Does not react with typhoid blood. Bacillus Enteritidis (Gartner1). This bacillus was cultivated by A. Gartner from the flesh of a cow slaughtered because of an intestinal disease, and from the spleen of a man that had been poisoned by eating meat obtained from it. The bacillus was subsequently encountered in other cases of meat-poi- soning by Karlinski and Lubarsch. The bacillus closely resembles the Bacillus coli com- munis. It is short and thick, is partly surrounded by a capsule, is actively motile, and stains irregularly with the ordinary solutions, but not by Gram's method. It has no spores. Upon gelatin plates it forms round, pale-gray, translucent colonies. It does not liquefy gela- tin. The deep colonies are brown and spherical. The 1 Kor respond, d. Allg. Arztl. Ver. von Thuring, 1888, 9. 33 5 14 PATHOGENIC BACTERIA. growth on agar-agar is similar to that of the colon bacil- lus. The organism produces no indol. It coagulates milk in a few days. Its fermentative powers have not been sufficiently studied. It reduces litmus. Upon potato it forms a yellowish-white, shining layer. The bacillus is pathogenic for mice, guinea-pigs, pig- eons, lambs, and kids, but not for dogs, cats, rats, or sparrows. The infection may be fatal, whether given subcutaneously or intraperitoneally, for mice and guinea- pigs, and may sometimes be fatal when the bacilli are ingested. The bacilli are found scattered throughout the organs in small groups, resembling those of the typhoid bacillus. At the autopsy a marked enteritis and swelling of the lymphatic follicles and patches, and occasional hemor- rhages, are found. The bacilli occur in the intestinal contents. The spleen is somewhat enlarged. The bacillus is differentiated from the colon bacillus chiefly by the absence of indol-production, by its ability to produce infection when ingested, and by the fact that it elaborates a toxic substance capable of producing sim- ilar symptoms to those seen in the infection. It may be distinguished from the Bacillus lactis aerogenes by its motility. It is with great difficulty separable from certain water bacteria ; but as far as is known its pathogenesis can be made use of for, assisting in the differentiation in doubtful cases. Bacillus Dysentericus (Shiga1). As the result of considerable investigation of the epi- demic dysentery which has been very prevalent in Japan, Shiga has come to the conclusion that a bacillus which he calls the Bacillus dysentericus is the specific cause. The organism is a short rod, having morphological characteristics identical with those of the typhoid and colon bacilli. Like the typhoid bacillus, it is prone to undergo involution. It is usually solitary, but may form 1 Centralbl.f. Bakt. u. Parasitenk., 1S98, xxiv., Nos. 22-24. BACILLI RESEMBLING TYPHOID BACILLUS. 515 pairs. When stained with methylene-blue the ends color deeper than the middle; and organisms from old, in- voluted cultures color irregularly. It does not stain by Gram's method, has no observable spores, is not motile, and has no demonstrable flagella. It does not liquefy gelatin. It grows slowly at ordinary temperatures; rapidly at the temperature of the body. The culture- media should be alkaline. Its death-point is 68° C, after twenty minutes' exposure. . The colonies upon gelatin plates are small, dewdrop- like in appearance, and upon microscopic examination are seen to be of regular outline and spherical form. By transmitted light they appear granular and of a yellowish color. The colonies do not spread out in a thin pellicle like those of the colon bacillus, and there are no essential differences between the superficial and deep colonies. The puncture culture in gelatin consists of crowded, rounded colonies forming a grayish-white growth along the puncture. There is no liquefaction. Upon the surface of agar-agar in the incubating-oven large, solitary colonies are evident at the end of twenty- four hours. They are of bluish-white color and rounded form. The surface appears moist. In the course of forty-eight hours a transparent border is observed about each colony, and the bacilli of which it is composed cease to stain evenly, presenting involution-forms. Glycerin agar-agar seems less well adapted to their growth than plain agar-agar. Blood-serum is not a suit- able medium. In gelatin and agar-agar containing sugars no gas is evolved. Upon boiled potato the young growth resembles that of the typhoid bacillus, but after twenty-four hours it becomes yellowish -brown, and at the end of a work forms a thick brownish-pink pellicle. In bouillon the bacillus grows well, clouding the liquid. No pellicle forms on the surface. The organism does not form indol. Acids are produced 516 PATHOGENIC BACTERIA. in moderate quantities after twenty-four hours. Milk is not coagulated. The blood-serum of those suffering from the dysentery or recovered from it causes a well-marked agglutinative reaction. This agglutination has been further studied by Flexner, and is thought to be specific and useful for diagnosis. By the progressive immunization of horses to an im- munizing fluid, the basis of which is a twenty-four hour old agar-agar culture dried in vacuo, Shiga has prepared, an antitoxic serum, with which, in 1898, 65 cases were treated, with a death-rate of 9 per cent. ; in 1899, 91 cases, with a death-rate of 8 per cent. ; in 1899, no cases, with a death-rate of 12 per cent. These results are very significant, as the death-rate in 2736 cases simultaneously treated without the serum averaged 34.7 per cent., and in consideration of the frequency and high death-rate of the disease, Japan alone, between the years 1878 and 1899, furnishing a total of 1,136,096 cases, with 275,308 deaths (a total mortality for the entire period of 24. 23 per cent.).1 The epidemic dysentery is apparently a different disease from the amebic form, the chief points of dissimilarity being that the extensive undermined ulcerations, de- scribed by Councilman and Lafleur, are rarely observed. The follicular, pustule-like ulcer is very uncommon; per- foration is unusual, the muscular coat offering strong re- sistance to the disease-process. Abscess of the liver is also rare in epidemic, though common in amebic, dysen- tery. It is not improbable that the bacillus of Shiga is identical with the Bacterium colt, variety dysenteries, of Celli, Fioca, and Scala.1 1 Public Health Reports, Jan. 5, 1900, vol. xv., No. I. 1 Hygien. Institut. Rom. Univ., 1895, anc* Centralbl. f. Bake. 11. Parasitenk.y 1899. BACILLI RESEMBLING TYPHOID BACILLUS. 517 Bacillus F^calis Alcaligenks ( Petruschky 1 ) . This bacillus lias been occasionally isolated from feces by Petruschky and others. It closely resembles the typhoid bacillus, being short and stout, with round ends ; forming spores ; staining with the usual dyes, but not by Gram's method, being actively motile, and hav- ing numerous flagella. It grows similarly upon gelatin, which it does not liquefy. It does not coagulate milk, produce gas, nor form indol. Its pathogenic powers are similar to those of the typhoid bacillus. It grows more luxuriantly than the typhoid bacillus upon potato, producing a brown color, and produces a strong alkali when grown in litmus-whey. Its cultures are not agglutinated by the typhoid serums. 1 Centralbl. fur Bakt. und Parasiienk., xix., 187. CHAPTER IV. YELLOW FEVER. Bacillus Icteroides (Sanarelli1). The bacteriology of yellow fever has been studied by Domingos Freire, Carmona y Valle, Sternberg, Havel- burg, and most recently by Sanarelli. Sternberg, at the Tenth International Medical Con- gress (Berlin, 1890), reported the study of 42 yellow fever autopsies in which aerobic and anaerobic cultures were made from the blood, liver, kidney, urine, stomach, and intestines, but the specific infectious agent was not found, and the most approved bacteriological methods failed to demonstrate the constant presence of any particular micro-organism in the blood and tissues of yellow fever cadavers. The micro-organism most frequently encoun- tered was the Bacillus coli communis. The most important micro-organism met with was Bacillus x (Sternberg), which was isolated by the culture- method from a considerable number of cases, and may have been present in all. It was not present in any of the control-experiments. It was very pathogenic for rab- bits when injected into the abdominal cavity. Sternberg says: " It is possible that this bacillus is concerned in the etiology of yellow fever, but no satisfactory evidence that this is the case has been obtained by experiments upon the lower animals, and it has not been found in such numbers as to warrant the inference that it is the veri- table infectious agent." The latest important researches upon yellow fever are those of Sanarelli and his followers.2 In studying the 1 Ann. de F Inst. Pasteur, June, 1897. 2 Brit. Med. Jour., July 3, 1897, and Ann. de I Inst. Pasteur, June, Sept., and Oct., 1897. 518 YELLOW FEVER. 519 cadavers of yellow fever Sanarelli found them either entirely sterile or universally invaded by certain microbic species, such as the Streptococcus pyogenes, the colon bacillus, the protei, etc., which cannot be the cause of the disease. In the second case he examined he was fortunate enough to find what he is satisfied is the specific microbe, the Bacillus icteroides. In 11 autopsies he never found the organism alone, but always associated with the bacteria mentioned above. The Bacillus icter- oides must be sought for in the blood and tissues, and not in the gastro-intestinal tract. In the latter it is never ySKi-r-;:-^ Fig. 116. — Bacillus icteroides (Sanarelli). found. The isolation of the specific microbe was possible only in 58 per cent, of the cases, and in some rare instances may be accomplished during life. The bacillus presents nothing morphologically charac- teristic. It is a small pleomorphous bacillus with rounded ends, usually united in pairs in the culture and in small groups in the tissues. It is 2-4 f* in length, and, as a rule, two or three times longer than broad (Fig. 116). The organism is actively motile, and has flagella. It does not form spores. It stains by the usual methods, but does not stain by Gram's method. It grows 520 PATHOGENIC BACTERIA. readily at ordinary temperatures, but best at 37 ° C. By employing suitable methods it can be found in the organs of yellow fever cadavers, usually united in little groups, always situated in the small capillaries of the liver, kid- ney, etc. The best method of demonstration is to keep a fragment of liver, obtained from a body soon after death, in the incubator at t>7° C. for twelve hours and allow the bacteria to multiply in the fresh tissue before examination. The bacillus can be cultivated upon the ordinary media. Upon gelatin plates it forms rounded, transpar- ent, granular colonies, which during the first three or four days present somewhat the appearance of leukocytes. The granular appearance becomes continuously more marked, and usually a central or peripheric nucleus, completely opaque, is seen. In time the entire colony becomes opaque, but does not liquefy gelatin. Stroke-cultures on obliquely solidified gelatin exhibit brilliant, opaque, little drops similar to drops of milk. In bouillon it develops slowly, without either pellicle or flocculi. The culture upon agar-agar is said to be characteristic. If grown at 370 C, the peculiar appearances of the colonies do not develop; but if the culture is kept at 200- 22° C, the colonies appear rounded, whitish, opaque, and prominent, like drops of milk. This appearance of the colonies shows well if the cultures are kept for the first twelve to sixteen hours at 370 C, and afterward at room- temperature, when the colonies will show a flat central nucleus, transparent and bluish, surrounded by a promi- nent and opaque zone, the whole resembling a drop of sealing-wax. Sanarelli refers to this appearance as con- stituting the diagnostic feature of Bacillus icteroides. It can be obtained in twenty-four hours. Upon blood serum the growth is very meagre. The growth upon potato corresponds to the classic description of that of the bacillus of typhoid fever. The bacillus is a facultative anaerobe. It slowly fer- YELLOW FEVER. 521 ments lactose, more actively ferments glucose and sac- charose, but is not capable of coagulating milk. In the cultures a small amount of indol is formed. It strongly resists drying, dies in water at 6o° C, and is killed in seven hours by the solar rays. It can live for a consider- able time in sea-water. The bacterium is pathogenic for the majority of the domestic animals. All mammals seem more or less sensitive to the pathogenic action of the bacillus; birds are often immune. Guinea-pigs are invariably killed by either intraperitoneal or subcutaneous injection of o. 1 can. White mice are killed in five days; guinea-pigs in eight to twelve days; rabbits in four to five days. The morbid changes present include splenic tumor, hy- pertrophy of the thymus, and adenitis. In the rabbit there are, in addition, nephritis, enteritis, albuminuria, hemoglobinuria, and hemorrhages into the body-cavities. The dog is the most susceptible animal. When it is injected intravenously the disease-process that results is almost immediately manifested with such violent symp- toms and such complex lesions as to recall the clinical and anatomical picture of yellow fever in the human being. The most prominent symptom in experimental yellow fever in the dog is vomiting, which begins directly after the penetration of the virus into the blood and con- tinues for a long time. Hemorrhages appear after the vomiting, the urine is scanty and albuminous, or there is suppression, which shortly precedes death. Once grave jaundice was observed. At the necropsy the lesions met are highly interesting, and are almost identical with those observed in man. Most conspicuous is the profound steatosis of the liver. The liver-cells, even when examined fresh, appear com- pletely degenerated into fat, this appearance correspond- ing to that found in fatal cases of yellow fever. The same result may be obtained by injecting the liver di- rectly or through the abdominal wall. The kidneys are the seat of acute parenchymatous nephritis, sometimes 522 PATHOGENIC BACTERIA. with marked fatty degeneration. The whole digestive tract is the seat of hemorrhagic gastro-enteritis comparable in intensity only to poisoning by cyanid of potassium. Experiments upon monkeys were also of interest, in- asmuch as they demonstrated the possibility of obtaining fatty degeneration more extensive than is observed in man. In one case the liver was transformed into a mass of fatty substance similar to wax. Goats and sheep are also very sensitive to the icteroid virus, and the lesions described also occur in them. The death of a yellow fever victim is the result of one of three causes: i. It may be due to the specific infection principally, when the Bacillus icteroides is found in the cadaver in a certain quantity and in a state of relative purity. 2. It may be due to the septicemias established during the course of the disease, the cadaver then presenting an almost pure culture of the other microbes. 3. It may be due in large measure to renal insufficiency, when the cadaver is found nearly sterile. The black vomit is due to the action of gastric acidity upon the blood which has extravasated in the stomach in consequence of the toxic products of the Bacillus icte- roides. The Bacillus icteroides produces a toxin the result of whose action corresponds to the essential symptoms of the disease. Animals immune to the infection, or only par- tially susceptible to it, are not much affected by the toxin. Susceptible animals, such as dogs, are profoundly affected. Ten to fifteen minutes after injecting the toxin the animals experience a general rigor; abundant lachryma- tion begins, followed by continued vomiting, first of food, then of mucus. In a short time the animals lie help- less and extended. Hematuria frequently occurs. If the dose be moderate, the dog recovers quickly from the violent attack; but if the quantity of toxin be very large or repeated on successive days, it finally succumbs, pre- senting the anatomical lesions already described as due to infection. YELLOW FEVER. 523 The proofs of the specificity of the Bacillus icteroides are not limited to the animal experiments quoted. Sana- relli also adduces five experimental inoculations upon men. These inoculations were not made with the bac- teria— /. e. were not infection experiments — but were made with the filtered sterile toxin, whose action could be more easily controlled. " The injection of the filtered cultures in relatively small doses reproduced in man typical yellow fever, accompanied by all its imposing anatomical and symptomatological retinue. The fever, congestions, hemorrhages, vomiting, steatosis of the liver, cephalalgia, collapse — in short, all that complex of symp- tomatic and anatomical elements which in their combina- tion constitute the indivisible basis of the diagnosis of yellow fever. This fact is not only striking evidence in favor of the specific nature of the Bacillus icteroides, but it places the etiological and pathologic conception of yel- low fever on an altogether new basis." The discovery of the Bacillus icteroides, and especially of its toxin, entirely changes our view of the pathology of the disease. Instead of being a disease of the gastro- intestinal tract, as one would conclude from the symp- toms, "all the symptomatic phenomena, all the functional alterations, all the anatomical lesions of yellow fever, are only the consequence of an eminently steatogenous, emetic, and hemolytic action of the toxic substances manufactured by the Bacillus icteroides." Readers interested in the study of yellow fever and the relationship of the Bacillus icteroides to the disease should not fail to read the critical papers upon the sub- ject by Novy.1 The mode by which the Bacillus icteroides enters the body to produce the disease has not been made out. The digestive and respiratory tracts are the most likely routes. Sanarelli points out that when it happens that a mould develops near the Bacillus icteroides, the products of 1 Medical News, Sept., 1898, pp. 331 and 360. 524 PATHOGENIC BACTERIA. material exchange of this hyphomycete or the transfor- mation effected by it, are sufficient to nourish the ba- cillus and enable it to live and multiply, whereas it would be otherwise condemned to a more or less early death. There seems to be no particular mould possessed of this power, as of six experimented upon all were capable of it. Sanarelli is of the opinion that in the holds of .ships and in damp places generally the presence of moulds favors the development of the Bacillus icteroides. About the same time that Sanarelli published his researches, Havelburg announced l the discovery of an entirely different bacillus. Without entering into a de- scription of Havelburg' s bacillus, which seems to be far from established in importance, it may be classified as an interesting member of the colon group of bacilli. In a lengthy and interesting review and comparison of Sanarelli's and his own work, Sternberg2 concludes that the Bacillus icteroides of Sanarelli is identical with the Bacillus x, which he had discovered in yellow fever cadavers as early as 1888. In a later paper3 Sanarelli discusses the validity of Sternberg's claim to priority of discovery, and points out a sufficient number of differences in the original descrip- tions of the organisms to establish conclusively the in- dividuality of the Bacillus icteroides. It would seem, from a careful consideration of the recent literature, that Havelburg had very little ground for considering his bacillus specific, and that it is not possible for Sternberg to establish the identity of the Bacillus x with the Bacillus icteroides, while at the same time Sanarelli's descriptions and arguments are convinc- ingly in favor of the accuracy of his own work and the specificity of his bacillus. Sanarelli's labors have not ceased with his careful study of the Bacillus icteroides, but have been carried into the 1 Ann. de r Inst. Pasteur, 1897. 2 Centra/6/, filr Bakt. und Parasitenk., Sept. 6, 1897, Bd. xxii., Nos. 6 and 7. 3 Ibid., Bd. xxii., Nos. 22 and 23, p. 668. YELLOW FEVER. 525 important field of serum-therapy. By careful manipula- tion he has succeeded in immunizing the horse and ox to large doses of the bacillus, injecting into a vein so as to prevent the intense local reaction, and has found that the serum of these animals has the power to protect guinea- pigs from lethal doses of the bacillus. He hopes that the serum will also be efficacious in the treatment of yel- low fever in the human being. Wasdin and Geddings, in the "Report of the Com- mission of Medical Officers detailed by authority of the President to Investigate the Cause of Yellow Fever," Washington, 1899, reach the following conclusions : 1. That the micro-organism discovered by Prof. Gui- seppe Sanarelli, of the University of Bologna, Italy, and named by him the " Bacillus icteroides," is the cause of yellow fever. 2. That yellow fever is naturally infectious to certain animals, the degree varying with the species ; that in some of the rodents local infection is very quickly fol- lowed by blood infection ; and that while in dogs and rabbits there is no evidence of this subsequent invasion of the blood, monkeys react to the infection the same as man. 3. That infection takes place by way of the respiratory tract, the primary colonization in this tract giving rise to the earlier manifestations of the disease. 4. That in many cases of the disease, probably a majority, the primary infection or colonization in the lungs is followed by a secondary infection, or a secondary colonization of this organism in the blood of this patient. This secondary infection may be complicated by coin- stantaneous passage of other organism into the blood, or this complication may arise during the last hours of life. 5. There is no evidence to support the theory advanced by Prof. Sanarelli that this disease is primarily a septi- cemia, inasmuch as cases do occur in which the Bacillus icteroides cannot be found in the blood or organs in which it might be deposited therefrom. 6. That there exists no causal relationship between the bacillus "*" of Sternberg and this highly infectious 526 PATHOGENIC BACTERIA. disease; and that this bacillus ".r" is frequently found in the intestinal contents of normal animals and of man, as well as in the urine and the bronchial secretion. 7. That, so far as your commission is aware, the Bacillus icteroides has never been found in the body other than of one infected with yellow fever ; and that whatever may be the cultural similarities between this and other micro-organisms, it is characterized by a spec- ificity which is distinctive. 8. That the Bacillus icteroides is very susceptible to influences injurious to bacterial life and that its ready control by the processes of disinfection, chemical and mechanical, is assured. 9. That the Bacillus icteroides produces in vitro as well as in vita a toxin of the most marked potency, and that from our present knowledge, there exists a reasonable possibility of the ultimate production of an antiserum more potent than that of Sanarelli. Archinard and Woodson and Archinard l found in 32 out of 39 autopsies a bacillus which quite accurately corresponded with the Bacillus icteroides. Careful studies of the agglutinating effects of the serum of yellow fever and other febrile affections were made upon this bacillus and the Bacillus icteroides, resulting in the observation that in 80 per cent, of the cases the blood of yellow fever cases or recent convalescents caused agglutinations of both bacilli in dilutions of 1 : 40. The blood of typhoid and dengue fevers does not cause the agglutinations ex- cept in rare instances. Agramonte,2 on the other hand, does not believe that the specific germ of yellow fever is yet discovered. His studies of Bacillus icteroides convince him that it is not concerned in the etiology of the disease, as he failed to find it in the blood of 16 out of 23 cases which he inves- tigated, and declares that he found it in cases other than yellow fever. 1 Ar. Y. Med. Journal, Jan. 28, 1899. 1 Medical News, Feb. 10, 1900, vol. lxxvi., No. 6. CHAPTER V. CHICKEN- CHOLERA. Bacillus Cholkr^f. Gallinarum. The barnyards of Europe, and sometimes of America, are occasionally visited by an epidemic disease which affects pigeons, turkeys, chickens, ducks, and geese, and causes almost as much destruction among them as the occasional epidemics of cholera and small-pox produce among men. Rabbit-warrens are also at times seriously affected by the epidemic. When fowls are ill with the disease, they fall into a condition of weakness and apathy which causes them to remain quiet, seemingly almost paralyzed, and ruffle up the feathers. The eyes are closed shortly after the illness begins, and the birds gradually fall into a stupor from which they do not awaken. The disease leads to a fatal termination in twenty-four to forty-eight hours. During its course there is profuse diarrhea, the very frequent fluid, slimy, grayish-white discharges containing numerous micro- organisms. The bacilli which are responsible for this disease were first observed by PerroncitoMn 1878, and afterward thor- oughly studied by Pasteur. They are short, broad bacilli with rounded ends, sometimes united to each other, with the production of moderately long chains (Fig. 117). Pasteur at first regarded them as cocci, because when stained with a penetrating anilin dye the poles stain intensely, but a narrow space between them remains almost uncolored. This peculiarity is very marked, and sharp observation is required to observe the outline of the intermediate substance. The bacillus does not form spores, and does not stain by Gram's method. When 1 Archiv f. Wissenschaftliche und Praklische Thierheilkunde, 1879. 527 528 PA THOGENIC BA CTERIA. examined in the living condition it is found to be non- motile.1 The bacillus readily succumbs to the action of heat and dryness. The cultures upon gelatin plates after about two days appear as irregular, small, white points. The deep colonies reach the surface slowly, and do not attain any considerable size. The gelatin is not lique- fied. The microscope shows the colonies to be irregularly Fig. 117. — Bacillus of chicken-cholera, from the heart's hlood of a pigeon; x 1000 (Frankel and Pfeiffer). rounded disks with distinct smooth borders. The color is yellowish-brown, and the contents are granular. Some- times there is a distinct concentric arrangement. In gelatin puncture-cultures a delicate white line occurs along the entire path of the wire. When viewed through a lens this line is seen to consist of aggregated mi- nute colonies. Upon the surface the development is 1 Most authorities state that the bacillus is not motile, but Thoinot and Mas- selin assert that it is so. Precis de Microbie, 2d ed., 1893. CHICKEN CHOLERA. 529 much more marked, so that the growth resembles a nail with a pretty good-sized flat head. If, instead of a punc- ture, the inoculation be made upon the surface of ob- liquely solidified gelatin, a much more pronounced growth takes place, and along the line of inoculation a dry, granular coating is formed. This growth is quite similar to that upon agar-agar and blood-serum, which growths are white, shining, rather luxuriant, and devoid of char- acteristics. No growth occurs in the absence of oxygen. Upon potato no growth occurs except at the incubation temperature. It is a very insignificant, yellowish-gray, translucent film. The introduction of cultures of this bacillus into the tissues of chickens, geese, pigeons, sparrows, mice, and rabbits is sufficient to produce fatal septicemia. Feeding chickens, pigeons, and rabbits with material infected with the bacillus is also sufficient to produce the disease with pronounced intestinal lesions. Guinea-pigs usually seem immune, though they succumb to very large doses, especially when given intraperitoneally. The autopsy shows that when the bacilli are intro- duced subcutaneously a true septicemia results, with the addition of a hemorrhagic exudate and gelatinous infil- tration at the seat of inoculation. The liver and spleen are enlarged; circumscribed, hemorrhagic, and infiltrated areas occur in the lungs ; the intestine shows an intense inflammation with red and swollen mucosa, and oc- casional ulcers following small hemorrhagic spots. Peri- carditis is of frequent occurrence. The bacilli are found in all the organs. If, on the other hand, the disease has been produced by feeding, the bacilli are chiefly to be found in the intestine. Pasteur found that when pigeons were inoculated into the pectoral muscles, if death did not come on rapidly, portions of the muscle {sequestrcz) underwent degeneration and appeared anemic, indurated, and of a yellowish color. The bacillus of chicken-cholera is one whose peculiar- ities can be made use of for protective vaccination. 34 53° PA THOGENIC BA CTERTA. Pasteur discovered that when cultures are allowed to remain undisturbed for several months, their virulence is greatly lessened, and new cultures planted from these are also attenuated. When chickens are inoculated with such cultures, no other change occurs than a local in- flammatory reaction by which the birds are protected against virulent bacilli. From this observation Pasteur worked out a system of protective vaccination in which fowls can first be inoculated with very weak, then with stronger, and finally with highly virulent cultures, with a resulting protection and immunity. Unfortunately, the method is too complicated to be very practical. Use has, however, been made of the ability of this bacillus to kill rabbits, and in Australia, where they are pests, they are being exterminated by the use of bouillon cul- ture. It is estimated that two gallons of bouillon culture will destroy 20,000 rabbits irrespective of infection by contagion. The bacillus of chicken-cholera seems not only to be specific for that disease, but seems able, when properly introduced into various other animals, to produce several different diseases. Indeed, no little confusion has arisen in bacteriology by the description of what is now pretty generally accepted to be this very bacillus under the various names of bacillus of rabbit-septicemia (Koch), Bacillus cuniculicida (Fliigge), bacillus of swine-plague (LofHer and Schiitz), bacillus of " Wildseuche" (Hiippe), bacillus of ' ' Biiffelseuche ' ' (Oriste-Armanni), etc. CHAPTER VI. HOG-CHOLERA. Bacillus Suipestifer (Salmon and Smith1). The bacillus of hog-cholera was first found by Salmon and Smith,1 but was for a long time confused with the bacillus of "swine-plague," which it closely resembles and with which it frequently occurs. It is a member of the group of which the Bacillus coli communis may be taken as a type. Since the careful studies of Smith,1 however, the claims of the discoverers that the bacillus of hog cholera is a separate and specific organism can hardly be doubted. Hog-cholera, or "pig typhoid," as the English call it, is a common epidemic disease of swine, which at times kills 90 per cent, of the infected animals, and thus causes immense loss to breeders. Salmon estimates that the annual losses from this disease in the United States range from $10,000,000 to $25,000,000. The disease is particularly fatal to young pigs. The symptoms are not very characteristic, and the animals often die suddenly without having appeared particularly ill, or after seeming ill but a few hours. The symptoms consist of fever (io6°-io7° F.), unwillingness to move, and more or less loss of appetite. The animals may ap- pear stupid and dull, and have a tendency to hide in the bedding and remain covered by it. The bowels may be normal or constipated at the beginning of the attack, but later there is generally a liquid and fetid diarrhea, abundant, exhausting, and persisting to the end. The eyes are congested and watery, the secretion drying and 1 Reports of the Bureau of Animal Industry, 1885-91. 2 Centra/6/, fur Bait, und Parasitenk., Bd. ix., Nos. 8, 9, and 10, March 2, 1897. 531 532 PATHOGENIC BACTERIA. gluing the lids together. The breathing is rapid, and there may be cough. Occasionally there is an eruption with crusts or scabs of various sizes on the skin, which is often congested. The animal becomes weak, stands with arched back and drawn abdomen, and walks with a weak, tottering gait. The course of this disease varies from one or two days to two or three weeks. hX. post-mortem examination petechias, ecchymoses, and extravasations of blood into the tissues are found to be com- mon and form one of the principal changes in the acute Fig Bacillus of hog-cholera, showing flagella. form of the disease. The spleen is enlarged to two or four times its normal size, and is soft and engorged with blood. The extravasations of blood are common in the lym- phatic glands, beneath the serous membranes of the thorax and abdomen, and particularly along the intes- tines; on the surface of the lungs and kidneys and in their substance. The contents of the intestine are some- times covered with clotted blood. In the subacute form of the disease the principal changes are found in the large intestine, and consist of ulcers which appear as circular, slightly projecting masses varying in color from HOG-CHOLERA. 533 yellowish to black. Occasionally these ulcers are slightly depressed in outline. When cut across they are found to consist of a firm, solid growth extending nearly through the intestinal wall. They are most frequent in the cecum, upper half of the colon, and on the ileocecal valve. In the chronic form of the disease the spleen is rarely enlarged. "In hog-cholera the first effect of the disease is believed to be upon the intestines, with secondary inva- sion of the lungs." The most characteristic lesions of the disease are the petechias and ecchymoses, the ulcerations of the large intestine (Fig. 118), and the collapse and occasional bron- chopneumonic changes in the lung. The kidneys are nearly always affected, the urine con- taining albumin and tube-casts. The specific bacillus of hog-cholera was secured by Smith from the spleens of more than 500 hogs. It occurs in all the organs and has also been cultivated from the urine. The organisms appear as short rods with rounded ends, 1. 2-1. 5 // long and 0.6-0.7 il *n breadth. They are very actively motile. No spore-prod 11 ction has ever been observed. In general the bacillus resembles in appearance that of typhoid fever. It stains readily by the ordinary methods, but not by Gram's method. The bacilli possess numerous long flagella, easily demonstrable by the usual methods of staining (Fig. 119). No trouble is experienced in cultivating the bacilli, which grow well in all the media. Upon gelatin plates the colonies become visible in twenty-four to forty-eight hours; the deeper ones spher- ical with sharply defined borders. The surface is brown- ish by reflected light, and is without markings. They are rarely larger than 0.5 mm. in diameter and are homo- geneous throughout. The superficial colonies have little tendency to spread upon the gelatin. Their borders may 534 PATHOGENIC BACTERIA. be circular and rounded, or irregular. They are said rarely to reach a greater diameter than 2 mm. The gela- tin is not liquefied. There is nothing distinctly charac- teristic about the appearance of the colonies. Upon agar-agar the superficial colonies attain a diam- Fig. 119. — Ulceration of the intestine in a typical case of swine-fever (Crookshank). eter of 4 mm. and have a gray translucent appearance with polished surface. They are round and slightly arched. In gelatin punctures the growth takes the form of a nail with a flat head. There is nothing characteristic about it. The growth in the puncture shows it to be an optional anaerobe. HOG-CHOLERA. 535 Linear cultures upon agar-agar present a translucent, rather circumscribed, grayish, smeary layer. Upon potato a yellowish coating is formed, especially when the culture is kept in the thermostat. Bouillon made with or without pepton is clouded in twenty-four hours. When the culture is allowed to stand for a couple of weeks without being disturbed a thin surface-growth can be observed. Milk is an excellent culture-medium, but is not visibly changed by the growth of these bacteria. Its reaction remains alkaline. The hog-cholera bacillus is a copious gas-producer, capable of breaking up sugars into C02, H, and an acid, which, formed late, eventually checks its further devel- opment. No indol and no phenol are formed in the culture-media. The bacillus is hardy. Smith found it vital after being kept dry for four months. It ordinarily dies sooner, how- ever. The thermal death-point is 540 C, maintained for sixty minutes. The bacillus is markedly pathogenic for animals. Small quantities introduced subcutaneously into rabbits or mice kill them in from seven to twelve days. The animal appears quite well for three or four days, then begins to sit quietly in the cage and eat but little, or refuses to eat at all, until death takes place. In Smith's experiments one-four-millionth of a cubic centimeter of a bouillon culture injected subcutaneously into a rabbit was sufficient to cause its death. Before death the temperature abruptly rises 2°-3° C, and re- mains high until death. Larger quantities may kill in five days. Injected intravenously in small doses the ba- cillus may cause death in forty-eight hours. When the animal is subjected to a postmortem exam- ination the spleen is found enlarged, firm, and dark red in color. The liver is found to contain small yellowish- white necrotic areas which sometimes occur in one, some- times in several acini, and not infrequently surround the 536 PATHOGENIC BACTERIA. interlobular veins. The kidneys are acutely inflamed and the urine is albuminous. The heart-muscle is spotted, gray, and fatty. In the intestinal tract the pic- ture of the disease will be found to vary according to its duration. The contents of the small intestine are yellowish, watery, and mucous; Peyer's glands are enlarged. In the neighborhood of the pylorus, ecchymoses and exten- sive extravasations of blood are common. The bacilli are found in all of the organs. The house mouse is very susceptible to the disease; guinea-pigs much less so, -^ c.cm. of a virulent cul- ture often being required to kill them. Pigeons are still more refractory, and Smith found that YA c.cm. of a bouillon culture injected into the breast-muscles was required to kill them. In spite of the fact that hog-cholera is a disease of swine, and that it is from dead swine that the bacilli are obtained, these animals are not very easily affected arti- ficially. They show no symptoms when injected subcu- taneously, but almost invariably die after intravenous injection of 1-2 c.cm. of a virulent culture. Smith found that feeding with 200-300 c.cm. of a bouillon culture after a day's fasting, or with small quan- tities administered daily, would also cause death, with a widespread diphtheritic inflammation of the stomach and colon. Feeding with the organs of dead, hogs produces the same lesions as the administration of the culture. As early as 1886 Salmon and Smith found it possible to produce, in both very and partly susceptible animals, immunity to hog-cholera by gradually accustoming them to increasing doses of the bacteria. DeSchweinitz iso- lated from cultures of the bacteria two toxic substances, a ptomain (sucholo-toxin) and an albumose (sucholo-albu- min), together with cadaverin and methylamin. With these substances he seems to have been able to produce immunity. Selander and Metschrrikoff found that im- munity could be produced more quickly by the use of HOG-CHOLERA. 537 blood of infected rabbits exposed to 580 C. This blood was found to be exceedingly toxic. DeSchweinitz ' found that the introduction of progress- ingly increased amounts of cultures into cows caused the development in them of an antitoxic substance capable of protecting guinea-pigs from the disease. After several years of treatment, some horses which I attempted to immunize for antitoxin formation failed to give serum powerful enough to be of use. To protect a rabbit against fatal infection required several cubic centi- meters of the blood. Working in my laboratory, Pitfield2 has found that after a single injection of a sterilized bouillon culture of the bacillus into the horse, the serum, which has origin- ally slight agglutinative reactive power, is so changed as to show a decided reaction. If the horse be immunized to large doses of such sterile cultures, the serum reaction becomes so marked that with a dilution of 1 : 10,000 a typical reaction occurs in sixty minutes. According to this experiment, in doubtful cases the use of this reaction should greatly facilitate the differen- tiation of the bacillus of hog-cholera from similar ba- cilli. 1 Cenlralbl. f. Bakt. u. Parasitenk., xx., p. 573. 2 Microscopical Bulletin, 1897, p. 35. CHAPTER VII. SWINE-PLAGUE. Bacillus Suisepticus. The bacillus of swine-plague, or the Bacillus suisepti- cus of Loffler and Schiitz,1 and Salmon and Smith,2 so closely resembles that of hog-cholera that it is easily confounded with it, and, indeed, at one time, they were thought to be identical. The species has, however, suf- ficient well-marked characters to make its differentiation clear (Fig. 120). Swine-plague is a rather common and exceedingly Fig. 120. — Bacillus of swine-plague (from photograph by E. A. de Schweinitz). fatal disease. It occurs alone or in combination with hog-cholera {q. v.), and because of the lack of suffi- ciently well-characterized symptoms — sick hogs appear- ing more or less alike — it is often mistaken for that disease. The confusion resulting from the mixed cases makes it impossible to determine exactly how fatal swine- plague may be in uncomplicated cases. 1 Arbeiten aus dem Kaiserlichen Gesundheitsamtes, I. 2 Zeitschrift fur Hygiene, x. 538 SWINE-PLAGUE. 539 The symptoms of swine-plague, while closely resem- bling those of hog-cholera, may differ from them in the existence of cough, swine-plague being prone to affect the lungs and oppress the breathing, which becomes frequent, labored, and painful, and associated with frequent cough, while hog-cholera chiefly presents intestinal symptoms. The course of the disease is usually rapid, a fatal result often occurring in one or two days. At autopsy the lungs are often found inflamed, and contain numerous small, pale, necrotic areas, and some- times large cheesy masses one or two inches in diameter. Inflammations of the serous membranes affecting the pleura, pericardium, and peritoneum, and associated with fibrinous inflammatory deposits on the surfaces, are com- mon. There may be congestion of the mucous mem- brane of the intestines, particularly of the large intestine, or the disease in this region may be an intense croupous inflammation with the formation of a fibrinous exudative deposit on the surface. A hemorrhagic form of the disease is said to be com- mon in Europe, but, according to Salmon, is rare in the United States. The bacillus of swine-plague much resembles that of hog-cholera, and not a little that of chicken-cholera. It is a short organism, rather more slender than its con- geners, not possessed of flagella, and is incapable of move- ment and produces no spores. Its vitality is low, and it is easily destroyed. Salmon says that it soon dies in water or by drying, and that the temperature for its growth must be more constant and every condition of life more favorable than for the hog-cholera germ. This germ is said to be widely distributed in nature, and is probably present in every herd of swine, though not pathogenic except when its virulence has been increased or the resistance of the animals diminished by some un- usual conditions. In its growth the bacillus of swine-plague is an optional anaerobic organism. 54° PATHOGENIC BACTERIA. In general, its appearance in culture-media is very similar to that of the bacillus of hog-cholera. Kruse, however,1 points out that when the bacillus grows in bouillon the liquid remains clear on account of the for- mation of a flocculent, stringy sediment. Upon ordi- nary acid potato the bacillus does not grow, but if the reaction of the medium be alkaline a grayish-yellow patch is formed. In its growth in milk slight acidity is pro- duced, but the milk is not coagulated and the litmus color added to it is not decolorized. The bacillus stains by the ordinary methods, some- times only at the poles, then resembling very closely the bacillus of chicken-cholera. It is not colored by Gram's method. The pathogenesis, while similar to that of the hog- cholera bacillus, presents some marked differences, espe- cially in regard to the seat of the local manifestations, to which attention has already been called, and in the duration of the disease, which is much shorter. There is also considerable resemblance to the bacillus of chicken- cholera in pathogenesis, but the local reaction following injection of the culture partakes of the nature of a hemor- rhagic edema, which is not present in chicken-cholera, and the cases often exhibit fatty metamorphosis of the liver. Rabbits, mice, and small birds are all very susceptible to the disease, generally dying of septicemia in twenty- four hours; guinea-pigs are less susceptible, except the very young animals, which die without exception. Chick- ens are more immune, but usually succumb to large doses. Hogs die after subcutaneous injection of the bacilli, and suffer from marked edema at the point of injection, and septicemia. If injected into the lung, a pleuropneumonia with multiple necrotic areas in the lung follows. In these cases the spleen is not much swollen, there is slight gastro-intestinal catarrh, and the bacilli are present every- where in the blood. Animals cannot be infected by feeding. 1 Fliigge's Mikroorganismen, p. 419, 1896. CHAPTER VIII. TYPHUS MURIUM. Bacillus Typhi Murium (Loffler1). The Bacillus typhi murium (Fig. 121), which created havoc among the mice in his laboratory, causing most of them to die, was discovered by Loffler in 1889. It is a short organism, somewhat resembling the bacillus of chicken-cholera. It is rather variable in its dimen- sions, and often grows into long, flexible filaments. No Fig. 121. — Bacillus typhi murium, from agar-agar; x iooo (Itzerott and Niemann). sporulatiou has been observed. It is a motile organism, with numerous flagella, like those of the typhoid-fever bacillus. It stains well with the ordinary dyes, but rather better with Loffler' s alkaline methylene blue. Upon gelatin plates the deep colonies are at first round, slightly granular, transparent, and grayish. Later they become yellowish-brown and granular. Superficial col- onies are similar to those of the typhoid bacillus. In 1 Centralbl.f. Bake. u. Parasitenk., xi., p. 129. 541 542 PATHOGENIC BACTERIA. gelatin punctures there is no liquefaction. The growth takes place upon the surface principally, where a grayish- white mass slowly forms. Upon agar-agar a grayish-white development devoid of peculiarities occurs. Upon potato a rather thin whitish growth may be observed after a few days. The bacillus grows well in milk, with the production of an acid reaction, but without coagulation. The organism is pathogenic for mice of all kinds, which succumb in from one to two days when inoculated subcutaneously, and in eight to ten or twelve days when fed upon material containing the bacillus. The bacilli multiply rapidly in the blood- and lymph-channels, and cause death from a general septicemia. Isomer expressed the opinion that this bacillus might be of use in ridding infested premises of mice, and the results of its use for this purpose have been highly satis- factory. He has succeeded in ridding a field so infested as to be useless for agricultural purposes by saturating some bread with bouillon cultures of the bacillus and distributing it near the holes inhabited by the mice. The bacilli that were eaten by the mice not only killed them, but also infected others which ate the dead bodies of the first victims, and so the extermination progressed until scarcely a mouse remained in the field. In discuss- ing the practical applicability of the employment of cul- tures of this bacillus for the destruction of field-mice, Brunner1 calls attention to certain conditions that are requisite for a satisfactory result, (i) It is necessary, first of all, to attack rather extensive areas of the invaded territory, and not to attempt to destroy the mice of a small field into which an indefinite number of fresh animals may immediately come from the surrounding fields. The country-people, who are the sufferers, should combine their efforts so as to extend the benefits widely. (2) The preparation of the cultures is a matter of im- 1 Centralbl. f. Bakt. u Parasitenk., Jan. 19, 1898, Bd. xxiii., No. 2. TYPHUS MURIUM. 543 portance. Agar-agar cultures are best, as being most readily transportable. They are broken up in water and well stirred, and the liquid poured upon a large num- ber of small pieces of broken bread. These are next to be distributed with reasonable care. Instead of being carelessly scattered over the ground, they should be dropped into the fresh mouse-holes, and pushed suffici- ently far in to escape the effects of sunlight upon the bacilli. Attention should be paid to holes in walls, under railway tracks, etc. and other places where mice live in greater freedom from disturbance than in the fields. (3) The attempted eradication of the mice should be begun at a time of year when the natural food is not plenty. By observing these precautions the mice can be eradicated with certainty, usually in a period of time not exceeding eight to twelve days. For this purpose, in the course of two years, no less than 250,000 cultures were distributed from the Bacteriological Laboratory of the Tierarznei Institut in Vienna. The bacilli are not pathogenic for the animals, such as the fox, weasel, ferret, etc. that feed upon the mice, do not affect man in any way, and so seem to occupy a useful place in agriculture by destroying the little but almost invincible enemies of the grain. CHAPTER IX. MOUSE -SEPTICEMIA. Bacillus Murisepticus (Koch1). In 1878, during his investigations upon the infectious traumatic diseases, Koch observed that when a minute amount of putrid blood or of meat-infusion was injected into mice the animals died of a septicemia caused by the multiplication in their blood of a minute bacillus to which he gave the name "Bacillus der Mausesepticamie" (Fig. 122). Fig. 122. — Bacillus of mouse-septicemia, from the blood of a mouse; x 1000 (Frankel and Pfeiffer). In 1885 the bacillus was again brought into promi- nence by L,6ffler and Schiitz, who found a very similar, 1 Wundinfektionskrankheiten, 1S78. 544 MOUSE-SEPTICEMIA. 545 perhaps identical, organism in the erysipelatous disease which attacks the swine of many parts of Europe. There seem to be certain slight morphological and developmental differences between these two organisms, but Baumgarten, Gunther, Sternberg, and others have regarded them as insufficient for the formation of sepa- rate species, and have boldly described the organisms as identical, while Lorenz has shown that immunity pro- duced in the rabbit by one bacillus protects against the other. The described differences are, indeed, so very small that I think it well to follow in the path of the ob- servers mentioned, pointing out in the description such points of difference as may arise. The bacilli are extremely minute, measuring about i.o x 0.2 fi (Sternberg). Fliigge, Frankel, and Eisenberg find the Bacillus erysipelas suis somewhat shorter and stouter than that of mouse-septicemia : there seems to be a division of opinion upon this point. Sporulation has been described by some observers, but nothing definite seems to be known upon this point. Motility is ascribed by some (Schottelius and Frankel) to the Bacillus erysipelas suis, and is denied to the bacillus of mouse-septicemia by others. The truth seems to be that the motility of both organisms is a matter of doubt. No flagella have been demonstrated upon the bacillus. It grows quite well both at the room-temperature and at the temperature of incubation. It can grow well with or without oxygen, but perhaps flourishes a little better without than with it. It is killed by a temperature of 52 ° C. in fifteen minutes. The colonies upon gelatin plates can first be seen on the second or third day, Fig. 123.— Colony then appearing as transparent grayish of the baclllus of 1 .,•■ 1 ■, j r mouse-septicemia: X specks with irregular borders, from go K which many branched processes extend (Fig. 123). Frankel describes them as resembling in shape the familiar branched cells occupying the lacunae 35 546 PATHOGENIC BACTERIA. of bone. When further developed the colonies flow together and give the plate a cloudy gray appearance. The gelatin is not liquefied, but is gradually softened and its evaporation thus aided. In gelatin puncture-cultures the growth is quite cha- racteristic, and the tendency of the bacillus to grow anaerobically is well shown (Fig. 124). The develop- Fig. 124. — Bacillus of mouse-septicemia: gelatin puncture-culture three and a half days old (Gunther). ment takes place all along the line of puncture, but is more marked below than at the surface. The growth takes place in a peculiar form, resembling superimposed disks, each disk separate from its neighbors and consist- ing of an area of clouded grayish gelatin reaching almost to the walls of the tube. This growth develops slowly, and causes a softening rather than an actual liquefaction of the gelatin. Upon agar-agar and blood-serum a very delicate, trans- parent grayish line develops along the path of the needle. It does not grow upon potato. The bacillus grows at the room temperature, but much better at the temperature of the incubator. The disease affects quite a variety of animals, notably hogs, rabbits, mice, white rats, pigeons, and sparrows. MOUSE-SEPTICEMIA. 547 The guinea-pig, which is generally the victim of labora- tory experiments, is not susceptible to it. Field- and wood-mice, cattle, horses asses, dogs, cats, chickens, and geese are immune. When mice are inoculated with a pure culture they soon become ill, lose their appetite, mope in a corner, and are not readily disturbed. As the disease becomes worse they assume a sitting posture with the back much bent; the eyelids are glued together by adhesive pus; and when death comes to their relief, in the course of forty to sixty hours after inoculation, they remain sitting in the same characteristic position. When the ears of rabbits are inoculated with the bacillus from cases of erysipelas suis, a violent inflam- matory edema and distinct redness occurs, much re- sembling erysipelas. This lesion gradually spreads, in- volves the head, then the body of the animal, and ulti- mately causes death. When swine are affected, they are dull and weak, and have a kind of paralytic weakness of the hind quarters. The temperature is elevated ; red patches appear upon the skin and swell and become tender. Death follows in two or three days. Sixty per cent, of the diseased animals die. In all animals the anatomical changes are much alike. The disease proves to be a septicemia, and the bacilli can be found in all the organs, especially the lungs and spleen. They are few in number in the streaming blood. As the organisms stain well by Grain's method, this stain is of great value for their discovery in the tissues, and can be highly recommended. Most of the bacilli occupy the capillary blood-vessels ; many of them are enclosed in leucocytes. The organs in such cases do not appear distinctly abnormal, except the spleen, which is considerably enlarged. The mesenteric and other lymphatics are also enlarged, and the gastric and intestinal mucous membranes are usually inflamed and mottled. The bacilli also occupy the intestinal con- 548 PATHOGENIC BACTERIA. tents, and Kitt, who discovered them in this position, points out that the infection of swine probably takes place by the entrance, along with the food, of the fecal matter of diseased animals into the alimentary apparatus of others. Pasteur, Chamberland, Roux, and others have worked upon a protective vaccination based upon the attenuation of the virulence of the organism by passing it through rabbits. Two vaccinations are said to be necessary to produce immunity. The vaccinated animals, however, may be a source of infection to others, and should always be isolated. ' Klemperer in 1892 found that the blood- serum of immunized rabbits would save infected mice into which it was injected. Lorenz in 1894 found an antitoxic substance in the blood of rabbits immunized to the disease. The effect of its injection into other animals is, however, only a temporary immunity. Later1 he found it possible to protect hogs against the disease by injecting them first with a serum obtained from a hog immunized in the ordinary manner described by Pasteur, afterward with a feeble culture of the bacillus, and finally with viru- lent cultures. The strength of the serum should be determined by injecting varying quantities of it into mice infected with definite amounts of a culture of known virulence. The immunity produced by Lorenz lasted for a year. 1 Centralbl.f. Bakt. u. Parasitenk., Jan., 1896, p. 168. CHAPTER X. RELAPSING FEVER. Spirillum Okkkmeikri (Obermeier '). As loug ago as 1873, Obermeier discovered that a flexible spiral organism, about o. 1 f* in diameter and from 20-40 ft in length, could be observed in the blood of patients suffering from relapsing fever. Although many of the best bacteriologists of our day have occupied themselves with the study of this spiril- lum, we really have, at present, very little more know- ledge than that given us by Obermeier. FlG. 125. — Spirochteta febris recurrentis; x 650 (Heim). The spirilla (Fig. 125) are generally very numerous, are long, slender, and flexible (spirochseta), and possess a vigorous movement by flagella. The ends are rather pointed. The spirillum stains well by ordinary methods, but not by Gram's method. It seems to be a strict parasite, and has never been cultivated artificially. Of the pathogenesis of the organism there can be no doubt, as it is invariably present in relapsing fever and 1 Centralbl.f. d. med. Wusenschaft., 1873. 549 550 PATHOGENIC BACTERIA. undergoes a peculiar cycle of changes according to the stage of the disease. During the pyrexia the organisms are found in the blood in active movement, swimming both by rotation on the long axis and by undulation. As soon as the crisis comes on they are found to be with- out motion, most of them enclosed in leucocytes and seemingly dead. The recurrence of the paroxysm has suggested to many that spores are formed in the spiril- lum, but no one has been successful in proving that this is the case. Koch, Carter, and Soudakewitch have all succeeded in giving the disease to monkeys, and Munch and Moczutkowsky have gone further and have produced it in men by introducing into them blood from diseased patients. Soudakewitch finds that the removal of the spleen causes the disease to terminate fatally in monkeys. CHAPTER XI. BUBONIC PLAGUE. Bacillus Pestis Bubonic* (Yersin,1 Kitasato*). Plague, or malignant polyadenitis, is an acute infec- tious febrile disease of an intensely fatal nature, charac- terized by inflammation of the lymphatic glands, marked cerebral and vascular disturbance, and the presence of the specific bacillus in the lymphatic glands and blood. The bubonic plague is' an extremely fatal disease, whose ravages in the hospital in which Yersin made his observations carried off 95 per cent, of the cases. The death-rate varies in different epidemics from 50 to 90 per cent. In the epidemic at Hong Kong in 1894 the death- rate was 93.4 per cent, for Chinese ; yy per cent, for Indians ; 60 per cent, for Japanese ; 100 per cent, for Eurasians, and 18.2 percent, for Europeans. It affects both men and animals, and is characterized by sudden onset, high fever, prostration, delirium, and the occur- rence of lymphatic swellings — buboes — affecting chiefly the inguinal glands, though not infrequently the axillary, and sometimes the cervical, glands. Death comes on in severe cases in forty-eight hours. If the case is of longer duration, the prognosis is said to be better. Autopsy in fatal cases reveals the characteristic enlargement of the lymphatic glands, whose contents are soft and sometimes purulent. Wyman in his very instructive pamphlet, "The Bu- 1 Ann. de V 'Inst. Pasteur, 1894, 9. 1 Preliminary notice of the bacillus of bubonic plague, Hong Kong, July 7, 1894. 551 552 PATHOGENIC BACTERIA. bonic Plague" (Government Printing Office, Washington, D. C. , 1900), finds it convenient to divide plague into {a) bubonic or ganglionic ; (b) septicemic ; and (c) pneu- monic forms. Of these the bubonic form is most frequent and the pneumonic form most fatal. The infection usually takes place through some periph- eral lesion, but may occur by inhalation of the specific organisms. The bacillus of bubonic plague (Fig. 126) seems to have met an independent discovery at the hands of •« r*1 ■*■•%. Fig. 126. — Bacillus of bubonic plague (Yersin). Yersin and Kitasato in the summer of 1894, during the activity of the plague then raging at Hong-Kong. There seems to be but little doubt that the micro-organisms described by the two observers are identical. The bacillus is short and thick — a cocco-bacillus, as some call it — with round ends. Its size is small {zft in length) and its form is subject to considerable variation. It not infrequently occurs in chains of four or six or even more, and is occasionally encapsulated. It shows active Brownian movements, which probably led Kitasato to consider it motile, while Yersin did not. Gordon ! found that some at least of the bacilli have flagella. It 1 Centralbl. f. Bakt. u. Parasitenk., Sept. 6, 1897, Bd. xxii., Nos. 6 and 7, p. 170. BUBONIC PLAGUE. 553 is an aerobic organism. No spores are formed. It stains well by the usual methods ; not by Gram's method. When stained the organism appears darker at the ends than at the centre, so as to resemble a dumb-bell ordiplo- coccns. The bacilli sometimes appear vacuolated and in old cultures show a variety of involntion-forms. Kitasato has compared the bacillus to that of chicken cholera. In his studies of plague, Ogata1 states that while Kitasato found the bacillus which he described in the blood of cadavers, Yersin seldom found his bacillus in Fig. 127. — Bacilli of plague and phagocytes; x 800. From human lymphatic gland (Aoyama). the blood, but always in the enlarged lymphatic glands. Kitasato's bacillus retains the color when stained by Gram's method; Yersin's does not. Kitasato's bacillus is motile; Yersin's, non-motile. The colonies of Kita- sato's bacillus when grown upon agar are round, irreg- ular, grayish-white with a bluish tint, and resemble glass- wool when slightly magnified; Yersin's bacillus forms white, transparent colonies with iridescent edges. Ogata, in the investigation of the cases that came into his hands, 1 Centralbl. f. Bakt. u. Parasitenk., June 24, 1 897, Bd. xxi., Nos. 20 and 21. 554 PATHOGENIC BACTERIA. found a bacillus that resembles that of Yersin, but not that of Kitasato, and it is certain that the description of Yersin is the more correct of the two. In the Japan Times, Tokio, Nov. 28, 1899, Kitasato explains that his investigations being made upon cadavers that were partly putrefied, he was led to believe that the bacillus first invaded the blood. Later studies upon living subjects showed him the error of this view and the cor- rectness of Yersin's observation that the bacilli first mul- tiply in the lymphatics. The studies of Kitasato and Yersin show that in blood drawn from the finger-tips and in the softened contents of the glands the bacillus may be demonstrable. When cultures are made from the blood or softened contents of the buboes the bacillus may be obtained in pure culture, and is found to develop upon artificial culture-media. In bouillon a diffuse cloudiness results from the growth, as observed by Kitasato, though in Yersin's observations the culture more nearly resembled erysipelas cocci, and contained zooglea attached to the sides and at the bottom of the tube of nearly clear fluid. According to Haffkine,1 when an inoculated bouillon culture is allowed to stand perfectly at rest, on a solid shelf or table, a characteristic appearance develops. In from twenty-four to forty-eight hours, the liquid remain- ing limpid, flakes appear underneath the surface, forming little islands of growth, which in the next twenty-four to forty-eight hours grow down into a long stalactite-like jungle, the liquid always remaining clear. In four or six days the islands are still more compact and solidified. If the vessel be disturbed, the islands fall like snow and are deposited at the bottom, leaving the liquid above clear. Upon gelatin plates at 22 ° C. the colonies may be ob- served in twenty-four hours by the naked eye. They are pure white or yellowish-white, spherical in the deep gela- tin, flat upon the surface, and are about the size of a 1 Brit. Med. Jour., June 12, 1897, p. 1461. BUBONIC PLAGUE. 555 pin's head. The gelatin is not liquefied. The borders of the colonies are, upon microscopic examination, found to be sharply defined and to become more granular as their age increases. The superficial colonies occasionally are surrounded by a fine, semi-transparent zone. In gelatin puncture-cultures the development is scant. The medium is not liquefied ; the growth takes place in the form of a fine duct, little points being seen on the surface and in the line of puncture. Sometimes fine filaments project into the gelatin from the central puncture. Upon agar-agar the bacilli grow freely but slowly, the colonies being whitish in color, with a bluish tint by re- flected light, and first appearing to the naked eye when cultivated from the blood of an infected animal after about thirty-six hours' incubation at 370 C. Under the microscope they appear moist, with rounded, uneven edges. The small colonies are said to resemble little tufts of glass-wool; the larger ones have large round centres. Microscopic examination of the bacilli grown upon agar-agar reveals the presence of long chains resem- bling streptococci. Upon glycerin agar the development of the colonies is slower, though in the end the colonies attain a larger size than those grown upon plain agar. Klein x says that the colonies develop quite readily upon gelatin made from beef-bouillon (not infusion), appearing in twenty-four hours, at 200 C, as small, gray, irregularly rounded dots. Magnification shows the col- onies to be serrated at the edges and made up of short, oval, sometimes double bacilli. Some colonies contrast markedly with their neighbors in that they are large, round, or oval, and consist of longer or shorter, straight or looped threads of bacilli. The appearance was much like that of the young colonies of the Proteus vulgaris. At first these were regarded as contaminations, but later he was led to believe that their occurrence was character- 1 Centralbl. f. Bakt. und Parasitenk., July 10, 1897, xxi., Nos. 24 and 25. 556 PATHOGENIC BACTERIA. istic of the plague bacillus. The peculiarities of these colonies cannot be recognized after forty-eight hours. Involution-forms on partly desiccated agar-agar not con- taining glycerin are said by Haff kine to be characteristic. The microbes swell up and form large, round, oval, pea- or spindle-shaped or biscuit-like bodies, which may attain twenty times the normal size and in growing gradually lose the ability to take up the stain. Such involution- forms are not seen in liquid culture. Hankin and Leumann l recommend for the differential diagnosis of the plague bacillus the addition of 2.5-3.5 per cent, of salt to the agar-agar. When transplanted from ordinary agar-agar to the salt agar-agar the involu- tion-forms which are so characteristic of the plague ba- cillus form with exceptional rapidity. In bouillon with this high precentage of salt the stalactite formation is very beautiful and characteristic. Upon blood-serum the growth at the temperature of the incubator is luxuriant. It forms a moist layer of a yellowish-gray color, and is unaccompanied by liquefac- tion of the serum. Upon potato no growth occurs at ordinary tempera- tures. When the potato is stood away for a few days in the incubator a scanty, dry, whitish layer develops. Abel found the best culture-medium to be 2 per cent, alkaline pepton solution with 1 or 2 per cent, of gelatin, as recommended by Yersin and Wilson. The bacillus develops under conditions of aerobiosis and anaerobiosis. In glucose-containing media it does not form gas. No indol is formed. Ordinarily the culture- medium is acidified by the development of an acid that persists for three weeks or more. By frequent passage through animals of the same species the bacillus increases very much in virulence. Curiously enough, however, the observations of Knorr, substantiated by Yersin, Calmette and Borrel, show that 1 Centralbl, f. Bakt. u. Parasitenk., Oct., 1897, Bd. xxii., Nos. 16 and 17, p. 438. BUBONIC PLAGUE. 557 the bacillus made virulent by frequent passage through mice is not increased in virulence for rabbits.1 Mice, rats, guinea-pigs, rabbits, monkeys, dogs, and cats are all susceptible to inoculation. During epidemics the purely herbivorous animals usually escape, though oxen have been known to die of the disease. When blood, lymphatic pulp, or pure cultures are inoculated into them, the animals become ill in from one to two days, according to their size and the virulence of the bacillus. Their eyes become watery, they begin to show disinclina- tion to take food or to make any bodily effort, the tem- perature rises to 41. 50 C, they remain quietly in a corner of the cage, and die with convulsive symptoms in from two to seven days. If the inoculation was intravenous, there is no lymphatic enlargement, but if it was sub- cutaneous, the nearest lymph-nodes are always enlarged, and, in cases with delayed death, suppurated. The bacilli are found everywhere in the blood, but not in very large numbers. Devell2 has found that frogs are susceptible to the disease. Wyssokowitz and Zabolotmy3 found monkeys to be highly susceptible to plague, especially when inoculated subcutaneously. When so small an inoculation was made as a puncture with a pin dipped in a culture of the bacillus, the puncture being made in the palm of the hand or sole of the foot, the monkeys always died in from three to seven days. In these cases the local edema observed by Yersin did not occur. They point out the interest attaching to infection through so insignificant a wound and without local lesions. According to Yersin, an infiltration or watery edema can be observed in a few hours about the point of inocula- tion. The autopsy shows the infiltration to be made up of a yellowish gelatinous exudation. The spleen and 1 Ann. de V Inst. Pasteur, July, 1895. * Centralbl. f. Bakt. u. Parasitenk., Oct. 12, 1 897. 3 Ann. de I Inst. Pasteur, Aug. 25, 1897, xi.,8, p. 665. 558 PATHOGENIC BACTERIA. liver are enlarged, the former often presenting an appear- ance much like an eruption of miliary tubercles. Some- times there is universal swelling of the lymphatic glands. Bacilli are found in the blood and in all the internal organs. Very often there are eruptions during life, and upon the inner abdominal walls there are petechias and occasional hemorrhages. The intestine is hyperemic, the adrenals congested. There are often sero-sanguinolent effusions into the serous cavities. Klein x found that the intraperitoneal injection of the bacillus into guinea-pigs is of diagnostic value, produc- ing in twenty-four to forty-eight hours a thick cloudy peritoneal exudate rich in leukocytes and containing characteristic chains of the plague bacillus. Animals fed upon cultures or upon the flesh of other animals dead of the disease became ill and died with typical symptoms. When Klein inoculated animals with the dust of dwelling-houses in which the disease had occurred, some died of tetanus, one from plague. Many rats and mice in which examination showed the charac- teristic bacilli died spontaneously in Hong-Kong. Yersin showed that flies also die of the disease. Mace- rating and crushing a fly in bouillon, he not only suc- ceeded in obtaining the bacillus from the medium, but infected an animal with it. Nuttall,2 in reviewing Yersin's fly-experiment, found the statement true, and showed that flies fed with the cadavers of plague-infected mice died in a variable length of time. Large numbers of plague bacilli were found in their intestines. He also found that bed-bugs allowed to prey upon infected animals took up large numbers of the plague bacilli and retained them for a number of days. These bugs did not, however, infect healthy animals when allowed, subsequently, to feed upon them. Nuttall is not, however, satisfied that the number of his experiments upon this point was great enough to be conclusive. ' Centralbl. f. Bakt. u. Parasitenk., xxi., No. 24, July 10, 1897, p. 849. 2 Ibid., Aug. 13, 1897. BUBONIC PLAGUE. 559 < tgata found that the plague bacillus existed in the bodies of fleas found upon diseased rats. One of these he crushed between sterile object-glasses and introduced into the subcutaneous tissues of a mouse, which died in three days with typical lesions of the plague, a con- trol-animal remaining well. Some guinea-pigs taken for experimental purposes into a plague district, and kept carefully isolated, died spontaneously of the disease, presumably because of insect infection. The animal most prone to spontaneous infection seems to be the rat, and there is much evidence in support of the view that it aids in the spread of epidemics. At several of the Asiatic plague districts and at Santos the ap- pearance of plague among the inhabitants was preceded by a large mortality among the rats, some of which when ex- amined showed buboes and had died of plague-septicemia. It is rather improbable that men become infected with plague through the bites of the fleas leaving the bodies of plague-destroyed rats, as was once supposed. Galli- Valerio1 thinks the fleas of the mouse and rat are incapa- ble of living upon man and do not bite him, and that it is only the Pulex irritans, or human flea, that is capable of transmitting the disease from man to man. Yersin found that when cultivated for any length of time upon culture-media, especially agar-agar, the viru- lence was rapidly lost and the bacillus eventually died. On the other hand, when constantly inoculated from animal to animal the virulence of the bacillus is much increased. The bacillus probably attenuates readily. Kitasato found that it did not seem able to withstand desicca- tion longer than four days ; but Rappaport (quoted by Wyman) found that they remained alive when kept dry upon woollen threads at 200 C. for twenty-three days, and Yersin found that although it could be secured from the soil beneath an infected house at a depth of 4-5 cm., the virulence of such bacilli was lost. 1 Centralbl. f. Bakt. u. Parasitenk., Jan. 6, 1900, xxvii., No. I, p. 1. 560 PATHOGENIC BACTERIA. Kitasato found that the bacillus was killed by two hours' exposure to 0.5 per cent, carbolic acid, and also by exposure to a temperature of 8o° C. for five minutes. Ogata found that the bacillus was instantly killed by 5 per cent, carbolic acid, and in fifteen minutes by 0.5 per cent, carbolic acid. In o. 1 per cent, sublimate solution it is killed in five minutes. According to Wyman, the bacillus is killed by exposure to 55° C. for ten minutes. The German Plague Com- mission found that the bacilli were killed by exposure to direct sunlight for three or four hours ; and Bowhill x found that they are killed by drying at ordinary room temperatures in about four days. It seems possible to make a diagnosis of the disease in doubtful cases by examining the blood, but it is admitted that a good deal of bacteriologic practice is necessary for the purpose. Abel finds that the blood may yield fallacious results because of the rather variable appearance of the bacilli, which are sometimes long and easily mistaken for other bacteria. He deems the best tests to be the inoculation of broth-cultures and subsequent inoculation into ani- mals, which he advises should have been previously vaccinated against the streptococcus. Plague bacilli persist in the urine a week after convalescence. Wilson, of the Hoagland Laboratory, found the thermal death-point of the organism was one or two degrees higher than that of the majority of pathogenic bacteria of the non-sporulating variety, and that, unlike cholera, the influence of sunlight and desiccation cannot be relied upon to limit its viability. Kitasato' s experiments first showed that it is possible to bring about immunity to the disease, and Yersin, working in India, and Fitzpatrick, in New York, have successfully immunized large animals (horses, sheep, goats). The serum of these immunized animals con- tains an antitoxin capable not only of preventing the dis- 1 Manual of Bacteriological Technique and Special Bacteriology, 1899, p. 197. BUBONIC PLAGUE. 561 ease, but also of curing it in mice and guinea-pigs and probably in man. Haffkine in his experiments followed the line of pre- ventive inoculation as employed against cholera. Bouil- lon cultures were used in which floating drops of butter were employed to make the islands of plague bacilli float. The cultures were grown for a month or so, suc- cessive crops of the island-stalactite growth as it formed having been precipitated by agitating the tube. In this manner there was obtained an "intense extra-cellular toxin" containing large numbers of the bacilli. The culture was killed by exposure to a temperature of 700 C. for one hour, and the mixture used in doses of about 3 c.cm. as a preventive inoculation. The most interesting collection of statistics, showing in a most convincing manner the importance of the Haff- kine prophylactic, is that of Leumann of Hubli. The figures, together with a great deal of interesting informa- tion upon the subject, can be found in the paper upon " A Visit to the Plague Districts in India" by Barker and Flint.1 The immunity conferred by the Haffkine prophylactic in doses of 1 c.cm. is of considerably longer duration, lasting about a month. The preparation must not be used if the person have already been exposed to infection, and is possibly in the incubation stage of the disease, as it contains the toxins of the disease, and greatly intensi- fies the existing condition. When injected into healthy persons it always produces fever, local swelling, and ma- laise. Wyssokowitz and Zabolotny,2 whose studies have already been quoted, used 96 monkeys in the study of the value of the "plague-serums," and found that when the treatment is begun within two days from the time of inoculation the animals can be saved, even though symptoms of the disease are marked. After the second day the treatment cannot be relied upon. The 1 New York Medical Journal, Feb. 3, 1900. * I.oc. cit. 36 562 PATHOGENIC BACTERIA. dose necessary was 20 c.cm. of a serum having a potency of 1 : 10. If too little serum was given, the course of the disease was slowed, the animal improved for a time and then suffered a relapse, and died in from thirteen to seventeen days. The serum also produced immunity, but of only ten to fourteen days' duration. Immunity lasting three weeks was conferred by inoculating a mon- key with an agar-agar culture heated to 6o° C. If too large a dose of such a culture was given, however, the animal was enfeebled and remained susceptible. Of Yersin's serum, which is prepared by immunizing horses in the usual manner to the toxins and cultures of the bacillus, 5 c.cm. doses have been found to confer an immunity lasting for about a fortnight. Larger doses confer a longer immunity. For the treatment of the de- veloped disease enormous doses of 50 and even 100 c.cm. seem to be necessary to produce the desired results, evi- dently indicating that the serums thus far obtained are weak. CHAPTER XII. TETRAGENUS. Micrococcus Tktragenus (Gafifky1). There can sometimes be found in the normal saliva, more commonly in tuberculous sputum, and still more commonly in the cavities of tuberculosis pulmonalis, a large micrococcus grouped in fours and known as the Micrococcus tetragenus (Fig. 128). It was discovered by Fig. 128. — Micrococcus tetragenus in pus from a white mouse; x 615 (Heim). Gaffky. It sometimes occurs in the pus of acute ab- scesses, and may be of importance in connection with the pulmonary abscesses which so often complicate tu- berculosis. The cocci are rather large, measuring about i // in diameter. In cultures they show no particular arrange- ment among themselves, but in the blood and tissues of animals they commonly appear arranged in groups of four surrounded by a transparent gelatinous capsule. The organism stains well by ordinary methods, and 1 Archives filr Chirurgie, 28, 3. 563 564 PATHOGENIC BACTERIA. most beautifully by Gram's method, by which it can be best demonstrated in tissues. Upon gelatin plates small white colonies are produced in from twenty-four to forty-eight hours. Under the microscope they are found to be spherical or elongate (lemon-shaped), finely granular, and lobulated like a raspberry or a mulberry. When superficial they form white, elevated, rather thick masses 1-2 mm. in diameter (Fig. 129). In gelatin punctures a large white surface-growth Fig. 129. — Micrococcus tetragenus: colony twenty-four hours old upon the sur- face of an agar-agar plate; x' ioo (Heim). rakes place, but very scant development occurs in the puncture, where the small spherical colonies generally remain isolated. Upon the surface of agar-agar spherical white colonies are produced. They may remain isolated or may become confluent. Upon potato a luxuriant thick, white growth occurs. The growth upon blood-serum is also abundant, espe- cially at the temperature of the incubator. It has no distinctive peculiarities. The introduction of tuberculous sputum or of a most minute quantity of a pure culture of this coccus into white mice generally causes a fatal septicemia. TE TRA GENUS. 565 The organisms are found in small numbers in the heart's blood, but are numerous in the spleen, lungs, liver, and kidneys. House-mice and field-mice are comparatively immune ; dogs and rabbits are also highly resistant. Guinea-pigs sometimes die from general infection, though sometimes local abscesses may be the only result of subcutaneous inoculation. The tetragenococci, when present in the cavities, prob- ably hasten the tissue-necrosis in tuberculosis pulmonalis, and may aid in the formation of abscesses of the lung and contribute to the production of the hectic fever. An interesting contribution to their relationship to human pathology is made by Lartigau,1 who succeeded in demonstrating that the tetracoccus may be the cause of a pseudomembranous angina, three cases of which came under his observation. 1 Phila. Med. Journal, April 22, 1899. CHAPTER XIII. INFLUENZA. Bacillus Influenzae (R. Pfeiffer1). Notwithstanding a large number of bacteriologic examinations conducted for the purpose of determining the cause of influenza, it was not until 1892, after the great epidemic, that there was found simultaneously by Canon and Pfeiffer a bacterium which conformed, at least in large part, to the requirements of specificity. The observers mentioned found the same organism — one in the blood of influenza patients, the other in the purulent bronchial discharges. The specific organisms (Fig. 130) are bacilli, very small in size, having about the same diameter as the bacillus "J . t :* v# •- .* *-» * «... ■» *^-< » • II.'** Fig. 130. — Bacillus influenzae, from a gelatin culture; x iooo (Itzerott and Niemann). of mouse-septicemia, but only about half as long (0.2 by 0.5 fx). They are usually solitary, but may be united in chains of three or four elements. They stain rather \ Deutsche tried. Wochenschrift, 1892, 2; Zeitschrift JUr Hygiene, 13. 566 INFLUENZA. 567 poorly, except with such concentrated penetrating stains as carbol-fuchsin and Loffler's alkaline methylene blue, and even with these the bacilli stain more deeply at the ends than in the middle, so that they appear not a little like diplococci. For the demonstration of the bacilli in the blood Canon recommends a rather complicated method. The blood is spread upon clean cover-glasses in the usual way, thor- oughly dried, and then fixed by immersion in absolute alcohol for five minutes. The stain which seems best is Czenzynke's : Concentrated aqueous solution of methylene blue, 40 ; o. 5 per cent, solution of eosin in 70 per cent. alcohol, 20 ; Distilled water, 40. The cover-glasses are immersed in this solution, and kept in the incubator for three to six hours, after which they are washed in water, dried, and then mounted in Canada balsam. By this method the erythrocytes are stained red, the leucocytes blue, and the bacillus, which is also blue, appears as a short rod or ofteu as a dumb-bell. Sometimes large numbers of the bacilli are present ; sometimes very few can be found after prolonged search. They are often enclosed within the leucocytes. It really is not necessary to pursue so tedious a staining method for demonstrating the bacilli, for they stain quite well by ordinary methods. They do not stain by Gram's method. The bacillus is non-motile, and, so far as is known, does not form spores. Its resisting powers are very re- stricted, as it speedily succumbs to drying, and is cer- tainly killed by an exposure to a temperature of 6o° C. for five minutes. It will not grow at any temperature below 280 C. The bacillus does not grow in gelatin or upon ordinary agar-agar. Upon glycerin agar-agar, after twenty-four hours in the incubator, minute colorless, transparent, 568 PA THOGENIC BA CTERIA . drop-like cultures may be seen along the line of inocula- tion. They do not look unlike condensed moisture, and Kitasato makes a special point of the fact that the colo- nies never become confluent. The colonies may at times be so small as to require a lens for their discovery. In bouillon a scant development occurs, small whitish particles appearing upon the surface, subsequently sink- ing to the bottom and causing a "woolly" deposit there. While the growth is so delicate in these ordinary media, the bacillus grows quite well upon culture-media contain- f^sgrj^jj* Fig. 131. — Bacillus of influenza; colonies on blood agar-agar; low magnifying power (Pfeiffer). ing hemoglobin or blood, and can be transferred from culture to culture many times before it loses its vitality. It cannot be positively proven that this bacillus is the cause of influenza, but from the fact that the bacillus can be found only in cases of influenza, that its presence corresponds with the course of the disease in that it is present as long as the purulent secretions last, and then disappears, and that Pfeiffer was able to demonstrate its presence in all cases of uncomplicated influenza, his con- clusion that the bacillus is specific is certainly justifiable. INFLUENZA. 569 The bacillus is pathogenic for certain of the laboratory animals, the guinea-pig in particular being subject to fatal infection. The dose required to cause death of a guinea-pig varies considerably, in the immunization ex- periments of Deline and Kole1 ^ of a 24-hour old culture being fatal in twenty- four hours. They found that the toxicity of the culture does not depend upon a soluble toxin, but in something retained in the bod- ies of the bacilli. The outcome of the researches, which .-'-' &$& 7& Fig. 132. — Bacillus of influenza; cover-glass preparation of sputum from a case of influenza, showing the bacilli in leukocytes; highly magnified (Pfeiffer). were made most scientifically and painstakingly, was the total failure to produce immunity. Increasing doses of the cultures injected into the peritoneum re- sulted in enabling the animals to resist rather more than a fatal dose, but never enabled them to main- tain vitality when large doses were administered. This discovery is in exact harmony with the familiar clinical observation that, instead of an individual being immune after an attack of influenza, he is as susceptible as before, if not more so. 1 Zeitschrift fur Hygiene, etc., Bd. xxiv., 1897, Heft. 2. 570 PA THOGENIC BA CTERIA . A. Catanni, Jr.1 trephined rabbits and injected influ- enza toxin into their brains, at the same time trephining control-animals, into some of whose brains he injected water. The results were that animals thus receiving' 0.5-1 mgr. of the living culture constantly died in twenty-four hours with all the nervous symptoms of the disease, dyspnea, paralysis beginning in the posterior extremities and extending over the whole body, clonic convulsions, stiffness- of the neck, etc. Control-animals injected with a variety of pathogenic bacteria in the same manner never manifested similar symptoms. The virulence of the bacillus was also observed to increase rapidly when transplanted from brain to brain. Wynekoop2 has successfully employed, for diagnosti- cating influenza and isolating the bacillus, a culture- outfit similar to that used for diphtheria-diagnosis, except that the serum contains more hemoglobin. The swab is used to secure secretions from the pharynx and tonsils, and from the bronchial secretions of patients with in- fluenza, then rubbed over the blood-serum. In many such cultures the minute colonies corresponding to those of the influenza bacillus were found. Those most isolated were picked up with a wire and transplanted to bouillon, from which fresh blood-serum was inoculated and pure cultures secured. Carbol-fuchsin was found most useful for staining the bacilli. An interesting observation made by Wynekoop was that influenza and diphtheria bacilli sometimes co- exist in the throat, and that influenza bacilli are present in the sore-eyes of those in the midst of household epi- demics of influenza. 1 Zeilschrift filr Hygiene, etc., 1896, Bd. xxiii. 2 Bureau and Division Reports, Department of Health, City of Chicago, Jan., 1899. CHAPTER XIV. MEASLES. In 1892, Canon and Pielicke,1 after the investigation of fourteen cases of measles, reported the discovery of a specific bacillus in the blood in that disease. The organism is quite variable in size, sometimes being quite small and resembling a diplococcus, some- times larger, and occasionally quite long, so that one bacillus may be as long as the diameter of a red blood- corpuscle. The discovery was made by means of a peculiar method of staining, as follows : The blood is spread in a very thin, even layer upon perfectly clean cover-glasses, and fixed by five to ten minutes' immersion in absolute alco- hol. These glasses are then placed in a stain consisting of Concentrated aqueous solution of methylene blue, 40 ; o. 25 per ct. solution of eosin in 70 per ct. alcohol, 20 ; Distilled water, 40, and stood in the incubator at 370 C. for from six to twenty-four hours. The bacilli do not all stain uni- formly. The discoverers of the bacillus claim to have made it grow several times in bouillon, but failed to induce a growth upon other media. The bacilli do not stain by Gram's method ; they seem to have motility ; no spores were observed. They were found not only in the blood, but also in the secretions from the nose and eyes. They are said to persist through- out the whole course of the disease, even occasionally being found after the fever subsides. 1 Berliner klin. Wochenschrift, 1892, 377. 571 572 PATHOGENIC BACTERIA. Czajrowski asserts that the bacillus can be cultivated upon various albuminous media except gelatin and agar. On glycerin agar-agar, especially with the addition of hematogen, and on blood-serum, they should grow in three or four days with an appearance like that of dew- drops. Under the microscope the colonies are structure- less. Mice die of a septicemia after a subcutaneous in- oculation. An interesting field for experimentation has been opened by Behla,1 who seems to have successfully inocu- lated a sucking-pig with measles by introducing some of the nasal secretion from a case of measles into the nose, which had been prepared to receive it by scratching with a wire. 1 Centralbl.f. Bakt. u. Parasitenk., Oct. 24, 1896, Bd. xx., Nos. 16 and 17, p. 36. CHAPTER XV. MALTA FEVER. Micrococcus Melitensis (Bruce1). Working in Malta in 1887 Brnce succeeded in finding in every fatal case of Malta fever a micrococcus which could readily be isolated in pure cultures from the spleen, liver, and kidney, which grew readily and when injected into monkeys produced all the phenomena of the disease. The serum from cases of Malta fever also caused aggluti- native phenomena when allowed to act upon the micro- organism. The Micrococcus melitensis is described as of a round or slightly oval form and measures about o. 3 fx in diame- ter. It is usually single, sometimes in pairs, but never in chains. When viewed in the hanging drop it is said to exhibit active " molecular" movements, but is proba- bly not motile. The organism stains well with aqueous solutions of gentian violet, but not by Gram's method. The best medium for cultivation is said to be ordinary agar-agar. After inoculating either by a stroke or punct- ure from an organ of a fatal case of Malta fever the tubes should be kept at 37 ° C. No growth appears for several days. At length, however, minute pearly-white spots appear scattered around the point of puncture and along the needle path. After some weeks the colonies grow larger and join to form a rosette-like colony, while the needle track becomes strongly marked, solid looking, and yellow-brown in color, with serrated edges. After a lapse of some months the growth remains restricted in area and its color deepens to buff. When the sloping surface of the agar-agar is examined by transmitted light, the appearance of the colonies is somewhat different. At the end of nine or ten days, if 1 Practitioner, xxxiv., p. 161. 573 574 PATHOGENIC BACTERIA. kept at 2)7° C., some of the colonies have a diameter of 2 to 3 mm. They are round in shape, with an even con- tour, slightly raised above the surface of the agar-agar, and smooth and shining in appearance. On holding up the tube and examining such colonies by transmitted light the centre of each is seen to be yellowish in color, while the periphery appears bluish-white. On looking at the same colonies by reflected light, no appearance of yellow can be seen ; they then appear to be milky-white in color. The separate colonies on the surface of the agar-agar do not extend indefinitely, and after a couple of months are found to be no larger than hemp-seeds. When kept at 250 C. the colonies do not become visi- ble to the naked eye before the seventh day ; at 37° C, about the third or fourth day. There is scarcely any growth in gelatin and no lique- faction of the medium takes place. No growth takes place on boiled potato. Plate-cultures are not adapted to the study of the organism because of the extreme slowness of its growth. The micro-organism seems to be constantly absent from the circulating blood. Hughes has, however, cul- tivated it from the heart's blood of a dead monkey. Bruce not only succeeded in securing the micro- organism from the cadavers of Malta fever, but has also obtained it during life by splenic puncture. The micro-organism is not pathogenic for mice, guinea- pigs, or rabbits, but is fatal to monkeys when agar-agar cultures suspended in water are injected beneath the skin. The natural occurrence of the micrococcus and the sources of contagion are unknown, and, as Bruce points out, would be very difficult to determine because of the high temperature at which development takes place, the extreme slowness of its growth, and the absence of well- marked morphological, cultural, or pathogenic charac- teristics by which it could. be recognized. 1 See Lancet, 1897, i., p. 656. D. MISCELLANEOUS. CHAPTER I. SYMPTOMATIC ANTHRAX. Bacillus Anthracis Symptomatic!. "Symptomatic anthrax," charbon symptomatique, Rauschbrand, "quarter-evil," and "black-leg" are the various names applied to a peculiar disease of cattle com- mon during the summer season in the Bavarian Alps, Baden, Schleswig-Holstein, and some parts of the United States, characterized by the occurrence of irregular, em- physematous, crepitating subcutaneous pustules. Dis- eased areas are also found in the muscles, and are most common over the quarters, hence the name "quarter- evil." When incised the affected tissues have a dark color and contain a dark, bloody serum. The micro-organismal nature of the disease had been suspected from an early date, but until the work of Faser and Bollinger x the disease was confounded with anthrax. Still later, Arloing, Thomas, Cornevin, and Kitasato studied the disease, and succeeded in demon- strating the specific micro-organism, which Kitasato successfuly cultivated upon artificial media. The bacillus which the results of these labors brought to light is a rather large individual (3-5 /1 in length, 0.5-0.6 fj- in breadth) with rounded ends. The bacilli are occasionally united in twos, but are never united in long chains (Fig. 133). They are actively motile (Thoinot and Masselin say scarcely at all motile) when examined in the hanging drop, but after a short time, perhaps because of the exposure to the oxygen required in the hanging-drop preparation, the movement is lost and the bacilli die. When stained by Loffler's method a con- siderable number of flagella can be demonstrated. Large 1 Berliner Thierarzthicher Wochenschiift, 1878. 575 576 PATHOGENIC BACTERIA. oval spores are found ; by their presence they distort the bacilli in which they occur, causing them to assume a spindle shape (Clostridium), or, when two are united and a spore occupies one of them, a drumstick shape. In- FlG. 133. — Bacillus of symptomatic anthrax, containing spores, from an agar- agar culture; x 1000 (Frankel and Pfeiffer). volution-forms are exceedingly common in old cul- tures, and are of enormous size and of granular appear- ance. The bacillus can be stained with the ordinary aqueous solutions of the anilin dyes, but will not retain the color by Gram's method or Weigert's fibrin method. It can be colored in sections of tissue with Loffler's solution, and can be observed in the blood without staining shor.tly after death. The spores, which can be stained by ordinary methods, are quite resistant to the action of heat and disinfect- ants, and withstand the effects of drying for a consider- able length of time. The bacillus of symptomatic anthrax (Fig. 134) is a strictly anaerobic, parasitic bacterium. It grows at tem- peratures above 180 C, but best at 370 C. .V > MPTOMA TIC ANTHRAX. 577 The artificial cultivation which was achieved by Kitasato is not more difficult than that of other an- aerobic organisms. In gelatin containing i to 2 per cent, of glucose or 5 per cent, of gly- cerin the organism develops quite well, the exact appearance depending somewhat upon the method by which it was planted. If the bacteria are dispersed through the culture -medium, the little colonies will appear in the lower parts of the tube as nearly spherical or slightly irreg- ular, clouded, liquefied areas con- taining bubbles of gas. If, on the other hand, the inoculation is made by a deep puncture, a stocking - shaped liquefaction forms along the whole lower part of the puncture, leads to considerable gas-production, and finally causes the liquefaction of all the gelatin except a thin superficial stratum, acid odor is given off by the cultures. When the bacteria grow anaerobically in Esmarch tubes, the colonies are irregularly club-shaped or spheri- cal, with a tangled mass of delicate projecting filaments visible upon microscopic examination. In agar-agar the development is similar to that in gelatin. The gas-production is marked, the liquefaction of course absent, and the same acid odor pronounced. The bacillus also develops quite well in bouillon, the bacillary masses sinking to the bottom in the form of whitish flakes, while the gas-bubbles collect at the top. In this medium the virulence is unfortunately soon lost. 37 »34- ■■■■■Kn •Bacillus of symp- A nprnliar tomal'c anthrax: four-days-old culture in glucose-gelatin (Fran- kel and Pfeiffer). 578 PATHOGENIC BACTERIA. Milk also seems to be a favorable culture-medium. The development of the bacilli is unaccompanied by coagulation. The virulence of the organism is soon lost in all culture-media, but it is said that the virulence of the culture can be much increased by the addition to it of 20 per cent, of lactic acid. When susceptible animals are inoculated with a minute portion of a pure culture in a little subcutaneous pocket, such as is described in connection with tetanus and malignant edema, the bacilli proceed to grow, pro- duce the well-known affection, and lead to a certainly fatal outcome. Cows seem to be the most susceptible animals, especially those between six mouths and four years old ; sheep and goats are also sometimes affected. Curiously enough, animals that are immune to malig- nant edema are seemingly more susceptible to Rausch- brand. Of the laboratory animals, the guinea-pig is most susceptible ; swine, dogs, and rabbits are very slightly susceptible ; horses, goats, and birds are im- mune. The virulence of the bacillus is capable of ready attenuation by exposure to heat, by previous exposure of its spores to heat, or by drying combined with ex- posure to increased temperature. The inoculation of animals with the attenuated bacilli causes a very mild affection, followed by complete immunity to the viru- lent organisms. Upon this principle the "protective vaccination" is based. Kitt has shown that dried mus- cle from an infected animal is more efficacious for the purpose of immunizing than the cultures themselves. The method for preparing these so-called " vaccines" is very simple. A calf is inoculated with a virulent culture in the most muscular parts of its body. As soon as it dies the "black spongy muscular tissue is dissected out, cut in small pieces, and dried in an oven at about the body-temperature. When dry the muscle is firmly ground, then heated for the purpose of attenuating the SYMPTOMATIC ANTHRAX. 579 virulence of the contained bacilli. Originally two inocu- lations, one of pulverized muscle heated for six or seven hours to ioo°-io4° C, and a week later one of the pow- der heated to 85°-90° C. were used. Most observers are now agreed that a single injection of the muscle attenu- ated by six or seven hours' exposure to 850 C. will suffice for perfect immunization. The muscle-powder is simply distributed in a convenient quantity of water and injected hypodermically. The immunity to symptomatic anthrax seems, how- ever, to be one of degree, for Arloing, Cornevin, and Thomas found that when the bacillus was introduced into the animal body simultaneously with a 20 per cent, solution of lactic acid, either the virulence of the bacil- lus or the resistance of the tissues was so changed that natural immunity was destroyed- and the bacteria allowed to develop and produce the disease. Roger found also that refractory animals, like the rabbit, mouse, pigeon, and chicken, could be made susceptible by the combined injection of the Rauschbrand bouillon, the Bacillus pro- digiosus, Proteus vulgaris, and other harmless organisms. When the guinea-pig is inoculated with the bacillus of symptomatic anthrax, it dies in from twenty-four to thirty-six hours. The post-mortem examination shows a bloody serum at the point of inoculation, and the mus- cles are dark red or black, like those of the "black-leg" of cattle. No changes are apparent in the internal organs. The bacilli are at first found near the point of inocula- tion in the inflammatory exudations only, but soon after death, being motile, they spread to all parts of the body. The peculiarities of symptomatic anthrax point to the entrance of the bacteria into the animal body through wounds, but the occurrence of epidemics at certain geo- graphical points, known technically as "Rauschbrand stations," suggests that infection may also take place through the respiratory and alimentary tracts. At first thought, as Frankel points out, one might imagine that an animal dead of quarter-evil and the dis- 580 PA THOGENIC BA CTERIA . charges from its body might be harmless, as compared, for example, with the cadavers and discharges of anthrax, because of the purely anaerobic method of the growth of the bacillus of symptomatic anthrax and the rapidity of its death in the presence of oxygen. This is, however, un- true, for the rapid development of a permanent form in the resisting spores of the bacillus makes the pollution of the soil exceedingly dangerous for cows who subse- quently browse upon it. That the spores are of great vitality is shown by the well-known laboratory method of keeping them on hand for experimental purposes, dried in the muscular tissue of a diseased animal. Every precaution should be exerted to have the affected animals isolated, and their cadavers disinfected and de- stroyed or buried in such a manner that subsequent infection is impossible. Statistical results of Guillod and Simon, based upon 3500 protective inoculations, show a distinct reduction of the death-rate from 5-20 per cent, in unprotected animals to 0.5-2 per cent, in protected animals. CHAPTER II. MALIGNANT EDEMA. Bacillus (Edematis Maligni (Koch1), The chief contaminating organism in the preparation of pure cultures of the tetanus bacillus is a large slender bacillus almost as large as that of anthrax, but with rounded ends and an individual motility accomplished by means of flagella attached to its ends and sides (Fig. 135). It is a strictly anaerobic bacterium, and was Fig. 135. — Bacillus of malignant edema, from the body -juice of a guinea-pig inoculated with garden-earth; x 1000 (Frankel and Pfeiffer). originally described by Pasteur2 (1875) as the Vibrion septique. It grows well at the room-temperature, as well as at the temperature of the incubator, produces oval central spores, and, because of its association with a spe- cific edema in certain animals, is known as the Bacillus oedema maligni. 1 Mittheilungen aus dem Kaiserl. Gesttndheitsamtes, I. 53. 1 Bull. Acad. Med., 1877 and 1881. 581 5^2 PATHOGENIC BACTERIA. The organism is widely distributed in nature, being almost always present in garden-earth. It is also found in various dusts, in the waste water from houses, and sometimes in the intestinal canals of animals. When introduced beneath the skin this bacillus proves pathogenic for a large number of animals — mice, guinea- pigs, rabbits, horses, dogs, sheep, goats, pigs, calves, chickens, and pigeons. Cattle seem to be immune. Gunther points out that the simple inoculation of the bacillus upon an abraded surface is insufficient to pro- duce the disease, because the oxygen which is, of course, abundant there is detrimental to its growth. When an experimental inoculation is performed, a small subcu- taneous pocket should be made, and the bacilli introduced into it in such a manner as not to be in contact with the air. If the inoculated animal be a mouse, guinea-pig, or rabbit, in about forty-eight hours it sickens and dies. The autopsy shows a general subcutaneous edema con- taining immense numbers of the bacilli. In the blood the bacilli are few or cannot be found, because of the oxygen which it contains. The great majority of them occupy the subcutaneous tissue, where very little oxygen is present and the conditions of growth are therefore good. If the animal is allowed to remain undisturbed for some time after death, the bacilli spread to the circulatory sys- tem and reach all the organs. Brieger and Hhrlich x have reported two cases of malig- nant edema in man. Both cases were typhoid-fever patients injected with musk, and developed the edema in consequence of impurity of the therapeutic agent. Grigorjeff and Ukke 2 have observed a most interesting case of typhoid fever with intestinal ulcerations, through which infection by the bacillus of malignant edema took place. The case was characterized by interstitial crepita- tion of the subcutaneous tissue of the neck and breast, gas- 1 Berliner klin. Wochenschrift, 1882, No. 44. 1 Milit'dr-medizin. Jour., 1898, p. 323. MALIGNANT EDEMA. 583 bubbles in the muscles, and a transformation of the entire liver into a spongy, porous mass of a gray-brown color. The spleen was enlarged, soft, and contained a few gas- bubbles. The kidneys were enlarged and had yellow stripes in the cortical substance. No case is reported, however, in which healthy men have been infected with the disease. Cornevin declares that the passage of the bacillus through the white rat diminishes its vir- ulence, and that the animals of various species that recover from this milder affection are subse- quently immune to the virulent organisms. The bacillus of malignant edema stains well with ordi- nary cold aqueous solutions of the anilin dyes, but not by Gram's method. The organism is not a difficult one to secure in pure culture, as has been said, generally con- taminating tetanus cultures and being much more easy to se- cure by itself than its congener. It is most easily obtained from the edematous tissues of guinea- IG'/3 '— • . . ° . nant edema growing in glucose pigs and rabbits inoculated with gelatin (Frankd and Pfeiffer). garden-earth. The colonies which develop upon the surface of gela- tin kept free of oxygen appear to the naked eye as small shining bodies with liquid grayish-white contents. They gradually increase in circumference, but do not change their appearance. Under the microscope they appear filled with a tangled mass of long filaments which under 584 PATHOGENIC BACTERIA. a high power exhibit individual movement. The edges of the colony have a fringed appearance, much like the hay or potato bacillus. In gelatin tube-cultures the characteristic growth can- not be observed in a puncture, because of the air which remains in the path of the wire. The best preparation is made by heating the gelatin to expel the air it may contain, inoculating while still liquid, then replacing the air by hydrogen, and sealing the tube. In such a tube the bacilli develop near the bottom. The appearance of the growth is highly typical, as globular circumscribed areas of cloudy liquefaction result (Fig. 136), and may con- tain a small amount of gas. In gelatin to which a little grape-sugar has been added the gas-production is marked. The gas is partly inflammable, partly C02. A distinct odor accompanies the gas-production, and is especially noticeable in agar-agar cultures. CHAPTER III. GASEOUS EDEMA. This very interesting micro-organism was carefully described by Welch, and subsequently studied by Welch and Nuttall,1 and Welch and Flexner.2 It is probably identical with the Bacillus phlegmone emphysematose of Frankel. It was first secured by Welch from the body of a man dying suddenly of aneurysm with a peculiar condition of gaseous emphysema of the subcu- taneous tissue and internal organs, and a copious forma- tion of gas in the veins and arteries. The blood was thin and watery, of a lac-color, and contained large num- bers of large and small gas-bubbles. The blood-altera- tion was associated with a change in its coloring-matter, which dissolved out of the corpuscles and stained the tissues a deep red. The blood was found to contain many bacilli, which were also obtained from it and the various organs, especially in the neighborhood of the gas-bubbles, in nearly pure culture. The bacillus is a large organism, measuring 3-5 // in length, about the thickness of the anthrax bacillus, with ends slightly rounded, or, when joined, square (Fig. 137). It occurs chiefly in pairs and in irregular masses, but not in chains, in this particular differing very markedly from the anthrax bacillus. In culture-media the bacillus is usually straight, with slightly rounded ends. In old cultures the rods may be slightly bent, and involution- forms occur. When several bacilli are joined together the opposed ends are square-cut. The bacillus varies somewhat in size, especially in length, in different cul- ture-media. It usually appears thicker and more vari- 1 Bull, of the John Hopkins Hospital, July and Aug., 1892, vol. viii., No. 24. 2 Jour, of Exper. Med., Jan., 1896, vol. i., No. I. 585 586 PATHOGENIC BACTERIA. able in length in artificial cultures than in the blood of animals and of man. The bacilli occur singly, in pairs, in clumps, and sometimes in short chains. When united, an angle is often formed. The bacillus is non-motile in both the ordinary hanging- drops and in anaerobic culture. No mention is made of the presence of flagella. The organism stains well with the ordinary stains, and retains the color well in Grain's method. When stained with methylene blue a granular or vacuolated appearance Fig. 137. — Bacillus aerogenes capsulatus (from photograph by Prof. Simon Flexner). is sometimes observable, due to the presence of unstained dots in the protoplasm. Usually in the body-fluids and often in cultures the ba- cilli are surrounded by distinct capsules — clear, unstained zones. To demonstrate this capsule to the best advan- tage, Welch and Nuttall devised the following special stain: a cover is thinly spread with the bacilli, dried, and fixed without over-heating. Upon the surface prepared, glacial acetic acid is dropped for a few moments, then al- lowed to drain off, and at once replaced by a strong aque- ous solution of gentian violet, which is poured off and renewed several times until the acid has been replaced by GASEOUS EDEMA. 587 the stain. The specimen is then examined in the color- ing-solution, after soaking up the excess with filter paper, the thin layer of coloring fluid not interfering with a clear view of the bacteria and their capsules. After mounting in Canada balsam the capsules are not nearly so distinct. The width of the capsule varies from one-lialf to twice the thickness of the bacillus. Its outer margin is stained, leaving a clear zone immediately around the bacillus. It was at first thought that the bacillus produced no spores, but Dunham ' found that spores were produced upon blood-serum, and especially upon Loffler's blood- serum bouillon mixture. The spores resist desiccation and exposure to the air for ten months. They stain readily in hot solutions of fuchsin in anilin water, and are not decolorized by a moderate exposure to the action of 3 per cent, solution of hydrochloric acid in absolute alcohol. They are oval, and are usually situated near the middle of the bacillus, which is distended because of the large size of the spore and bulges at the sides. The bacillus is anaerobic. It grows upon all culture- media, both at the room-temperature and at the tempera- ture of incubation, best at the latter. The bacillus grows in ordinary neutral or alkaline gelatin, but better in gela- tin containing glucose, in which the characteristic gas- production is marked. Soft gelatin, made with 5 instead of 10 per cent, of the crude gelatin, is said to be better than the ordinary medium. There is no distinct liquefaction, but in 5 per cent, gelatin there is sometimes a softening that can be best demonstrated by tilting the tube and observing that the gas-bubbles change their position, as well as by noticing that the growth tends to sediment. In making agar-agar cultures careful anaerobic precau- tions must be observed. The tubes should contain con- siderable of the medium, which should be boiled and freshly solidified before using. The implantation should be deeply made with a long wire. The growth takes 1 Bull, of the Johns Hopkins Hospital, April, 1897, p. 68. 588 PATHOGENIC BACTERIA. place slowly unless such tubes are placed in a Buchner's jar. The deeper colonies are the largest. Sometimes the growth only takes place within 10-12 mm. of the surface, at others within 3-4 cm. of it. After repeated cultivation the organism seems to become somewhat accustomed to the presence of oxy- gen, and will grow higher up in the tube than when freshly secured from animal tissue (see Fig. 138). The colonies seen in the culture- media are grayish-white or brownish- white by transmitted light, and some- times exhibit a central dark dot. At the end of twenty-four hours the larger colonies do not exceed 0.5-1.0 mm. in diameter, though they may subse- quently attain a diameter of 2-3 mm. or more. Their first appearance is as little spheres or ovals, more or less flattened, with rather irregular con- tours, due to the presence of small projecting prongs, which are quite distinct under a lens. The colonies may appear as little irregular masses with projections. After several days or weeks, single, well-separated colonies may attain a large size and be surrounded by pro- jections, either in the form of little knobs or spikes or of fine branchings — hair-like or feathery. Their ap- pearance has been compared to thistle-balls or powder-puffs and to thorn-apples. When the growth takes place in the puncture the feathery projections are continuous. Bubbles of gas Fig. 138. — Bacillus aerogenes capsulatus, with gas-production (from photograph by Prof. Si- mon Flexner). GASEOUS EDEMA. 589 make their appearance in plain agar as well as in sugar- agar, though, of course, less plentifully. They first ap- pear in the line of growth; afterward throughout the agar, often at a distance from the actual growth. Any fluid collecting about the bubbles or at the surface of the agar-agar may be turbid from the presgnce of bacilli. The gas-production is more abundant at incubation- than at room-temperatures. The agar-agar is not liquefied by the growth of the bacillus, but is often broken up into fragments and forced into the upper part of the tube, by the excessive gas-pro- duction. In its growth the bacillus produces acid in considerable amount. In bouillon growth does not occur in tubes exposed to the air, but when the tubes are placed in Bnchner's jars, or kept under anaerobic conditions, it occurs with abun- dant gas-formation, especially in glucose-bouillon, with the formation of a frothy layer on the surface. The growth is very rapid in its development, the bouillon becoming clouded in two to three hours. After a few days the bacilli sediment and the bouillon again becomes clear. The reaction of the bouillon becomes strongly acid. In milk the growth is rapid and luxuriant under anaerobic conditions, but does not take place in cul- tures exposed to the air. The milk is coagulated in from twenty-four to forty-eight hours, the coagulum being either uniform or firm, retracted, and furrowed by gas-bubbles. When litmus has been added to the milk it becomes decolorized when the culture is kept without oxygen, but turns pink when it is exposed to the air. The bacillus will also grow upon potato when the tubes are enclosed in an anaerobic apparatus. There is a copious gas-development in the fluid at the bottom and sides of the tube, so that the potato becomes surrounded by a froth. After complete absorption of the oxygen a 59° PATHOGENIC BACTERIA. thin, moist, grayish-white growth takes place upon the surface of the potato. The vital resistance of the organism is not great. Its thermal death-point was found to be 580 C. after ten minutes' exposure. Cultures made by displacing the air with hydrogen are less vigorous than those in which the oxygen is absorbed from the air by pyrogallic acid. It was found that in the former class of cultures the bacillus generally died in three days, while in the absorption ex- periments it was kept alive at the body-temperature for one hundred and twenty-three days. It is said to live longer in plain than in sugar-agar. To keep the cultures alive it has been recommended to seal the agar-agar tube after two or three days' growth. It is believed that the natural habitat of the bacterium is the soil, but there is reason to think that it occurs in the intestine at times, and it may occasionally be found upon the skin. The pathogenic powers of the bacillus are limited, and while in some cases it seems to be the cause of a fatal outcome in infected cases, its power to do mischief in the body seems to depend upon the pre-existence of other depressing and devitalizing conditions predisposing to its growth. Being anaerobic, the bacilli are unable to live in the circulating blood, but they grow in old clots and in cav- ities, such as the uterus, etc., where but little oxygen ever enters, and from such areas enter the blood and are distributed. In support of these views Welch and Nuttall cite the result of inoculation into healthy and diseased rabbits. When a healthy rabbit is injected with 2^ c.cm. of a fresh sugar-bouillon into the ear-vein it generally recov- ers without any evident symptoms. One of their rabbits was pregnant, and at time of injection was carrying two dead embryos. After similar injection with but 1 c.cm. of the culture it died in twenty-one hours. It seems that the bacilli were first able to secure a foothold in the dead GASEOUS EDEMA. 59 l embryos, and there multiply sufficiently to bring about death later on. After the death of the animal, when the blood is no longer oxygenated, the bacilli grow rapidly with a marked gas-production, which in some cases is said to have caused the bodies to swell to twice their normal size. The result of injection into guinea-pigs does not differ very much from that observed in rabbits. Gaseous phlegmons are sometimes produced. Pigeons when inoculated subcutaneously in the pec- toral region frequently succumb. Following the injec- tion there is gas-production that causes the tissues of the chest to become emphysematous. The bird generally dies in from seven to twenty-four hours, but may live. Intraperitoneal inoculation of animals sometimes causes fatal purulent peritonitis. The infection as seen in man generally occurs from wounds into which dirt has been ground, as in the case of a compound, comminuted fracture of the humerus, with fatal infection, reported by Dunham, or in wounds and injuries in the neighborhood of the perineum. Among the twenty-three cases reported by Welch and Flexner1 we find wounds of the knee, leg, hip, and fore- arm, ulcer of the stomach, typhoid ulcerations of the in- testine, strangulated hernia with operation, gastric and duodenal ulcer, perineal section, and aneurism, as con- ditions in which external or gastro-intestinal infection occurred. Dobbin, P. Ernst, Graham Stewart and Baldwin, and Kronig have met cases of puerperal sepsis and sepsis fol- lowing abortion caused by the bacillus, or in which it played an important role. The symptoms following infection are quite uniform. There are usually redness and swelling of the wound, with rapid elevation of temperature and rapid pulse. The wound is usually more or less emphysematous, and dis- charges a thin, dirty, brownish, offensive fluid which con- x Jour, of Exper. Med., vol. I, No. I, Jan., 1896. 592 PATHOGENIC BACTERIA. tains gas-bubbles and is sometimes frothy. Occasionally the patients recover, especially when the infected part is susceptible of amputation, but death is a more common outcome. After death the body begins to swell almost immediately; it may attain twice its normal size and be unrecognizable. Upon palpation a peculiar crepitation can be felt in the subcutaneous tissue nearly everywhere, and the presence of gas in the blood-vessels is easy of demonstration. The gas is inflammable, and as the bub- bles ignite explosive sounds are heard. At the autopsy the gas-bubbles are found in most of the internal organs, sometimes so numerously as to justify the German term " Schaumorgane " (frothy-organs). The liver especially is apt to show this frothy con- dition. When the tissues from such a case are hardened and examined microscopically it is found that the bub- bles appear as open spaces in the tissue, the "borders of which are lined with large numbers of the gas bacillus. There are also clumps of bacilli without gas-bubbles, but surrounded by tissue, whose nuclei show a disposition to fragment or disappear, and whose cells and fibers show signs of disintegration and fatty change. In discussing these changes Ernst1 concluded that they were ante- mortem and due to the irritation caused by the bacillus. The gas-production he regarded as postmortem. In the internal organs the bacillus is usually found in pure culture, but in the wound it is generally mixed with other bacteria. On this account it is difficult to estimate just how much of the damage before death is the result of the activity of the gas bacillus. That gas-production after death has nothing to do with pathogenesis during life is shown by injecting into the ear-vein of a rabbit a liquid culture of the gas bacillus, allowing about five minutes' time for the distribution of the bacilli through- out the circulation, and then killing the rabbit. In a few hours the rabbit will swell and his organs and tissues will be riddled with the gas-bubbles. 1 Virchow's Archiv, Bd. 133, Heft ii. GASEOUS EDEMA. 593 At times, however, as in a case of Graham Stewart and Baldwin, there is no doubt that the bacillus produces gas in the tissues of the entire body during life. These observers, in a case of abortion with subsequent infection, found the patient "emphysematous from the top of her head to the soles of her feet" several hours before death. In this case, in which the bacillus was found in pure culture, it would indeed be difficult to doubt that the fatal issue was due to the bacillus aerogenes capsulatus. Williams l has observed the presence of the bacillus in a case of suppurative. pyelitis. Whether the fatal termi- nation of the cases is due to the presence of gas in the vessels, or partly to that and partly to some toxic prop- erty it possesses, has not been worked out as yet. It would seem, however, to have a toxic property from the fact that the onset of the infection is first shown bv the occurrence of chill, pyrexia, and rapid pulse, as well as from the necrotic changes produced by the clumps of bacilli upon the surrounding cells of the tissues in which they occur. 1 Johns Hopkins Hospital Bulletin, April, 1896, p. 66. 38 CHAPTER IV. BACILLUS PROTEUS VULGARIS (HAUSER1)- This bacillus was first found by Hauser in decompos- ing animal infusions, generally in company with two closely allied forms, Proteus mirabilis and Proteus Zen- keri, which, as the experiments and observations of San- felice and others show, may be identical with or represent / Fig. 139. — Swarming islands of proteus bacilli on the surface of gelatin; x 650 (Hauser). attenuated forms of it. According to Kruse, it is quite probable that the old species called. Bacterium termo was largely made up of the proteus. The bacilli are very variable in size and shape — pleo- morphic— and are named proteus from this peculiarity. Some forms differ very little from cocci, some are more 1 Ueber Faulnissbakterien, Leipzig, 1885. 594 BACILLUS PROTEUS VULGARIS. 595 like the colon bacteria in shape, others are found as very long filaments, and occasionally sporulina-forms are met with. True spirilla-forms are never found. All the forms mentioned may be met with in cultures of the same organism. The diameter of the bacillus is usually about 0.6 /i, but the length varies from 1.2 ft or less to 4 // or more. No spores are formed. The organisms are actively motile. The long filaments frequently form loops and tangles. Flagella are present usually in large num- ber; upon one of the longer bacilli as many as one hun- dred have been counted. Involution-forms are frequent in old cultures. The bacilli stain well by the ordinary methods. Gram's method is irregular in action, but usually fails to color the bacteria. Upon gelatin plates a typical phenomenon is observed in connection with the development of the colonies, but for the most advantageous observation the gelatin used for making the cultures should contain only 5 per cent, of gelatin instead 10 per cent, as ordinarily used. Kruse1 describes the phenomenon as follows: "at the temperature of the room rounded, saucer-shaped depressions, with a whitish central mass surrounded by a lighter zone, are quickly formed. Under low magnification the center of the growth is seen to be surrounded by radiations extend- ing in all directions into the solid gelatin, and made up of chains of bacilli. Between the radiations and the granular center motile bacteria are seen in active motion. Upon the surface the colony extends as a thin patch, consisting of a layer of bacilli arranged in threads, sending numerous projections from the periphery. Occa- sionally filaments are found in the surroundings. Under certain conditions the wandering of the processes can be directly observed under the microscope. It depends not only upon the culture-medium, but, in part, upon the culture itself. Entire groups of bacilli or single threads, bv gradual extension and circular movement, detach themselves from the colony and wander about upon the 1 Fliigge's Mikroorganismtn. 596 PA THOGENIC BA CTERIA . plate. Often from the radiated central part of the colony- peculiar zooglea are formed, having a sausage- or screw- shape, or wound in spirals like a corkscrew. The younger colonies, which have not yet reached the surface of the gelatin, are more compact, rounded or nodular, later covered with hair, and then becoming radiated and like the superficial colonies." When the culture-medium is more concentrated, or the culture one that has been frequently transplanted, the phenomenon is much less marked and sometimes does not take place at all. Puncture-cultures in gelatin are not at all character- istic. They show a rapid stocking-like liquefaction of the gelatin, extending so as to take in the entire gelatin in the tube in a few days. Anaerobic cultures do not liquefy. Upon agar-agar the bacillus grows with the production of a moist, thin, transparent, rapidly extending layer which probably rarely reaches the sides of the tube. Upon agar-agar plates the wandering of the colonies is also said to occur. Upon potato the growth is in the form of a dirty-look- ing, smeary patch. In culture-media containing either grape- or cane-sugar fermentation occurs both in the presence and in the absence of oxygen. Milk-sugar is not decomposed. When grown in milk the medium is coagulated. In its growth the bacillus usually produces a strong alkaline reaction. Indol and phenol are formed from the peptone of the culture-media. Nitrates are reduced to nitrites, and then partly reduced to* NH3. In most culture-media not containing sugar the bacillus pro- duces a very disagreeable odor. It is a question whether the Bacillus proteus is to be ranked among the pathogenic bacteria. Small doses of it are harmless for the laboratory animals; in large doses it produces abscesses. A toxic substance undoubtedly results from the metabolism of the organism, and is the BACILLUS PROTEUS VULGARIS. 597 cause of death in cases in which considerable quantities are injected into the peritoneal cavity or blood-vessels. The bacilli do not seem able to multiply in the animal body in health, but can do so when there has been pre- vious injury to its tissues or when associated with patho- genic bacteria. In such cases, if it be enabled to grow in considerable quantity, its toxin may cause pronounced symptoms. By various observers the proteus has been secured in culture from cases of wound and puerperal in- fections, purulent peritonitis, endometritis, and pleurisy. When the local lesion in which it grows is small, as in endometritis, the danger of toxemia is slight, but when spread over large areas, as the peritoneum, may prove serious. It is quite probable that in some of the cases in which ^>lood-infection with the pfoteus has been found after death it did not exist previously, as the researches of Bordoni-Uffreduzzi have shown that the proteus quite regularly enters the tissues after death. While thus apparently unable to keep up an indepen- dent existence in the tissues during life, and important in "the body only in conjunction with other bacteria, the proteus seems able to grow abundantly in urine and to produce primary inflammation of the bladder when in- troduced spontaneously or experimentally into that viscus. The inflammatory process may extend from the bladder to the kidney, and so prove quite serious. The Bacillus proteus has also been found in acute in- fectious jaundice and in acute febrile icterus, or Weil's disease. CHAPTER V. WHOOPING-COUGH. IT is only recently that the bacteriology of whooping- cough has begun to assume definiteness, and even yet there is no certainty that any of the various described bacteria play any specific part in its etiology. In all diseases of the respiratory apparatus the discharges are almost certain to be so contaminated with the nasal and oral bacteria as to make the isolation from them of a single probably specific organism a matter of difficulty, and its original recognition a matter of genius. Of historical interest are the researches and observa- tions of Deichler, Kurloff, Szemetzchenko, Cohn, Neu- mann, Ritter and Afanassiew. Those of Kurloff and Afanassiew are of especial importance because they opened the way for the recent studies of Koplik l and those of Czaplewski and Hensel.2 Koplik and Czaplewski and Hensel worked entirely independently of each other, and while the bacterium studied by the former differs in several points from that of the latter, Czaplewski and Hensel have claimed to see in Koplik's work a confirma- tion of their own. Koplik studied 16 cases of whooping-cough. The sputum was collected in sterile Petri dishes, in which it was allowed to stand for an hour or so in order that it should break up into mucous fragments. When the clear viscid expectoration from uncompli- cated cases of whooping-cough is allowed to stand for an 1 Centralbl. f. Bait. u. Parasiienk., Sept. 15, 1897, xxii., Nos. 8 and 9, p. 222. 2 Deutsche med. Wock., 1897, No. 57, p. 586, and Centralbl. f. Bait. u. Parasiienk., Dec. 22, 1897, xxii., Nos. 22, 23, p. 641. 598 WHOOPING-COUGH. 599 hour or so it separates into a fluid portion and a mass of whitish, opalescent, irregularly formed flakes or frag- ments. These were selected for study, and were trans- planted by means of a platinum-wire hook to the cul- ture-media. Czaplewski and Hensel used a rather better technique than this, and secured purity of the bacteria in the flakes by transferring them to a test-tube contain- ing pepton solution and violently agitating the tube to wash off foreign bacteria. After washing, the flakes were sown upon culture-media. Hydrocele-fluid was found most useful as a culture- fluid, but particles of sputum were planted upon all the culture-media, and attempts to cultivate bacteria from them were conducted both aerobically and anaerobically. In 13 out of the 16 cases the same bacillus (x) was iso- lated. The organism when. stained and examined micro- scopically appeared as a remarkably short and delicate bacillus, shorter and more slender than the diphtheria bacillus, measuring about 0.8-1.7 fx in length and about 0.3-0.4 ft in breadth. When stained it appeared some- what granular, and so resembled somewhat the diphtheria bacillus. Old cultures presented similar involution-forms to those seen in old cultures of the diphtheria bacillus. In general the bacillus resembles the organism found by Afanassiew l and others in cover-glass specimens of whooping-cough sputum, but diners in that spores were seen several times. In pure cultures on coagulated hydrocele-fluid the ba- cillus forms a finely granular layer of pearl-white color. On agar-agar the cultures are opaque, pearl-white, and occur as a thin layer. The colonies upon agar-agar are whitish by reflected light, and straw-yellow or deeper olive-green by trans- mitted light. They are of an irregularly rounded shape and are granular. In gelatin puncture-cultures the growth resembles that of the streptococcus, forming along the track of the wire 1 St. Petersburger med. Woch., 1887, Nos. 39-42. 600 PATHOGENIC BACTERIA. a line of finely granular, non-liquefying'colonies. Upon the surface of the gelatin the growth expands so as to form the so-called u nail-growth. " The colonies upon gelatin have an irregularly circular form, appear white or straw-yellow by reflected light and olive-green by transmitted light, and are granular. They do not liquefy and do not grow to large colonies. In bouillon after twenty-four hours there was a faint clouding of the liquid and subsequently a sedimentation of the bacteria in small clusters. After a week or so the surface of the medium is covered with a delicate pellicle, which grows thicker with the passage of time. The bacillus grows quite well anaerobically. It is motile. The bacillus is pathogenic for mice, but does not pro- duce characteristic symptoms in any of the experiment- animals. In discussing the results of Koplik's work, and com- paring it with their own, which very shortly preceded it, Czaplewski and Hensel suggest that the bacillus is better described as a bacterium than as a bacillus. The finely granular ("fein punktiertes ") appearance described by Koplik, in their observations seems to consist of a deeper staining at the poles of the cells. The growths on gelatin and on Lofner's blood-serum mixture correspond m every way. The agar-agar growths are similar, though a slight difference in color is noted, and is attrib- uted to a difference in the quality of the medium used. The bouillon culture differs, the description of Czaplewski and Hensel being as follows: at the end of a day at 37 ° C. the bouillon is scarcely clouded. At the bottom of the tube is a sharply defined, lentil-like sediment, which arises in the form of slimy threads when the fluid is whirled about, and mixes with the fluid when ener- getically shaken. Neither bacillus grows on potato. Koplik's bacillus was also peculiar in that it was motile. Regarding Koplik's bacillus as identical with their own, Czaplewski and Hensel do not agree with him in believ- WHOOPING-COUGH. 601 ing it to be the same as that described by Afanassiew, and by comparison found the latter to be a much larger, shorter, more elongate bacillus. Czaplewski and Hen- sel's studies embraced 44 cases of whooping-cough, in which the bacillus was isolated 18 times; 5 cases of bronchitis, which subsequently developed whooping- cough, in all of which it was found; and 1 case of rhinitis and bronchitis which developed whooping-cough, and in which it was found on three different occasions. From the preceding, it will be seen that many scholars have labored to detect the specific organism of this dis- ease. At present several agree upon the presence of«a certain bacillus in the expectorated matter; but none of them have yet succeeded in producing the disease or any modification of it in the lower animals. The specificity is, therefore, a matter of much doubt, and rests solely upon the constancy of the presence of the micro-organ- ism in the sputum. INDEX. Abscess, tuberculous, 314 Acids and alkalies, production of, by bacteria, 55 Actinomyces bovis, 356 Actinomycosis, 356 bacillus of, 356. See also Strepto- thrix actinomyces. communication of, to man, 356 in man, section of liver in, 361 Adhesion preparation for preservation of colonies, 210 Administration of antitoxins, 137 Aerobic bacteria, 45 Aerogenic bacteria, 51, 56 Agar-agar, 191 bacterial growths, 213 blood, 193 glycerin, as a culture-medium, 193 preparation, 192 Ravenel's method, 192 Agglutination, 88 cause, 89 reaction, 89 Air as a factor in causation of suppura- tion, 246 bacteria in, 228 determination of, 229 quantitative methods of estimat- ing, 229 Hesse's, 229 Petri's, 230 Sedgwick and Miquel's modification of, 231 bacteriologic examination of, 228 Hesse's apparatus for collecting bacteria from, 229 of sick-room, sterilization of, 177 Alexin, 101 Alexocytes, 102 Alkali-albuminate, Deycke's, as cul- ture-medium, 196 Amphitricha bacteria, 31 Anaerobic bacteria, 45 cultivation, 216 methods, 216-220 Anaerobics, optional, 45 Anesthetic leprosy, 342 Anilin dyes for staining, 146 Animal inoculation, pure cultures secured by, 211 Animals, communication of gonorrhea to, 269 experimentation upon, 221 inoculation of, with bacteria, 221 tuberculin test for, 320 Anjeszky's method for staining spores, '57 Anthracin, 462 Anthrax, 455 antitoxin of, 464 bacillus of, 456. See also Bacilltts anthracis. cause of death from, 462 immunity to, experiments in destruc- tion of, 464 in cattle, how acquired, 464 inoculation with, 461 means of protecting animals against, 463, 464 spores of, 456, 457 symptomatic, 575 bacillus of, 575. See also Bacil- lus anthracis symptomatici. precautions to be observed in, 580 protective vaccination for, 578 statistics of, 580 Antimicrobic phenomena, 137 powers of blood-serum in forced immunity, 123 Antiphthisin, 327 Antipneumococcic serum, 286 Antisepsis, discovery of, 27 early, 24 Antiseptic value of reagents, determi- nation of, 241 Antiseptics, definition of, 163 influence of, on growth of bacteria, 49 relative value of, 172 t Antistreptococcus serum. 2m Antitoxic powers of blood-serum in forced immunity, 123 theory of immunity, 142 Antitoxin, 118, 123 action of, upon bacteria, 132 upon toxin, 132-137 603 604 INDEX. Antitoxin, action of, upon toxin, chemic action theory, 132 combining ferment theory, 136 cytic stimulation theory, 134 chemic nature of, 131 mode of administration, 137 of anthrax, 464 of diphtheria, 408. See also Diph- theria, antitoxin of origin of, 127 an enzyme produced in culture, 129 product of cytic activity, 130 " Seiten-ketten Theorie," 130 theories, 127-130 toxins in a modified form, 127 serum of bacillus dysentericus, 516 of diphtheria, preparation of, for therapeutic purposes, 413 of hog-cholera, 537 of tetanus, 378-382 of yellow fever, 525 specific action of, 130 theory of immunity, 105 Antitubercle serum, Maragliano's, 327 Antituberculin, 328 Arnold's steam sterilizer, 166 Aromatics, production of, by bacteria, 57 Arthrospores, 36 formation of, 36 resisting power of, 36 Ascococci, 38 Aseptic surgery, 171 Asiatic cholera, spirillum of, 421. See also Spirillum cholera Asiat- ic a. Association, influence of, on growth of bacteria, 47 Atmosphere, germs in dust in, 20 presence of bacillus tuberculosis in, 308 Autoclave for rapid sterilization, 167 Trillat, 173 Bacilli, 38 diagram illustrating morphology of, 38 forms of, 38 morphology of, 390 motility of, 31 in colonies, 32 multiplkation of, 38 resembling bacillus anthracis, 465 bacillus diphtherise, 418 .bacillus tetani, 382 bacillus tuberculosis, 332 bacillus typhosus, 504 Bacillus aerogenes capsulatus, 585. See also Bacillus of gaseous edema. Bacillus anthracis, 456 bacilli resembling, 465 channels of entrance, 460 cultures of, 458-460 on gelatin plate, 456 discovery of, 25 methods of diminishing virulence of, 463 resisting powers of, 463 staining of, 457 to show spores, 457 susceptibility of, to heat, cold, etc., 463 toxic products of, 462 symptomatica 575 cultures of, 577 morphology of, 575 staining of, 575, 576 virulence of, 578 capsulatus mucosus as cause of pneumonia, 290 growth of, 289 choleras gallinarum, 527. See also Bacillus of chicken-cholera. coli communis, 266, 504 and bacillus typhosus, resem- blance between, 471-475 cause of cholera infantum, 51 1 cultures of, 505-507 determination of, 511 differentiation of, from bacillus typhosus, 513 distribution of, 504 immunization against, 5 10 in yellow fever, 518 morphology of, 505 motility of, 505 pathogeny of, 507-5 11 penetration of intestinal tissues by, 507 production of ammonia by, 506 of indol by, 507 resistance to germicides, 507 staining of, 505 varieties of, 512 virulence of, 508 coli immobilis, 505 colon, 504. See also Bacillus coli communis. comma, 422 discovery of, 28 der Mausesepticamie, 544 diphtherise, 390 bacilli resembling, 418 colony on agar-agar, 396 cover-glass preparations, 392 cultivation of, 392 differentiation from pseudodiph- theria bacillus, 419 INDEX. 605 Bacillus diphtheriae, discovery of, 28 dotation of, in throat of patients, 406 entrance of, into internal organs,398 growth of, 394 relation of, to diphtheria, 403 staining of, 391 Lower's method, 391 Neisser's method, 391 susceptibility of different animals to, 404 toxalbumin of, 407 toxin elaborated by, 407 dysentericus, 514-516 antitoxin serum of, 516 enteritidis, 513 fsecalis alcaligenes, 517 icteroides, 519 cultures of, 520 discovery of, 28 morphology of, 519 pathogenesis of, 521 specificity in man, 523 staining of, 518 toxin of, 522 influenzae as cause of influenza, 568 cultures of, 567 demonstration of, 567 discovery of, 28 isolation of, 570 morphology of, 566 pathology of, 569 staining of, 566, 570 toxin, effects of, when injected into animals, 570 Klebs-Loffler, 390. See also Ba- cillus diphtheria:. leprae, 336 discovery of, 28 growth of, 338 inoculation with, 340 isolation of, 338 mode of infection, 341 relation of, to tubercle bacillus, 341 staining of, 340 mallei, 344 discovery of, 28 growth of, 347 isolation of, 346 methods of making cultures, 346 Strauss', 346 staining of, 348 in sections of tissue, 349 Kiihne's method, 349 Loffler's method, 348 of blue pus, 262. See also Bacillus pyocyaneus. of bubonic plague, 552 culture of, 554, 556 Bacillus of bubonic plague, differential diagnosis of, 556 discovery of, 28, 55 2 inoculation of animals with, 557 morphology of, 552 pathogenesis of, 557, 558 thermal death-point of, 560 vitality of, 559 of chicken-cholera, 527 cultures of, 528 inoculation with, 529 morphology of, 527 usa of, to kdl rabbits, 530 of G5rtner, 513 of gaseous edema, 585 colonies of, 588 cultures of, 587 gas-production by, 589 after death, 592 infection of man by, 591 inoculations with, 590, 591 morphology of, 585 natural habitat of, 590 origin of, 585 pathogeny of, 590 spores of, 587 staining of, 586 vital resistance of, 590 of glanders, 344. See also Bacillus mallei. of Havelburg, 524 of hog-cholera, 531, 533 cultures of, 533 morphology of, 533 pathogeny of, 535 vitality of, 535 of Lustgarten, 35 1. See also Bacil- lus of syphilis. of malignant edema, 581 cultures of, 583 inoculation of animals with, 582 staining of, 583 of measles, 571 cultures of, 572 morphology of, 571 staining of, 571 of mouse-septicemia, 544 cultures of, 545 inoculation with, 547 morphology of, 545 pathogenesis of, 547 staining of, 547 cedematis maligni, 581. See also Bacillus of malignant edema. of Petruschky, 517 of pneumonia, 265, 279. See also Diplococcus lanceolatus. of Sanarelli, 519. See also Bacillus icteroides. 6o6 INDEX. Bacillus of Shiga, 514-516 of swine-plague, 538 cultures of, 540 morphology of, 539 pathogenesis of, 540 staining of, 540 of syphilis, 351 characteristic appearance of colo- nies, 354 growth of, 353, 354 staining of, 351, 352 in sections of tissue, 355 Lustgarten's method, 351 DeGiacomi's modification of, 352 of Van Neissen, 353. See also Bacillus of syphilis. of whooping-cough, 598 cultures of, 599 morphology of, 599 of yellow fever, 519. See also Bacillus icteroides. pestis bubonicse, 552. See also Bacillus of bubonic plague. pneumoniae of Friedlander, 287. See also Bacillus capsulatus mu- cosus. proteus vulgaris, 594 colonies of, 594, 595 cultures of, 595, 596 discovery of, 594 found in human body, 597 morphology of, 594 pathogeny of, 596 staining of, 595 pseudodiphtheria, 405, 418 differentation from bacillus diph- therise, 419 pseudotuberculosis, 334, 335 pyocyaneus, 262 formation of crystals in agar-agar growth, 263 growth of, 262 pathogenic powers of, 264 presence of, in human economy, 264 pyogenes fcetidus, 266 rhinoscleromatis, 368 smegmatis, 333 cultivation of, 333 suipestifer, 531, 533 suisepticus, 538. See also Bacillus of swine -plague. tetani, 371 bacilli resembling, 382 channels of entrance into body, 376 colony on gelatin, 373 cultivation of, 375 discovery of, 28 Bacillus tetani, distribution of, in nature, 375 glucose-gelatin culture of, 372 growth of, 374 in manure, 376 isolation of, 372 Kitasato's method, 373 puncture-culture of, 372 resisting powers of, 373 tuberculosis, 292 avium, 329 bacillus resembling, 332 bovis, 329 channels of entrance, 310 gastro-intestinal tract, 311 placenta, 310 respiratory tract, 310 sexual apparatus, 312 wounds, 312 chemotactic property of dead bacilli, 313 cover-glass preparation, adhesive, 307 detection of, in urine, 301 discovery of, 28 growth of, influence of light on. 308 on gelatin, 306 on glycerin agar-agar, 306 on potato, 306 on simple media, 307 hygienic precautions, 309 inoculation with, susceptibility of birds to, 332 in sections of tissue, staining of, 299, 302 Ehrlich's method, 302 Unna's method, 302 in sputum, 294 demonstration of, 296-299 Ehrlich's method, 297 Gabbett's method, 299 Koch-Ehrlich's method, 297 Ziehl's method, 298 Kitasato's method of obtaining from, 305 Koch's observations upon toxic products of, 317 method of making cultures, 303 motility of, 295 oxygen required for development c of, 308 presence in the atmosphere, 308 relation of, to bacillus leprae, 341 relative number of, in expectora- tion of consumptives, 300 staining of, 952 Koch's method, 296 Ehrlich's modification, 296 INDEX. 607 Bacillus typhi ahdominalis, 466. See also Bacillus typhosus. murium, 541 cultures of, 541 pathogenesis of, 524, 525 staining of, 541 use of, to destroy mice, 524 typhosus, 266, 466 and bacillus coli communis, re- semblance between, 471-475 bacilli resembling, 504 channels of entrance into body, 475-478 cultures of, 469-475 differentiation of, from colon bacil- lus, 573 discovery of, 28 distribution of, in nature, 468 rlagella of, staining of, 467 immunization of animals to, 482 in blood in roseola?, 479 isolation of, hindrance to, 475 morphology of, 466 motility of, 467 presence of, in discharges, 476, 477 pyogenic power of, 478 resisting powers of, 469, 470 staining of, 467 in sections, 467 toxin of, 479, 480 X, 518 Bacteremias, 455 Bacteria, 29 ability of, to live in tissues, 63 absence of, from normal body-juices and tissues, 43 action of antitoxin on, 132 aerobic, 45 aerogenic, 51, 56 amphitricha, 31 anaerobic, 45 cultivation of, 216 Botkin's apparatus for, 2f9 Buchner's method, 217 Esmarch's method, 217 Frinkel's method, 217, 218 Gruber's method, 217 Hesse's method, 216, 217 Kitasato's method, 218 Koch's method, 216 Liborius' method, 216 methods generally employed, 218 Novy's jars for, 219, 220 Roux's method, 219 Smith's fermentation-tube, 216 Weil's method, 218 and spores, differences between, 35 avenue of infection by, 70 Bacteria, biology of, 43 carmin as a stain for, 146 changes in cell-walls, 30 chemical analysis of, 29 chromogenic, 51, 53 cilia of, 30 classification of, 41 Cohn's morphologic, 42 collodion capsules for growing of, 225 colonies of, 203 appearances of, 208, 209 microscopic examination of, 207 preservation of, 210 color-producing, 51, 53 combining power of, with nitrogen, 59 cover-glass preparations, 147 Gram's method, 155 cultivation of, 183 cultures of, 202 determination of pathogenic powers, 60 of thermal death-point, 240, 241 development of, in liquid culture- media, 211 difficult to stain, 152 Loffler's stain for, 152 distribution of, 43 division of, into groups, 85 effects of number of, in infection, 69 elimination of, from blood, 87 Esmarch's instrument for counting colonies of, in tubes, 235 examination of, 144 fate of, in dead body, 182 flagella of, 30 gas-producing, 51, 56 growth of, conditions influencing, 45 antiseptics, 49 association with other bacteria, 47 electricity, 47 light, 46 moisture, 46 movement, 47 nutriment, 45 oxygen, 45 reaction, 46 temperature, 48 jr-rays, 49 hematoxylon as a stain for, 146 Heyroth's instrument for counting colonies of, in Petri's dishes, 234 higher forms of, 40 characteristics of, 40 in air, 228 determination of, 229 6o8 INDEX. Bacteria in air, Hesse's apparatus for collecting, 229 quantitative methods of estimating, 229. See also Air, bacteria in, quantitative methods of estimating. in gelatin, 211, 212 microtome sections, 212 in hydrant-water, 235 in milk, 59 as cause of enterocolitis, 59 of '.* summer complaint," 59 inoculation of animals with, 221 in pump-water, 235 in river-water, 235 in soil, 238, 239 estimation of, 238 in stomach, 76 internal structure suggested by stain- ing, 30 intestinal, 77 disposition of, at death, 81 introduction of, into lymphatics, 223. in water, 233 filtration as means of diminishing number in, 236 quantitative estimation of, 233, 234 liquefaction of blood-serum by, 215 of gelatin by, 54 locomotory powers of, 31 lophotricha, 31 manner of maintaining temperature of, for growth, 215 means of entrance into circulation, 87 measurement of, 162 methods of cultivation, 202 Esmarch tubes, 206 Petri's dishes, 206 plate-culture, 203 of imbedding, 150 celloidin, 150 glycerin-gelatin, 151 paraffin, 150 of observing, 144 monotricha, 31 morphology of, 36 multiplication of, 33 not stained by Gram's method, 154 occasionally parasitic, 50, 63 of acute inflammatory diseases, 246 of chronic inflammatory diseases, 292 of fermentation, 51 of putrefaction, 51 of specific diseases, 246 of uterus, 77 of vulva, 78 on plates, Wolfhiigel's apparatus for counting colonies of, 233 pathogenic, 51, 60 Bacteria, pathogenesis of, forms of, 83 peptonization of milk by, 59 peritricha, 31 phlogistic, 85 phosphorescent, 51, 57 photogenic, 51, 57 photographing, 162 production of acids and alkalies by, 55 of aromatics by, 57 of disease by, 60 of enzymes by, 61 of gases by, 56 of odors by, 57 of phosphorescence by, 57 of pigment by, 53 purely parasitic, 50, 65 saprophytic, 63 recognition of, 227 reduction of nitrites by, 57 determination of, 58 relative pathogenic powers of, 63 results of vital activity in, 51 saprogenic, 51 scheme to illustrate relationship of, to tissue-lesions, 64 septic, 85 size of, 23 staining of, 148, 149 Ehrlich's solution for, 152 in sections of tissue, 149 Gram's method, 152 obviation of precipitate in, 154 LSfner's method, 151 Pfeiffer's method, 151 stock-solutions for, 148 structure of, 30 thermophilic, 48 toxic, 85 unit of measurement for, 32 unstained, method of examining, 145 virulence of, 66 zymogenic, 51 Bacterial growths on agar-agar, 213 on litmus-milk, 214 on milk, 214 on potato, 213 Bactericidal substances, distribution of, 103 Bacteriologic examination of air, 228 of soil, 238 * of water, 223 Bacteriology, history of, 17 biologic contributions, 1 7 chemical contributions, 22 medical contributions, 24 surgical contributions, 24 objects and aims of, 221 Bacterium, 39 INDEX. 609 Bacterium, definition, 29 Bain fijca/ettr, 161 Bairn reducteur <•/ rcinforcateur, 161 Bain tensibiiisattur, 101 extract, preparation of bouillon culture-medium from, 188 Beggiatoa, 40 Benches, glass, for use in plate-culture, 205 liinary division, 2>3 Biologic contributions to history of bac- teriology, 15 Biology of bacteria, 43 Birds, susceptibility of, to inoculations with tubercle bacilli, 332 " Black-leg," 575 Black vomit in yellow fever, cause of, 522 Blood agar-agar as culture-medium, 193 elimination of bacteria from, 87 germicidal action of, as productive of immunity, 141 Blood-serum as culture-medium, 194 addition of neutrose to, 196 advantages of, 194 alkaline, 196 preparation, 194 Koch's apparatus for coagulating and sterilizing, 195 liquefaction of, by bacteria, 215 mixture, Loffler's, 196 therapy, 28 Blue pus, bacillus of, 262. See also Bacillus pyocyaneus. Body, surfaces of, as avenue of infec- tion, 79 Boer's table of relative antiseptic power of methyl violet, 1 72 Botkin's apparatus for making anae- robic cultures, 219 Bouillon as basis of other culture-media. 189 as culture-medium, 187 preparation, 187 from beef-extract, 188 de panse, 410 method of making " pure" cultures in, 209 sugar, as culture-medium, 189 Bovine tuberculosis, 329 Brieger's tetanin, 37S typhotoxin, 480 Bubonic plague, 551 bacillus of, 552. See also Bacil- lus of bubonic plague. diagnosis of, 560 immunity to, 560-562 means of infection in man, 559 89 Bubonic plague, serums of, 561 toxin of, 561 Buchner's method for cultivation of anaerobic bacteria, 217 Bunge's method for staining flagella, 160 Cabot's method of treating hydropho- bia, 389 Carbol-fuchsin stain, 156 Carmin as a stain for bacteria, 146 Catarrhal pneumonia, 290. See also Pneumonia, catarrhal. Catgut, disinfection of, 175 Celloidin, method of imbedding, 150 Cells, lepra, 342 Cerebrospinal meningitis, 274 diplococcus of, 274. See also Dip- lococcut intracellularis men- ingitidis. Charbon symptomatique, 575 Chemical contributions to history of bacteriology, 22 Chicken-cholera, 527 bacillus of, 527. See also Bacillus of chicken-cholera. pathogenesis of, 529 Cholera, 421 Asiatic, spirillum of, 421. See also Spirillum cholera Asiatica. spread of, 421, 422 chicken-, 527. S,ee also Chicken- cholera. hog-, 531. See also Hog-cholera. immunity from, Haffkine's method, 434 of animals and certain men to, 430 infantum caused by bacillus coli com- munis, 511 spirillum, spirilla resembling, 421 Von Pettenkoffer's theory of, 431 Chromogenic bacteria, 51, 53 Cilia, 30 Circulation, means of entrance of bac- teria into, 87 Cladothrix, 40 Classification of bacteria, 41 Cohn's morphologic, 42 Clostridium, 34 Clothing of sick-room, disinfection of, 179 Coagulation of blood-serum, Koch's apparatus for, 195 Cocci, 36, 250, 253 diagram illustrating morphology of,37 distribution of, in nature, 250 morphology of, 36 Cohn's morphologic classification of bacteria, 42 6io INDEX. Collodion capsules, making of, 225 use of, for growing bacteria, 225 Color-producing bacteria, 5 1, 53 Comma bacillus, 422 discovery of, 28 Concentrated tuberculin, 320 Conidia, 40 Conjunctiva as source of infection, 74 Consumptives, expectoration of, relative number of tubercle bacilli in, 300 Cotton, sterile, use of, in bacteriologic work, 165 Cover-glass, cleansing of, 147 forceps, Stewait's, 148 preparations for examination of bacteria, 147 Gram's method for, 155 Croupous pneumonia, 279 Cultivation of bacteria, 183 Culture, definition, 202 Culture-media, 183 agar-agar, 191 alkaline blood-serum, 196 blood agar-agar, 193 blood-serum, 194 bouillon, 187 desirable qualities of, 183 Deycke's alkali-albuminate, 196 Dunham's solution, 199 gelatin, 190 glycerin agar-agar, 193 liquid, apparatus for filling tubes with, 189 best means of keeping, 188 development of bacteria in, 211 litmus-milk, 198 Lofner's blood-serum mixture, 196 manner of expressing reaction of, 186 method of determining degree of reaction for, 185 solutions for, 185 milk, 198 peptone*solution, 199 Petruschky's whey, 199 potato-juice, 198 potatoes, 197 standard reaction of, 187 sterilization of, 166 sugar bouillon, 189 Culture, plate-, 202, 203. See also Plate-cultures. present use of the word, 202 stroke-, 210 Cultures, 202 anaerobic, various methods for mak- ing, 216-220 methods employed to secure, 202 puncture- 209 Cultures, puncture-, gelatin, various forms, 212 " pure," 202 method of making, 208 in bouillon, 209 in gelatin, 209 secured by animal inoculation, 211 study of, 202 Culture-tubes, method of filling, 189 of inoculating, 204 Cup, pasteboard, for receiving infec- tious sputum, 178, 309 Czaplewski and Hensel's method of washing sputum, 305 Death-point, thermal, determination of, 240, 241 Defensive proteids, 101 De Giacomi's modification of Lust- garten's method for staining bacillus of syphilis, 352 Dejecta, disinfection of, 176, 178 Deycke's alkali-albuminate as culture- medium, 196 Digestive apparatus as source of infec- tion, 75, 81. See also Infec- tion, sources of, digestive ap- paratus. Diluted tuberculin, 320 Diplococci, 36 morphology of, 36 Diplococcus intracellularis meningiti- dis, 274 channels of infection, 277 cultivation of, 275 distribution in nature, 278 Park's method for obtaining, from lumbar puncture, 276 results of animal inoculation with, 277 staining of, 275 vitality of, 276 lanceolatus, 279 capsulated, 281 colony of, on gelatin, 283 discovery of, 28 for purposes of study, 282 growth of, 282 other lesions caused by, 287 susceptibility of animals to inocu- lation with, 285 virulence of, 282-284 of cerebrospinal meningitis, 275. See also Diplococcus intra- cellularis meningitidis. of mumps, 271—273 Diphtheria, 390 antitoxic serum, determination of strength of, 413, 414 INDEX. ()i i Diphtheria, antitoxic serum, determina- tion of strength of, Ehrlich's method, 414-416 dried. 4I3 in treatment of, 413-41 8 preparation of, 413 preservation of, 413 antitoxin, 40S immunization of animals for pro- duction of, 410 paralysis after, 417 preparation of, 408 association of other organisms with, 400 bacillus of, 390. See also Bacillus diphtheria. bacteriologic diagnosis, 393 coagulation-necrosis of, 403 contagiousness of, 407 in man and in animals, 404, 405 mixed infections, 402 pseudomembrane of, 402 relation of bacillus diphtherias to, 403 Disease, germ theory of, 25 production of, by bacteria, 60 Diseases, acute inflammatory, bacteria of, 246 chronic inflammatory, bacteria of, 292 infectious, study of, 24 phlogistic, bacteria of, 246 specific, bacteria of, 246 Disinfection, 163, 171 and sterilization, 163 by formaldehyde, 173, 174, 177 apparatus for, 173 of catgut, 175 of dejecta, 176, 178 of hands, 171 of instruments, etc., 171 of living-rooms by formaldehyde, 180 of objects used by surgeon, 171 of sick-chamber, 176 air of, 177 clothing, 179 dejecta, 178 furniture and walls, 180 patient, 181 of silk ligatures, 1 75 of silkworm gut, 175 of skin/174 of syringes, 222 use of sulphur in, 177 Disposition of intestinal bacteria at death, 81 Dunham's solution as a culture-medium, 199 Smith's modification of, 200 test for indol in, 200 Dust in atmosphere, germs in, 20 Ear, external, as source of infection, 78 Early antisepsis, 24 Edema, gaseous, 585 bacillus of, 585. Sec also Bacil- lus of gaseous edema. symptoms of, in man, 591 malignant, 581 bacillus of, 581. See also Bacil- lus of malignant edema. in man, 582 Ehrlich's method for demonstration of tubercle bacilli in sputum, 297 for staining tubercle bacilli in sections of tissue, 302 for testing diphtheria-antitoxin, 414-416 modification of Koch's method of staining tubercle bacilli, 296 solution for staining, 152 Ehrlich-Wasserman lateral chain theory of immunity, 142 Electricity, influence of, on growth of bacteria, 47 Eisner's method of differentiating be- tween bacillus typhosus and bacillus coli communis, 471 Endospores, 34 Enterocolitis, bacteria in milk as cause of, 59 Enzymes, production of, by bacteria, 61 tryptic, 55 Epidemic parotitis, 270. See also Mumps. Epitoxoids, 415 Erysipelas, streptococcus of, 259 Esmarch's instrument for counting colonies of bacteria in tubes, 235 method for cultivation of anaerobic bacteria, 217 tubes for cultivation of bacteria, 206 Exaltation of immunity, Ml Examination, bacteriologic, of air, 228 of soil, 238 cf water, 233 of bacteria, cover-glass preparations, 147 Exhaustion theory of immunity, 139 Experimentation upon animals, 221 External ear as source of infection, 78 Farcin du bceuf, 367 streptothrix of, 367 Fermentation, 22, 5 1 Fermentation-tube, Smith's, 56 Fever, malta, 573. See also Malta fever. 6l2 INDEX. Fever, relapsing, 545. See also Relaps- ing fever. typhoid, 466. See also • Typhoid fever. yellow, 518. See also Yellow fever. Filter, sand-, for examination of air, Petri's, 230 Filters, Kitasato's, 170 Pasteur-Chamberland, 168 porcelain, in sterilization, 169 Reichel's, 170 sterilization of, 170 Filtration as means of diminishing num- ber of bacteria in water, 236 rapid, of toxins, apparatus for, 1 71 Fiocca's method for staining spores, 157 Fission, 33 results of, ^^ Flagella, 30 methods of staining, 158 Bunger's, 160 Loffler's, 158 Pitfield's, 160 Van Ermengem's, 161 Gorden's modification of, 162 Flora of vagina, 78 Forceps, cover-glass, Stewart's, 148 Formaldehyde as a germicide, 173, 178 disinfection by, 173, 174, 177 apparatus for, 173 of living-rooms by, 180 regenerator, sanitary, 173 " Formalin," 174 germicidal power of, 174, 177 Fowl-tuberculosis, 329 Frankel's instrument for obtaining earth from depths, 238 method for cultivation of anaerobic bacteria, 217, 218 for quantitative estimation of bac- teria in soil, 238 Friedlander's bacillus pneumoniae, 287. See also Bacillus capsulatus mucosus. Fungi, 41 division of, 41 Furniture of sick-room, disinfection of, 180 Gabbett's method for demonstration of tubercle bacilli in sputum, 299 Gartner's bacillus, 513 Gaseous edema, 595- See also Edema, gaseous. Gases, production of, by bacteria, 56 Smith's method for determina- tion, 56 Gas-producing bacteria, 51 » 56 Gastro-intestinal tract as channel for entrance of tubercle bacilli, 3H Gelatin as a culture-medium, 190 advantages of, 190 bacteria in, 211, 212 glycerin-, method of imbedding, 151 growth, microtome sections cf, 212 liquefaction of, by bacteria, 54 puncture-cultures, various forms of, 212 Generation, spontaneous, doctrine of, 17 Germ theory of disease, 25 Germicidal action of blood, productive of immunity, 14 1 power of " formalin," 174, 177 relative, of methyl violet, 172 of pyoktanin, 172 value of reagents, determination of, 242-245 Germicides, antiseptic values of, 172 definition, 163 list of, 172 Germs in dust in atmosphere, 20 Glanders, 344 bacillus of, 344. See also Bacillus mallei. Glass bench, 205 Glassware, sterilization of 164 Glycerin agar-agar as a culture-medium, 193 . Glycerin-gelatin, method of imbedding, 151 " Golden " staphylococcus, 249-254 Gonococci, cultivation of, 267 Gonococcus, 266 as cause of gonorrhea, 269 discovery of, 28 in urethral pus, 267 Gonorrhea, communication of, to ani- mals, 269 micrococcus of, 266 Gorden's modification of Van Ermen- gem's method for staining flagella, 162 Gram's method for cover-glass prepara- tions, 155 for staining bacteria, bacteria not stained by, 154 in sections of tissue, 152 solution, 153 Growth of bacteria, conditions in- fluencing, 45. See also Bac- teria, growth of, conditions influencing. Gruber's method for cultivation of anaerobic bacteria, 217 INDEX. 613 Hakfkink's method for cholera im- munity, 434 toxin of bubonic plague, 561 H mils, disinfection of, 171 Hanging-drop method of examining living micro-organisms, 144 Havelburg's bacillus, 524 Hematoxylon as a stain for bacteria, 146 Henscl and Czaplewski's method of washing sputum, 305 Hesse's apparatus for collecting bacteria from air, 229 method for cultivation of anaerobic bacteria, 216, 217 quantitative method for estimating bacteria in air, 229 Heyroth's instrument for counting col- onies in Petri dishes, 234 Hiss's culture-media for differentiating bacillus typhosus from allied forms, 472 History of bacteriology, 17 biologic contributions, 17 chemical contributions, 22 medical contributions, 24 surgical contributions, 24 Hog-cholera, 531 antitoxin serum of, 537 bacillus of, 531, 533. See also Bacillus of hog-cholera. immunity to, 536 lesions of, 532, 533, 535 pathology of, 532 symptoms, 531 HSgyes' method of treating hydro- phobia, 388 Hot-air sterilizer, 165 Humor theory of immunity, 99 Hydrophobia, 384 and tetanus, parallelism existing be- tween, 385 cure for. 387 incubation-period of, 384 treatment of, Cabot's method, 389 Hogyes' method, 388 Pasteur's system, 387 with bile, 388 Imbedding, methods of, 150. See also Bacteria, methods of imbed- ding. Immunity, 90 acquired, 72, 90, III accidentally, III, 112 aberrant diet, 1 13 causes, 112 infection, 112 modified infection, 113 Immunity, acquired, experimentally, active, 1 14 passive, 118 theories of, 139-142 antitoxin, 142 exhaustion, 139 germicidal action of blood, 141 lateral chain, 142 phagocytosis, 1 40 retention, 140 action of cells on leukocytes in, 97 of leukocytes in, 96 active, 90, 114 inoculation, 114 intoxication, 117 vaccination, 1 1 5 activity of cells in, 94 of humors, 99 and susceptibility, 90 blood-serum therapy, 28 duration, 122 exaltation of, in forced, 112, 121 antimicrobic powers of blood-serum in, 123 antitoxic powers of blood-serum in, 123 germicidal power of blood, 99 modification of, 107 natural, 72, 90, 93 explanation of, 94 influence of race, 93 passive, 90 antitoxins, 118 inert particles, 121 tissue suspensions, 118 produced by saprophytic bacteria, 116 reduction of, 107 depressing hygienic conditions, 107 diseased conditions, 1 10 effect of drugs, 109 exposure to cold, 108 fatigue, 108 mixed infections, 1 10 noxious gases, 107 operative manipulations, 109 peculiarities of diet, 108 traumatic injury, III relative condition of, 92 standard of, 92 theories of, antitoxin, 105 humor, 99 phagocytosis, 94 Immunity-reaction, 433 Immuni/.ation. 112, 121 Immunizing unit, 413, 414 Incubating-oven, new model, 214 614 INDEX. "Indicator" for culture-media, 184 phenolphthalein, 184 Indol, 57, 200 production of, by spirillum cholera Asiatica, 429 test for, 200 Infection and intoxication, 62 avenue of, 70, 79 digestive apparatus, 81 mucous membranes, 80 placenta, 83 respiratory apparatus, 80 skin, 79 surfaces of the body, 79 conditions modifying results of, 66 subject of infection, 72 infecting bacterium, 66 necessary for, 63, 78 definition, 62 effects of number of bacteria on, 69 influence of injury on, 72 of vital condition on, 72 immunizing, 112 modified, immunizing, 113 special phenomenon of, 88 agglutination, 88 sources of, 72 conjunctiva, 74 digestive apparatus, 75-77 external ear, 78 mouth, 75 nose, 74 respiratory passages, 74 sexual apparatus, 78 skin, 73 time of, 62 Infections, mixed, 66 Infectious diseases, study of, 24 sputum, pasteboard cup for reception of, 178, 309 Influenza, 566 bacillus of, 566. See also Bacillus influenza. "Infusorial life," 19 Injection of bacteria into animals, 221 Inoculation, 1 14 of animals with bacteria, 221 of rabbit, method of, 223 Instruments, etc., disinfection of, 171 sterilization of, 164, 175 Intermittent sterilization, 167 Intestine as source of infection, 77 bacteria in, 77 Intoxication and infection, 62 Introduction, 17 Johnston's (Wyatt) method for diag- nosis of typhoid fever, 496 Kashida's method for differentiating between bacillus typhosus and bacillus coli communis, 472 Kitasato's filter, 170 method for cultivation of anaerobic bacteria, 218 for isolation of bacillus tetani, 373 for obtaining tubercle bacilli from sputum, 305 mouse-holder, 224 " Klatsch praparat," 210 Klebs-Loffler bacillus, 390. See also Bacillus diphtheria. Kny-Sprague steam sterilizer, 167 Koch's apparatus for coagulating and sterilizing blood-serum, 195 bacteriologic syringe, 222 method for cultivation of anaerobic bacteria, 216 for determination of germicidal value of reagents, 242 for staining tubercle bacilli, 296 Ehrlich's modification of, 296 new tuberculin, 321, 326 observations on toxic products of tubercle bacillus, 317 quantitative method for estimation of bacteria in water, 234 steam sterilizer, 166 tuberculin, 320 Koch-Ehrlich's method for demon- stration of tubercle bacilli in sputum, 297 Kruse's explanation of " Pfeiffer's phe- nomenon," 139 Kuhne's carbol-mefhylene-blue, 349 method for staining bacillus mallei, 349 Latent tuberculosis, 317 " Lateral chain theory " of immunity, 142 of origin of antitoxin, 130 Lepra bacillus, 336. See also Bacillus lepra. cells, 342 Leprosy, 336 anesthetic, 342 bacillus of, 336. See also Bacillus lepra. nodular, 342 tubercular, 343 Leptothrix, 39, 40 Leuconostoc, 38 Levelling apparatus for pouring plate- cultures, 203 Liborius' method for cultivation of anaerobic bacteria, 216 INDEX. 6i5 I iborios* tube for cultivation of anaer- obic bacteria, 2l8 l.ifc low forms of, resistance of, 49 otaneous generation of, doctrine of, 17 Ligatures, catgut, disinfection of, 175 silk, disinfection of, 175 silkworm-gut, disinfection of, 175 Light, influence of, on growth of bac- teria, 46 Liquefaction of gelatin by bacteria, 54 Liquid culture-media, apparatus for fill- ing tubes with, 189 best means of keeping, 1S8 Liquids, sterilization of, 169 Listerism, 27 Litmus-milk as a culture-medium, 198 bacterial growths on, 214 Liver, section of, in actinomycosis in man, 361 Lobar pneumonia, 279. See also Pneu- monia, lobar. Locoinotory powers of bacteria, 31 Loffiet's alkaline methylene-blue stain, 152 blood-serum mixture, 196, 393 method for staining bacillus diph- theria?, 391 bacillus mallei, 348 bacteria in sections of tissue, 151 flagella, 158 to detect spirillum of cholera in drinking water, 433 Lophotricha bacteria, 31 Lungs, tuberculosis of, PI. II. Lustgarten's bacillus, 351. See also Bacillus of syphilis. method for staining bacillus of syphilis, 351 De Giacomi's modification, 352 Lymphatics, introduction of bacteria into, 223 Madura-foot, 363. See also Myce- toma. Malignant edema, 581. See also Edema, malignant. Mallein, 350 manufacture of, 350 Malta fever, 573 micrococcus of, 573. See also Micrococcus melitensis. Manure, bacillus tetani in, 376 Maragliano's antitubercle serum, 327 Marmorek's antistreptococcus serum, 261 Martin's bouillon de pause, 410 Measles, 57 1 bacillus of, 571. See also Baoillus of measles. Meat infusion, 187 Measurement of bacteria, 32, 162 Media, simple, growth <>f tubercle bacillus on, 307 Medical contributions to history of bac- teriology, 24 Merismopedia, 37 Metachromatic granules, 30 Methods generally employed for culti- vation of anaerobic bacteria, 218 Methyl violet, relative germicidal power of, 172 Meyer's bacteriologic syringe, 222 Micophylaxins, 107 Micosozins, 107 Micrococci, 36 morphology of, 36 Micrococcus gonorrhoeae, 266 melitensis, 573 cultures of, 573 discovery of, 573 morphology of, 573 pathogeny of, 574 staining of, 573 Pasteuri, discovery of, 28 tetragenus, 266, 563 cultures of, 564 morphology of, 563 pathogenesis of, 565 staining of, 563 Micromillimeter, 32 Micro-organisms, living, hanging-drop examination of, 144 Microtome sections of gelatin growth 212 Milk as a culture-medium, 198 bacteria in, 59 bacterial growths on, 214 litmus-, as a culture-medium, 198 peptonization of, by bacteria, 59 Miquel and Sedgwick's modification of Petri's method for quantita- tive estimation of bacteria in air, 231 Mixed infections, 66 pneumonias, 291 Modification of immunity, 107 Moisture, influence of, on growth of bacteria, 46 Monotricha bacteria, 31 Morphology of bacteria, 36 Mouse-holder, 224 Mouse-septicemia. 544 bacillus of, 544. See also_ Bacillus of mouse-septicemia. 6x6 INDEX. Mouth as source of infection, 75 Movement, influence of, on growth of bacteria, 47 Mucous membranes as avenues of in- fection, 80 Mumps, 270 diplococcus of, 271-273 Mycetoma, 363, PI. III. causes of, 364 distribution of, 366 microscopic study of diseased tissue, 365 streptothrix of, 363. See also Strep- tot hrix Madura : Mycoderma, 211 Myconostoc, 39 Mycoprotein, 29 composition, 29 Neisser's method for staining bacillus diphtherise, 391 Nitrate broth, 58 Nitrites, reduction of, by bacteria, 57 Nitrogen, combination of, with bacteria, 59 Nodular leprosy, 342 Nose as source of infection, 74 Novy's jars for cultivation of anaerobic bacteria, 219, 220 Nutriment, influence of, on growth of bacteria, 45 Obermeier's spirillum, 549 Objects injured by dry heat, sterilization of, 164, 171 Odors, production of, by bacteria, 57 Ophidiomonas, 39 Optional anaerobics, 45 Oxygen, influence on growth of bac- teria, 45 Oxytuberculin, 326 Paraffin, method of imbedding, 150 Paralysis after use of diphtheria anti- toxin, 417 Parasites, 50 Parasitic bacteria, occasionally, 50, 63 purely, 50, 65 Park's method for obtaining diplococ- cus of cerebrospinal menin- gitis from lumbar puncture, 276 Parotitis, epidemic, 270. See also Mumps. Pasteur-Charnberland filter, 168 Pasteur's treatment of hydrophobia, 387 Pathogenesis, bacterial, forms of, 83 Pathogenic bacteria, 51, 60 determination of, 60 Patient, disinfection of, 181 Penis, bacteria of, 78 Peptone solution as culture-medium, 199 Smith's modification of, 200 test for in do I in, 200 Peptonization of milk by bacteria, 59 Peritricha bacteria, 31 Petri dishes for cultivation of bacteria, 206 Heyroth's instrument for counting colonies of bacteria in, 234 Petri's quantitative method for estimat- ing bacteria in air, 230 Sedgwick and Miquel's modification of, 231 sand-filter for air-examination, 230 Petruschky's bacillus, 517 whey as culture-medium, 199 as means of differentiating between acid and alkaline products, 199 Pfeiffer's method for staining bacteria in sections of tissue, 15 1 " Pfeiffer's phenomenon," 138 Kruse's explanation of, 139 Phagocytosis theory of immunity, 95, 140 Phenolphthalein as "indicator" for culture-media, 184 Phenomena, antimicrobic, 137 " Phenomenon, Pfeiffer's," 138 Kruse's explanation of, 1 39 special, of infection, 88 Phlogistic bacteria, 85 diseases, bacteria of, 246 Phosphorescence, production of, by bac- teria, 57 Phosphorescent bacteria, 51, 57 Photogenic bacteria, 51,57 Photographing bacteria, 162 Phyloxins, 106 Pied de Madura, 363. See also, My- cetoma. Pigment, production of, by bacteria, 53 " Pig typhoid," 531. See also Hog- cholera. Piorkowski's culture-media for differen- tiating bacillus typhosus from allied forms, 474 Pitfield's method for staining flagella, 160 Placenta as avenue of infection, 83 for tubercle bacilli, 310 Plague, bubonic, 551. See also Bu bonic plague. Plague-serums, value of, 561 Plate-cultures, 202, 203 apparatus, 203 for pouring, 203 INDEX. 617 Plate-cultures, media used, 205 method, 204 pure, method of making, 208 Pneumobactllus, 287. See also Ba- . cillus capsulatus mucosum. Pneumococcal, 265, 279. See also Diplococcus lanceolatus. Pneumonia, 279 bacillus of, 265, 279. See also, Dip- lococcus lanceolatus. catarrhal, 290 bacillus capsulatus mucosus as cause of, 290 croupous, 279 immunity from, 285 lobar, 279 bacillus capsulatus mucosus as cause of, 290 treatment by antipneumococcic se- rum, 286 tubercular, 291 Pneumonias, mixed, 291 Porcelain filter in sterilization, 169 Potato, bacterial growths on, 213 Potatoes as culture-medium, 197 preparation, 197 Potato-juice as culture-medium, 198 Proteids, defensive, ioi Protoxoids, 415 Pseudodiphtheria bacillus, 405, 418. See also Bacillus pseudodiph- theria. " Pseudotetanus bacillus," 382 Pseudotuberculosis, 334 Puncture-cultures, 209 gelatin, various forms, 212 Putrefaction, 22, 51 Pyocyanase, 61 Pyoktanin, relative germicidal power of, 172 Quantitative determination of bac- teria in air, 229. See also Air, bacteria in, quantitative methods of estimating. in water, 233, 234 " Quarter-evil," 575 Rabbits, intravenous injection into, 223 Rabies, 384. See also Hydrophobia, Rauschbrand, 575 stations, 579 Ravenel's method for preparation of agar-agar for culture-medium, 192 Reaction, influence of, on growth of bacteria, 46 standard, of culture-media, 187 Reagents, determination of antiseptic value of, 241 of germicidal value of, 242-245 Recognition of bacteria, 227 Reduction of immunity, 107. See also Immunity, reduction of. Regenerator, sanitary formaldehyde, 173 Reichel's filter, 1 70 Relapsing fever, 549 spirillum of, 549. See also Spiril- lum of relapsing fever. Resistance of low forms of life, 49 Respiratory apparatus as avenue of in- fection, 80 passages as sources of infection, 74 tract as a channel for entrance of tubercle bacilli, 310 Retention theory of immunity, 140 Rhinoscleroma, 368 bacillus of, 368 Roux's bacteriologic syringe, 222 method for cultivation of anaerobic bacteria, 219 R, tuberculin-, 321-326 Sanarei.li's bacillus, 520. See also Bacillus icteroides. Sapremia, 63 Saprogenic bacteria, 5 1 Saprophytes, 44, 50 Saprophytic bacteria, purely, 63 Sarcina, 37 Scarlatina, streptococci in blood in, 258, 259 " Schaumorgane," 592 Scheme to illustrate relationship of bacteria to tissue lesions, 64 Sedgwick and Miquel's modification of Petri's quantitative method for estimating bacteria in air, 231 Sedgwick's expanded tube for air-ex- amination, 231 " Seiten-ketten Theorie " of immunity, 142 of origin of antitoxin, 130 Semmel-cocci, 37 Septic bacteria, 85 Serum, antipneumococcic, 286 antistreptococcus, 261 antitoxic, for tetanus, 378-382 antitubercle, Maragliano's, 327 Serums, "keeping" qualities of, 418 Sexual apparatus as channal of infec- tion, 78 for tubercle bacilli, 312 Shiga's bacillus, 514-516 Siberian pest, 455. See also Anthrax. 6i8 INDEX. Sick-chambers, disinfection of, 176. See also Disinfection of sick- chambers. ventilation of, 177 Size of bacteria, 33 Skin as avenue of infection, 73, 79 disinfection of, 174 Smegma bacillus, 333. See also Bacil- lus smegmatis. Smith's fermentation-tube, 56 for cultivation of anaerobic bac- teria, 216 method for determination of gas pro- duced by bacteria, 56 of isolating tubercle bacilli, 304, 305 modification of Dunham's solution, 200 Soil, bacteria in, 238, 239 Sozins, 106 Specific action of antitoxins, 130 Spirilla, 39 diagram illustrating morphology of, 39 forms of, 39 resembling cholera spirillum, 421, 436 Spirillum aquatilis, 451 Berolinensis, 445 cultures of, 446 Bonhoffi, 449 cholera Asiatica, 421 characteristics of, 426 colonies of, on gelatin, 426 cultures of, 425-428 detection of, iu drinking water, 433 in feces, 433 differentiation of, in cultures, 433 discovery of, 28, 422 distribution of, 429 from bouillon culture, 424 gelatin-puncture culture of, 427 growth of, 422 inoculation forms of, 424 production of indol by, 429 resisting powers of, 432 showing flagella, 423 spirilla resembling, 436 spores of, 424 staining of, 425 toxic products of metabolism of, 429 Danubicus, 447 Denecke, 440 cultures of, 440-442 pathogenesis of, 442 Dunbar, 447 Spirillum, Finkler and Prior, 436 cultures of, 436-439 pathogenesis of, 439 staining of, 439 Gamaleia, 442 cultures of, 443-445 pathogenesis of, 445 staining of, 443 Milleri, 451 Obermeieri, 549 discovery of, 26, 28 of relapsing fever, 549 discovery of, 26 pathogenesis of, 549 staining of, 549 terrigenus, 452 Weibeli, 450 I. of Wernicke, 448 II. of Wernicke, 448 Spiromonas, 39 Spirulina, 39 Splenic fever, 455. See also Anthrax. Spores, 34 and bacteria, differences between, 35 difficulty of staining, 35 formation of, 34 methods of staining, 156 Anjeszky's, 157 Fiocca's, 157 resisting power of, 35 Sporulation, 7,^, 34 diagram illustrating, 34 Sputum, Czaplewski and Hensel's method of washing, 305 infectious, cup for reception of, 178, 309 Kitasato's method of obtaining tu- bercle bacillus from, 305 tubercle bacillus in, 294 demonstration of, 296-299 Staining, anilin dyes for, 146 bacteria in sections of tissue, 149. See also Bacteria, staining of, in sections of tissue. cover-glass specimens, 148 Ehrlich's solution for, 152 of bacteria, 148, 149 as means of revealing internal structure, 30 of flagella, 158. See also Flagella, met/tods of staining. of spores, 156. See also Spores, methods of staining. difficulty of, 35 stock-solutions for, 148 Stapylococci, 37, 249, 250 Staphylococcus citreus, 254 epidermidis albus, 248 "golden," 249-254 INDKX. 619 Staphylococcus pyogenes albus, 248-254 aureus, 249-254 Sterilization, 163 and disinfection, 163 intermittent, 167 of blood-serum, Koch's apparatus for, 195 of culture-media, 166 of filters, 170 of glassware, 164 of instruments, etc., 164, 175 of liquids, 169 of objects injured by dry heat, 164, 171 rapid, 168 autoclave for, 167 use nf cotton plugs in, 165 of porcelain filter, 169 Sterilizer, Arnold's steam, 166 hot-air, 1 65 Kny-Sprague steam, 167 Koch's steam, 167 Sternberg's method for determination of germicidal value of re- agents, 243 Stewart's cover-glass forceps, 148 " Stichcultur," 210 Stock-solutions of dyes for staining, 148 Stomach as source of infection, 76 bacteria in, 76 Strauss' method for making bacillus mallei cultures, 346 Streptococci, 37 in blood in scarlatina, 258, 259 in intestinal canal of infants, 258 vitality in culture, 256 Streptococcus conglomeratus, 255 diffusus, 255 erysipelatis, 259 growth of, 260 inoculation with, as a therapeutic measure, 261 longus, 254 pyogenes, 254 growth of, 255 in internal diseases, 257 staining of, 255 virulence of, 256 Strepto-diplococci, 37 Streptothrix, 40 actinomyces, 356 growth of, 358 on blood-serum, 359 manner of entrance into body, 360 virulence of, when grown on ar- tificial media, 359 farcinica, 367 Madura1, 363 growth of, 364 " Strichcultur," 210 Stroke-culture, 210 Study of cultures, 202 Sulphur fumes as disinfectant, 177 " Summer complaint," bacteria in milk as cause of, 5c) Suppuration, air as factor in causation of, 246 causes of, 247 Surgery, aseptic, 171 Surgical contributions to history of bac- teriology, 24 Swine-cholera, susceptibility of animals to, 540 Swine-fever, ulceration of intestine in, 534 Swine-plague, 538 bacillus of, 538. See also Bacillus of siuine-plague. lesions of, 539 symptoms of, 539 Symptomatic anthrax, 575 Syntoxoids, 415 Syphilis, 351 bacillus of, 351. See also Bacillus of syphilis. Syringe, bacteriologic, Koch's, 222 Meyer's, 222 Roux's, 222 Syringes, disinfection of, 222 Temperature, influence of, on growth of bacteria, 48 manner of maintainance, for bacte- rial growth, 215 Tetanin, 378 Tetano-toxin, 378 Tetanus, 37 1 and hydrophobia, parallelism existing between, 385 antitoxic serum for, 378-382 bacillus of, 371. See also Bacillus tetani. bottle, 37s susceptibility of animals to, 377 Tetracocci, 37 Tetragenococci, 563. See also Micro- coccus tetragenus. Tetragenus, 563 micrococcus of, 563. See also Micro- coccus tetragenus. Thermal death-point, determination of, 240, 241 Thermophilic bacteria, 48 Thiothrix, 40 Tongue, wooden, 357, 362 TO-tubercuh'n, 323 Toxalbumin of diphtheria, 407 Toxic bacteria, 85 620 INDEX. Toxids, 415 Toxin elaborated by bacillus diph- theriae, 407 by bacillus icteroides, 522 tetano-, 378 Toxins, 86 action of antitoxin on, 132-137. See also Antitoxin, action upon toxin. apparatus for injection of, into horses, by gravity, 412 for rapid nitration of, 171 Toxophylaxins, 107 Toxosozins, 107 Trillat autoclave, 173 TR-tuberculin, 321-326 Tryptic enzymes, 55 Tubercle bacillus, 292. See also Bacil- lus tuberculosis. from cow, showing tubercle bacilli, 293 Tubercles, development of, 315 Tubercular leprosy, 343 pneumonia, 291 Tuberculin, 318 action of, 320 concentrated, 320 diluted, 320 effects of injections of, 319 Koch's, 320 new, 321-326 preparation, 318 R-, 321-326 test for animals, 320 TO-, 323 TR-, 321-326 Tuberculosis, 292 bacillus of, 292. See also Bacillus tuberculosis. bovine, 329 fowl-, 329 latent, 317 macroscopic lesions of, 312 of lung, PI. II. pseudo-, 334 universality of, 292 Tuberculous abscess, 314 Typhoid fever, 466 bacillus of, 266, 466. See also Bacillus typhosus. comparative immunity of animals to, 480 injections of sterilized cultures in, 502 inoculation experiments on animals, 480 intestinal perforation in, 477 Johnston's method for diagnosis of, 496 Typhoid fever, treatment of, by extracts of internal organs, 502 virulent character of discharges, 503 Widal reaction in, 484-502. See also Widal reaction. serum, action of, 483 Typhotoxin, 480 Typhus murium, 541 bacillus of, 541 . See also Bacillus typhi murium. Unna's method for staining tubercle bacilli in sections of tissue, 302 Unstained bacteria, method of examin- ing, 145 Urine, detection of tubercle bacilli in, 301 Uterus, bacteria of, 78 Vaccination, 113, 115 for symptomatic anthrax, 578 Vagina, flora of, 78 Van Ermengem's method for staining flagella, 161 Gorden's modification of, 162 Van Neissen's bacillus, 353. See also Bacillus of syphilis. Ventilation of sick-chamber, 177 Vibrio, 39 Metschnikowi, 442 proteus, 436 Schuylkiliensis, 452-454 tyrogenum, 440 " Vibrioneusepticcemia," 445 Virulence, 66 Vital activity in bacteria, results of, 51 Von Pettenkoffer's theory of cholera, 43 1 Vulva, bacteria of, 78 Walls of sick-room, disinfection of, 180 Water, bacteria in, 233 filtration as means of diminishing number in, 236 quantitative estimation of, 233, 234 drinking-, examination of, 236 hydrant-, bacteria in, 235 pump-, bacteria in, 235 river-, bacteria in, 235 Weil's method for cultivation of anaero- bic bacteria, 218 Whooping-cough, 598 bacillus of, 598. See also Bacillus of whooping- cough. bacillus X in, 599 Widal reaction, 484-502 INDEX. 621 Widal reaction, agglutinating power, re- lation of, to immunity, 492 transmission of, from parent to child, 490 substance, chemic nature of, 491 distribution of, 489 relation of, to leukocytes, 492 clinical value of serum diagnosis, 502 condition of culture, 499-501 culture method, 496 duration of, 487 table of, 488 rapid method, 496 relation of, to germicidal activity of blood, 489 slow method, 496 specific nature of, 494 technic of, 495 time of development, 484 Wolfhiigel's apparatus for counting colonies of bacteria upon plates, 233 Wooden tongue, 357, 362 Wool-sorters' disease, 461. See also Anthrax. Wounds as channels of entrance for tubercle bacilli. 312 Wynekoop's culture-outfit for isolating bacillus influenzae, 570 X bacillus, 518 A'-rays, influence of, on bacteria, 49 growth of Yellow fever, 518 antitoxin serum for, 525 bacillus coli communis in, 518 bacillus of, 518. See also Bacillus icteroides. bacillus of Havelburg in, 524 bacillus X in, 518 cause of black vomit in, 522 of death in, 522 pathology of, 523 Yersin's serum for plague, 562 ZlKHL's method for demonstration of tubercle bacilli in sputum, 298 Zooglea, 2 1 1 Zymogenic bacteria, 51 CATALOGUE OF THE MEDICAL PUBLICATIONS OP W- B, SAUNDERS & CCX, No. 925 WALNUT STREET, PHILADELPHIA. Arranged Alphabetically and Classified under Subjects. THE books advertised in this Catalogue as being sold by subscription are usually to be obtained from travelling solicitors, but they will be sent direct from the office of pub- lication (charges of shipment prepaid) upon receipt of the prices given. All the other books advertised are commonly for sale by booksellers in all parts of the United States ; but books will be sent to any address, carriage prepaid, on receipt of the published price. 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AN AMERICAN TEXT-BOOK OF THE DISEASES OF CHILDREN. Second Edition, Revised. By 65 Eminent Contributors. Edited by Louis Starr, M. D., Con- sulting Pediatrist to the Maternity Hospital, etc. ; assisted by Thomp- son S. Westcott, M. D., Attending Physician to the Dispensary for Diseases of Children, Hospital of the University of Pennsyl- vania. In one handsome imperial octavo volume of 1244 pages, profusely illustrated. Cloth, $7.00 net; Sheep or Half Morocco, $8.00 net. Sold by Subscription. •'This is far and away the best text-book on children's diseases ever published in the English language, and is certainly the one which is best adapted to American readers. We congratulate the editor upon the result of his work, and heartily commend it to the attention of every student and practitioner." — American Journal of the Medical Sciences. AN AMERICAN TEXT-BOOK OF DISEASES OF THE EYE, EAR, NOSE, AND THROAT. By 58 Prominent Specialists. Edited by G. E. de Schweinitz, M.D., Professor of Ophthalmology in the Jefferson Medical College, Phila- delphia ; and B. Alexander Randall, M.D., Professor of Diseases of the Ear in the University of Pennsylvania. Imperial octavo, 1251 pages ; 766 illustrations, 59 of them in colors. Cloth, $7.00 net ; Sheep or Half Morocco, $8.00 net. Sold by Subscription. Illustrated Catalogue of the'" American Text-Books ** seat free upon application. 6 Medical Publications of W. B. Saunders & Co. AN AMERICAN TEXT-BOOK OF GENITO-URINARY AND SKIN DISEASES. By 47 Eminent Specialists and Teachers. Edited by L. Bolton Bangs, M. D., Professor of Genito- Urinary Surgery, University and Bellevue Hospital Medical College, New York : and W. A. Hard- away, M. D., Professor of Diseases of the Skin, Missouri Medical College. Imperial octavo volume of 1229 pages, with 300 engravings and 20 full-page colored plates. Cloth, $7.00 net; Sheep or. Half Morocco, $8.00 net. Sold by Subscription. " This volume is one of the best yet issued of the publisher's series of ' American Text- Books.' The list of contributors represents an extraordinary array of talent and extended experience. The book will easily take the place in comprehensiveness and value of the half dozen or more costly works on these subjects which have heretofore been necessary to a well-equipped library." — New York Polyclinic. AN AMERICAN TEXT-BOOK OF GYNECOLOGY, MEDICAL AND SURGICAL. Second Edition, Revised. By 10 of the Leading Gynecologists of America. Edited by J. M. Baldy, M. D., Professor of Gynecology in the Philadelphia Polyclinic, etc. Handsome imperial octavo volume of 718 pages, with 341 illus- trations in the text, and 38 colored and half-tone plates. Cloth, $6.00 net; Sheep or Half Morocco, $7.00 net. Sold by Subscription. " It is practical from beginning to end. Its descriptions of conditions, its recommen- dations for treatment, and above all the necessary technique of different operations, are clearly and admirably presented. . . . It is well up to the most advanced views of the day, and embodies all the essential points of advanced American gynecology. It is destined to make and hold a place in gynecological literature which will be peculiarly its own." — Medical Record, New York. AN AMERICAN TEXT-BOOK OF LEGAL MEDICINE AND TOXI- COLOGY. Edited by Frederick Peterson, M.D., Clinical Professor of Mental Diseases in the Woman's Medical College, New York; Chief of Clinic, Nervous Department, College of Physicians and Surgeons, New York ; and Walter S. Haines, M.D., Professor of Chemistry, Pharmacy, and Toxicology in Rush Medical College, Chicago. In Preparation. AN AMERICAN TEXT-BOOK OF OBSTETRICS. By 15 Eminent American Obstetricians. Edited by Richard C. Nor- ris, M.D.; Art Editor, Robert L. Dickinson, M.D. One handsome imperial octavo volume of 1014 pages, with nearly 900 beautiful colored and half-tone illustrations. Cloth, $7.00 net; Sheep or Half Morocco, $8.00 net. Sold by Subscription. " Permit me to say that your American Text-Book of Obstetrics is the most magnificent medical work that I have ever seen. I congratulate you and thank you for this superb work; which alone is sufficient to place you first in the ranks of medical publishers." — Alexander J. C. Skene, Professor of Gynecology in the Long Island College Hospital, Brooklyn, N. Y. " This is the most sumptuously illustrated work on midwifery that has yet appeared. In the number, the excellence, and the beauty of production of the illustrations it far surpasses every other book upon the subject. This feature alone makes it a work which no medical library should omit to purchase." — British Medical Journal. " A* an authority, as a book of reference, as a ' working book ' for the student or prac- titioner, we commend it because we believe there is no better." — American Journal of the Medical Sciences. Illustrated Catalogue of the ** American Text-Books n sent free upon application* Medical Publications of W. B. Saunders & Co. AN AMERICAN TEXT-BOOK OF PATHOLOGY. Edited by Ludvig Hektoen, M. P.. Professor of General Pathology and of Morbid Anatomy in the University of Pennsylvania ; and David RiESMAN, M. D., Demonstrator of Pathological Histology in the University of Pennsylvania. In preparation. AN AMERICAN TEXT-BOOK OF PHYSIOLOGY. By 10 of the Leading Physiologists of America. Edited by William H. Howell, Ph.D., M.D., Professor of Physiology in the Johns Hop- kins University, Baltimore, Md. Second edition, revised and enlarged, in two volumes. " We can commend it most heartily, not only to all students of physiology, but to every physician and pathologist, as a valuable and comprehensive work of reference, written by men who are of eminent authority in their own special subjects." — London Lancet. ** To the practitioner of medicine and to the advanced student this volume constitutes, we believe, the best exposition of the present status of the science of physiology in the English language." — American Journal of the Medical Sciences. AN AMERICAN TEXT-BOOK OF SURGERY. Third Edition. By ii Eminent Professors of Surgery. Edited by William W. Keen, M.D., LL.D., and J. William White, M.D., Ph.D. Handsome im- perial octavo volume of 1230. pages, with 496 wood-cuts in the text, and 37 colored and half-tone plates. Thoroughly revised and enlarged, with a section devoted to " The Use of the Rontgen Rays in Surgery." Cloth, $7-°° net; Sheep or Half Morocco, #8.00 net. •' Personally, I should not mind it being called THE Text-Book (instead of A Text- Book) , for I know of no single volume which contains so readable and complete an account of the science and art of Surgery as this does." — EDMUND Owen, F.R.C.S., Member 0/ the Board of Examiners of the Royal College of Surgeons, England. " If this text-book is a fair reflex of the present position of American surgery, we must admit it is of a very high order of merit, and that English surgeons will have to look very carefully to their laurels if they are to preserve a position in the van of surgical practice."— London Lancet. AN AMERICAN TEXT-BOOK OF THE THEORY AND PRACTICE OF MEDICINE. By 12 Distinguished American Practitioners. Edited by William Pepper, M.D., LL.D., Professor of the Theory and Practice of Medi- cine and of Clinical Medicine in the University of Pennsylvania. Two handsome imperial octavo volumes of about 1000 pages each. Illus- trated. Prices per volume : Cloth, $5.00 net ; Sheep or Half Morocco, $6.00 net. Sold by Subscription. M I am quite sure it will commend itself both to practitioners and students of medicine, and become one of our most popular text-books." — Alfred Loomis, M.D., LL.D., Pro- fessor of Pathology and Practice of Medicine, University of the City of New York. " We reviewed the first volume of this work, and said : * It is undoubtedly one of the best text-books on the practice of medicine which we possess.' A consideration of the second and last volume leads us to modify that verdict and to say that the completed work is in our opinion the best of its kind it has ever been our fortune to see." — New York Medicai Journal. Illustrated Catalogue of the * American Text-Books " sent free upon application. 8 Medical Publications of W. B. Saunders & Co. AN AMERICAN YEAR-BOOK OF MEDICINE AND SURGERY. A Yearly Digest of Scientific Progress and Authoritative Opinion in all branches of Medicine and Surgery, drawn from journals, monographs, and text-books of the leading American and Foreign authors and investigators. Arranged with critical editorial comments, by eminent American specialists, under the general editorial charge of George M. Gould, M.D. Volumes for 1896, '97, '98, and '99. One imperial octavo volume of about 1200 pages. Cloth, $6.50 net ; Half Morocco, $7.50 net. Year-Book of 1900 in two volumes — Vol. L, including General Medicine; Vol. II., General Surgery. Prices per volume: Cloth, $3.00 net; Half Morocco, $3.75 net. Sold by Subscription. " It is difficult to know which to admire most — the research and industry of the distin- guished band of experts whom Dr. Gould has enlisted in the service of the Year-Book, or the wealth and abundance of the contributions to every department of science that have been deemed worthy of analysis. ... It is much more than a mere compilation of abstracts, for, as each section is entrusted to experienced and able contributors, the reader has the advant- age of certain critical commentaries and expositions . . . proceeding from writers fully qualified to perform these tasks. ... It is emphatically a book which should find a place in every medical library, and is in several respects more nseful than the famous 'Jahrbiicher' of Germany." — London Lancet. ABBOTT ON TRANSMISSIBLE DISEASES. The Hygiene of Transmissible Diseases ; their Causation, Modes of Dissemination, and Methods of Prevention. By A. C. Abbott, M.D., Professor of Hygiene and Bacteriology, University of Pennsylvania ; Director of the Laboratory of Hygiene. Octavo volume of 311 pages, containing a number of charts and maps, and numerous illustrations. Cloth, $2.00 net. THE AMERICAN POCKET MEDICAL DICTIONARY. [See D or I and' s Pocket Dictionary, page 12.] ANDERS' PRACTICE OF MEDICINE. Third Revised Edition. A Text-Book of the Practice of Medicine. By James M. Anders, M.D., Ph.D., LL.D., Professor of the Practice of Medicine and of Clinical Medicine, Medico-Chirurgical College, Philadelphia. In one handsome octavo volume of 1292 pages, fully illustrated. Cloth, $5.50 net; Sheep or Half Morocco, $6.50 net. " It is an excellent book, — concise, comprehensive, thorough, and up to date. It is a credit to you ; but, more than that, it is a credit to the profession of Philadelphia — to us." James C. Wilson, Professor of the Practice of Medicine and Clinical Medicine, Jefferson Medical College, Philadelphia. ASHTON'S OBSTETRICS. Fourth Edition, Revised. Essentials of Obstetrics. By W. Easterly Ashton, M.D., Pro- fessor of Gynecology in the Medico-Chirurgical College, Philadelphia. Crown octavo, 252 pages; 75 illustrations. Cloth, $1.00 net; inter- leaved for notes, $1.25 net. [See Saunders1 Question- Compends, page 23.] " Embodies the whole subject in a nut-shell. We cordially recommend it to our read ers." — Chicago Medical Times. Medical Publications of W. B. Saunders & Co. 9 BALL'S BACTERIOLOGY. Third Edition, Revised. Essentials of Bacteriology ; a Concise and Systematic Introduction to the Study of Micro-organisms. By M. V. Ball, M.D., Bacteriol- ogist to St. Agnes' Hospital, Philadelphia, etc. Crown octavo, 218 pages; 82 illustrations, some in colors, and 5 plates. Cloth, $1.00; interleaved for notes, $1.25. [See Saunders' Question- Compends, page 23.] ' • The student or practitioner can readily obtain a knowledge of the subject from a perusal of this book. The illustrations are clear and satisfactory." — Medical Record, New York. BASTIN'S BOTANY. Laboratory Exercises in Botany. Bv Edson S. Bastin, M.A., late Prof, of Materia Medica and Botany, Philadelphia College of Phar- macy. Octavo volume of 536 pages, with 87 plates. Cloth, §2.00 net. «• It is unquestionably the best text-book on the subject that has yet appeared. The work is eminently a practical one. We regard the issuance of this book as an important event in the history of pharmaceutical teaching in this country, and predict for it an unquali- fied success." — Alumni Report to the Philadelphia College of Pharmacy. BECK ON FRACTURES. Fractures. By Carl Beck, M.D., Surgeon to St. Mark's Hospital and the New York German Poliklinik, etc. 225 pages, 170 illustrations. Cloth, $3.50 net. BECK'S SURGICAL ASEPSIS. A Manual of Surgical Asepsis. By Carl Beck, M.D., Surgeon to St. Mark's Hospital and the New York German Poliklinik, etc. 306 pages; 65 text-illustrations, and 12 full-page plates. Cloth, $1.25 net. " An excellent exposition of the * very latest ' in the treatment of wounds as practised by leading German and American surgeons." — Birmingham (Eng.) Medical Review. " This little volume can be recommended to any who are desirous of learning the details of asepsis in surgery, for it will serve as a trustworthy guide." — London Lancet. BOISLINIERE'S OBSTETRIC ACCIDENTS, EMERGENCIES, AND OPERATIONS. Obstetric Accidents, Emergencies, and Operations. By L. Ch. Boisliniere, M.D., late Emeritus Professor of Obstetrics, St. Louis Medical College. 381 pages, handsomely illustrated. Cloth, $2.00 net. " A manual so useful to the student or the general practitioner has not been brought to our notice in a long time. The field embraced in the title is covered in a terse, interesting way." — Yale Medical Journal. BROCKWAY'S MEDICAL PHYSICS. Second Edition, Revised. Essentials of Medical Physics. By Fred J. Brockwav, M.D., Assistant Demonstrator of Anatomy in the College of Physicians and Surgeons, New York. Crown octavo, 330 pages ; 155 fine illustrations. Cloth, $1.00 net ; interleaved for notes, $1.25 net. [See Saunders' Question- Compends, page 23.] "We know of no manual that affords the medical student a better or more concise exposition of physics, and the book may be commended as a most satisfactory presentation of those essentials that are requisite in a course in medicine." — New York Medical Journal. 10 Medical Publications of W. B. Saunders & Co. BUTLER'S MATERIA MEDICA, THERAPEUTICS, AND PHAR- MACOLOGY. Third Edition, Revised. A Text=Book of Materia Medica, Therapeutics, and Pharma- cology. By George F. Butler, Ph.G., M.D., Professor of Materia Medica and of Clinical Medicine in the College of Physicians and Surgeons, Chicago; Professor of Materia Medica and Therapeutics, Northwestern University, Woman's Medical School, etc. Octavo, 874 pages, illustrated. Cloth, $4.00 net; Sheep, $5.00 net. " Taken as a whole, the book may fairly be considered as one of the most satisfactory of any single-volume works on materia medica in the market," — Journal of the American > Medical Association. CERNA ON THE NEWER REMEDIES. Second Edition, Revised. Notes on the Newer Remedies, their Therapeutic Applications and Modes of Administration. By David Cerna, M.D., Ph.D., formerly Demonstrator of and Lecture* on Experimental Therapeutics in the University of Pennsylvania; Demonstrator of Physiology in the Medical Department of the University of Texas. Rewritten and greatly enlarged. Post-octavo, 253 pages. Cloth, $1.00 net. " The appearance of this new edition of Dr. Cerna's very valuable work shows that it is properly appreciated. The book ought to be in the possession of every practising physi- cian."— New York Medical Journal. CHAPIN ON INSANITY. A Compendium of Insanity. By John B. Chapin, M.D., LL.D., Physician-in-Chief, Pennsylvania Hospital for the Insane ; late Physi- cian-Superintendent of the Willard State Hospital, New York ; Hon- orary Member of the Medico-Psychological Society of Great Britain, of the Society of Mental Medicine of Belgium. i2mo, 234 pages, illustrated. Cloth, $1.25 net. " The practical parts of Dr. Chapin's book are what constitute its distinctive merit. We desire especially to call attention to the fact that on the subject of therapeutics of insanity the work is exceedingly valuable. It is not a made book, but a genuine condensed thesis, which has all the value of ripe opinion and all the charm of a vigorous and natural style." — Philadelphia Medical Journal. CHAPMAN'S MEDICAL JURISPRUDENCE AND TOXICOLOGY. Second Edition, Revised. Medical Jurisprudence and Toxicology. By Henry C. Chapman, M.D., Professor of Institutes of Medicine and Medical Jurisprudence in the Jefferson Medical College of Philadelphia. 254 pages, with 55 illustrations and 3 full-page plates in colors. Cloth, $1.50 net. "The best book of its class for the undergraduate that we know of." — Nevu York Medical Times. CHURCH AND PETERSON'S NERVOUS AND MENTAL DISEASES. Second Edition. Nervous and Mental Diseases. By Archibald Church, M. D., Professor of Clinical Neurology, Mental Diseases, and Medical Juris- prudence in the Northwestern University Medical School, Chicago ; and Frederick Peterson, M. D., Clinical Professor of Mental Dis- eases, Woman's Medical College, N. Y. ; Chief of Clinic, Nervous Dept., College of Physicians and Surgeons, N. Y. Handsome octavo volume of 843 pages, profusely illustrated. Cloth, $5.00 net; Half Morocco, $6.00 net. Medical Publications of W. B. Saunders <£- Co. 11 CLARKSON'S HISTOLOGY. A Text-Book of Histology, Descriptive and Practical. By Arthur Clarkson, M.B., CM. Edin., formerly Demonstrator of Physiology in the Owen's College, Manchester; late Demonstrator of Physiology in Yorkshire College, Leeds. Large octavo, 554 pages; 22 engravings in the text, and 174 beautifully colored original illustra- tions. Cloth, strongly bound, $4.00 net. "The work must be considered a valuable addition to the list of available text books, and is to be highly recommended." — New York Medical Journal. "This is one of the best works for students we have ever noticed. We predict that the book will attain a well-deserved popularity among our students." — Chicago Medical Recorder. CLIMATOLOGY. Transactions of the Eighth Annual Meeting of the American Climatological Association, held in Washington, September 22-25, 1 89 1. Forming a handsome octavo volume of 276 pages, uniform with remainder of series. (A limited quantity only.) Cloth, $1.50. COHEN AND ESHNER'S DIAGNOSIS. Second Edition, Revised. Essentials of Diagnosis. By Solomon Solis-Cohen, M.D., Pro- fessor of Clinical Medicine and Applied Therapeutics in the Philadel- phia Polyclinic ; and Augustus A. Eshner, M.D., Professor of Clinical Medicine in the Philadelphia Polyclinic. Post-octavo, 417 pages; 55 illustrations. Cloth, $1.00 net. [See Saunders1 Question- Compends, page 23.] " We can heartily commend the lx>ok to all those who contemplate purchasing a 'com- pend.' It is modern and complete, and will give more satisfaction than many other works which are perhaps too prolix as well as behind the times." — Medical Review, St. Louis. CORWIN'S PHYSICAL DIAGNOSIS. Third Edition, Revised. Essentials of Physical Diagnosis of the Thorax. By Arthur M. Corwin, A.M., M. D., Demonstrator of Physical Diagnosis in Rush Medical College, Chicago ; Attending Physician to Central Free Dis- pensary, Department of Rhinology, Laryngology, and Diseases of the Chest, Chicago. 219 pages, illustrated. Cloth, flexible covers, $1.25 net. " It is excellent. The student who shall use it as his guide to the careful study of physical exploration upon normal and abnormal subjects can scarcely fail to acquire a good working knowledge of the subject." — Philadelphia Polyclinic. "A most excellent little work. It brightens the memory of the differential diagnostic signs, and it arranges orderly and in sequence the various objective phenomena to logical solution of a careful diagnosis." — Journal of Nervous and Mental Diseases. CRAGIN'S GYN/ECOLOGY. Fourth Edition, Revised. Essentials of Gynaecology. By Edwin B. Cragin, M. D., Lecturer in Obstetrics, College of Physicians and Surgeons, New- York. Crown octavo, 200 pages; 62 illustrations. Cloth, $1.00 net; interleaved for notes, Si. 2 5 net. [See Saunders1 Question- Compends, page 23.] " A handy volume, and a distinct improvement on students' compends in general. No author who was not himself a practical gynecologist could have consulted the students needs so thoroughly as Dr. Cragin has done." — Medical Record, New York. 12 Meaical Publications of W. B. Saunders & Co. CROOKSHANK'S BACTERIOLOGY. Fourth Edition, Revised. A Text-Book of Bacteriology. By Edgar M. Crookshank, M.B., Professor of Comparative Pathology and Bacteriology, King's College, London. Octavo volume of 700 pages, with 273 engravings and 22 original colored plates. Cloth, $6.50 net; Half Morocco, $7.50 net. *' To the student who wishes to obtain a good resume" of what has been done in bacteri- ology, or who wishes an accurate account of the various methods of research, the book may be recommended with confidence that he will find there what he requires." — London Lancet. Da COSTA'S SURGERY. Second Ed., Revised and Greatly Enlarged. Modern Surgery, General and Operative. By John Chalmers DaCosta, M. D., Professor of Practice of Surgery and Clinical Surgery, Jefferson Medical College, Philadelphia ; Surgeon to the Philadelphia Hospital, etc. Handsome octavo volume of 911 pages, profusely illus- trated. Cloth, $4.00 net; Half Morocco, $5.00 net. "We know of no small work on surgery in the English language which so well fulfils the requirements of the modern student." — Medico- Chirurgical Journal, Bristol, England. DE SCHWEINITZ ON DISEASES OF THE EYE. Third Edition, Revised. Diseases of the Eye. A Handbook of Ophthalmic Practice. By G. E. de Schweinitz, M.D., Professor of Ophthalmology in the Jefferson Medical College, Philadelphia, etc. Handsome royal octavo volume of 696 pages, with 256 fine illustrations and 2 chromo-litho- graphic plates. Cloth, $4.00 net ; Sheep or Half Morocco, $5.00 net. " A clearly written, comprehensive manual. One which we can commend to students as a reliable text-book, written with an evident knowledge of the wants of those entering upon the study of this special branch of medical science." — British Medical Journal. " A work that will meet the requirements not only of the specialist, but of the general practitioner in a rare degree. I am satisfied that unusual success awaits it." — William Pepper, M.D., Professor of the Theory and Pi-actice of Medicine and Clinical Medicine, University of Pennsylvania. DORLAND'S DICTIONARY. Third Edition, Revised. The American Pocket Medical Dictionary. Containing the Pro- nunciation and Definition of all the principal words and phrases, and a large number of useful tables. Edited by W. A. Newman Dorland, M. D., Assistant Demonstrator of Obstetrics, University of Pennsylvania; Fellow of the American Academy of Medicine. 518 pages ; handsomely bound in full leather, limp, with gilt edges and patent index. Price, $1.00 net; with thumb index, $1.25 net. DORLAND'S OBSTETRICS. A Manual of Obstetrics. By W. A. Newman Dorland, M.D., Assistant Demonstrator of Obstetrics, University of Pennsylvania; Instructor in Gynecology in the Philadelphia Polyclinic. 760 pages; 163 illustrations in the text, and 6 full-page plates. Cloth, $2.50 net. " By far the best book on this subject that has ever come to our notice." — American Medical Review. " It has rarely been our duty to review a book which has given us more pleasure in its perusal and more satisfaction in its criticism. It is a veritable encyclopedia of knowledge, a gold mine of practical, concise thoughts." — American Medico-Surgical Bulletin. Medical Publications of W. B. Saunders & Co. 13 FROTHING HAM'S GUIDE FOR THE BACTERIOLOGIST. Laboratory Guide for the Bacteriologist. By Langdon Froth- ingham, M.D.V., Assistant in Bacteriology and Veterinary Science, Sheffield Scientific School, Yale University. Illustrated. Cloth, 75 cts. '* It is ■ convenient and useful little work, and will more than repay the outlay neces- sary for its purchase in the saving of time which would otherwise be consumed in looking up the various points of technique so clearly and concisely laid down in its pages. "— Ameri- can Medico-Surgical Bulletin. GARRIGUES' DISEASES OF WOMEN. Third Edition, Revised. Diseases of Women. By Henry J. Garrigues, A.M., M.D., Pro- fessor of Gynecology in the New York School of Clinical Medicine ; Gynecologist to St. Mark's Hospital and to the German Dispensary, New York City, etc. Handsome octavo volume of 783 pages, illus- trated by 367 engravings and colored plates. Cloth, #4.00 net; Sheep or Half Morocco, $5.00 net. '* One of the best text-books for students and practitioners which has been published in the English language ; it is condensed, clear, and comprehensive. The profound learning and great clinical experience of the distinguished author find expression in this book in a most attractive and instructive form. Young practitioners to whom experienced consultants may not be available will find in this book invaluable counsel and help." — Thad. A. Reamy, M.D., LL.D., Professor of Clinical Gynecology, Medical College of Ohio. GLEASON'S DISEASES OF THE EAR. Second Edition, Revised. Essentials of Diseases of the Ear. By E. B. Gleason, S.B., M.D. , Clinical Professor of Otology, Medico-Chirurgical College, Philadelphia ; Surgeon-in-Charge of the Nose, Throat, and Ear Depart- ment of the Northern Dispensary, Philadelphia. 208 pages, with 114 illustrations. Cloth, $1.00 net; interleaved for notes, $1.25 net. [See Saunders1 Question- Compends, page 23.] " It is just the book to put into the hands of a student, and cannot fail to give him a useful introduction to ear-affections ; while the style of question and answer which is adopted throughout the book is, we believe, the best method of impressing facts permanently on the mind. " — Liverpool Medico- Chirurgical Journal. GOULD AND PYLE'S CURIOSITIES OF MEDICINE. Anomalies and Curiosities of Medicine. By George M. Gould, M.D., and Walter L. Pyle, M.D. An encyclopedic collection of rare and extraordinary cases and of the most striking instances of abnormality in all branches of Medicine and Surgery, derived from an exhaustive research of medical literature from its origin to the present day, abstracted, classified, annotated, and indexed. Handsome im- perial octavo volume of 968 pages, with 295 engravings in the text, and 12 full-page plates. POPULAR EDITION : Cloth, $3.00 net ; Half Morocco, $4.00 net. " One of the most valuable contributions ever made to medical literature. Ii is, so far as we know, absolutely unique, and every page is as fascinating as a novel. Not alone for the medical profession has this volume value : it will serve as a book of reference for all who are interested in general scientific, sociologic, or medico-legal topics." — Brooklyn Medical Journal. "This is certainly a most remarkable and interesting volume. It stands alone among medical literature, an anomaly on anomalies, in that there is nothing like it elsewhere in medical literature. It is a book full of revelations from its firsi to its last page, and cannot but interest and sometimes almost horrify its readers." — American Medico- Surgical Bulletin. 14 Medical Publications of W. B. Saunders & Co. GRAFSTROM'S MECHANO-THERAPY. A Text=Book of Mechano-Therapy (Massage and Medical Gym- nastics). By Axel V. Grafstrom, B. Sc, M. D., late Lieutenant in the Royal Swedish Army ; late House Physician City Hospital, Black- well's Island, New York. i2mo, 139 pages, illustrated. Cloth, $1. 00 net. GRIFFITH ON THE BABY. Second Edition, Revised. The Care of the Baby. By J. P. Crozer Griffith, M.D., Clini- cal Professor of Diseases of Children, University of Pennsylvania ; Physician to the Children's Hospital, Philadelphia, etc. i2mo, 404 pages, with 67 illustrations in the text, and 5 plates. Cloth, $1.50 net. " The best book for the use of the young mother with which we are acquainted. . . . There are very few general practitioners who could not read the book through with advan- tage."— Archives of Pediatrics. "The whole book is characterized by rare good sense, and is evidently written by a master hand. It can be read with benefit not only by mothers but by medical students and by any practitioners who have not had large opportunities for observing children." — Ameri- can Journal of Obstetrics. GRIFFITH'S WEIGHT CHART. Infant's Weight Chart. Designed by J. P. Crozer Griffith, M. D. , Clinical Professor of Diseases of Children in the University of Penn- sylvania, etc. 25 charts in. each pad. Per pad, 50 cents net. GROSS, SAMUEL D., AUTOBIOGRAPHY OF. Autobiography of Samuel D. Gross, M. D., Emeritus Professor of Surgery in the Jefferson Medical College, Philadelphia, with Remi- niscences of His Times and Contemporaries. Edited by his Sons, Samuel W. Gross, M.D., LL. D., and A. Haller Gross, A.M. Pre- ceded by a Memoir of Dr. Gross, by the late Austin Flint, M.D. Two handsome volumes, over 400 pages each, demy octavo, gilt tops, with Frontispiece on steel. Price per volume, $2.50 net. HAMPTON'S NURSING. Second Edition, Revised and Enlarged. Nursing: Its Principles and Practice. By Isabel Adams Hamp- ton, Graduate of the New York Training School for Nurses attached to Bellevue Hospital ; late Superintendent of Nurses and Principal of the Training School for Nurses, Johns Hopkins Hospital, Baltimore, Md. 12 mo, 512 pages, illustrated. Cloth, $2.00 net. " Seldom have we perused a book upon the subject that has given us so much pleasure as the one before us. We would strongly urge upon the members of our own profession the need of a book like this, for it will enable each of us to become a training school in him- self."— Ontario Medical Journal. HARE'S PHYSIOLOGY. Fourth Edition, Revised. Essentials of Physiology. By H. A. Hare, M.D., Professor of Therapeutics and Materia Medica in the Jefferson Medical College of Philadelphia. Crown octavo, 239 pages. Cloth, #1.00 net; inter- leaved for notes, #1.25 net. [See Saunders' Question- Compends, page 23.] " The best condensation of physiological knowledge we have yet seen." — Medical Record, New York. Medical Publications of W. B. Saunders & Co. 16 HART'S DIET IN SICKNESS AND IN HEALTH. Diet in Sickness and in Health. By Mrs. Ernest Hart, formerly Student of the Faculty of Medicine of Paris and of the London School of Medicine for Women ; with an Introduction by Sir Henry Thompson, F.R.C.S., M.D., London. 220 pages. Cloth, $1.50 net. " We recommend it cordially to the attention of all practitioners ; both to them and to their patients it may be of the greatest service." — New York Medical Journal. HAYNES* ANATOMY. A Manual of Anatomy. By Irving S. Havnes, M.D., Adjunct Professor of Anatomy and Demonstrator of Anatomy, Medical Depart- ment of the New York University, etc. 680 pages, illustrated with 42 diagrams in the text, and 134 full-page half-tone illustrations from original photographs of the author's dissections. Cloth, $2.50 net. 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Cloth, $2.00 net. KEEN'S OPERATION BLANK. Second Edition, Revised Form. An Operation Bl»nk, with Lists of Instruments, etc., Required in Various Operations. Prepared by W. W. Keen, M.D., LL.D., Professor of the Principles of Surgery in Jefferson Medical College, Philadelphia. Price per pad, blanks for fifty operations, 50 cents net. Medical Publications of W. B. Saunders t£ Co, 17 KEEN ON THE SURGERY OF TYPHOID FEVER. The Surgical Complications and Sequels of Typhoid Fever. By Wm. \V. KEEN, M.D., LL.D., Professor of the Principles of Sur- gery and of Clinical Surgery, Jefferson Medical College, Philadelphia; Corresponding Member of the Societe de Chirurgie, Paris ; Honorary Member of the Societe Beige de Chirurgie, etc. Octavo volume of 386 pages, illustrated. Cloth, $3.00 net. " This is probably the first and only work in the English language that gives the reader a clear view of what typhoid fever really is, and what it does and can do to the human organism. 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Containing also Venereal Diseases, Surgi- cal Landmarks, Minor and Operative Surgery, and a complete de- scription, with illustrations, of the Handkerchief and Roller Bandages. By Edward Martin, A.M., M.D., Clinical Professor of Genito- Urinary Diseases, University of Pennsylvania, etc. Crown octavo, 342 pages, illustrated. With an Appendix on the preparation of the materials used in Antiseptic Surgery, etc., and a chapter on Appendicitis. Cloth, $1.00 net; interleaved for notes, $1.25 net [See Saunders' Question- Compends, page 23.] "Contains all necessary essentials of modern surgery in a comparatively small space. Its style is interesting, and its illustrations are admirable." — Medical and Surgical Reporter. Medial Publications of W. B. Saunders .1- Co. 19 McFARLAND'S PATHOGENIC BACTERIA. Second Edition, Re- vised and Greatly Enlarged. Text-Book upon the Pathogenic Bacteria. By Joseph McFar- i AND, M. D.. 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[See Saunders' Question- Compends, page 22.] "This work, already excellent in the old edition, has been largely improved by revi- sion."— American Practitioner and News. MORRIS, WOLFF, AND POWELL'S PRACTICE OF MEDICINE. Third Edition, Revised. Essentials of the Practice of Medicine. By Henry Morris, M.D., late Demonstrator of Therapeutics, Jefferson Medical College, Phila- delphia; with an Appendix on the Clinical and Microscopic Examina- tion of Urine, by Lawrence Wolff, M.D. , Demonstrator of Chemistry, Jefferson Medical College, Philadelphia. Enlarged by some 300 essen- tial formulae collected and arranged by William M. Powell, M.D. Post-octavo, 488 pages. Cloth, $1.50 net. [See Saunders' Question- Compends, page 22.] " The teaching is sound, the presentntion graphic ; matter full as can be desired, ends, page 21.] "Contains the gist of all the best works in the department to which it relates."— American Practitioner and News. PRINGLE'S SKIN DISEASES AND SYPHILITIC AFFECTIONS. Pictorial Atlas of Skin Diseases and Syphilitic Affections (American Edition). Translation from the French. Edited by J. J. Pringle, M.B., F.R.C.P., Assistant Physician to the Middlesex Hospital, London. Photo-lithochromes from the famous models in the Museum of the Saint-Louis Hospital, Paris, with explanatory wood- cuts and text. In 12 Parts. Price per Part, $3.00. Complete in one volume, Half Morocco binding, $40.00 net. " I strongly recommend this Atlas. The plates are exceedingly well executed, and will be of great value to all studying dermatology." — Stephen Mackenzie, M.D. "The introduction of explanatory wood-cuts in the text is a novel and most important feature which greatly furthers the easier understanding of the excellent plates, than which nothing, we venture to say, has been seen better in point of correctness, beauty, and general merit." — New York Medical Journal. PR YOR— PELVIC INFLAMMATIONS. The Treatment of Pelvic Inflammations through the Vagina. By W. R. Pryor, M.D., Professor of Gynecology in New York Poly- clinic. i2mo, 248 pages, handsomely illustrated. Cloth, $2.00 net. " This subject, which has recently been so thoroughly canvassed in high gynecological circles, is made available in this volume to the general practitioner and student. Nothing is too minute for mention and nothing is taken for granted ; consequently the book is of the utmost value. The illustrations and the technique are beyond criticism." — Chicago Medical Recorder. PYE'S BANDAGING. Elementary Bandaging and Surgical Dressing. With Direc- tions concerning the Immediate Treatment of Cases of Emergency. For the use of Dressers and Nurses. By Walter Pye, F.R.C.S., late Surgeon to St. Mary's Hospital, London. Small i2mo, with over 80 illustrations. Cloth, flexible covers, 75 cents net. " The directions are clear and the illustrations are good." — London Lancet. " The author writes well, the diagrams are clear, and the book itself is small and port* able, although the paper and type are good." — British Medical Journal. RAYMOND'S PHYSIOLOGY. A Manual of Physiology. By Joseph H. Raymond, A.M., M.D., Professor of Physiology and Hygiene and Lecturer on Gynecology in the Long Island College Hospital ; Director of Physiology in the Hoagland Laboratory, etc. 382 pages, with 102 illustrations in the text, and 4 full -page colored plates. Cloth, #1.25 net. " Extremely well gotten up, and the illustrations have been selected with care. The text is fully abreast with modern physiology." — British Medical Journal. Saunders' Ananf d in Qrtion ^ Answer Form. Question Compends npHE MOST COMPLETE AND BEST ILLUSTRATED SERIES OF COMPENDS EVER ISSUED. Now the Standard Authorities in Medical Literature with Students and Practitioners in every City of the United States and Canada. ^ OVER 175,000 COPIES SOLD. ^ THE REASON WHY. They are the advance guard of "Student's Helps" — that DO help. They are the leaders in their special line, well and authoritatively written by able men, who, as teachers in the large colleges, know exactly what is wanted by a student preparing for his examinations. The judgment exercised in the selection of authors is fully demonstrated by their professional standing. Chosen from the ranks of Demonstrators, Quiz-masters, and Assistants, most of them have become Professors and Lecturers in their respective colleges. Each book is of convenient size (5x7 inches), containing on an average 250 pages, profusely illustrated, and elegantly printed in clear, readable type, on fine paper. The entire series, numbering twenty-three volumes, has been kept thoroughly revised and enlarged when necessary, many of the books being in their fifth and sixth editions. TO SUM UP. Although there are numerous other Quizzes, Manuals, Aids, etc. in the market, none of them approach the " Blue Series of Question Compends ; ' ' and the claim is made for the following points of excellence : 1. Professional distinction and reputation of authors. 2. Conciseness, clearness, and soundness of treatment. 3. Quality of illustrations, paper, printing, and binding. Any cf these Compends will be mailed on receipt of price (see next page for List). Saunders' v^uestion-Compend Oeries* Price, Cloth, $1.00 net per copy, except when otherwise ordered. "Where the work of preparing students' manuals is to end we cannot say, but the Saunders Series, in our opinion, bears off the palm at present."— New }"orJk Medical Record. 1. ESSENTIALS OF PHYSIOLOGY. By H. A. Hare, M.D. Fourth edition, revised and enlarged. 2. ESSENTIALS OF SUROERY. By Edward Martin, M. D. Seventh edition, revised, with an Appendix And a chapter on Appendicitis. 3. ESSENTIALS OF ANATOMY. By Chari.es B. Nancrede, M.D. Sixth edition, thoroughly revised and enlarged. 4. ESSENTIALS OF MEDICAL CHEMISTRY, ORGANIC AND INORGANIC. By Lawrence Wolff, M.D. Fifth edition, revised. 5. ESSENTIALS OF OBSTETRICS. By W. Easterly Ashton, M.D. Fourth edition, revised and enlarged. 6. ESSENTIALS OF PATHOLOGY AND MORBID ANATOMY. By C. E. Armand Semple, M.D. 7. ESSENTIALS OF MATERIA MED1CA, THERAPEUTICS, AND PRE- SCRIPTION-WRITING. By Henry Morris, M.D. Fifth edition, revised. 8,9. ESSENTIALS OF PRACTICE OF MEDICINE. By Henry Morris, M.D. An Appendix on Urine Examination. By Lawrence Wolff, M.D. Third edition, enlarged by some 300 Essential Formulae, selected from eminent authorities, by Wm. M. Powell, M.D. (Double number, $1.50 net.) 10. ESSENTIALS OF GYN/ECOLOGY. By Edwin B. Cragin, M.D. Fourth edition, revised. 11. ESSENTIALS OF DISEASES OF THE SKIN. By Henry W. Stelwagon, M.D. Fourth edition, revised and enlarged. 12. ESSENTIALS OF MINOR SURGERY, BANDAGING, AND VENEREAL DISEASES. By Edward Martin, M.D. Second ed., revised and enlarged. 13. ESSENTIALS OF LEGAL MEDICINE, TOXICOLOGY, AND HYGIENE. By C. E. Armand Semple, M.D. 14. ESSENTIALS OF DISEASES OF THE EYE, NOSE, AND THROAT. By Edward Jackson, M.D., and E. B. Gleason, M.D. Second ed., revised. 15. ESSENTIALS OF DISEASES OF CHILDREN. By William M. Powell, M. D. Second edition. 16. ESSENTIALS OF EXAMINATION OF URINE. By Lawrence Wolff, M.D. Colored " Vogel Scale." (75 cents net.) 17. ESSENTIALS OF DIAGNOSIS. By S. Solis Cohen, M.D., and A. A. Eshner, M.D. Second edition, thoroughly revised. 18. ESSENTIALS OF PRACTICE OF PHARMACY. By Lucius E. Sayrk. Second edition, revised and enlarged. 20. ESSENTIALS OF BACTERIOLOGY. By M. V. Ball, M.D. Third edidon, revised. 21. ESSENTIALS OF NERVOUS DISEASES AND INSANITY. By John C. Shaw, M.D. Third edition, revised. 22. ESSENTIALS OF MEDICAL PHYSICS. By Fred J. Brockway, M.D. Second edition, revised. 23. ESSENTIALS OF MEDICAL ELECTRICITY. By David D. Stewart, M.D., and Edward S. Lawrance, M.D. 24. ESSENTIALS OF DISEASES OF THE EAR. By E. B. Gleason, M.D. Second edition, revised and greatly enlarged. Pamphlet containing specimen pages, etc sent free upon application. m S aunders' r c , tor otudents New Series and of Manu ^t„ Practitioners, ' I vriAT there exists a need for thoroughly reliable hand-books on the leading branches of Medicine and Surgery is a fact amply demonstrated by the favor with which the SAUNDERS NEW SERIES OF MANUALS have been received by medical students and practitioners and by the Medical Press. These manuals are not merely condensations from present literature, but are ably written by well-known authors and practitioners, most of them being teachers in representative American colleges. Each volume is concisely and authoritatively written and exhaustive in detail, without being encumbered with the introduction of "cases," which so largely expand the ordinary text-book. These manuals will therefore form an admirable collection of advanced lectures, useful alike to the medical student and the practitioner: to the latter, too busy to search through page after page of elaborate treatises for what he wants to know, they will prove of inestimable value ; to the former they will afford safe guides to the essential points of study. The SAUNDERS NEW SERIES OF MANUALS are conceded to be superior to any similar books now on the market. No other manuals afford so much infor- mation in such a concise and available form. A liberal expenditure has enabled the publisher to render the mechanical portion of the work worthy of the high literary standard attained by these books. Any of these Manuals will be mailed on receipt of price (see next page for List). Saunders' New Series of Manuals* VOLUMES PUBLISHED. PHYSIOLOGY. 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Chapman, M.D. Professor of Insti- tutes of Medicine and Medical Jurisprudence in the Jefferson Medical College of Phila- delphia. Illustrated. Cloth. $1.50 net. SYPHILIS AND THE VENEREAL DISEASES. By James Nevins Hyde, M.D., Professor of Skin and Venereal Diseases, and Frank H. Montgomery, M.D., Lecturer on Dermatology and Genito-Urinary Diseases in Rush Medical College, Chicago. Second edition, thoroughly revised and greatly enlarged. PRACTICE OF MEDICINE. By George Roe Lockwood, M.D., Professor of Practice in the Woman's Medical College of the New York Infirmary ; Instructor in Physical Diagnosis in the Medical Department of Columbia College, etc. Illustrated. Cloth, $2.50 net. MANUAL OF ANATOMY. By Irving S. Haynes, M.D., Adjunct Professor of Anatomy and Demonstrator of Anatomy, Medical Department of the New YorK University, etc. Beautifully illustrated. Cloth, $2.50 net. MANUAL OF OBSTETRICS. By W. A. Newman Dorland, M.D., Assistant Demonstrator of Obstetrics, University of Pennsylvania ; Chief of Gynecological Dis- pensary, Pennsylvania Hospital, etc. Profusely illustrated. Cloth, $2.50 net DISEASES OF WOMEN. By J. Bland Sutton, F. R.C. S., Assistant Surgeon to Middlesex Hospital and Surgeon to Chelsea Hospital, London; and Arthur E. Giles, M. D., B. Sc. Lond., F.R.C.S. Edin., Assistant Surgeon to Chelsea Hospital, London. Handsomely illustrated. Cloth, $2.50 net. VOLUMES IN PREPARATION. NERVOUS DISEASES. By Charles W. Burr, M.D., Clinical Professor of Nervous Diseases, Medico-Chirurgical College. Philadelphia ; Pathologist to the Orthopaedic Hospital and Infirmary for Nervous Diseases; Visiting Physician to the St. Joseph Hospital, etc. *»* There will be published in the same series, at short intervals, carefully-prepared work* on various subjects by prominent specialists. Pamphlet containing specimen pages, etc sent free upon application. 28 Medical Publications of W. B. Saunders & Co. SAUNDBY'S RENAL AND URINARY DISEASES. Lectures on Renal and Urinary Diseases. By Robert Saundby, M.D. Edin., Fellow of the Royal College of Physicians, London, and of the Royal Medico-Chirurgical Society ; Physician to the General Hospital ; Consulting Physician to the Eye Hospital and to the Hos- pital for Diseases of Women; Professor of Medicine in Mason College, Birmingham, etc. Octavo volume of 434 pages, with numerous illus- trations and 4 colored plates. Cloth, $2.50 net. " The volume makes a favorable impression at once. The style is clear and succinct. We cannot find any part of the subject in which the views expressed are not carefully thought out and fortified by evidence drawn from the most recent sources. The book may be cordially recommended." — British Medical Journal. SAUNDERS' MEDICAL HAND-ATLASES. For full description of this series, with list of volumes and prices, see page 2. " Lehmann Medicinische Handatlanten belong to that class of books that are too good to be appropriated by any one nation." — Journal of Eye, Ear, and Throat Diseases. " The appearance of these works marks a new era in illustrated English medical works." — The Canadian Practitioner. SAUNDERS' POCKET MEDICAL FORMULARY. Sixth Edition, Revised. By William M. Powell, M.D., Attending Physician to the Mercer House for Invalid Women at Atlantic City, N. J. Containing 1800 formulae selected from the best-known authorities. With an Appen- dix containing Posological Table, Formulae and Doses for Hypo- dermic Medication, Poisons and their Antidotes, Diameters of the Female Pelvis and Foetal Head, Obstetrical Table, Diet List for Various Diseases, Materials and Drugs used in Antiseptic Surgery, Treatment of Asphyxia from Drowning, Surgical Remembrancer, Tables of Incompatibles, Eruptive Fevers, Weights and Measures, etc. Hand- somely bound in flexible morocco, with side index, wallet, and flap. $1.75 net. " This little book, that can be conveniently carried in the pocket, contains an immense amount of material. It is very useful, and, as the name of the author of each prescription is given, is unusually reliable." — Medical Record, New York. SAYRE'S PHARMACY. Second Edition, Revised. Essentials of the Practice of Pharmacy. By Lucius E. Sayre, M.D., Professor of Pharmacy and Materia Medica in the University of Kansas. Crown octavo, 200 pages. Cloth, $1.00 net; interleavec for notes, #1.25 net. [See Saunders1 Question- Compends, page 21.] "The topics are treated in a simple, practical manner, and the work forms a very usefuJ student's manual." — Boston Medical and Surgical Journal. SCUDDER'S FRACTURES. The Treatment of Fractures. By Chas. L. Scudder, M.D., As- sistant in Clinical and Operative Surgery, Harvard Medical School. Octavo, 433 pages, with nearly 600 original illustrations. Cloth, #4.50 net. Medical Publications of W. B. Saunders & Co. 27 SEMPLE'S LEGAL MEDICINE, TOXICOLOGY, AND HYGIENE. Essentials of Legal Medicine, Toxicology, and Hygiene. By C. E. Armand Semple, B. A., M. B. Cantab., M. R. C. P. Lond., Physician to the Northeastern Hospital for Children, Hackney, etc. Crown octavo, 212 ]>ages ; 130 illustrations. Cloth, $1.00 net; inter- leaved for notes, $1.25 net. [See Saunders1 Question- Compends, page 21.] 11 No general practitioner or student can afford to be without this valuable work. The subjects are dealt with by a masterly hand." — London Hospital Gazette. SEMPLE'S PATHOLOGY AND MORBID ANATOMY. Essentials of Pathology and Morbid Anatomy. By C. E. Armand Semple, B.A., M.B. Cantab., M.R.C.P. Lond., Physician to the Northeastern Hospital for Children, Hackney, etc. Crown octavo, 1 74 pages; illustrated. Cloth, $1.00 net; interleaved for notes, $1.25 n^t. [See Saunders1 Question- Compends, page 21.] " Should take its place among the standard volumes on the bookshelf of both student and practitioner." — London Hospital Gazette. SENN'S GENITO-URINARY TUBERCULOSIS. Tuberculosis of the Genito-Urinary Organs, Male and Female. By Nicholas Senn, M.D., Ph.D., LL.D., Professor of the Practice of Surgery and of Clinical Surgery, Rush Medical College, Chicago. Handsome octavo volume of 320 pages, illustrated. Cloth, $3.00 net. •* An important book upon an important suliject, and written by a man of mature judg- ment and wide experience. The author has given us an instructive book upon one of the most important subjects of the day." — Clinical Reporter. " A work.which adds another to the many obligations the profession owes the talented author." — Chicago Medical Recorder. SENN'S SYLLABUS OF SURGERY. A Syllabus of Lectures on the Practice of Surgery, arranged in conformity with »« An American Text-Book of Surgery." By Nicholas Seen, M. D., Ph.D., Professor of the Practice of Surgery and of Clinical Surgery, Rush Medical College, Chicago. Cloth, $1.50 net. " This syllabus will be found of service by the teacher as well as the student, the work being superbly done. There is no praise too high for it. No surgeon should be without it. " — New York Medical Times. SENN'S TUMORS. Second Edition, Revised. Pathology and Surgical Treatment of Tumors. By N. Senn, M.D, Ph.D., LL.D., Professor of Surgery and of Clinical Surgery, Rush Medical College ; Professor of Surgery, Chicago Polyclinic ; Attending Surgeon to Presbyterian Hospital ; Surgeon-in-Chief, St. Joseph's Hospital, Chicago. Second Edition, Thoroughly Revised. Oc- tavo volume of 718 pages, with 478 illustrations, including 12 full-page plates in colors. Prices: Cloth, $5.00 net; Half Morocco, $6.00 net. " The most exhaustive of any recent book in English on this subject. It is well illus- trated, and will doubtless remain as the principal monograph on the subject in our language for some years. The book is handsomely illustrated and printed, and the author has given & notable and lasting contribution to surgery." — Journal of the American Medical Association. 28 Medical Publications of W. B. Saunders & Co. SHAW'S NERVOUS DISEASES AND INSANITY. Third Edition, Revised. Essentials of Nervous Diseases and Insanity. By John C. Shaw, M.D., Clinical Professor of Diseases of the Mind and Nervous System, Long Island College Hospital Medical School; Consulting Neurologist to St. Catherine's Hospital and to the Long Island College Hospital. Crown octavo, 186 pages; 48 original illustrations. Cloth, #1.00 net ; interleaved for notes, $1.25 net. [See Saunders' Question- Compends, page 21.] "Clearly and intelligently written." — Boston Medical and Surgical Journal. "There is a mass of valuable material crowded into this small compass." — American Medico- Surgical Bulletin. STARR'S DIETS FOR INFANTS AND CHILDREN. Diets for Infants and Children in Health and in Disease. By Louis Starr, M.D., Editor of "An American Text-Book of the Diseases of Children." 230 blanks (pocket-book size), perforated and neatly bound in flexible morocco. #1.25 net. The first series of blanks are prepared for the first seven months of infant life ; each blank indicates the ingredients, but not the quantities, of the food, the latter directions being left for the physician. After the seventh month, modifications being less necessary, the diet lists are printed in full. Formulae for the preparation of diluents and foods are appended. STELW AGON'S DISEASES OF THE SKIN. Fourth Ed., Revised. Essentials of Diseases of the Skin. By Henry W. Stelwagon, M.D., Clinical Professor of Dermatology in the Jefferson Medical College, Philadelphia; Dermatologist to the Philadelphia Hospital; Physician to the Skin Department of the Howard Hospital, etc. Crown octavo, 276 pages; 88 illustrations. Cloth, $1. 00 net; inter- leaved for notes, $1.25 net. [See Saunders' Question- Compends, page 21.] '• The best student's manual on skin diseases we have yet seen." — Times and Register. STENGEL'S PATHOLOGY. Second Edition. A Text-Book of Pathology. By Alfred Stengel, M.D., Professor of Clinical Medicine in the University of Pennsylvania ; Physician to the Philadelphia Hospital ; Physician to the Children's Hospital, etc. Handsome octavo volume of 848 pages, with nearly 400 illustrations, many of them in colors. Cloth, $4.00 net; Half Morocco, $5.00 net. STEVENS' MATERIA MEDICA AND THERAPEUTICS. Second Edition, Revised. A Manual of Materia Medica and Therapeutics. By A. A. Stevens, A.M., M.D., Lecturer on Terminology and Instructor in Physical Diagnosis in the University of Pennsylvania; Professor of Pathology in the Woman's Medical College of Pennsylvania. Post- octavo, 445 pages. Flexible leather, $2.00 net. '.' The author has faithfully presented modern therapeutics in a comprehensive work, and, while intended particularly for the use of students, it will be found a reliable guide and sufficiently comprehensive for the physician in practice." — University Medical Magazine. Medical Publications of W. B. Saunders & Co. 29 STEVENS' PRACTICE OF MEDICINE. Fifth Edition, Revised. A Manual of the Practice of Medicine. By A. A. Stevens, A.M., M. D., Lecturer on Terminology and Instructor in Physical Diagnosis in the University of Pennsylvania ; Professor of Pathology in the Woman's Medical College of Pennsylvania. Specially intended for students preparing for graduation and hospital examinations. Post- octavo, 519 pages; illustrated. Flexible leather, $2.00 net. " The frequency with which new editions of this manual are demanded bespeaks its popularity. It is an excellent condensation of the essentials of medical practice for the student, and maybe found also an excellent reminder for the busy physician." — Buffal* Medical Journal. STEWART'S PHYSIOLOGY. Third Edition, Revised. A Manual of Physiology, with Practical Exercises. For Students and Practitioners. By G. N. Stewart, M.A., M.D., D.Sc, lately Examiner in Physiology, University of Aberdeen, and of the New Museums, Cambridge University ; Professor of Physiology in the Western Reserve University, Cleveland, Ohio. Octavo volume of 848 pages; 300 illustrations in the text, and 5 colored plates. Cloth, $3.75 net. " It will make its way by sheer force of merit, and amply deserves to do so. It is one of the very best English text-books on the subject." — London Lancet. "Of the many text-books of physiology published, we do not know of one that so nearly comes up to the ideal as does Prof. Stewart's volume." — British Medical Journal. STEWART AND LAWRANCE'S MEDICAL ELECTRICITY. Essentials of Medical Electricity. By D. D. Stewart, M.D., Demonstrator of Diseases of the Nervous System and Chief of the Neurological Clinic in the Jefferson Medical College; and E. S. Lawrance, M. D., Chief of the Electrical Clinic and Assistant Demon- strator of Diseases of the Nervous System in the Jefferson Medical College, etc. Crown octavo, 158 pages; 65 illustrations. Cloth, $1.00 net; interleaved for notes, $1.25 net. [See Saunders1 Question- Compends, page 21.] " Throughout the whole brief space at their command the authors show a discriminating knowledge of their subject." — Medical News. STONEY'S NURSING. Second Edition, Revised. Practical Points in Nursing. For Nurses in Private Practice. By Emily A. M. Stoney, Graduate of the Training-School for Nurses, Lawrence, Mass.; late Superintendent of the Training-School for Nurses, Carney Hospital, South Boston, Mass. 456 pages, illustrated with 73 engravings in the text, and 8 colored and half-tone plates. Cloth, $1.75 net. " There are few books intended for non-professional readers which can be so cordially endorsed by a medical journal as can this one." — Therapeutic Gazette. " This is a well-written, eminently practical volume, which covers the entire range of private nursing as distinguished from hospital nursing, and instructs the nurse hdw best to meet the various emergencies which may arise, and how to prepare everything ordinarily needed in the illness of her patient." — American Journal of Obstetrics and Diseases of Women and Children. " It is a work that the physician can place in the hands of his private nurses with thf assurance of benefit." — Ohio Medical Journal. 30 Medical Publications of W. B. Saunders & Co. STONEY'S MATERIA MEDICA FOR NURSES. Materia Medica for Nurses. By Emily A. M. Stoney, Graduate of the Training-School for Nurses, Lawrence, Mass. ; late Superintendent of the Training-School for Nurses, Carney Hospital, South Boston, Mass. Handsome octavo volume of 306 pages. Cloth, #1.50 net. The present book differs from other similar works in several features, all of which are 'ntended to render it more practical and generally useful. The general plan of the contents !ollows the lines laid down in training-schools for nurses, but the book contains much use- ful matter not usually included in works of this character, such as Poison-emergencies, Ready Dose-list, Weights and Measures, etc., as well as a Glossary, defining all the terms used in Materia Medica, and describing all the latest drugs and remedies, which have been generally neglected by other books of the kind. SUTTON AND GILES' DISEASES OF WOMEN. Diseases of Women. By J. Bland Sutton, F.R.C.S., Assistant Surgeon to Middlesex Hospital, and Surgeon to Chelsea Hospital, London; and Arthur E. Giles, M.D., B.Sc. Lond., F.R.C.S. Edin., Assistant Surgeon to Chelsea Hospital, London. 436 pages, hand- somely illustrated. Cloth, $2.50 net. "The text has been carefully prepared. Nothing essential has been omitted, and its teachings are those recommended by the leading authorities of the fay."— Journal of the American Medical Association. THOMAS'S DIET LISTS. Second Edition, Revised. Diet Lists and Sick=Room Dietary. By Jerome B. Thomas, M.D., Visiting Physician to the Home for Friendless Women and Children and to the Newsboys' Home ; Assistant Visiting Physician to the Kings County Hospital. Cloth, #1.25 net. Send for sample sheet. THORNTON'S DOSE=BOOK AND PRESCRIPTION=WRITING. Dose=Book and Manual of Prescription=Writing. By E. Q. Thornton, M.D., Demonstrator of Therapeutics, Jefferson Medical College, Philadelphia. 334 pages, illustrated. Cloth, $1.25 net. "Full of practical suggestions; will take its place in the front rank of works of this sort." — Medical Record, New York. VAN VALZAH AND NISBET'S DISEASES OF THE STOMACH. Diseases of the Stomach. By William W. Van Valzah, M.D. , Professor of General Medicine and Diseases of the Digestive System and the Blood, New York Polyclinic ; and J. Douglas Nisbet, M.D., Adjunct Professor of General Medicine and Diseases of the Digestive System and the Blood, New York Polyclinic. Octavo volume of 674 pages, illustrated. Cloth, $3.50 net. " Its chief claim lies in its clearness and general adaptability to the practical needs of the general practitioner or student. In these relations it is probably the best of the recent special works on diseases of the stomach." — Chicago Clinical Review. VECKI'S SEXUAL IMPOTENCE. The Pathology and Treatment of Sexual Impotence. By Victor G. Vecki, M.D. From the second German edition, revised and en- larged. Demi-octavo, 291 pages. Cloth, $2.00 net. The subject of impotence has seldom been treated in this country in the truly scientific spirit that it deserves. Dr. Vecki's work has long been favorably known, and the German uook has received the highest consideration. This edition is more than a mere translation, tor, although based on the German edition, it has been entirely rewritten in English. Medical Publications of W. B. Saunders & Co. 31 VIERORDT'S MEDICAL DIAGNOSIS. Fourth Edition, Revised. Medical Diagnosis. By Dr. Oswald Vierordt, Professor of Medi- cine at the University of Heidelberg. Translated, with additions, from the fifth enlarged German edition, with the author's permission, by Francis H. Stuart, A. M., M. D. Handsome royal octavo volume of 603 pages; 194 fine wood-cuts in text, many of them in colors. Cloth, $4.00 net; Sheep or Half Morocco, $5.00 net. ** Rarely is a book published with which a reviewer can find so little fault as with the volume before us. Each particular item in the consideration of an organ or apparatus, which is necessary to determine a diagnosis of any disease of that organ, is mentioned ; nothing seems forgotten. The chapters on diseases of the circulatory and digestive apparatus and nervous system are especially full and valuable. The reviewer would repeat that the book is one of the best — probably the best — which has fallen into his hands." — University Medical Magazine. WATSON'S HANDBOOK FOR NURSES. A Handbook for Nurses. By J. K. Watson, M.D., Edin. Ameri- can Edition, under supervision of A. A. Stevens, A.M., M.D., Lecturer on Physical Diagnosis, University of Pennsylvania. i2mo, 413 pages, 73 illustrations. Cloth, $1.50 net. WARREN'S SURGICAL PATHOLOGY. Second Edition. Surgical Pathology and Therapeutics. By John Collins Warren, M.D., LL.D., Professor of Surgery, Harvard Medical School. Hand- some octavo, 832 pages ; 136 relief and lithographic illustrations, 33 in colors ; with an Appendix on Scientific Aids to Surgical Diagnosis, and a series of articles on Regional Bacteriology. Cloth, $5.00 net; Half Morocco, $6.00 net. " A most striking and very excellent feature of this book is its illustrations. Without exception, from the point of accuracy and artistic merit, they are the best ever seen in a work of this kind. Many of those representing microscopic pictures are so perfect in their coloring and detail as almost to give the beholder the impression that he is looking down the barrel of a microscope at a well-mounted section." — Annals of Surgery. WOLFF ON EXAMINATION OF URINE. Essentials of Examination of Urine. By Lawrence Wolff, M.D., Demonstrator of Chemistry, Jefferson Medical College, Philadelphia, etc. Colored (Vogel) urine scale and numerous illustrations. Crown octavo. Cloth, 75 cents net. [See Saunders1 Question- Compends, page 21.] " A very good work of its kind — very well suited to its purpose." — Times and Register. WOLFF'S MEDICAL CHEMISTRY. Fifth Edition, Revised. Essentials of Medical Chemistry, Organic and Inorganic. Containing also Questions on Medical Physics, Chemical Physiology, Analytical Processes, Urinalysis, and Toxicology. By Lawrence Wolff, M. D. , Demonstrator of Chemistry, Jefferson Medical College, Philadelphia, etc. Crown octavo, 222 pages. Cloth, gi.oo net; inter- leaved for notes, $1.25 net. [See Saunders1 Question- Compends, page 21.] ' ' The scope of this work is certainly equal to that of the best course of lectures on Medical Chemistry." — Pharmaceutical Era. CLASSIFIED LIST Medical Publications OF W. B. SAUNDERS & COMPANY, 925 "Walnut Street, Philadelphia. ANATOMY, EMBRYOLOGY, HISTOLOGY. Clarkson — A Text-Book of Histology, 1 1 Haynes — A Manual of Anatomy, . . . 15 Heisler — A Text- Book of Embryology, 15 Nancrede — Essentials of Anatomy, . . 20 Nancrede — Essentials of Anatomy and Manual of Practical Dissection, ... 20 Semple — Essentials of Pathology, . . 27 BACTERIOLOGY. Ball — Essentials of Bacteriology, ... 8 Crookshank — A Text-Book of Bacteri- ology, 12 Frothingham— Laboratory Guide, . . 13 Levy and Klemperer's Clinical Bacte- riology, 17 Mallory and Wright — Pathological Technique, 18 McParland — Pathogenic Bacteria, . . iq CHARTS, DIET-LISTS, ETC. Griffith — Infant's Weight Chart, ... 14 Hart — Diet in Sickness and in Health, . 15 Keen — Operation Blank, 17 Laine — Temperature Chart, . . . 17 Meigs — Feeding in Early Infancy, . . 19 Starr — Diets for Infants and Children, . 28 Thomas — Diet-Lists, 30 CHEMISTRY AND PHYSICS. Brockway — Essentials of Medical Phys- ics, 9 Wolff — Essentials of Medical Chemistry, 31 CHILDREN. An American Text-Book of Diseases of Children, . . 5 Griffith — Care of the Baby, 14 Griffith — Infant's Weight Chart, ... 14 Meigs — Feeding in Early Infancy, . . 19 Powell — Essentials of Dis. of Children, 21 Starr — Diets for Infants and Children, . 28 DIAGNOSIS. Cohen and Eshner— Essentials of Di- agnosis, 11 Corwin — Physical Diagnosis, .... 11 Macdonald — Surgical Diagnosis and Treatment, : . 18 Vierordt — Medical Diagnosis, .... 31 DICTIONARIES. Dorland — Pocket Dictionary, .... 12 Keating — Pronouncing Dictionary, . . 16 Morten — Nurse's Dictionary, .... 20 EYE, EAR, NOSE, AND THROAT. An American Text- Book of Diseases of the Eye, Ear, Nose, and Throat, . 5 De Schweinitz — Diseases of the Eye, . 12 Gleason — Essentials of Dis. of the Ear, 13 Jackson — Manual of Diseases of Eye, . 16 Jackson and Gleason — Essentials of Diseases of the Eye, Nose, and Throat, 16 Kyle — Diseases of the Nose and Throat, 1 7 GENITO-URINARY. An American Text-Book of Genito- urinary and Skin Diseases, 6 Hyde and Montgomery — Syphilis and the Venereal Diseases, 15 Martin — Essentials of Minor Surgery, Bandaging, and Venereal Diseases, . 18 Saundby — Renal and Urinary Diseases, 26 Senn — Genito-Urinary Tuberculosis, . 27 Vecki — Sexual Impotence, 30 GYNECOLOGY. American Text- Book of Gynecology, 6 Cragin — Essentials of Gynecology, . . II Garrigues — Diseases of Women, ... 13 Long — Syllabus of Gynecology, ... 17 Penrose — Diseases of Women, .... 20 Pryor — Pelvic Inflammations, .... 34 Sutton and Giles — Diseases of Women, 30 MATERIA MEDICA, PHARMACOL- OGY, AND THERAPEUTICS. An American Text-Book of Applied Therapeutics, . . .' 5 Butler — Text-Book of Materia Medica, Therapeutics and Pharmacology, ... 10 Cerna — Notes on the Newer Remedies, 10 Griffin — Materia Med. and Therapeutics, 14 Morris — Essentials of Materia Medica and Therapeutics, 19 Saunders' Pocket Medical Formulary, 26 Sayre — Essentials of Pharmacy, ... 26 Stevens — Essentials of Materia Medica and Therapeutics, 28 Stoney — Materia Medica for Nurses, . . 30 Thornton — Dose- Book and Manual of Prescription-Writing, 30 MEDICAL JURISPRUDENCE AND TOXICOLOGY. Chapman — Medical Jurisprudence and Toxicology, 10 Semple — Essentials of Legal Medicine, Toxicology, and Hygiene, 27 Medical Publications of W. B. Saunders & Co. 88 NERVOUS AND MENTAL DISEASES, ETC. Burr — Nervous Diseases, 9 Chapin — Compendium of Insanity, . . 10 Church and Peterson — Nervous and Mental Diseases, 10 Shaw — Essentials of Nervous Diseases and Insanity 28 NURSING. Griffith— The Care of the Baby, ... 14 Hampton — Nursing, 14 Hart — Diet in Sickness and in Health, 15 Meigs — Feeding in Early Infancy, . . 19 Morten — Nurse's Dictionary 20 Stoney — Materia Medica for Nurses, . . 30 Stoney — Practical Points in Nursing, . 29 Watson — Handbook for Nurses, ... 31 OBSTETRICS. An American Text-Book of Obstetrics, 6 Ashton — Essentials of Obstetrics, Boisliniere — Obstetric Accidents, Dorland — Manual of Obstetrics, Hirst — Text-Book of Obstetrics, Norris — Syllabus of Obstetrics, . PATHOLOGY. An American Text-Book of Pathology, Mallory and Wright — Pathological Technique, Semple — Essentials of Pathology and Morbid Anatomy, Senn — Pathology and Surgical Treat- ment of Tumors, Stengel — Text- Book of Pathology, . . Warren— Surgical Pathology and Thera- peutics, PHYSIOLOGY. An American Text-Book of Physi- ology Hare — Essentials of Physiology, . . . Raymond — Manual of Physiology, . . Stewart — Manual of Physiology, . . . PRACTICE OF MEDICINE. An American Text-Book of the The- ory and Practice of Medicine, .... An American Year- Book of Medicine and Surgery, Anders — Text-Book of the Practice of Medicine, Lockwood — Manual of the Practice of Medicine, Morris — Essentials of the Practice of Medicine, Stevens— Manual of the Practice of Medicine, SKIN AND VENEREAL. An American Text-Book of Genito- urinary and Skin Diseases, Hyde and Montgomery — Syphilis and the Venereal Diseases, 15 20 Martin — Essentials of Minor Surgery, Bandaging, and Venereal Diseases, . 18 Pringle— Pictorial Atlas of Skin Dis- eases and Syphilitic Affections, ... 21 Stelwagon— Essentials of Diseases of the Skin 2g SURGERY. An American Text- Book of Surgery, 7 An American Year-Bookof Medicine and Surgery, 8 Beck — Fractures, 9 Beck — Manual of Surgical Asepsis, . . 9 DaCosta — Manual of Surgery, .... 12 International Text-Book of Surgery, . 15 Keen— Operation Blank, 17 Keen — The Surgical Complications and Sequels of Typhoid Fever, 17 Macdonald — Surgical Diagnosis and Treatment, 18 Martin — Essentials of Minor Surgery, Bandaging, and Venereal Diseases, . 18 Martin — Essentials of Surgery, .... 18 Moore — Orthopedic Surgery, 19 Nancrede — Principles of Surgery, . . 20 Pye — Bandaging and Surgical Dressing, 21 Scudder — Treatment of Fractures, . . 26 Senn — Genito-Urinary Tuberculosis, . 27 Senn — Syllabus of Surgery, 27 Senn — Pathology and Surgical Treat- ment of Tumors, 27 Warren — Surgical Pathology and Ther- apeutics, 31 URINE AND URINARY DISEASES. Saundby — Renal and Urinary Diseases, 26 Wolff — Essentials of Examination of Urine 31 MISCELLANEOUS. Abbott — Hygiene of Transmissible Dis- eases, 8 Bastin — Laboratory Exercises in Bot- any, 9 Gould and Pyle — Anomalies and Curi- osities of Medicine, 13 Grafstrom — Massage 14 Keating — How to Examine for life Insurance, ...» 16 Rowland and Hedley — Archives of the Roentgen Ray, 21 Saunders' Medical Hand-Atlases. . 2, 3, 4 Saunders' New Series of Manuals, 24, 25 Saunders* Pocket Medical Formulary, 26 Saunders' Question-Compends, . . 22, 23 Senn — Pathology and Surgical Treat- ment of Tumors ■ 27 Stewart and Lawrance — Essentials of MedieaF-F^WtricUy, 29 Thornton — Dose-BooV~anjI_Manual of Prescription-Writing, 30 Van Valzah and Nisbet— Diseases of the Stomach 3° BOOKS JUST ISSUED. THE AMERICAN ILLUSTRATED MEDICAL DICTIONARY. For Students and Practitioners. A Complete Dictionary of the Terms used in Medi- cine and the Allied Sciences, with a large number of Valuable Tables and Numerous Handsome Illustrations. Edited by W. A. Newman Dorland, M. D., Editor of the American Pocket Medical Dictionary. Handsome large octavo, 800 pages, bound in full limp leather, and printed on thin paper of the finest quality, forming a handy volume, only \]^ inches thick. This is an entirely new and unique work, intended to meet the need of practitioners and students for a complete, up-to-date dictionary of moderate price. The book is designed to furnish a maximum amount of matter in a minimum space and at the lowest possible cost. It contains double the material in the ordinary students' dictionary, and yet. by the use of a clear, condensed type and thin paper of the finest quality, is only 1% inches in thickness. It is bound in full flexible leather, and is just the kind of a book that a man will want to keep on his desk for constant reference. The book makes a special feature of the newer words, and defines hundreds of important terms not to be found in any other dictionary. It is especially full in the matter of tables, containing more than a hundred of great practical value. A new feature is the inclusion of numerous handsome illustrations, many of them in colors, drawn and engraved specially for this book. These have been chosen with great care and add infinitely to the value of the work. The book will appeal to both practitioners and students, since, besides a complete vocabulary, it gives to the more important subjects extended consideration of an encyclopedic character. BOHM, DAVIDOFF, AND HUBER'S HISTOLOGY. A Text-Book of Human Histology. Including Microscopic Technic. By Dr. A. A. Buhm and Dr. M. von Davidoff, of Munich, and G. C. Huber, M. D., Junior Professor of Anatomy and Histology, University of Michigan. FRIEDRICH AND CURTIS ON THE NOSE, THROAT, AND EAR. Rhinology, Laryngology, and Otology in their Relations to General Medicine. By Dr. E. P. Friedrich, of the University of Leipsig. Edited by H. Holbrook Curtis, M. D., Consulting Surgeon to the New York Nose and Throat Hospital. LEROY'S HISTOLOGY. The Essentials of Histology. By Louis Leroy, M. D., Professor of Histology and Pathology, Vanderbilt University, Nashville, Tennessee. OGDEN ON THE URINE. Clinical Examination of the Urine. By J. Bergen Ogden, M. D., Assistant in Chemistry, Harvard Medical School. Handsome octavo volume of over 408 pages, with 54 illustrations and 1 1 full-page plates, many in colors. PYLE'S PERSONAL HYGIENE. A Manual of Personal Hygiene. Edited by Walter L. Pyle, M. D., Assist- ant Surgeon to Wills Eye Hospital, Philadelphia. Octavo volume of 344 pages, fully illustrated. SALINGER AND KALTEYER'S MODERN MEDICINE. Modern Medicine. By Julius L. Salinger, M. D., Demonstrator of Clinical Medicine, Jefferson Medical College, and F. J. Kalteyer, M. D., Assistant Demon- strator of Clinical Medicine, Jefferson Medical College. Handsome octavo volume of over 800 pages, fully illustrated. STONEY'S SURGICAL TECHNIC FOR NURSES. Surgical Technic for Nurses. By Emily A. M. Stoney, late Superintendent of the Training-School for Nurses, Carney Hospital, South Boston, Massachusetts. i PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET UNIVERSITY OF TORONTO LIBRARY i QR 4-6 M25 1900 McFarland, Joseph A text-book upon the pathogenic Bacteria BioMed