< WAN ‘i . ees co cN ~ OC A ‘ SN cS SS SS AN — AAs ‘ SSS \ x me TREES WS \ SSR oO SSS OSG WAS SERA RARE CC We AES SS AS x Cornell University Library QR 41.M87 T he fun i 24 000 225 627 vet 3 19 ANTHONY VAN LEEUWENHOEK Who first saw bacteria THE FUNDAMENTALS OF BACTERIOLOGY BY CHARLES BRADFIELD MORREY, B.A., M.D. PROFESSOR OF BACTERIOLOGY AND HEAD OF THE DEPARTMENT IN THE OHIO STATE UNIVERSITY, COLUMBUS, OHIO ILLUSTRATED WITH 165 ENGRAVINGS AND 6 PLATES LEA & FEBIGER PHILADELPHIA oor NEW YORK OR 4 | Na, 5594 N87 7 CoryYyricut LEA & FEBIGER 1917 mors TO GRACE HAMILTON MORREY AMERICAN PIANIST PREFACE. AN experience of nearly twenty years in the teaching of Bacteriology has convinced the author that students of this subject need a comprehensive grasp of the entire field and special training in fundamental technic before specializing in any particular line of work. Courses at the, University are arranged on this basis. One semester is devoted to General Bacteriology. During the second semester the student has a choice of special work in Pathogenic, Dairy, Soil, Water, or Chemical Bacteriology. A second year may be devoted to advanced work in any of the above lines, to Immunity and Serum Therapy, or to Pathogenic Protozoa. This text-book is intended to cover the first or introductory semester’s work, and requires two class-room periods per week. Each student is compelled to take two laboratory periods of three hours per week along with the class work. The outline of the laboratory work is given at the end of the text. Results attained seem to justify this plan. A text-book is but one of many pedagogical mechanisms and is not intended to be an encyclopedia of the subject. The author makes no claim to originality of content, since the facts presented are well known to every bacteriologist, though the method of presentation is somewhat different from texts in general. During the preparation of this work he has made a thorough review of the literature of Bacteri- ology, covering the standard text-books as well as works of reference and the leading periodicals dealing with the sub- ject. Thus the latest information has been incorporated. No attempt has been made to give detailed references in a work of this character. vi . PREFACE The photomicrographs are original except where otherwise indicated and are all of a magnification of one thousand diameters where no statement to the contrary appears. These photographs were made with a Bausch & Lomb Pro- jection Microscope fitted with a home-made camera box. Direct current arc light was used and exposures were five to ten seconds. Photographs of cultures are also original with a few indicated exceptions. All temperatures are indicated in degrees centigrade. For use of electrotypes or for prints furnished the author is indebted to the following: A. P. Barber Creamery Supply Company, Chicago, Ill.; Bausch & Lomb Optical Company, Rochester, N. Y.; Creamery Package Manufacturing Com- pany, Chicago, IIl.; Davis Milk Machinery Company, N. Chicago, Ill.; Mr. C. B. Hoover, Superintendent of Sewage Disposal Plant,:Columbus, O.; Mr. C. P. Hoover, Super- intendent of Water Filtration Plant, Columbus, O.; The Hydraulic Press Manufacturing Company, Mt. Gilead, O.; Loew Manufacturing Company, Cleveland, O.; Metric Metal Works, Erie, Pa.; Sprague Canning Machine Company, Chicago, Ill.; U. S. Marine Hospital Service.; Wallace and Tiernan Company, New York City, N. Y. For the preparation of many cultures and slides, for great assistance in the reading of proof and in the preparation of the index, Miss Vera M. McCoy, Instructor in Bacteriology, deserves the author’s thanks. _ The author trusts that the book will find a place in College and University courses in Bacteriology. C. B. M. CoLumBus, 1917. ‘ CONTENTS. Historical Introduction—Spontaneous Generation—Causation of Disease—Putrefaction and Fermentation—Study of Forms— Chronological Table. . . 2. . 2. 2... eee OU CHAPTER I. Position of Bacteria—Relationships to Algee—Yeasts—Molds— Protozoa . . i 4st) ewe. trae 3 z 34 PAR TL. MORPHOLOGY. CHAPTER II. Cell Structures—Cell Wall—Protoplasm—Plasmolysis—Plasmop- tysis—Nucleus—Vacuoles—Capsules—Metachromatic Granules —Flagella—Spores . & & gg yg 4339 CHAPTER III. Cell Forms—Coccus—Bacillus—Spirillum . . 49 CHAPTER IV. Cell Groupings . . . ....... r sc es, “452 CHAPTER V. Classification——Migula’s < - 2. .'% . . 56 vill CONTENTS PART II. PHYSIOLOGY. CHAPTER VI. Occurrence—General Conditions for Growth—Moisture—Tem- perature—Light—Oxygen—Osmotic Pressure — Electricity — X-rays and Radium Emanations — Pressure — Mechanical Vibration .. 61 CHAPTER VII. Chemical Environment—Reaction of Medium—Chemical Com- position 70 CHAPTER VIII. Chemical Environment (Continued)—General Food Relation- ships—Metabolism 75 CHAPTER IX. Physiological GHEE Eee Ea of Carbohydrates—Split- ting of Fats 2 82 CHAPTER X. Physiological Activities (Continued)—Putrefaction of Proteins— Cycles of Nitrogen, Carbon, Sulphur, Phosphorus 91 CHAPTER XI. : Physiological Activities (Continued)—Production of Acids, Gases, Esters, Alcohols, Aldehydes, Aromatic Compounds—Phosphor- escence—Chromogenesis— Reduction— Oxidation — Production of Heat—Absorption of Free ee Nutrition of Green Plants . ee oe we 98 CHAPTER XII. Physiological Activities (Continued)—Production of Enzymes— Discussion on Enzymes—Toxins—Causation of Disease 109 CONTENTS CHAPTER NII. Disinfection — Sterilization — Disinfectants — Physical Agents— Pasteurization 7 CHAFTER XIV. Disinfection and Sterilization (Continued)—Chemical Agents CHAPTER XV. Disinfection and Sterilization (Continued)—Choice of Agent— Practical Sterilization and Disinfection PART Ty. THE STUDY OF BACTERIA. CHAPTER XVI. Culture Media—Broth, Gelatin, Agar, Potatoes, Milk, Blood Serum - CHAPTER XVII. Methods of Using Culture Media—Culture Tubes—Plates— Anaérobic Cultures—Vignal Tubes—Fermentation Tubes— Deep Culture Tubes—Novy Jars—Inoculation of Culture Media dele, i CHAPTER XVIII. Isolation of Bacteria in Pure Culture—Dilution—Plating—Streak- ing—Barber Apparatus—Aids in Isolation—Heat—Selective Antiseptics—Selective Food—Indicators—Animal Inoculation CHAPTER XIX. Study of the Morphology of Bacteria—Bacteriological Microscope —Hanging Drop Slides—Staining—Gram’s Method—Spores— Acid-fast Bacilli—Capsules—Flagella é i Be «4 ix 118 14t 151 157 168 178 184 x CONTENTS CHAPTER XX. Study of the Physiology of Bacteria—Temperature—Incubators— Thermal Death Point—Oxygen Relationships—Study of Physi- ological Activities—Appearance of Growth on Culture Media . 196 CHAPTER XXI. Animal Inoculation—Material for Bacteriological Examination . 210 PART IV. GENERAL PATHOGENIC BACTERIOLOGY. ' CHAPTER XXII. Introduction — Infection — Acute Infection — Chronic Infection —Specific—N peer Postulates—Virulence—Sus- ceptibility . 2. 2. 2... owe ee ee 2. (218 CHAPTER XXIII. Pathogenic Bacteria Outside the Body—As lea Facultative Saprophytes—Latent—Carriers . . - 218 CHAPTER XXIV. Channels of Infection—Skin—Mucose—Respiratory Tract—Ali- mentary Tract—Dissemination in the Body—Paths of Elimi- nation—Specificity of Location. . . . ‘ a 222 CHAPTER XXV. Immuni ty—Natural—Artificial—Active—Passive—Production of Immunity—Vaccine—Antiserum . . . . . . . . . 228 CHAPTER XXVI. Theories of Immunity — Pasteur — Chauveau — Baumgirtner — Metchnikoffi—Ehrlich—Principles of Ehrlich’s Theory . . . 234 CONTENTS CHAPTER XXVII. Ehrlich’s Theory (Continued)—Receptors of the First Order— Antitoxin—Antienzyme—Preparation of Antitoxins—Units CHAPTER XXVIII. Ehrlich’s Theory (Continued)—Receptors of the Second Order— Agglutinins—Agglutination Reaction—Precipitins—Precipitin Test CHAPTER XXIX. Ebrlich’s Theory (Continued)—Receptors of the Third Order— Cytolysins — Amboceptor — Complement — Anti-amboceptors —Antisnake Venoms—Failure of Cytolytic Serums in Practice— Complement-fixation Test enn Cees CHAPTER XXX. Phagocytosis—Opsonins—Opsonic Index—Bacterial Vaccines— Preparation of—Use of—Aggressins fou ee ASR ‘: CHAPTER XXXI. Anaphylaxis—Author’s Theory—Tuberculin Test—Summary 238 242 . 248 256 264 BACTERIOLOGY. HISTORICAL INTRODUCTION. BacTERIOLOGY as a science is a development of the latter half of the nineteenth century. It may be said to have begun with Koch’s proof that Bacterium anthracis is the cause of anthrax in 1876. Nevertheless this discovery of Koch’s was preceded by numerous observations and experiments which led up to it. Some of this work was done in attempt- ing to disprove the old “spontaneous generation” theory as to the origin of organisms; some in searching for the causes of disease and some in the study of fermentation and putre- faction. SPONTANEOUS GENERATION. Speculation as to the first origin of life is as old as history and doubtless older. Every people of antiquity had its own legends, as for example, the account in Genesis. This ques- tion never can be definitely settled, even though living matter should be made in the laboratory. The doctrine of the “spontaneous origin” of particular animals or plants from dead material under man’s own observation is a somewhat different proposition and may be subjected to experimental test. The old Greek philosophers believed it. Anaximander (B.c. 610-547) taught that some animals are derived from moisture. Even Aristotle (B.c. 384-322) said that “animals sometimes arise in soil, in plants, or in other animals,” 7. e., spontaneously. It can be stated that this belief was general from his day down through the Dark and Middle Ages and later. Cardano (a.p. 1501- 1576) wrote that water gives rise to fish and animals and is 2 18 HISTORICAL INTRODUCTION also the cause of fermentation. Van Helmont (1578-1644) gives directions for making artificial mice. Kircher (1602- 1680) describes and figures animals produced under his own eyes by water on plant stems. However, many thinkers of the seventeenth century doubted the truth of this long-established belief. Francesco Redi (1626-1698) made a number of experiments which tended to prove that maggots did not arise spontaneously in meat, as was generally believed, but developed only when flies had an opportunity to deposit their eggs on the meat. It seems that by the latter part of this century the idea that organisms large enough to be seen with the naked eye could originate spontaneously was generally abandoned by learned men. : The work of Leeuwenhoek served to suspend for a time the subject of spontaneous generation, only to have it revived more vigorously later on. He is usually called “The Father of the Microscope,” though the compound microscope was invented probably by Hans Zansz or his son Zacharias, of Holland, about 1590. Leeuwenhoek used a simple lens, but his instruments were so much more powerful that they opened up an entirely new and unknown world. Anthony van Leeuwenhoek (1632-1723) was apprenticed to a linen draper, and accumulated a comfortable fortune in this business. He became interested in the grinding of spectacle lenses, then an important industry in Delft, Holland, where he lived, and did a great deal of experimental work in this line, mainly for his own enjoyment. Finally he succeeded in making a lens so powerful that he could see in water and various infusions very minute living bodies never before observed. Leeuwenhoek contributed 112 papers to the Royal Society of Great Britain, the first in 1673, many of them accompanied by such accurate descriptions and drawings, for example a paper submitted September 12, 1683, that there is no doubt that he really saw bacteria and was the first to do so (Fig. 1). Rightly may he be styled “The Father of Bacteriology,” if not of the microscope. He says in one paper: “With the greatest astonishment I observed that everywhere through the material which I SPONTANEOUS GENERATION 19 was examining were distributed animalcules of the most microscopic dimness which moved themselves about in a remarkably energetic way.” Thus he considered these liv- ing objects to be animals, from their motion, and this belief held sway for nearly two hundred years. Leeuwenhoek was a pure observer of facts and made no attempt at speculation, but his discoveries soon started the theorists to discussing the origin of these minute organisms. Most observers, as was probably to be expected, believed that they arose spontaneously. Needham, in 1749, described the development of microédrganisms around grains of barley Fic. 1.—The first drawings of bacteria by Leeuwenhoek. The dotted line - C-D indicates movement of the organism. in water. Bonnet, in 1768, suggested that probably Need- ham’s animalcules came from ova in the liquid. The Abbot Spallanzani, in 1769, called attention to the crudeness of Needham’s methods and later, in 1776, attempted to dis- prove spontaneous origin by heating infusions of organic material in flasks and then sealing them. His critics raised the objections that heating the liquids destroyed their ability to support life, and that sealing prevented the access of fresh air which was also necessary. The first objection was disproved by the accidental cracking of some of the flasks which thereafter showed an abundant growth. This 20 HISTORICAL INTRODUCTION accident seemed also to support the second objection, and Spallanzani did not answer it. Though Spallanzani’s experi- ments failed to convince his opponents, they led to important practical results, since in Francois Appert, 1810, applied them to the preserving of fruits, meats, etc., and in a sense started the modern canning industry. From Spallanzani to Schultze, there were no further experiments to prove or disprove spontaneous generation. Schultze, in 1836, attempted to meet the second objection to Spallanzani’s experiment, 7. ¢., the exclusion of air, by draw- ing air through his boiled infusions, first causing it to bubble Fic. 2.—Schultze’s experiment. The set of bulbs next to the face con- tained KOH and the other set concentrated H2SO.. Air was drawn through at frequent intervals from May until August but no growth developed in the boiled infusion. through concentrated sulphuric acid to kill the “germs” (Fig. 2). His flasks fortunately showed no growths, but his critics claimed that the strong acid changed the proper- ties of the air so that it would not support life. Schwann, in 1837, modified this experiment by drawing the air through a tube heated to destroy the living germs (Fig 3). His experiments were successful but the “spontaneous genera- tion” theorists raised the same objection, 7. e., the change in the air by heating. Similar arguments were brought forward, also to the use of cotton plugs as filters by Schroeder SPONTANEOUS GENERATION 21 and Dusch in 1859 (Fig. 4). It remained for Chevreuil and Pasteur to overcome this objection in 1861 by the use of Fic. 3.—Schwann’s experiment. After boiling, as shown in the diagram, and cooling, air was drawn into the flask by aspiration while the coiled tube was kept hot with the flame. Fic. 4.—Schroeder and Dusch’s experiment. The aspirating bottle drew the air through the flask after it had been filtered by the cotton in the tube 22 HISTORICAL INTRODUCTION Fic. 5.—Pasteur’s flask. Fic. 6.—Tyndall’s box. One side is removed to show the construction. The bent tubes at the top are to permit a free circulation of air into the interior. The window at the back has one corresponding in the front (removed). Through these the beam of light sent through from the lamp at the side was observed. The three tubes received the infusion and were then boiled in an oil bath. The pipette was for filling the tubes. (Popular Science Monthly, April, 1877.) CAUSATION OF DISEASE 23 flasks with long necks drawn out to a point and bent over. These permitted a full access of air by diffusion but kept out living germs, since these cannot fly but are carried mechanically by air currents or fall of their own weight (Fig. 5). Hoffman, the year before (1860), had made similar experiments but these remained unnoticed. The Pasteur flasks convinced most scientists that “spontaneous genera- tion” has never been observed by man, though some few, notably Dr. Charlton Bastian, of England, vigorously sup- ported the theory from the early seventies until his death in November, 1915. John Tyndall, in combating Bastian’s views, showed that boiled infusions left open to the air in a closed box through which air circulated did not show-any growth of organisms provided the air was so free of particles that the path of a ray of light sent through it from side to side could not be seen (Fig. 6). Or if such sterilized infusions were exposed to dust-free air, as in the high Alps, the majority showed no growth, while all infusions in dusty air did show an abun- dance of organisms. Tyndall’s experiments confirmed those of Pasteur and his predecessors and showed that the organ- isms developed from ‘germs’ present in the air falling into the liquids and not spontaneously. CAUSATION OF DISEASE. Apparently the first writer on this subject was Varo, about B.c. 70, who suggested that fevers in swampy places were due to invisible organisms. Fracastorius (1484-1553), in a work published in 1546, elaborated a theory of “disease germs” and “direct and indirect contagion” very similar to modern views, though based on no direct pathological knowl- edge. Nevertheless Kircher (mentioned already) is usually given undeserved credit for the “contagium vivum”’ theory. In 1657 by the use of simple lenses he observed “worms” in decaying substances, in blood and in the pus from bubonic plague patients (probably rouleaux of corpuscles in the blood, certainly not bacteria in any case). Based on these observations and possibly also on reading the work of 24 HISTORICAL INTRODUCTION Fracastorius, his theory of a “living cause” for various dis- eases was published in 1671, but received little support. The discoveries of Leeuwenhoek which proved the exist- ence of microscopic organisms soon revived the “contagium vivum”’ idea of Kircher. Nicolas Andry in a work pub- lished in 1701 held such views. Lancisi in 1718 advanced the idea that “animalcules” were responsible for malaria, a view not proved until Laveran discovered the malarial para- site in 1880.1. Physicians ascribed the plague which visited Southern France in 1721 to the same cause, and many even went so far as to attribute all disease to animalcules, which brought the theory into ridicule. Nevertheless, the ‘“con- tagium vivum” theory survived, and even Linnaeus in his Systema Nature (1753-6) recognized it by placing the organisms of Leeuwenhoek, the contagia of diseases and the causes of putrefaction and fermentation in one class called “Chaos.” Plenciz, a prominent physician and professor in the Vienna Medical School, published in 1762 a work in which he gave strong arguments for the “living cause” theory for trans- missable diseases. He taught that the agent is evidently transmitted through the air and that there is a certain period of incubation pointing to a multiplication within the body. He also believed that there was a specific agent for each disease. His writings attracted little attention at the time and the “contagium vivum’’ theory seems to have been almost lost sight of for more than fifty years. Indeed, Oznam, in 1820, said it was no use to waste time in refuting hypotheses as to the animal nature of contagium. Isolated observers were, however, keeping the idea alive, each in his own locality. In 1787 Wollstein, of Vienna, showed that the pus from horses with glanders could infect other horses if inoculated into the skin. Abilgaard, of Copenhagen, made similar experiments at about the same time. In 1797 Eric Viborg, a pupil of Abilgaard’s, published experiments in which he showed the infectious nature not 1Sir H. A. Blake has called attention to the fact that the ‘mosquito - theory” of malaria is mentioned in a Sanscrit manuscript of about the 6th century A.D. 4 CAUSATION OF DISEASE 25 only of the pus but also of the nasal discharges, saliva, urine, etc., of glandered horses. Prevost’s discovery of the cause of grain rust (Puccinia graminis) in 1807 was the first instance of an infectious disease of plants shown to be due to a microscopic plant organism, though not a bacterium in this case. In 1822 Gaspard showed the poisonous nature of material from infected wounds by injecting it into animals and caus- ing their death. Bearing on the “contagium vivum”’ theory was the rediscovery of the “itch mite” (Sarcoptes scabier) by Renucci (1834), an Italian medical student. This had been declared several hundred years before but had been lost sight of. Chevreuil and Pasteur, in 1836, showed that putre- faction did not occur in meat protected from contamination, and suggested that wound infection probably resulted from entrance of germs from without. Bassi, investigating a dis- ease of silkworms in Italy, demonstrated that a certain mold-like fungus (Botrytis bassiana) was the cause in 1837. This was the first instance of a microscopic vegetable organ- ism proved to be capable of causing disease in an animal. Boehm, in 1838, observed minute organisms in the stools of cholera patients and conjectured that they might have a causal connection with the disease. The fungous nature of Favus, a scalp disease, was recognized by Schénlein in 1839, and the organism was afterward called ‘‘Achorion schoen- leinat.”” Berg, in 1839-41, showed that Thrush is likewise due to a fungus, “ Oidium albicans.” These discoveries led Henle, in 1840, to publish a work in which he maintained that all contagious diseases must be _ due to living organisms, and to propound certain postulates (afterward restated by Koch and now known as “Koch’s postulates”) which must be demonstrated before one can be sure that a given organism is the specific cause of a given disease. The methods then in vogue and the instruments of that period did not enable Henle to prove his claims, but he must be given the credit for establishing the “contagium vivum” theory on a good basis and pointing the way for men better equipped to prove its soundness in after years. In 1842-43 Gruby showed that Herpes tonsurans, a form 26 HISTORICAL INTRODUCTION of ringworm, is due to the fungus Trichophyton tonsurans. Klencke, in 1843, produced generalized tuberculosis in a rab- bit by injecting tuberculous material into a vein in the ear, but did not carry his researches further. Liebert identified the Peronospora infestans as the cause of one type of potato rot in 1845. The skin disease pityriasis. (tinea) versicolor was shown to be due to the Microsporon furfur by Eichstedt in 1846. Pollender, in 1849, and Davaine and Rayer, in 1850, inde- pendently observed small rod-like bodies in the blood of sheep and cattle which had died of splenic fever (anthrax). That Egyptian chlorosis, afterward identified with Old World “hookworm disease,” is caused by the Ankylosto- mum duodenale was shown by Griesinger in 1851. In the same year the Schistosomum hematobium was shown to be the cause of the “Bilharzia disease’? by Bilharz. Kiichen- meister discovered the tapeworm, Tenia solium, in 1852, Cohn, an infectious disease of flies due to a parasitic fungus (Empusa musce) in 1855, and Zenker the Trichinella spiralis in trichinosis of pork (“measly pork”) in 1860. The organ- isms just mentioned are, of course, not bacteria, but these discoveries proved conclusively that leving things of one kind or another, some large, most of them microscopic, could cause disease in other organisms and stimulated the search for other ‘‘living contagiums.”’ In 1863 Davaine, already men- tioned, showed that anthrax could be transmitted from animal to animal by inoculation of blood, but only if the blood contained the minute rods which he believed to be the cause. In 1865 Villemin repeatedly caused tuberculosis in rabbits by subcutaneous injection of tuberculous material and showed that this disease must be infectious also. In this same year Lord Lister introduced antiseptic methods in surgery. He believed that wound infections were due to microérganisms getting in from the air, the surgeon’s fingers, etc., and without proving this, he used carbolic acid to kill these germs and prevent the infection. His pioneer experiments made modern surgery possible. In this year also, Pasteur was sent to investigate a disease, Pebrine, which was destroying the silkworms in Southern France. SIR JOSEPH LISTER ROBERT KOCH CAUSATION OF DISEASE 27 He showed the cause to be a protozoan which had been seen previously by Cornalia and described by Niageli under the name Nosema bombycis and devised preventive measures. This was the first infectious disease shown to be due to a proto- zoan. In 1866 Rindfleisch observed small pin-point-like bodies in the heart muscle of persons who had died of wound infection. Klebs, in 1870-71, published descriptions and names of organisms he had found in the material from simi- lar wounds, though he did not establish their causal rela- tion. Bollinger, in 1872, discovered the spores of anthrax and explained the persistence of the disease in certain dis- tricts as due to the resistant spores. In 1873 Obermeier observed in the blood of patients ‘suffering from recurrent fever long, flexible spiral organisms which have been named Sptirocheta obermeiert. Lésch ascribed tropical dysentery to an ameba, named by him Ameba colt, in 1875. Finally, Koch, in 1876, isolated the anthrax bacillus by means of gelatin plates (first used by Emil Chr. Hansen in his studies on veast), worked out the life history of the organism and reproduced the disease by the injection of pure cultures and recovered the organism from the inoculated animals, thus establish- ing beyond reasonable doubt its causal relationship to the disease. This was the first instance of a bacterium proved to be the cause of a disease in animals. Pasteur, working on the disease at the same time, confirmed all of Koch’s find- ings, though his results were published the next year, 1877. Bollinger determined that the Actinomyces bovis (Strepto- thriz bovis) is the cause of actinomycosis in cattle in 1877. Woronin in the same year discovered a protozoan (Plasmo- diophora brassice) to be the cause of a disease in cabbage plants, the first proved instance of a unicellular animal caus- ing a disease in a plant. In 1878 Koch published his researches on wound infection in which he showed beyond question that microérganisms are the cause of this condi- tion, though Pasteur, in 1837, had suggested the same thing and Lister had acted on the theory in preventing infection. These discoveries, especially those of Koch, immediately attracted world-wide attention and stimulated a host of workers, so that within the next ten years most of the bac- 28 HISTORICAL INTRODUCTION teria which produce disease in men and animals were iso- lated and described. It is well to remember that the first specific disease of man proved to be caused by a bacterium was tuberculosis, by Koch in 1882. Progress was greatly assisted by the introduction of anilin dyes as suitable stains for organisms by Weigert in 1877, by Koch’s application of special technic and solid cultures for isolation and study, and the great improvements in the microscope by Prof. Abbé, of Jena. Laveran’s discovery of the malarial parasite in 1880 turned attention to protozoa as the causes of disease and led to the discovery of the various piroplasmoses and irypanosemiases in man and the lower animals. Pasteur’s protective inoculations in chicken cholera and anthrax directed attention to the possibility of using bac- teria or their products as a specific protective or curative means against particular diseases. This finally led to the dis- covery of diphtheria antitoxin by Behring, and independently by Roux, in 1890, a discovery which opened up the wide field of immunity which is so persistently cultivated at the ' present time. While the causation of disease by bacteria has probably attracted most attention, especially in the popular mind, it should not be forgotten that this is but one of the numerous ways in which these organisms manifest their activities, and in a sense it is one of their least-important ways, since other kinds are essential in many industries (dairying, agriculture) and processes (sewage purification) and are even indispen- sable for the very existence of all green plants and hence of animals, including man himself. PUTREFACTION AND FERMENTATION. The idea that there is a certain resemblance between some infectious diseases and the processes of putrefaction and fermentation seems to have originated during the dis- cussion on spontaneous generation and the “contagium vivum” theory which followed Leeuwenhoek’s discoveries. Plenciz (1762) appears to have first formulated this belief LOUIS PASTEUR PUTREFACTION AND FERMENTATION 29 in writing. He considered putrefaction to be due to the “animalcules” and said that it occurred only when there was a coat of organisms on the material and only when they increased and multiplied. Spallanzani’s experiments tended to support this view since his infusions did not “spoil” when boiled and sealed. Appert’s practical appli- cation of this idea has been mentioned. Thaer, in his Principles of Rational Agriculture, pub- lished in the first quarter of the nineteenth century, expressed the belief that the “blue milk fermentation” was probably due to a kind of fungus that gets in from the air, and stated that he had prevented it by treating the milk cellars and vessels with sulphur fumes or with “ oxygenated hydrochloric acid” (hypochlorous acid). In 1836 Chevreuil and Pasteur showed that putrefaction did not occur in meat protected from contamination. In 1837 Caignard-Latour, in France, and Schwann, in Germany, ‘independently showed that alcoholic fermentation in beer and wine is due to the growth of a microscopic plant, the yeast, in the fermenting wort. C.J. Fuchs described the organism which is commonly called the “blue milk bacillus”’ in 1841 and conjectured that the souring of milk was prob- ably bacterial in origin. It remained for Pasteur to prove this in 1857. During the following six or seven years Pas- teur also proved that acetic acid fermentation, as in vinegar making, butyric acid fermentation (odor of rancid butter and old cheese) and the ammoniacal fermentation of urea, so noticeable around stables, were each due to different species of bacteria. Pasteur also, during the progress of this work, discovered the class of organisms which can grow in the absence of free oxygen—the anaérobic bacteria. There is no question that Pasteur from 1857 on did more to lay the foundations of the science of bacteriology than any other one man. Influenced by Pasteur’s work von Hesseling, in 1866, stated his belief that the process of cheese ripening, like the souring of milk, was associated with the growth of fungi, and Martin also, in 1867, stated that cheese ripening was a process which was akin to alcoholic, lactic and butyric fermentations. Kette, in 1869, asserted the probability of 30 HISTORICAL INTRODUCTION Pasteur’s researches furnishing a scientific basis for many processes of change in the soil. In 1873 Schlésing and Miintz showed that nitrification must be due to the action of microérganisms, though the discovery of the particular ones remained for Winogradsky in 1889. Thus the belief that fermentation and putrefaction are due to microdrgan- isms was as well established by the early eighties of the last century as that similar organisms are the causes of infectious diseases. STUDY OF FORMS. An important part of the scientific knowledge of living organisms is dependent on a study of their forms and rela- tionships. As has been stated, Leeuwenhoek considered bacteria to be “animalcules’” because they showed inde- pendent movement. But little attention was paid to, the natural history of these animalcules for nearly a hundred years after Leeuwenhoek. During the last quarter of the eighteenth century, however, workers busied themselves chiefly with the discovery and description of new forms. Among these students were Baron Gleichen, Jablot, Lesser, Reaumur, Hill and others. Miiller, of Copenhagen, in 1786 published the first attempt at classification, a most impor- tant step in the study of these organisms. Miiller intro- duced the terms Monas, Proteus and Vibrio which are still in use. Ehrenberg, in his work on Infusoria, or the organisms found in infusions, published in 1838, introduced many generic names in use at present, but still classed the bacteria with protozoa. Joseph Leidy, the American geol- ogist, who took great interest in natural history in general, considered that the ‘‘vibrios” of previous writers were plants and not ‘“‘animalcules.’’ He seems to have been the first to have made this distinction (1849). Perty (1852) recognized the presence of spores in some of his organisms. Ferdinand: Cohn (1854) classed the bacteria among plants. Niégeli (1857) proposed the name “Schizomycetes” or “fission fungi,” which is still retained for the entire class of bacteria. Cohn . in the years 1872-1875 established classification on a mod- ern basis and added greatly to the knowledge of morphology STUDY OF FORMS 31 and natural history of bacteria. He described spore forma- tion and the development of spores into active bacteria, and showed the close relationships as well as differences between the bacteria and the lower alge. Robert Koch was a pupil of Cohn. An examination of the accompanying chronological table will show how the investigations and discoveries in con- nection with “spontaneous generation,” the “contagium vivum” theory and putrefaction and fermentation must have been mutually suggestive: “1546. Fracastorius, disease germs theory and direct and indirect contagion. 1671. Kircher, “contagium vivum”’ theory. ° 1675. Leeuwenhoek, first saw bacteria, ‘‘animalcules.”’ 1701. Andry, “animalcules” cause of diseases. 1718. Lancisi, “animalcules” cause of malaria. 1749. Needham, described development of organisms in water around barley grains. 1762. Plenciz, arguments for “living cause” theory and that “animalcules” cause putrefaction. 1768. Bonnet, suggested that probably Needham’s organ- isms came from germs in the liquid. 1776. Spallanzani, boiled and sealed infusions. 1786. Miiller, first classified “animalcules.” 1787. Wollstein, glanders pus infectious. 1797. Viborg, transmitted glanders repeatedly. 1807. Prevost, grain rust, Puccinia graminis. 1810. Appert, directions for ‘canning.’ 1822. Gaspard, infectiousness of material from wounds. 1834. Renucci, itch—itch mite (Sarcoptes scabvet). 1836. Schultze, air through acid to kill “germs.” 1837. Chevreuil and Pasteur, protected meat did not putrefy; suggested wound infection due to entrance of germs from without. 1837. Caignard-Latour, Schwann, alcoholic fermentation —yveast. 1837. Schwann, air through heated tubes to kill germs. 1837. Bassi, muscardine of silkworms, Botrytis bassiana. 32 1838. 1838. 1839. HISTORICAL INTRODUCTION Boehm, cholera, saw organisms in stools (not the cause). Ehrenberg, study of forms. Schénlein, Favus, Achorion schoenleinit. 1839-41. Berg, Thrush, Oidiwm albicans. 1840. 1841. Henle, theory of contagious diseases. Fuchs, bacterial cause of blue milk. 1842-43. Gruby, Herpes tonsurans, Trichophyton ton- 1843 1845 1846. 1849. 1849. 1850. 1851. 1851. 1852. 1852. 1854. 1855. 1857. 1857. 1860. 1861. 1863. 1865. 1865. 1865. 1866. 1866. 1867. 1869. surans. Klencke, inoculations of tuberculous material into rabbit. Liebert, a potato rot, Peronospora infestans. . Eichstedt, Pityriasis versicolor, Microsporon furfur. Leidy, Joseph (American geologist), considered “‘vibrios” to be plants. Pollender, Anthrax, saw rods in blood. Davaine and Rayer, Anthrax , saw rods in blood. Griesinger, Egyptian chlorosis, Ankylostoma duo- denale. Bilharz, Bilharzia disease, Schistosomum hematobium. Kiickenmeister, tapeworm, Tenia soliwm. Perty, saw spores in bacteria. Cohn, classed bacteria as plants. Cohn, Disease of Flies, Empusa musce. Nageli, named bacteria, Schizomycetes. Pasteur, lactic, acetic, butyric acid fermentation. Zenker, Trichinosis, Trichinella spiralis. Pasteur, disproof of spontaneous generation. Davaine, transmitted anthrax by blood injections. Pasteur, Pebrine of silkworms, Nosema bombycis. Villemin, repeatedly transmitted tuberculosis to rabbits. Lister, introduced antisepsis in surgery. Rindfleisch, Pyemia, organisms in the pus. Von Hesseling, cheese ripening. De Martin, cheese ripening akin to alcoholic fer- mentation. Kette, Pasteur’s researches scientific basis for many processes in the soil. 1871. 1872. STUDY OF FORMS 30 Klebs, Pyemia, organisms in the pus. Bollinger, spores in anthrax. 1872-75. Cohn, definite classification. 1873. 1873. 1875. Obermeier, Recurrent fever, Spirocheta obermeiert. Schlésing and Miinz, nitrification due to organisms. Lésch, Amebic dysentery, Ameba colt. 1875-76. Tyndall, germs in the air. 1876. 1877. 1877. 1877. 1878. 1881. Robert Koch, Anthrax, Bacteriwm anthracis. Bollinger, Actinomycosis, Actinomyces bovis (Strep- tothriz bovis). Weigert, used anilin dyes for staining. Woronin, Cabbage disease, Plasmodiophora brassice. Koch, wound infections, bacterial in origin. Koch, gelatin plate cultures. Abbé, improvements in the microscope. CHAPTER I. POSITION—RELATIONSHIPS. Bacteria are considered to belong to the plant kingdom not because of any one character they possess, but because they most nearly resemble organisms which are generally recognized as plants. While it is not difficult to distinguish between the higher plants and higher animals, it becomes almost, if not quite, impossible to separate the lowest forms of life. It is only by the method of .resemblances above mentioned that a decision-is finally reached. It has even been proposed to make a third class of organisms neither plants nor animals but midway between in which the bac- teria are included, but such a classification has not as yet been adopted. In many respects the bacteria are most nearly related to - the lowest alg@, since both are unicellular organisms, both reproduce by transverse division and the forms of the cell are strikingly similar. The bacteria differ in one important respect, that is, they do not contain chlorophyl, the green coloring matter which enables all plants possessing it to absorb and break up carbon dioxide in the light, and hence belong among the fungi. Bacteria average much smaller than even the smallest alge. Bacteria are closely con- nected with the fission yeasts and the yeasts and torule. All are unicellular and without chlorophyl. The bacteria, as has been stated, reproduce by division but the others characteristically by budding or gemmation, though the fission yeasts also by division. There is a certain resemblance to the molds in their absence of chlorophyl. But the molds grow as branching threads and also have special fruiting organs for producing spores as a means of reproduction, neither of which charac- teristics is found among the true bacteria. The higher POSITION—RELATIONSHIPS 35 thread bacteria do show true branching and rudimen- tary fruiting bodies (Streptothrix) and appear to be a link connecting the true bacteria and the molds. Fic. 7.— NaOH, using phenolphthalein as an indi- eator; make titrations at same time from blank. The difference gives the amount of acid produced. The titration should be done after boiling to drive off any CO: present in the culture. (7) Generic nomenclature shall begin with the year 1872 (Cohn’s first important paper). . ; Species nomenclature shall begin with the year 1880 (Koch's discovery of the pour plate method for the separation of organisms). (8) Chromogenesis shall be recorded in standard color terms. TABLE I. A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM. (GROUP NUMBER.) LOO: 2oxis 239 ¢ eae s Endospores produced. 200. Endospores not produced 10. ..Aérobic (strict) 2 © —_.___. suoysofanby :juassa10qsp ‘asowunjd ‘snoyia ‘ayoydod ‘peppsq ‘utwofipf ‘amjound jo eury “poasds-apin ‘paysysal :yuppungo ‘Ayunss {MOI aowpims :t070q 7D 783q ‘doz 7D 7s2q ‘wiofiun YpMoIN) “qeIS Tes IpRaes Ve ut ajzeduioo ‘po Ur sulgeq woljejenbry “pausa.8 ‘paniq ‘pauappas ‘paumolg ‘pefins8 WUNIpeyy se Wunere e. Sane cutie (g) Stseuadom0Iy -asoontiaa ‘as0bns ‘painozuos ‘yjooums ‘Aqdeigodoy, “snoaanjaso ‘ynp. ‘buruajsy6 ‘ai1ysn'y “zZaauos ‘pasos ‘asnffa ‘yous ‘GYMOIZ JO UOTBAa[AL ‘pwoziys ‘yusrsasogip ‘asounjd ‘Bur -ppasds ‘papvag ‘aypjnurysa ‘wsofiyyfy ‘YIMoIZ Jo WI0T ‘quppungn ‘3zbsspou. ‘fyuvos ‘ajqisaUs BOS “wnJas-poolg S,JaqyeoT “pausaib ‘pang ‘pauappas ‘paunosg ‘paftosd ‘umIpay "aqqttg ‘snoI70109 ee ‘ Source______--. as cemnwes Group No. ()ascsscesweg ye eececes DETAILED @ moderate, strong, absent, BRIEF CHARACTERIZATION. ; Mark + or O, and when two terms NOTE -Underacore requir occur on a line erase the one which glossary of terms on opposite does not apply unless both apply. ements DOK MOY 2 ENG BT I. MORPHOLOGY (2 free nitrogen...........- aa 3 r Caen ites ae RD. = Diameter over Iu 1. Mapetaiine Cells, Mediumnedium, slight. Bi Chains, filaments Diss pessesce rascal cea ag era ae short rods, medium, slight 3 — chains, filaments, comm ‘ ae ‘clostridium, cuneate, cla’wth in bouillon, stated in a eee Limits OUI Ze ces cote, # igang oe bY en Dees as By Zooglea, Pseudozooglea ize of Majority....... i derate, h i Ends, mnie truncate, ue atpmnodegete, tong g Motile 2 rientation ; ‘ Involution forms H 10) minutes’ exposure in RV ONO: angene Bib Short ae is adapted to growth of Gram's stain ; 2. Sporaieid, peas for growth... ... ° C.; or Cloudy, turbid age ‘daw C., 25° C., 30° C., 87° C., | Ring gee aMaeeecare: $ a SC, SOEs short Top prowth.......... °C. & Pellicle Limits of Size.......... = growth... eae C. ; Agar Orientation sistant to drying. Sediment Hanging-block ee oe (salt and crushed ice or Shining 1ON +... 3. ede ndospores, ¢ in thinly sown agar plates; 3 put fee Form, round, elliptical, el ime 15 minutes), sensitive, & | < | Wrinkled Pool imits of Size.......... n i Size of Majority.......°°°7 7" I eee — ve thick, a pe 5 Round. orangium wal eren. 7 ermination, equatorial," """ g 3 Proteus-like stretching, = = ‘"7ttt : & a izoi 4, Flagella, No........- Ati diastase, invertase, pectase, i) ai eee trichiate. How Stained peroxidase, lipase, catalase, & | Filamentous 5, Capsules, present on... ia a Zooglea, neudlaxpoelee a Curled nae - Involution Forms, on... =) 70 © Stebcie Bonstignee 7” 218075 #58 4 tera Gems s wee 5 34 Surface growth =e 1:10 watery fuchsin, g H | On | Needle growth : Loeffler’s alkaline meth 8 bares Special Stains, - 5 Moderate, absent . J co a B Ist 2 ee sea Neisser..........0-00+ g a Be © | Discolored 1 . eo — used a z = Be Starch destroyed £ Il. CULTURAL FEATURES 3 2 2 s g Grows at 37° C. 1. Agar Stroke. g E a & 2 Grows in Cohn’s sol. ——| Growth, invisible, scanty, - an Uschi eae! Form of enaniih, filiform, Grows in Uschinsky’s sol. | Pp , arbor: t, rh Gelatin (4 Elevation of growth, flat, 6a © = Lustre, alis tening, dull, cre an EI 3 Blood-serum ‘opography, smooth, cont & ao A Opies! characters, opaqu. S Hs an escent. Chromogenesis (8)...... ! a = 7 Grice, absent, me reser | g | Acid curd onsistency, slimy, butyr¢ i & a aceous, brittle. | I Rennet curd eco Medium grayed, browned, ___! | | Z| Casein peptonized 2. Potato. SS i ea nad gr oo 6. | ! | 3 Indol ©) ‘orm of growth, filiform, | * plumose, arborescent, rhi. | B Hydrogen sulphide fa Elevation of growth, flat, ¢ | | Ammonia () Lustre, glistening, dull, ere 3 z Topography, snes cont: 3 Nitrates reduced (5) \ omogenesis (8)....... . uble, soluble: other solve Bluorescent | Odor, absent, decided, reser Luminous | Consistency, slimy, but: - | Animel.patheran onizaon GROWTH ON DIFFERENT CULTURE MEDIA. 201 Among the cultural appearances the following are of most importance: | Fic. 142.—Broth cultures x 3. broth cloudy from growth of organisms; 3, broth slightly cloudy with a deposit in bottom; 4, broth slightly cloudy with a heavy membrane at the surface. 1, uninoculated transparent broth; 2, In broth cultures the presence or absence of growth on the surface and the amount of the same. Whether the 202 STUDY OF THE PHYSIOLOGY OF BACTERIA broth is rendered cloudy or remains clear, and whether there is a deposit at the bottom or not (Fig. 142). An abundant surface growth with little or nothing below indicates a strict aérobe, while a growth or deposit at bottom and a clear or nearly clear medium above an anaérobe. These appear- Fig. 143.—A fili- Fic. 144.—A beaded Fic. 145.—A vill- form stab or punc- stab or puncture cul- ous stab or puncture ture culture. x . ture. > }. culture. xX 3. ances are for the first-few days only of growth. If the broth is disturbed, or after the culture stands for several days many surface growths tend to sink to the bottom. So an actively motile organism causes in general a cloudiness, especially if the organism is a facultative anaérobe, which tends to clear up by precipitation after several days when GROWTH ON DIFFERENT CULTURE MEDIA 203 the organisms lose their motility. Non-motile facultative anaérobes usually cloud the broth also, but settle out more rapidly than the motile ones. Fie. 146 Fig. 147 Fic. 148 Fria. 149 Fia. 146.—Crateriform liquefaction of gelatin. xX 3. Fig. 147.—Funnelform liquefaction of gelatin. X 3. Fic. 148.—Saccate liquefaction of gelatin. x 4. Fic. 149.—Stratiform liquefaction of gelatin. X 4. In gelatin and agar punctures the oxygen relationship is shown by surface growth for aérobes, growth near the bottom of the puncture for anaérobes, and a fairly uniform growth all along the line of inoculation for facultative 204 STUDY OF THE PHYSIOLOGY OF BACTERIA anaérobes. In the case of these last organisms, a preference for more or less oxygen is indicated by the approach to the aérobic or anaérobic type of growth. Fie. 150.—Filiform Fie. 151.—Filiform, Fic. 152.—Beaded slope culture. X 4. slightly spreading, slope culture. x 3. slope culture. x 3. Along the line of puncture the commonest types are filiform (Fig. 143), which indicates a uniform growth; beaded (Fig. 144), or small separate colonies; villous (Fig. 145), deli- cate lateral outgrowths which do not branch; arborescent, tree-like growths branching laterally from the line. In agar these branchings are usually. short and stubby, or technically, papilate. GROWTH ON DIFFERENT CULTURE MEDIA 205 Further, in the gelatin puncture the liquefaction which occurs is frequently characteristic. It may be crateriform (Fig. 146), a shallow saucer at the surface; or funnel-shaped Fig. 153 Fic. 154 Fig. 153 Fic. 156 Fig. 153.—Effuse slope culture. x 3. Fic. 154.—Rhizoid slope culture. X 2., Fic. 155.—Rugose slope culture. X 3. Fic. 156.—Verrucose slope culture. x 3. (Fig. 147); or it may be of uniform width all along the puncture, 7. e., saccate (Fig. 148); or it may be stratiform, (Fig. 149), 7. e., the liquefaction extends to the sides of the tube and proceeds uniformly downward, 206 STUDY OF THE PHYSIOLOGY OF BACTERIA \ On agar, potato and blood serum slope tubes the amount of growth, its form and elevation, the character of the sur- Fic. 158.—A rhizoid colony on a plate. Natural size. GROWTH ON DIFFERENT CULTURE MEDIA 207 face, and the consistency should be carefully noted, and in some few cases the character of the edge. Figures 150 to 156 show some of the commoner types. Fic. 159.—Ameboid colonies on a plate. X 3. Fic. 160.—Large effuse colony on a plate. The edge is lacerated. Inci- dentally the colony shows the rate of growth for six successive days. X 3. 208 STUDY OF THE PHYSIOLOGY OF BACTERIA Fic. 161.—Colony with edge entire Fic. 162.—Colony with edge as seen under the low-power objective. coarsely granular as seen under xX 100. thelow-power objective. 100. Fic. 163.—Colony with edge Fic. 164.—Colony with edge rhiz- curled as seen under the low-power oid as seen under the low-power objective. X 100. objective. X 100. Fic. 165.—A sinall deep rhizoid colony as seen under the low-power objective. X 100, GROWTH ON DIFFERENT CULTURE MEDIA 209 On agar and gelatin plates made so that the colonies are well isolated, the form of the latter, the rate of their growth, the character of the edge and of the surface, the elevation and the internal structure as determined by a low-power lens are often of almost. diagnostic value. Also in the case of the gelatin plates, the character of the liquefaction is important. Figs. 157 to 165 show some of the commoner , characteristics to be noted. 14 CHAPTER XXI. ANIMAL INOCULATION. ANIMAL inoculation has been referred to (1) as a method of assisting in the preparation of pure cultures of patho- genic organisms; (2) as a means of testing the poisonous properties of substances produced in bacterial cultures; (3) in order to test the ability of an organism to cause a disease; (4) for the production of various antibodies; it may be added (5) that some bacteria produce in the smaller experimental animals lesions which do not occur in animals naturally infected, but which nevertheless are characteristic for the given organism. The best illustration is the testicular reac- tion of young male guinea-pigs to intraperitoneal injections of glanders bacilli. Experimental animals are also inocu- lated (6) to test the potency of various bacterial and other biological products, as toxins, antitoxins, ete. Guinea-pigs are the most widely used experimental animals because they are easily kept and are susceptible to so many diseases on artificial inoculation. Rabbits are used very largely also, as are white mice. For special purposes white rats, pigeons, goats and swine are necessary. For commercial products horses (antitoxins) and cattle (small- pox vaccine) are employed. In the study of many human diseases the higher monkeys and even the anthropoid apes are necessary, since none of the lower animals are susceptible. The commonest method of animal inoculation is undoubt- edly the subcutaneous. This is accomplished most readily with the hypodermic needle. The skin at the point selected (usually in guinea-pigs the lateral posterior half of the abdominal surface, in mice the back near the root of the tail) is pinched up to avoid entering the muscles and the needle quickly inserted. Clipping the hairs and washing with an antiseptic solution should precede the inoculation as routine practice. Frequently a small “skin pocket’ is all that is needed. The hair is clipped off, the skin pinched up with small forceps and a slight snip with sharp scissors ANIMAL INOCULATION 211 is made. The material may be inserted into this pocket with a heavy platinum needle. Cutaneous inoculation is made by shaving the skin and rubbing the material onto the shaved surface or scratching with a scalpel or special searifier, but without drawing blood, and then rubbing in the material to be inoculated. Intravenous injections are made with larger animals. In rabbits the posterior external auricular is a convenient vein. In larger animals the external jugular is used. Intraperitoneal, -thoracic, -cardiac, -ocular, -muscular injec- tions, and injections into the parenchyma of internal organs are accomplished with the hypodermic needle. In the case of the first two, injury to contained organs should be care- fully avoided. Intracardiac injection, or aspiration of the heart to secure blood, requires considerable practice to be successful without causing the death of the animal at once through internal hemorrhage. In subdural injections into the cranial cavity it is necessary to trephine the skull first, while such injections into the spinal canal may be accom- plished between tlie vertebra with needles longer and stronger than the usual hypodermic needle. Occasionally animals are caused to inhale the organisms, or are fed cultures mixed with the feed. SECURING AND TRANSPORTING MATERIAL FROM ANIMALS FOR BACTERIOLOGICAL EXAMINATION. If the site of the lesion is readily accessible from the exterior, material from the /:ving animal should be collected with sterile instruments and kept in sterile utensils until the necessary tests can be made. Testing should be done on material as soon after collection as possible, in all cases, to avoid the effects of “decomposition” bacteria. If the blood is to be investigated it may be aspirated from a peripheral vein with a sterile hypodermic syringe of appro- priate size or allowed to flow through a sterile canula into sterile receptacles. The site of the puncture should be shaved and disinfected before the instrument is introduced. Discharges of whatever kind should likewise be collected in sterile receptacles and examined as soon as may be. 212 ANIMAL INOCULATION If internal organs are to be examined it is best to kill a moribund animal than to wait for death, since after death, and in severe infections even sometimes before, the tissues are rapidly invaded by saprophytic bacteria from the ali- mentary and respiratory tracts which complicate greatly the isolation of the specific organism. Hence the search for specific bacteria in carcasses or on organs several hours after death is frequently negative. Animal inoculation with such material is very often followed by sepsis or septicemia in a few hours, so that the specific organism has no opportunity to manifest itself. In securing material for cultures from internal organs it is a good plan’ to burn the surface of the organ with a gas or alcohol flame, or to sear it with a hot instrument to kill surface organisms, then make the incision or puncture through the burned area and secure material from the inte- rior of the organ. Such punctures made with a stiff platinum needle frequently give pure cultures of the organism sought. Slides may be made from such material and culture media inoculated at once Since a bacteriological diagnosis depends most commonly on growing the organisms, it is evident that material sent for examination must never be treated with an antiseptic or pre- servative. If decomposition is to be feared the only safe pro- cedure is to pack the material in ice and forward in this way. Tuberculous material from the parenchyma of internal organs may be forwarded in a preservative (not formalin, since this makes it very difficult to stain the bacteria) as in this special case a very positive diagnosis may be made by staining alone. Even here it is better to pack in ice in order that the diagnosis by staining may be confirmed by inoculating the living organisms into guinea-pigs. In the case of material from a rabid animal and many protozoal diseases the rule against preservatives is not abso- lute, since staining is a reliable diagnostic means. Even in these cases it is often desirable to inoculate animals, hence, as before stated, it is best to make it a uniform practice to pack material for examination in ice and use no preservatives. PART IV. GENERAL PATHOGENIC BACTERI- OLOGY. CHAPTER NNXII. INTRODUCTION. PaTHOGENIC Bacteriology treats of the unicellular micro- organisms which are responsible for disease conditions, i. e¢., pathological changes in other organisms. Hence not only are bacteria considered, but also other low vegetable forms, as yeasts and molds, likewise protozoa insofar as they may be pathogenic. For this reason the term pathogenic “Microbiology” has been introduced to include all these organisms. It is largely for the reason that the methods devised for the study of bacteria have been applied to the investigation of other microérganisms that the term “bac- teriology” was extended to cover the entire field. The general discussion in this chapter is intended to include, therefore, niicroérganisms of whatever kind pathogenic to animals. The term pathogenic as applied to an organism must be understood in a purely relative sense, since there is no single organism that can cause disease in all of a certain class, but each is limited to a more or less narrow range. Some form of tuberculosis attacks nearly all vertebrates, but no other classes of animals and no plants. Lockjaw or tetanus attacks most mammals, but not any other vertebrates naturally. Typhoid fever affects human beings; hog cholera, swine, etc. Diseases which are due to unicellular pathogenic micro- organisms are called infectious diseases, while if such diseases 214 INTRODUCTION are transmitted under natural conditions from organism to organism they are spoken of as contagious diseases. Most infectious diseases are contagious but not all. Tetanus is a good illustration of a non-contagious infectious disease. There are very few such diseases. When a unicellular microérganism gains entrance into the body and brings about any pathological changes there the result is an infection. Undoubtedly many pathogenic organ- isms get into the body but never manifest their presence by causing disease conditions, hence do not cause an infection. It is the pathological conditions which result that constitute the infection and not the mere invasion. The time that elapses between the entrance of the organ- ism and the appearance of symptoms is called the period of incubation and varies greatly in different diseases. The term infestation is used to denote pathological condi- tions due to multicellular parasites. Thus an animal is infested (not infected) with tapeworms, roundworms, lice, mites, etc. Many of these conditions, probably all, are contagious, 2. ¢., transmissable naturally from animal to animal. The word contagious has been used in & variety of ways to mean communicated by direct contact, communi- cated by a living something (contagium) that might be car- ried to a distance and finally communicable in any manner, transmissible. The agency of transmission may be very roundabout—as through a special tick in Texas fever, a mosquito in malaria, etc., or by direct personal contact, as generally in venereal diseases. After all, though exactness is necessary, it is better to learn all possible about the means of transmission of diseases, than quibble as to the terms to be used. An infectious disease may be acute or chronic. An acute infection is one which runs for a relatively short time and is “self-limited,” so-called, 7. e., the organisms cease to mani- fest their presence after a time. In some acute infections the time is very short—German measles usually runs five or six days. Typhoid fever may continue eight to ten weeks, sometimes longer, yet it is an acute infectious dis- ease. It is not so much the time as the fact of self-limita- tion that characterizes acute infections. INTRODUCTION 215 In chronic infections there is little or no evidence of limi- tation of the progress of the disease which may continue for. years. Tuberculosis is usually chronic. Leprosy in man is practically always so. Glanders in horses is most commonly chronic; in mules and in man it is more apt to be acute. Many infections begin acutely and later change to the chronic type. Syphilis in man is a good illustration. The differences between acute and chronic infections are partly due to the nature of the organism, partly to the num- ber of organisms introduced and the point of their intro- duction and partly to the resistance of the animal infected. An infectious disease is said to be specific when one kind of organism is responsible for its manifestations—as diph- theria due to the Bacterium diphtherie, lockjaw due to Bacillus tetani, Texas fever due to the Piroplasma bigeminum, etc. It is non-specific when it may be due to a variety of organisms, as enteritis (generally), bronchopneumonia, wound infections. Henle, as early as 1840, stated certain principles that must be established. before a given organism can be accepted as the cause of a specific disease. These were afterward restated by Koch, and have come to be known as “ Koch’s postulates.”” They may be stated as follows: 1. The given organism must be found in all cases of the disease in question. 2. No other organism must be found in all cases. 3. The organism must, when obtained in pure culture, reproduce the disease in susceptible animals. 4. It must be recovered from such animals in pure cul- ture and this culture likewise reproduce the disease. These postulates have not been fully met with reference to any disease, but the principles embodied have been applied as far as possible in all those infections which we recognize as specific, and whose causative agent is accepted. In many recognized infectious and contagious diseases no organism has been found which is regarded as the specific cause. In some of these the organism appears to be too small to be seen with the highest powers of the microscope, hence they are called “ultramicroscopic” organisms. Because these agents pass through the finest bacterial filters, they are also 216 INTRODUCTION frequently called “filterable.” The term “virus” or “ filter- _able virus” is likewise applied to these “ ultra-microscopic” and “‘filterable” agents. The term primary infection is sometimes applied to the first manifestation of a disease, either specific or non- specific, while secondary refers to later developments. For example, a secondary general infection may follow a primary wound infection, or primary lung tuberculosis be followed by secondary generalized tuberculosis, or primary typhoid fever by a secondary pneumonia. Where several organisms seem to be associated simultaneously in causing the condition then the term mixed infection is used— in severe diphtheria, streptococci are commonly associated with the Bacterium diphtherie. In many cases of hog- cholera, mixed infections in the lungs and in the intestines are common. Wound infections are usually mized. Auto- infection refers to those conditions in which an organism commonly present in or on the body in a latent or harmless condition gives rise to an infectious process. If the Bacillus coli normal to the intestine escapes into the peritoneal cavity, or passes into the bladder, a severe peritonitis or cystitis, respectively, is apt to result. ‘Boils’ and “pimples’’ are frequently autoinfections. Such infections are also spoken of as endogenous to distinguish them from those due to the entrance of organisms from without—exogenous infec- tions. Relapses are usually instances of autoinfection. Those types of secondary infection where the infecting agent is transferred from one disease focus to another or several other points and sets up the infection there are sometimes called metastases. Such are the transfer of tubercle bacilli from lung to intestine, spleen, etc., the for- mation of abscesses in internal organs following a primary surface abscess, the appearance of glanders nodules through- out various organs following pulmonary glanders, etc. The characteristic of a pathogenic microérganism which indicates its ability to cause disease is called its virulence. Ii slightly virulent, the effect is slight, if highly virulent, the effect is severe, may be fatal. On the other hand, the characteristic of the host which indicates its capacity for infection is called susceptibility. INTRODUCTION 217 If slightly susceptible, infection is slight, if highly suscep- tible, the infection is severe. Evidently the degree of infection is dependent in large measure on the relation between the virulence of the in- vading crganism and the susceptibility of the host. High virulence and great susceptibility mean a severe infection; low virulence and little susceptibility, a slight infection; while high virulence and little susceptibility or low viru- lence and great susceptibility might mean a moderate infec- tion varying in either direction. Other factors influencing the degree of infection are the number of organisms intro- duced, the point where they are introduced and various conditions. These will be discussed in another connection (Chapter XXV). The study of pathogenic bacteriology includes the thor- ough study of the individual organisms according to the methods already given (Chapters XVIII-XXJ) as an aid to diagnosis and subsequent treatment, bacteriological or other, in a given disease. Of far greater importance than the treatment, which in most infectious diseases is not specific, is the prevention and ultimate eradication of all infectious diseases. To accomplish these objects involves further a study of the conditions under which pathogenic organisms exist outside the body, the paths of entrance into and elimina- tion from the body and those agencies within the body itself which make it less susceptible to infection or overcome the infective agent after its introduction. That condition of the body itself which prevents any manifestation of a virulent pathogenic organism after it has been once introduced is. spoken of asimmunity in the modern sense. Immunity is thus the opposite of susceptibility and may exist in varying degrees. That scientists are and have been for some years in posses- sion of sufficient knowledge to permit of the prevention and eradication of most, if not all, of our infectious diseases can scarcely be questioned. The practical application of this knowledge presents many difficulties, the chief of which is the absence of a public sufficiently enlightened to permit the expenditure of the necessary funds. Time and educative effort alone can surmount this difficulty. It will probably be years yet, but it will certainly be accomplished. CHAPTER XXIII. PATHOGENIC BACTERIA OUTSIDE THE BODY. PATHOGENIC bacteria may exist. outside the body of the host under a variety of conditions as follows: I. In or on inanimate objects or material. (a) As true saprophytes. (b) As facultative saprophytes. (c) Though obligate parasites, they exist in a latent state. II. In or on other animals, or products from them: (a) Sick themselves. (b) Recovered from illness but carrying the organisms. (c) Never sick with the disease but carrying the organisms. (d) Serving as necessary intermediate hosts for cer- tain stages of the parasite—this applies to protozoal diseases only, as yet. I. (a) The bacilli of tetanus and malignant edema are . widely distributed. There is no evidence that their entrance into the body is at all necessary for the continuation of their life processes, or that one case of either of these diseases ever has any connection with any other case; they are true saprophytes. Manifestly it would be futile to attempt to prevent or eradicate such diseases by attacking the organ- ism in its natural habitat. Bacillus botulinus, which causes a type of meat poisoning in man, does not even multiply in the body, but the disease symptoms are due to a soluble toxin which is produced during its growth outside the body. (b) Organisms. like the bacterium of anthrax and the bacillus of black-leg from their local occurrence seem to be distributed from animals infected, though capable of living a saprophytic existence outside the body for years. These can no more be attacked during their saprophytic existence PATHOGENIC BACTERIA OUTSIDE THE BODY 219 than those just mentioned. Doubtless in warm seasons of the year and in the tropics other organisms pathogenic to animals may live and multiply in water or in damp soil where conditions are favorable, just as the cholera organism in India, and occasionally the typhoid bacillus in témperate climates do. (c) Most pathogenic organisms, however, when they are thrown off from the bodies of animals, remain quiescent, do not multiply, in fact always tend to die out from lack of all that is implied in a ‘favorable environment,” food, moisture, temperature, light, etc. Disinfection is sometimes effective in this class of diseases in preventing new cases. II. (a) The most common infectious diseases of animals are transmitted more or less directly from other animals of the same species. Human beings get nearly all their dis- eases from other human beings who are sick; horses, from other horses; cattle, from other cattle; swine, from swine, etc. Occasionally transmission from one species to another occurs. Tuberculosis of swine most frequently results from feeding them milk of tuberculous cattle or from their eating the droppings of such cattle. Human beings contract anthrax from wool, hair and hides of animals dead of the disease, or from postmortems on such animals; glanders from horses; tuberculosis (in children) from tuberculous milk; bubonic plague from rats, etc. The mode of limiting this class of diseases is evidently to isolate the sick, dis- infect their discharges and their immediate surroundings, sterilize such products as must be handled or used, kill dangerous animals, and disinfect, bury properly, or destroy their carcasses. (b) This class of “carriers” offers one of the most difficult problems in preventing infectious diseases. A perfectly healthy individual may give off dangerous organisms and infect others for years. Typhoid carriers have been known to do so for fifty-five years. Cholera, diphtheria, menin- gitis and other carriers are well known in human practice. The difficulty in detecting such individuals is obvious. Carriers among animals have not been so frequently demon- strated, but there is every reason for thinking that hog- 2290 PATHOGENIC BACTERIA OUTSIDE THE BODY cholera, distemper, roup, influenza and other carriers are common. Carriers furnish the explanation for many of the so-called “spontaneous” outbreaks of disease among men and animals. (c) In this class come the “accidental carriers” like flies, fleas, lice, bed-bugs, ticks and other biting and_blood- sucking insects, vultures, buzzards, foxes, rats and carrion- eating animals generally; pet animals in the household, ete. Here the animals are not susceptible to the given disease but become contaminated with the organisms and then through defilement of the food or drink, or contact with individuals or with utensils pass the organisms on to the susceptible. Some biting and blood-sucking insects transmit the organisms through biting infected and non-infected ani- mals successively. The spirilloses and trypanosomiases seem to be transmitted in this way, though there is evidence accumulating which may place these diseases in the next class. Anthrax is considered in some instances to be trans- mitted by flies and by vultures in the southern United States. Typhoid transmission by flies is well established in man. Why not hog-cholera from farm to farm by flies, English sparrows, pigeons feeding, or by turkey buzzards? Though this would not be easy to prove, it seems reasonable. Preventing contact of such animals with the discharges or with the carcasses of those dead of the disease, destruc- tion of insect carriers, screening and prevention of fly ee ing are obvious protective measures. (d) In this class come certain diseases for which sag: lar insects are necessary for the parasite in question, so that certain stages in its life history may be passed therein. The most certain means for eradicating such diseases is the destruction of the insects concerned. Up to the present no bacterial disease is known in which this condition exists, unless Rocky Mountain spotted fever and typhus fever shall prove to be due to bacteria. Such diseases are all due to protdézoa. Among them are Texas fever, due to Piroplasma bigeminum in this country which has been eradicated in entire districts by destruction of the cattle tick (Margaropus annulatus). Piroplasmoses in South Africa among cattle and horses, PATHOGENIC BACTERIA OUTSIDE THE BODY 221 and in other countries are transmitted in similar ways. Prob- ably many of the diseases due to spirochetes and trypano- somes are likewise transmitted by necessary insect inter- mediaries. In human medicine the eradication of yellow fever from Panama and Cuba is due to successful warfare against a certain mosquito (Stegomyia). So the freeing of large areas in different parts of the world from malaria follows the destruction of the mosquitoes. The campaign against disease in animals and man from insect sources must be considered as still in its infancy. The full utiliza- tion of tropical lands depends largely on the solution of this problem. CHAPTER XXIV. PATHS OF ENTRANCE OF PATHOGENIC ORGANISMS, OR CHANNELS OF INFECTION. 1. The Skin.—If the skin is healthy there is no oppor- tunity for bacteria to penetrate it. It is protected not only by the stratified epithelium, but also in various animals, by coats of hair, wool, feathers, etc. The secretion pressure of the healthy sweat and oil glands acts as an effective bar even to motile bacteria. Nevertheless a very slight injury only is sufficient to give normal surface parasites and other pathogenics, accidentally or purposely brought in contact with it, an opportunity for more rapid growth and even entrance for general infection. Certain diseases due to higher fungi are characteristically “skin diseases” and rarely become general—various forms of Favus, Trychophyton in- fections, etc. A few disease organisms, tetanus, malignant edema, usually get in through the skin; others, black-leg, anthrax, quite commonly; and those diseases transmitted by biting and blood-sucking insects, piroplasmoses, trypano- somiases, spirilloses, scarcely in any other way. Defective secretion in the skin glands from other causes, may permit lodgment and growth of bacteria in them or in the hair follicles. ‘“ Pimples” and boils in man and local abscesses occa- sionally in animals are illustrations. Sharp-edged and freely bleeding wounds are less liable to be infected than contu- sions, ragged wounds, burns, etc. The flowing blood washes out the wound and the clotting seals it, while there’ is less material to be repaired by the leukocytes and they are free THE SKIN 223 to care for invading organisms (phagocytosis). Pathogenic organisms, especially pus cocci, frequently gain lodgment in the milk glands and cause local (mastitis) or general infection. _B. Mucose directly continuous with the skin and lined with stratified epithelium are commonly well protected thereby and by the secretions. (a) The external auditory meatus is rarely the seat of even local infection. The tympanic cavity is normally sterile, though it may become infected by extension through the Eustachian tube from the pharynx (otitis media). (b) The conjunctiva is frequently the seat of localized, very rarely the point of entrance for a generalized infection, except after severe injury. (c) The nasal cavity, on account of its anatomical structure retains pathogenic organisms which give rise to local infec- tions more frequently than other mucose of its character. These may extend from here to middle ear, neighboring sinuses, or along the lymph spaces of the olfactory nerve into the cranial cavity (meningitis). Acute coryza (‘‘colds” in man) is characteristic. Glanders, occasionally, is primary in the nose, as is probably roup in chickens, leprosy in man. The meningococcus and the virus of poliomyelitis pass from the nose into the cranial cavity without local lesions in the former. (d) The mouth cavity is ordinarily protected by its epi- thelium and secretions, though the injured mucosa is a common source of actinomycosis infection, as well as thrush. In foot-and-mouth disease no visible lesions seem necessary to permit the localization of the unknown infective agent. (e) The tonsils afford a ready point of entrance for ever- present micrococct and streptococci whenever occasion offers (follicular tonsilitis, “quinsy”), and articular rheumatism is not an uncommon sequel. The diphtheria bacillus charac- teristically seeks these structures for its development. Tubercle and anthrax organisms occasionally enter here. (f) The pharynx is the seat of localized infection as in miacrococcal, streptococcal and diphtherial “sore throat” in human beings, but both it and the esophagus are ‘rarely infected in animals except as the result of injury. 224. ENTRANCE OF PATHOGENIC ORGANISMS (g) The external genitalia are the usual points of entrance for the venereal organisms in man (gonococcus, Treponema pallidum, and Ducrey’s bacillus). The bacillus of contagious abortion and probably the trypanosome of dourine are commonly introduced through these channels in animals. C, Lungs.—The varied types of pneumonia due to many different organisms (tubercle, glanders, influenza, plague bacilli, pneumococcus, streptococcus, micrococcus and many others) show how frequently these organs are the seat of a localized infection, which may or may not be general. Whether the lungs are the actual point of entrance in these cases is a question which is much discussed at the present time, particularly with reference to tuberculosis. The mucous secretion of the respiratory tract tends to catch incoming bacteria and other small particles and the ciliary movement along bronchial tubes and trachea tends to carry such material out. ‘‘ Foreign body pneumonia’’ shows clini- cally, and many observers have shown experimentally that microérganisms may reach the alveoli even though the exchange of air between them and the bronchioles and larger bronchi takes place ordinarily only by diffusion. The pres- ence of carbon particles in the walls of the alveoli in older animals and human beings and in those that breathe dusty air. for long periods indicates strongly, though it does not prove absolutely, that these came in with inspired air. On the other hand, experiment. has shown -that tubercle bacilli introduced into the intestine may appear in the lungs and cause disease there and not in the intestine. It is probably safe to assume that in those diseases which are transmitted most readily through close association though not neces- sarily actual contact, the commonest path is through the lungs, which may or may not show lesions (smallpox, scar- let fever, measles, chicken-pox, whooping-cough, pneumonic plague in man, lobar and bronchopneumonias and influenza in man and animals, some cases of glanders and tubercu- losis). On the other hand, the fact that the Bacillus typhosus and. Bacillus coli may cause pneumonia when they evi- dently have reached the lung from the intestinal tract, and the experimental evidence of lung tuberculosis above men- DISSEMINATION OF ORGANISMS 225 tioned show that this route cannot be excluded in inflam- mations of the lung. D. Alimentary Tract.—The alimentary tract affords the ordinary path of entrance for the causal microbes of many of the diseases of animals and man, since they are carried into the body most commonly and most abundantly in the food and drink. (a) The stomach is rarely the seat of local infection, even in ruminants, except as the result of trauma. The character of the epithelium in the rumen, reticulum and omasum in ruminants, the hydrochloric acid in the abomasum and in the stomachs of animals generally are usually sufficient pro- tection. Occasionally anthrax “pustules” develop in the gastric mucosa. (The author saw nine such pustules in a case of anthrax in a man.) (b) The intestines are frequently the seat of localized infections, as various “choleras” and “dysenteries” in men and many animals, anthrax, tuberculosis, Johne’s disease. Here doubtless enter the organisms causing “hemorrhagic septicemias” in many classes of animals, and many others. These various organisms must have passed through the stomach and the question at once arises, why did the HCl not destroy them? It must be remembered that the acid is present only during stomach digestion, and that liquids taken on an “empty stomach” pass through rapidly and any organisms present are not subjected to the action of the acid. Also spores generally resist the acid. Other organisms may pass through the stomach within masses of undigested food. The fact that digestion is going on in the stomach of ruminants practically all the time may explain the relative freedom of adult animals of this class from ““choleras” and ‘“dysenteries.” Dissemination of Organisms.—Dissemination of organisms within the tissues occurs either through the lymph channels or the bloodvessels or both. If through the lymph vessels only it is usually much more restricted in extent, or much more slowly disseminated, while blood dissemination is characterized by the number of organs involved simul- taneously. 15 226 ENTRANCE OF PATHOGENIC ORGANISMS PATHS OF ELIMINATION OF PATHOGENIC MICRO- ORGANISMS. I. Directly from the point of injury, This is true in infected wounds open to the surface, skin glanders (farcy), black-leg, surface anthrax; exanthemata in man and animals [scarlet fever (?), measles (?), smallpox; hog erysipelas, foot- and-mouth disease]. Also in case of disease of mucous mem- branes continuous with the skin—from nasal discharges (glanders), saliva (foot-and-mouth disease), material coughed or sneezed out (tuberculosis, influenza, pneumonias), ure- thral and vaginal discharges (gonorrhea and syphilis in man, contagious abortion and dourine in animals), intestinal dis- charges (typhoid fever, “choleras,” “dysenteries,” anthrax, tuberculosis, Johne’s disease). Material from nose, mouth and lungs may be swallowed and the organisms passed out through the intestines. II. Indirectly through the secretions and the excretions where the internal organs are involved. The saliva of rabid animals contains the ultra-microscopic virus of rabies (the sympathetic ganglia within the salivary glands, and pan- creas also, are affected in this disease as well as the cells of the central nervous system). The gall-bladder in man is known to harbor colon and typhoid bacilli, as that of hog-- cholera hogs does the virus of this disease. It may harbor analogous organisms in other animals, though such knowl- edge is scanty. The kidneys have been shown experimen- tally to excrete certain organisms introduced into the circu- lation within a few minutes (micrococci, colon and typhoid bacilli, anthrax). Typhoid bacilli occur in the urine of typhoid-fever patients in about 25 per cent. of all cases and the urine of hogs with hog cholera is highly virulent. Most observers are of the opinion, however, that under natural conditions the kidneys do not excrete bacteria unless they themselves are infected. The milk both of tuberculous cattle and tuberculous women has been shown to contain tubercle bacilli even when the mammary glands are not involved. LOCATION OF INFECTIVE ORGANISMS 227 SPECIFICITY OF LOCATION OF INFECTIVE ORGANISMS. It is readily apparent that certain disease organisms tend to locate themselves in definite regions and the question arises, Is this due to any specific relationship between organ- ism and tissue or not? Diphtheria in man usually attacks the tonsils first, gonorrhea and syphilis the external geni- tals, tuberculosis the lung apex (in man most commonly), “choleras” the small intestine, “dysenteries” the large intestine, influenza the lungs. In these cases the explana- tion is probably that the points attacked are the places where the organism is most commonly carried, with no specific relationship, since all of these organisms (Asiatic cholera, excepted) also produce lesions in other parts of the body when they reach them. On the other hand, the virus of hydrophobia attacks nerve cells, leprosy frequently singles out nerves, glanders bacilli introduced into the abdominal cavity of a young male guinea-pig cause an inflammation of the testicle, malarial parasites and piroplasms attack the red blood corpuscles, etc. In these cases there is apparently a real chemical relationship, as there is also between the toxins of bacteria and certain tissue cells (tetanus toxin and nerve cells). Whether “chemotherapy” will ever profit from a knowledge of such chemical relationships remains to be developed. CHAPTER XXV. IMMUNITY. Immunity, as has already been stated, implies such a con- dition of the body that pathogenic organisms after they have been introduced are incapable of manifesting them- selves, are unable to cause disease. The word has taken the place of the earlier term, resistance, and is the opposite of susceptibility. The term must be understood always in a relative sense, since no animal is immune to all pathogenic organisms, and conceivably not entirely so to anyone, since there is no question that a sufficient number of bacteria of any kind might be injected into the circulation to kill an animal, even though it did it purely mechanically. Immunity may be considered with reference to a single individual or to entire divisions of the organic world, with all grades between. Thus plants are immune to the diseases affecting animals; invertebrates to vertebrate diseases; cold- blooded animals to those of warm blood; man is immune to most of the diseases affecting other mammals; the rat to anthrax, which affects other rodents and most mammals; the well-known race of Algerian sheep is likewise immune to anthrax while other sheep are susceptible; the negro appears more resistant to yellow fever than the white; some. few individuals in a herd of hogs always escape an epizootic of hog cholera, etc. Immunity within a given species is modified by a number of factors—age, state of nutrition, extremes of heat or cold, fatigue, excesses of any kind, in fact, anything which tends to lower the “normal healthy tone” of an animal also tends to lower its resistance. Children appear more susceptible to scarlet fever, measles, whooping-cough, etc., than adults; young cattle more frequently have black-leg than older ones IMMUNITY 229 (these apparently greater susceptibilities may be due in part to the fact that most of the older individuals have had the diseases when young and are immune for this reason). Ani- mals weakened by hunger or thirst succumb to infection more readily. Frogs and chickens are immune to tetanus, but if the former be put in water and warmed up to and kept at about 37°, and the latter be chilled for several hours in ice-water, then each may be infected. Pneumonia fre- quently follows exposure to cold. The immune rat may be given anthrax if first he is made to run in a “squirrel cage” until exhausted. Alcoholics are far less resistant to infec- tion than temperate individuals. “Worry,” mental anguish, tend to predispose to infection. The following outlines summarize the different classifi- cations of immunity so far as mammals are concerned for the purposes of discussion: Immunity. : | 1. Inherited through the germ cell or cells. i (a) By having the A. Congenital A : I. Natural disease in utero. | 2. Acquired in utero. } (b) By absorption of immune _ sub- | B. Acquired by having the disease. stances from the mother. II. Artificial—acquired through human agency by: 1. Introduction of the organism or its products. 2. Introduction of the blood serum of an immune animal. Immunity. I. Active—due to the introduction of the organism or due to the intro- duction of the products of the organism. A. Naturally by having the disease. B. Artificially. 1. By introducing the organism: 1. Passage through another animal. (a) Alive and virulent. 2. Drying. 7 ; 3. Growing at a higher temperature. () ee 4. Heating the cultures. reduced py 5. Treating with chemicals. (c) Dead. erie 6. Sensitizing. 7. Cultivation on artificial media. 2. By introducing the products of the organism. II. Passive—due to the introduction of the blood serum of an actively immunized animal. 230 IMMUNITY Immunity present in an animal and not due to human interference is to be regarded as natural immunity, while if brought about by man’s effort it is considered artificial. Those cases of natural immunity mentioned above which are common to divisions, classes, orders, families, species or races of organisms and to those few individuals where no special cause is discoverable, must be regarded as instances of true inheritance through the germ cell as other char- acteristics are. All other kinds of immunity are acquired. Occasionally young are born with every evidence that they have had a disease in utero and are thereafter as immune as though the attack had occurred after birth (“smallpox babies,” “hog-cholera pigs’). Experiment has shown that immune substances may pass from the blood of the mother to the fetus 7m utero and the young be immune for a time after birth (tetanus). It is a familiar fact that with most infectious diseases recovery from one attack confers a more or less lasting immunity, though there are marked excep- tions. Active Immunity.—By active immunity is meant that which is due to the actual introduction of the organism, or in some cases of its products. The term active is used because the body cells of the animal immunized perform the real ‘work of bringing about the immunity as will be discussed later. In passive immunity the blood serum of an actively immunized animal is introduced into a second animal, which thereupon becomes immune, though its cells are not concerned in the process. The animal is passive, just as a test-tube, in which a reaction takes place, plays no other part than that of a passive container for the reagents. In active immunity the organism may be introduced in what is to be considered a natural manner, as when an ani- mal becomes infected, has a disease, without human intér- ference. Or the organism may be purposely introduced to bring about the immunity. For certain purposes the intro- duction of the products of the organism (toxins) is used to bring about active immunity (preparation of diphtheria and tetanus antitoxin from the horse). The method of produc- ing active immunity by ,the artificial introduction of the ACTIVE IMMUNITY 231 organism is called vaccination, and a vaccine must therefore contain the organism. Vaccines for bacterial diseases are frequently called bacterins. The use of the blood serum of an immunized animal to confer passive immunity on a second animal is properly called serum therapy, and the serum so used is spoken of as an antiserum, though the latter word is also used to denote any serum containing any kind of an antibody (Chapters XXVIJ-XXXI). Ina few instances both the organism and an antiserum are used to cause both active and passive immunity (serum-simultaneous method in immunizing against hog cholera). In producing active immunity the organism may be intro- duced (a) alive and virulent, but in very small doses, or in combination with an immune serum, as just mentioned for hog cholera. The introduction of the live virulent organism alone is done only experimentally as yet, as it is obviously too dangerous to do in practice, except under the strictest control (introduction of a single tubercle bacillus, followed by gradually increasing numbers—Barber and Webb). More commonly the organisms are introduced (b) alive but with their virulence reduced (“attenuated’’) in one of several ways: (1) By passing the organism through another animal as is the case with smallpox vaccine derived from a calf or heifer. (2) By drying the organism, as is done in the preparation of the vaccine for the Pasteur treatment of rabies, where the spinal cords of rabbits are dried for varying lengths of time— one to four days, Russian method, one to three days, German method, longer in this country. (It is probable that the passage of the “fixed virus” through the rabbit is as impor- tant in this procedure as the drying, since it is doubtful if the “fixed virus” is pathogenic for man.) (8) The organism may be attenuated by growing at a temperature above the normal. This is the method used in preparing anthrax vac- cine as done by Pasteur originally. (4) Instead of growing at a higher temperature the culture may be heated in such a way that it is not killed but merely weakened. Black-leg vaccines are made by this method. (5) Chemicals are some- times added to attenuate the organisms, as was formerly done in the preparation of black-leg vaccine by Kruse’s 232 IMMUNITY method in Germany. This method is no longer used to any great extent. (6) Within the past few years the workers in the Pasteur Institute in Paris have been experimenting with vaccines prepared’ by treating living virulent bacteria with antisera (‘sensitizing them”) so that they are no longer capable of causing the disease when introduced, but do cause the production of an active immunity. The method has been used with typhoid fever bacilli in man and seems to be successful. It remains to be tried out further before its worth is demonstrated (the procedure is more compli- cated and the chance for infection apparently much greater than by the use of killed cultures). (7) Growing on artifi- cial culture media reduces the virulence of most organisms after a longer or shorter time. This method has been tried with many organisms in the laboratory, but is not now used in practice. The difficulties are that the attenuation is very uncertain and that the organisms tend to regain their virulence when introduced into the body. In producing active immunity against many bacterial diseases the organisms are introduced (c) dead. They are killed by heat or by chemicals, or by using both methods (Chapter XXX). When the products of an organism are introduced the resulting immunity is against the products only and not against the organism. If the organism itself is introduced there results an immunity against it and in some cases also against the products, though the latter does not necessarily follow. Hence the immunity may be antibacterial or anti- toxic or both. Investigation as to the causes of immunity and the various methods by which it is produced has not resulted in the dis- covery of specific methods of treatment for as many dis- eases as was hoped for at one time. Just at present progress in serum therapy appears to be at a standstill, though vac- cines are giving good results in many instances not believed possible a few years ago. As a consequence workers in all parts of the world are giving more and more attention to the search for specific chemical substances, which will destroy invading parasites and not injure the host (Chemotherapy). ACTIVE IMMUNITY 233 Nevertheless, in the study of immunity very much of value in the treatment and prevention of disease has been learned. Also much knowledge which is of the greatest use in other lines has been accumulated. Methods of diagnosis of great exactness have resulted, applicable in numerous diseases. Ways of detecting adulterations in foods, particularly foods from animal sources, and of differentiating proteins of varied origin, as well as means of establishing biological relation- ships and differences among groups of animals through “Immunity reactions” of blood serums have followed from knowledge gained by application of the facts or the methods of immunity research. Hence the study of “immunity problems”’ has come to include much more than merely the study of those factors which prevent the development of disease in an animal or result in its spontaneous recovery. A proper understanding of the principles of immunity neces- sitates a study of these various features and they will be considered in the discussion to follow. 4 CHAPTER XXVI. THEORIES OF IMMUNITY. Pasteur and the bacteriologists of his time discovered that bacteria cease to grow in artificial culture media after a time, because of the exhaustion of the food material in some cases and because of the injurious action of their own products in other instances. These facts were brought for- ward to explain immunity shortly after bacteria were shown to be the cause of certain diseases. Theories based on these observations were called (1) “Exhaustion Theory’’ of Pasteur, and (2) “ Noxious Retention Theory’ of Chauveaw respec- tively. The fact, soon discovered, that virulent pathogenic bacteria are not uncommonly present in perfectly healthy animals, and the later discovery that immunity may be conferred by the injection of dead bacteria have led to the abandonment of both these older ideas. The (3) “ Unfavor- able Environment” theory of Baumgirtner, i. e., bacteria do not grow in the body and produce disease because their surroundings are not suitable, in a sense, covers the whole ground, though it is not true as to the first part, as was pointed out above, and is of no value as a working basis, since it offers no explanation as to what the factors are that constitute the “unfavorable environment.” Metchnikoff brought forward a rational explanation of immunity with his (4) “Cellular or Phagocytosis Theory.” As first pro- pounded it based immunity on the observed fact that cer- tain white blood corpuscles, phagocytes, engulf and destroy bacteria. Metchnikoff has since elaborated the original theory to explain facts of later discovery. Ehrlich soon after published his (5) “Chemical or Side-chain Theory” which seeks to explain immunity on the basis of chemical substances in the body which may in part destroy pathogenic organ- isms or in part neutralize their products; or in some instances PAUL EHRLICH THEORIES OF IMMUNITY 235 there may be an absence of certain chemical substances in the body cells so that bacteria or their products cannot unite with the cells and hence can do no damage. At the present time it is generally accepted, in this coun- try at least, that Ehrlich’s theory explains immunity in many diseases as well as many of the phenomena related to immunity, and in other diseases the phagocytes, frequently assisted by chemical substances, are the chief factors. Spe- cific instances are discussed in Pathogenic Bacteriologies which should be consulted. It is essential that the student should be familiar with the basic ideas of the chemical theory, not only from the standpoint of immunity, but also in order to understand the principles of a number of valuable methods of diagnosis. The chemical theory rests on three fundamental physi- ological principles: (1) the response of cells to stimuli, in this connection specific chemical ‘stimuli, (2) the presence within cells of specific chemical groups which combine with chemi- cal stimuli and thus enable them to act on the cell, which groups Ehrlich has named receptors, and (3) the “over- production” activity of cells as announced by Weigert. 1. That cells respond to stimuli is fundamental in physi- ology. These stimuli may be of many kinds as mechanical, electrical, light, chemical, etc. Chemical stimuli are well illustrated along the digestive tract. That the chemical stimuli in digestion may be more or less specific is shown by the observed differences in the enzymes of the pancreatic juice dependent on the relative amounts of carbohydrates, fats, or proteins in the food, the specific enzyme in each case being increased in the juice with increase of its corresponding foodstuff. The cells of the body, or certain of them at least, seem to respond in a specific way when proteins or substances closely related to them are brought into direct contact with them, that is, without having been subjected to digestion in the alimentary tract, but injected directly into the blood or lymph stream. Cells may be affected by stimuli in one of three ways: if the stimulus is too weak, there is no effect (in reality there is no “stimulus” acting); if the stimulus is too strong, the cell is injured, may be 236 THEORIES OF IMMUNITY destroyed; if the’ stimulus is of proper amount then it excites the cell to increased activity, and in the case of specific chemical stimuli the increased activity, as mentioned for the pancreas, shows itself in an increased production of what- ever rs called forth by the chemical stimulus. In the case of the proteins and related bodies, the substances produced by the cells under their direct stimulation are markedly specific for the particular substance introduced. 2. Since chemical action always implies at least two bodies to react, Ehrlich assumes that in every cell which is affected by a chemical stimulus there must therefore be a chemical group to unite with this stimulus. He further states that there must be as many different kinds of these groups as there are different kinds of chemicals which stimu- late the cell. Since these groups are present in the body cells primarily to take up different kinds of food material, Ehrlich calls them receptors. Since these groups must be small as compared with the cell as a whole, and must-be more or less on the surface and unite readily with chemical substances he further speaks of them as “side-chains” after the analogy of compounds of the aromatic series especially. The term receptors is now generally used. As was. stated above, the effect of specific chemical stimuli is to cause the production of more of the particular substance for which it is specific and in the class of bodies under discussion, proteins and their allies, the particular product is these cell receptors with which the chemical may unite. 3. Weigert first called attention to the practically con- stant phenomenon that cells ordinarily respond by doing more of a particular response than is ctually called for by the stimulus, that there is always an “overproduction” of activity. In the case of chemical stimuli this means an increased production of the specific substance over and above the amount actually needed. Whenever a cell accumulates an excess of products the normal result is that it excretes them from its own substance into the surrounding lymph, whence they reach the blood stream to be either carried to the true excretory organs, utilized by other cells or remain . for a longer or shorter time in the blood. THEORIES OF IMMUNITY 237 To recapitulate, Ehrlich’s theory postulates specific chemi- cal stimuli, which react with specific chemical substances in the body cells, named receptors, and that these receptors, according to Weigert, are produced in excess and hence are excreted from the cell and become free receptors in the blood and lymph. These free receptors are the various kinds of antibodies, the kind depending on the nature of the stimulus, the substance introduced. Any substance which when introduced into the body causes the formation of an anti- body of any kind whatsoever is called an antigen, 7. e., anti (body) former. If the three fundamental principles just discussed are thoroughly understood, the theory of the formation of different kinds of antibodies should not be difficult to comprehend. CHAPTER XXVII. RECEPTORS OF THE FIRST ORDER. ANTITOXINS—ANTIENZYMES. TuE general characteristics of toxins have been described (Chapter XII). It has been stated that they are more or less specific in their action on cells. In order to affect a cell it is evident that a toxin must enter into chemical combina- tion with it. This implies that the toxin molecule possesses a chemical group which can combine with the cell. This group is called the haptophore or combining group. The toxic or injurious portion of the toxin molecule is likewise spoken of as the toxophore group. When a toxin is intro- duced into the body its haptophore group combines with suitable receptors in different cells of the body. If not too much of the toxin is given, instead of injuring, it acts as a chemical stimulus to the cell in the manner already described. The cell in response produces more of the specific thing, which in this instance is more receptors which can combine with the toxin, 7. e., with its haptophore group. If the stim- ulus is kept up, more and more of these receptors are pro- duced until an excess for the cell accumulates, which excess is excreted from the individual cell and becomes free in the blood. These free receptors have, of course, the capacity to combine with toxin through its haptophore group. When the toxin is combined with these free receptors, it cannot combine with any other receptors, e. g., those in another cell and hence cannot injure another cell. These free recep- ' tors constitute, in this case, antitoxin, so-called because they can combine with toxin and hence neutralize it. Antitoxins are specific—that is, an antitoxin which will combine with the toxin of Bacillus tetani will not combine with that of Bacterium diphtherie, or of Bacillus botulinus, or of any other toxin, vegetable or animal. ANTITOXINS—ANTIENZY MES 239 When a toxin is kept in solution for some time or when it is heated above a certain temperature (different for each toxin) it loses its poisonous character. It may be shown, however, that it is still capable of uniting with antitoxin, and preventing the latter from uniting with a fresh toxin. This confirms the hypothesis that a toxin molecule has at least two groups: a combining or haptophore, and a poison- ing or toxophore group. A toxin which has lost its poisonous property, its toxophore group, is spoken of as a toxoid. The theory of antitoxin formation is further supported by the fact that the proper introduction of toxoid, the haptophore group, and hence the real stimulus, can cause the production of antitoxin to a certain extent at least. The close relationship between toxins and enzymes has already been pointed out. This is still further illustrated by the fact that when enzymes are properly introduced into the tissues of an animal there is formed in the animal an antienzyme specific for the enzyme in question which can prevent its action. The structure of enzymes, as composed of a haptophore, or uniting, and a zymophore or digesting (or other activity) group, is similar to that of toxins, and enzymoids or enzymes which can combine with the substance acted on but not affect it further have been demonstrated. These free-cell receptors, antitoxins or antienzymes, which are produced in the body by the proper introduction of toxins or enzymes, respectively, have the function of com- bining with these bodies but no other action. As was pointed out above, this is sufficient to neutralize the toxin or enzyme and prevent any injurious effect since they can unite with nothing else. Since these receptors are the simplest type which has.been studied as yet, they are spoken of by Ehrlich as receptors of the first order. Other antibodies which are likewise free receptors of the first order and have the func- tion of combining only have been prepared and will be referred to in their proper connection. They are mainly of theoretical interest. Ehrlich did a large part of his work on toxins and anti- toxins with ricin, the toxin of the castor-oil bean, abrin, from the jequirity bean, robin from the locust tree, and with 240 RECEPTORS OF THE FIRST ORDER the toxins and antitoxins for diphtheria and tetanus. Anti- toxins have been prepared experimentally for a large number of both animal and vegetable poisons, including a number for bacterial toxins. The only ones which, as yet, are of much practical importance are antivenin for snake poison, (not a true toxin, however, see p. 252), antipollenin (sup- posed to be for the toxin of hay fever) and the antitoxins for the true bacterial toxins of Bactertwm diphtherie and Bacillus tetani. The method of preparing antitoxins is essentially the same in all cases, though differing in minor details. For commer- cial purposes large animals are selected, usually horses, so that the yield of serum may be large. The animals must, of course, be vigorous, free from all infectious disease. The first injection given is either a relatively small amount of a solution of toxin or of a mixture of toxin and antitoxin. The animal shows more or less reaction, increased temper- ature, pulse and respiration and frequently an edema at the point of injection, unless this is made intravenously. After several days to a week or more, when the animal has recov- ered from the first injection, a second stronger dose is given, usually with less reaction. Increasingly large doses are given at proper intervals until the animal may take several hundred times the amount which would have been fatal if given at first. The process of immunizing a horse for diph- theria or tetanus toxin usually takes several months. Varia- tions in time and in yield of antitoxin are individual and not predictable in any given case. After several injections a few hundred cubic centimeters of blood are withdrawn from the jugular vein and serum from this is tested for the amount of antitoxin it contains. When the amount is found sufficiently large (250 “units” at least for diphtheria per c.c.) then the maximum amount of blood is collected from the jugular with sterile trocar and canula. The serum from this blood with the addition of an antiseptic (0.5 per cent. phenol, tricresol, etc.) constitutes ‘“‘antidiphtheritic serum,” or “antitetanic serum,” etc. All sera which are put on the market must conform to definite standards of strength expressed in “units” as determined ANTITOXINS—ANTIENZY MES 241 by the U. S. Hygienic Laboratory. In reality a “unit” of diphtheria antitoxin in the United States is an amount equivalent to 1 c.c. of a given solution of a standard diph- theria antitorin which is kept at the above-mentioned laboratory. This statement, of course, gives no definite idea as to the amount of antitoxin actually in a “unit.” Specifi- cally stated, a “unit” of antitoxin contains approximately the amount which would protect a 250-gram guinea-pig from 100 minimum lethal doses of diphtheria toxin, or protect 100 guinea-pigs weighing 250 grams each from one minimum lethal dose each. The minimum lethal dose (M. L. D.) of diphtheria toxin is the least amount that will kill a guinea- pig of the size mentioned within four days. Since toxins on standing change into toxoids to a great extent, the amount of antitoxin in a “unit,” though protecting against 100 M.L. D., in reality would protect against about 200 M. L. D. of toxin containing no toxoid. The official unit for tetanus antitoxin is somewhat dif- ferent, since it is standardized against a standard toxin which is likewise kept ‘at the Hygienic Laboratory. The unit is defined as “ten times the amount of antitoxin necessary to protect a 350 gm. guinea-pig for 96 hours against the standard test dose’ of the standard toxin. The standard test dose is 100 M. L. D. of toxin for a 350 gm. guinea-pig. To express it another way, one could say that a “unit” of tetanus anti- toxin would protect one thousand 350 gm. guinea-pigs from 1 M. L. D. each of standard tetanus toxin. Various methods have been devised for increasing the amount of antitoxin in 1 c.c. of solution by precipitating out portions of the blood-serum proteins and at the same time concentrating the antitoxin in smaller volume. It is not considered necessary in a work of this character to enter into these details nor to discuss the process of standardizing antitoxin so that the exact content of “units” per c.c, may -be known. 16 CHAPTER XXVIII. RECEPTORS OF THE SECOND ORDER. AGGLUTININS. CHARRIN and Rogers appear to have been the first (1889) to observe the clumping together of bacteria (Pseudomonas pyocyanea) when mixed with the blood serum of an animal immunized against them. Gruber and Durham (1896) first used the term “agglutination” in this connection and called the substance in the blood-serum “agglutinin.” Widal (1896) showed the importance of the reaction for diagnosis by testing the blood serum of an infected person against a known culture (typhoid fever). _ It is now a well-known phenomenon that’ the proper injec- tion of cells of any kind foreign to a given animal will lead to the accumulation in the animal’s blood of substances which will cause a clumping together of the cells used when suspended in a suitable liquid. The cells settle out of such suspension much more rapidly than they would otherwise do. This clumping is spoken of as “agglutination” and the substances produced in the animal are called “agglutinins.” If blood cells are injected then “hemagglutinins” result; if bacterial cells “bacterial agglutinins” for the particular organism used as “ glanders agglutinin” for Bacteriwm mallet, “abortion agglutinin” for Bacterium abortus, ‘typhoid agglutinin” for Bacillus typhosus, ete. The phenomenon may be observed either under the micro- scope or in small test-tubes, that is, either microscopically or macroscopically. In this case the cells introduced, or more properly, some substances within the cells, probably protein in nature, act _ as stimuli to the body cells of the animal injected to cause them to produce more of the specific cell receptors which AGGLUTININS 243 respond to the stimulus. The substance within the intro- duced cell which acts as a stimulus (antigen) to the body cells is called an “agglutinogen.” That “agglutinogen’”’ is present in the cell has been shown by injecting animals exper- imentally with extracts of cells (bacterial and other cells) and the blood serum of the animal injected showed the presence of agglutinin for the given cell. It will be noticed that the receptors which become the free agglutinins have at least two functions, hence at least two chemical groups. They must combine with the foreign cells and also bring about their clumping together, their agglutination. Hence it can be stated technically that an agglutinin possesses a haptophore group, and an agglutinating group. The formation of agglutinin in the body for different bac- teria does not as yet appear to be of any special significance in protecting the animal from the organism, since the bac- teria are not killed, even though they are rendered non- motile, if of the class provided with flagella, and are clumped together. The fact that such bodies are formed, however, is of decided value in the diagnosis of disease, and also in the identification of unknown bacteria. In many bacterial diseases, agglutinins for the particular organism are present in the blood serum of the affected animal. Consequently if the blood serum of the animal be mixed with a suspension of the organism supposed to be the cause of the disease and the latter be agglutinated, one is justified in considering it the causative agent, provided cer- tain necessary conditions are fulfilled. In the first place it must be remembered that the blood of normal animals fre- quently contains agglutinins (“normal agglutinins’) for many different bacteria when mixed with them in full strength. Hence the serum must always be diluted with physiological salt solution (0.85 per cent.). Further; closely related bacteria may be agglutinated to some extent by the same serum. It is evident that if they are closely related, their protoplasm must contain some substances of the same kind to account for this relationship. Since some of these substances may be agglutinogens, their introduction into the animal body will give rise to agglutinins for the related 244 RECEPTORS OF THE SECOND ORDER cells, as well as for the cell introduced. The agglu- tinins for the cell introduced will be formed in larger quan- tity, since a given bacterial cell must contain more of its own agglutinogen than that of any other cell. By diluting the blood serum from the animal to be tested the agglutinins for the related organisms (so-called “coagglutinins’”’) will become so much diminished as to show no action, while the agglutinin for the specific organism is still present in an amount sufficient to cause its clumping. Agglutinins are specific for their particular agglutinogens, but since a given blood serum may contain many agglutinins, the serwm’s specificity for a given bacterium can be determined only by diluting it until this bacterium alone is agglutinated. Hence the necessity of diluting the unknown serum in varying amounts when testing against several known bacteria to determine for which it is specific, 7. e., which is the cause of the disease in the animal. Just as an unidentified disease in an animal may be deter- mined by testing its serum as above described against known kinds of bacteria, so unknown bacteria isolated from an animal, from water, etc., may be identified by testing them against the blood sera of different animals, each of which has been properly inoculated with a different kind of known bac- teria. If the unknown organism is agglutinated by the blood of one of the animals in high dilution, and not by the others, evidently the bacterium is the same as that with which the animal had been inoculated, or immunized, as is usually stated. This method of identifying cultures of bacteria is of wide application, but is used practically only in those cases where other methods of identification are not readily applied, and especially where other methods are not sufficient, as in the “intestinal group” of organisms in human practice. The diagnosis of disease in an animal by testing its serum is also a valuable and much used procedure. This is the method of the “ Widal”’ or “ Gruber-Widal”’ test for typhoid fever in man and is used in veterinary practice in testing for glanders, contagious abortion, etc. In some cases a dilu- tion of the serum of from 20 to 50 times is sufficient for PRECIPITINS 245 diagnosis (Malta fever), in most cases, however, 50 times is the lowest limit. Evidently the greater the dilution, that is, the higher the “titer,” the more specific is the reaction. PRECIPITINS. Since agglutinins act on bacteria, probably through the presence of substances protein in nature within the bacterial cell, it is reasonable to expect that if these substances be dissolved out of the cell, there would be some reaction between their (colloidal) solution and the same serum. As a matter of fact Kraus (1897) showed that broth cultures freed from bacteria by porcelain filters do show a precipi- tate when mixed with the serum of an animal immunized against the particular bacterium and that the reaction is specific under proper conditions of dilution. It was not long after Kraus’s work until the experiments were tried of “immunizing” an animal not against a bacterium or its filtered culture, but against (colloidal) solutions of proteins, such as white of egg, casein of milk, proteins of meat and of blood serum, vegetable proteins, etc. It was ascertained that in all these ‘cases the animal’s serum contains a sub- stance which causes a precipitate with solutions of the pro- tein used for immunization. The number of such precipi- tating serums that have been made experimentally is very large and it appears that protein from any source when properly introduced into the blood or tissues of an animal will cause the formation of a precipitating substance for its solutions. This substance is known, technically as a ““precipitin.”’ The protein used as antigen to stimulate its formation, or some part of the protein molecule (hapto- phore group), which acts as stimulus to the cell is spoken of as a “precipitinogen,”’ both terms after the analogy of “agglutinin” and “agglutinogen.” In fact the specific pre- cipitation and agglutination are strictly analogous phenom- ena. Precipitins act on proteins in (colloidal) solution and cause them to settle out, agglutinins act on proteins within cells which cells are in suspension in a fluid and cause the cells to settle out. Ehrlich’s theory of the formation of 246 RECEPTORS OF THE SECOND ORDER precipitins is similar to that of agglutinitis, and need not be » repeated. Substitute the corresponding words in the theory of formation of agglutinins as above given and the theory applies. _ The precipitin reaction has not found much practical use in bacteriology largely because the “agglutination test’’ takes its place as simpler of performance and just as ac- curate. The reaction is, however, generally applicable to filtrates of bacterial cultures and could be used if needed. The so-called “mallease” reaction in glanders is an instance. Precipitins find their greatest uséfulness in legal medi- cine and in food adulteration work. As was noted above, if animals, rabbits for example, are immunized with the blood of another animal (human beings) precipitins are developed which are specific for the injected blood with proper dilution. This forms an extremely valuable means of determining the kind of blood present in a given spot shown by chemical and spectroscopic tests to be blood, and has been adopted as a legal test in countries where such rules of procedure are applied. Similarly the test has been used to identify the different kinds of meat in a sausage, and different. kinds of milk in a mixture. An extract of the sausage is made and tested against the serum of an animal previously treated with extract of horse meat, or hog meat, or beef, etc., the specific precipitate occurring with the specific serum. Such reactions have been obtained where the protein to be tested was diluted 100,000 times and more. Biological relationships and differences have been detected by the reaction. Human immune serum shows no reaction with the blood of any animals except to a slight extent with that of various monkeys, most with the higher, very slight with the lower Old World and scarcely any with New World ‘monkeys. It is a fact of theoretical interest mainly that if agglutinins and precipitins themselves be injected into an animal they will act as antigens and cause the formation of antiagglu- tinins or antiprecipitins, which are therefore receptors of the first order since they simply combine with these immune bodies to neutralize their action, have only a combining or ‘ PRECIPITINS 247 haptophore group. Also if agglutinins or precipitins be heated to the proper temperature they may retain their combining power but cause no agglutination or precipita- tion, 2. e., they are converted into agglutinoid or precipi- tinoid respectively after the analogy of toxin and toxoid. Precipitins like agglutinins possess at least two groups— a combining or haptophore group and a precipitating (some- times called zymophore) group. Hence they are somewhat more complex than antitoxins or antienzymes which have a combining group only. For this reason Ehrlich classes agglutinins and precipitins as receptors of the second order. CHAPTER XXIX. RECEPTORS OF THE THIRD ORDER. CYTOLYSINS. BrroreE Koch definitely proved bacteria capable of caus- ing disease several physiologists had noted that the red cor- puscles of certain animals were destroyed by the blood of other animals (Creite, 1869, Landois, 1875), and Traube and Gescheidel had shown that freshly drawn blood destroys bacteria (1874). It was not until about ten years afterward that this action of the blood began to be investigated in connection with the subject of immunity. Von Fodor (1885) showed that saprophytic bacteria injected into the blood are rapidly destroyed. Fliigge and his pupils, especially Nuttall in combating Metchnikoff’s theory of phago- cytosis, announced in 1883, studied the action of the blood on bacteria and showed its destructive effect (1885-87). Nuttall also showed that the blood lost this power if heated to 56°. Buchner (1889) gave the name ‘“‘alexin” (from the Greek “to ward off”) to the destroying substance and showed that the substance was present in the blood serum as well as in the whole blood, and that when the serum lost its power to dissolve, this could be restored by adding fresh blood. Pfeiffer (1894) showed that the destructive power of the blood of animals immunized against bacteria (cholera and typhoid) was markedly specific for the bacteria used. He introduced a mixture of the blood and the bacteria into the abdominal cavity of the immunized animal or of a normal one of the same species and noted the rapid solution of the bacteria by withdrawing portions of the peritoneal fluid and exam- ining them (“Pfeiffer’s phenomenon”). Belfanti and Car- bone and especially Bordet (1898) showed the specific dis- solving action of the serum of one animal on the blood cor- CYTOLYSINS 249 puscles of another animal with which it had been injected. Since this time the phenomenon has been observed with a great variety of cells other than red blood corpuscles and bacteria—leukocytes, spermatozoa, cells from liver, kidney, brain, epithelia, etc., protozoa, and many vegetable cells. It is therefore a well-established fact that the proper injection of an animal with almost any cell foreign to it will lead to the blood of the animal injected acquiring the power to injure or destroy cells of the same kind as those introduced. The destroying power of the blood has been variously called its “cytotoxic” or “cytolytic” power, though the terms are not strictly synonymous since “cytotoxic” means ‘‘cell poisoning” or ‘injuring,’ while “cvtolytic”’ means “cell dissolving.” The latter term is the one gen- erally used and there is said to be present in the blood a specific “‘cvtolysin.”” The term is a general one and a given cytolysin is named from the cell which is dissolved, as a bacteriolysin, a hemolysin (red-corpuscle-lysin), epitheliolysin, nephrolysin (for kidney cells), ete. If the cell is killed but not dissolved the suffix “ cidin’’ or “toxin” is frequently used as “bacteriocidin,” “spermotoxin,”’ “neurotoxin,” etc. The use of the term ‘“‘cytolysin” is also not strictly cor- rect, though convenient, for the process is more complex than if one substance only were emploved. As was stated above, the immune serum loses its power to dissolve the cell if it is heated to 55° to 56° for half an hour, it is inactivated. But if there be added to the heated or inactivated serum a small amount of normal serum (which contains only a very little cytolytic substance, so that it has no dissolving power when so diluted) then the mixture again becomes cytolytic. It is evident then that in cytolysis there are two distinct substances involved, one which is present in all serum, normal or immune, and the other present only in the immune cytolytic serum. Experiment has shown that it is the substance present in all serum that is the true dissolving body, while the immune substance serves merely to unite this body to the cell to be destroyed, 7. e., to the antigen. Since the immune body has therefore two uniting growps, one for the dissolv- ing substance and one for the cell to be dissolved, Ehrlich 250 RECEPTORS OF THE THIRD ORDER calls it the “amboceptor.”’ He also uses the word “ comple- ment” to denote the dissolving substance, giving the idea that it completes the action of dissolving after it has been united to the cell by the amboceptor, thus replacing Buchner’s older term “alexin” for the same dissolving body. AMBOCEPTORS. The theory of formation of amboceptors is similar to that for the formation of the other types of antibodies. The cell introduced contains some substance, probably protein, which acts as a chemical stimulus to some of the body cells provided with proper receptors so that more of these special receptors are produced, and eventually in excess so that they become free in the blood and constitute the free ambocep- tors. It will be noticed that these free receptors differ from either of the two groups already described in that they have two uniting growps, one for the antigen (cell introduced) named cytophil haptophore, the other for the complement, complementophil haptophore. Hence amboceptors are spoken of as receptors of the third order. They have no other func- tion than that of this double combining power. The action which results is due to the third body—the complement. It will be readily seen that complement must possess at least two groups, a combining or haptophore growp which unites with the amboceptor, and an active group which is usually called the zymophore or toxophore group. Comple- ments thus resemble either toxins, where the specific cell (antigen) is injured or killed, or enzymes, in case the cell is likewise dissolved. This action again shows the close rela- tion between toxins and enzymes. Complement may lose its active group in the same way that toxin does and becomes then complementoid. Complement is readily destroyed in blood or serum by heating it to 55° to 56° for half an hour, and is also destroyed spontaneously when serum stands for a day or two, less rapidly at low temperature than at room temperature. ji “ Amboceptors appear to be specific in the same sense that agglutinins are. That is, if a given cell is used to immunize COMPLEMENTS 251 an animal, the animal’s blood will contain amboceptors for the cell used and also for others closely related to it. Jmmun- ization with spermatozoa or with epithelial or liver cells gives rise to amboceptors for these cells and also for red blood corpuscles and other body cells. A typhoid bactericidal serum has also some dissolving effect on colon bacilli, ete. Hence a given serum may contain a chief amboceptor and a variety of “coamboceptors,”’ or one amboceptor made up of a number of “partial amboceptors” each specific for its own antigen (“amboceptorogen’’). Amboceptors may com- bine with other substances than antigen and complement, as is shown by their union with lecithin and other “lipoids,” though these.substances seem capable of acting as comple- ment in causing solution of blood corpuscles. COMPLEMENTS. As to whether complements are numerous, as Ehrlich claims, or there is only one complement, according to Buch- ner and others, need not be discussed here. In the practi- cal applications given later as means of diagnosis it is appar- ent that all the complement or complements are capable of uniting with at least two kinds of amboceptors. If complement be injected into an animal it may act as an antigen and give rise to the formation of anticomplement which may combine with it and prevent its action and is consequently analogous to antitoxin. If amboceptors as antigen are injected into an animal there will be formed by the animal’s cells antiamboceptors. As one would expect, there are two kinds of antiamboceptors, one for each of its combining groups, since it has been stated that it is always the combining group of any given antigen that acts as the cell stimulus. Hence we may have an ‘“‘anticytophil ambo- ceptor” or an “anticomplementophil amboceptor.”’ These antiamboceptors and the anticomplements are analogous to antitoxin, antiagglutinin, etc., and hence are receptors of the first order. 1 252. RECEPTORS OF THE THIRD ORDER ANTISNAKE VENOMS. A practical use of antiamboceptors is in antisnake venoms. Snake poisons appear to contain only amboceptors for differ- ent cells of the body. In the most deadly the amboceptor is specific for nerve cells (cobra), in others for red corpuscles, or for endothelial cells of the bloodvessels (rattlesnake). The complement is furnished by the blood of the individual bitten, that is, in a sense the individual poisons himself, since he furnishes the destroying element. The antisera contain antiamboceptors which unite with the amboceptor introduced and prevent it joining to cells and thus binding the complement to the cells and destroying them. With this exception these antibodies are chiefly of theoretical interest. FAILURE OF CYTOLYTIC SERUMS. The discovery of the possibility of producing a strongly bactericidal serum in the manner above described aroused the hope that such sera would prove of great value in passive immunization and serum treatment of bacterial diseases. Unfortunately such expectations have not been realized and no serum of this character of much practical importance has been developed as yet (with the possible exception of Flex- ner’s antimeningococcus serum in human practice. What hog cholera serum is remains to be discovered). The reasons for the failure of such sera are not entirely clear. The following are some that have been offered: (1) Amboceptors do not appear to be present in very large amount so that relatively large injections of blood are neces- sary, which is not without risk in itself. (2) Since the com- plement is furnished by the blood of the animal to be treated, there may not be enough of this to unite with a sufficient quantity of amboceptor to destroy all the bacteria present, hence the disease is continued by those that escape. (3) Or the complement may not be of the right kind to unite with the amboceptor introduced, since this is derived from the blood of a heterologous (‘other kind”) species. In hog- cholera serum, if it is bactericidal, this difficulty is removed COMPLEMENT-FIXATION TEST 253 by using blood of an homologous (“same kind’’) animal. Hence Ehrlich suggested the use of apes for preparing bac- tericidal sera for human beings. (4) The bacteria may be localized in tissues (lymph glands), within cavities (cranial, peritoneal), in hollow organs (alimentary tract), etc., so that it is not possible to get at them with sufficient serum to destroy all. This difficulty is obviated by injecting directly into the spinal canal when Flexner’s antimeningococcus serum is used. (5) Even if the bacteria are dissolved it does not necessarily follow that their endotoxins are destroyed. These may be merely liberated and add to the danger of the patient, though this does not appear to be a valid objec- tion. (6) Complement which is present in such a large excess of amboceptor may just as well unite with amboceptor which is not united to the bacteria to be destroyed as with that which is, and hence be actually prevented from dissolv- ing the bacteria. Therefore it is difficult to standardize the serum to get a proper amount of amboceptor for the com- plement present. COMPLEMENT-FIXATION TEST. Although little practical use has been made of bactericidal sera, the discovery of receptors of this class and the peculiar relations between the antigen, amboceptor and complement have resulted in developing one of the most delicate and accurate methods for the diagnosis of disease and for the recognition of small amounts of specific protein that is in use today. This method is usually spoken of as the “complement-fixa- tion” or the ‘“complement-deviation test” (““Wassermann test” in syphilis) and is applicable in a great variety of mi- crobial diseases, but it is of practical importance in those ‘diseases only where other methods are uncertain—syphilis in man, concealed glanders in horses, contagious abortion in cattle, ete. The principle is the same in all cases. The method depends, as indicated above, on the ability of complement to combine with at least two amboceptor-antigen systems, 254 RECEPTORS OF THE THIRD ORDER ! and on the further fact that if the combination with one amboceptor-antigen system is once formed, it does not dis- sociate so as to liberate the complement for union with the second amboceptor-antigen system. If an animal is infected with a microdrganism and a part of its defensive action con- sists in destroying the organisms in its blood orlymph, then it follows from the above discussion of cytolysins that there will be developed in the blood of the animal amboceptor specific for the organism in question. If the presence of this specific amboceptor can be detected, the conclusion is war- ranted that the organism for which it is specific is the cause ‘of the disease. Consequently what is sought in the “com- plement-fixation test” is a specific amboceptor. In carrying out the test blood serum from the suspected animal is col- lected, heated at 56° for half an hour to destroy any comple- ment it contains and mixed in definite proportions with the specific antigen and with complement. The antigen is an extract of a diseased organ (syphilitic fetal liver, in syphilis), a suspension of the known bacteria, or an extract of these bacteria. Complement is usually derived from a guinea- pig, since the serum of this animal is higher in complement than that of most animals. The blood of the gray rat con- tains practically as much. If the specific amboceptor is present, that is, if the animal is infected with the suspected disease, the complement will unite with the antigen-ambo- ceptor system and be “fixed,” that is, be no longer capable of uniting with any other amboceptor-antigen system. No chemical or physical means of telling whether this union has occurred or not, except as given below, has been discovered as yet, though doubtless will be by physicochemical tests, nor can the combination be seen. Hence an “indicator,” as is so frequently used in chemistry, is put into the mixture of antigen-amboceptor complement after it has been allowed to stand in the incubator for one hour to permit the union to become complete. The “indicator” used is a mixture of sheep’s corpuscles and the heated (‘inactivated’) blood serum of a rabbit which’ has been injected with sheep’s blood corpuscles and therefore contains a hemolytic ambo- ceptor specific for the corpuscles, which is capable also of ~ COMPLEMENT-FIXATION TEST 255 uniting with complement. The indicator is put into the first mixture and the whole is again incubated for at least two hours and examined. If the mixture is clear and color- less with a deposit of red corpuscles at the bottom, that would mean that the complement had been bound to the first complex, since it was not free to unite with the second sheep’s corpuscles (antigen)—rabbit serum (hemolytic ambo- ceptor) complex—and destroy the corpuscles. Hence if the complement is bound in the first instance, the specific ambo- ceptor for the first antigen must have been present in the blood, that is, the animal was infected with the organism in question. Such a reaction is called a “ positive’ test. On the other hand, if the final solution is clear but of a red color, that would mean that complement must have united with the corpuscles—hemolytic amboceptor system—and destroyed the corpuscles in order to cause the clear red solution of hemoglobin. If complement united with this system it could not have united with the first svstem, hence there was no specific amboceptor there to bind it; no specific amboceptor in the animal’s blood, means no infection. Hence a red solution is a “negative test.” In practice all the different ingredients must be accurately tested, standardized and used in exact quantities, and a test must also be run as a control with a known normal blood of an animal of the same species as the one examined. The complement-fixation test might be applied to the determination of unknown bacteria, using the unknown cul- ture as antigen and trying it with the sera of different animals immunized against a variety of organisms, some one of which might prove to furnish specific amboceptor for the unknown organism and hence indicate what it is. The test used in this way has not been shown to be a practical necessity and hence is rarely employed. It has been used, however, to detect traces of unknown proteins, particularly blood-serum proteins, in medicolegal cases in exactly the above outlined manner and is very delicate and accurate. CHAPTER XXX. PHAGOCYTOSIS—OPSONINS. Ir has been mentioned that Metchnikoff, in a publication in 1883, attempted to explain immunity on a purely cellular basis. It has been known since Haeckel’s first observation in 1858 that certain of the white corpuscles do engulf solid particles that may get into the body, and among them bac- teria. Metchnikoff at first thought that this engulfing and subsequent intracellular digestion of the microdrganisms were sufficient to protect the body from infection. The later discoveries (discussed in considering Ehrlich’s theory of immunity) of substances present in the blood serum and even in the blood plasma which either destroy the bacteria or neutralize their action have caused Metchnikoff to modify his theory to a great extent. He admitted the presence of these substances, though giving them other names, but ascribed their formation to the phagocytes or to the same organs which form the leukocytes—lymphoid tissue generally, bone marrow. It is not within the province of this work to attempt to reconcile these theories, but it may be well to point out that Ehrlich’s theory is one of chemical substances and that the origin of these substances is not an essential part of the theory, so that the two theories, except in some minor details, are not necessarily mutually exclusive. Sir A. E. Wright and Douglas, in 1903, showed that even in those instances where immunity depends on phagocytosis, as it certainly does in many cases, the phagocytes are more or less inactive unless they are aided by chemical substances present in the blood. These substances act on the bacteria, not on the leukocytes, and change them in such a way that they are more readily taken up by the phagocytes. Wright proposed for these bodies the name opsonin, derived from a ELIE METCHNIKOFF PHAGOCYTOSIS—OPSONINS 257 Greek word signifying “to prepare a meal for.’ Neufeld and Rimpau at about the same time (1904), in studying immune sera, observed substances of similar action in these sera and proposed the name bacteriotropins, or bacteriotropic substances. There is scarcely a doubt that the two names are applied to identical substances and that Wright’s name opsonin should have preference. The chemical nature of opsonins is not certainly deter- mined, but they appear to be a distinct class of antibodies and to possess two groups, a combining or haptophore and a preparing or opsonic group and hence are similar to anti- bodies of Ehrlich’s second order—agglutinins and _precipi- tins. Wright also showed that opsonins are just as specific as agglutinins are—that is, a micrococcus opsonin prepares micrococci only for phagocytosis and not streptococci or any other bacteria. Wright showed that opsonins for many bacteria are present in normal serum and that in the serum of an animal which has been immunized against such bacteria the opsonins are increased in amount. Also that in a person infected with certain bacteria the opsonins are either increased or diminished, depending on whether the progress of the infec- tion is favorable or unfavorable. The opsonic power of a serum normal or otherwise is determined by mixing an emulsion of fresh leukocytes in normal saline§solution with a suspension of the bacteria and with the serum to be tested. The leukocytes must first be washed in several changes of normal salt solution to free them from any adherent plasma or serum. The mixture is incubated for about fifteen min- utes and then slides are made, stained with a good differ- ential blood stain, Wright’s or other, and the average num- ber of bacteria taken up by at least fifty phagocytes taken in order in a field is determined by counting under the microscope. The number so obtained Wright calls the phagocytic index of the serum tested. The phagocytic index of a given serum divided by the phagocytic index of a nor- mal serum gives the opsonic index of the serum tested. Assuming the normal opsonic index to be 1, Wright asserts that in healthy individuals the range should be not more 17 258 PHAGOCYTOSIS—OPSONINS | than from 0.8 to 1.2, and that an index below 0.8 may show a great susceptibility for the organism tested, infection with ‘the given organism if derived from the individual, or improper dosage in case attempts have been made to immunize by using killed cultures, vaccines, of the organism. On the occasion of the author’s visit to Wright’s clinic (1911) he stated that he used the determination of the opsonic index chiefly as a guide to the dosage in the use of vaccines. Most workers outside the Wright school have failed to recognize any essential value of determinations of the opsonic index in the use of vaccines. Some of the reasons for this are as follows: The limit of error in phagocytic counts may be as great as 50 per cent. in different series of fifty, hence several hundred must be counted, which adds greatly to the tediousness and time involved; the variation in apparently healthy individuals is frequently great, hence the “normal” is too uncertain; finally the opsonic index and the clinical course of the disease do not by any means run parallel. Undoubtedly the method has. decided value in the hands of an individual who makes opsonic determina- tions his chief work, as Wright’s assistants do, but it can scarcely be maintained at the present time that such deter- minations are necessary in vaccine therapy. Nevertheless that opsonins actually exist and that they play an essential part in phagocytosis, and hence in immunity, is now generally recognized. BACTERIAL VACCINES. Whether determinations of opsonic index are useful or not is largely a matter of individual opinion, but there is scarcely room to doubt that Wright has conferred a lasting benefit by his revival of the use of dead cultures of bacteria, bacterial vaccines, both for protective inoculation and for treatment. It is perhaps better to use the older terms “vaccination” and “vaccine” (though the cow, vacca, is not concerned) than to use Wright’s term “opsonic method” in this connection, bearing in mind that the idea of a vaccine is that it contains the causative organism of the infection as indicated on p. 231. BACTERIAL VACCINES 259 As early as 1880 Touissant proposed the use of dead cul- tures of bacteria to produce immunity. But because injec- tions of such cultures were so frequently followed by abscess formation, doubtless due to the high temperatures used to kill the bacteria, the method was abandoned. Further, Pas- teur and the French school persistently denied the possi- bility of success with such a procedure, and some of them even maintain this attitude at the present time. The suc- cesses of Wright and the English school which are being repeated so generally wherever properly attempted, leave no doubt in the unprejudiced of the very great value of the method and have unquestionably opened a most promising field both for preventive inoculation and for treatment in many infectious diseases. That the practice is no more universally applicable than are immune serums and that it has been and is still being grossly overexploited is undoubted. The use of a vaccine is based on two fundamental prin- ciples. The first of these is that the cell introduced must not be in a condition to cause serious injury to the animal by its multiplication and consequent elaboration of injurious sub- stances. The second is that, on the other hand, it must con- tain antigens in such condition that they will act as stimuli to the body cells to produce the necessary ’ antibodies, whether these be opsonins, bactericidal substances, or anti- endotoxins. In the introduction of living organisms there is always more or less risk of the organism not being sufh- ciently attenuated and hence of the possibility of its pro- ducing too severe an infection. In using killed cultures, great care must be exercised in destroying the organisms, so that the antigens are not at the same time rendered inactive. Hence in the preparation of bacterial vaccines by Wright’s method the temperature and the length of time used to kill the bacteria are most important factors. This method is in gen- eral to grow the organisms on an agar medium, rub off the culture and emulsify in sterile normal salt solution (0.85 per cent. NaCl). The number of bacteria per c.c. is deter- mined by staining a slide made from a small volume of the emulsion mixed with an equal volume of human blood drawn from the finger and counting the relative number of hac- 260 PHAGOCYTOSIS—OPSONINS teria and of red blood corpuscles. Since the corpuscles are normally 5,000,000 per c.mm., a simple calculation gives the number of bacteria. The emulsion of bacteria is then diluted so that a certain number of millions shall be contained in each c.c., ‘‘standardized”’ as it is called, then heated to the proper temperature for the necessary time and it is ready for use. A preservative, as 0.5 per cent. phenol, tri- cresol, etc., is added unless the vaccine is to be used up at once. The amounts of culture, salt solution, etc., vary. with the purpose for which the vaccine is to be used, from one or two agar slant cultures and a few c.c. of solution, when a single animal is to be treated, to bulk agar cultures and liters of solution as in preparing antityphoid vaccine on a large.scale. . Agar surface cultures are used so that there will be as little admixture of foreign protein as possible (see Anaphy- laxis, p. 264 et seq.). Normal saline solution is isotonic with the body cells and hence is employed as the vehicle. Vaccines are either “autogenous” or ‘“‘stock.’”’ An “autog- enous” vaccine is a vaccine that is made from bacteria derived from the individual or animal which it is desired to vaccinate and contains not only the particular organism but the particular strain of that organism which is responsible for the lesion. Stock vaccines are made up from organisms like the infective agent in a given case but derived from some other person or animal or from laboratory cultures. Commercial vaccines are “stock” vaccines and are usually “polyvalent” or even “mixed.” EIcHSTED, 26 Electric milk purifier, 139, 140 Electricity, 68, 138 Elements, chemical, in bacteria, 71 Elimination_of pathogenic organ- isms, 226 paths of, gall-bladder, 226 kidneys, 226 saliva, 226 urine, 226 Empusa muce, 26 Endo-enzymes, 114 Endogenous infection, 216 _Endoplasm, 39 Endotoxins, 116 Energy relationships of bacteria, Ensilage, 87 Enteritis, 215 Entire edge, 208 Entrance, paths of, of pathogenic organisms, 222 ‘Enzyme, final test for, 111 Enzymes, 109-114, 268 activating, 113 and toxins compared, 114, 115 as catalyzers, 111 chief characteristics of, 111 classification of, 112, 113 co-, 110 277 Enzymes, coagulating, 112 name of, .112 oxidizing, 113 produced by all cells, 112 production of, 109 reducing, 113° solar to living organisms, 109, 11 splitting, 112 Enzymoid, 239 Epitheliolysin, 249 Equatorial germination of spore, 45 Erysipelas, hog, 226 Essential structures, 39 Essentials of a culture medium, 158 Esters, production of, 98 Ether as disinfectant, 150 Eubacteria, 59 Examination, bacteriological, mate- rial for, 211 Exanthemata, 226 yee theory of immunity, 23: Exo-enzymes, 114 Exogenous infection, 216 Exotoxins, 116 Experimental animals, 210 Explanation of natural immunity, 216 External auditory meatus as chan- nel of infection, 223 genitalia, 227 ; as channels of infection, 224 Eyes in cheese, 85 F Factors affecting disinfectants, 151, 152 modifying immunity, 228 Facultative, 66 aérobe, 66 anaérobe, 66 parasite, 76 Failure of cytolytic serums, 2o2 of vaccines, 261 Fats in bacteria, 73 splitting of, 90 Favus, 25 Feces, bacteria in, 62 Fermentation, 82 acetic acid, 29, 88 ‘ acid, 82 278 Fermentation, alcoholic, 29, 89 ammoniacal, 29 and putrefaction, 28 blue milk, 29 . butyric acid, 29, 88 gaseous, 82 lactic acid, 85-88 of carbohydrates, 82 tubes, 68, 174 Ferments, organized, 114 unorganized, 114 Fever, Malta, 245 recurrent, 27 scarlet, 224, 226 Texas, 214, 215, 220 typhoid, 214, 226 yellow, 221 Filament, 52 Filiform growth, 204 Film, fixing of, 191 preparation of, 190 Filter, Berkefeld, 142 candles, 141 Pasteur-Chamberland, 142 sprinkling, 104 Filterable organisms, 216 virus, 216 Filtration, 140, 141 air, 141 water, 141 First order, receptors of, 239 Fixation test, complement, 253-255 Fixed, 254 Fixing of film, 191 Flagella, position of, 42, 43 staining of, 194 Flagellum, 42 Flash process, 133 Fleas as carriers, 220 _ FLEXNER, 253 Flies as carriers, 220 disease of, 26 Frisean, 248 Fopor, von, 248 Food taken in by diffusion, 36 uses of, 75 Foot-and-mouth disease, 223, 226 Foreign-body pneumonia, 224 Formaldehyde and steam disinfec- tion, 156 as disinfectant, 148-150 Formation of agglutinins, theory 0 of antibodies, 116 INDEX Formation of precipitins, Ehrlich’s theory, 245 | . of spores, 45. conditions for, 48 Forms, cell, 49, 55 degeneration, 51 growth, 51 involution, 51 study of, 30 Foxes as carriers, 220 FRACASTORIUS, 23 Free receptors, 237 spore, 45 Fucus, C. J., 29 Fuchsin, anilin, 189 carbol, 189 Functions of agglutinin, 243° Fungi, bacteria classed as, 34 Funnel-shaped liquefaction, 205 G GaBBeEt’s blue, 190 method of staining, 193 Gall-bladder as a path of elimina- tion, 226 Gas, natural, 84 production of, 98 Gaseous fermentation, 82 GaASPARD, 25 Gelatin culture medium, 161-163 liquefaction of, 92 plates first used, 27 General conditions for growth, 62 infections, vaccines in, 261 Generation, spontaneous, 17 Generic names introduced, 30 Genitalia, external, 227 as channels of infection, 224 Gentian violet, anilin, 18 aqueous, 189 German measles, 214 Germination of spore, 45. bipolar, 46 equatorial, 45 oblique, 45 polar, 45 Germs, disease, 23 GESCHEIDEL, 248 Glanders, 215, 223, 224, 226, 227, 253 infectious, 24 mallease, reaction in, 246 INDEX Glands, mammary, 226 salivary, 226 GLEICHEN, 30 Globulin in bacteria, 73 Glycerin broth, 159 Glycerinized potato, 166 Gonococcus, 224 Gonorrhea, 226, 227 Good health, 269 Grain rust, 25 Gram-negative, 192 positive, 192 Gra m’s method of staining, 191 solution, 192 Granular, coarsely, edge, 208 Granules, metachromatic, 42 Neisser, 42 polar, 42 Grape juice, pasteurization of, 129, 132 Grass bacilli, 193 one plants, nitrogen nutrition of, GRIESINGER, 26 Group, agglutinating, 243 BAD VOPHEs 238, 239, 243, 247, 250 precipitating, 246 toxophore, 250 zymophore, 239, 247, 250 Groupings, cell, 52, 55 Growth, appearance of, in identifi- cation, 200 arborescent, 204 beaded, 204 filiform, 204 forms, 51 papillate, 204 villous, 204 GrusER, 242 Gruber-Widal test, 244 Grusy, 25 H HAECKEL, 256 Hanging drop slide, 185 Hansen, Emin Cur., 27 Haptophore, complementophil, 250 cytophil, 250 group, 238, 239, 243, 247, 250 Harness, etc., disinfection of, 155 | Health, good, 269 279 Heat, 119-135 dry, 121 moist, 121 production of, 104 Heated serum, 248 Hemagglutinin, 242 Hemicellulose, 72 Hemolysin, 249 Hemolytic amboceptor, 254 Hemorrhagic septicemias, 225 HENLE, 25, 215 Hericourt, 264 Herpes tonsurans, 25 HEssELine, von, 29 Heterologous, 252 Heterotrophic, 75 Hitt, 30 Horrman, 23 Hog cholera, 220, 226, 230 erysipelas, 226 Homologous, 253 Hookworm disease, 26 Host, 76 Hot beds, 105 Hydrochloric acid, 225 Hydrogen peroxide as disinfectant, 145, 150 uses of, 78 Hydrophobia, 227 Hydrostatic pressure, 69 Hypochlorites as disinfectant, 145 I Ick-cREAM poisoning, 93 Identification, appearance of growth in, 200 of blood, 246 of meat, 246 of milk, 246 physiological activities in, 199 Immunity, 228 acquired, 230 active, 230 production of, 230-232 antibacterial, 232 antitoxic, 232 artificial, 230 cellular theory of, 234 chemical theory of, 234 definition of, 213 Ehrlich’s theory of, fundamental principles of, 235 280 Immunity, Ehrlich’s theory of, summary of, 237 exhaustion. theory of, 234 inherited, 230 natural, 230 explanation of, 269 noxious retention theory of, 234 outlines of, 229 passive, 230 phagocytosis theory of, 234 relative, 228 perumepioaultaneous method in, side-chain theory of, 234 summary of, 268 theories of, 234 to protein, 265, 267 unfavorable environment theory of, 234 Inactivated, 249 Incubation, "period of, 214 Incubator, cold, 198 Incubators, 196 Index, chronological, 31 opsonic, 257, 258 phagocytic, 257 Indicator, 254 Indol, 93 Infection, 214 auto-, 216 channels of, 222 endogenous, 216 exogenous, 216 mixed, 216 primary, 216 secondary, 216 wound, 25, 27, 215 caused by bacteria, 27 Infections, general, vaccines in, 261 localized, vaccines in, 261 Infectious diseases, 213 Infective organisms, specificity of location of, 227 Infestation, 214 Infested, 214 Influenza, 224, 226, 227 Infusoria, 30 Inherited immunity, 230 Inoculation, 210 by feeding, 211 by inhalation, 211 cutaneous, 211 intracardiac, 211 intramuscular, 211 INDEX Inoculation, intra-ocular, 211 intraperitoneal, 211 intraspinal, 211 intrathoracic, 211 intravenous, 211 needles, 176 of culture medium, 170, 176 subcutaneous, 210 subdural, 211 Inoculations, protective, first, 26 Instruments, surgical, disinfection of, 153 Intestinal discharges, 226 Intestine, large, 227 small, 227 Intestines as channel of infection, 25 Intracardiac inoculation, 211 Intramuscular inoculation, 211 Intra-ocular inoculation, 211 + Intraperitoneal inoculation, 211 Intraspinal inoculation, 211 Intrathoracic inoculation, 211 Intravenous inoculation, 211 Invasion, 214 Invertase, 112 Involution forms, 51 Iodine as disinfectant, 145 Tron bacteria, 75, 78 uses of, 78 Isolation ‘of bacteria, 178-183 aids in, 182, 183 J JABLOT, 30 Jack o’lanterns, 94 Jar, Novy, 175 Johne’s disease, 225, 226 K Karte, 29 Kidneys as path of elimination, 226 Kinase, 11 Kircuer, 18, 23 Kzss, 27 ‘ KLENCKE, 26 Koc, Ropert, 17, 27, 31, 215, 248 Koch’s postulates, 215 Kraus, 245 Kross, 231 KUcHENMEISTER, 26 INDEX \ L. Lacrrate edge, 207 Lactacidase, 113 Lactic acid fermentation, 85-88 Lancis1, 24 Lanpots, 248 Large intestine, 227 Latour, 2 ‘Laveran, 24, 28 Lecithin as complement, 251 LEEUWENHOEK, ANTHONY VAN, 18 Legumes, 106 Leiwy, Joserx, 30 Leprosy, 215, 223, 227 Lesser, 30 Lethal dose, minimum, 241 Leukocytes, washing of, 257 Lice as carriers, 220 Lizsert, 26 Light, 65, 136 production of, 99 Linnagus, 24 Lipase, 112 Liquefaction, crateriform, 205 funnel-shaped, 205 of gelatin, 92 saccate, 205 stratiform, 205 Liquid manure, disinfection of, 155 media, 158 Lister, 26 Litmus milk, 161 Lobar pneumonia, 224 Lecines infections, vaccines in, Location, specificity of, of infective organisms, 227 Loeffler’s blood serum, 166 blue, 189 Léscu, 27 Loop needles, 177 Lopotrichic, 43 Lung, apex of, 227 Lungs, 227 as channels of infection, 224 Lysol as disinfectant, 148 M McCoy, Vera, 148 Macrococcus, 49 Macroscopic agglutination, 242 i 281 Malaria, 24, 214, 220, 227 Malarial parasite, 28 Mallease reaction in glanders, 246 Mallein test, 267 Malignant edema, 218, 222 Malta fever, 245 Mammary gland, 226 Manure, liquid disinfection of, 155 Margaropus annulatus, 220 Martin, 29 Mass cultures, 172 Material for bacteriological exami- nation, 211 Maximum conditions, 62 Measles, 224, 226 German, 214 Q Measly pork, 26 Measurement of bacteria, 37 Meat broth, 159 identification of, 246 poisoning, 93 Meatus, external auditory, as chan- nel of infection, 223 Mechanical vibration, 69 Media, liquid, 158 selective, 182, 183 solid, 158 synthetic, 158 Medium, agar, 163, 164 blood serum, 166, 167 culture, 157 essentials of, 158 inoculation of, 170, 176 gelatin, 161-163 large quantities of, 172 potato, 164-166 reaction of, 70 synthetic, 167 Meningitis, 223 Meningococcus, 223 Lae chloride as disinfectant, Merismopedia, 54 Metabiosis, 92 Metabolism of bacteria, 75-81 relative to man, 81 Metachromatic granules, 42 Metastases, 216 Metatrophic, 75 Metcanikorr, 234, 256 Methods, antiseptic, 26 of obtaining pure cultures, 178- 8 Microbiology, 213 282 INDEX Micrococci, 226 N Micrococcus, 49, 59, 223, 224 pyogenes aureus, 148 N&ceru1, 30 Micromillimeter, 37 Micron, 37 Microscope, bacteriological, 184 improvements in, 28 invented, 18 Microscopic agglutination, 242 Microspira, 60 comma, 63 Microsporon furfur, 26 Migula’s classification, 59, 60 Milk, 161 blue, bacillus of, 27 fermentation 1 in, 29 digestion of, 91 Blend as channel of infection, identification of, 246 litmus, 161 pasteurization of, 129, 132, 133, 134, 135 purifier, electric, 139, 140 souring of, 29 tuberculous, 226 Minimum conditions, 62 lethal dose, 241 Mixed infection, 216 vaccine, 260 Mixotrophic, 75 M.L. D., 241 Moist heat, 121 Moisture, 63 Molds, relation to en 34 Monas, introduced, 30 Monotrichic, 42 Mordants, 188 Morphology of bacteria, 39-55 Mosquito theory of malaria, 24 Motile bacteria, 42 Motion, Brownian, 44 Mouth cavity as channel of infec- tion, 223 Movement, Brownian, 44 rate of, in bacteria, 42 37 Mu, 37 Mucose as channels of infection, 223 Miter, 30 Miwnrz, 30 Mycoproteid, 72 Myxomycetes, 36 Names, generic, introduced, 30 Nasal cavity as channel of infec- tion, 223 discharges, 226 Natural gas, 84 immunity, 230 explanation of, 269 NrErEpHaM, 19 Needles, inoculation, 176 loop, 17 platinum, 176 straight, 176 Negative phase, 262, 263 test, 255 Neisser granules, 42 Nephrolysin, 249 NEUFELD, 257 Neurotoxin, 249 Nichrome wire, 177 Nitrate broth, 161 Nitric bacteria, 103 Nitrification, 103 due to organisms, 30 Nitrifying organisms, 30 Nitrogen, absorption of, 105 circulation of, 96 nutrition of green plants, 107 uses of, 78 Nitrous bacteria, 102 Non-pathogenic, 76 Non-specific disease, 215 Normal agglutinins, 243 Nosema bombycis, 27 Novy jar, 175 Noxious retention theory of im- munity, 234 Nuclein, 40 in bacteria, 73 Nucleus, 40 Nutrition of green plants, nitrog- enous, 107 Norra, 248 Oo OBERMEIER, 27 Objective, oil immersion, 184, 185 Oblique germination of spore, 45 Occurrence of bacteria, 51 Oidium albicans, 25 INDEX Oil immersion objective, 184, 185 Opsonic index, 257, 258 value of, 258 power, 257 Opsonin, 256 Opsonins, 256-263, 268 antibodies of second order, 257 specificity of, 257 Optimum conditions, 62 Order, first, receptors of, 239 second, receptors of, 242, 247 third, receptors of, 248, 250 Organisms, anaérobic, cultivation of, 172-176 dissemination of, in the body, 225 » filterable, 216 meee specificity of location, 22 nitrifying, 30 pathogenic, elimination of, 226 paths of entrance, 222 ultramicroscopic, 215 Organized ferments, 114 Osmotic pressure, 68, 137 Otitis media, 223 Orto, 264 Outlines of immunity, 229 Overproduction activity of cells, 235, 236 Oxidation, 102 Oxidizing enzymes, 113 Oxygen, 66 as disinfectant, 144 compressed, 67 pressure, 66, 67 relations, determination of, 198 uses of, 77 Oznam, 24 Ozone, 67, 138 as disinfectant, 145 P PANCREAS, 226 Papillate growth, 204 Parasite, 76 facultative, 76 of malaria, 28 strict, 76 Partial amboceptor, 251 Passive immunity, 230 Pasteur, 21, 25, 26, 27, 28, 29, 231, 234 283 Pasteur flask, 22, 23 treatment of rabies, 231 Pasteur-Chamberland filter, 142 Pasteurization, 127-135 continuous, 129 of beer, 129, 133 of grape juice, 129, 132 of milk, 129-132, 133, 134, 135 of wine, 129 Pathogenic, 76 bacteria, definition of, 213 outside body, 218 pastoncley reasons for study, 21 organisms, elimination of, 226 paths of entrance, 222 Paths of elimination, gall-bladder, 226 kidneys, 226 saliva, 226 urine, 226 of entrance of pathogenic organ- isms, 222 Pebrine, 26 Peptone solution, Dunham’s, 161 Period of incubation, 214 Peritrichic, 43 Peronospora infestans, 26 Perty, 30 Petri dishes, 164 Petroleum, 84 PFEIFFER, 248 phenomenon, 248 Phagocytes, 234 Phagocytic index, 257 Phagocytosis, 256-263, 268 theory of immunity, 234 Pharynx as channel of infection, 223 Phase, negative, 262, 263 positive, 263 Phenol as disinfectant, 147 Phenomena, anaphylactic, 267 Phenomenon, Arthus’s, 264 Pfeiffer’s, 248 Phosphate rock, 103 Phosphine, 94 Phosphorescence, 99 Phosphorus, circulation of, 96 uses of, 79 Photogenesis, 99 Physical agents for disinfection, 119-143 284 Physiological activities, definition of, 76 use in identification, 199 Physiology of bacteria, study of, 196-209 Pigment, production of, 100 Pimples, 216, 222 Piroplasma bigeminum, 215, 220 Piroplasmoses, 220, 222 Piroplasms, 227 von Pirquet, 264 Pityriasis versicolor, 26 Plague, 224 Planes of division, 53 . determination of, 54 Planococcus, 59 Planosarcina, 59 Plasmodiophora brassice, 27 Plasmolysis, 39 Plasmoptysis, 40 Plate colonies, study of, 209 cultures, 164, 172 Plates, dilution, 178 gelatin, first used, 27 Platinum needles, 176 Plectridium, 47 Prienciz, 24, 28 Plugs, cotton, 168 Pneumococcus, 224 Pneumonia, broncho-, 224 foreign body, 224 lobar, 224 Pneumonias, 226 Poisoning, cheese, 93 ice-cream, 93 meat, 93 Polar germination of spore, 45 granules, 42 Poliomyelitis, 223 PoLLENDER, 26 Polyvalent vaccine, 260 Pork, measly, 26 Position of bacteria, 34 of flagella, 42, 43 of spore, 46, 47 Positive phase, 263 test, 255 Postulates, Koch’s, 215 Potato, glycerinized, 166 medium, 164-166 rot, 26 Power, opsonic, 257 Practical sterilization and disinfec- tion, 152-156 INDEX Pragmidiothrix, 60 Precipitatory group, 246 Precipitin, 245 Precipitinogen, 245 Precipitins, 245-247 anti-, 246 ; compared with agglutinins, 245 formation of, Ehrlich’s theory, 245 ' uses of, 246 Preparation of antitoxin, 240 of bacterial vaccine, 259, 261 of film, 190 Preservative in vaccine, 260 Pressure, hydrostatic, 69 osmotic, 68, 137 oxygen, 66, 67 steam, 124 7 Preventatives, stock vaccines as, 260 Prevost, 25 Primary infection, 216 Production of acid, 98 of active immunity, 230-232 of antibodies, place of, 269 of aromatic compounds, 99 of enzymes, 109 of esters, 98 of gas, 98 , of heat, 104 of light, 99 of pigment, 100 of toxins, 114 Prophylaxis, 264 Protamin in bacteria, 73 Protease, 112 Protective inoculations, first, 28 Protein immunity, 265, 267 split products, 266 Proteins, putrefaction of, 91-97 Proteus, introduced, 30 Protoplasm, 39 Prototrophic, 75 | Protozoa related to bacteria, 37 Pseudomonas, 59 pyocyanea, 116, 242 radicicola, 106 Ptomaines, 92 Puccinia graminis, 25 Punctiform colony, 206 Puncture cultures, 169 Pure culture, 157 methods of obtaining, 178-183 Purin bases in bacteria, 73 INDEX Putrefaction, 25, 91 and fermentation, 28 of proteins, 91-97 Q Quick lime as disinfectant, 146 Quinsy, 223 R RaBieEs, 226 Pasteur treatment of, 231 Rabiger’s method of staining, 194 Radiations, 69 Radium, 69 Rashes, serum, 264 Rate of division, 80 of movement in bacteria, 42 Rats as carriers, 220 Rattlesnake, 252 RayYErR,.26 Reaction, anaphylactic, use of, 268 ease, in glanders, 246 of medium, 70 surface, 81 Reactions, biochemical, definition of, 76 ReEaumour, 30 Receptor, 238 Receptors, 235, 236 free, 237 of the first order, 239 of the second order, 242, 247 of the third order, 248, 250 Recurrent fever, 27 Red corpuscles, 227 Reni, Francesco, 18 Reducing actions, 101 enzymes; 113 Relapses, 216 Relationships of bacteria, 30 Renucct, 25 Resistance of spores, 47 Retarder, 131 Rheumatism, 223 Rhizoid colony, 206, 208 edge, 208 Rhodobacteriacee, 60 , RicHEt, 264 Ricin, 239 Rimpav, 257 285 RINDFLEISCH, 27 Ringworm, 26 Ripening of cheese, 29 of cream, 86 Robin, 239 Rock, phosphate, 103 Rods in blood, 26 - Roesrs, 242 Rooms, disinfection of, 153 Root tubercles, 105 RosENAv, 264 Rot, potato, 26 Roup, 223 Rovx, 28 Rust, grain, 25 SaccaTe liquefaction, 205 Saliva as path of elimination, 226 Salivary glands, 226 Salt rising bread, 84 Saprogenic, 91 Saprophilic, 92 Saprophyte, 76 Sarcina, 29, 54 lutea, 67 Sarcoptes scabiei, 25 Sauer kraut, 87 Scarlet fever, 224, 226 Scavengers, bacteria as, 95 Scuicx, 264 Schistosomum hematobium, 26 Schizomycetes, first used, 30 ScHONLEIN, 25 ; Scuésine and Miwnrz, 30 ScHROEDER and Dvusca, 20 experiment, 20, 21 ScHuttzeE, 20 experiment, 20 Scuwann, 20, 29 experiment, 20, 21 Sea, bacteria in, 61 Sealing air-tight, 19 Second order, receptors of, 242, 247 Secondary infection, 216 Selective media, 182, 183 Sensitization, 265 Sensitized, 265 vaccine, 232 Septicemias, hemorrhagic, 225 Serum, antitetanic, 240 blood, necessity for diluting, 244 286 Serum, diphtheritic, 240 heated, 248 rashes, 264 sickness, 264, 267 Serum-simultaneous method in im- munity, 231 Serums, cytolytic, failure of, 252 Shape of spore, 46 Sickness, serum, 264, 267 Side-chain theory of immunity, 234 Side-chains, 236 Silkworm disease, 25, 26 Size of bacteria, 37 Skatol, 93 Skin as channel of infection, 222 diseases, 222 ° Slant cultures, 170 Slide, cleaning of, 191 hanging drop, 185 Slope, cultures, 170 Small intestine, 227 Smallpox, 224, 226, 230, 231 vaccine, 231 Smitu, THEOBALD, 264 Snake venoms, anti-, 252 Sneezing, 226 Soap as disinfectant, 148 Sodium hypochlorite as disinfec- tant, 146 Soil, bacteria in, 61 Soils, acid, 70 Solid media, 158 Solution, Gram’s, 192 stock, of stains, 189 Sore throat, 223 Sound, 69 Sour mash distilling, 87 Source of complement, 254 Souring of milk, 29 SPALLANZANI, 19 Specific amboceptor, 254 chemical stimuli of cells, 235 disease, 215 Specificity of agglutinins, 244 of amboceptors, 250 5 of location of infective organisms, 227 of opsonins, 257 Spermotoxin, 249 Spherotilus, 60 Split products of protein, 266 Splitting enzymes, 112 of fats, 90 Spirillacee, 60 INDEX Spirilloses, 220, 222 Spirillum, 50, 60 Spirosoma, 60 Spirocheta, 60 Spirocheta obermeiert, 27 Spirochete, 50 Spirochetes, 221 Spoiling of canned goods, 48 Spontaneous combustion, 104 generation, 17 Spore capsule, 45 definition of, 48 free, 45 germination of, 45 of anthrax, 27 position of, 46, 47 shape of, 46 Spores, 44 cause of spoiling of canned goods, 48 conditions for formation of, 48 first recognized, 30 formation of, 45 resistance of, 47 staining of, 192 two, in bacterium, 47 Sprinkling filters, 104 Stab cultures, 169 Stables, disinfection of, 153-155 Stain, anilin-fuchsin, 189 gentian violet, 189 aqueous gentian violet, 189 carbol-fuchsin, 189 Gabbet’s blue, 190 Loeffler’s blue, 189 Staining bottles, 190 for cell forms, 195 groupings, 194 Gabbet’s method, 193 Gram’s method, 191 of acid-fast bacilli, 193 of bacteria, 188-195 of capsules, 194 of flagella, 194 of spores, 192 Rabiger’s method, 194 reasons for, 188 re of physiological activity, 11 Welch’s method, 194 Ziehl-Neelson method, 193 Stains, stock solutions, 189 Standard antitoxin, 241 test dose, 241 INDEX Standard toxin, 241 en of vaccines, 259, Staphylococcus, 54 Srarin, W. A., 181 Steam, 122 ear perneleenite disinfection, 1 sterilizers, 122 under pressure, 124 Stegomyia, 221 Sterile, 119 Sterilization, 118 ond disinfection, practical, 152, 56 discontinuous, 121 Sterilizers, steam, 122 Stimuli, chemical, effect of, 236 of cells, 235 specific chemical, 235 Stock cars, disinfection of, 155 solutions of stains, 189 vaccine, 260 ‘ as preventatives, 260 in treatment, 260 a as channel of infection, 25 Straight needles, 176 ‘ Stratiform liquefaction, 205 ~ Streptobacillus, 52 Streptococcus, 53, 59, 223, 224 Streptospirillum, 52 Streptothrizx bovis, 27 Strict parasite, 76 Structures, accidental, 40 cell, 39 essential, 39 Study of bacteria, 157 of bacterium, necessary steps for, 158 of forms, 30 of pathogenic bacteriology, rea- sons for, 217 of plate colonies, 209 of physiology of bacteria, 196-209 Subcutaneous inoculation, 210 Subdural inoculation, 211 Substances, cytolytic, 268 Substrate, 111 Sugar broth, 160 Sulphur bacteria, 94 circulation of, 97 deposits of, 104 uses of, 78 287 Summary of Ehrlich’s theory of immunity, 237 of immunity, 268 Surface reaction, 81 urgical instruments, disinfection of, 153 Susceptibility, 216 Symbionts, 76 Symbiosis, 76 Synthetic media, 158 medium, 167 Syphilitic antigen, 254 Syphilis, 215, 226, 227, 253 T Tapeworm, 26 Temperature, 64 Tenia solium, 26 Test, complement-deviation, 253 complement-fixation, 253-255 dose, standard, 241 for enzymes, 111 for toxins, 115 Gruber-Widal, 244 mallein, 267 negative, 255 positive, 255 tuberculin, 267 Wassermann, 253 Widal, 244 Testicle, 227 Tetanus, 214, 218, 222, 227 Tetracoccus, 54 Tetrad, 54 Texas fever, 214, 215, 220 THarr, 29 Theories of immunity, 234 Theory, cellular, of immunity, 234 chemical, of immunity, 234 contagium vivum, 23, 24 Ehrlich’s, of formation of precipi- tins, 245 of immunity, fundamental principles of, 235 summary of, 237 exhaustion, of immunity, 234 for use of vaccines, 261 mosquito, of malaria, 24 noxious retention, of immunity, 234 of anaphylaxis, 266 of formation of agglutinins, 242 288 Theory, phagocytosis, of immu- nity, 234 side-chain, of immunity, 234 unfavorable environment, of im- munity, 234 . Thermal decaieciih, 65 determination of, 198 Thermophil, 65 Thiobacteria, 60 Thiothrix, 60 Third order, receptors of, 24g 250 |. Thread, 52 Thrush, 25, 223 Ticks as carriers, 220 Titration, 160 Tonsillitis, 223 Tonsils, 227 as channels of infection, 223 ToOUISSANT, 259 Toxin, 227, 238 final test for, 115 standard, 241 Foxe gnd enzymes compared, 114, characteristics of, 114, 115 other than bacterial, 115 production of, 114 true, 116 Toxoid, 239 Toxophore group, 250 Tract, alimentary, as channel of infection, 225 TRAUBE, 248 Treatment of rabies, Pasteur, 231 stock vaccines in, 260 Treponema pallidum, .224 Trichinella spiralis, 26 Trichinosis, 26 Trichophyton tonsurans, 26 Tropical dysentery, 27 True toxins, 116 Trypanosomes, 221 Trypanosomiases, 220, 222 Tubercle bacilli, chemical analysis of, 74 Tubercles, root, 105 Tuberculin test, 267 Tuberculosis, 215, 228, 224, 225, 226, 227 due to bacteria, 28 infectious nature of, 26 Klencke’s experiment, 26 Villemin’s experiment, 26 Tuberculous milk, 226 INDEX Tubes, culture, 168 deep, 174 fermentation, 168, 174 Vignal, 173 Two spores in bacterium, 47 TYNDALL, JOHN, 23 Tyndall’s box, 22, 23 Typhoid fever, 214, 226 transmission by flies, 220 U ULTRAMICROSCOPIC organisms, 215 Ultra-violet rays, 138 Unfavorable environment theory of immunity, 234 Unit, definition of, 241 of antitoxin, 241 Unorganized ferments, 114 Unwashable articles, disinfection of, 155 Urea, decomposition of, 95 Urease, 113 Urethral discharges, 226 Urine as path of elimination, 226 bacteria in, 62 Use of agelutinins, 243, 244 of anaphylactic reaction, 268 Uses of carbon, 77 of food, 75 of hydrogen, 78 of iton, 78 of nitrogen, 78 of oxygen, 77 of phosphorus, 79 of precipitins, 246 of sulphur, 78 of vaccines, theory of, 261 Vv VaccInaTIon, 231 Vaccine, 231 anthrax, 231 autogenous, 260 bacterial preparation of, 259, 261 black-leg, 231 mixed, 260 polyvalent, 260 preservative in, 260 sensitized, 232 smallpox, 231 INDEX Vaccine, stock, 260 Vaccines, bacterial, 258 dosage of, 262 failure of, 261 m general infections, 261 in localized infections, 261 standardization of, 259, 260 stock, as preventatives, 260 in treatment, 260 theory for use of, 261 Vacuoles, 41 Vaginal discharges, 226 Value of opsonic index, 258 Varo, 23 VaucHAN, 266 Vehicles, disinfection of, 155 Venoms, antisnake, 252 Visors, Eric, 24 Vibration, mechanical, 69 Vibrio, 50 introduced, 30 Vignal tubes, 173 VILLEMIN, 26 Villous growth, 204 Vinegar, 88 Virulence, 216 Virus, filterable, 216 Vultures as carriers, 220 Ww Watt, cell, 39 composition of, 72 Washable articles, disinfection of, 155 Washing of leukocytes, 257 Wassermann test, 253 Water, bacteria in, 61 filtration, 141 Wess and BarBeEr, 231 WEIGERT, 28, 235 289 Welch’s method of staining, 194 Whooping-cough, 224 Wat, 242 test, 244 ' Widal-Gruber test, 244 Will 0’ the wisp, 94 Wine, pasteurization of, 129 WInoGRADSEY, 30, 60 Wire baskets, 168 nicrome, 177 WOLLSTEIN, 24 Worontn, 27 Wound infection, 25, 27, 215 caused by bacteria, 27 Wricat, 256 x X-rays, 69 Y YeEast, 29 relation to bacteria, 34 Yellow fever, 221 Z Zansz, Hans and Zacwarias, 18 ZENKER, 26 Ziehl-Neelson method of staining, 193 Zooglea, 41 Zootoxins, 116 Zymase, 113 Zymases, 113 Zymogen, 113 Zymophore group, 239, 247, 250 < a AN a “ 8 ~ SAN RS \ SS AN . ‘ AC ‘ ON ww x a . x WEN SS ~~ LENG ‘ SANS “ x SAS ‘ . ASS a SRNNS x“ oH ae : RAN S Cae —— \ a SASS SA “ a \ a a ae < ANE ‘ . AA ‘ ANS A ‘ . ss i ans AN Sh ‘ \ \ aS c \ SR . SSA SAN ~ Te \ a \Y AS Sy \ Be. 3 \ WS 5 ‘ . RS RRR SR S a _. AS uM MS \ | CO