MEDICAL SCHOOL LIIB1RAIKV Gift of Glanvilie Y. Rusk, M.D. A TEXT-BOOK OF ACTERIOLOGY BY GEORGE M. STERNBERG, M.D., LL.D. SURGEON- GENERAL U.S. ARMY EX-PRESIDENT AMERICAN PUBLIC HEALTH ASSOCIATION; HONORARY MEMBER OP THE EPIDEMIOLOGICAL SOCIETY OP LONDON, OP THE ROYAL ACADEMY OP MEDICINE OP ROME, OP THE ACADEMY OP MEDICINE OF RIO DE JANEIRO, OP THE SOCIETE FRANCAISE D'HYGIENE, ETC., ETC. ILLUSTRATED BY HELIOTYPE AND CHROMO-LITHOGRAPHIC PLATES AND TWO HUNDRED ENGRAVINGS. NEW YORK WILLIAM WOOD AND COMPANY 1896 COPYRIGHT BY .1.1 AM WOOD & COMPANY, 189C. PREFACE. THE writer's Manual of Bacteriology, published in 1892, has been very favorably received both in this country and abroad, but its use- fulness has no doubt been to some extent restricted by the size and expense of the volume. The following is an extract from the preface of the Manual : " A Manual of Bacteriology, therefore, which fairly represents the present state of knowledge, will consist largely of a statement of facts established by experimental data, and cannot fail to be of value to physicians and to advanced students of bacteriology as a work of reference. The present volume is an attempt to supply such a man- ual, and at the same time a text-book of bacteriology for students and guide for laboratory work. That portion of the book which is printed in large type will, it is hoped, be found to give an accurate and sufficiently extended account of the most important pathogenic bacteria, and of bacteriological technology, to serve as a text-book for medical students and others interested in this department of science. The descriptions of non-pathogenic bacteria, and of the less important or imperfectly described species of pathogenic bacteria, are given in smaller type." For the benefit of students of medicine and others who do not care especially for the detailed descriptions of non-pathogenic bacteria and the extensive bibliography contained in the Manual, this TEXT-BOOK OF BACTERIOLOGY is now published. It comprises that portion of the Manual above referred to as printed in large type, revised to include all important additions to our knowledge of the pathogenic bacteria since the original date of publication. TABLE OF COISTTEOTS. PART FIRST. CLASSIFICATION, MORPHOLOGY, AND GENERAL BACTERIOLOGICAL TECHNOLOGY. PAGE I. HISTORICAL, 3 II. CLASSIFICATION, 10 III. MORPHOLOGY, 20 IV. STAINING METHODS, 25 V. CULTURE MEDIA 37 VI. STERILIZATION OF CULTURE MEDIA, 50 VII. CULTURES IN LIQUID MEDIA, 60 VIII. CULTURES IN SOLID MEDIA, 67 IX. CULTIVATION OF ANAEROBIC BACTERIA, 78 X. INCUBATING OVENS AND THERMO-REGULATORS, . .86 XI. EXPERIMENTS UPON ANIMALS, 94 XII. PHOTOGRAPHING BACTERIA, 101 PART SECOND. GENERAL BIOLOGICAL CHARACTERS. I. STRUCTURE, MOTIONS, REPRODUCTION, 115 II. CONDITIONS OF GROWTH, 122 III. MODIFICATIONS OF BIOLOGICAL CHARACTERS, . . . .126 IV. PRODUCTS OF VITAL ACTIVITY, 130 V. PTOMAINES AND Tox ALBUMINS, .... . . 143 VI. INFLUENCE OF PHYSICAL AGENTS, .149 VII. ANTISEPTICS AND DISINFECTANTS (GENERAL ACCOUNT OF THE ACTION OF), 160 VIII. ACTION OF GASES AND OF THE HALOID ELEMENTS UPON BAC- TERIA, .168 IX. ACTION OF ACIDS AND ALKALIES, .... .176 X. ACTION OF VARIOUS SALTS, XI. ACTION OF COAL-TAR PRODUCTS, ESSENTIAL OILS, ETC., . 193 XII. ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS, . 204 XIII. PRACTICAL DIRECTIONS FOR DISINFECTION, . . . .208 ri TABLE OP CONTENTS. PART THIRD. PATHOGENIC BACTERIA. PAGE I. MODES OF ACTION, 221 1 1 . CHANNELS OP INFECTION 229 III. SUSCEPTIBILITY AND IMMUNITY, 233 IV. I'VOOENIC BACTERIA 275 V. I:\.-TKIM \ IN CROUPOUS PNEUMONIA, 300 VI. PATHOGENIC MIOROCOCCI NOT DESCRIBED IN SECTIONS IV. AND V., 322 VII. THE BACILLUS OF ANTHRAX, 339 VMI. THE BACILLUS OF TYPHOID FEVER, .... .349 IX r.v< iKitiA IN DIPHTHERIA, 371 X. BACTERIA IN INFLUENZA 387 X I . BACILLI IN CHRONIC INFECTIOUS DISEASES 392 XII. BACILLI WHICH PRODUCE SEPTICAEMIA IN SUSCEPTIBLE ANI- MALS 428 X 1 1 [. PATHOGENIC AEROBIC BACILLI NOT DESCRIBED IN PREVIOUS SECTIONS, 461 XIV. PATHOGENIC ANAEROBIC BACILLI, . ... 531 XV. PATHOGENIC SPIRILLA, .... .... 549 XVI. BACTERIA IN INFECTIOUS DISEASES, .... 571 PART FOURTH. SAPROPHYTES. I. BACTERIA IN THE AIR 623 1 1 BACTERIA IN WATER, 636 III i'.ACTERIA IN THE SOIL, 652 IV. BACTERIA OF THE SURFACE OF THE BODY AND OF EXPOSED Mucous MEMBRANES 658 v BACTERIA OF THE STOMACH AND INTESTINES, .... 668 vi I:\.TKIM.V OF CADAVERS AND OF PUTREFYING MATERIAL FROM VARIOUS SOURCES 674 vil. BACTERIA IN ARTICLES OF FOOD, 677 INDEX, .683 LIST OF ILLUSTRATIONS. PAGE 1. Staphylococci, . . . . . . . . . 21 2. Zoogloea 21 3. Ascococcus, 21 4. Streptococci, 21 5 Tetrads, . . 22 6. Packets— sarcina, . . . 22 7. Bacilli, 23 8. Involution forms, ........... 23 9. Chains formed by binary division, ........ 23 10. Spirilla, 24 11. Cladothrix 24 12. Flagella, 24 13. Platinum wire in glass handle, ........ 25 14. Flask for drawing off blood serum, . 38 15. Method of forcing blood serum into test tube, 38 16. Suction pipette, 38 17. Hot- water funnel, 42 18. Karlinski's agar filter, . . . . 44 19. Unna's agar filter, 45 20. Glass dishes for preserving potato cultures, 48 21. Test tube for sterilizing potato, . 48 22. Shape of potato for test-tube culture, 48 23. Hot air oven, . 52 24. Koch's steam sterilizer, . 53 25. Koch's steam sterilizer, 53 26. Arnold's steam sterilizer, ... 54 27. Miincke's steam sterilizer, ......... 54 28. Koch's apparatus for coagulating blood serum, ..... 55 29. Miincke s steam sterilizer and coagulator, 56 30. Pasteur Chamberlain filter, 57 31. Pasteur- Chamberlain filter without metal case 58 32. Modified Pasteur-Chamberlain filter, 59 33. Erlenmeyer flask 6* 34. Flask used by Pasteur, 61 35. Platinum wire loop, 62 36. Platinum needle, 63 37. Sternberg's bulb, • 38. Fermentation tube, 66 39. Method of making stick culture, 6? iji LIST OF ILLUSTRATIONS. PAGE o. 68 40. Sloping surface of culture medium, 40A. Growth of non-liquefying bacteria in gelatin stick cultures, . 41. Growth of same along line of puncture, ... ^ 42. Growth of liquefying bacilli, ... ^ 43. Colonies of bacteria, 73 44. Apparatus for gelatin plates, . . ^ Ksmarch roll tube 46. (See Fig. 15). 7S 47 Mode of development of a facultative anaerobic bacillus, . 48. Mode of development of strict anaerobic in long stick culture, . . 78 49. Exhausted-air flask for liquid media, 50. Method of displacing air with hydrogen, ™ 51. Salomonson's tube 52. Frftnkel's method of cultivation 58. Sternberg's method of cultivation 54. Sternberg's method of cultivation 55. Buchner's method of cultivation, 56. Hydrogen generator, Hydrogen apparatus for plate cultures . 58. Incubating oven 59. Thermo-regulator for gas 60. Moitessier's pressure regulator, r,i. Mica screen for flame, 62. Koch's device for cutting off flame 63. Heichert's thermo regulator, 64. Bohr's thermo-regulator 65. MQncke's thermo- regulator 66. Sternberg's thermo-regulator 67. Gas valve for the same 68. D'Arsonval's incubating apparatus, 69. Roux's incubating oven and thermo-regulator, 70. Koux's thermo-regulator 71. Koch's syringe 7J. Sternberg's glass syringe 78. Pringle's photomicrographic apparatus, . 74. Sternberg's photomicrographic apparatus for gas, 75. Spores of bacilli 76. Method of germination of spores, 77. Apparatus for cultivating anaerobic bacilli l^-> Bacillus of mouse septicaemia in leucocytes from blood of mouse, . ~~><> 79. Staphylococcus pyogenes aureus, 80. Gelatin culture of Staphylococcus pyogenes aureus, . . . 27 :» 81. Vertical section through a subcutaneous abscess caused by inoculation with staphylococd in the rabbit 281 88. Pus containing streptococci, 2*7 88. Streptococcus of erysipelas in nutrient gelatin, 288 84. Section from margin of un erysipelatous inflammation, showing strepto- cocci in lymph spaces, 289 85. Gouococci 2J»."> 86. Gonococcus in gonorrhoea! pus 2JW 87. Gonorrhoea 1 conjunctivitis, second day of sickness, . . . 298 LIST OF ILLUSTRATIONS. ix FIG. PAGE 88. Friedlander's bacillus, 308 89. Friedlander's bacillus; stick culture in gelatin, 309 90. Micrococcus pneumonise crouposae, 313 91. Micrococcus pneumonias crouposae, 313 92. Micrococcus pneumoniae crouposae, 313 93. Micrococcus pneumoniae crouposae, showing capsule, . . . 314 94. Single colony of Micrococcus pneumoniae crouposae upon agar plate, . 315 95. Micrococcus pneumoniae crouposae in blood of rabbit inoculated with sputum, 319 96. Microcoecus of progressive tissue necrosis in mice, .... 328 97. Micrococcus of pyaemia in rabbits, .* 334 98. Micrococcus tetragenus, 326 99. Streptococcus of mastitis in cows, 333 100. Bacillus anthracis, showing development of long threads in convoluted bundles, 340 101. Bacillus anthracis, showing formation of spores, 341 102. Culture of Bacillus anthracis in nutrient gelatin, . . . . . 342 103. Colonies of Bacillus anthracis upon gelatin plates, .... 343 104. Bacillus anthracis in liver of mouse 346 105. Bacillus anthracis in kidney of rabbit, 347 106. Bacillus of typhoid fever; colonies in stained sections of spleen, . . 353 107. Bacillus of typhoid fever; colonies in stained sections of spleen, . . 352 108. Bacillus typhi abdominalis, 358 109. Bacillus typhi abdominalis, 358 110. Bacillus typhi abdominalis, showing flagella, 359 111. Single colony of Bacillus typhi abdominalis in nutrient gelatin, . . 360 112. Bacillus typhi abdomiualis; stick culture in nutrient gelatin, . . 360 113. Section through wall of intestine, showing invasion by typhoid bacilli, 364 114. Bacillus diphtheriae, 375 115. Colonies of Bacillus diphtheriae in nutrient agar, ..... 376 116. Bacillus tuberculosis, .......... 394 117. Bacillus tuberculosis in sputum, ........ 395 lib. Section through a tuberculous nodule in the lung of a cow, showing two giant cells, 397 119. Tubercle bacilli from surface of culture upon blood serum, . . . 400 120. Culture of tubercle bacillus upon glycerin-agar, 402 121. Limited epithelioid- celled tubercle of the iris, 408 122. Section of a recent lepra nodule of the skin, 414 123. Bacillus mallei, 417 124. Section of a glanders nodule, ......... 417 125. Section through a glanders nodule in liver of field mouse, . . . 420 126. Migrating cell containing syphilis bacilli, ...... 423 127. Pus from hard chancre containing syphilis bacilli, 423 128. Bacillus of rhinoscleroma in lymphatic vessels of the superficial part of tumor, ............. 425 129. Bacillus septicaemiae haemorrhagicae in blood of a rabbit, . . . 439 130. Bacillus septicaemiae haemorrhagicae; stick culture in nutrient gelatin, . 431 131. Bacillus of Schweineseuche, 431 132. Colonies of bacillus of swine plague, ....... 431 133. Bacillus of Schweineseuche in blood of rabbit, 433 134. Bacillus of hog cholera, ... ... . 435 OF ILLUSTRATION-. PAGE 185. Bacillus of mouse septictemia in leucocytes from blood of mouse, 186. Bacillus of rouget, • Ilacillus of mouse septicaemia ; culture in nutrient gelatin, . 188. Bacillus of mouse septicaemia; single colony in nutrient gelatin, . m Section of diaphragm of a mouse dead from mouse septicaemia, . 140. Bacillus cavicida Havaniensis, 141. Bacillus crassus sputigenus ™ Proteushominiscapsulatus, *5* 148. Bacillus capsulatus, 144. Bacillus hydrophilus fuscus, ... 145. Culture of Bacillus hydrophilus fuscus in nutrient gelatin, . 146. Bacillus coli communis, 1 5:u illus coli communis in nutrient gelatin, 46* 148. A port ion of the growth shown in Fig. 147, 465 149. Bacillus lactis aSrogenes, 472 150. Bacillus acidiformuns 4^4 151. Culture of Bacillus acidiformans in nutrient gelatin, .... 4^4 152. Bacillus cuniculicida Havaniensis 4^5 158. Colonies of Bacillus cuniculicida Havaniensis, 476 154. Colonies of Bacillus cuniculicida Havaniensis 476 155. Bacillus pyocyanus, 479 156. Proteus vulgaris 485 157. " Swarming islands" from a culture of Proteus mirabilis, . . . 490 158. Spiral zooglcea from a culture of Proteus mirabilis, .... 490 159. Bacillus gradlis cadaveris 516 160. Colonies of B;u ill us gracilis cadaveris, . 516 161. Tetanus bacillus 532 162. Tetanus bacillus, 532 168. Culture of Bacillus tetani in nutrient gelatin, 533 164. Bacillus ocdematis maligni, 538 165. Bacillus cedematis maligni, 538 166. Cultures of. Bacillus cedematis maligni in nutrient gelatin, . . . 539 Hi?. Bacillus cadaveris, 541 168. Bacillus cadaveris 541 169. Bacillus of symptomatic anthrax, 542 170. Bacillus of symptomatic anthrax, ........ 542 171. Culture of bacillus of symptomatic anthrax, 543 172. Spirillum Obermeieri, 550 178. Spirillum Obermeieri, 550 174. Spirillum choleras Asiatic®, 552 175. Spirillum cholerae Asiaticae, :>.YJ 176. Colonies of Spirillum cholera Asiaticae 553 177. Spirillum cholerae Asiaticae, 553 178. Cultures of Spirillum cholerae Asiatic;r in nutrient gelatin, . . . 554 179. Spirillum cholera; Asiaticae, 554 180. Colonies in nutrient gelatin of Spirillum cholerae Asiaticae, Spirillum tyrogenum, and Spirillum of Finkler and Prior, .... 555 181. Section through mucous membrane of intestine from cholera cadaver, 559 188. Spirillum of Finkl.-r -md Prior 562 188. Colonies of Spirillum of Finkler and Prior, 5r,-j 184. Spirillum of Finkler and Prior; culture in gelatin, .... 562 LIST OF ILLUSTRATIONS. xi FIG- PAGE 185. Spirillum tyrogenum, , 553 186. Colonies of Spirillum tyrogenum, 563 187. Spirillum Metschnikovi, 564 188. Penicillum glaucum, .......... 624 189. Miquel's aeroscope, 625 190. Hesse's aeroscope, 627 191. Miquel's flask, 629 192. Straus and Wiirtz's soluble filter, 629 193. Petri's sand filter, 630 194. Sugar filter, 631 195. Sedgwick and Tucker's apparatus, 631 196. Sternberg's vacuum tube, 637 197. Lepsius' apparatus for collecting water at various depths, . . . 638 198. Koch's plate method, 639 199. Smear preparation from liver of yellow-fever cadaver, .... 675 200. Bacillus cadaveris grandis, 675 PART FIRST. CLASSIFICATION, MORPHOLOGY, AND GENERAL BACTERIOLOGICAL TECHNOLOGY. I. HISTORICAL. II. CLASSIFICATION. III. MORPHOLOGY. IV. STAINING METHODS. V. CULTURE MEDIA. VI. STERILIZATION OP CULTURE MEDIA. VII. CULTURES IN LIQUID MEDIA. VIII. CULTURES IN SOLID MEDIA. IX. CULTIVATION OF ANAEROBIC BAC- TERIA. X. INCUBATING OVENS AND THERMO REGU- LATORS. XI. EXPERIMENTS UPON ANIMALS. XII. PHOTOGRAPHING BACTERIA. PAET FTEST. I. HISTORICAL. IT is probable that Leeuwenhoeck, " the father of microscopy/' observed some of the larger species of bacteria in faeces, putrid in- fusions, etc., which he examined with his magnifying glasses (1675), but it was nearly a century later before an attempt was made to de- fine the characters of these minute organisms and to classify them (O. F. Miffler, 1773). In the absence of any reliable methods for obtaining pure cultures, it is not surprising that the earlier botanists, in their efforts to classify microorganisms, fell into serious errors, one of which was to include under the name of infusoria various motile bacteria. These are now generally recognized as vegetable organisms, while the Infusoria are unicellular animal organisms. Ehrenberg (1838), under the general name of Vibrioniens, de- scribes four genera of filamentous bacteria, as follows : 1. Bacterium — filaments linear and inflexible ; three species. 2. Vibrio — filaments linear, snake-like, flexible ; nine species. 3. Spirillum — filaments spiral, inflexible ; three species. 4. Spirochcete — filaments spiral, flexible ; one species. These vibrioniens were described by Ehrenberg as " filiform ani- mals, distinctly or apparently polygastric, naked, without external organs, with the body uniform and united in chains or in filiform series as a result of incomplete division/' Dujardin (1841) also placed the vibrioniens of Ehrenberg among the infusoria, describing them as "filiform animals, extremely slen- der, without appreciable organization, and without visible locomotive organs." Charles Robin (1853) suggested the relationship of Ehrenberg's vibrioniens with the genus Leptothrix, which belongs to the algae ; and Davaine (1859) insisted that the vibrioniens are vegetable organ- HISTORICAL. nearly allied to the algae. His classification will be found in the " Dictionnaire Encyclop. des Sciences Medicales," art. " Bac- teriej* " (1868). This view is also sustained by the German botanist Cohn and is now generally accepted. Spallan/ani, in 177»>, endeavored to show by experiment that the generally received theory of the spontaneous generation of micro- organisms in organic liquids was not true. This he did by boiling putrescible liquids in carefully sealed flasks. The experiment was n <>t always successful, but in a certain number of instances the liquids were sterilized and remained unchanged for an indefinite Period. The objection was raised to these experiments that the oxy- gen of the air was excluded by hermetically sealing the flasks, and it was claimed, in accordance with the views of Gay-Lussac, that free admission of this gas was essential for the development of fer- mentation. This objection was met by Franz Schulze (1836), who admitted air to boiled putrescible liquids by drawing it through strong sulphuric acid, in which suspended microorganisms were destroyed. He thus demonstrated that boiled solutions, which, when exposed to the air without any precautions, quickly fell into putrefaction, remained un- changed when freely supplied with air which had been passed through an agent capable of quickly destroying all living organisms con- tain. M! in it. Schwann (1839) demonstrated the same fact by another method. Air was freely admitted to his boiled liquids through a tube which -> heated to a point which insured the destruction of suspended microorganisms. The same author is entitled to the credit of hav- ing first clearly stated the essential relation of the yeast plant — lioromyces cereuisice — to the process of fermentation in saccha- rine liquids, which results in the formation of alcohol and carbonic acid. Helmholtz, in 1843, repeated the experiments of Schwann with calcined air, and arrived at similar results — i.e., he found that the free admission of calcined air to boiled organic infusions did not pro- t IHTS with pure cultures of the bacillus, which were shown to have the same pathogenic effects as had been obtained in inoculation ex- periments with the blood of an infected animal. The next demonstration of the causal relation of a parasitic mi- 'Toorpmirtin t<> an infectious malady was made by Pasteur, who de- v..t,.,l several years to the study of an infectious disease of silkworms threatened to destroy the silk industry of France— pebrine. HISTORICAL. 7 In 1873 Obermeier, a German physician, announced the discov- ery, in the blood of patients suffering from relapsing fever, of a mi- nute, spiral, actively motile microorganism — the Spirochcete Ober- meieri — which is now generally recognized as the specific infectious agent in this disease. The very important work of Koch upon traumatic infectious diseases was published in 1878. In 1879 Hansen reported the discovery of bacilli in the cells of leprous tubercles, and subsequent researches have shown that this bacillus is constantly associated with leprosy and presumably bears an etiological relation to the disease. In the same year (1879) Neisser discovered the " gonococcus " in gonorrhoeal pus. The bacillus of typhoid fever was first observed by Eberth, and independently by Koch, in 1880, but it was not until 1884 that Gaff- ky's important researches relating to this bacillus were published. In 1880 Pasteur published his memoir upon fowl cholera, and the same year appeared several important communications from this pioneer in bacteriological research upon the "attenuation" of the virus of anthrax and of fowl cholera and upon protective inocula- tions in these diseases. In 1880 the present writer discovered a pathogenic micrococcus, which he subsequently named Micrococcus Pasteuri, and which is now generally recognized as the usual agent in the production of acute croupous pneumonia — commonly spoken of as the " diplococ- cus pneumoniae," but described in the present volume under the name of Micrococcus pneumonice crouposce. In 1881 several important papers by Koch and his colleagues ap- peared in the first volume of the " Mittheilungen " published by the Imperial Board of Health of Germany. The following year (1882) Koch published his discovery of the tubercle bacillus. The same year Pasteur published his researches upon the disease of swine, known in France as rouget. The same investigator (Pasteur) also published in 1882 his first communication upon the subject of rabies. Another important discovery was made in 1882 by the German physicians Loffler and Schiitz, viz., that of the bacillus of glan- ders. Koch published his discovery of the cholera spirillum — " comma bacillus "—in 1884. The same year (1884) Loffler discovered the diphtheria bacillus. Another important publication during the same year was that of Rosenbach, who, by the application of Koch's methods, fixed defi- HISTORICAL. nit<-ly the characters of the various microorganisms found in pus 1 1 « .ni acute abscesses, etc. The tetanus bacillus was discovered in 1884 by Nicolaier, a stu- dent in the laboratory of Prof. Fliigge, of Gottingen. That this bacillus is the cause of tetanus in man has been demonstrated by the subsequent researches of numerous investigators. For an exact knowl- edge of its biological characters we are especially indebted to Kitasato. So far as human pathology is concerned, no important pathogenic microorganism was discovered after the year 1884 until the year 1892. After numerous unsuccessful researches by competent bacteriologists, a bacillus was discovered by Pfeiffer, of Berlin, and independently by Canon, which is believed to be the specific cause of influenza. In 1894 the distinguished Japanese bacteriologist, Kitasato, dur- ing a visit to China made for the purpose, discovered the bacillus of the bubonic plague of the Orient. Recent experimental evidence appears to justify the conclusion that infectious pleuro-pneumonia of cattle is due to a bacillus dis- covered by Arloing— his Pneumobacillus liquefaciens bovis. Finally we may refer to the recent discovery of the antitoxins of diphtheria and of tetanus as one of the most important events in the history of bacteriology and of scientific medicine. The name of Behr- ing has the first place in connection with this discovery. Having briefly passed in review some of the principal events in the progress of our knowledge in this department of scientific investi- gation, it will be of interest to students to know something more of the literature of bacteriology. Important papers have appeared in medical and scientific journals in all countries, and research work of value has been done by enthusiastic investigators of nearly every nation. The brilliant pioneer work done by Pasteur and by Koch has attracted to them many pupils and has made France and Germany the leading countries in this line of investigation. The very great advantages of Koch's methods of research, introduced at the com- mencement of the last decade, have attracted many students from various parts of the world to Berlin, and to other cities of Germany where instruction was to be obtained from some of Koch's earlier pupils. But to-day bacteriological laboratories have been established in all parts of the world, and it is no longer necessary to go to Ger- many to obtain such instruction. The literature of the subject is, however, largely in the German and French languages. We can only refer here to such periodicals as are principally devoted to bac- t.-riological research work. The Zeitxchrift ///> Ifi/giene has been published since 1886, and contains numerous valuable papers, contributed for the most part by the pupils of Koch and of Fliigge, who are the editors of the journal. HISTORICAL. 9 The Annales de VInstitut Pasteur is a monthly journal which has been published since 1888. It is edited by Duclaux, and contains many important papers and reviews, as well as the statistics of the Pasteur Institute relating to preventive inoculations against hydro- phobia. The Annales de Micrography is a monthly journal, published in Paris. The principal editor is Miquel. The Centralblatt fur Bakteriologie und Parasitenkunde is a weekly journal which has been published by Gustav Fischer, of Jena, since 1887. The editors are Uhlworm, of Cassel; Loffler, at present professor at Greifswald; and Leuckart, professor at Leipzig. The Journal of Pathology and Bacteriology is published monthly in Edinburgh and London. It dates from 1892. A most important work for students of bacteriology is the Jahres- bericht of Baumgarten, which has been published since 1885 by Harald Bruhn, Braunschweig, Germany. This gives a brief abstract of nearly every paper of importance relating to the subject which has been published during the year. II. CLASSIFICATION. THE earlier naturalists— Ehrenberg (1838), Dujardin (1841)— placed the bacteria among the infusoria; but they are now recog- nized as vegetable microorganisms, differing essentially from the infusoria, which are unicellular animal organisms. One of the prin- cipal points in differentiating animal from vegetable organisms among the lowest orders of living things is the fact that animal organisms receive food particles into the interior of the body, assimi- lating the nutritious portion and subsequently extruding the non- nutritious residue ; vegetable organisms, on the other hand, are nourished through the cell wall which encloses their protoplasm, by organic or inorganic substances held in solution. Ehrenberg (1838), under the name of vibrioniens, established four gen- era, as follows: 1. Bacterium — filaments linear and inflexible. 2. Vibrio — filaments linear, snake-like, flexible. 3. Spirillum — filaments spiral, inflexible. 4. Spirochcete — filaments spiral, flexible. Duiardin (1841) united the two genera Spirillum and Spirochcete of Ehrenberg, and added to the description of the generic characters as fol- lows: 1. Bacterium — filaments rigid, with a vacillating movement. 2. Vibrio — filaments flexible, with an undulatory movement. 3. Spirillum — filaments spiral, movement rotatorv. It will be seen that this classification leaves no place for the motionless bacilli, such as the anthrax bacillus and many others, and does not include the spherical bacteria, now called micrococci. " The classification of Davaine (18t>8) provides for the motionless, fila- m< ntous bacteria, but does not include the micrococci. This author first insisted that the vibrioniens of Ehrenberg are truly vegetable organisms, allied to the algae. He makes four genera, as follows: Filaments straight or bent, ( Moving spontane- I Rigid Bacterium. but not in a spiral, ously, \ Flexible Vibrio. ( Motionless, . Bacteridium. Filaments spiral, Spirillum. Following Davaine, the French bacteriologists frequently speak of the motionless anthrax bacillus as la bacttridie. llotVinaii in 1869 included in his classification the spherical bacteria, and pointed out the fact that motility could not be taken as a generic char- acter, us it was not constant in the same species and depended to some ex- tent upon temperature conditions, etc. CLASSIFICATION. H Having determined that the bacteria are truly vegetable organ- isms, the attention of botanists has been given to the question as to what class of vegetable organisms they are most nearly related to. There are decided differences of opinion in this regard. While Da- vaine, Rabenhorst, and Cohn insist upon their affinities with the algae, Robin, Nageli, and others consider them fungi. One of the principal characters which distinguish the algae from the fungi is the presence of chlorophyll in the former and its absence in the latter. Now, the bacteria are destitute of chlorophyll, and in this regard resemble the fungi; yet in others their affinities with the Palmellacece and Oscillatoriacece are unmistakable. It is not necessary, how- ever, that we should consider them as belonging to either of these classes of the vegetable kingdom. By considering theni a distinct class of unicellular vegetable organisms, under the general name of bacteria, we may avoid the difficulties into which the botanists have fallen. We must refer briefly, however, to the classification proposed by some of the leading German botanists. Nageli, placing the bacteria among the lower fungi, which give rise to the decomposition of organic substances, divides these into three groups : 1. The Mucorini, or mould fungi. 2. The Saccharomycetes, or budding fungi, which produce alcoholic fer- mentation in saccharine liquids. 3. The Schizomycetes, or fission fungi, which produce putrefactive pro- cesses, etc. Cohn, under the name of Schizophytes, has grouped these low vegetable organisms, whether provided or not with chlorophyll, into two tribes hav- ing the following characters : 1. GL.EOGENES— cells free or united into glairy families by an intercel- lular substance. 2. NEMATOGENES— cells disposed in filaments. In the first tribe he has placed the genera Micrococcus (Hallier), Bacte- rium (Dujardin), Merismopedia (Meyer), Sarcina (Goodsir), and Ascococcus (Billroth), with various genera of unicellular algae containing chlorophyll. In the second tribe we have the genera Bacillus (Cohn), Leptothrix (Kg.), Vibrio (Ehr.), Spirillum (Ehr.), Spirochcete (Ehr.), Streptococcus (Billr.), Cladothrix (Cohn), and Streptothrix (Cohn), associated with gen- era of green filamentous algae. The German botanist Sachs unites the fungi and the algae into a single group, the Thallophytes, in which he establishes two parallel series, one in- cluding those containing chlorophyll, and the other without, as follows: THALLOPHYTES. Forms with chlorophyll. Forms without chlorophyll. Class! I. — Protophytes. A. Cyanophycese (Oscillatoria- A. Schizomycetes (Bacteria), ceae, etc.). B. Palmellacese. B. Saccharomycetes. 12 CLASSIFICATION. Zopf. who insists upon the polymorphism of these low organisms, divides tho h;trt«-n.-» into four groups: Genera. Streptococcus, 1. O»rcocE,«.— Up to the pre- | Merismopedia, sent time, only known in the form of \ Sarcina, cocci. Micrococcus, Ascococcus. 2. BACTERIACE^E.— Have for the part spherical, rod-like, and filamentous forms ; the first (cocci) Bacterium, Spirillum, Vibrio, may be wanting ; the last are not j Leuconostoc, different at the two extremities; fila- Bacillus, ments straight or spiral. Clostridium. :\. LEPTOTRICHE.E. — Spherical, ] Crenothrix rod-shaped, and filamentous forms; Beaaiatoa ' the last show a difference between the Phragmidiothrix, two extremities ; filaments straight | Levtothrix < u-s i >i ral ; spore formation not known. J 4. CLADOTRICHE^E. — Spherical, } rod-shaped, filamentous, and spiral | forms ; the filamentous form pre- |- Cladothrix. sents pseudo-branches ; spore forma- | tion not known. The main objection to this classification is that it assumes a pleomorph- ism for the bacteria of the second group — Bact^riaceae — which has only been established for a few species, and which appears not to be general among the rod shaped and spiral bacteria. De I&arv diviaes the bacteria into two principal groups, one including those which form endospores, and the other those which are reproduced by arthrospores. But our Knowledge is yet too imperfect to make this classifi- cation of value, and the same may be said of Hueppe's recent attempt at classification, in which the mode of reproduction is a principal feature. The classification of Baumgarten (1890) appears to us to have more practical value, and, with slight modifications, we shall adopt it in the present volume. This author divides the bacteria into two principal groups, as follows : GROUP I. Species relatively monomorphous. GROUP II. Species pleomorphous. I lie first group includes the micrococci, the bacilli, and the ' ilia; the second group the spirulina of Hueppe, leptotrichece '/"pf), and cl(ul<>trirln>cocci, numerous well-established species which the most expert microscopist could not differentiate by the use of the microscope alone ; the same is true of the rod-shaped bacteria. The assump- tion often made by investigators who are not sufficiently impressed with this fact, that two microorganisms from different sources, or even from the same source, are the same because stained prepara- tions examined under the microscope look alike, has led to serious errors and to much confusion. As an example of what is meant we may refer to the pus organisms. Before the introduction of Koch's 44 plate method" micrococci had been observed in the pus of acute abscesses. Some of these were grouped in chains — streptococci— and some were single, or in pairs, or in groups of four ; but whether these were simply different modes of grouping in a single species, or whether the chain micrococci represented a distinct species, was not determined with certainty. That there were in fact four or more distinct species to be found in the pus of acute abscesses was not suspected until Rosenbach and Passet demonstrated that this is the case, and showed that not only is the streptococcus a distinct species, but that among the cocci not associated in chains there are three species which are to be distinguished from each other by their color when grown on the surface of a solid culture medium. One of these has a milk-white color, one is of a lemon-yellow color, while the third i golden-yellow. Those microorganisms which form pigment are called chromo- genes, or chromogenic ; those which produce fermentations are spoken of as zymogenes, or zymogenic ; those which give rise to dis- ease processes in man or the lower animals are denominated patho- genes, or pathogenic. We cannot, however, classify bacteria under the three headings chromogenes, zymogenes, and pathogenes, for some of the chromogenic species are also pathogenic, as are some <>t the zymogenes. These characters must therefore be considered separately as regards each species, and in studying its life history and • I languishing characters we determine whether it is chromogenic or non-chrontogenic ; whether it produces special fermentations ; and whether it is or is not pathogenic when inoculated into the 1 "Wl ir Animals. In making the distinction between pathogenic • M.I non-pathogenic microorganisms we must remember that a certain species may be pathogenic for one animal and not for an- other. Thus the anthrax bacillus, which is fatal to cattle, sheep, rabbits, guinea-pigs, and mice, does not kill white rats ; the bacillus of mouse septicaemia kills house mice, but field mice are fully im- mune from its pathogenic effects ; on the other hand, the bacillus of glanders is fatal to field mice but not to house mice. CLASSIFICATION. 15 Again, it must be remembered that pathogenic power also de- pends, to a greater or less extent, upon the dose injected into an animal as compared to its body weight. Some pathogenic organ- isms in very minute doses give rise to a fatal infectious malady ; others are only able to overcome the vital resisting power of the tissues and fluids of the body when introduced into the circulation, or into the subcutaneous tissue or abdominal cavity, in considerable amounts. Some pathogenic bacteria invade the blood ; others mul- tiply only in certain tissues of the body ; and others again multiply in the intestine and by the formation of poisonous products which are absorbed show their pathogenic power. Another classification of the bacteria relates to the environment favorable to their development. Thus we speak of saprophytic and parasitic bacteria, or of SAPROPHYTES and PARASITES. The saprophytes are such as exist independently of a living host, obtaining their supply of nutriment from dead animal or vegetable material and from water containing organic and inorganic matters in solution. The strict parasites, on the other hand, depend upon a living host, in the body of which they multiply, sometimes without injury to the animal upon which they depend for their existence, but frequently as harmful invaders giving rise to acute or chronic infec- tious diseases. Microorganisms which ordinarily lead a saprophy- tic existence, but which can also thrive within the body of a living animal, are called facultative parasites. Thus the leprosy bacillus, which is only found in leprous tissues, is a strict parasite ; while the typhoid bacillus, the cholera spirillum, etc. , are facultative parasites, inasmuch as they are capable of maintaining an independent exist- ence, for a time at least, external to the bodies of living animals. It seems probable that the pathogenic organisms which are only known to us to-day as strict parasites were, at some time in the past, saprophytes, which gradually became accustomed to a parasitic mode of existence, and, under the changed conditions of their envi- ronment, finally lost the power of living in association with other saprophytes exposed to variations of temperature, etc. The tubercle bacillus, for example, is known to us only as a parasite which has its habitat in the lungs, lymphatic glands, etc. , of man and of certain of the lower animals. But we are able to cultivate it in artificial media external to the body ; and it is in accord with modern views relating to the development of species to suppose that at some time in the past it was able to lead a saprophytic existence. Not to admit this forces us to the conclusion that, at some time subsequent to the appearance of man and the lower animals in which it is now found as a parasite, it was created with its present biological characters, which restrict it to a parasitic existence in the bodies of these ani- ],; CLASSIFICATION. mals. and that, consequent!}', the immense destruction of human life \vhidi has resulted from its parasitic invasion of successive genera- tions was designed when it was created. The opposite view is sup- ported by numerous facts which show that these low organisms, like those higher in the scale, are subject to modifications as a result of changed conditions of environment, and that such modifications, in the course of time, may become well-established specific characters. Again, the bacteria may be grouped into aerobic and anaerobic species. This is a very important distinction, which was first estab- lish. M! by Pasteur, who found that certain bacteria will only grow when freely supplied with oxygen, while others absolutely decline to grow in the presence of this gas. The latter, which are spoken of as .s///V/ (tuaerobics, maybe cultivated in a vacuum or in an atmo- sphere of hydrogen. Those species which grow either in the pre- sence of oxygen or when it is excluded are called facultative an- a&robics, Certain bacteria produce a peptonizing ferment which has the power of liquefying gelatin. This has led to the classification of those microorganisms of this class which grow in Koch's flesh-pep- tone-gelatin as liquefying and non-liquefying bacteria. Again, we speak of them as motile or non-motile. It is evident that these biological characters, although all-im- portant in the definition of species, cannot serve us in an attempt to establish natural genera ; for the lines are not sharply drawn between the saprophytes and the parasites, the aerobics and the anaerobics, etc. , inasmuch as we have facultative parasites and facultative an- aerobics which we cannot include in either class, and which yet do not form a distinct class by themselves. We therefore adhere to the morphological classification, although this is open to criticism. For example, among the rod-shaped organisms which we call bacilli and describe under the generic name Bacillus there are some which multiply by binary division only, while others form endogenous re- productive bodies known as spores. Certainly so important a differ- ence in the mode of reproduction should be sufficient to separate these rod-shaped organisms into two natural groups or genera. As heretofore stated, the German bacteriologist Hueppe has at- t' mpted a classification based upon the mode of reproduction, in which h<- makes two groups, or "tribes," one in which reproduction occurs by tl m formation of endogenous spores— " endospores "— the other in which it occurs by the formation of " urtliroworrx." ' The latter group includes all of those bacteria in which no other mode of multiplication is known than that by binary division, which is com- 111011 to ;l11 ln tin- present state of our knowledge this classification 1 An account of this mode of reproduction is given on page 19. CLASSIFICATION. 17 is scarcely to be considered of practical value, inasmuch as the ques- tion of spore formation is still undetermined for a large number of species. In the following table we shall give the characters of the dif- ferent genera which have been described by recent botanists and bacteriologists, arranged under the three headings, MICROCOCCI, BACILLI, SPIRILLA. Where we doubt the propriety of maintaining a distinct generic name upon the supposed distinguishing characters, the description will be printed in small type. MICROCOCCI. General Characters. — Spherical bacteria which are reproduced by binary division ; usually without spontaneous movements ; do not form endogenous spores. (According to some authors, certain cells, known as arthrospores, may be distinguished by their greater size and refractive power, and these are supposed to have greater resist- ance to desiccation than the ordinary cocci resulting from binary division, and to serve as reproductive bodies.) Some micrococci are not precisely round, but are somewhat oval in form ; and when in process of division the cocci, necessarily, are more or less elongated in one diameter before a complete separation into two spherical ele- ments has occurred. MiCROCOCCUS. — Division in one direction ; cocci single, in pairs, or accidentally associated in irregular groups ; sometimes held to- gether in irregular masses by a transparent, glutinous, intercellular substance. (Micrococci belonging to this genus are frequently de- scribed as " staphylococci," and Staphylococcus is used by Rosen- bach as a generic name for the pus cocci described by him, which are solitary or associated in irregular groups, as above described. ) Ascococcus. — Cocci associated in globular or lobulated, zoogloea masses by a rather firm intercellular substance. LEUCONOSTOC. — Cocci, solitary or in chains, surrounded by a thick, gelatinous envelope and forming zoogloea of cartilaginous consistence. STREPTOCOCCUS. — Division in one direction only ; cocci associ- ated in chains. Diplococcus. — Division in one direction only ; cocci associated in pairs. Association in pairs is common to all of the micrococci, inasmuch as they multiply by binary division. When such association has rather a per- manent character, it is customary to speak of the microorganism as a diplo- coccus, but we doubt the propriety of recognizing this mode of association as a generic character. MERISMOPEDIA. — Division in two directions, forming groups of four, which remain associated in a single plane — "tetrads." SARCINA. — Division in three directions, forming packets of eight jg CLASSIFICATION. or more elements, which remain associated in more or less regular cubical masses. BACILLI. General Characters.— Rod-shaped and filamentous (not spiral) bacteria in which there is no differentiation between the extremities of the rods ; reproduction by binary division in a direction trans- verse to the long axis of the rods, or by binary division and the for- mation of endogenous spores ; rigid or flexible ; motile or non-motile. BACILLUS. — Characters as given above. Bacterium.— This genus, established by Dujardin, is now generally abandoned, the species formerly included in it being transferred to the genus Bacillus. As defined by Cohn, the generic characters were : Cells cylindri- cal or elliptical, free or united in pairs during their division, rarely in fours, never in chains, sometimes in zooglcea (differing from the zooglcea of spherical bacteria by a more abundant and firmer intercelluar substance), having spontaneous movements, oscillatory and very active, especially in media rich in alimentary material and in presence of oxygen. Clostridium.— Rod-shaped bacteria which form large, endogenous, and usually oval spores ; these are centrally located, and during the stage of spore formation the rods become fusiform. SPIRILLA. General Characters. — Curved rods or spiral filaments ; rigid or flexible ; reproduction by binary division, or by binary division and the formation of endogenous spores (or by arthrospores ?) ; move- ments rotatory in the direction of the long axis of the filaments. SPIRILLUM. — Characters as above. Spirochcete.— Flexible, spiral filaments; movements rotatory. Vibrio. — Filaments flexible, straight or sinuous; movements sinuous. A considerable number of bacteria which are usually seen as short, curved rods, but which may grow out into long, spiral filaments, are described by so MM- authors under the generic name Vibrio, e.o., the so-called "comma bacillus" of Koch—" Spirillum cholerae Asiatic*' ; the spirillum of Finkler and Prior — " Vibrio proteus" ; the spirillum described by Gameleia — " Vibrio M' tschnikovi/'etc. These microorganisms have not the characters which distin^ruislK'd the genus Vibrio as established by Ehrenberg, and we prefer to follow Fliigge in describing them under the generic name Spirillum. The pathogenic bacteria now known belong to one or the other of the above-described genera, and the attention of bacteriologists has been given chiefly to the study of micrococci, bacilli, and spirilla. But the botanists place among the bacteria certain other forms which are found in water, and which, in a systematic account of this class of microorganisms, demand brief attention at least. These are in- cluded in Baumgarten's second group, which includes the pleomor- phous bacteria. Spun LIN A (Hueppe). — The vegetative cells are sometimes rod- shaiH.«d and sometimes spiral ; in suitable media they may grow out CLASSIFICATION. 19 into long, straight, wavy, or spiral filaments. These filaments may break up into cocci-like reproductive elements — " arthrospores. " LEPTOTRICHE^E (Zopf). — The vegetative cells present rod-shaped and spiral forms, and grow out into straight, wavy, or spiral fila- ments ; these may show a difference between the two extremities, of base and apex. Cocci-like reproductive bodies are formed by seg- mentation of the rod-shaped elements in these filaments. In some of the species the segments are enclosed in a common sheath. Sub- genera: LEPTOTHRIX, BEGGIATOA, CRENOTHRIX, PHRAGMIDIO- THRIX (for generic characters see page 12). CLADOTRICHE^E (Zopf). — The vegetative cells are rod-shaped or spiral, and grow out into straight or spiral filaments, which may present pseudo-ramifications. A single genus, CLADOTHRIX (see page 12). III. MORPHOLOGY. IN the present chapter we shall give a general account of iiu? morphology, modes of grouping, and dimensions of the bacteria. The standard of measurement used by bacteriologists is the micro- millimetre, or the one-thousandth part of a millimetre. This is represented by the Greek letter ^. One /* (micromillimetre) is equal to about one-twenty-five-thousandth of an English inch. The spherical bacteria, or micrococci, differ greatly in size, and also in the mode of grouping when, as a result of binary division, they remain associated one with another. The smallest may mea- sure no more than O.l/*, while some of the larger species are from one to two p in diameter. The enormous number of these minute organisms which may be contained in a small drop of a pure culture may be easily estimated in a rough way. Compare a single micro- coccus, for example, with a sphere having a diameter of one-twenty- fifth of an inch. If our micrococcus is one of the larger sort, having a diameter of one //, it would take a chain of one thousand to reach across the diameter of such a sphere, and its mass, as compared to the larger sphere, would be as 1 to 523,600,000. The number of cocci in a milligramme of a pure culture of Staphy- lococcus pyogenes aureus has been estimated by Bujwid, by count- ing, at 8,000,000,000. Not only do different species differ in dimensions, but consider- able differences in size may be recognized in the individual cocci in a pure culture of the same species. On the other hand, there are numerous species which so closely resemble each other in size and mode of association that they cannot be differentiated by a micro- scopic examination alone, and we must depend upon other characters, such an color, growth in various culture media, pathogenic power, etc., to decide the question of identity or non-identity. When in active growth the micrococci necessarily depart from a typical spherical form just before dividing, and under these circum- stanrrs may U'of a short or long oval. When division lias taken place, if the two members of a pair remain associated they are often mure or less flattened at the jHniit of contact (Fig. 1, a). MORPHOLOGY. 21 When in a culture the cocci are for the most part associated in pairs (Fig. 1, d ) , we speak of the organism as a diplococcus. The staphylococci are characterized by the fact that, for the most part, the individual cocci in a culture are solitary (Fig. 1, b). But, inasmuch as multiplication occurs by binary division, we also have pairs and occasionally a group of four — probably from the accidental apposition of two pairs (Fig. 1, c) ; or they may be associated in grape- 00 oo 80 FIG. 1. like bunches ; and after staining and mounting a preparation we find the cells associated in irregular groups. This results from the fact that they are surrounded by a glutinous material which causes them to adhere to each other (Fig. 1, e). A mass of cocci held together in Fio. 2. FIG. 3. FIG. 4. this way by a transparent, glutinous, intercellular substance is spoken of as a zooglcea (Fig. 2). In the genus Ascococcus the intercellar substance is quite firm and the zooglcea are in the form of spherical or irregularly lobulated masses surrounded by a resistant envelope of jelly-like material (Fig. 3). When, as a result of division in one direction only, the cocci 22 MORPHOLOGY. remain united in chains (Fig. 4, a), they are described as streptococci, and are sometimes spoken of as in chaplets or in torula chains. In such chains we frequently find the evidence of recent division of the cocci, as shown by the grouping of the elements of the chain into pairs (Fig. 4, b). When division occurs habitually in two directions, groups of four result, which are spoken of as tetrads. This is the distinguishing character of the genus Merismopedia. In these groups of four the individual cocci are often flattened at the points of contact, as in Fig. 5, 6. We also find pairs and groups of three in pure cultures of species belonging to this genus, as shown in Fig. 5, c. In these, transverse division has not yet occurred in one or in both elements of a pair. This association of micrococci in tetrads seems to be main- tained, in some species at least, by the fact that each group of four is enclosed in a jelly-like capsule. The extent of this capsule differs in the same species under different circumstances; as a rule, it is most apparent when a culture has been made in a liquid medium. Some of SB 88 OQ on rjf) CD **f 00 SB e Fio. 5. the diplococci have a similar capsule. The jelly-like substance does not stain well with the aniline colors and is seen as a transparent halo around the stained cocci. Some authors (Frankel and Pfeiffer) believe that this capsule is formed by the swelling up of the cell membrane as a result of the imbibition of water. When division occurs in three directions packets of eight or more elements are formed. This mode of association characterizes the genus Sarcina. The "packet form "is best seen in an un- stained preparation from a fresh culture, in which a little material >u>l»«'Mil.Ml in water is examined under a comparatively low-power objective — one-sixth (Fig. G). Among the bacilli there is room for a wider range of morphologi- cal characters. They differ not only in dimensions and in modes of grouping, but in form. The relation of the transverse to the longi- tudinal diameters affords a great variety of forms, varying from a short oval element to a slender rod or elongated filament. But it must he remembered that \ve may h;ive short rods and long filaments in a pure culture of the same bacillus— the typhoid bacillus, for MORPHOLOGY. 23 example. There are also considerable differences in the transverse diameter of bacilli belonging to the same species when cultivated in different media, or even in the same medium, although, as a rule, the transverse diameter is tolerably uniform in pure cultures. Again, the form of the extremities of the rods is to be observed (Fig. 7). This may be square, or the corners may be slightly rounded, or the extremities may be quite round or lance-oval, or the outlines of the rod may be spindle-shaped from the formation of OO c=x=> 00 CO a large central spore — "clostridium" — or one end may be dilated from the formation of a large terminal spore. In old cultures we frequently find irregular forms due to swellings and constrictions, which probably occur in bacilli which have but little vitality or are already dead. These are spoken of as involution forms (Fig. 8). The bacilli multiply by binary division in a direction transverse to the longitudinal axis, and, as a result of such binary division, long Fio. 9. chains in which the elements remain associated may be formed (Fig. 9) ; or the rods may be for the most part solitary or united in pairs. Like the micrococci, the bacilli are sometimes surrounded by a gelatinous envelope or capsule. They may also be united by a glutinous material into zoogloea masses. Bacilli which under certain conditions are seen as short rods may, under other circumstances, grow out into long filaments, and these may be associated in bundles or in tangled masses. The spirilla differ from the bacilli in the form of the rods and fila- 24 MORPHOLOGY. mente, which are curved or spiral. The shorter elements in a pure culture may be simply curved, as in a, Fig. 10, while the spiral form becomes apparent in those which are longer, and we may have one or several turns of the spiral (Fig. 10, b). The spiral form may be but slightly marked (Fig. 10, c), or the turns may be close and deep as in a corkscrew (Fig. 10, d). Again, the curved filaments may be short and rigid, or long and flexible (Fig. 10, e). In the genus Cladothrix, which is placed by botanists among the bacteria, the filaments appear to branch ; but this branching is only apparent, and there is no true dichotomous branching in this class of microorganisms. The false branching of Cladothrix <1n'hotoma, Cohn, is shown in Fig. 11. The fact that some of the larger species of bacilli and spirilla are provided with slender, whip- like appendages called flagella has been known for many years, and it has for some time been suspected that all of the motile organisms Fio. 10. FIG. 11. Fio. 12. of this class are provided with similar appendages and that these are organs of locomotion. Recently, by improvements in methods of staining, Loftier has demonstrated the presence of flagella in many species in which they had heretofore escaped observation. They are sometimes single, at the ends of the rods (Fig. 12, a); or there may be several at the extremity of a single rod (Fig. 12, &); again, they are seen in considerable numbers around the periphery of the rod (Fig. 12, c). The bacilli and spirilla sometimes contain in the interior of the cells granules of different kinds. These may appear like little oil tJ n >ps or they may be more opaque. In the genus Beggiatoa grains of sulphur are found in the interior of the cells. Again, we may find vacuoles in the protoplasm ; or, in stained preparations, deeply M;tin»- preparation sufficiently to coagulate the albu- m -n. in order that it may not be washed off in the subsequent stain- ing process. This is best done, in accordance with Koch's directions for the preparation of tuberculous sputum, by passing the cover glass, held in slender forceps, rather quickly through the flame of an alcohol lamp three times in succession. In this operation it must ba remembered that too much heat will destroy the preparation, while too little will fail to accomplish the object in view — coagu- lation of the albumen. In passing the cover glass through the thine the smeared side is to be held upward. The time required will be about three seconds for passing it three times as directed ; but this will vary according to the intensity of the flame, and some little experience is necessary in order to obtain the best results. The operation of " fixing," or coagulating the albumen, may also be effected by exposure in a dry-air oven, heated to 120° to 130° C., for a few minutes (two to ten minutes), as directed by Ehrlich. Bacteria simply suspended in distilled water adhere very well to the cover glass when treated as directed, but if they have been taken from a liquefied gelatin culture the film is very apt to be washed away during the staining process. This is best avoided by taking as little as possible of the gelatin medium and suspending the bacteria to be examined in a drop of water, which dilutes the gelatin and washes it away from the surface of the cells. Smear Preparations. — In various infectious diseases bacteria are found in the blood and tissues of the body, and their presence may be demonstrated by making what is called a smear preparation. A little drop of blood may be spread upon the thin glass cover, or it may be brought in contact with the freshly cut surface of one of the vascular organs, as the liver or spleen. It is especially desirable that the material used for such a preparation be small in amount and dis- tributed evenly in a very thin layer. In Germany it is the custom, in making smear preparations, to press the material between two glass covers, which are then separated by sliding them apart, thus leaving a thin layer upon each. This answers very well, but the writer pre- fers to spread the material by drawing across the face of the cover glass the end of a well-ground and polished glass slide. This method i- « 'specially useful for spreading blood in a uniform layer, in which the corpuscles are evenly distributed and retain their normal form. A very small drop of blood is placed near one edge of the cover glass, which is placed u]x>n a smooth surface ; the glass slide is held at a very acute angle and is gently drawn across the cover glass, without any pressure. Most bacteriologists make their preparations upon tlu» covor 'glass. STAINING METHODS. 27 as above described, but the writer has for a number of years made his mounts of bacteria upon the glass slide, and believes that this method has some advantages for every-day work. The thin glass covers required when a preparation is to be examined with an im- mersion objective of high power, are easily broken and often dropped from the fingers or forceps. When the material to be examined is spread and dried directly upon the glass slide, the operation is at- tended with less difficulty and fewer accidents and the results are quite as good. In this case the slide is held in the fingers during the various steps in the operation of distributing, drying, and staining, while the thin glass cover must be held in delicate forceps. Contact Preparations. — When a dry and clean cover glass is brought in contact with a colony or surface culture we may often obtain a very pretty preparation, showing the bacteria in a single layer, and preserving the arrangement, as regards growth, which characterizes the species. Similar preparations may sometimes be obtained from the surface of liquid cultures, when the bacteria grow upon the surface as a thin film. The cover glass is to be gently brought into contact with this surface growth, which adheres to it and is dried and stained by the usual methods. Stain ing of the dried film is quickly effected by using an aqueous solution of one of the aniline colors above mentioned. For general use the writer prefers a solution of f uchsin, on account of the prompt- ness of its staining action, and because, in preparations for permanent preservation, it is not as likely to fade as methylene blue or gentian violet. It is also a better color than blue or violet in case a photo- micrograph is to be made from the preparation. It is best to keep on hand saturated alcoholic solutions of the staining agents named, and to make an aqueous solution whenever required by the addition of a few drops to a little water in a watch glass or test tube ; for the aqueous solutions do not keep well on ac- count of the precipitation of the dye as a fine powder, which ren- ders the solution opaque. The addition of ten per cent of alcohol to the aqueous solution will, however, prevent this precipitation ; but, as a rule, freshly prepared solutions are the best. These should be filtered before use. We may place a few drops of the filtered solution upon the dried film on the slide or cover glass, or the thin cover may be floated upon a little of the solution in a watch glass. In some cases it is best to use heat to expedite the staining, and this may be done by holding the slide or the watch glass over the flame of an alcohol lamp until steam commences to be given off. If the heating is carried too far the preparation is likely to be spoiled by the precipitation of the staining agent. As a rule, heating will not be necessary, and when an aqueous solution of fuchsin (one part to 28 STAINING METHODS. one hundred of water) is used most bacteria are stained within a few seconds to a minute. At the end of this time the staining solu- tion is to be washed away by means of a gentle stream of water, or by moving the cover glass about in a vessel containing distilled water. Decolorization. — It often happens that the albuminous material associated with the bacteria which we propose to examine is stained so deeply as to obscure the view of these ; and, generally, we will obtain more satisfactory preparations by the use of a decolorizing agent, by which the background is cleared up and the outlines of the cells more clearly defined. The agents chiefly used for this purpose are alcohol, diluted acids, and solution of iodine with potassium iodide (Gram's solution). Koch recommends a solution containing sixty parts of alcohol to forty parts of water. The cover glass is to be quickly passed through this solution two or three times. Some bacteriologists pre- fer to use absolute alcohol. Or we may use dilute acetic acid (one-half to one per cent) or very dilute hydrochloric acid (ten drops to half a litre of water). For decolorizing preparations containing the tubercle bacillus strong solutions of the mineral acids are employed (one part of ni- tric or of sulphuric acid to three parts of water). Gram's solution contains one part of iodine and two parts of potassic iodide in three hundred parts of water. Special directions will be given for the use of these agents when we give an account of the staining methods most useful for the various pathogenic organisms. Double Staining. — After decolorizing the background of albu- minous material we may again stain this with a contrast stain, such as eosin or vesuvin. In mounts made from pure cultures, either liquid or solid, a single stain, for the bacteria only, is all that we require, and our aim is to have the background as free as possi- ble from any material which would obscure the view. After staining, decolorizing, and washing the preparation the cover glass or slide is again dried by exposure to the air or gentle heat, and is then ready for the permanent mounting in Canada bal- sam. If the bacteria have been stained upon the slide, a small drop of balsam dissolved in xylol is placed in the middle of the prepara- tion and a clean, thin glass cover applied. If it is the intention to make the microscopical examination with an immersion objective of high power, or to make photomicro- graphs from it, only the thinnest glass covers should be used — one- two-hundredths of an inch or less. If the preparation is not intended for permanent preservation, STAINING METHODS. 29 the examination may be made without drying the surface upon which the stained bacteria are spread, the water taking the place of balsam in a permanent mount ; or we may dry the film and use a drop of cedar oil between the slide and cover. While simple aqueous solutions of the aniline colors, when freshly prepared, will promptly stain most bacteria, certain agents may be added to these which aid in the preservation of the solution, or which act as mordants, and are useful in special cases. We shall only give here a few of the standard solutions which are most frequently employed by experienced bacteriologists : 1. Aniline-Gentian-Violet (Ehrlich). * Saturated alcoholic solution of gentian violet, . . 5 cc. Aniline water, . . . . . . . 100 cc. 2. Aniline-Methyl-Violet (Ehrlich- Weigert). Saturated alcoholic solution of methyl violet, . . 11 cc. Absolute alcohol, ...... 10 cc. Aniline water, ....... 100 cc. Aniline water for the above solutions is prepared by shaking in a test tube one part of aniline oil with twenty parts of distilled water, and, after allowing it to stand for a short time, filtering the saturated aqueous solution through a moistened filter. If the solution is not perfectly transparent it should be filtered a second time. 3. Carbol-Fuchsin (ZiehPs solution). Fuchsin, ......... 1 gm. Alcohol, 10 cc. Dissolve and add 100 cc. of a five-per-cent solution of carbolic acid. 4. Alkaline Blue Solution (Loffler's solution). Saturated solution of methyl ene blue, ... 30 cc. Solution of caustic potash of 1 : 10, 000, . 100 cc. These solutions keep better than the simple aqueous solutions, but after having been kept for a time they are likely to lose their staining power as a result of the precipitation of the aniline color. The following special methods of staining cover-glass prepara- tions will be found useful in certain cases: Gram's Method.— The dried film upon a slide or coyer glass is stained with an aqueous solution of methyl violet or with aniline- gentian-violet solution (No. 1); it is then placed in the iodine solution for a minute or two (iodine one part, potassic iodide two parts, water 30 STAINING METHODS. thrtM» hundred parts); then washed in alcohol, dried, and, if for per- manent preservation, mounted in balsam. METHODS OP STAINING THE TUBERCLE BACILLUS. — Numerous methods of staining the tubercle bacillus in sputum dried upon a cover glass have been proposed, but we shall only give here two or three of the most approved methods, either one of which may be relied upon for satisfactory results if carefully followed. 1. The Ehrlich- Weigert Method. — Place in a watch glass a little of the aniline-methyl-violet solution (No. 2); float upon the surface of this the cover glass with the dried film downward ; heat over a small flame until it begins to steam, then allow it to stand for from two to five minutes ; decolorize in a tray cont lining one part of nitric acid to three parts of water— the cover glass, held in forceps, is gently moved about in the decolorizing solution for a few seconds. It is then washed off in sixty-per-cent alcohol to remove the remaining blue color — this usually takes but a second or two — and then in water. For a contrast stain a saturated aqueous solution of vesuvin may be used, a few drops being left upon the cover glass for five minutes. The stained preparation is then washed, dried, and mounted in balsam. 2. The Ziehl-Neelson Method. — Float the cover glass upon the carbol-fuchsin solution (No. 3) ; heat gently until steam commences to rise — from three to five minutes' time will usually be sufficient ; wash off in water, and decolorize in nitric or sulphuric acid, twenty- five-per-cent solution, then in sixty-per-cent alcohol for a very short time to remove remaining color from albuminous background; wash well in water and mount in Canada balsam. 3. Friedlander's Method. — Spread and dry the sputum upon the slide ; fix by passing the slide three times through the flame of an alcohol lamp or Bunsen burner ; place upon the dried film three or four drops of carbol-fuchsin (No. 3); heat gently over a flame until steam is given off ; wash in a dish of distilled water ; drain off excess of water, and add a few drops of the following decolorizing solution : Acid, nitric, pure, . . . . 5 cc. Alcohol (eighty per cent), . . . .to 100 cc. —usually the preparation will be decolorized in about half a minute ; wash in water ; add a few drops of an aqueous solution of methylene blue a- ;i contrast stain ; allow the stain to act for about five minutes, without heating ; wash again in water, dry, and mount in balsam, or for a temporary mount use a drop of cedar oil. 1. <;r one cubic centimetre alcoholic solution of methyl violet.) STAINING METHODS. 33 No. 2. A one-per-cent solution of caustic soda. No. 3. A solution of sulphuric acid of such strength that one cubic centimetre is exactly neutralized by one cubic centimetre of the soda solution. According to Loffler, solution No. 1 is just right for staining the flagellum of Spirillum concentricum, but for certain other bacteria it is necessary to add to this some of No. 2 or of No. 3. Thus, for the cholera spirillum from half a drop to a drop of the acid solution is added to sixteen cubic centimetres of No. 1. For the bacillus of typhoid fever one cubic centimetre of No. 2 is added to sixteen cubic centimetres of No. 1. Bacillus subtilis requires twenty-eight to thirty drops of No. 2 ; the bacillus of malignant oedema thirty-six to thirty-seven drops, etc. This method has not been very successful in the hands of other bacteriologists, and improvements in the technique have been made since it was first published. Van Ermengem (1893) points out the fact that. a principal condition of success is that the cover glasses shall be absolutely clean. He boils them in a mixture composed of potas- sium bichromate, sixty grammes; concentrated sulphuric acid, sixty grammes ; water, one hundred grammes. After coming from this they are thoroughly washed in water, then in absolute alcohol, and then dried in an upright position under a bell-jar. Recent agar cultures (ten to eighteen hours) are preferred, and the suspension in water should be very much diluted so that in the cover-glass preparation the bacteria are well isolated. The cover glass, held between the fingera, is passed three times through a flame. A drop of the follow- ing solution is then placed upon it: Osmic acid two-per-cent solution, one part ; solution of tannin (ten to twenty-five per cent) two parts. This is allowed to act for about five minutes at a temperature of 50° to 60° C. — or half an hour at the room temperature. After careful washing with water and alcohol the cover glass is immersed for a few seconds in a bath containing one-quarter to one-half per cent of nitrate of silver. Then without washing it is placed for a short time in the following: Gallic acid, five grammes; tannin, three grammes; fused potassium acetate, ten grammes; distilled water, three hundred and fifty grammes. It is then returned to the silver bath and kept there, with constant movement of the bath, until this commences to turn black. It is then thoroughly washed in water, dried, and mounted in balsam. Pitfield (1895) has devised a much simpler method which he de- scribes as follows : "The method consists in the use of but a single solution, which is at once mordant and stain. The solution should be made in two parts, which are filtered and mixed. 3 34 STAINING METHODS. Saturated aqueous solution of alum, . .10 c.c. Saturated alcoholic solution of gentian -violet, . . 1 c.c. B. Tannicacid, .....-• 1 g™- Distilled water, 10 c.c. "The solutions should be made with cold water, and immediately after mixing the stain is ready for use, " The cover slip is to be carefully cleaned, the grease being burned off m a flame, and after it has cooled the bacteria are spread upon it, well diluted in water, care being taken to exclude culture medium. After the preparation has been thoroughly dried in the air it should be held over the name with the fingers (the preparation need not be fixed) as Loftier has directed. After- ward the stain is gradually poured on the slip and heated gently, bringing the fluid almost to a boil ; the slip covered with the hot stain should then be laid aside for one minute, then washed in water and mounted. ** If the filtered stain is used, a second stain of aniline water containing gentian-violet had better be used, which should be applied but a moment and thru washed off, thus leaving a clean field, showing only bacteria lightly stained, with their flagella still more lightly colored." METHODS OF STAINING BACTERIA IN TISSUES. — The solutions re- commended for staining cover-glass preparations are also used in staining bacteria in thin sections of the various organs, in which they are found in certain infectious diseases; but, in general, a longer time is required to stain sections, and it is best not to hasten the process by the use of heat. To obtain good thin sections, the material, cut in small cubes, must be very thoroughly hardened in absolute alcohol. The piece selected for cutting may be attached to a cork by the use of melted glycerin jelly, which is hardened by placing the cork and attached piece of tissue in alcohol. This an- swers for well-hardened pieces of liver, kidney, etc. , but the hollow viscera and tissues of loose structure will require embedding in paraffin or celloidin. Any well-made sledge microtome will answer t • >r cutting the sections, if the knife is properly sharpened. The sec- tions should, of course, be cut under alcohol, and they can scarcely be too thin when the object is to demonstrate the presence or ab- sence of bacteria. Very thin sections may be cut dry by embedding in paraffin having a melting point of 50° C. In this case the knife is set at a right angle to the material to be cut, and the sections are spread out upon and attached to the glass slide for staining. One of the most useful solutions for staining tissues is Loffler's alkaline solution of methylene blue (No. 4). A freshly-prepared so- lution \\ill stain sections in four or five minutes. Superfluous color is removed by immersing the sections in diluted alcohol or in a one- half-|K'r-(vnt solution of acetic acid for a few seconds. The sections STAINING METHODS. 35 are dehydrated in absolute alcohol, cleared up with oil of cedar, and mounted in a drop of cedar oil for examination, or in balsam if they are to be preserved. Gram's method may be used as directed for cover-glass prepara- tions, the sections being first stained in aniline-gentian-violet solu- tion (No. 1), then washed in water, or in aniline water as recently (1892) recommended by Botkin, then decolorized in the iodine solu- tion (see page 29). The sections when decolorized are again washed in water, dehydrated in absolute alcohol, cleared in cedar oil, and mounted in balsam. Weigert's Method. — This is a modification of Gram's method in which the sections are dehydrated by the use of aniline oil. The stained section, after having been washed, is transferred to a clean glass slide, the excess of water is removed by the use of filtering paper, and the iodine solution is placed upon it in sufficient quantity to cover the entire section. When sufficiently' decolorized this is re- moved in the same way. The section is then dehydrated by placing a few drops of aniline oil upon it, removing this with filtering paper, and repeating the operation once or twice. The aniline oil must then be completely removed by the use of xylol, after which the sec- tion is mounted in balsam. Kuhne's Method.— The object of this method is to prevent the removal of the color from stained bacteria in sections during the treatment which such sections usually receive before they are ready for mounting — i.e., during the washing and dehydrating processes usually employed. For staining, Kiihne prefers a methylene-blue solution prepared as follows: Methylene blue, 1.5 parts; absolute alcohol, ten parts; triturate in a watch glass and add gradually one hundred parts of a solution of carbolic acid containing five parts in one hundred of water. The section is placed in this solution for about half an hour, then washed in water and decolorized in a weak solution of hydrochloric acid — ten drops to five hundred grammes of water. This part of the operation must be conducted very carefully, and usually thin sections will only require to be dipped in the acid solution for an instant, after which they must be at once immersed in a solution of lithium — eight drops of a saturated solution of carbonate of lithium in ten grammes of water. They are then allowed to remain in a bath of distilled water for a few minutes, after which they are dipped into absolute alcohol, which Kiihne colors by the addition of methylene blue. The sections are then placed in aniline oil which contains a little methylene blue in solution, where they are dehydrated without the color being extracted from the stained bacteria present. The aniline-oil blue solution is prepared by adding an ex- cess of dry methylene blue to a small quantity of clarified aniline oil. The undissolved pigment settles to the bottom, and a few drops of the colored solution are added to a little aniline oil in a watch glass to make the colored dehydrating bath. The section is next washed out in pure aniline oil— not colored — after which every trace of aniline oil is to be removed by the use * xylol. The section is cleared up in turpentine and mounted in balsam. Ziehl-Neelson Method, for the tubercle bacillus in tissues. — Leave the sections for fifteen minutes in carbol-fuchsin solution (No. 3) ; decolorize in sulphuric or nitric acid, twenty-five-per-cent 36 STAINING METHODS. solution; wash in sixty-per-cent alcohol; place in a saturated aque- ous solution of methylene blue for contrast stain; wash, dehydrate, and mount in balsam. The following1 method of staining sections for the purpose of demon- strating bacteria present in the tissues is recommended by Pregl (1891) as a substitute for the method of Kuhne. The results are said to be excellent, and it is much simpler and more expeditious. The sections are made from tissues embedded in paraffin, and are attached to clean glass slides with albumen-glycerin. Or they may be attached to a cover glass by the following method when not embedded in paraffin : The sections, completely dehydrated, are taken out of absolute alcohol on a thin glass cover, upon which they are extended ; a piece of filter paper is applied to the side of the cover glass to absorb the alcohol, and before the section is completely dry a drop of aceton-celloidin solution is placed upon it by means of a glass rod. The cover glass is now moved about in the air to promote rapid evaporation of the alcohol, and is then placed in water. The section now remains attached to the cover glass during subsequent manipulations. The aceton-celloidin solution referred to is prepared by adding celloidin in small, dry pieces to aceton until a concentrated solution is obtained. A large drop of this added*to five cubic centimetres of absolute alcohol makes a suitable solution for use. This must be kept in a glass-stoppered bottle, and will require to be frequently renewed, as it is not suitable for use after hav- ing absorbed moisture from the air. The aceton as obtained from dealers contains considerable water and must be dehydrated by adding to it red-hot sulphate of copper. The sections, attached to a slide or cover glass by one of the methods mentioned, are stained with Kuhne's carbol-methylene-blue solution, which is drooped upon them from a pipette. Usually they will be sufficiently stained at the end of half a minute to a minute, but in some cases a longer time and the application of heat will be desirable. They are then washed in water and immediately placed in fifty-per-cent alcohol, where they remain until the sections have a Dale-blue color with a greenish tinge. They are now completely dehydrated in absolute alcohol and subsequently cleared up in xylol. STAINING SECTIONS OF GELATIN STICK CULTURES.— Fischl, Weigert, and Neisser have given an account of methods for staining stick cultures in gelatin of non-liquefying bacteria. The object of this is to show the mode of growth and the association of individual cells in undisturbed cultures. Neisser gives the following directions : The gelatin cultures are inoculated, by several punctures, with the microorganism to be studied. When the development is deemed sufficient the cylinder of gelatin is removed from the test tube by gently warming its walls. It is then placed for several days- one to eight, according to its size and thickness— in a one-per-cent solution of bichromate of potassium. While in this solution it must be exposed to the light, which causes a change in the gelatin, rendering it insoluble. The gelatin cylinder is thoroughly washed and then hardened in alcohol, first of seventy per cent, and then of ninety -six percent. It is then cut into suit- able pieces, and these are attached to a cork in the usual manner and placed for twenty-four hours in absolute alcohol. Thin sections may now be made witli a microtome, and these are attached to a glass slide and stained by < tram s or Weigerfs method or by the use of Loffler's solution (No. 4). The •olorization should he effected by the use of alcohol and not with an acid ution. When Gram's method %is used decolorize by the alternate use of alcohol and oil of cloves. Clear the preparation with oil of bergamot. V. CULTURE MEDIA. To obtain a satisfactory knowledge of the biological characters of the different species of bacteria, it is necessary to isolate them in " pure cultures " and to study their growth in various culture media. By a pure culture we mean a cultivation containing a single species only ; and to be absolutely sure that we have a pure culture it is desirable that all of the bacteria in a culture shall be the progeny of a single cell. The methods of obtaining pure cultures will be given later. At present we propose to give an account of the various cul- ture media commonly employed by bacteriologists, and the methods of preparing them for use. By a natural culture medium we mean one which, as obtained in nature, contains the necessary pabulum for the development of one or more species of bacteria. An artificial culture medium is one which is prepared artificially by adding nutritive material to water. A sterile medium is one which does not contain any living micro- organisms. We may obtain natural media in a sterile condition, but artificial media require sterilization, as they are infallibly contami- nated with living " germs " from the atmosphere during the process of preparing them. Sterilization is usually effected by heat. For- ceps, glass tubes, etc. , may be sterilized by passing them through the flame of an alcohol lamp or Bunsen burner. NATURAL CULTURE MEDIA. — The most important natural cul- ture medium is blood serum, which may be obtained from one of the lower animals — preferably from oxen or calves. This is to be collected in a sterilized jar, with every precaution to insure cleanli- ness, at the moment of slaughtering the animal. Or the blood of a calf, sheep, or dog may be collected at the laboratory by a carefully conducted operation, in which the femoral or carotid artery is con- nected with a sterilized glass tube leading into a sterilized receptacle; such as a Woulf 's bottle, into one neck of which a cotton plug has been placed to permit the air to escape as the bottle fills with blood through a tube which is secured in the other neck. When blood is passed directly from an artery into a sterilized receptacle the serum will not subsequently require sterilization. The writer is in CULTURE MEDIA. the habit of collecting it in this way, and, after the serum has sepa- rated, of drawing it off in little flasks having a long neck, as shown in Fig. 14. The neck of the flask, previously sterilized by heat, is slipped into the Woulf s bottle beside the cotton plug, the bulb (a) having been previously gently heated to expand the contained air. As the heated air cools a partial vacuum is formed and the clear serum mounts into the little flask. One after another is filled in this way, and each one is hermetically sealed in the flame of a lamp -a Fio. 14. Fio. 15. Fio. 18. as soon as it is withdrawn. The sterile blood serum may be pre- served indefinitely in this way, and may be used as a liquid culture me?'U™ -« *ht httle flask' or it may be transferred to a test tube and solidified by heat whenever a solid blood-serum medium is re- ft, $£!*& °f PreservinS bl<*>d serum and other liquid edia m hese httle flasks is in the fact that they may be preserved .definitely without becoming contaminated or drying up, and that Uiey are easily transported, while a liquid medium in a test tube must be kept upright. The contents of one of these flasks are readily CULTURE MEDIA. 39 transferred to a test tube by breaking off the sealed extremity with sterile forceps and slipping it past the cotton plug, which must be partly withdrawn for the purpose. Upon applying gentle heat to the bulb its contents are forced out into the test tube (Fig. 15). Blood serum which is collected without these special precautions will require sterilization by heat, for which directions will be given later. To obtain the clear serum from blood collected as above directed, the jars containing it are set aside in a cool place in order that a firm clot may form, care being taken not to shake them. After the clot has formed they may be transported to the laboratory, where they are placed in an ice box or in a cool cellar for from twenty-four to forty-eight hours. By this time the serum has separated from the clot, and it may be transferred to sterilized test tubes by means of a suction pipette (Fig. 16), or may be distributed in little flasks as above directed. M ilk is largely used as a culture medium, and is especially useful in studying the biological characters of various microorganisms, as shown by their causing coagulation of the casein, or otherwise ; or an acid or alkaline reaction of the liquid ; or peptonization of the precipitated casein, etc. In the udder of healthy cows milk is quite sterile, and by proper precautions it may be drawn into sterilized flasks without any contamination and kept indefinitely without un- dergoing coagulation or any other change. But in practice it is easier to sterilize it in test tubes or small flasks by the use of heat than to obtain it in a sterile condition from the udder of the cow. Urine has been used to some extent as a culture medium, and many bacteria multiply in it abundantly, although, on account of its acid reaction, other species fail to grow in it. As contained in the healthy bladder it is sterile, but the mucous membrane of the mea- tus urinarius always contains numerous bacteria upon its surface, and some of these are sure to be carried away with the current when urine is passed. A culture fluid which the writer has found extremely useful, in tropical countries where it is to be obtained, is the transparent fluid contained in the interior of unripe cocoanuts — called agua coco by the Spaniards. In countries where the cocoanut is indigenous this cocoanut water is largely used as a refreshing drink. It contains about four per cent of glucose in solution, together with some vege- table albumen and salts. Some microorganisms multiply in it with- out appropriating the glucose, while others split this up, producing an abundant evolution of carbon dioxide and giving to the fluid a very acid reaction. The following are the results of an analysis 40 CULTURE MEDIA. made for me by Dr. L. L. Van Slyke in the chemical laboratory of Johns Hopkins University : The weight of the fluid obtained from H\ nuts averaged 339.1 grammes. The specific gravity averaged 1.02285. The amount of water averaged 95 per cent ; the amount of inorganic ash, 0.618 per cent; the amount of glucose, 3.97 per cent ; the amount of fat, 0.119 per cent ; the amount of albuminoids, 0.133 percent. As this fluid is contained in a germ-proof receptacle, no steriliza- tion is required when it is drawn off with proper precautions in the little flasks heretofore described. Hydrocele fluid has been used as a culture medium, and many bacteria multiply in it abundantly. Other natural culture media are found in the animal and vege- table kingdoms, which are used, either cooked or raw, as solid sub- strata upon which bacteria may be cultivated. One of the most use- ful of these is the potato, which is a favorable medium for the de- velopment of numerous species, and upon which (cooked) many of them present characters of growth which are so distinctive as to aid greatly in the differentiation of species. Other tubers, roots, or fruits may also be used as solid media, or their juices extracted and employed as liquid media. Cooked fish and meats of various kinds are also suitable media for certain spe- cies— e.g., the phosphorescent bacteria grow very well upon the sur- face of boiled fish, and in a dark room give off a bright, phosphores- cent light. Eggs, sterilized by boiling, have been used by some bacteriolo- gists, especially for the cultivation of anaerobic species. ARTIFICIAL CULTURE MEDIA.— A great variety of liquid media have been employed by bacteriologists, the most useful of which are infusions of beef or mutton, with the addition of a little peptone. But Pasteur has shown that some species of bacteria will grow in a medium which does not contain any albuminous material, nitrogen being obtained from salts containing ammonia. Pasteur's solution, which is rarely used at present, contains : Distilled water, one hundred parts ; cane sugar, ten parts ; tartrate of ammonia, one part, with the addition of the ashes from one gramme of yeast. Cohn modified this by leaving out the cane sugar, which favors the development of moulds. These fluids are not, however, in- tended for general use in the cultivation of bacteria, but to demon- si rate certain facts relating to their physiology. Infusions of meat, or " flesh water," are made by chopping fine lean U-ef MI- mutton (,,11,' pound) and covering it with water (one litre). This is placed in an ice chest for twenty-four hours, and the CULTURE MEDIA. 41 aqueous extract is then obtained by filtration through muslin by pressure. This extract is cooked, filtered, and carefully neutralized by the addition of a solution of carbonate of sodium, which is added drop by drop. Usually we add to this one-half per cent of chloride of sodium. The addition of ten grammes of peptone to a litre of this meat infusion constitutes the flesh-peptone solution which is largely used in the preparation of solid culture media, to be described hereafter. The addition of five per cent of glycerin to the above infusion makes a useful liquid medium for the cultivation of the tubercle ba- cillus (Roux and Nocard). The liquid should be again neutralized after adding the glycerin, which commonly has an acid reaction. Bouillon is made by cooking the chopped meat — one pound in a litre of water — for about half an hour in a large glass flask or an enamelled iron kettle. The filtered bouillon is then carefully neu- tralized with sodium carbonate, and again boiled for an hour to pre- cipitate all coagulable albuminoids. It is again filtered and dis- tributed in test tubes or small flasks, in which it is subsequently sterilized. For certain pathogenic bacteria a bouillon made from the flesh of a fowl or of a rabbit is preferable to beef bouillon. Flesh infusion may also be made from one of the standard beef extracts, such as Liebig's (five grammes to a litre of water). Various vegetable infusions may also be used as culture media, such as yeast water, potato water, infusion of hay, of barley, or of wheat, of dried fruits, beer wort, etc. SOLID CULTURE MEDIA. — The introduction of solid culture media, and especially the use of gelatin and agar-agar, as first recommended by Koch (1881), for the isolation and differentiation of species, was a most important advance in bacteriological technology. We are concerned here only with the composition and preparation of these media. Flesh-Peptone-Gelatin. — This is made by adding ten per cent of the best French gelatin to the flesh-peptone solution above de- scribed. This is the standard gelatin medium, but more or less gelatin may be added to serve a special purpose. Thus, in Havana during the summer months the writer used a medium containing twenty per cent of gelatin, because when but ten per cent was used the gelatin was liquefied by the normal temperature of the atmo- sphere. Teii-per-cent gelatin, of good quality and carefully pre- pared, will stand a temperature of 20° to 22° C. (68° to 71. 6°, F.) without melting. When twenty per cent of gelatin is used the melting point is about 8° C. higher. It must be remembered that exposure to a boiling temperature reduces the melting point of gela- tin. It is therefore desirable to accomplish the operations of cook- 42 CULTURE MEDIA. ing and sterilizing in as short a time as is practicable. The French gelatin used comes in thin sheets ; this is broken up and added to the flesh-peptone solution. Usually we prepare a litre of nutrient gelatin at one time, and for this quantity one hundred grammes of gelatin will be required for the standard preparation (ten per cent). It is well to allow it to soak for a time in the liquid before applying heat for the purpose of dissolving it. Then apply gentle heat until it is completely dissolved. The gela- tin of commerce usually has an acid reaction, and it will be necessary to carefully neutralize the medium after it has been added. A slightly alkaline reaction is usually no disadvantage, but certain pathogenic bacteria will not grow when there is a trace of acid present. The m next step consists in clarifying the nutrient medium. It is allowed tooool to about 50° C., and an egg, previously broken into one hundred grammes of water, is gradually added while stirring the liquid with a -lass rod. A whole egg is used for a litre of the solu- Heat is again applied and the solution is kept at the boiling point for about ten minutes, during which time the egg albumen is precipitated and carries down with it all insoluble particles, which without this clarifying process would have interfered with the trans- parency of the medium, even when carefully filtered. The hot oiution is thru filh'red. A hot-water funnel (Fig. 17) is usually •mploy«Ml. as tlu» -olatin solution does not pass through filtering paper very rapidly, and when cooled to near the point of solidifying ceases to pass. CULTURE MEDIA. 43 The advantages of the gelatin medium are that it is perfectly transparent, that it is easily melted for making "plates/' and that many bacteria exhibit in it special characters of growth by which they may be differentiated from others which resemble them in form. The principal disadvantage is the low melting point, which prevents us from making use of this medium for cultivating bacteria in an in- cubating oven at a higher temperature than about 22° C. for ten-per- cent gelatin. This disadvantage is overcome by using agar-agar instead of gelatin. This is prepared in Japan and other Eastern countries from certain species of gelatinous algae. It comes to us in the form of bundles of dried strips, which form a stiff jelly when dissolved in water in the proportion of one to two per cent. This jelly remains solid at a temperature of 40° C. and above. It was first employed by Hesse, one of Koch's collaborators in the office of the imperial board of health of Berlin. Koch, who was in search of a trans- parent jelly which would stand the temperature required for the cul- tivation of certain pathogenic bacteria (37° to 38° C.), quickly recog- nized its value and introduced it into general use. The agar-agar jelly is more difficult to filter than the gelatin medium, and some skill is required in order to obtain a transparent solution. It will bear long boiling without losing its quality of forming a stiff jelly. From ten to twenty grammes are added to a litre of flesh infusion, or we may make a peptonized agar in accor- dance with the following formula which is given by Salomonson : Add to one litre of distilled water five grammes Liebig's extract, thirty grammes peptone, five grammes cane sugar, fifteen grammes agar. Cook for an hour, render slightly alkaline, and cool to below 60° C. Clarify and cook again for an hour or more. Glycerin-agar is made by adding five per cent of glycerin to the peptonized agar made by the above formula or by the use of the flesh-peptone infusion. This is a very favorable medium for the cul- tivation of the tubercle bacillus — first used by Roux and Nocard. Agar-gelatin, a medium which has recently come into favor and is said to be very useful, as it resembles gelatin in transparency and has a considerably higher melting point than ten-per-cent gelatin, is made by adding fifty grammes of gelatin and 7. 5 grammes of agar to a litre of flesh-peptone solution. Care should be taken not to cook this longer than is necessary. In making all of these agar culture media the main difficulties encountered result from the difficulty of dissolving the agar and the slowness with which the solution passes through filtering paper. These difficulties are best met as follows : Break up the sticks of agar into small fragments and allow them to soak in cold water for twenty- 44 CULTURE MEDIA. four hours. Pour off the water and add the flesh-peptone solution. Boil for several hours until the agar is completely dissolved. Neu- tralize hy adding gradually a solution of carbonate of soda (or render slightly alkaline). Filter. The last operation is the most troublesome, and various plans have been proposed to avoid the tedious filtration through filtering paper in a hot- water filter. A method which gives satisfactory re- sults is to place the filter containing the hot agar solution, and the flask which is to receive the filtrate, in a steam sterilizing apparatus, where it is left in an atmosphere of streaming steam until the filtra- Fio. 18. tion is completed. Or the solution may be put in a tall jar and left in the steam sterilizer for several hours until it is clear as a result of sedimentation. The clear solution is then obtained by decantation. Or by conducting the operation in a tall cylindrical vessel, and al- lowing sedimentation to occur in the steam sterilizer and the agar subsequently to solidify by cooling, the cylinder of jelly may be re- moved from the jar and the part containing the sediment can be cut away. The transparent portion is then melted again and distributed in test tubes for use. In the present volume we frequently refer to the nutrient medium madr hy adding one to two per cent of apir-agar to the standard tl< >h-peptone solution as ** nutrient agar '' or simply as " agar." CULTURE MEDIA. 45 The following method of filtering agar has recently (1890) been proposed by Karlinsky. It is a modification of the method previously described by Jakobi and depends upon the use of pressure. In Fig. 18, a is a cylindrical vessel of tin, which is closed above by a perforated rubber cork, through which is passed a glass tube, b. This is enclosed in a larger tin cylinder, c, which contains water, which may be kept hot by placing an alcohol lamp under the pro- jecting arm d. The central cylinder has a tube, e, passing through the bottom of the hot-water cylinder, and which is provided with a FIG. 10. stopcock for drawing off the filtered solution. Before pouring the hot agar solution into the cylinder a, a cotton filter about ten centi- metres thick is placed at the bottom of this cylinder and hot water is poured upon it while the stopcock of the outlet tube is open. This washes out the cotton and prepares the filter for the agar solution. The apparatus is supported upon a tripod, not shown in the figure. Filtration is said to occur rapidly when the air in the central cylinder is compressed by means of the hand bellows attached to the tube b. Unna (1891) has devised a filtering apparatus for agar which is shown in Fig. 19. In this the pressure of steam is utilized. A hollow 4»; CULTURE MEDIA. sphere of copper, supported upon a tripod, is so constructed that an upper hemispherical segment can be removed to give access to the interior. An opening at the bottom contains a perforated rubber cork, through which the stem of an enamelled iron funnel passes. A simple filter of filtering paper is used in this funnel, and this is filled to a depth of two centimetres with well-burned kieselgur (dia- tomaceous earth in which the organic matter has been destroyed by heat). The hot solution of agar is poured into the funnel, and hot water into the space between it and the copper vessel ; this must not come too near the top of the funnel — not nearer than three centi- metres. The hemispherical cover is then secured in its place by means of a clamp screw shown in the figure. By placing a Bunsen burner under the projecting arm the water is made to boil and a sufficient steam pressure secured. A small stopcock attached to the cover of the copper vessel permits the escape of steam if the pressure is too great. According to Unna, solutions containing as much as three per cent of agar can be filtered by means of this apparatus, and a litre of two-per-cent agar will pass through it in about two hours. Schultz' Rapid Method of Preparing Nutrient Agar- Agar.— Place one thousand five hundred cubic centimetres of water in an en- amelled iron pot; add eighteen grammes of agar-agar, broken in small pieces, and place upon a gas stove ; boil for half an hour ; add while boiling two grammes of Liebig's extract of beef ; remove from fire and cool to 60° C. ; then add ten grammes of dry peptone, five grammes of sodium chloride, and the contents of one egg beaten up in a sufficient quantity of water to supply that lost by evaporation ; neut- ralize the mixture by the addition of dilute hydrochloric acid ; boil again for five or ten minutes; filter through white filter paper. If the filtrate is not entirely clear add to it the albumen of a second egg and boil until this is coagulated ; then filter again. Ahvays mois- ten the filter with water before filtering solutions containing gelatin or agar-agar. When the process is completed the amount of filtered culture medium should be about one thousand cubic centi- metres. For serial purposes various substances are added to the above- described solid and liquid media. A favorable addition for the .urn >wth of a considerable number of bacteria is from one to three per cent of glucose. The phosphorescent bacteria grow best in a medium < ontaining two to three per cent of sodium chloride. The addition of three to four per cent of potassium nitrate is made in conducting experiments designed to test the reducing power of certain bacteria, by which this salt is decomposed with the production of nitrites. Acids are also added in various proportion to test the ability of bacteria under investigation to grow in an acid medium. From CULTURE MEDIA. 47 1 : 2,000 to 1 : 500 of hydrochloric acid may be used for this purpose. The addition of litmus to milk or other culture media is fre- quently resorted to for the purpose of ascertaining whether acids or alkalies are developed during the growth of bacteria under investi- gation. The addition of aniline colors which are variously changed by the products of growth of certain species has also been resorted to in the differentiation of species. Various disinfecting agents, such as carbolic acid, etc. , have also been used for the same purpose, and it has been shown by experiment that some bacteria will grow in a medium containing such agents in a proportion which would entirely restrain the development of others. The soluble silicates which form a jelly-like mass have been proposed as a culture medium for certain bacteria which do not grow in the usual media. Kiihne (1890), Winogradsky (1891), and Sles- kin (1891) have made experiments which indicate that this medium has considerable value. Winogradsky uses in the preparation of his silicate jelly the following salts : Ammonium sulphate, . . . . 0.4 gramme. Magnesium sulphate, . . . . 0.05 Potassium phosphate, .... 0.1 Calcium chloride, .... a trace. Sodium carbonate, . . . 0.6 to 0.9 gramme. Distilled water, . . . . 100 grammes. To this he adds a solution of silicic acid. According to Kiihne, a solution containing 3.4 per cent of silicic acid and having a specific gravity of 1.02 may be preserved in a liquid condition. To this the salts are added in greater or less amount, according to the consis- tence desired. Sleskin states that a suitable jelly is formed by the addition of 1.15 to 1.45 per cent of the salts, and recommends that concentrated, sterilized solutions be added to the acid. He dissolves separately, in as little water as possible, the sulphates, the potassium phosphate and sodium carbonate, and the calcium chloride. The use of a culture medium containing an extract from the je- quirity seeds has been recommended by Kaufmann (1891), who has found, by experimenting upon various bacteria, that such a medium is useful in differentiating species. Thejequirity solution, which may be used as a liquid medium or may be employed in the preparation of nutrient gelatin or agar, is prepared as follows : Ten grammes of jequirity seeds are bruised in a mortar and the shells removed ; they are then placed in one hun- dred cubic centimetres of water and cooked for two hours in the steam sterilizer ; after allowing the infusion to cool it is filtered. The fil- tered liquid has a pale-yellow color and a neutral or slightly alkaline 48 CULTURE MEDIA. reaction. Certain bacteria grow in this solution without producing any change in its color ; others, which produce an acid reaction, cause it to be decolorized ; others, which produce an alkaline reac- tion of the medium, change the color to green. Cooked Potato. — Schroter first used cooked potato as a culture medium for certain chromogenic bacteria (1872), and Koch subse- quently called attention to the great value of potato cultures for differentiating species. His plan of preparing potatoes is as follows : Sound potatoes are chosen in which the epidermis is intact. These are thoroughly washed and scrubbed with a brush to remove all dirt. The " eyes" and any bruised or discolored spots are removed with a sharp-pointed knife. They are again thoroughly washed in water, and are then placed for an hour in a bath containing mercuric chloride in the proportion of 1 : 500, to thoroughly disinfect the surface. They are then placed in a steam sterilizer for about three-quarters of an hour, and after an interval of twenty-four hours A Fro. 80. Flo. 21. Fio. 22. are again steamed for fifteen minutes. It is well to wrap each potato in tissue paper before placing it in the bichloride bath, and to leave it in this protecting envelope until it is placed in the glass dish CULTURE MEDIA. 49 in which it is preserved from contamination by atmospheric germs after being inoculated with some particular microorganism. Just before such inoculation the potato is cut in halves with a sterilized (by heat) table knife. The bacteria to be cultivated are placed upon the cut surface and the potato is preserved in a glass dish (Fig. 20). A more convenient method, and one which secures the potato more effectually from atmospheric organisms, is to cut a cylinder, about an inch in diameter, from a sound potato, by means of a tin instru- ment resembling a cork borer or apple corer. This cylinder is cut obliquely into two pieces having the form shown in Fig. 22, and each piece is placed in a large test tube having a cotton air filter, in which it is sterilized. This method, first employed by Bolton, has been slightly modified by Roux, who recommends that a receptacle for catching the water which separates during the sterilizing process be formed by making a constriction around the test tube an inch above its lower extremity. This is done by the use of a blowpipe. The cylinder of potato rests upon the constricted portion of the tube, as shown in Fig. 21. Sometimes a potato paste is employed. The potatoes are boiled for an hour and the skins removed, after which they are mashed with a little sterilized water, placed in suitable plates, and sterilized by exposure for half an hour on three successive days in the steam sterilizer. Bread paste may be made in the same way, and is a very favorable medium for the growth of certain bacteria and also for the common moulds. VI. STERILIZATION OF CULTURE MEDIA. A MOST important part of bacteriological technology consists in the sterilization of the various culture media employed. A sterile medium is essential for maintaining a pure culture, and we can only obtain an exact knowledge of the biological characters of a species by studying its growth in various media, its physiological reactions, its pathogenic power, etc., independently of all other microorgan- isms— i.e., in pure cultures. We may sterilize a culture medium either by heat or by filtration through a substance which does not permit bacteria to pass. The last-mentioned method is useful for certain special purposes ; but, in general, sterilization of culture media, and of the vessels in which they are preserved, is effected by heat. The scientific use of heat as an agent for sterilizing our culture media depends upon a knowledge of the thermal death-point of the various microorganisms which are liable to be present in them, and upon various facts relating to the manner in which heat is applied. All this has been determined by experiment, and before giving practical directions for sterilization it will be well to consider the experimental data upon which our methods are based. As a rule, bacteria which do not form spores are killed at a com- paratively low temperature. Thus, in a series of experiments made by the writer upon the thermal death-point of various pathogenic organisms, the pus cocci were found to be the most resistant, and all of these were killed by exposure for ten minutes to a temperature of 62° C. (143.6° F.). There are several species of bacteria known, however, which not only are not killed by this temperature, but are able to grow and multiply at a temperature of 65° to 70° C. (Miquel, Van Tieghem, Globig). But it is safe to say that exposure to a boiling temperature for a minute or two will infallibly destroy all microorganisms in the absence of spores, when they are in a moist condition or moist heat is used — i.e., when they are directly ex- posed to the action of boiling water or of steam. The power of dry heat to destroy microorganisms in a desiccated condition is a differ- ent matter and will require special consideration. STERILIZATION OF CULTURE MEDIA. 51 The spores of bacilli have a much greater resisting power, and the vitality of some of these reproductive bodies, from known spe- cies, is not destroyed by a boiling temperature maintained for sev- eral hours. Thus Globig found that the spores of a certain bacillus from the soil — his " red potato bacillus " — required six hours' exposure to streaming steam in order to destroy it. Steam under pressure, at a temperature of 115° C., killed it in half an hour ; at 125° C. in five minutes. This extreme resisting power is exceptional, however, and many spores are destroyed in a few minutes by the boiling tem- perature of water. In practice we assume that some of the more resistant spores, which are frequently present in the atmosphere, may have fallen into our culture material, and to insure its sterilization we subject it to a temperature which can be depended upon to destroy these ; or we resort to the method of discontinuous heating. This method was first employed by Tyndall (1877), and is now in general use in the bacteriological laboratories of Germany, having been adopted by Koch and his pupils ; while in France a single sterilization by means of steam under pressure, securing a higher temperature, is still the favorite method with many. In the method by discontinuous heating we subject the culture material for a short time to the temperature of boiling water, thus destroying all bacteria in the vegetative stage. After an interval, usually of twenty-four hours, we repeat the operation for the pur- pose of destroying those which in the meantime have developed from spores which may have been present. Again the material is put aside, and after twenty-four hours it is again heated to the boiling point. This is usually repeated from three to five times. The object in view is to kill the growing bacteria which are de- veloped from spores which were present ; and, as a matter of expe- rience, we find that this method of sterilization is more reliable than a single prolonged boiling, unless this be effected at a higher tem- perature than that of boiling water at the ordinary pressure of the atmosphere. Discontinuous heating is especially useful for the sterili- zation of liquids which would be injured by prolonged boiling — as is the case with solutions of gelatin — or which are coagulated by the boiling temperature. By means of a water bath, the temperature of which is regulated automatically, we may conduct the operation at any desired degree. Thus in sterilizing blood serum we use a temperature a little below that at which coagulation occurs (about 70° C.). Test tubes, flasks, and apparatus of various kinds are commonly sterilized by dry heat in a hot-air oven. This is usually made of sheet iron, with double walls, and shelves for supporting the articles £2 STERILIZATION OP CULTURE MEDIA. to be sterilized. The form shown in Fig. 23 is commonly used in bacteriological laboratories. It must be remembered that a much higher temperature is re- quired for the destruction of microorganisms when dry heat is em- ployed than is the case with moist heat. The experiments of Koch and Wolffhugel (1881) show that a temperature of 120° to 128° C. (248° to 262° F.) is required to destroy the spores of mould fungi, and micrococci or bacilli in the absence of spores. For the spores of ba- cilli a temperature of 140° C. (284° F.), maintained for three hours, was required. In practice we usually maintain a temperature of about 150° C. Fio.28. (302° F.) for an hour or more; and it is customary to sterilize all test tubes and flasks, which are to be used as receptacles for culture media, in the hot-air sterilizer. This procedure could no doubt, how- ever, be dispensed with in many cases and reliance be placed upon the sterilization of the flask, together with its contents, in the steam sterilizer, especially with such culture media as are not injured by long exposure to a boiling temperature — e.g., bouillon and agar-agar. When we propose to cultivate aerobic bacteria, or such as require oxygen for their development, a cotton air filter is placed in the mouth of each test tube and flask before it is sterilized in the hot-air oven. This is a loose plug of cotton, pushed into the neck of the flask for an inch or more, and projecting from its mouth for a short distance. These cotton filters should fill the tube completely and STERILIZATION OF CULTURE MEDIA. 53 uniformly, but should not be packed so closely that there is difficulty is removing them. Steam Sterilizers. — Steam at the ordinary pressure of the atmo- sphere has the same temperature as boiling water, and in practice is preferable to a water bath for several reasons. The form of steam sterilizer adopted by Koch, after extensive experiments made in col- laboration with Loffler and Gaffky, is now generally used in bacte- riological laboratories. This is shown in Fig. 24. It consists of a cylindrical vessel of zinc which is covered with a jacket of felt. The cover, also covered with non-conducting material, has an aper- ture at the top for the escape of steam. A glass tube, which is in communication with the interior of the vessel, serves to show the FIG. 24. FIG. 25. height of the water when the apparatus is in use. The bottom of the cylindrical vessel should be of copper. A Bunsen burner having three jets will commonly be required to keep the water in ebullition and the upper part of the steam sterilizer filled with "live steam, " which should escape freely from the aperture in the cover to insure a temperature of 100° C. in the steam chamber. A perforated zinc or copper shelf in the interior of the cylinder serves to support the flasks, etc., which are to be sterilized. Usually they are lowered into the cylinder in a light wire basket, or tin pail with perforated bottom, of proper diameter to slip easily into the sterilizer. Fig. 25 is a sectional view of this sterilizer. The steam sterilizer shown in Fig. 26 ' is an American invention, 1 The Arnold steam sterilizer, manufactured at Rochester, N. Y. 54 STERILIZATION OF CULTURE MEDIA. which answers the purpose admirably, and which has the advantage of getting up steam very quickly and also of using comparatively little gas. The use of steam under pressure, by which higher temperatures are obtained, requires a more expensive apparatus, made on the principle of Papin's digester. The form manufactured by Miincke is one of the best. This is shown in Fig. 27. It is provided with a pressure gauge and a safety valve. A single sterilization in this ap- paratus, at a temperature of 115° C., for half an hour, will usually Fio. 26. FIQ. 27. suffice, and for liquid culture media or for agar-agar this method is entirely satisfactory ; but a gelatin medium which is exposed to this temperature loses its property of forming a jelly at 20° to 22° C., and consequently its value as a solid culture medium. In practice the simpler form of apparatus in which streaming steam is used will be found to answer every requirement. To insure sterilization with this it is customary to resort to discontinuous heating, as heretofore described. The standard flesh-peptone-gelatin medium should, as a rule, be subjected to a temi>erature of 100° C. for ten minutes, at intervals of twenty-four hours, four days in succession. Bouillon, H«-*h int'iiMMiis. and a^ar-agiir jolly may be steamed for an hour at a time two or three days in succession. STERILIZATION OF CULTURE MEDIA. 55 It is always advisable to test the sterilization of culture material before making use of it. This is done by placing it for a few days in an incubating oven at 30° to 35° C. If a considerable quantity of material in test tubes has been prepared at one time, it will be suffi- cient to put a few tubes in the incubating oven to test sterilization. Failure to make this test often leads to serious complications in experimental investigations. A laboratory sometimes becomes in- fected with resistant spores, which are not all destroyed by the usual methods of sterilization, and these may not develop until some time has elapsed after the supposed sterilization. Sterilization of Blood Serum. — Blood serum which has been collected in test tubes or small flasks, as heretofore directed, is FIG. 28. sterilized in a water bath at 60° C. (140° F.) by the method of dis- continuous heating. It is usually left in the hot-water bath for about an hour, and this is repeated, at intervals of twenty-four hours, for five to seven days. This rather tedious process may be avoided by collecting the serum in the first instance with proper precautions to prevent it from becoming contaminated with atmospheric organ- isms. A special apparatus was devised by Koch for sterilizing blood serum, but an improvised hot-water bath which is regulated to a temperature of 60° C. by an automatic thermo-regulator will answer the purpose. After being sterilized the serum is solidified by careful exposure to a temperature of about 68° C., which causes it to co- agulate, forming a transparent, jelly-like mass. When coagulated at a higher temperature it becomes opaque. The time required for this operation varies from half an hour to an hour, and it is best to remove the tubes from the receptacle in which they are exposed to 56 STERILIZATION OF CULTURE MEDIA. heat as soon as the serum is solidified. Koch's apparatus for coagu- lating blood serum is shown in Fig. 28. It is customary to place the test tubes in an oblique position, so that a large surface may be ex- posed upon which to cultivate the tubercle bacillus or whatever microorganism may be under investigation. A form of apparatus designed for both sterilizing and coagulating blood serum is shown in Fig. 29. It is manufactured by Miincke in accordance with the directions of Hueppe, and special precautions have been taken to se- cure a uniform temperature in all parts of the air chamber. We FIG. 29. may remark that since it has been shown by Roux and Nocard that the tubercle bacillus grows very well in agar-agar jelly to which five per cent of glycerin has been added, blood serum is not so largely used as a culture medium in bacteriological laboratories. Sterilization by Filtration.— This method is especially useful for separating the soluble substances contained in a liquid culture of bacteria from the living cells. It has been demonstrated that several e most important pathogenic bacteria produce toxic substances during their growth which may cause the death of susceptible ani- mdependently of the living bacteria; and this demonstration STERILIZATION OF CULTURE MEDIA. 57 has been made either by sterilizing a pure culture by means of heat, or by separating the bacteria from the culture liquid by filtration. Some of these toxic products of bacterial growth are destroyed by a comparatively low temperature ; the method of sterilization by fil- tration is therefore very important in researches relating to the composition and pathogenic power of these soluble products. Pas- teur, in his earlier experiments, used plaster of Paris as a filter, and Fig. 30. subsequently resorted to the use of unglazed porcelain, through which a liquid may be forced by pressure, but which does not per- mit of the passage of suspended particles, however small. As the porcelain filter is the most reliable and convenient for accomplishing the object in view, we shall not describe other methods of filtration which have been proposed and successfully used. The porcelain used is a very fine paste, manufactured at Sevres, which is moulded into cylinders (bougies) of the form proposed by Chamber- land and baked at a high temperature. 58 STERILIZATION OF CULTURE MEDIA. In Fig. 30 the Pasteur-Chamberland filter is shown as arranged for the filtration of water. A is the hollow porcelain cylinder, which is enclosed in a metal case, D. The metal case is tightly clamped against a projecting shoulder at the lower part of the porcelain filter, a ring of rubber being interposed to secure a tight joint. When water under pressure is admitted to the space E, between the cylin- der of porcelain and the metal case, it slowly filters through, and, running down the inner wall of the filter, escapes at B into a recep- tacle placed to receive it. If we fill the space E with a liquid cul- ture of bacteria and apply sufficient pressure (one or two atmo- spheres), a clear filtrate is obtained which is entirely sterile if the porcelain filter is sound and made of proper material. After the Fio. 31. filter has been in use for some time, however, it may permit the pas- sage of bacteria, and it will be necessary to subject it to a high tem- perature for the purpose of destroying all organic matter contained in the porous porcelain. We may use the Chamberland filter without a metal case by im- mersing it in a cylindrical glass vessel containing the liquid to be fil- tered, as shown in Fig. 31. The porcelain cylinder is connected with an aspirator bottle, a, and a small Erlenmeyer flask, 6, is interposed to catch tlic lilt rate \vhen it overflows from the interior of the filter. Of course all the necessary precautions must be taken with refeivmv to the sterilization of the interior of the bougie, of the flask b, and of the rubber tube connecting the two. Another arrangement of the Pasteur-Chamberland filter for labora- tory purposes is shown in Fig. 32. In this form of apparatus a STERILIZATION OF CULTURE MEDIA. 59 receptacle, R, is provided for the liquid to be filtered, and a pump for compressing air is attached to it by a rubber tube. Instead of this pump, water pressure may be used indirectly by attaching a strong bottle to the water supply and allowing it to fill slowly with water, and at the same time to force out the air through a tube connected with the filtering apparatus. For this purpose the bottle, having a capacity of a quart or more, should be provided with a rubber stop- per through which two short tubes are passed. One of these is con- nected with the water supply and the other with the filter. Of course this is only practicable when a water supply with sufficient pressure is available. FIG. 32. As a rule, filtration cannot be substituted with advantage for ster- ilization by heat in the preparation of culture media. Albuminous liquids pass through the filter with difficulty, and the process of sterilization by discontinued heating will usually prove more satis- factory than filtration, which requires extreme precautions to pre- vent accidental contamination of the filtered liquid. Moreover, the filter may change the composition of the medium passed through it by preventing the passage of colloid and albuminous material in so- lution. Thus, in an attempt to separate blood corpuscles from the serum by filtration through a Chamberland filter, the writer obtained a transparent liquid which did not coagulate by heat — i. e. , the albu- minous constituents of the serum did not pass through the filter. VII. CULTURES IN LIQUID MEDIA. PRIOR to the introduction of gelatinous media by Koch in 1881 f cultures were made in various organic liquids, and these are still largely used, being for certain purposes preferable to solid media. The method of preparing and sterilizing the flesh infusions and other organic liquids commonly used has already been given. We are here concerned with the various modes of using these nutritive liquids in cultivating bacteria. Flasks and tubes of various forms have been employed by differ- ent investigators, but the most useful receptacle for liquid as well as for solid culture media is the ordinary test tube. These are care- fully cleaned, plugged with a cotton air filter, sterilized in the hot-air oven at 150° C., and are then ready to receive the filtered liquid. Usually the tube should not be filled to more than one-third to one- half of its capacity. Sterilization of the culture liquid is then effected by placing the tubes in the steam sterilizer for half an hour on three successive days. Before using, the tubes should be placed for a few days in an incubating oven at 30° to 35° C. to test the sterilization. This is especially important with liquid media, for if a single living spore is present it may give rise to an abundant progeny, which will be distributed through the liquid in association with the species which has been planted. In solid cultures, on the contrary, such a spore would give rise to a colony, which by its locality and characters of growth would probably be recognized as different from the species planted, and consequently accidental. This is the great danger in the use of liquid media ; imperfect sterilization, or accidental contami- nation by atmospheric germs, may lead the inexperienced student into serious errors resulting from the assumption that the micro- organisms present in his cultures are all derived from the seed he planted. On 1h«» other hand, liquid media are more convenient than solid when it is t lie intention to isolate by filtration the soluble products of hartrrial growth; for injection into animals to test pathogenic power; for experiments on the germicidal or antiseptic power of chemical agents, etc. CULTURES IN LIQUID MEDIA. 61 For larger quantities of liquid than can be held in an ordinary test tube the small flasks with a flat bottom, known as Erlenmeyer flasks, are very convenient (Fig. 33). In his earlier researches Pasteur used flasks and tubes of various forms, which served a useful purpose, but have been displaced in his laboratory by the simpler form of apparatus shown in Fig. 34. This is a little flask having a cover which is ground to fit the neck. This cover is drawn out above into a narrow tube which admits oxygen to the flask through a cotton air filter. To obtain access to the interior of the flask for the purpose of introducing bacteria to start a culture, or to obtain material for microscopical examina- tion, the cover is detached at the ground joint by a gentle twisting motion. There is much less danger that a sterile culture liquid will become FIG. 33. FIG. 34. contaminated during the momentary removal of the cover from one of these little flasks, or of the cotton plug from a test tube, than is usually supposed. Abundant laboratory experience demonstrates that such contamination by bacteria floating in the atmosphere rarely occurs. The spores of mould fungi are commonly more abundant in the air, but even these do not very frequently fall into the culture liquid when the tube is opened to inoculate it with the bacteria it is proposed to cultivate. This inoculation is best made with a platinum wire, bent into a loop at the free extremity, and sealed fast into the end of a glass rod (Fig. 35). This is sterilized in the flame of a Bunsen burner or alcohol lamp by bringing the platinum wire to a red heat and passing the end of the glass rod which carries it through the flame several times. With this instrument we may transfer a little drop from a culture to the sterile fluid in another frt CULTURES IX LIQUID MEDIA. tube for the purpose of starting a new culture. Or we may start a pure culture from a drop of blood taken from the veins of an animal which has been inoculated with anthrax, or any similar infectious disease in which the blood is invaded by a bacterial parasite. But if we have not a pure culture to start with our liquid media do not afford us the means of obtaining one ; and if two or more bacteria which resemble each other in their morphology are associated in such a culture we cannot differentiate them, and are likely to infer that we have a pure culture of a single microorganism when this is not really the case. But if we have pure stock to start with we may maintain pure cultures in liquid media without any special difficulty. Various characters of growth, etc., are to be observed in culti- vating different microorganisms in liquid media. Thus some grow at the surface in the form of a thin film or membranous layer — " my- coderma " — while others are distributed uniformly through the liquid, rendering it opalescent or more or less milky and opaque ; others, again, form little flocculi which are suspended in the transparent Fro. 35. fluid. Usually, when active growth has ceased, the bacteria fall to the bottom of the tube as a more or less abundant, white or colored, pulverulent or glutinous deposit. In some cases the liquid is colored with a soluble pigment formed during the growth of the bacteria, and usually this is formed most abundantly at the surface, where there is free access of oxygen. The reaction of the medium is often changed as a result of the growth of bacteria in it. From being neu- tral it may become decidedly alkaline or acid in its reaction. These changes may be observed by adding a litmus solution before sterili- zation of the culture medium, and observing the change of color when an acid-producing bacterium is under cultivation. The re- ducing power of bacteria upon various aniline colors may also be MU died ; also their power to break up various organic substances shown by the evolution of gas or other volatile products which may be collected, or by substances which remain in solution and can !><> studied by ordinary chemical methods. Drop Cultures. — When we desire to study the life history of a microorganism and to witness its development from spores, for ex- ample, its motions, etc., the method of cultivation in a hanging drop CULTURES IN LIQUID MEDIA. 63 of culture fluid, attached to a thin glass cover and suspended over a circular excavation ground out of a glass shoe, is very useful. Such a drop culture may be left under the microscope and kept under observation for hours or days. pieveat the inoculation of the drop of culture liquid with any other bacteria than those which are to be studied. The smiJMdk form of moist chamber for drop cultures consists of an ordinary glass shoe having a concave depression, about fifteen in diameter, ground out in its centre. This and the thin r. having been sterflhed by exposure in the hot-air oven at ISO0 CL for an hoar or more, or by passing them through the flame of an alcohol lamp, are ready for use. The cover glass is held in sterile forceps, and a little drop of the culture fluid containing the bacterium to be studied is transferred to its centre by means of the pfatjnmn loop heretofore described. It is best to spread the drop out as thin as possible, and it may be inoculated, from a pure cul- 36) after it has been placed upon he hollow place in the glass to prevent the entrance of air and attach EtOa VMBBIM HOMnd (he mtuspm of flba by attaching a glass to the centre of a glass elide by In Ranvier s moist chamber there is a central eminence sur- by a groove ground into the glass slide, and the drop of above. Tins affords a more satisfactory view under the micro- TheA*tkor*C«lt«ri> JfefltodL— In a paper read at 1681, the writer described a method of conducting culture 64 CULTURES IN LIQUID MEDIA. berg's bulbs/' as they are sometimes called, are that a culture me- dium may be preserved in them indefinitely and that they are easily transported from place to place; whereas test tubes, Pasteur's flasks, and similar receptacles must be kept upright, and after a time the culture liquid in them is changed in its composition by evaporation. They are also liable to be contaminated by the entrance of mould fungi when kept in a damp place. The spores of these fungi, falling upon the surface of the cotton air filter, germinate, and the myce- lium grows down through the cotton into the interior of the tube, where a new crop of spores is quickly formed. It is, therefore, a convenience to have sterile culture liquids always ready for use in a receptacle which can be packed in a box and transported from place to place ; but for every-day use in the laboratory the ordinary Fio. 37. test tube, with its cotton air filter, is the most economical and conve- nient receptacle for culture liquids as well as for solid media. With reference to the method of making and using these little flasks, I quote from a paper published in the American Journal of the Medical Sciences in 1883 :J The culture flasks employed contain from one to four fluidrachms. They are made from glass tubing of three- or four- tenths inch diameter, and those which the writer has used in his numerous experiments have all been ** home-made." It is easier to make new flasks than to clean old ones, and they are thrown away after being once used. Bellows operated by foot, and a flame of considerable size — gas is preferable — will be required by one who proposes to construct these little flasks for himself.9 After a little practice they are made rapidly ; but as a large number are required, the time and labor expended in their preparation are no slight matter. After blowing a bulb at the extremity of a long glass tube, of the diameter mentioned, this is provided with a slender neck, drawn out in the flame, and the end of this 1 " The Germicide Value of Certain Therapeutic Agents," op. cit., vol. clxx. " A glass-blower ought to make them for two or three dollars per hundred. CULTURES IN LIQUID MEDIA. 65 is hermetically sealed. Thus one little flask after another is made from the same piece of tubing- until this becomes too short for further use. To intro- duce a culture liquid into one of these little flasks, heat the bulb slightly, break off the sealed extremity of the tube and plunge it beneath the surface of the liquid (Fig. 37). The quantity which enters will of course depend upon the heat employed and the consequent rarefaction of the enclosed air. Ordinarily the bulb is filled to about one-third of its capacity with the cul- ture liquid, leaving it two-thirds full of air for the use of the microscopic plants which are to be cultivated in it. ... Sterilization is effected by heat after the liquid has been introduced and the neck of the flask hermetically sealed in the flame of an alcohol lamp. Sterilization may be effected by boiling for an hour in a bath of paraffin or of concentrated salt solution, by which a temperature considerably above that of boiling water is secured. The writer is in the habit of preparing a considerable number of these flasks at one time, and leaving them, in a suit- able vessel filled with water, for twenty- four hours or longer on the kitchen stove.1 To inoculate the liquid contained in one of these little flasks with mi- croorganisms from any source, the end of the tube is first heated to destroy germs attached to the exterior; the extremity is then broken off with steril- ized (by heat) forceps; the bulb is very gently heated, so as to force out a little air, and the open end is plunged into the liquid containing the organ- ism to be cultivated (or into a vein, or one of the solid viscera of an animal dead from an infectious germ disease, such as anthrax). Inoculation from one tube to another may also be effected by means of the ordinary platinum wire needle. Before the introduction of Koch's plate method for isolating bac- teria in pure cultures, certain methods had been proposed, and em- ployed to some extent, which at present have a historical value only. Thus Klebs (1873) proposed to take from a first culture in which two or more species were associated a minute quantity, by means of a capillary tube, and with this to inoculate a second culture. By re- peating this procedure several times he expected to exclude all except the species which was present in the greatest abundance and which multiplied most rapidly in the medium employed. The method by dilution, first employed with precision by Brefeld (1872) in obtaining pure cultures of mould fungi, and subsequently by Lister for the isolation of bacteria, consists in so diluting a minute quantity of the mixed culture that the number of bacteria in the dilu- tion may be less than one for each drop of the liquid. If now a single drop be added to each of a series of tubes containing a small quantity of sterile bouillon, some of the inoculations made may give a pure culture, as the drop may have contained but a single vege- tative cell. Another method of obtaining a pure culture in liquid media, when several microorganisms are associated which have a different ther- 1 Where a steam sterilizer is at hand they will be most conveniently sterilized in the usual way, by subjecting them to the boiling temperature for an hour at a time on three successive days. 5 66 CULTURES IN LIQUID MEDIA. mal death-point, consists in the application of heat and thus destroy- ing all except the most resistant species. This method is especially applicable when one of the species, only, forms spores. By subject- ing the mixed culture to a temperature which is sufficient to destroy all the vegetative cells in it, the more resistant spores are left and, under favorable conditions, may subsequently vegetate and give us a pure culture of the species to which they belong. Fermentation. — The development of certain bacteria is attended with an evolution of gas, especially in media containing grape sugar or glycerin. For the determination of the quantity and kind of gas produced by a given micro- organism the fermentation tube recommended by Theobald Smith has special advantages. This is a bent tube (Eihorn's) supported upon a glass base as shown in the accompanying figure taken from the catalogue of Eimer & Amend. The graduation shown upon the up- right arm is not essential for ordinary labora- tory work. A liquid culture medium containing one to two per cent of grape sugar is usually used. This is introduced into the upright arm of the fermentation tube, where it is held by atmospheric pressure. A cotton plug is placed in the opening of the short and bulbous arm of the tube, which is intended as a receptacle for the culture liquid when it is forced out of the closed arm by the accumulation of gas at its upper extremity. Fio. 38. VIII. CULTURES IN SOLID MEDIA. THE introduction of solid culture media in 1881 by the famous German bacteriologist, Robert Koch, inaugurated a new era in the progress of our knowledge relating to the bacteria. His methods enable us to obtain pure cultures with ease and certainty, and to study the morphological and biological characters of each species free from the complications which led to so much error and confusion before these methods were introduced. We have already given an account of the method of preparing and sterilizing the various solid culture media, and are here concerned with the manner in which they are used and the special advantages which they afford. Koch's flesh-peptone-gelatin, which contains ten per cent of gelatin, is a transparent jelly which liquefies at from 22° to 24° C. It is a favorable culture medium for a great number of bacteria, and many species show de- finite characters of growth in this medium which serve to differentiate them. One of the most prominent of these characters depends upon the fact that some bacteria liquefy gelatin and others do not. This is made apparent when we make stick cultures — also called "stab cultures." This is the usual manner of inoculating a solid culture medium, and is illustrated in Fig. 39. A platinum needle, consisting of a piece of platinum wire inserted into a glass rod which serves as a handle, is passed through the flame of an alcohol lamp to sterilize it. When cooled, which occurs very quickly, the point is introduced into the ma- terial containing the bacteria to be planted in the gelatin medium. We may obtain our seed for a pure culture FlG ^ from a single colony, from another stick culture, from the blood of an infected animal, etc. The point of the needle is then carried into the sterilized jelly, as shown in the figure, care being taken to introduce it in the central line and in a direction parallel 68 CULTURES IN SOLID MEDIA. with the sides of the tube. It is best always to hold the tube in- verted during the inoculation, and not to remove the cotton air filter until we are ready to make it. The cotton plug is then returned to its place and the platinum needle again brought to a red heat to destroy any bacteria which remain attached to it. Sometimes it is an advantage to have the culture medium with a FIG. 40. sloping surface, as shown in Fig. 40. We may then draw the nee- dle over the surface in a longitudinal direction, and by this means distribute the seed in a line along which development will take place. The characters of growth in these stick cultures in gelatin are very various. Non-liquefying bacteria may grow only on the sur- face, as at a, Fig. 40A ; or both on the surface and along the line of puncture, as at b; or only at the bottom, as at c. In the first case the microorganism is aerobic— that is, it requires oxygen, and grows only in the presence of this gas. In the second case it is not strictly aerobic, but may grow either in the presence of oxygen CULTURES IN SOLID MEDIA. 69 or in its absence — a facultative anaerobic. In the third case the microorganism is an anaerobic, which cannot grow in the presence of oxygen, and consequently does not grow upon the surface of the culture medium or along the upper portion of the line of puncture. Again, we have differences as to the character of growth upon the surface or along the line of puncture. The surface growth may be a little mass piled up at the point where the needle entered the gela- tin ; or it may form a layer over the entire surface, and this may be thin or thick, dry or moist, viscid or cream-like, and of various colors — green, blue, red, or yellow, of different shades — or more fre- quently of a milk-white color. -JC k The growth along the line of puncture also differs greatly with different species. We may have a number of scattered spherical colonies (a, Fig. 41), and these may be translucent or opaque ; or we may have little tufts, like moss, projecting from the line of puncture (6, Fig. 41) ; or slender, filamentous branches may grow out into the gelatin (c, Fig. 41). The liquefying bacilli also present different characters of growth. Thus liquefaction may take place all along the line of puncture, forming a long and narrow funnel of liquefied gelatin (a, Fig. 42) ; or we may have a broad funnel, as at b ; or a cup-shaped cavity, as at c; or the upper liquefied portion may be separated from that which is not liquefied by a horizontal plane surface, as at d. fO CULTURES IN SOLID MEDIA. The characters of growth in agar-agar jelly are not so varied, but this medium possesses the advantage of not liquefying at a tem- perature of 35° to 38° C., which is required for the development of certain pathogenic bacteria. Variations in mode of growth are also manifested in nutrient agar similar to those referred to as pro- duced by non-liquefying bacteria in flesh-peptone-gelatin. These relate to the surface growth and to growth along the line of punc- ture. One character not heretofore mentioned consists in the for- mation of gas bubbles in stick cultures either in gelatin or agar. Colonies. — If we melt the gelatin or agar in a test tube, pour the liquid medium into a shallow glass dish previously sterilized, FIG. 42. and allow it to cool while properly protected by a glass cover, we will have a broad surface of sterile nutrient material. If now we ex- pose it to the air for ten or fifteen minutes, and again cover it and put it aside for two or three days at a favorable temperature, we can scarcely fail to have a number of colonies upon the surface of the culture medium, which have been developed from atmospheric germs which were deposited upon it during the exposure. Each of these colonies, as a rule, is developed from a single bacterium or spore, and consequently the little mass, visible to the naked eye, which we call a colony, is a pure culture of a particular species. In this ex- periment we are more apt to have colonies of mould f uiigi than of bacteria, but the principle is the same, viz. , that a colony developed fn>ni a single i^rm is a pure culture. By touching our platinum CULTURES IN SOLID MEDIA. 71 needle, then, to such a colony, which is quite independent of, and well separated from, all others, we may make a stick culture in gela- tin or agar, and preserve the pure culture for further study. This is a most important advantage which pertains to the use of solid culture media. It is a singular fact that, as a rule, colonies of bac- teria which lie near each other do not grow together, but each re- mains distinct. If there are but few colonies, each one, haying plenty of room, may grow to considerable size ; if there are many and they are crowded, they remain small, but are still independent colonies. Now, these colonies differ greatly in their appearance and char- acters of growth, according to the species (Fig. 43). Some are spherical, and these may be translucent or opaque, or they may have an opaque nucleus surrounded by a transparent zone. Again, the Fro. 43.— Colonies of Bacteria. outlines may be irregular, giving rise to amoeba-like forms, or to a fringed or plaited margin, or the form may be that of a rosette, etc. ; or the colony may appear to be made up of overlapping scales or masses, or of tangled filaments; or it may present a branching growth. In the case of liquefying bacteria, when the colonies have developed in a gelatin medium they commonly do not at once cause liquefaction of the gelatin, but at the end of twenty-four hours or more the gelatin about them commences to liquefy and they are seen in a little funnel of transparent liquefied gelatin ; or in other cases little opaque drops of liquefied gelatin are seen, which, as the liquefaction extends, run together. All of these characters are best studied under a low-power lens, with an amplification of five to twenty diameters ; and by a careful observation of the differences in the form and development of colonies we are greatly assisted in the differentiation of species. Single, isolated colonies do not always contain a single species, for they are not always developed from a single cell. We may have 72 CULTURES IN SOLID MEDIA. deposited upon our plate, exposed as above described, a little mass of organic material containing two or more different bacteria, and this would serve as the nucleus of a colony from which we could not obtain a pure culture. Koch's Plate Method.— In the experiment above described, colonies were obtained from air-borne germs which were deposited upon the surface of our gelatin medium. By Koch's famous " plate method " we obtain colonies of any particular microorganism which we desire to study, or of two or more associated bacteria which we desire to study separately in pure cultures. Evidently, when we have obtained separate colonies of different bacteria upon the sur- face of a solid culture medium, we can easily obtain a pure culture of each by inoculating stick cultures from single colonies. To obtain separate colonies we resort to the ingenious method of Koch. Three test tubes containing a small quantity of nutrient gelatin (or of agar) are commonly employed. The tubes are num- bered 1, 2, and 3. The first step consists in liquefying the nutrient jelly by heat, and it will be well for beginners to place the tubes in a water bath having a temperature of about 40° C. (104° F.) for the purpose of keeping the culture material liquid, and at the same time at a temperature which is not high enough to destroy the vitality of the bacteria which are to be planted. We next, by means of a platinum- wire loop or the platinum needle used for stick cultures, introduce into tube No. 1 a small amount of the culture, or material from any source, containing the bacteria under investigation. Care must be taken not to introduce too much of this material, and it must be remembered that the smallest visible amount may contain many millions of bacteria. The reason for using three tubes will now be apparent. It is usually impossible to introduce a few bac- teria into tube No. 1, but we effect our object by dilution, as follows : With the platinum- wire loop we take up a minute drop of the fluid in tube No. 1, through which the bacteria have been distributed by stirring, and carry it over to tube No. 2. Washing off the drop by stirring, we may repeat this a second or third time — this is a matter of judgment and experience ; often it will suffice to carry over a single ose (the German name for the platinum- wire loop). Next we carry over one, or two, or three ose from tube No. 2 to tube No. 3. By this procedure we commonly succeed in so reducing the num- ber of bacteria in tube No. 3 that only a few colonies will develop upon the plate which we subsequently make from it; or it may happen that the dilution has been carried too far and that no colonies de- velop upon the plate made from this tube, in which case we are likely to get what we want from tube No. 2. The next step is to pour the liquid gelatin upon sterilized glass plates, which are num- CULTURES IN SOLID MEDIA. 73 bered to correspond with the tubes. The plates used by Koch are from eight to ten centimetres wide and ten to twelve centimetres long. They must be carefully cleaned and sterilized in the hot-air oven, at 150° C., for two hours. They may be wrapped in paper be- fore sterilization, or placed in a metal box especially made for the purpose. In order that the liquid gelatin may be evenly distributed upon the plate the apparatus shown in Fig. 44 is used. This con- sists of a glass plate, g, supported by a tripod having adjustable feet. By means of the spirit level / the glass plate is adjusted to a hori- zontal position. A sterilized glass plate is placed in the glass tray, shown in the figure, and the gelatin from one of the tubes is care- fully poured upon it and distributed upon its surface with a steril- ized glass rod, care being taken not to bring it too near the edge of the plate. The glass tray in then covered until the gelatin has cooled sufficiently to become solid, after which plate No. 1 is re- moved and plates Nos. 2 and 3 are made in the same way. In Fio.44. order to save time it is customary to fill the glass tray shown in the figure with ice water, to place a second glass support upon it, and upon this the sterilized glass plate upon which the liquid gelatin is poured. This is protected by a glass cover, as before, until the gela- tin becomes solid. The three plates, prepared as directed, are put aside in a glass jar of the form shown in Fig. 44, one being supported above the pther by a bench of sheet zinc or glass. Petri's Dishes. — A modification of the plate method of Koch, which has some advantages, consists in the use of three small glass dishes of the same form as the larger one used by Koch to contain the plates. These dishes of Petri are about ten to twelve centime- tres in diameter and one to 1.5 centimetres high, the cover being of the same form as the dish into which the gelatin is poured. These dishes take less room in the incubating oven than the larger glass jar used in the plate method, and they do not require the use of a levelling apparatus. The colonies also may be examined and counted, if desired, without removing the cover, and consequently 74 CULTURES IN SOLID MEDIA. without the exposure which occurs when a plate prepared by Koch's method is under examination. In agar-agar cultures or in gelatin cultures of non-liquefying bacteria made in Petri's dishes, we may examine and count colonies, without removing the cover, by inverting the dish. In pouring the liquefied gelatin from the test tubes in which the dilution has been made into sterilized Petri's dishes, care must be taken to first sterilize the lip of the test tube by passing it through the flame of a lamp. We may at the same time burn off the top of the cotton plug, then remove the remaining portion with forceps, when the lip has cooled, for the purpose of pouring the liquid into the shallow dish. Von Esmarch' s Roll Tubes. — Another very useful modification of Koch's plate method is that of von Esmarch. Instead of pouring the liquefied gelatin or agar medium upon plates or in shallow FIG. 45. dishes, it is distributed in a thin layer upon the walls of the test tube containing it. This is done by rotating the tube upon a block of ice or in iced water. Esmarch first used a tray containing iced water, and to prevent the wetting of the cotton filter a cap of thin rubber was placed over the end of the tube. It is more convenient to turn the tubes upon a block of ice having a horizontal flat surface, in which a shallow groove is first made by means of a test tube con- taining hot water (Fig. 45). Or, in the winter, we may turn the tube under a stream of cold water from the city supply — i.e., from a faucet in the laboratory. A little practice will enable the student to distribute the culture medium in a uniform layer on the walls of the test tube, and as soon as it is quite solidified these may be placed a>i«lr for tin- development of colonies from the bacteria which had l>eeii introduced. When roll tubes are made from the agar jelly it is l»est to place the tubes in a nearly horizontal position, for if placed upright at once the film of jelly is likely to slip from the walls of the CULTURES IN SOLID MEDIA. 75 tube. This is due to the fact that a little fluid is pressed out of the jelly, probably by a slight contraction while cooling. If the tubes are slightly inclined from the horizontal the film does not slip and the fluid accumulates at the bottom. After a day or two they may be placed in an upright position. These roll tubes possess several advantages. They are quickly made and take but little space in the incubating oven, and the film of jelly is protected from contamination by atmospheric germs. When colonies have formed we may examine them through the thin walls of the tube, either with a pocket lens or a low-power objective. In making a stick culture from a single colony in one of these roll tubes, we invert the tube, remove the cotton air filter, and pass the point of a sterilized platinum needle up to the selected colony. In the same way we obtain material for microscopical examination. Streak Cultures. — In his earlier experiments with solid culture media Koch made " streak cultures" by drawing the point of a plati- num needle, charged with bacteria, over the surface of a gelatin or agar plate ; and this method is still useful in certain cases. If we draw the needle over the moist surface several times in succession the greater number of bacteria will be deposited in the first streak, and in the second or third single cells are likely to be left at such intervals from each other that each will develop an independent colony. If the streaks were made with impure stock we may thus succeed in getting separate colonies of the several bacteria contained in it, so that this method may be employed for obtaining pure cul- tures. But for this purpose it is much inferior to the plate method, and it is chiefly used for observing the growth of bacteria on the sur- face of solid culture media. Thus we commonly make a streak upon the surface of cooked potato or solidified blood serum in studying the development of various bacteria on these culture media. Cultures upon Blood Serum. — The use of blood serum as a solid medium is practically restricted to stick cultures and streak cultures, for we cannot substitute it for the gelatin and agar media in making plates and roll tubes. This is because it only becomes solid at a temperature which would be fatal to most bacteria (70° C.), and when once made solid by heat cannot again be liquefied. Its use is, therefore, restricted mainly to the cultivation of bacteria for which it is an especially favorable medium. It may be used, however, in combination with a gelatin or agar medium. For this purpose it is most conveniently kept in a fluid condition in the little flasks hereto- fore described (" Sternberg's bulbs "). The gelatin or agar jelly in test tubes is liquefied by heat and cooled in a water bath to about 40° C. The desired amount of ste- rile blood serum is then forced into each tube by passing the slender 76 CULTURES IN SOLID MEDIA. neck of the little flask along the side of the cotton filter (see Fig. 46) and applying gentle heat to the bulb. The slender neck is first ste- rilized by passing it through a flame, and the point is broken off with sterile forceps. After inoculating the liquefied medium in the test tubes in the usual manner we may make plates or roll tubes. Cultures on Cooked Potato.— The method of preparing pota- toes for surface cultures has already been given (page 48). It was in using them that Koch first got his idea of the importance of solid media, which led to his introduction of the use of gelatin and agar- agar and the invention of the plate method. By means of streak FIG. 46. cultures upon potato he had succeeded in obtaining isolated colonies and pure cultures. We now use the potato chiefly for the purpose of differentiating species. Some bacteria grow on the surface of cooked potato and some do not. Those which do present various characters of growth. Thus we have differences as to color, as to rapidity of growth, as to the character of the mass formed — thick or thin, viscid, moist or dry, restricted to line of inoculation or ex- tending over the entire surface, etc. Instead of using a cut section of the potato in the manner here- tofore described, we may make a puree by mashing the peeled and cooked tubers and distributing the mass in Erlenmeyer flasks. After CULTURES IN SOLID MEDIA. 77 thorough sterilization by steam the culture medium is ready for use. In the same way other vegetables, or bread, etc. , may be used for special purposes, and especially for cultures of the mould fungi. Potatoes usually have a slightly acid reaction, and on this ac- count certain bacteria will not grow upon them. This acid reaction is not constant and differs in degree, and as a result we may have decided differences in the growth of the same species upon different potatoes. To overcome this objection the writer has sometimes neu- tralized the cones of potato in test tubes (see Fig. 21, page 48) by first boiling them in water containing a little carbonate of soda. The liquid is poured off after they have been in the steam sterilizer for half an hour, and they are returned for sterilization. Salomonson's Method of cultivation in capillary tubes has a his- torical value only since the introduction of Koch's plate method. The following modifications of Koch's plate cultures have recently been introduced : Kruse (1894) pours the liquefied gelatin or agar into Petri dishes, and after it is solidified brushes the surface with a sterilized camel's- hair brush which has been dipped into water containing in suspen- sion—properly diluted— the bacteria to be studied. By this procedure surface colonies only are obtained. Von Freudenreich (1894) prefers to pour the contents of the test tube upon the surface of the sterile medium, in Petri dishes. The fluid is allowed to run off by placing the Petri dish in a vertical position, and this is subsequently placed in the incubating oven in an inverted position — i.e., with cover below. To obtain satisfactory plates with well-separated, superficial colonies it may be necessary to use two or three dilutions, made in sterilized water in the usual way — i.e., from one tube to another, by means of the platinum wire having a loop at its extremity. IX. CULTIVATION OF ANAEROBIC BACTERIA. PASTEUR (1861) first pointed out the fact that certain species of bacteria not only grow in the entire absence of oxygen, but that for some no growth can occur in the presence of this gas. Such bacteria are found in the soil, and in the intestines of man and the lower ani- mals. The cultivation of "strict anaerobics" calls for methods by which oxygen is excluded. The "facultative anaerobics '' grow Fio. 47. FIG. 48. either in the presence or absence of oxygen. There are various gra- dations in this regard, from the strictly aerobic species which re- • piiro an abundance of oxygen and will not grow in its absence, to the strictly anaerobic species which will not grow if there is a trace of oxygen in the medium in which we propose to cultivate them. Among the most interesting pathogenic bacteria which are strictly anaerobic are the bacillus of tetanus, the bacillus of malignant and tin- ku-illus of symptomatic anthrax* CULTIVATION OF ANAEROBIC BACTERIA. 79 If we make an inoculation of one of the species which is not strictly anaerobic into a test tube containing nutrient gelatin or agar- agar, we may have a development all along the line of puncture, and this may be more abundant below, as in Fig. 47. But when we make a long stick culture with a strict anaerobic the development occurs only near the bottom of the line of puncture (Fig. 48). We may then, if we have a pure culture to start with, propagate these anaerobic bacilli in long stick cultures. It is best to use tubes which have been recently sterilized, as boiling expels the air from the culture medium ; and a very slender needle should be used in making the inoculation. To prevent the absorption of oxygen a layer of sterilized olive oil may be poured into the tube after the in- oculating puncture has been made, or it may be filled up with agar jelly which has been cooled to about 40° C. Roux has proposed to prevent the absorption of oxygen by the culture medium by plant- ing an aerobic bacterium — Bacillus subtilis — upon the surface, after making a long stick culture with the anaerobic species. The agar jelly is first boiled and quickly cooled ; the inoculation is then made with a slender glass needle ; some sterile agar cooled to 40° C. is poured into the tube, and when this is solid the aerobic species is planted upon the surface. The top of the test tube is then closed hermetically and it is placed in the incubating oven. The aerobic species exhausts the oxygen in the upper part of the tube by its growth on the surface of the culture medium, and the anaerobic species grows at the bottom of the tube. To obtain material for a new culture or for microscopical examination the test tube is broken near its bottom. Cultures in liquid media may be made by exhausting the air in a suitable receptacle or by displacing it with hydrogen gas. The first-mentioned method has been largely used in Pasteur's laboratory, but methods in which hydrogen gas takes the place of atmospheric air in the culture tube are more easily applied and require simpler apparatus. The flask shown in Fig. 49 may be used in connection with an air pump. The sterile culture liquid is first introduced into a long-necked flask and inoculated with the anaerobic bacillus to be cultivated. The neck of the flask is then drawn out in a flame at c. The open end is then connected with a Sprengle's pump or some other apparatus for exhausting the air. The flask is placed in a water bath at 40° C. , which causes ebullition at the diminished pres- sure, and the exhaustion is continued for about half an hour. The narrow neck is then sealed at c by the use of a blowpipe flame. The flask shown in Fig. 49, which can be made from a test tube, may also be used in connection with a hydrogen apparatus. In this case a slender glass tube is passed into the flask, as shown in Fig. 80 CULTIVATION OF ANAEROBIC BACTERIA. 50, and this is connected with a hydrogen apparatus by a rubber tube, The hydrogen is allowed to bubble through the culture liquid in a full stream for, ten to fifteen minutes, in order that all of the oxygen in the flask may be removed by displacement. Then. while the gas is still flowing, the flask is sealed at a with a blow- pipe flame, the hydrogen tube being left in position and melted fast to the flask. Some little #kill is required in the successful perform- ance of the last step in this procedure, and it will be easier for those Fio. 49. TIG. 51. who are not skilful in the use of the blowpipe to use Salomonson's tube, shown in Fig. 51. In this, hydrogen is admitted through the arm b, and escapes through the cotton plug a. The vertical tube is sealed at c while the^as is flowing, and then the horizontal tube at b. /•V///AW* Method. — Instead of these tubes specially made for the purpose, an ordinary test tube may be used, as recommended by Krankel. This is closed by a soft rubber cork through which two Ljlass tuhes pass— one, reaching nearly to the bottom of the test tube, CULTIVATION OF ANAEROBIC BACTERIA. 81 for the admission of hydrogen, which passes through the liquefied culture medium ; and the other a short tube for the escape of the gas. The outlet tube is sealed in the flame of a lamp while the gas is freely flowing, and after sufficient time has elapsed to insure the complete expulsion of atmospheric oxygen — which, when the hydro- gen flows freely, requires about four minutes (Frankel) — melted paraffin is applied freely to the rubber stopper to prevent leakage of the hydrogen and entrance of oxygen. A roll tube may then be made after the manner of Esmarch, and, after colonies have de- veloped, the anaerobic culture will appear as shown in Fig. 52. To isolate anaerobic bacteria in pure cultures it is well to make a FIG. 58. FIG. 53. series of dilutions as heretofore described for aerobic cultures ; we will then usually obtain isolated colonies in tube No. 2 or No. 3 of a series, and by removing the rubber stopper we may transplant bac- teria from these colonies to deep stick cultures in nutrient gelatin or agar. The Writer's Method. — The following simple method has been successfully employed by the writer: Three Esmarch roll tubes are prepared as is usual for aerobic cul- tures. The cotton air filter, or a portion of it, is then pushed down the tubes for a short distance, as shown at a, Fig. 53. A section of a soft rubber stopper carrying two glass tubes is then pushed into the 6 82 CULTIVATION OF ANAEROBIC BACTERIA. test tube for about half an inch, as shown at b, Fig. 53. The space above the cork is then filled with melted sealing wax, which I have found to prevent leakage better than paraffin, which contracts upon cooling. The test tube is inverted while hydrogen is passed through the tube c, and by reason of its levity the gas quickly passes through the cotton air filter and displaces the oxygen in the test tube (Fig. 54). After allowing the gas to flow for a few minutes the outlet tube is first sealed in a flame and then the inlet tube. As the cotton filter is interposed between the rubber stopper and the culture mate- rial, no special precautions need be taken for the sterilization of the rubber cork and the glass tubes which it carries. Fio. 54. FIG. 55. This method is more convenient than that previously described, and the only objection to it is that the oxygen is not completely re- moved from the film of solid gelatin or agar attached to the walls of the test tube. But by passing the hydrogen for a long time it would seem that by diffusion the oxygen remaining in this thin layer would be gotten rid of. At all events, this method will serve for all except the very strict anaerobics. Method of Esmarch.—ThQ following method has been proposed by Esmarch : Three roll tubes are made in the usual way, and into these liquid gelatin, that is nearly cooled to the point of becoming solid, is poured. This fills the tube without melting the layer of CULTIVATION OP ANAEROBIC BACTERIA. 83 gelatin, previously cooled upon its walls, which contains the bacteria under investigation. When the anaerobic colonies have developed the test tube must be broken to get at them, or the cylinder of gela- tin may be removed by first warming the walls of the tube. Another method, recommended by Liborius, consists in distri- buting the bacteria in test tubes nearly filled with nutrient gelatin or agar which has been recently boiled to expel air. Colonies of anaero- bic bacteria will develop near the bottom of such a tube, while the aerobic species will only grow near the surface. The cylinder of jelly is removed by heating the walls of the tube, and sections are } a FIG. 56. made with a sterilized knife for the purpose of "obtaining material from individual colonies for further cultures, etc. Koch and his pupils are in the habit of testing the aerobic char- acter of bacteria in plate cultures by covering the recently made plates with a thin sheet of mica which has been sterilized by heat. The strictly aerobic species do not grow under such a plate ; but, according to Liborius, the exclusion of oxygen is not sufficiently complete for the growth of strict anaerobics. Buchner's Method consists in the removal of oxygen by means of pyrogallic acid. The anaerobic species under investigation is planted in recently boiled agar jelly in a small test tube. This is placed in a larger tube having a tightly fitting rubber stopper, as shown in Fig. 55. The small tube is supported by a bent-wire 84 CULTIVATION OF ANAEROBIC BACTERIA. stand, and in the lower part of the large tube are placed ten cubic centimetres of a ten-per-cent solution of caustic potash, to which one gramme of pyrogallic acid is added. The absorption of the oxygen takes some time, but, according to Buchner, it is finally so complete that strict anaerobics grow in the small tube. In practice, cultivation in an atmosphere of hydrogen will be found the most convenient method, and for this any form of hydro- gen generator may be used. The writer is in the habit of using the form shown in Fig. 56. A perforation a quarter of an inch in diameter is drilled through the bottom of a wide-mouthed bottle. Some fragments of broken glass are then put into the bottle, f orm- FIQ. 57. ing a layer two or three inches thick. Upon this is placed a quan- tity of granulated zinc. This bottle has a tightly fitting cork, through which passes a metal tube having a stopcock. The bottle is placed in a glass jar containing diluted sulphuric acid (one part by weight of sulphuric acid to eight parts of water). The acid, ris- ing through the perforation in the bottom of the bottle, when it comes in contact with the zinc gives rise to an abundant evolution • •I hydrogen, which escapes by the tube a when the stopcock is open. When this is closed the gas forces the acid back from con- tact with the zinc. To remove any trace of oxygen present the #as may be passed through a solution of pyrogallic acid in caustic potash. Evidently plates prepared by Koch's method, or Esmarch roll CULTIVATION OF ANAEROBIC BACTERIA. 85 tubes, may be placed in a suitable receiver and the air exhausted, or hydrogen substituted for atmospheric air. Such an apparatus for hydrogen has been devised by Blucher and is shown in Fig. 57. A glass dish, A, contains a smaller dish, B, which has a diameter of about seven centimetres. The small dish is kept in its position in the centre of the larger one by the wire ring, having three project- ing arms, which is shown in the figure. The culture medium con- taining the anaerobic bacteria to be cultivated is poured into the small dish and the glass funnel D is put in position. This is held in its place by a weight of lead which encircles the neck of the fun- nel at F. A mixture of glycerin and water (twenty to twenty -five per cent) is poured into the dish A to serve as a valve to shut off the atmospheric air from the interior of the funnel D. Hydrogen gas is introduced through the tube E, which is connected by a rub- ber tube with a hydrogen apparatus. A somewhat similar apparatus has been devised by Botkin, in which the hydrogen is admitted beneath a bell jar covering small glass dishes containing the culture medium. We believe that in practice the writer's method (page 81), in which Esmarch roll tubes are first made, will be found more convenient than either of the last- mentioned methods of preserving plates in an atmosphere of hydro- gen ; or roll tubes may be prepared in the way usually practised in cultivating aerobic bacteria, and these may be placed in a suitable receptacle which can be filled with hydrogen. X. INCUBATING OVENS AND THERMO-REGULATORS. THE saprophytic bacteria generally, and many of the pathogenic species, grow at the ordinary temperature of occupied apartments (20° to 25° C.) ; but some pathogenic species can only be cultivated at a higher temperature, and many of those which grow at the " room temperature " develop more rapidly and vigorously when kept in an incubating oven at a temperature of 35° to 38° C. Every bacteriological laboratory should therefore be provided with one or more brood ovens provided with thermo-regulators to maintain a constant temperature. These incubating ovens are made with dou- ble walls surrounding an air chamber. The space between the dou- ble walls is filled with water, which is usually heated by a small gas flame. The gas passes through the thermo-regulator, and its flow is automatically controlled for any temperature to which this is ad- justed. The exterior of the incubating oven is covered with felt or asbestos to prevent the loss of heat by radiation. A simple and cheap form which answers every purpose is shown in Fig. 58. The quadrangular box with double walls should be made of zinc or cop- per. An outer metal door covered with non-conducting material, and an inner door of glass, give access to the interior space ; and a thermometer introduced through an aperture in the top (Fig. 58, b) shows the temperature of this space when the door is closed. The stopcock e permits the drawing off of the water from the space be- tween the double walls, and the glass tube d shows the height of the water, as it is connected with the space containing it. The thermo-regulator passes through an aperture at one side of the oven into the water, the temperature of which controls the flow of gas. The ordinary thermo-regulator is shown in Fig. 59 as manufac- tured by Rohrbeck. A glass receptacle, shaped like an ordinary test tube, has an arm, c, for the escape of the gas, which enters by the bent tube a, which passes through a perforated cork and is ad- justable up and down. Tube a is connected with the gas supply and tube c with the burner by means of rubber tubing. A glass parti- extending downward as a tube, aid to act with great precision. It is a modification of Reichert's FIG. 65. Fio. 66. regulator. Its mode of action will be readily understood by a refe- rence to the figure. A thermo-regulator which gives very accurate results, which ,-nv not influenced by differences in pressure, is that invented by the Fio. 67. writer over twenty years ago. The regulating thermometer may ""tain mercury only, or air and mercury, as shown in the thermo- regulator for gas ( Kifr 50). In the simplest form a large bulb con- aming mercury is used, and a platinum wire is hermetically sealed a glass so as to have contact with the mercury (Fig. (5(1, ft). INCUBATING OVENS AND THERMO-REGULATORS. 91 Another platinum wire passes down the tube of the thermometer, b, and is adjustable for any desired temperature. The gas passes through a valve which is controlled by an electro-magnet. A simple form of valve is shown in Fig. 67. The bent tube a is con- nected with the gas supply by a piece of rubber tubing. The up- right arm of this tube is enclosed in a larger tube, fr, having an out- Fio. 68. FIG. 69. let, e, which is connected with the burner under the incubating oven. The upper end of this larger tube is closed by means of a piece of sheet rubber, which prevents the escape of gas. When this is depressed by means of the lever c, the flow of gas through the valve is arrested. The lever c has attached to it the armature d, and is operated by an electro-magnet under the control of the regu- lating thermometer. 92 INCUBATING OVENS AND THERMO-REGULATORS. When the thermometer is immersed in a water bath the tem- perature of which it is desired to regulate, and the proper electric connections are made, it acts as a circuit breaker. When the de- sired temperature is reached the mercury in the tube of the ther- mometer touches the wire b (Fig. 66), an electric circuit is com- pleted, and the valve is closed, shutting off the gas supply and preventing the temperature from going any higher. When contact is broken in the thermometer tube the valve opens and permits the gas to flow again. A small opening, o (Fig. 67), permits the con- stant flow of a sufficient amount of gas to prevent the flame from being extinguished. In practice, however, it is better to have a small side jet of gas, quite, independent of that which passes through the valve, which burns constantly and relights the principal jet when Fio. 70. the valve is opened. This apparatus is very well adapted for regu- lating the temperature of a water bath with precision, but for gene- ra 1 use in collection with incubating ovens the ordinary gas regu- lator is preferable, on account of the trouble connected with keeping a galvanic battery in order when it is required to act at frequent intervals " on a closed circuit," for weeks and months together. The incubating apparatus of D'Arsonval is shown in Fig. 68. It is a cylindrical vessel of copper having double walls, and is provided with the thermo-regulator of D'Arsonval, by which very accurate regulation is maintained at any desired temperature. In its form this apparatus is not as convenient as are the brood ovens made in the form shown in Fig. 58, with a swinging door which givrs easy access to the interior, which is provided with one or more shelves upon which the cultures are placed. Various modifications INCUBATING OVENS AND THERMO-REGULATORS. 93 of this simple and convenient incubating oven are manufactured by Robrbeck and by Muncke, of Berlin. The apparatus of D'Ar- sonval, and other forms in favor at the French capital, may be ob- tained from Wiesnegg, of Paris. The last-named manufacturer also supplies the incubating oven and thermo-regulator described by Roux (1891). This is shown in Fig. 69. The regulator is formed of two metallic bars, One of steel and the other of zinc ; these are soldered together in the shape of a letter U ; the regulator is seen in position in the cut (Fig. 69). The most dilatable metal (zinc) is on the outside. When the temperature is raised the arms of the U ap- proach each other, and the reverse when it falls. The method by which regulation is effected is shown in Fig. 70. The U-shaped regulator is placed vertically, and one of its branches, A, is firmly fixed to the wall of the incubating oven ; the other, free arm car- ries a horizontal bar which projects through the wall of the incu- bator in an opening which permits it to move freely under the influ- ence of a change in the temperature within. The end of this projecting bar is turned up at a right angle and the screw p passes through it ; this can be fixed at any desired point by means of the nut e. The end of the screw p rests against the stem of a conical brass valve which controls the flow of gas. The valve is closed by a spiral spring and opened by the screw p under the control of the thermo-regulator. In the absence of gas incubating ovens may be heated by a small petroleum lamp, and various devices have been invented for control- ling the temperature. Reichenbach describes an apparatus for this purpose in the Centralblatt fiir Bakteriologie, Vol. XV., p. 847, 1894. Dr. Borden of the U. S. Army has also invented a thermo- regulator to be used in connection with a petroleum lamp. In the absence of any regulating apparatus an incubating oven may be kept at a tolerably uniform temperature by personal supervision — adjusting the flame of the lamp and its distance from the bottom of the oven ac- cording to the changes in the external temperature. For most bac- teria a variation of several degrees is not important, so long as the temperature is not allowed to rise above 37° to 38° C. The typhoid bacillus, the diphtheria bacillus, the anthrax bacillus, the pus cocci, and most saprophytic bacteria grow at the ordinary room temperature, and may therefore be cultivated without any form of incubating oven or thermo-regulator. XL EXPERIMENTS UPON ANIMALS. THE pathogenic power of various bacteria has been demonstrated by injecting pure cultures into susceptible animals. As a rule, the herbivora are more susceptible than the carnivora, and this is per- haps to be explained in accordance with the theory of natural selec- tion. Carnivorous animals often feed upon the bodies of animals which have succumbed to infectious diseases, and upon dead animals in which putrefactive changes have commenced. In their struggles with each other they are wounded by teeth and claws soiled with in- fectious material which would cause a fatal disease if inoculated into the more susceptible herbivorous animals. As this has been going on for ages, we may suppose that, by survival of the fittest, a race tolerance has been acquired. The lower animals have their own in- fectious diseases, some of which are peculiar to certain species and some common to several. Asa rule, the specific infectious disea» - of man cannot be transmitted to lower animals, and man is not sub- ject to the diseases of the same class which prevail among animals. But certain diseases furnish an exception to this general rule. Thus tuberculosis is common to man and several of the lower animals : relapsing fever may by inoculation be transmitted to monkeys ; diphtheria may be transmitted to pigeons and guinea-pigs. On the other hand, anthrax and glanders may be contracted by man as a result of accidental inoculation or contact with an infected animal. Nearly allied species sometimes present very remarkable differ- ences as to susceptibility. Thus the bacillus of mouse septicaemia is fatal to house mice but not to field mice, while, on the other hand. field mice are killed by the bacillus of glanders and house mice are immune from this pathogenic bacillus. The animals most commonly used for testing the pathogenic po \\-rr <>f bacteria are the mouse, the guinea-pig, and the rabbit. Domestic fowls and pigeons arc also useful for certain experiments. The dog and the rat are of comparatively little use on account of their slight susceptibility. EXPERIMENTS UPON ANIMALS. 95 Inoculations are made directly into the circulation through a vein, into the subcutaneous connective tissue, or into one of the serous cavities — usually the peritoneal. The ordinary hypodermic syringe may be used in making injec- tions, but this is difficult to sterilize on account of the leather piston, and complications are liable to arise from its use which it is best to avoid. The best way to sterilize a piston syringe is to wash it thor- oughly with a solution of bichloride of mercury of 1 : 1,000, and then to remove every trace of bichloride by washing in alcohol. But one never feels quite sure that the most careful washing will insure steril- ization, and it is best to use a syringe which may be sterilized by FIG. 71. heat, such as that of Koch, shown in Fig. 71. In this the metal point and glass tube are easily sterilized in a hot-air oven. Fluid is drawn into the syringe and forced out of it by a rubber ball which has a perforation to be covered by the finger. The writer has for some years been in the habit of making injec- tions in animals with an improvised glass syringe. This is made from a piece of glass tubing in the same form as the collecting tubes heretofore described. A bulb is blown at one end of the tube, and the other end is drawn out to form a slender tube which serves as the FIG. 72. needle of the syringe (Fig. 72). By gently heating the bulb in an alcohol lamp and immersing the open end of the capillary tube in the fluid to be injected, this rises into the syringe as the expanded air cools. Having introduced the glass point beneath the skin or into the cavity of the abdomen of the animal to be injected, the contents of the tube are forced out by again heating the bulb by means of a small alcohol lamp. The glass point is easily forced through the thin skin of a mouse or of a young rabbit ; but for animals with a thicker skin it is necessary to cut through, or nearly through, the skin with some other instrument. A small pair of curved scissors answers very well for this purpose. 96 EXPERIMENTS UPON ANIMALS. Generally, in making injections into animals, it is customary to remove the hair for some distance around the point of inoculation with scissors and razor, and then to sterilize the surface by careful washing with a solution of bichloride of mercury. This precaution is necessary in researches in which pathogenic bacteria are being tested, in order to remove any possibility of accidental inoculation with germs other than those under investigation, and, as a conse- quence, a mistaken inference as to the pathogenic action of the spe- cies under investigation. But when we know the specific pathogenic power of a certain microorganism it is hardly necessary to take this precaution, as a few drops of culture will contain millions of the bac- teria, while contamination, if it occurs from the surface of the body, must be by a comparatively small number of bacteria, which are likely to be of a harmless kind which will have no influence on the result of the experiment. Instead of sterilizing the surface, the writer usually clips away a small portion of skin with curved scissors, not cutting deep enough to draw blood, but leaving a bare surface through which the point of the syringe can be introduced with very little danger of carrying Bac- teria into the connective tissue other than those contained in the syringe. In making injections into the peritoneal cavity care must be taken not to wound the liver or the distended stomach. The intestine is not very likely to be wounded, as it slips out of the way. By seizing a longitudinal fold of the abdominal wall and pushing the point of the syringe quite through it, and then releasing the fold and care- fully withdrawing the instrument until the point remains in the cavity, the danger of wounding the intestine will be reduced to a minimum. Injections into the circulation are made by exposing a vein and carefully introducing the needle of the syringe in the direction of tlir blood current. Care must of course be taken not to inject air. In the rabbit one of the large veins of the ear may be conveniently I" -net rated 1,\ t lie point of a hypodermic syringe without any piv- N i« -us dissection. The ear is first washed with a solution of bichloride of mercury or simply with warm water. The animal had better be full d.'tails with ivtVivmv PHOTOGRAPHING BACTERIA. 107 to the technique of making photomicrographs, but append an account of a form of apparatus which we have used with great satisfaction : "Photomicrography by Gaslight. — Those who have had much experience in making- photomicrographs will agree with me that one of the most essen- tial elements of success is the use of a suitable source of illumination. " Without question the direct light of the sun, reflected in a right line by the mirror of a heliostat, is the most economical and, in some respects, the most satisfactory light that can be used. But we cannot command this light at all times and places, and it often happens that, when we are ready to de- vote a day to making photomicrographs, the sun is obscured by clouds or the atmosphere is hazy. Indeed, in some latitudes and at certain seasons of the year a suitable day for the purpose is extremely rare. The use of sun- light also requires a room having a southern exposure and elevated above all surrounding buildings or other objects by which the direct rays of the sun would be intercepted. For these reasons a satisfactory artificial light is ex- tremely desirable. " The oxyhydrogeii lime light, the magnesium light, and the electric arc li^ht have all been employed as a substitute for the light of the sun, and all give satisfactory results. I have myself made rather extensive use of the 'lime light,' and think it the best substitute for solar light with which I am familiar. But to use it continuously, day after day, is attended with considerable expense, and the frequent renewal of the supply of gas which it calls for is an inconvenience which one would gladly dispense with. "These considerations have led some microscopists to use an oil lamp as the source of illumination, and very satisfactory photomicrographs with comparatively high power have been made with this cheap and convenient light. But in my experience the best illumination which I have been able to secure with an oil lamp has called for very long exposures when working with high powers, and, as most of my photomicrographs, of bacteria are made with an amplification of one thousand diameters, I require a more powerful illumination than I have been able to secure in this way. And especially so because of the fact that a colored screen must be interposed, which shuts off a large portion of the actinic rays, on account of the staining agent usually employed in making my mounts. The most satisfactory staining agents for the bacteria are an aqueous solution of fuchsin, or of methylene blue, or of gentian violet; and all of these colors are so nearly transparent for the actinic rays at the violet end of the spectrum that a satisfactory photographic contrast cannot be obtained unless we shut off these rays by a colored screen. " I am in the habit of using a yellow screen for my preparations stained with f uohsin or methylene blue, and have obtained very satisfactory results with the orthochromatic plates manufactured by Carbutt, of Philadelphia, and a glass screen coated with a solution of .tropaeolin dissolved in gelatin. " But with such a screen, which shuts off a large portion of the actinic light and increases the time of exposure three- or fourfold, the use of an oil lamp becomes impracticable with high powers, on account of the feeble- ness of the illumination. "These considerations have led me to experiment with gaslight, and the simple form of apparatus which I am about to describe is the result of these experiments. I have now had the apparatus in use for several months, during which time I have made a large number of very satisfactory photo- micrographs of bacteria from f uchsm-stained preparations with an amplifica- tion of one thousand diameters. My photographs have been made with the three-millimetre oil-immersion apochromatic objective of Zeiss and his pro- jection eyepiece No. 3. I use a large Powell and Lealand stand, upon the substage of which I have fitted an Abbe' con denser. The arrangement of the apparatus will be readily understood by reference to the accompanying figure. "A is the camera, which has a pyramidal bellows front supported by the 108 PHOTOGRAPHING BACTERIA. heavy block of wood B; this can be pushed back upon the baseboard which supports it, so as to allow the operator to place his eye at the eyepiece of the microscope. When it is brought forward an aperture of the proper size ad- mits the outer extremity of the eyepiece and shuts off all light except that coming through the objective. C is the microscope, and D the Abbe con- denser, supported upon the substage. E is a thick asbestos screen for pro- tecting the microscope from the heat given off by the battery of gas burners F. This asbestos screen has an aperture of proper dimensions to admit the light to the condenser D. The gas burners are arranged in a series, with the flat portion of the flame facing the aperture in trie asbestos screen E. The concave metallic mirror G is properly placed to reflect the light in the desired direction. I have not found any advantage in the use of a condens- ing lens other than the Abbe condenser upon the substage of the microscope. The focussing is accomplished by means of the rod I, which carries at one extremity a grooved wheel, H, which is connected with the fine adjustment screw of the microscope by means of a cord. " The focussing wheel J may be slipped along the rod I to any desired position, and is retained in place by a set screw. The rod I is supported Fio. 74. above the camera oy arms depending from the ceiling, or by upright arms attached to the baseboard. 44 1 have lost many plates from a derangement of the focal adjustment resulting from vibrations caused by the passing of loaded wagons in the street adjoining the laboratory in which I work. This has been overcome to a great degree by placing soft rubber cushions under the whole appa- ratus."1 I have recently (1805) seen a gaslight which I believe would prove to be a valuable substitute for ordinary street gas, and I judge that, owing to its superior brilliancy, a single jet would suffice to replace the five burners in a linear series which are shown in the above figure. The gas referred to is acetylene, which may now be obtained in a liquid form in strong metal cylinders. Reference has already been made to the use of an oil light, and for low powers an ordinary lamp with a flat wick may be used. That bacteria may be successfully photographed, with an amplification of one thousand diameters, by means of an oil lamp is shown by the beautiful photomicrographs made by Capt. W. C. Borden, Assistant Surgeon U. S. Army. At my request Dr. Borden has prepared the following detailed account of his method : 'From Johns Hopkins University Circulars, vol. ix., No. 81, p. 72. PHOTOGRAPHING BACTERIA. 109 DESCRIPTION OF APPARATUS FOR PHOTOMICROGRAPHY BY OIL LIGHT. The apparatus consists of a camera, hung- in a vertical position, of a microscope with substage condensers, suitable objectives and projection oculars, and a Laverne tri-wick, oil stereopticon with the projection objec- tive removed. Tfie Light. — After trying all kinds of lamps, I found that the best illu- mination could be obtained by using a tri-wick stereopticon with the pro- jection objective removed, the middle wick only being lighted. The large four-inch condensers serve to concentrate the light, while the double lantern body prevents the radiation of heat to the microscope and shuts off all radiat- ing light. Consequently the microscope does not become heated, and if the room is darkened the absence of extraneous light greatly aids in focussing on the camera screen. The oil light itself is quite yellow and so nearly mono- chromatic that with orthochromatic plates a color screen is seldom, if ever, required. After experimenting by taking photographs with and without a screen, I have found 110 particular difference in result even when photo- graphing difficult bacteria, and now seldom use one. If a screen is used a solution of bichromate of potash and sulphate of copper in dilute ammonia water placed in a trough between the lantern and microscope gives excellent results and does not materially lengthen the time of exposure. The lantern is placed about twelve inches in front of the microscope and with its central long axis in a plane which extends through the centre of the microscope mirror, the substage condenser, the objective, ocular and centre of camera. Microscope. — The microscope is used in the upright position. I have used this position rather than the horizontal for several reasons. The microscope is used on the work -table in an upright position, and in working when an object is found which it is desired to photograph, the microscope without changing adjustments has only to be carried to the photomicro- graphic apparatus, placed in position, correct adjustments of light made, the camera racked into contact and the exposure made. With a conveniently placed dark room the whole operation will occupy but a few minutes. The upright position is necessitated when liquid preparations, as colonies of bacteria floating on liquefied gelatin, are to be photographed, or when the microscope is used for clinical photomicrography, as in photographing uri- nary deposits in urine, blood corpuscles in Thoma blood counter, etc. In bacteriological work where the bacteria are stained on the coyer and after mounting the balsam is not quite dry, the cover is apt to slip if the micro- scope is used horizontally, but this does not occur with the microscope placed vertically. The horizontal position and long extension of camera is neces- sary for certain work, particularly where large pictures (i.e., over four inches in diameter) have to be taken, or where it is desired to obtain high am- plification by extension of camera rather than by high eyepiecing, or in photographing test diatoms with very high amplifications. For practical work, however, up to amplifications of one thousand diameters, and for photographs for illustration or reproduction, which are seldom required of over three and one-half or four inches in diameter, the upright position is much to be preferred 011 account of its ease of application and its practical advantages. Camera. — The upright position of the microscope necessitates a similar position for the camera. To allow easy working, the camera is hung on a rack -work attached to a rigid upright. * The upright is placed to the right of the microscope so that it will be out of the way while working. Both the upper and the lower ends of the camera are movable on the rack-work. The upper end, which carries the screen and plate-holder, is movable, in order that different amplifications within limits can be gotten with the same objective. The lower end is movable that it may be racked 110 PHOTOGRAPHING BACTERIA. up and out of the way and allow the operator to manipulate the microscope before attaching the camera. The bellows has an extension of two feet, measured from the eyepiece of the microscope to the focussing screen. This, with a two-millimeter objective and projection ocular 4, gives an amplifica- tion of one thousand diameters. With less extension of bellows and lower objectives amplifications ranging down to ten diameters may be obtained. In focussing, the operator can, by standing, observe the image on the screen with a focussing glass and manipulate the fine adjustment of the microscope with his hand without using a focussing rwers some form of substage condenser is necessary. This is due to the fact that the source of light must always be focussed on the object in order to give proper definition. In working with the objectives of four millimetres or lower, it will be found advantageous to use objectives of lower power as substage condensers, for it will be found that if placed in the substage for ordinary work they greatly improve the definition of objects. In fact it may be laid down as a general rule that whatever with a given light gives the best definition to the observer's eye will give the sharpest photographic iinairr. Consequently, in high-power work where a condenser is used it will seldom be necessary to change the microscope attachments when a photograph has to be taken; for in bacteriological work the Abbe condenser which gives good definition will, when properly adjusted, give good photographic defi- nition also, statements to the contrary notwithstanding. To photograph, place the microscope and lantern in position, light the centre wick of the lamp, place a ground glass between the lamp and camera, and focus the objective accurately on the object. The ground glass is used on I v to reduce the light which might otherwise injure the observer's eye. The ground glass is then removed, a fine wire screen placed close against the front of the lantern condenser, and by means of the substage condenser an imago of the screen is projected accurately on the object. This is very important, for it is necessary that the light should be accurately focussed on the object in < >rder to produce sharp definition. After focussing the light, the screen is removed and an opal glass is put, in its place. On looking through the eyepiece a clear sharp image of the object will be seen. If an Abbe con- denser is used the iris diaphragm of the condenser should now be carefully opened and dosed until such an aperture is obtained that to the observer's eve the object appears to the best advantage. The opal glass is now removed, the camera attached to the microscope, and the projected image focussed on the camera screen, preparatory to exposure. PHOTOGRAPHING BACTERIA. HI Plate Used.— Orthochromatic plates only should be used. Of these I use the Cramer rapid, isochromatic plate exclusively. With these when photo- graphing bacteria and using an amplification of one thousand diameters the exposure will vary from one and one-half to three minutes, two minutes being about the average. It is with these plates that I have found a color screen unnecessary, and since using them I have had no difficulty in photographing bacteria, for they are particularly sensitive to the yellow-colored oil light. Possibly other makes of orthochromatic plates might be found to work equally well, but the oil light works so very well with the Cramer isochro- matic that I have had no desire to try others. Development. — For development, I have obtained best results with for- mulas in which hydrochiiion either alone or with some other developing agent is used. The following gives excellent results, and I prefer it to other developers as it gives good clear negatives of sufficient contrast and gradation: No. 1. Water, ....... 10 ounces. Sodium sulphite, . .... 1 ounce. Potassium bromide, ..... 10 grains. Hydrochinon, . . . . . .30 grains. Metol, . . . . . . 40 grains. No. 2. Water, ....... 10 ounces. Sodium carbonate, ...... 300 grains. Use equal parts of No. 1 and No. 2. Development should be continued until sufficient density is obtained. In- tensification should be rarely required, for with proper exposure and develop- ment a good negative can usually be obtained. If intensification is neces- sary, after fixing and washing the plate, I prefer to use a saturated aqueous solution of bichloride of mercury, followed by washing, the application of dilute ammonia water, and a final washing. Students who desire to perfect themselves in the art of making photomicrographs are advised to first make themselves familiar with the technique of photography with a landscape or portrait camera, and not to undertake the more difficult task of photographing bac- teria until they know how to make a good negative and to judge whether an exposure has been too long or too short, etc. 1'LATH I. STERNBERG'S BACTERIOL' 3. *!&' I 4 I'LATl-; 11. STERNBERG'S BACTERIOLOGY • I'1 IK a. 4. I1' IK. 5. KM:. <',. PLATE I. PHOTOMICROGRAPHS OP BACTERIA MADE BY GASLIGHT. FIG. 1. — Streptococcus cadaveris, from a culture in agua coco; stained with f uchsin. x 1,000. (Steriiberg.) FIG. 2. — Streptococcus Ha vaniensis. x 1,000. From a photomicrograph. (Sternberg.) FIG. 3. — Bacillus cuniculicida Ha vaniensis, from peritoneal cavity of inoculated rabbit, showing leucocytes containing bacilli and free bacilli; stained with f uchsin. x 1,000. (Sternberg.) FIG. 4.— Bacillus cadaveris, smear preparation from yellow-fever liver kept for forty-eight hours in an antiseptic wrapping (Havana, 1889) ; stained with fuchsin. x 1,000. (Sternberg.) Note. — All of the above photomicrographs were made with the three- millimetre apochromatic horn. ol. im. objective and projection eye-piece of Zeiss. PLATE II. PHOTOGRAPHS OF COLONIES (IN ESMARCH ROLL TUBES) AND OF TEST-TUBE CULTURES. FIG. 1. — Colonies of Bacillus leporis lethalis, in gelatin roll tube, end of forty-eight hours at room temperature, x 5. (Sternberg.) FIG. 2. —Colonies of Bacillus coli similis in gelatin roll tube, end of twenty-four hours at 22° C. x 10. (Sternberg.) FIG. 3. — Stick culture of Bacillus coli similis in nutrient gelatin, end of seven days at 20° C. (Sternberg.) FIG. 4. — Stick culture of Bacillus intestinus motilis in nutrient gelatin, end of four days at 22° C. (Sternberg.) FIG. 5.— Stick culture of Bacillus leporis lethalis in nutrient gelatin, end of eight days at 22° C. (Sternberg.) FIG. 6. — Stick culture of Micrococcus tetragenus versatilis in nutrient gelatin, end of two weeks at 22 3 C. (Sternberg.) FIG. 7.— Colonies of Bacillus cuniculicida Ha vaniensis in gelatin roll tube, end of forty-eight hours at 21° C. x 10. (Sternberg.) FIG. 8. — Colonies of Bacillus coli communis in gelatin roll tube, end of forty-eight hours at 22° C. x 10. (Sternberg.) PART SECOND. GENERAL BIOLOGICAL CHARACTERS: INCLUDING AX ACCOUNT OF THE ACTION OF ANTISEPTICS AND GERMICIDES. I. STRUCTURE, MOTIONS, REPRODUCTION. II. CONDITIONS OF GROWTH. III. MODIFICATIONS OF BIOLOGICAL CHARACTERS. IV. PRODUCTS OF VITAL ACTIVITY. V. PTOMAINES AND TOXALBUMINS. VI. INFLUENCE OF PHYSICAL AGENTS. VII. ANTISEPTICS AND DISINFECTANTS — GENERAL ACCOUNT OF THE ACTION OF. VIII. ACTION OF GASES AND OF THE HALOID ELEMENTS UPON BACTERIA. IX. ACTION OF ACIDS AND ALKALIES. X. ACTION OF VARIOUS SALTS. XI. ACTION OF COAL-TAR PRO- DUCTS, ESSENTIAL OILS, ETC. XII. AC- TION OF BLOOD SERUM AND OTHER OR- GANIC LIQUIDS. XIII. PRACTICAL DIRECTIONS FOR DISINFECTION. PART SECOND. I. STRUCTURE, MOTIONS, REPRODUCTION. THE bacteria are unicellular vegetable organisms, and consist of a cell membrane enclosing transparent and apparently structureless protoplasm. The very varied biological characters which distin- guish different species make it evident, however, that there are es- sential differences in the living cell contents, although these differ- ences are not revealed by our optical appliances. And among the bacteria, as in the cells of higher plants and animals, the peculiar biological characters of a species are transmitted to the cellular pro- geny of each individual cell. These characters are, however, sub- ject to various modifications as a result of differing conditions of environment, as is the case with plants and animals higher in the scale of existence, and in this way more or less permanent varieties are produced. It is probable that among these lowly plants species are evolved more quickly, as a result of the laws of natural selec- tion, in the struggle for existence, than among those of more com- plex organization. Still, this has not been proved, and, on the ptljer hand, we have ample evidence that widely distributed species exist having very definite morphological and biological characters wnich enable us to recognize them wherever found. It has generally been supposed that these simple vegetable cells are destitute of a nucleus, but a recent author (Frankel) suggests the probability that a nucleus may exist, although it has not; been demonstrated. This suggestion is based upon the fact that in stain- ing bacteria very quickly it sometimes happens that a portion of the protoplasm is sharply differentiated by taking the stain more deeply than the remaining portion. Sjobring in 1892 made an investigation for the purpose of ascertaining the structure of bacterial cells. Various methods were employed, but the most satisfactory results were obtained by fixing with nitric acid, with or without alcohol, and without pre- 116 STRUCTURE, MOTIONS, REPRODUCTION. vious drying ; the preparations were then stained with carbol-meth- ylene-blue or carbol-f uchsin solution ; they were decolorized with nitric acid and examined in glycerin or in water. By this procedure the author named was able to demonstrate two kinds of corpuscles. One of these may be seen just inside the cell wall ; it stains deeply with the carbol-fuchsiii solution. The other lies in a position analo- gous to that occupied by the nucleus of vegetable cells higher in the scale, and resembles this both in its resting condition and in the process of indirect division. In his address before the International Medical Congress of Ber- lin (1890) Koch says : " We had not succeeded, in spite of the constantly improving methods of staining and in spite of the use of objectives with con- stantly increasing angles of aperture, in learning more with reference to the interior structure of the bacteria than was shown by the origi- nal methods of staining. Only very recently new methods of stain- ing appear to give us further information upon the structure of the bacteria, inasmuch as they serve to differentiate an interior portion of the protoplasm, which should probably be regarded as a nucleus, from an exterior protoplasmic envelope from which is given off the organ of locomotion, the flagellum." Although usually transparent, the protoplasm sometimes presents a granular appearance. The botanist Van Tieghem claims to have found chlorophyll grains in some water bacteria studied by him, and in the genus Beggiatoa grains of sulphur are found embedded in the protoplasm of certain species. The cell membrane in certain species appears to be very flexible, a-- may be seen in those which have a sinuous motion. It is not easily recognized under the microscope, but by the use of reagents which cause the protoplasm to contract may be demonstrated — e.g., by iodine solution. Outside of the true cell membrane a gelatinous envelope — so-called capsule — is sometimes seen. This may perhaps be, as claimed by some authors, nothing more than a jelly-like thick- ruing of the outer layers of the cell wall. This jelly-like material causes the cells to adhere to each other, forming zoogloea masses. I n some cases the growth upon the surface of a culture medium is extremely viscid , and may be drawn out into long threads when touched with a platinum needle, owing to the gebitinous intercellular substance by which the cells are surrounded. There is but little more to be said of the structure of these minute organisms, except to mention the fact that the motile species arc provided with slrn> fission or by the formation of endogenous spores, the protoplasm < >f the cells in a pure culture of any microorganism is simply a sepa- rated portion of the protoplasm of the progenitors of these cells. Some of the bacilli grow out into long filaments l>ef ore the forma- tion of spores occurs ; and these filaments may be associated in bun- dies or intertwined in irregular masses. At first the protoplasm of the STRUCTURE, MOTIONS, REPRODUCTION. 119 filaments is homogeneous, but after a time it becomes segmented, and later the protoplasm of each segment becomes condensed into a spherical or oval refractive body, which is the spore. For a time these are retained in a linear position by the cell membrane of the filament (Fig. 75, a), but this is after a while dissolved or broken up and the spores are set free. In liquid cultures they sink to the bottom as a pulverulent precipitate, and upon the surface of a solid medium they form a layer which is usually of a white or yellowish- white color, and which, when examined under the microscope, in old cultures is found to consist almost entirely of shining spherical or oval bodies which do not stain, by the ordinary methods, with the aniline colors. While many of the bacilli during the stage of spore formation grow out into long filaments, others do not, and one or more spores make their appearance in rods of the ordinary length which characterizes the species. These may be located in the centre of the rod or at one extremity (Fig. 75, 6). It sometimes occurs c- Fia. 75. that when a single central spore is formed the rod becomes very much enlarged in its central portion, assuming a spindle shape (Fig. 75, c); or one extremity may be enlarged, producing forms such as are shown in Fig. 75, d. Some of the smaller spherical spores mea- sure less than 0.5 yu in diameter, but they are, for the most part, oval bodies having a short diameter of 0. 5 to 1 /< and a long diame- ter of one to two /<, or even more. They are enveloped in a cellular envelope which, according to some observers, consists of two layers — an exosporium and an endosporium. The germination of spores has been studied by Prazmowski, Brefeld, and others. The process is as follows : By the absorption of water they become swollen and pale, losing their shining, refrac- tive appearance. Later a little protuberance is seen upon one side or at one extremity of the spore, and this rapidly grows out to form a rod which consists of soft-growing protoplasm enveloped in a membrane which is formed of the endosporium or inner layer of the cellular envelope of the spore. The outer envelope, or exosporium, is cast off and may be seen in the vicinity of the newly formed rod (Fig. 76). Sometimes the vegetative cell emerges from one extrem- 120 -IIMCirKK, MOTIONS, REPRODUCTION. ity of the oval spore, as shown at a, Fig. 70, arid in other species the exosporium is ruptured and the bacillus emerges from the side. as seen at b. The considerable resistance of these endogenous spores to desic- cation, to heat, and to various chemical agents is an important fact both from a biological and from a hygienic point of view, and will be fully considered in a subsequent chapter. The fact that certain bacilli and spirilla do not withstand a temperature of 80° to 90° C. , which does not destroy the vitality of known spores, leads to the in- ference that they do not form similar reproductive bodies. But re- productive elements of a different kind are described by some botan- ists as being produced during the development of these bacteria, and also of the micrococci. These are the so-called arthrospores. In the process of binary division certain cells in a chain may be ob- served to be somewhat larger than others and to refract light more strongly. The same may be true of certain cells in a culture in which the elements are not united in chains. These cells are believed a-- FIG. 76. by De Bary and others to have greater resisting power to desiccation than the remaining cells in the culture, and to serve the purpose of reproductive elements. It has generally been supposed that spore formation is most likely to occur when the pabulum for supporting the growth of the vegeta- tive form is nearly exhausted. But, as pointed out by Frankel, facts do not support this view, as many species form spores when condi- tions are most favorable for a continued development. An abundant supply of oxygen favors the formation of spores in aerobic species, and, in some instances at least, the temperature has an important in- fluence upon spore formation. Thus the anthrax bacillus does not form spores at temperatures below 20° C. or above 42° C. The very interesting fact has been demonstrated by Lehman and by Behring that a species which usually forms spores may be so modified by certain influences that it is no longer capable of spore production, and that such an asporogenous variety may be cultivated for an indefinite time without showing any return to the stage of spore formation. This was effected in Behring's experiments by cultivating the anthrax bacillus in a medium containing some agent detrimental to the vitality of the vegetative cells, but not in suffi- cient quantity to restrain their development. STRUCTURE, MOTIONS, REPRODUCTION. 121 The chemical composition of the bacterial cells has been inves- tigated by Nencki, Brieger, and others. Putrefactive bacteria culti- vated in a two-per-cent solution of gelatin, and which produced an abundant intercellular substance connecting the cells in zoogloea masses, were found by Nencki to have the following composition : Water, 84. 26 per cent ; solids, 5. 74 per cent, consisting of albumin 87.46 per cent, fat 6.41, ash 3.04, undetermined remnant 3.09. The albuminous substance, according to Nencki, is not precipitated by alcohol, and differs in its chemical composition from other known substances of this class. He calls it mykoprotein and gives the fol- lowing as its chemical composition : C, 52.33 percent; H, 7.55 per cent ; N, 14.75 per cent. It contains no sulphur and no phosphorus. The spores of the anthrax bacillus, according to Nencki, do not con- tain mykoprotein, but a peculiar albuminous substance which he calls anthrax-protein. Brieger analyzed a gelatin culture of Fried- lander's bacillus, with the following result : Water, 84.2 per cent ; solids, 5.8 per cent, containing 1.74 per cent of fats. After removal of the fat the solids gave an ash of 30.13 per cent ; this contains cal- cium phosphate, magnesium phosphate, sodium sulphate, and sodium chloride. The amount of nitrogen in the dried substance after re- moval of the fat was 9. 75. II. CONDITIONS OF GROWTH. BACTERIA only grow in presence of moisture, under certain condi- tions of temperature, and when supplied with suitable pabulum. As they do not contain chlorophyll, they cannot assimilate carbon diox- ide, and light is not favorable to their development. The aerobic species obtain oxygen from the air and cannot grow unless supplied with it. The anaerobic species, on the other hand, will not grow in the presence of oxygen, and must obtain this ele- ment, as they do carbon and nitrogen, from the organic material which serves them as food. As a class the bacteria are supplied with nutriment by the higher plants and animals, the dead tissues of which they appropriate, and which it is their function to decompose, releasing the organic ele- ments as simple compounds which may again be assimilated by the chlorophyll-producing plants. Water is essential for the development of bacteria, and many spe- cies have their normal habitat in the waters of the ocean, of lakes, and of running streams ; others thrive upon damp surfaces or in the interior of moist masses of organic material. Many species grow in- differently either in salt or fresh water, but it is probable that cer- tain species will be found peculiar to the waters of the ocean. Some of the water bacteria multiply in the presence of an exceedingly minute amount of organic pabulum, or even in distilled water. This is shown by the experiments of Bolton and others. The author named tested two species of water bacteria (Micrococcus aquatilis ,ind Bacillus erythrosporus) in the following manner: Ten cubic centimetres of distilled water in a test tube were infected with a si MM II quantity of a culture of one of these microorganisms. A drop from this tube was transferred to the same quantity of distilled water in a second tube, and from this to a third. The number of bacteria in this tube No. '.\ \VMS now ascertained by counting, and it was put a>i be capable of modification in this regard to such an extent that when cultivated for a time in a favor- able medium — bouillon with five per cent of glycerin — it will even grow in ordinary bouillon made from the flesh of a calf or a fowl (Roux). licdcf i'>n nf Medium. Sonic bacteria groxv readily in a medium having an acid reaction, while the slightest trace of aridity prevents the development of others. As a rule, the pathogenic species require a neutral «>r slightly alkaline culture medium. CONDITIONS OF GROWTH. 125 While many species grow in various media and under various conditions of temperature, etc.^ others are greatly restricted in this regard ; thus Bumm only succeeded in cultivating the gonococcus upon human blood serum, and even upon this was not able to carry it through a series of successive cultures. It is very probable that certain species can only grow in association with others which elaborate products necessary for their development. Substances favorable for the growth of a particular species may restrain its development if present in too large an amount. Thus the phosphorescent bacilli multiply abundantly in a nutrient solution containing 2.5 per cent of sodium chloride ; but this amount would restrain the development of some other species, and a considerable increase in the quantity of salt prevents the growth of all microor- ganisms. In the same way the addition of two per cent of glucose to culture solutions is favorable for the development of certain spe- cies, and especially for the anaerobic bacteria ; but a concentrated solution of the same substance prevents the growth of all bacteria. The influence of one species upon the growth of another has been studied by various bacteriologists, and especially by Sirotinin and by Freudeiireich. When several species are associated in the same culture one may take the precedence and the others may de- velop later ; or two or more species may develop at the same time ; or the growth of one species may prevent the development of an- other, either (a) by exhausting the pabulum necessary for its growth or (b) by producing substances which inhibit the development of an- other species or destroy its vitality. Freudeiireich found, as a result of his numerous experiments, that the following species cause a change in bouillon which renders it unfit for the growth of other species : Bacillus pyocyaneus, Bacil- lus cyanogenus, Bacterium phosphorescens, Bacillus prodigiosuf, Spi- rillum cholera? Asiaticne. The following species do not cause such a change in bouillon as to render it unfit for the growth of other spe- cies : Bacillus typhi abdomiiialis, Bacillus anthracis, Bacillus septi- caemias hsemorrhagicse, Spirillum tyrogeiium. The following have a decided antagonism : Bacillus pyogeiies foetidus prevents the growth of Spirillum cholera Asiaticse ; Micrococcus roseus prevents the growth of Micrococcus tetragenus. The cholera spirillum will not grow in sterilized cultures of Bacillus pyocyaneus, or in bouillon which has served for a previous culture of the same microorganism (Kitasato). Other bacteria which fail to grow in bouillon which has already served for the cultivation of the same species are Bacil- lus typhi abdominalis, Bacillus cyanogenus, 'Bacillus prodigiosus, Micrococcus roseus, etc. (Freudenreich). III. MODIFICATIONS OF BIOLOGICAL CHARACTERS. WE have already referred to the production of an asporogenous variety of the anthrax bacillus. This was effected by Behring by cultivation in media containing small amounts of hydrochloric acid, caustic soda, methyl violet, malachite green, and various other agents. This is only one of many instances of a change in biologi- cal characters due to changed conditions of environment. We have abundant experimental evidence that growth may occur under ad- verse conditions when the species is gradually habituated to these conditions. Thus the temperature limitations may be passed by suc- cessive cultivations at temperatures approaching these limits, and bacteria may grow in the presence of agents which in a given pro- portion have a complete restraining influence upon their develop- ment. For example, in the experiments of Kossiakoff, published in the Annales of the Pasteur Institute (vol. i.), it was found that the several species tested all became habituated to the presence of anti- septic agents in proportions which at first completely restrained their growth. This modification of biological characters is well shown in the case of the chromogenic bacteria, some of which only form pig- ment under exceptionally favorable conditions of growth. It has been shown by several observers that non-chromogenic varieties of some of the best known chromogenic species may be produced by special methods of cultivation. Thus Wasserzug obtained a i ion -chromogenic variety of the bacillus of green pus (Bacillus pyocyaneus) by the action of time added to that of antiseptics. He Bays : " These two actions combined have permitted me to obtain cultures which remained without color in a durable way, and in which, consequently, the chromogenic function was abolished by heredity/' In the case of a chromogenic bacillus obtained by the writer in Havana (my Bacillus Havanieiisis), a non-chromogenic vari- ety was obtained from a culture on nutrient agar which had been kept in a hermetically sealed glass tube for about a year. The variety preserved the morphological characters of the original stock, but, al- MODIFICATIONS OF BIOLOGICAL CHARACTERS. 127 though carried through successive cultures for a considerable period, did not regain its power to produce the brilliant carmine color which is the most striking character of the species. Katz, in cultivating the phosphorescent bacilli isolated by him from sea water at New South Wales, found that, after being propagated for some time in artificial media, their power to give off a phosphorescent light was diminished or temporarily lost. He also found that two species which when first cultivated did not liquefy gelatin, subsequently, after a year, caused liquefaction of the usual gelatin medium. Modification shown in Cultures. — When bacteria have been subjected to the action of heat or chemical agents, without having their vitality completely destroyed, they often show diminished vigor of growth. Cultures which would ordinarily show an abundant de- velopment within twenty-four hours may not commence to grow for several days. For this reason, in disinfection experiments, it is neces- sary to test the question of destruction of vitality by leaving the cul- tures for a week or more under favorable conditions as to tempera- ture. In plate cultures or Esmarch roll tubes a few colonies may develop in this tardy way, showing that there was a difference in the vital resisting power of the individual cells, some having survived while the majority were killed. This is well illustrated by Abbott's experiments upon the germicidal action of mercuric chloride as tested upon Staphylococcus pyogenes aureus. Irregularities in the results in experiments in which the conditions were identical having been no- ticed, Abbott inferred that this was due to a difference in the resist- ing power of individual cocci (arthrospores ?). By making cul- tures from colonies which developed from these more resistant cocci, and again exposing the micrococci in these cultures to mercuric chlo- ride in the proportion of 1:1,000 for a longer time and making new cultures from the surviving cocci, and so on, Abbott obtained cultures in which a majority of the cells survived exposure to a solution of the strength mentioned for ten to twenty minutes, whereas in his original culture most of the cocci were killed by this solution in five minutes. These changes in vital resisting power enable us to comprehend other modifications which can only be detected by chemical or bio- logical reactions. Thus the reducing power for various substances may be modified by changes in the conditions of environment. And among the pathogenic bacteria changes of a more or less permanent nature may be induced, which are shown by a modified degree of virulence when injected into susceptible animals. Attenuation of Virulence may be effected by several methods, all of which depend upon subjecting the cultures to prejudicial in- fluences of one kind or another. Pasteur first announced, in 1880, that the microbe of fowl cholera 128 MODIFICATIONS OF BIOLOGICAL CHARACTERS. could be modified by special treatment in such a manner that it no longer produced a fatal form of the disease. He found that the viru- lence was greatest when cultures were made from fowls which had died from a chronic form of the disease, and that this virulence was not lost by successive cultivations in chicken bouillon, repeated at short intervals. But when an interval of more than two months was allowed to elapse without renewing the cultures, the virulence was diminished and fewer deaths occurred in fowls inoculated with such cultures. This diminution of virulence became more marked in proportion to the length of time during which a culture solution containing the microbe remained exposed to the action of the atmo- sphere, and at last all virulence was lost as a result of the death of the pathogenic microorganism. When the virus was preserved in hermetically sealed tubes it did not undergo this modification, but re- tained its full virulence for many months. According to Pasteur, the various degrees of modification of virulence resulting from pro- longed exposure to the air may be preserved in successive cultures made at short intervals. Subsequent experiments with cultures of the anthrax bacillus gave similar results and enabled him to produce an ' ' attenuated virus " for his protective inoculations. In the case of the anthrax bacillus it was found that the spores retain their full virulence for years, and that the production of an at- tenuated virus required the exclusion of these reproductive elements. Cultivations were consequently made at a temperature of 43° to 43 ° C., at which point this bacillus is incapable of producing spores. Cultivation at this temperature for eight days gave an attenuated virus suitable for use in protective inoculations. Attenuation by Heat. — Toussaint has shown that a similar modi- fication of virulence may be produced by exposure for a short time to a temperature a little below that which destroys the vitality of the pathogenic organism. This is best accomplished, according to Chau- veau, in the case of the bacillus of anthrax, by exposure for eighteen minutes to a temperature of 50° C. Exposure to this temperature for twenty minutes is said to completely destroy the vitality of the bacillus. Attenuation by Antiseptic Agents. — The writer, in 1S80, ob- tained evidence that attenuation of virulence may result from ex- IM .sure to the action of antiseptic agents. In a series of experiments made to determine the comparative value of disinfectants, the blood of a rabbit recently dead from a form of septicaemia induced by the subcutaneous injection of my own saliva, and due to the presence of a micrococcus (Micrococcus pneumonue crouposie), was subjected to the action of various chemical agents, and subsequently injected into a rabbit to test the destruction of virulence. In the published report of these experiments the following statement is made : MODIFICATIONS OF BIOLOGICAL CHARACTERS. 129 1 'The most important source of error, however, and one which must be kept in view in future experiments, is the fact that a pro- tective influence has been shown to result from the injection of virus the virulence of which has been modified, without being entirely de- stroyed, by the agent used as a disinfectant." ' ' Sodium hyposulphite and alcohol were the chemical reagents which produced the result noted in these experiments ; but it seems probable that a variety of antiseptic substances will be found to be equally effective when used in proper proportion. Subsequent ex- periments have shown that neither of these agents is capable of de- stroying the vitality of the septic micrococcus in the proportion used (one per cent of sodium hyposulphite or one part of ninety -five-per- cent alcohol to three parts of virus), and that both have a restraining influence upon the development of this organism in culture fluids." ' Cultivation in the Blood of an Immune Animal. — It has been shown by the experiments of Ogata and Jasuhara that when the anthrax bacillus is cultivated in the blood of an immune animal, such as the dog or the white rat, its pathogenic power is modified so that it no longer kills susceptible animals and may be used as a vaccine. Pasteur had previously shown (1882) that the virus of rouget can be attenuated by passing it through rabbits. Recovery of Virulence. — Pasteur has shown that when the viru- lence of a pathogenic organism has been modified it may be re- stored by successive inoculations into susceptible animals. Thus in the case of the anthrax bacillus a culture which would not kill an adult guinea-pig may be inoculated into a very young animal of the same species with a fatal result ; and by inoculating the blood of this animal into another, and so on, the original virulence may be restored, so that a culture is obtained which will kill a sheep. In the same way the attenuated virus of fowl cholera may be restored to full vigor by inoculating a small bird— sparrow or canary— to which it is fatal. After several successive inoculations the virus resumes its original activity. In general, pathogenic virulence is increased by successive inocu- lations into susceptible animals, and diminished by cultivation in arti- ficial media under unfavorable conditions. Thus various pathogenic bacteria which have been cultivated in laboratories for a length of time are likely to disappoint the student if he makes inoculation ex- periments for the purpose of demonstrating their specific action as described in text books. 1 Quoted from " Bacteria," pages 207, 208, written in 1883. IV. PRODUCTS OF VITAL ACTIVITY. ALL living cells, animal or vegetable, while in active growth, appropriate certain elements for their nutrition from the pabulum with which they are supplied, and at the same time excrete certain products which, in some cases at least, it is their special function to produce. In the higher plants and animals specialized cells excrete substances which are injurious to the economy of the individual, and secrete substances which are required to maintain its existence. As an example in animals we may mention the excretion of urea by the epithelium of the kidneys, the retention of which is fatal to the individual, and the gastric secretion which is essential for its con- tinued existence. Among the higher plants we have an immense variety of substances formed in the cell laboratories, some of which are evidently useful for the preservation of the species, while others are perhaps to be considered simply as excretory products. The odorous volatile products given off by flowers are supposed to be useful to the plant in attracting insects by which cross-fertilization is effected. The various poisonous substances stored up in leaves and bark may serve to protect the plant from enemies, etc. The minute plants with which we are especially concerned also produce a great variety of substances, some of which may be useful to the species in the struggle for existence. Thus the deadly pto- maines produced by some of the pathogenic bacteria serve to para- lyze the vital resisting power of living animals and enable the para- sitic invader to thrive at the expense of its host. In the present section we shall consider in a general way these various products < >t bacterial growth. Pigment Production. — A considerable number of species arc distinguished by the formation of pigment of various colors and shades. We have all of the shades of the spectrum from violet to red. The color, as a rule, is only produced in the presence of oxy- gen, and when the pigment-producing microorganisms are massed upon the surface of a solid culture medium the pigment production is often limited to the superficial portion of the mass. In some cases a soluble pigment is formed which is absorbed by the transpa- ST KR N 1 5 KK (V S B A CTE R I OLD GY PIa^e HI. Fig.l. 2. 3. fig. 4-. fig.] . Sarcinalutea,a^ar culture Fig. 2. Bacillus prodi^iosus, a^ar culture. Fig. 3. Bacillus pyocyanus, agar culture. Fiq.4". Bacillus Havamensis, potato culture. PRODUCTS OF VITAL ACTIVITY. 131 rent culture medium, coloring especially the upper portion, in stick cultures in nutrient gelatin or agar. This is the case with Bacillus pyocyaneus, .which produces a blue pigment which has been isolated and carefully studied by Gessard and others. The pigment, which is called pyocyanin, is soluble in chloroform and crystallizes from a pure solution in long blue needles. Acids change the blue color to red, reducing substances to yellow. It resembles the ptomaines in its chemical reactions, being precipitated by platinum chloride and phosphomolybdic acid. In some media the color produced by the Bacillus pyocyaneus (bacillus of green pus) is a fluorescent green. The recent studies of Gessard show that this is a different pigment. According to this author, cultures in a two-per-cent solution of peptone give a beautiful blue tint, the production of which is hastened by adding to the liquid five per cent of glycerin. In nutrient gelatin and agar cultures a Fluorescent green color is developed, which, according to Gessard, is due to the presence of albumin. Peptone and gelatin are said to produce pyocyanin without the fluorescent-green pigment, and cul- tures in bouillon to give both this and pyocyanin. In milk the fluorescent-green color is first seen, but subsequently, when the ca- sein has been peptonized by a diastase produced in the culture, pyo- cyanin is also formed. Several other microorganisms are known which produce a fluorescent-green color, due probably to the same pigment as is produced by the bacillus of green pus in albuminous media. Babes claims to have obtained two pigments from cultures of the Bacillus pyocyaneus in addition to pyocyanin : one, soluble in alcohol, has by transmitted light a chlorophyll-green color, by reflected light it is blue; the other, insoluble in alcohol and chloroform, by trans- mitted light is of a dark orange-red, by reflected light a greenish- blue. In Gessard's latest publication (1891) he shows that the produc- tion of pyocyanin or of the fluorescent-green pigment does not de- pend alone upon the culture medium, but that there are different varieties of the Bacillus pyocyaneus. He has succeeded in producing four distinct varieties — one which produces both pyocyanin and fluorescence, one which produces pyocyanin alone, one which pro- duces the fluorescent-green pigment alone, and one which produces no pigment. The last-mentioned non-chromogenic variety was pro- duced by subjecting the second variety to the action of heat. A temperature of 57° maintained for five minutes destroyed the power to produce pigment without destroying the vitality of the bacillus, which was propagated through successive cultures without regaining this power. 132 PRODUCTS OF VITAL ACTIVITY. The well-known Bacillus prodigiosus (also described as a micro- coccus) produces a red pigment which is insoluble in water but solu- ble in alcohol. By the addition of an acid the color becomes car- mine and then violet, which is changed to yellow by an alkali. The color is said by Schottelius to be diffused in the young cells, and after the death of the cells to be present in their vicinity in the form of granules. The same author has shown that by subjecting the bacillus to special conditions a variety may be obtained which no longer produces pigment. The conditions which govern the formation of pigment in the chromogenic bacteria are determined with comparative facility be- cause the results of changed conditions are apparent to the eye ; in the case of products which are not colored the difficulties attending the study of these conditions are much greater, but the results are in many instances more important. The following are among the best known pigment-producing (chromogenic) bacteria : Staphylococcus pyogenes aureus (No. 1), Staphylococcus pyc- genes citreus (No. 3), Sarcina aurantiaca (No. 226), Sarcina lutea (No. 227), Bacillus cyanogenus (No. 257), Bacillus janthinus (No. 207), Bacillus fluorescens liquefaciens (No. 277), Bacillus indicus (-No. 283), Bacillus pyocyaneus (No. 95), Bacillus prodigiosus (No. 284), Spirillum rubrum (No. 429). Liquefaction of Gelatin. — Many species of bacteria, when planted in a medium containing gelatin, cause a liquefaction of the gelatin in the immediate vicinity of the growing microorganisms, while many others multiply abundantly in the same medium with- out liquefying the gelatin. This character, as first shown by Koch, is an important one in the differential diagnosis of species which re- semble each other in form and in other respects. It has no relation to pathogenic power, as some liquefying organisms are harmless sap- rophytes and some deadly disease germs, while, on the other hand, non-liquefying bacteria may be very pathogenic or quite innocent. Liquefaction is produced by a soluble peptonizing ferment formed during the growth of the cells. This is shown by the fact that if a liquefying organism is cultivated in bouillon and the living cells re- moved by filtration or killed by heat, the power of liquefying gelatin remains in the culture fluid. This was first observed by Bitter (1880) and independently by the writer in 1887. In experiments made to determine the thermal death-point of 'various bacteria the writer found that when cultures of liquefying species were subjected to a temperature which killed the microorganisms, a few drops of tin* culture added to nutrient gelatin which had been liquefied by heat prevented it from subsequently forming a solid jelly when cold. In a study of the ferments produced by bacteria which cause PRODUCTS OF VITAL ACTIVITY. 133 liquefaction of gelatin — "tryptic enzymes" — made by Fermi, in the laboratory of the Hygienic Institute of Munich (1891), the following results were obtained : The enzymes were not obtained pure, and their isolation from other proteids present in the cultures was found to be attended with great difficulties, but their ferment action was studied and was found to be influenced by various conditions. All were destroyed by a temperature of 70° C. , but the enzymes produced by various liquefying bacteria differed considerably as to the temperature which they were able to withstand. Some were de- stroyed by a temperature of 50° to 55° C. — Bacillus megatherium, Bacillus ramosus, Staphylococcus pyogenes aureus ; some by a tem- perature of 55° to 60° C. — Bacillus subtilis, Bacillus pyocyaneus, Ba- cillus fluorescens liquefaciens, Sarciua aurantiaca; some by 65° to 70° C. — Bacillus anthracis, Spirillum cholera? Asiaticse, Spirillum of Finkler and Prior, Spirillum tyrogenum. These enzymes, like the previously known pepsin, trypsin, and invertin, do not dialyze. Only a few of these bacteria enzymes acted upon fibrin, and no action was observed upon casein or upon egg albumen. Their liquefying action upon gelatin was prevented by the action of sulphuric acid, and to a less degree by nitric acid, but was not in- terfered with by acetic acid. The liquefying bacteria, as a rule, only produce enzymes when cultivated in a medium containing albumen. These enzymes are not produced by a solution of the protoplasm of dead bacterial cells, but are a product of the vital activity of liv- ing cells. Among the numerous liquefying bacteria known to bacteriolo- gists we may mention the following species as deserving the student's special attention : Staphylococcus pyogenes aureus (No. 1), Staphylo- coccus pyogenes albus (No. 2), Sarcina lutea (No. 227), Sarcina au- rantiaca (No. 226), Bacillus anthracis (No. 45), Bacillus pyocyaneus (No. 95), Bacillus subtilis (No. 379), Bacillus indicus (No. 283), Ba- cillus prodigiosus (No. 284), Spirillum cholera? Asiatics (No. 155), Spirillum of Finkler and Prior (No. 156), Proteus vulgaris (No. 97). Production of Acids. — Numerous bacteria give an acid reaction to the media in which they are cultivated, and the acids produced are various — lactic, acetic, butyric, propionic, succinic, etc. The power to produce an acid is well shown by adding to neu- tral or alkaline culture media a solution of litmus. The change in color due to the formation of an acid may be followed by the eye, and comparative tests may be made to aid in the differentiation of similar bacteria. 134 PRODUCTS OF VITAL ACTIVITY. A considerable number of bacteria are able to produce lactic acid from milk sugar and other carbohydrates. One of these is considered the special lactic-acid ferment — Bacillus acidi lactici — and is the usual cause of the acid fermentation of milk. Pure cultures of this bacillus introduced into sterilized milk or solutions of milk sugar, cane sugar, dextrin, or mannite, give rise to the lactic-acid fermentation, in which carbonic acid is also set free. The proc-ess requires free access of oxygen, and progresses most favorably at a temperature of 35° to 40° C., ceasing at about 45°. In milk, coagu- lation of the casein occurs within fifteen to twenty-four hours after adding a small quantity of a pure culture of the lactic-acid bacillus. This is not due, however, to the acid fermentation, but to a ferment resembling that of rennet, which is produced by many different bacteria, some of which do not produce an acid reaction of the milk. Among the bacteria which produce lactic acid from milk sugar we may mention the staphylococci of pus, Bacillus lactis aerogenes, and Bacillus coli communis. The formula showing the transformation of sugar into lactic acid is usually stated as follows : C8HjaO0 = 2(HC3H5O3). Acetic acid is also produced from dilute solutions of alcohol by the action of a special bacterial ferment, which accumulates upon the surface of the fluid as a mycoderma, consisting almost entirely of the Bacillus aceticus (Mycoderma aceti). Free access of oxygen is required, and a temperature of about 33° C. is most favorable to the process. According to Duclaux, the " Mycoderma aceti " oxi- dizes the alcohol, in solutions containing it, so long as any is present, and when it is exhausted it oxidizes the acetic acid previously formed by oxidation of the alcohol, producing from it carbon diox- ide and water. The formation of acetic acid from alcohol is shown by the follow- ing formula : Ethyl alcohol CH3.CHa.OH + O2 = CH8.COOH + H,O. Butyric acid is produced by a considerable number of bacteria, one of which, named Bacillus butyricus, has received the special at- tention of Prazmowski. This is strictly anaerobic. In solutions of M.ireh, dextrin, sugar, or salts of lactic acid, when oxygen is ex- cluded it produces butyric acid in considerable quantity, and at the •ame time carbon dioxide and hydrogen gas are set free. Duclaux gives the following formula of a solution containing lactate of lime in which the action of the butyric-acid ferment may be well studied : Water, . . . . . 8 to 10 litres. Lactate of lime (pure), .... 225 grammes. Phosphate of ammonia, .... 0.75 Phosphate of potash,. . . . . 0.4 Sulphate of magnesa, .... 0.4 Sulphate of ammonia, .... 0.2 PRODUCTS OF VITAL ACTIVITY. 135 This is introduced into a flask with two necks, such as is shown in Fig. 77. Having filled the flask with the culture liquid, the bent neck is dipped into a porcelain dish containing the same. Heat is then applied both to flask and dish, and the liquid in each is kept in ebullition for half an hour. By this means the air is completely driven out of the flask. This is now allowed to cool, while the fluid in the shallow dish is kept hot, so that the liquid mounting from it into the flask shall be free from air. When the flask is full it is transferred to an incubating oven heated to 25° to 30° C., and the bent tube is immersed in a dish containing mercury. The little funnel attached to the upright tube is then filled with carbon dioxide and a culture of the butyric-acid bacillus is introduced into the funnel. By turning the stopcock in the upright tube a little of the culture is FIG. 77. admitted to the flask without admitting any air. Fermentation com- mences very soon, as is seen by the bubbles of gas given off. The liquid loses its transparency and the lactic acid is gradually con- sumed, butyrate of lime taking the place of the lactate. Aerobic bacilli capable of producing butyric acid in culture solu- tions containing grape sugar or milk sugar have also been described by Liborius and by Hueppe. Fitz has shown that in culture solutions containing glycerin the Bacillus pyocyaneus produces butyric acid in addition to ethyl alcohol and succinic acid. Bacillus Fitzianus also produces some butyric acid in solutions containing glycerin, although the principal product of the fermentation caused by this microorganism is, according to Fitz, ethyl alcohol, twenty-nine grammes of which may be obtained from one hundred grammes of glycerin. 136 PRODUCTS OF VITAL ACTIVITY. Botkin (1892) has described a "Bacillus butyricus" (No. 40*,) which he has not been able to identify positively with the butyric- acid ferment described by Prazmowski. It is a widely distributed anaerobic bacillus, which he was able to obtain from milk or water containing it by placing it in the steam sterilizer for half an hour. The spores resisted this temperature and subsequently grew in anae- robic cultures, in a suitable medium, while all other bacteria and spores present were destroyed. The writer has described a bacillus which causes active acid fermentation in culture solutions containing glycerin. The aoid formed is volatile and is probably propionic acid — see Bacillus acidi- formans. The Caucasian milk ferment — Bacillus Kaukasicus — produces a variety of products in the fermented milk which is a favorite drink among the Caucasians. The principal ones are ethyl alcohol, lactic acid, and carbon dioxide, but in addition to these small quanti- ties of succinic, butyric, and acetic acids are formed. The inhabi- tants of the Caucasian mountains prepare this fermented drink in a very simple manner from the milk of cows or goats, to which they add the dried ferment collected from a receptacle in which the fermen- tation had previously taken place. Fliigge gives the following di- rections for the preparation of this drink : " Two methods may be employed. In the first the dry brown kefir-kdr- ner of commerce are allowed to lie in water for five to six hours until they swell; they are then carefully washed arid placed in fresh milk, which should be changed once or twice a day until the korner become pure white in color and when placed in fresh milk quickly mount to the surface — in twenty to thirty minutes. One litre of milk is then poured into a flask and a full tablespoonful of the prepared korner added to it. It is allowed to stand open for five to eight hours; the flask is then closed and kept at 18° C. It should be shaken every two hours. At the end of twenty four hours the milk is poured through a fine sieve into another flask, which must not be more than four-fifths full. This is corked and allowed to stand, being shaken from time to time. At the end of twenty four hours a drink is ob- tained which contains but little COa or alcohol. Usually it is not drunk until the second day, when, upon standing, two layers are formed, the lower milky, translucent, and the upper containing fine flakes of casein. When shaken it has a cream like consistence. On the third day it again becomes thin and very acid. "The second method is used when on** has a good kefir of two or three days to start with. Three or four parts of fresh cow's milk are added to one part of this and poured into flasks which are allowed to stand for forty- eight hours with occasional shaking When the drink is i-eady for use'a portion (one-fifth to one third) is left in tho flask as ferment fora fresh quantity of milk. The temperature should be maintained at about 18° O. ; but at the commencement a higher temperature is desirable. The korner snould be carefully cleaned from time to time and broken up to the size of j>«-as. The cleaned korner may be dried upon blotting paper in the sun or in the vicinity of a stove: when dried in the air they retain their power to germinate for a long time." Fermentation of urea. The alkaline fermentation of urino i< PRODUCTS OF VITAL ACTIVITY. 137 effected by various microorganisms, but chiefly by the Micrococcus ureae, the ferment action of which has been carefully studied by Pas- teur, Duclaux, and others. The change which occurs under the action of the living ferment was determined by the chemist Dumas as long ago as 1830, but it remained for Pasteur to show that this change depends upon the presence and vital activity of a living microorganism. The transformation of urea into carbonate of ammonia is shown by the following formula : COH4ST0 + 2H2O = CO2 + 2NH9 + H,0 - (NHJ.CO,. According to Van Tiegheni, Micrococcus ureae continues to grow in a liquid containing as much as thirteen per cent of carbonate of ammonia. It may be cultivated in an artificial solution of urea, with the addition of some phosphates, as well as in urine. The Bacillus ureae of Miquel has also the power of producing the alkaline fermentation of urine, but it does not thrive in so strong a solution of carbonate of ammonia. A different micrococcus — Micrococcus ureae liquefaciens — nas also been studied in Fliigge's laboratory which possesses the same power. According to Musculus, a soluble ferment may be isolated from urine which has undergone alkaline fermentation, which changes urea into carbonate of ammonia. He obtained it from urine containing con- siderable mucus, in a case of catarrh of the bladder. But Leube has shown that cultures of Micrococcus ureas from which the micrococ- cus was removed by filtration through clay do not induce alkaline fermentation. The soluble ferment obtained by Musculus must therefore be from some other source. Miquel has given special attention to the study of bacteria which produce alkaline fermentation in urine, and in addition to the spe- cies above mentioned has described the following : Urobacillus Pas- teuri, Urobacillus Duclauxi, Urobacillus Freudenreichi, Urobacillus Madcloxi, Urobacillus Schutzenbergi. Viscous fermentation. A special fermentation which occurs sometimes in wines, and in the juices of bulbous roots containing glucose, and in milk, is produced by various bacteria. One of these is a micrococcus which has been described by Conn under the name of Micrococcus lactis viscosus. The fermented juices become very viscous, owing to the formation of a gum-like product resembling dextrin; at the same time mannite and CO2 are produced. The gum-like substance, called viscose by Bechamp, is soluble in cold water and is precipitated by alcohol. Guillebeau (1892) has de- scribed a micrococcus and a bacillus which produce viscous fer- mentation in milk — Micrococcus Freudenreichi and Bacillus Hessi. 138 PRODUCTS OF VITAL ACTIVITY. A micrococcus producing viscous fermentation in milk has also been described by Schmidt-Miihlheim, and a bacillus by Loffler. Bacillus mesentericns vnlgatus also produces a similar change in milk. Mai'fth f/^.s-, CH4, is produced by the fermentation of cellulose, through the action of microorganisms the exact characters of which have not yet been determined. According to Tappeiner, there are two different fermentations of cellulose. The first occurs in a neu- tral one-per-cent flesh extract solution to which cotton or paper pulp has been added. The gases given off are CO, and CH4 and small quantities of H3S. The second fermentation occurs when an alkaline solution of flesh extract containing cellulose in suspension is used. The gases formed are CO, and H. In both cases small quantities of aldehyde, isobutyric acid, and acetic acid are produced. Hydrosulphuric acid, H2S. This gas is produced during the growth of certain bacteria. The conditions governing its develop- ment have been studied by Holschewnikoff, who experimented with two species, one isolated by himself and one by Lindeiiborn, named respectively Bacterium sulfureum and Proteus sulfureus. The first- mentioned bacterium, when inoculated into eggs, produced within three or four days an abundant quantity of H2S ; the other did not. Upon raw albumin both species produced but little, and 011 the yolk of egg a considerable amount of this gas. Upon cooked egg the action was the reverse. In peptone-bouillon the evolution of H2S was abundant ; in the absence of peptone, very slight. Putrefactive fermentation. The putrefactive decomposition of albuminous material of animal and vegetable origin is effected by a great variety of microorganisms and gives rise to the forma- tion of a great variety of products, some of which are volatile and are characterized by their offensive odors. According to Flugge, the first change which occurs consists in the transformation of the albu- mins into peptone, and this may be effected by a large number of different bacteria. Among the products of putrefactive fermenta- tion known to chemists are the following substances : Carbon diox- ide, hydrogen, nitrogen, hydroBulphuric acid (H2S), phosphoretted hydrogen (PHJ, methane, formic acid, acetic acid, butyric acid. valerianic acid, palmitic acid, crotonic acid, glycolic acid, oxalic acid, succinic acid, propionic acid, lactic acid, amidostearic acid, leucin, ammonia, ammonium carbonate, ammonium sulphide, tri- methylamine, propylamine, indol, skatol, tyrosin.neuridin, cadaverin. putrescin, cholin, neurin, peptotoxin. and various other volatile acids, ptomaines, etc. The special products of putrefaction vary according to the nature of the material, the conditions in which it is placed, and the micro- PRODUCTS OF VITAL ACTIVITY. 139 organisms present. One or the other of the bacteria concerned will take the precedence when circumstances favor its growth. Thus the aerobic bacteria cannot grow unless the putrefying material is freely exposed to atmospheric oxygen ; the anaerobic species require its exclusion. Some saprophytic bacteria grow at a comparatively low temperature, others take the precedence when the temperature is high ; some, no doubt, thrive only in presence of products evolved by other species, and are consequently associated with and depend- ent upon these species ; some are restrained in their growth sooner than others by the products evolved as a result of their own vital activity or that of associated organisms ; some grow in the presence of acids and give rise to an acid fermentation which wholly prevents the development of other species. At the outset putrefaction is often attended with the presence of several species of micrococci and certain large bacilli, which are displaced later by short motile bacteria belonging to a group which includes several bacilli formerly described under the common name of Bacterium termo. The malodorous volatile products of putrefaction are to a consid- erable extent produced by anaerobic species. For this reason these odors are more pronounced when masses of albuminous material undergo putrefaction in situations where the oxygen of the air has not free access or where it is displaced by carbon dioxide. The body of a dead animal, although freely exposed to the air, furnishes in its interior a suitable nidus for these anaerobic gas-forming spe- cies, and they may give rise to products of one kind, while aerobic species upon the surface of the mass induce different forms of putre- factive fermentation. In the bodies of living animals these anaero- bic microorganisms are constantly present in the intestine, and after death they quickly invade the body and multiply at its expense under favorable conditions as to temperature. The surface decom- position due to aerobic bacteria occurs later and is not attended with the same putrefactive odors, the products evolved being of a simpler chemical composition — CO2, HN3. No doubt these aerobic bacteria, by consuming the oxygen and forming an atmosphere of carbon dioxide, help to make the conditions favorable for the con- tinued development of the aiiaerobics in the interior of the organic • mass ; at the same time they find a suitable pabulum in some of the more complex products of decomposition occurring in the absence of oxygen. The gases produced in the interior of a putrefying mass are mainly CH4, H2S, and H. Many of the bacteria of putrefaction are facultative aiiaerobics — that is to say, they are able to multiply either in the presence of oxy- gen or in its absence. The products evolved by these differ, no 140 PRODUCTS OF VITAL ACTIVITY. doubt, arc, .nling to whether they are or are not supplied with atmos- pheric oxygen. The anaerobic bacteria concerned in putrefaction have as yet received comparatively little attention. Among the aerobics and facultative anaerobics the following are best known: Micrococcus foetidus, Bacillus saprogenes I., II., and III., Bacillus coprogenes- foetidus, Bacillus putrificus coli, Proteus vulgaris, Proteus Zenkeri, Proteus mirabilis, Bacillus pyogeues foetidus, Bacillus fluorescens liquefaciens, Bacillus pyocyaneus, Bacillus coli communis, Bacillus janthinus. Sithihh' /-V/ -im-iifti. — Several species of bacteria produce soluble ferments capable of changing starch into maltose, dextrin, etc. Hueppe has shown that the lactic-acid bacillus produces a diastase, and Miller obtained 1'nnn the human intestine a species which dis- -olves starch. Marcano, by filtering cultures of species capable of this ferment action through porcelain. \vas able to show that the effect is due to a soluble ferment, which must have been produced by the vital activity of the living microorganisms. Wortmann also obtained a diastase from culture liquids which was precipitated by alcohol ;ind again dissolved in water: in slightly acid solutions it promptly converted starch into glucose. This is said to be produced in culture liquids only when these do not contain albumin. In the presence of all mini n a peptonizing ferment was formed; in its ab- ••e. a diastase by which starch was dissolved to serve as pabulum for the bacteria present. These experiments were not made with pure cultures, and more exact researches in this direction are de- niable. A peptonizing ferment for gelatin is produced by a considerable number of bacteria, as stated under the heading "Liquefaction of Gelatin." The jellified albumin in cultures in blood serum is also liquefied by a peptoni/in- ferment prod need by certain species of ba.-- >'• authors also speak of a soluble ferment capable of inverting cane sugar or milk >n-ar. According to Hueppe. such a ferment iced by the Bacillus acidi lactici. A soluble ferment for cel- hlkMH is Mippo^-d by Flttgge to be produced by several species— •mOOg Others by Bacillus butyricus and by Vibrio ruguhi. Sev.-ral bacilli produce a soluble ferm.'iit capable of coa-'ulatiiiLC tin- 'MM»in of milk. l:""n.—The researches of Gayon. hupettit. and others show that certain bacteria are able to reduce nitrates with liberation qf ammonia and free nitrogen. This is effected in the absence of oxygen by anaerobic bacteria, and. PRODUCTS OF VITAL ACTIVITY. 141 among others, lay Bacillus butyricus. Certain aerobic bacteria also accomplish the same result. Thus Herseus obtained two species from water which reduced nitrates in a very decided manner. On the other hand, a number of species are known to oxidize ammonia, producing nitric acid. Schlosing and Miinz, as a result of numerous experiments, arrived at the conclusion that in the soil nitrification is effected by a single species. But it is doubtful whether they worked with pure cultures, and more recent researches show that several, and probably many, different bacteria possess this power. Accord- ing to Heraaus, the following species, tested by him, oxidize am- monia : Bacillus prodigiosus, the cheese spirillum of Deneke, the Finkler-Prior spirillum, the typhoid bacillus, the anthrax bacillus, the staphylococci of pus. The oxidation does not always go to the point of forming nitrates, but nitrites may be formed in the soil (Duclaux). Warrington states that certain bacteria which formed nitrates in a suitable culture medium produced only nitrites when, after an interval of four or five months, some of the culture was transferred to a solution containing muriate of ammonia. The same author states that the process of nitrification occurs only in the dark. The researches of Winogradsky, of the Franklands, and of Jor- dan show that the failure of earlier investigators to obtain the nitri- fying bacteria from the soil in pure cultures was due to the fact that these bacteria do not grow in the usual culture media. By the use of certain saline solutions the authors named have succeeded in iso- lating nitrifying bacteria in pure cultures, or nearly so. It is still uncertain whether these investigators have obtained the same bac- teria, but the microorganisms described by them, and obtained from widely distant sources, are similar in their morphological and bio- logical characters, and at least belong to the same group. In a com- munication made in 1891 Winogradsky arrives at the conclusion that the ferments which cause the oxidation of ammonia and pro- duction of nitrites are not capable of producing nitrates, but that other microorganisms are concerned in the oxidation of nitrites. In sterilized soil to which a pure culture of his nitromonas was added nitrites only were produced, and the presence of various microorganisms common in the soil did not result in the forma- tion of nitrates so long as the specific ferment was absent to which this second oxidation is ascribed (nitrifying bacillus of Winograd- sky). Phosphorescence. — Several different bacteria have been studied which, in pure cultures, give rise to phosphorescence in the medi- um in which they are cultivated. In gelatin cultures the light is sufficient in some instances to enable one to tell the time by a 11-.' PROMPTS OF VITAL ACTIVITY. watch in a |*?rfectly dark room, and such cultures have even been photographed by their own light. The phosphorescence is influenced by changes in the culture medium and by conditions of temperature, but we have no exact knowledge of the mode of its production. The Bacillus phosphores- < ins from sea water in the vicinity of the West Indies gives the must striking results, especially when planted upon the surf ace of cooked fish and placed in an incubating oven at 30° C. Two other species have been studied by Fischer — one obtained from the water of the- harl>or at Kiel, and the other a widely distributed species • •ailed by Fischer Bacterium phosphorescens. Katz (1801) has de- -« -rilied several species obtained by him from sea water and from phosphorescent fish in the markets at Sydney, New South Wales — Bacillus smaragdino-phosphorescens, Bacillus argenteo-phosphores- cens, Bacillus cyaneo-phosphorescens, Bacillus argenteo-phosphores- cens liquefaciens. V. PTOMAINES AND TOXALBUMINS. VARIOUS basic substances containing nitrogen, and in chemical constitution resembling the vegetable alkaloids, have been isolated by chemists from putrefying material and from cultures of the bac- teria concerned in putrefaction, and also from certain pathogenic species. Some of these ptomaines are non-toxic, and others are very poisonous in minute doses (toxines). The toxic substances sometimes developed in milk, cheese, sausage, etc., are also of this nature, and are doubtless produced by the action of microorganisms. The pathogenic power of the bacteria which cause various infectious diseases in man and the lower animals has also been shown to result from the production of toxic ptomaines or of toxalbumins. Selmi first gave the name ptomaines to cadaveric alkaloids isolated by him, and Panum subsequently called attention to the fact that poisonous basic substances of this class are contained in putrefying material. Ex- tended researches with reference to the ptomaines have since been made by numerous chemists, the most important being those of Berg- mann, Schmiedeberg, Zuelzer and Sonnenschein, Hager, Otto, Sel- mi, Brieger, Gautier and Etard, and Vaughan. For a full account of the history and chemical composition of the ptomaines the reader is referred to the valuable work of Vaughan and Novy (" Ptomaines and Leucomaines," Philadelphia, 1891). In the present volume we shall give a brief account only of some of the most important. NON-TOXIC PTOMAINES. Neuridin, C5HJ4N2. — This is one of the most common of the al- kaloids of putrefaction and was isolated by Brieger in 1884. It is obtained most abundantly from tissues containing gelatin. Very soluble in water, but insoluble in ether and absolute alcohol. Has a disagreeable odor. Cadaverin, C6HMN3. — Isomeric with iieuridin ; has a very dis- agreeable odor ; forms a thick, transparent, syrupy liquid ; is vola- tile, and can be distilled with steam without undergoing decomposi- tion. When exposed to the air the base absorbs carbon dioxide and 144 PTOMAINKS AND TOXALBl'MINS. form> a crystalline mass. Is produced in cultures of the cholera >j.ii ilium and of the spirillum of Finkler and Prior which have been kept for a month or more at 37° C. Putretcin, C4H1SN,.— A base resembling cadaverin and com- 1 1 1< mly associated with it. Obtained by Brieger from various sources, most abundantly from substances containing gelatin and in the in- »re advanced stages of putrefaction. It is obtained in the form of a hydrate, which is a transparent liquid having a boiling point of a bou t 1 :'».")0. With acids it forms crystalline salts. X'i/n'in. Ct A ,.N2.— Resembles cadaverin and is commonly as- B „ Mated with it in putrefying material. Isolated by Brieger. Met Ill/In mine, CHS.NH,.— Obtained by Brieger from putrefying ti-li and from old cultures of the cholera spirillum. Dinn'f In/la mine, (CH,),.NH. — Obtained by Brieger from putre- fying p -la tin and by Bocklisch from decomposing fish. Trt'im-f hi/him inc. (CH,)SN. — Obtained from various sources, and l»y Brieger from cultures of the cholera spirillum and of the strepto- •ns «»r pu-. TOXIC PTOMAINES. \ ••'iriii, CftH,,NO. —First obtained by Liebreich in 1865 as a • I. < ,.nijK>sition product of protagon from the brain. Obtained by Brieger from putrefying muscular tissue. When crystallized from an aqueous solution it forms five- or six-sided plates ; from an alco- holic solution it crystallizes in the form of needles (Liebreich). This -e, is toxic in small doses. In frogs the injection of a few milli- 1 1 lines produces paralysis of the extremities. Respiration is first a r posted and the heart stops in diastole. Atropine appears to be a physiological antidote to the toxic effects of neurin. In rabbits it produces profuse salivation. The pupil is contracted by the direct application of a concentrated solution. <'!„,/ in.. C.H..NO,.— First obtained from hog's bile by Strecker in 1862. Has been obtained by Brieger from various sources, in- « •hiding i Mil tn ITS of the cholera spirillum. It is also found widely di-trihuted in the vegetable kingdom. Maybe prepared from the s oik of eggs by the method of Diakonow. Cholin is obtained in the t'onn of a syrupy, alkaline liquid which combines with acids to form deliquescent salt-. At first this base was not supposed to have toxic properties. l»ut more recent researches have shown that in compara- tively large doses it produces symptoms resembling those caused by minute doses of m-nrin. Mu8carin, C,HUNO,.— This toxic principle of poisonous mush- • i ns has al so h, M n < >btained by Brieger f r< >m pu t ref ying fish. It may be produ.-.-d artiti.-ially hy tin- oxidation of oholin. In small doses it kilU rahhit< and frogs. In the rabbit it produces lacrymation and PTOMAINES AND TOXALBUMINS. 145 salivation, the pupil is contracted, and the animal dies in convul- sions. Frogs are completely paralyzed by the action of muscarin and die with arrest of the heart's action in diastole. Peptotoxin. — The exact composition of this ptomaine has not been determined. Brieger obtained it during the early putrefac- tion of proteid substances and also from the artificial digestion of fibrin. It is very poisonous for frogs, which become paralyzed and die within fifteen or twenty minutes after the subcutaneous injection of a few drops of a dilute solution. Rabbits also are killed by doses of half a gramme to a gramme, the symptoms being paralysis of the posterior extremities and stupor. Peptotoxin is soluble in water, but insoluble in ether or chloroform. It is not destroyed by boiling. Tyrotoxicon. — First obtained by Vaughan in poisonous cheese, and subsequently by the same chemist and others in poisonous milk and ice cream. Chemically tyrotoxicon is very unstable. It is de- composed when heated with water to 90° C. It is insoluble in ether. From sixteen kilogrammes of poisonous cheese Yaughan obtained 0. 5 gramme of the poison. The symptoms produced in man by eat- ing cheese or milk containing tyrotoxicon are vertigo, nausea, vomit- ing, and severe rigors, with pain in the epigastrium, cramps in the legs, griping pain in the bowels attended with purging, numbness and a pricking sensation in the limbs, and great prostration. Methyl-guanidin, C3H7N3. — Obtained by Brieger from putrefy- ing horseflesh which had been kept at a low temperature for several months. This base was previously known to chemists, having been obtained by the oxidation of creatin. By Bocklisch it has been ob- tained from impure cultures of the Finkler-Prior spirillum which had been kept for about a month. It is obtained as a colorless mass having an alkaline reaction, and which is quite deliquescent. Brie- ger gives the following account of the toxic action as tested on guinea-pigs in a dose of 0. 2 gramme : The respiration increases in rapidity, the pupils dilate to the extreme limit, the animal has copi- ous discharges of urine and faeces, the extremities become paralyzed, and at the end of about twenty minutes death occurs in convulsions. Mytilotoxin. — Obtained by Brieger from poisonous mussels. The toxic action resembles that of curare. Typhotoxtn, C7H17NO2. — Obtained by Brieger from bouillon cultures of the typhoid bacillus which had been kept for a week or more at a temperature of about 37.5° C. In mice and guinea-pigs this base produces salivation, rapid respiration, dilatation of the pupils, diarrhoea, and death in from twenty-four to forty-eight hours. It is believed by Brieger that the specific action of the typhoid bacil- lus is due to the production of this ptomaine. A base which is isomeric with typhotoxin has been obtained by 10 PTOMAINES AND TOXALBUMINS. Brieger from putrefying horseflesh which was kept at a low tempe- rature for several months. Unlike it, however, the free base has an acid reaction, while typhotoxin is strongly alkaline. It differs also in its physiological action, being more toxic and producing convul- sions ; the heart is arrested in diastole. Typhotoxin, on the other hand, does not induce convulsions and the heart is arrested in systole. Tetanin, C,,H,0N,O4.— Obtained by Brieger from impure cul- tures of the tetanus bacillus cultivated in bouillon in an atmosphere of hydrogen. (The tetanus bacillus is a strict anaerobic.) Obtained subsequently by the same chemist from the amputated arm of a pa- tient with tetanus. This base has been obtained, by crystallization from hot alcohol, in clear yellow plates which are not very soluble in water. The hydrochloride is a deliquescent salt which dissolves readily in alcohol. When injected into guinea-pigs or mice in rather large doses, tetanin first causes the animal to fall into a lethargic condition, followed by increased rapidity of respiration and tetanic convulsions. In guinea-pigs opisthotonos is induced, together with the characteristic tetanic convulsions as seen in animals suffering from tetanus. Three other toxic bases have been obtained by Brieger from cultures of the tetanus bacillus, which cause similar symptoms. One — tetanotoxin — is given by Brieger the formula C5HMN. A second base, the composition of which has not been determined, is called spasmotoxin. Cholera Ptomaines. — Brieger has obtained from pure cultures of the cholera spirillum several of the toxic ptomaines heretofore re- ferred to — cadaverin, putrescin, cholin, methyl-guanidin. In addi- tion to these he found two toxic substances which appear to be pe- culiar products of this microorganism. One induces cramps and muscular tremors in small animals, the other diarrhoea and symp- toms of collapse. Toxalbumins. — Researches by Brieger and Frankel (1890) show that very toxic substances of a different nature are present in cultures of some of the pathogenic bacteria ; these have been designated by the authors named "toxalburnins." Roux and Yersin had previously shown that filtered cultures of the diphtheria bacillus contain a toxic substance which produces paralysis ami death in ^ninea-pi-s and rabbits. This substance lias now been obtained in a pure state and its toxic action tested by the authors ti''st ii.-nm-d. It is destroyed by a temperature of On ('., but remains in an active condit i« .n in cultures which have been sterilized by seve- ral hours' exposure to a temperature of 50°, or in those which have IMM-II passed thro.,-!, a clay filter. It is not volatile, and differs essen- li;lll.v fr"m tll(1 i't"inainosand also from the soluble ferments. It was obtained as a snow-white, amorphous mass which was ex- PTOMAINES AND TOXALBUMINS. 14? tremely toxic in its action upon small animals. When injected into guinea-pigs in the proportion of two and one-half milligrammes to one kilogramme of body weight, it caused death after a considerable interval of time (from a few days to several weeks), during which the animal became emaciated and spreading abscesses and necrosis of the tissues occurred at the point of injection. This toxalbumin was obtained in a pure state by repeated precipitation from an aque- ous solution by means of alcohol. It is produced most abundantly in cultures containing albumin, and old cultures are more toxic than recent ones. Chemical analysis gave the following result : C 45, 35, H 7, 13, N 16, 33, S 1, 39, 'O 29, 80. The authors remark, however, that the chemical characters have not yet been fully determined. The same chemists have obtained toxic substances of a similar nature from cultures of the bacillus of typhoid fever, of the tetanus bacillus, of the Staphylococcus aureus, and of the cholera spirillum. Hankin had previously obtained a toxic "albumose" from cultures of the anthrax bacillus by precipitation with alcohol, drying, solu- tion in water, and filtration through porcelain ; and Christmas had obtained an albuminous substance from cultures of Staphylococcus aureus which produced pus formation when injected beneath the skin of rabbits or into the anterior chamber of the eye. According to Brieger and Frankel, these toxalbumins are divided into two principal groups, one of which is characterized by solubility in water, as in that produced by the diphtheria bacillus ; and one in which the albumin is insoluble or but slightly soluble, as is the case with those obtained from cultures of the typhoid bacillus, the cholera spirillum, and the Staphylococcus aureus. The toxalbumin from cholera cultures, obtained as pure as pos- sible and suspended in water, when injected under the skin of a guinea-pig, caused its death in two or three days. It was not, how- ever, toxic for rabbits, even when injected in considerable quantity. On the contrary, the toxalbumin of the typhoid bacillus, which is dissolved with difficulty in water, was more poisonous for rabbits than for guinea-pigs. When injected subcutaneously into rabbits death usually occurred in eight to ten days. No notable pathologi- cal changes were observed at the autopsy. The toxalbumin of Staphylococcus aureus killed rabbits and guinea-pigs within a few days, and in some cases at the end of twenty-four hours. The post-mortem appearances were necrosis or purulent breaking down of the tissues at the point of injection, with swelling and redness of the surrounding tissues and general inflam- matory appearances. The toxalbumin of anthrax cultures resembles that of the diphtheria bacillus in being soluble in water. It was obtained by Brieger from the organs of animals recently dead from 148 PTOMAINES AND TOXALBUMINS. anthrax. In a dry condition it has a grayish-white color and gives the reactions of albumins. The toxalbumin of the tetanus bacillus is also soluble in water. It is best obtained in bouillon cultures containing glucose. G. and F. Klemperer (1891) have announced their success in obtaining a toxalbumin from cultures of Micrococcus pneumonias crouposse ('diplococcus pneumonia*') ; this they propose to call pneu- motoxin. Koch's " Tuberculin."— This, is a glycerin extract of the toxic substances present in cultures of the tubercle bacillus. Crude tu- berculin is obtained from liquid cultures made in veal broth to which one per cent of peptone and four to five per cent of glycerin have been added. This culture liquid is placed in flasks and inoculated upon the surface with small masses from a pure culture of the tu- bercle bacillus. A tolerably thick and dry white layer is developed, which after a time covers the entire surface. At the end of six to eight weeks development ceases and the culture liquid is evaporated over a water bath to one-tenth its volume ; this, after being filtered, constitutes the crude tuberculin. By precipitation with sixty-per- cent alcohol Koch has obtained from this a white precipitate which has the active properties of the glycerin extract. This is soluble in water and in glycerin, and has the chemical reactions of an albumi- nous body. Zuelzer has (1801) reported his success in isolating atoxic sub- stance from tubercle cultures. The contents of tubes containing pure cultures of the bacillus are first treated with hot water acidulated with hydrochloric acid. This solution is filtered, evapo- rated, and then several times precipitated with platinum chloride. The double salt formed is decomposed by hydrosulphuric acid, after which the liquid is filtered and evaporated to dryness. A white, crystalline salt is thus obtained which is soluble in hot water. This salt was toxic for rabbits and guinea-pigs in doses of from one to three centigrammes. Death usually occurred in from two to four days. In guinea-pigs one centigramme injected subcutaneously caused, within a few minutes, a greatly increased frequency of respi- ration, an elevation of temperature, and protrusion of the eyeballs. Malle'in. — Kalwing, Preusse, and Pearson have obtained from cultures of the glanders bacillus a "lymph" which somewhat re- sembles the crude tuberculin of Koch. This was obtained by 1'ivussc l.y tivjitin^- <>1«1 j»ot;it< > cult mvs of thr glanders bacillus with glycerin and water. The extract was filtered several times and then sterilized in a steam stn-ili/er. This lymph injected into horses in- fected with glanders gives rise to a very decided elevation of tempe- ratuiv. \\ liilr iu horses free from this disease no such result follows. VI. INFLUENCE OF PHYSICAL AGENTS. Heat. — We have already seen (Section II. , Part Second) that the temperature favorable for the growth of most bacteria is between 20° and 40° C. ; that some species are able to multiply at the freezing tem- perature, and others at as high a temperature as 60° to 70° C. ; that, as a rule, the parasitic species require a temperature of 35° to 40°; and that low temperatures do not kill bacteria. Frisch (1877) exposed various cultures to a temperature of —87° C., which he obtained by the evaporation of liquid CO2, and found that micrococci and bacilli, after exposure to such a temperature, multi- plied abundantly when again placed in favorable conditions. Prud- den has also made extended experiments upon the influence of freezing. He found that while certain species resisted the freezing temperature for a long time, others failed to grow. Thus Bacillus prodigiosus did not grow after being frozen for fifty-one days ; Pro- teus vulgaris was killed in the same time, and a slender, liquefying bacillus obtained from Croton aqueduct water was killed in seven days. Staphylococcus pyogenes aureus withstood freezing for sixty- six days, a fluorescent bacillus from Hudson River ice for seventy- seven days, and the bacillus of typhoid fever for one hundred and three days. Cultures made at intervals showed, however, a dimi- nution in the number of bacteria. A similar diminution would per- haps have occurred in old cultures in which the pabulum for growth was exhausted, independently of freezing ; for bacteria, like higher plants, die in time — which varies for different species — as a result of degenerative changes in the living protoplasm of the cells, and con- tinued vitality in a culture depends upon continued reproduction. Repeated freezing and thawing was found by Prudden to be more fatal to the typhoid bacillus than continuous freezing. Cul- tures were sterilized by being thawed out at intervals of three days and again ref rozen, after repeating the operation five times. Cadeac and Malet kept portions of a tuberculous lung in a frozen condition for four months, and found that at the end of this time tuberculosis was still produced in guinea-pigs by injecting a small quantity of this material. ]50 INFLUENCE OF PHYSICAL AGENTS. In considering the influence of high temperatures we must take account of the very great difference in the resisting power of the vegetative cells and the reproductive elements known as spores, also of the fact as to whether dry or moist heat is used and the time of exposure. Dry Heat.— When microorganisms in a desiccated condition are exposed to the action of heated dry air, the temperature required for their destruction is much above that required when they are in a moist condition or when they are exposed to the action of hot water or steam. This was thoroughly demonstrated by the experiments of Koch and Wolffhugel (1881). A large number of pathogenic and non-pathogenic species were tested, with the following general result : A temperature of 78° to 123° C. maintained for an hour and a half (over 100° for an hour) failed to kill various non-pathogenic bacteria, but was fatal to the bacillus of mouse septicaemia and that of rabbit septicaemia. To insure the destruction of all the species tested, in the absence of spores, a temperature of 120° to 128° C., maintained for an hour and a half, was required. The spores of Bacillus anthracis and of Bacillus subtilis resisted tins temperature and required to insure their destruction a tempera- ture of 140° C! maintained for three hours. This temperature was found to injure most objects requiring disinfection, such as clothing and bedding. But the lower temperature which destroys micro- ;;inisms in the absence of spores (120° C. = 248° F.) can be used for disinfecting articles soiled with the discharges of patients with cholera, typhoid fever, or diphtheria, as the specific germs of these « 1 1 Ceases do not form spores. It is probable also that it may be safely usersinii- t«> li\ tli<> tlnM-mal atli-j>oint of the virus of lydrophobia, I obtained, through the kindness of Dr. H. C. Ernst, a rabbit which had been inoculated, by the method of trephining, with material lch came originally from Pasteur's laboratory. The rabbit sent me showed the first symptom of paralytic rabies on the eighth day after inocu- It died on the eleventh day (March 2d, 1887), and I at once pro- MMM to i,,ak«> th«- f.,Ilmvii,ir ••xp.'miiont : A portion of the medulla was removed and thoroughly mixed with K,?lfl? I?18,,?*8 written Brieger has isolated a toxalbumin from cultures of the diphtheria bacillus which is destroyed by a temperature of 60° C., but resists 50°. INFLUENCE OF PHYSICAL AGENTS. 153 sterilized water. The milky emulsion was introduced into four capillary tubes, such as had been used in my experiments heretofore recorded. Two of these tubes were then placed for ten minutes in a water bath, the tem- perature of which was maintained at 60° C. Four rabbits were now inocu- lated by trephining, two with the material exposed to 60° C. for ten min- utes, and two with the same material from the capillary tube not so exposed. The result was as definite and satisfactory as possible. The two control rabbits were taken sick, one on March 10th and one on the llth ; both died with the characteristic symptoms of paralytic rabies on the third day. The two rabbits inoculated with material exposed to 60° C. remained in perfect health. On the 26th of March one of these rabbits was again inoculated, by trephining, with material from the medulla of a rabbit just dead from hydrophobia. This rabbit died from paralytic rabies on the 8th of April. Its companion remains in perfect health. "A second experiment was made in the same way on the 14th of March. Two rabbits were inoculated with material exposed for ten minutes to a temperature of 50° C. ; two with material exposed to 55° C. ; and two con- trol rabbits with material not so exposed. One of the rabbits inoculated with material exposed to 50° C., and one of the control rabbits, died on the 25th; the other rabbit inoculated with the material exposed to 50°, the other control, and one inoculated with material exposed to 55°, on the 26th. The second rabbit inoculated with material exposed to 55° died five days later with the characteristic symptoms of the disease. These experiments show, then, that the virus of hydrophobia is destroyed by a temperature of 60° C., and that 55° C. fails to destroy it, the time of exposure being ten minutes."1 The experimental data given show that the pathogenic bacteria tested and different kinds of virus are all killed by a temperature of 60° C. or below ; some, like the cholera spirillum and Micrococcus pneumonias crouposse, failing to grow after exposure to as low a tem- perature as 52° C. for four minutes. By extending the time a still lower temperature will effect the same result. Thus, according to Chauveau, the anthrax bacillus is killed by twenty minutes7 exposure to a temperature of 50° C. ; and Brieger sterilizes cultures of the diphtheria bacillus, to obtain the soluble toxalbumin produced in them, by exposure for several hours to 50° C. A temperature of 60° has been found to decompose the toxalbumin. The non-pathogenic bacteria tested have, as a rule, a higher thermal death-point — 58° C. for Bacillus prodigiosus, 64° C. for Sarcina lutea, etc. It is a remarkable fact that certain bacteria not only are not de- stroyed at higher temperatures than this, but are able to multiply at a temperature of 65° to 70° C. Thus Miquel, in 1881, found in the waters of the Seine a motionless bacillus which grew luxuriantly in bouillon at a temperature of 69° to 70° C. Van Tieghem has also cultivated several different species at about the same temperature, and more recently Globig has obtained from the soil several species which grow at temperatures ranging from 50° to 70° C. The resisting power of spores to heat also varies in different spe- cies ; but the spores of known pathogenic bacteria are quickly de- stroyed by a temperature of 100° C. (212° F.). In the writer's experi- 1 Report of the Committee on Disinfectants (op. cit.), p. 147. l.VJ INFLUENCE OF PHYSICAL AGENTS. ments the spores of Bacillus anthracis and of Bacillus alvei failed to grow after exposure to a temperature of 100° C. for four minutes, and only a few colonies developed after two minutes' exposure to this temperature. The thermal death-point of spores of the " wurtzel ba- cillus " and of Bacillus butyricus (of Hueppe) was the same — 100° C. for four minutes. Schill and Fischer, in 1884, made a number of experiments to de- termine the thermal death-point of Bacillus tuberculosis. They found that five minutes' exposure to a temperature of 100° C. in steam destroyed the vitality of the bacillus in sputum in five min- utes. When the time was reduced to two minutes a negative result from inoculation was obtained in two guinea-pigs, but one inoculated at the same time became tuberculous. My own experiments and those of Yersin, made since, lead me to think that there may have been some cause of error in this experiment of Schill and Fischer, and that the thermal death-point of the spores of Bacillus tuber- culosis is considerably below the boiling point of water. I inoculated guinea-pigs with tuberculous sputum subjected for ten minutes to the following temperatures : 50°, 60°, 70°, 80°, 90° C. The animal inoculated with material exposed to 50° died from tuberculosis at the end of seven weeks. None of the others developed tuberculosis. Yersin exposed an old culture in glycerin bouillon, in which many of the bacilli contained spores—" tres nettes" — to the following tem- peratures : 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 100° C. " At the end of ten days the bacilli heated to 55° gave a culture in glycerin bouillon ; those exposed to 60° grew after twenty-two days ; none of the bacilli heated above 70° gave any development. This experiment, repeated a great number of times, has always given us the same re- sult." Voelsh, who has studied the same question, reports as the result of his experiments that the tubercle bacillus in sputum was not destroyed by heating to 100° C. Further experiments will be re- quired to reconcile these contradictory results. While the spores of the pathogenic bacteria mentioned are de- stroyed by the boiling point of water within a few minutes, certain non-pathogenic species resist this temperature for hours. Thus Qlobig obtained a bacillus from the soil the spores of which required five and one-half to six hours' exposure to streaming steam for their destruction. These spores survived exposure for three-quarters of an hour in st. -am under pressure at from 109° to 113° C. They were de- stroyed, however, by exposure for twenty-five minutes in steam at 113° to 11U°, and in two minutes at 127°. In the practical application of steam for disinfecting purposes it must be remembered that, while steam under pressure is more effec- tive than streaming steam, it is scarcely necessary to give it the pre- INFLUENCE OF PHYSICAL AGENTS. 155 ference, in view of the fact that all known pathogenic bacteria and their spores are quickly destroyed by the temperature of boiling water ; and also that superheated steam is less effective than moist steam. When confined steam in pJpes is ' ' superheated " it has about the same germicidal power as hot dry air at the same temperature. This is shown by the experiments of Esmarch, who found that an- thrax spores were killed in streaming steam in four minutes, but were not killed in the same time by superheated steam at a, tempera- ture of 141° C. Desiccation. — Cultures of bacteria kept in a moist condition re- tain their vitality for a considerable time, which varies greatly with different species. The writer has found that a culture of the typhoid bacillus preserved in a hermetically sealed glass tube retained its vitality for eighteen months, as did also Bacillus prodigiosus, Bacil- lus cavicida, and some others. According to Kitasato, the cholera spirillum may be preserved in a moist state for seven months ; other bacteria die out in a month or two, but, as a rule, vitality is preserved for several months at least. Spores in a desiccated condition preserve their vitality for a great length of time. But desiccation is quickly fatal to some of the pathogenic bacteria, and especially so to the cholera spirillum. Koch, in his earlier experiments, found that his " comma bacillus" did not grow after being dried upon a cover glass for three hours. Kitasato, in experiments made since, found that a bouillon culture dried upon a thin glass cover was incapable of development after three hours' time, but that cultures in nutrient agar or gelatin survived for two days, probably on account of the thicker layer formed and the longer time required for complete desiccation. Pfuhl has found that the typhoid bacillus dried upon a cover glass retains its vitality for eight to ten weeks, and Lofner states that the diphtheria bacillus re- sists desiccation for four or five months. Cadeac and Malet pro- duced tuberculosis in guinea-pigs by injecting material from the lung of a tuberculous cow which had been kept in the form of a dried powder for nearly five months ; at a later date the virulence was lost. Light. — Downes and Blunt, in a communication made to the Royal Society of London in 1877, first called attention to the fact that light has an injurious effect upon bacteria, and that cultures may be sterilized by exposure to direct sunlight. Tyndall, in experiments made in the clear sunlight of the Alps, verified the fact that the development of bacteria was restrained in cultures during their exposure, but failed to obtain evidence that vitality was destroyed. In 1885 Duclaux took up the subject with pure cultures of various 156 INFLUENCE OF PHYSICAL AGENTS. bacteria, and showed that by prolonged exposure to direct sunlight the spores of various bacilli lose their capacity to germinate. About the same time Arloing published his researches upon the influence of light upon the development of anthrax spores. He found that the anthrax bacillus was not restrained in its growth by diffused lamp- light, but its growth was retarded by an intense gaslight. Spore formation was more abundant in darkness than in red light, and more abundant in red than in white light. When a screen was interposed between the culture and the source of light, consisting of an aqueous solution of hgematoglobin, the growth of the bacilli and of spores was much more luxuriant than in white light. In yellow light it was less abundant than in red. The blue and violet rays were still less favor- able for the growth of the bacillus and the development of spores. The pathogenic power of cultures was not especially influenced by exposure to white gaslight. In subsequent experiments with sun- light Arloing found that two hours of exposure to the July sun suf- ficed to destroy the vitality of anthrax spores, but that a considerably longer exposure (twenty-six to thirty hours) was necessary when the spores had been allowed to germinate in a suitable culture medium. Cultures which were not exposed long enough to destroy the vitality of the bacilli were retarded in their growth, and subsequent exposure for a shorter time (nine to ten hours) completely sterilized them. Cultures which were weakened in their reproductive energy by ex- posure to sunlight were also "attenuated" as to their pathogenic power and could be used as a vaccine in protective inoculations. Ac- cording to Arloing, the effect produced results from the action of the full sunlight and cannot be obtained by the use of monochromatic light. The experiments of Strauss seemed to give support to the view advanced by Nocard that in Arloing's experiments spores did not really exhibit a less degree of resisting power than the vegetating Iwicilli, but that in fact they commenced to vegetate before they were killed. Strauss placed anthrax spores in sterilized distilled water and in txmillnn, and found that, under the same conditions of exposure, the bouillon cultures were sterilized in direct sunlight in nine hours, while the spores suspended in distilled water grew when trans- ferred to a suitable medium. This was accounted for on the suppo- >iti«>n that the bouillon furnishes the necessary pabulum for the de- velopment of the spores and that distilled water does not. Arloing combats this view and has published additional experi- ments which seem to disprove it. He placed small flasks containing anthrax spores in bouillon in the direct rays of the sun in February. Some of the flasks were placed upon a block of ice which reduced the temperature to 4° C. ; the others were not so placed, and the tempe- INFLUENCE OF PHYSICAL AGENTS. 157 rature, in the open air where all were exposed, was 11° C. All of the spores failed to grow after an exposure of four hours. When exposed in water the time of exposure was longer. Roux has shown that the light also has an effect upon the culture medium, and that sterilized bouillon which has been exposed to direct sunlight for some hours restrains the development of anthrax spores subsequently introduced into it, but not of the growing bacilli. His experiments show that access of oxygen is a necessary factor in the sterilization of cultures by sunlight. In the experiments of Momont (1892) dry anthrax spores were found to resist the action of light for a long time, but moist spores, freely exposed to the air, failed to grow after forty-four hours' ex- posure to sunlight. In the absence of spores, anthrax bacilli in a moist condition, when freely exposed to the air, failed to grow after exposure to sunlight for half an hour to two hours ; but in the ab- sence of air the same bacilli were not destroyed at the end of fifty hours' exposure. Geisler (1892), in experiments made upon the typhoid bacillus, found that all portions of the solar spectrum except the red rays ex- ercised a restraining influence upon the development of this bacillus. The electric light gave a similar result. The most decided effect was produced by rays from the violet end of the spectrum. The restrain- ing influence appears, fro.ni the researches of Geisler, not to be due solely to the direct action of light upon the development of the bacilli, but also to changes induced in the gelatin culture medium employed in his experiments. In his address before the International Medical Congress of Berlin, 1890, Koch states that the tubercle bacillus is killed by the action of direct sunlight in a time varying from a few minutes to several hours, depending upon the thickness of the layer exposed. Diffused day- light also has the same effect, although a considerably longer time of exposure is required — when placed close to a window, from five to seven days. Dieudonne (1894), in experiments upon Bacillus prodigiosus and Bacillus fluorescens putidus, found that direct sunlight in March, July, and August killed these bacilli in one and one-half hours, in November in two and one-half hours. Diffuse daylight in March and July restrained development after three and one-half hours' ex- posure (in November four and one-half hours), and completely de- stroyed vitality in from five to six hours. Ward's experiments (1892-1894) show that the blue and violet rays have decided germicidal power, while the rays at the red end of the spectrum are comparatively inert. This corresponds with results previously reported by Arloing. 158 INFLUENCE OF PHYSICAL AGENTS. In the writer's experiments on the cholera spirillum (1892) test tubes, containing sterile bouillon inoculated with one or two ose of a pure culture, were sterilized by two hours' exposure to direct sunlight (in December). Dieudonne (1894) found that the electric arc light destroyed his test organisms (Bacillus prodigiosusand Bacillus fluorescens putidus) in eight hours. The same result was accomplished by the incandes- cent light in eleven hours. In view of these facts we may conclude, with Duclaux, that sun- light is one of the most potent and one of the cheapest agents for the destruction of pathogenic bacteria, and that its use for this purpose is to be remembered in making practical hygienic recommendations. The popular idea that the exposure of infected articles of clothing and bedding in the sun is a useful sanitary precaution is fully sus- tained by the experimental data relating to the action of heat, desic- cation, and sunlight. Electricity. — Cohn and Mendelssohn, in 1879, attempted to de- termine the effect of the galvanic current upon bacteria. Cultures were placed in U -tubes through which a constant current was passed. A feeble current was found to be without effect. A strong current from two elements, maintained for twenty-four hours, restrained de- velopment in the vicinity of the positive pole, but this was probably due to the highly acid reaction which the culture liquid acquired. When a current from five elements was used for twenty-four hours the liquid was sterilized, but this may have been due to the decided changes produced in the chemical composition of the culture liquid rather than to the direct action of the galvanic current. The same may be said of the similar results obtained in later ex- periments by Apostoli and Laquerriere, and by Prochownick and Spaeth. The last-mentioned investigators found that the positive pole had a more decided effect than the negative, and that the effect de- pended upon the intensity and duration of the current. A current of lit ty milliamperes passed for a quarter of an hour did not kill Staphy- lococcus pyogenes aureus, but a current of sixty milliamperes main- tained for the same time did. The spores of Bacillus anthracis required a current of two hundred to two hundred and thirty milli- amperes during an hour or two. In these experiments the cultures in gelatin were attached to the strips of platinum serving as the two poles, and these were immersed in a solution of sodium chloride. As chlorine was disengaged at the positive pole, the germicidal action is attributed to this gas rather than to the direct action of the current upon the living microorganisms. The more recent researches of Spilker and Gottstein, made with an induction current from a dynamo machine, are more valuable in INFLUENCE OF PHYSICAL AGENTS. 159 estimating the power of this agent to destroy the vitality of bacteria. The current was passed through a spiral wire which was wrapped around a test tube of glass, containing the microorganism to be tested, suspended in distilled water. In a first experiment Bacillus prodigi- osus, suspended in sterilized distilled water and contained in test tubes having a capacity of two hundred and fifty cubic centimetres, was subjected to a current having an energy of 2.5 amperes X 1.25 volts for twenty-four hours. The temperature did not go above 30° C. No development occurred when the microorganism tested was subsequently planted in nutrient gelatin. Further experiments gave a similar result. It was found that stronger currents were effective in shorter time ; but in no case was sterilization effected in less than an hour. Pressure. — D'Arsonval and Charrin (1894) submitted a culture of Bacillus pyocyaneus to a pressure of fifty atmospheres, under car- bon dioxide. At the end of four hours cultures could still be ob- tained, but the bacillus had lost its power of pigment production. A few colonies were developed after six hours' exposure to this pressure ; but after twenty-four hours no development occurred. Agitation. — Meltzer (1894) has shown that the vitality of bacteria is destroyed by protracted and violent shaking, which causes a molec- ular disintegration of the cells. VII. ANTISEPTICS AND DISINFECTANTS. GENERAL ACCOUNT OF THE ACTION OF. THE term antiseptic is used by some authors to designate an which destroys the vitality of the microorganisms which pro- duce septic decomposition, and others of the same class. We prefer to restrict the use of the term to those agents which restrain the de- velopment of such microorganisms without destroying their vitality. The complete destruction of vitality is effected by germicides or dis- infectants. Material containing the germs of infectious diseases is infectious material, and we disinfect it by the use of agents which destroy the living disease germs or pathogenic bacteria which give it its infecting power. Such an agent is a disinfectant. But we ex- tend the use of this term to germicides in general — that is, to those agents which kill non-pathogenic bacteria as well as to those which destroy disease germs. All disinfectants are also antiseptics, for agents which destroy the vitality of the bacteria of putrefaction ar- rest the putrefactive process ; and these agents, in less amount than is required to completely destroy vitality, arrest growth and thus act as antiseptics. But all antiseptics are not germicides. Thus a concentrated solution of salt or of sugar will prevent the putrefac- tive decomposition of organic material, animal or vegetable ; but these agents do in»t destroy the vitality of the germs of putrefaction. In a certain degree of concentration they are antiseptics and are largely u>.-.l f.,r the preservation of meats and vegetables. In the same way many mineral salts in solutions of various strengths act as antisep- tics, and some of these in still stronger solutions are disinfectants. Thus mercuric chloride, when introduced into a culture solution in the proportion of 1 : 300,000, will restrain the development of anthrax spores, but to insure the destruction of these spores a solution of 1 : 1,000 must be used. As a rule, the difference between restraining action — antiseptic— and germicidal power — disinfectant — is not so great as this. We give below some recent determinations by Boer which illustrate this point, the test organism being the bacillus of typhoid fever in a culture in bouillon twenty-four hours old : ANTISEPTICS AND DISINFECTANTS. Restrains. Kills. 1 2100 1 -300 1 : 1550 1 • 500 Silver nitrate 1 : 50000 1 -4000 1 : 6000 1 • 250 Carbolic acid .... 1 :400 1 -200 Method of Determining Antiseptic Value. — To determine the restraining or antiseptic power of an agent for a particular micro- organism, the agent is dissolved in a definite proportion in a suitable culture medium, which is then inoculated with a pure culture of the test organism and placed in favorable circumstances — as to tempera- ture— for its growth. At the same time a control experiment is made by placing another portion of the same culture medium, inocu- lated with the same microorganism, in the same conditions, but with- out the addition of the antiseptic agent. If development occurs in the control experiment and not in the culture medium containing the antiseptic, the failure to grow must be attributed to the presence of this agent. Having made a preliminary experiment, we are guided by the result in further experiments to determine the exact amount required to restrain development under the same conditions. Or we may make a series of experiments in the first instance. The problem being, for example, to determine the antiseptic value of carbolic acid for the typhoid bacillus, we may add this agent to a definite amount of bouillon in test tubes in the proportion of 1 : 100, 1 : 200, 1 : 300, 1 : 400, 1 : 500. In experiments with volatile agents the bouillon, in test tubes or small flasks, must be sterilized in ad- vance, and the antiseptic agent introduced by means of a sterilized pipette with great care to prevent the accidental contamination of the nutrient medium. In experiments with non-volatile agents it will be best to sterilize the culture medium after the antiseptic has been added. Next we inoculate the liquid in each flask with a pure cul- ture of the test organism. The flasks are then placed in an incubat- ing oven at 35° to 37° C. At the same time a control, not containing any carbolic acid, is placed in the oven. At the end of twenty-four hours the control will be found to be clouded, showing an abundant multiplication of the bacillus. Taking the result of Boer above given, we would expect to find all of the solutions clear except that contain- ing 1 : 500. This too might remain clear for some days and finally ** break down/' for experience shows that when we pass the point at which a permanent restraining influence is exerted there may be a temporary restraint or retardation of development. For this reason we must continue the experiment for a considerable time — not less 11 ]62 ANTISEPTICS AND DISINFECTANTS. than two weeks. Having found that 1 :400 and below prevents development, and 1 . 500 does not, we may make further experiments to determine the antiseptic power within narrower limits ; but this is hardly necessary from a practical point of view. In these experiments the result will be influenced by several cir- cumstances, as follows : (a) By the composition of the nutrient medium. This is a very important factor, especially in determining the antiseptic value of certain metallic salts. The presence of a considerable quantity of albumin, for example, reduces greatly the antiseptic power of mercuric chloride, silver nitrate, creolin, etc. The presence of a sub- stance chemically incompatible, as, for example, sodium chloride in testing nitrate of silver, will of course neutralize antiseptic action. (b) The nature of the test organism. Within certain limits an antiseptic for one microorganism of this class restrains the devel- opment of all, but there are wide differences in the ability of differ- ent species to grow in the presence of different chemical agents. Some grow readily in the presence of a considerable amount of free acid, others are restrained by a slightly acid reaction of the medium in which they are placed. The Bacillus acidi lactici, for example, can thrive in the presence of a considerable amount of the acid which is a product of its growth, but there is a limit to its power of developing in the presence of this and other acids. So, too, Mi- crococcus urese, which causes the alkaline fermentation of urine, grows in the presence of a considerable amount of carbonate of am- monia, but is finally restrained in its growth by this alkaline salt. The following determinations by Boer show the difference in the antiseptic power of hydrochloric acid for certain pathogenic bacte- ria : Bacillus of anthrax (without spores), 1 : 3,400 ; diphtheria bacil- lus, 1 : 3,400 ; glanders bacillus, 1 : 700 ; typhoid bacillus, 1 : 2,100 ; cholera spirillum, 1 :5,500. It will be noted that the cholera spiril- lum is restrained in its growth by about one-eighth the amount of hydrochloric acid which is required to prevent the development of the bacillus of glanders. The typhoid bacillus has a special tole- rance for carbolic acid, etc. (c) The temperature at which the experiment is made. At the temperature most favorable for growth a greater proportion of the antiseptic agent is required than at unfavorable temperatures- lower or higher. (d) The restraining influence for spores is much greater than for the vegetative form of bacteria. Methods of Determining Germicide Value.— The disinfecting power of a chemical agent is determined by allowing it to act for a given time, in a definite proportion, on a pure culture of a given ANTISEPTICS AND DISINFECTANTS. 163 microorganism, and then testing the question of loss of vitality by culture experiments or by inoculations of infectious disease germs into susceptible animals. The test by cultivation is the most reliable, but in making it several points must be kept in view. Naturally the conditions must be such as are favorable for the growth of the particular microor- ganism which serves as the test ; and we must allow a considerable time for the development of the test organism, for it often happens that its vital activity has been weakened without being completely destroyed, and that growth will occur after an interval of several days, while in the control experiment it has perhaps been seen at the end of twenty-four hours. Another most important point is the fact that some of the disinfecting agent is necessarily carried over with the test organisms when these are transferred to a nutrient medium to ascertain whether they will grow, and this may be in sufficient amount to restrain their development and lead to the mis- taken inference that they have been killed. This is especially true of mercuric chloride, which restrains the development of spores in very minute amounts. Spores which have been subjected to its ac- tion in comparatively strong solutions, when transferred to a culture medium may fail to grow because of the restraining influence of the mercuric chloride carried over at the same time. For this rea- son liquid cultures are to be preferred in experiments of this kind. When the test organisms are planted in a solid culture medium the chemical agent is left associated with them ; in a liquid culture, on the other hand, it is diluted, and the microorganisms, being distri- buted through the nutrient medium, have the disinfecting agent washed from their surface. In the case of mercuric chloride, how- ever, the experiments of Geppert show that the agent is so attached to spores which have been subjected to its action that ordinary washing does not suffice. Moreover, spores which have been ex- posed to the action of mercuric chloride without being killed are re- strained in their growth by a much smaller proportion of the corro- sive sublimate than is required for spores not so exposed — according to Geppert, by 1 part in 2,000,000. Geppert therefore proposes, in experiments with this agent, to neutralize the mercuric chloride which remains attached to the test organisms by washing these in a solution of ammonium sulphide, by which the sublimate is preci- pitated as an inert sulphide. With most agents simple dilution will serve the purpose of pre- venting an erroneous inference from the restraining influence of the chemical agent being tested. If we carry, by means of a platinum loop, one or two ose into five to ten cubic centimetres of bouillon, the dilution will usually be beyond the restraining influence of the 1G4 ANTISEPTICS AND DISINFECTANTS. germicidal agent ; but we may carry the dilution still further, to be on the side of safety, by inoculating a second tube containing the same amount of sterile bouillon from the first, carrying over in the same way one or two ose. We will still be very sure to have a considerable number of the microorganisms to test the question of the destruction of vitality. Instead of bouillon we may use liquefied flesh-peptone-gelatin, which gives us the same advantage as to dilu- tion of the disinfecting agent ; and after inoculating two tubes as above indicated, we may make Esmarch roll tubes by turning them upon a block of ice. The development of colonies will show that there was a failure to disinfect ; their absence, after a proper inter- val, will be evidence of the germicidal action of the agent employed. Koch's Method.— In 1881 Koch published his extended experi- ments made to determine the germicidal power of various chemical agents as tested upon anthrax spores. His method consisted in ex- posing silk threads, to which the dried spores were attached, in a solution of the disinfecting agent, and at intervals transferring one of these threads to a solid culture medium. The precaution was taken to wash the thread in distilled water when the agent tested was supposed to be likely to restrain development. In these experiments a standard solution of the disinfecting agent was used, and the time of exposure was varied from a few hours to many days. The Writer's Method. — In the writer's experiments, made in 1 880 and subsequently, a different method has been adopted. The time has been constant — usually two hours — and the object has been to find the minimum amount of various chemical agents which would destroy the test organisms in this time ; and instead of sub- jecting a few of the test organisms attached to a silk thread to the action of the disinfecting agent, a certain quantity of a recent cul- ture— usually five cubic centimetres — has been mixed with an equal quantity* of a standard solution of the germicidal agent. Thus five cubic centimetres of a 1 : 200 solution of carbolic acid would be added to five cubic centimetres of a recent culture of the typhoid bacillus, for example, and after two hours' contact one or two ose would be introduced into a suitable nutrient medium to test the question <>t disinfection. In the case given the result obtained would be set down as the action of a solution of carbolic acid in the proportion of 1 : 400, for the 1 : 200 solution was diluted by the addi- i u equal quantity of the culture. other experimenters have adopted still a different method. In- stead of using a considerable and definite quantity of a culture con- taining the test organism, they introduce one or two ose from such a culture into a solution containing a given proportion of the disin- fectant ; then after exposure for a given time the nutrient medium is inoculated. ANTISEPTICS AND DISINFECTANTS. 165 These different methods give results which cannot be directly compared one with another, for to obtain corresponding results we must have identical conditions. Test by Inoculation into Susceptible Animals. — In testing the action of disinfectants upon anthrax spores and other infectious dis- ease germs, we may inoculate the microorganisms, after exposure to the disinfectant, into a susceptible animal. This method was adopted by the writer in a series of experiments in 1881, but he has not since employed it, for reasons set forth in his paper giving an account of these experiments. "First. The test organism maybe modified as regards repro- ductive activity without being killed; and in this case a modified form of disease may result from the inoculation, of so mild a character as to escape observation. Second. An animal which has suffered this modified form of the disease enjoys protection, more or less perfect, from future attacks, and if used for a subsequent experiment may, by its immunity from the effects of the pathogenic test organism, give rise to the mistaken assumption that this had been destroyed by the action of the germicidal agent to which it had been sub- jected."1 In experiments to determine the value of an agent as a disinfec- tant, no matter by what method, the following conditions, which in- fluence the result, should be kept in view : (a) The difference in vital resisting power of different species of bacteria. As a rule, the pathogenic species have rather less re- sisting power than the common saprophytes, and the micrococci have greater resisting power than many of the bacilli. The differ- ence in the vital resisting power of some of the best known patho- genic species is shown in the following table, which we have made up from determinations made by Boer — cultures in bouillon twenty- four hours old ; time of exposure, two hours. Hydrochloric Acid. Caustic Soda. Chloride of Gold and Sodium. Nitrate of Silver. Carbolic Acid. Anthrax bacillus . . . 1 • 1100 1 • 450 1 :8000 1 • 20000 1:300 Diphtheria bacillus Glanders bacillus Typhoid bacillus 1 :700 1 :200 1 :300 - 1 : 300 1 : 150 1 :190 1 :1000 1:400 1 :500 1 :2500 1 : 4000 1 :4000 1 :300 1 :300 1 : 200 Cholera spirillum 1 : 1350 1 :150 1 : 1000 1 :4000 1 :400 (b) The presence or absence of spores. The reproductive ele- ments known as spores have a far greater resisting power to chemi- cal agents, as well as to heat, than have the vegetative cells. In 1 Quoted from article on " Germicides and Disinfectants/' in " Bacteria," p. 212, 1(;(; ANTISEPTICS AND DISINFECTANTS. practical disinfection, therefore, it is important to know what disease germs form spores and what do not. The following are known to form spores : The bacillus of anthrax, the bacillus of tetanus, the bacillus of malignant oedema, the bacillus of symptomatic anthrax, the bacillus of foul brood (infectious disease of bees). The following, so far as is known, do not form spores : The pus cocci (Staphylo- coccus pyogenes albus, aureus, and citreus, and Streptococcus pyo- genes), the micrococcus of pneumonia, the bacillus of typhoid fever, the bacillus of glanders, the bacillus of diphtheria, the spirillum of cholera, the spirillum of relapsing fever. Many agents which kill the growing bacteria are incapable of destroying the vitality of spores, and others only do so in much stronger solutions or after a long exposure to their action. (c) The number of bacteria to be destroyed. This is an essen- tial factor which has often been overlooked by those making experi- ments. To destroy the bacteria carried over to five cubic centimetres of distilled water by means of a platinum loop, is a very different matter from destroying the immensely greater number in five cubic centimetres of a recent bouillon culture. (d) The nature and quantity of associated material. The oxidizing disinfectants, like permanganate of potash and chloride of lime, not only act upon the bacteria, destroying them by oxidation, but upon all organic matter with which they come in contact, and at the same time the disinfecting agent is destroyed in the chemical reaction, which is a quantitative one. The presence, therefore, of organic material in association with the bacteria is an important factor, and if this is in excess the disinfectant may be neutralized before the living bacteria are destroyed. Other substances which precipitate the disinfecting agent in an insoluble form, or decompose it . must of course have the same effect. Thus the presence of sodium chloride in a culture medium would be an important circumstance if nitrate of silver was the agent being tested, as the insoluble chloride would be precipitated. And in the case of mercuric chloride and certain other metallic salts the presence of albumin very materially influences the result. Van Ermengem states that the cholera spiril- lum in bouillon is destroyed in half an hour by mercuric chloride in the proportion of 1 : 00,000, while in blood serum 1 : 800 was required t«. destroy it in the same time. (e) The time of exjxttturv is also an important factor. Some agents act very promptly, while others require a considerable time to effect the destruction of bacteria exposed to their action. Thus a solution "of chloride of lime containing 0.12 per cent destroys the typhoid bacillus and the cholera spirillum in five minutes, and tin- anthrax bacillus iu one minute (Nissen). On the other hand, ANTISEPTICS AND DISINFECTANTS. 167 quicklime (milk of lime) requires a contact of several hours to in- sure the destruction of pathogenic bacteria. (/) The temperature at which the exposure is made has a material influence upon the result. This is shown by the experi- ments of Henle and of Nocht. As a general rule germicidal activ- ity increases in direct proportion to the increase in temperature from 20° C. upward. (g) The degree of dilution of the disinfecting agent is also a matter of importance . This is especially true of solutions of acids and alkalies. When a silk thread to which bacteria are attached is suspended in an acid solution the essential point is the degree of acidity, and not the quantity of acid in the entire solution. But if a solution of permanganate of potash, or any other active oxidizing agent, is used, the principal question is not the degree of dilution, but the amount of the disinfecting agent present in the solution used. A grain of potassium permanganate dissolved in two fluidounces of distilled water would probably kill just as many bacteria as if it were dissolved in half a fluidounce, although the time required for disinfection might be longer. From what has been said it is evident that the simple statement that a certain agent is a germicide in a certain proportion has but little scientific value, unless we are made acquainted with the condi- tions under which its germicidal action has been tested. VIII. ACTION OF GASES AND OF THE HALOID ELEMENTS UPON BACTERIA. Oxygen. — Free oxygen is essential for the development of a large number of species of bacteria — aerobics ; and it completely prevents the growth of others — anaerobics. Many bacteria, even when freely exposed in a desiccated condition to the action of atmospheric oxygen, retain their vitality for a long time. The gradual loss of pathogenic power which Pasteur has shown occurs in cultures of the anthrax bacillus and the micrococcus of fowl cholera, is ascribed by him to exposure to oxygen, and as proof of this he states that cultures kept in hermetically sealed tubes do not lose their virulence in the same degree. But other circumstances may influence the result. Thus some of the products of growth which accumulate in culture fluids have an injurious effect upon the vitality of the bacteria which pro- duced them, and in time may cause a complete destruction of vitality. In cultures exposed to the air these products would be in a more concentrated solution from the gradual evaporation of the culture liquid. It must also be remembered that light in the presence of oxygen is a germicidal agent. The experiments of Frankel show that the aerobic bacteria grow abundantly in the presence of pure oxygen, and some species even more so than in ordinary air. Micrococcus prodigiosus, however, appeared to be unfavorably affected by pure oxygen, inasmuch as it did not produce pigment so readily as when cultivated in ordinary air. Nascent oxygen is a very potent germicidal agent, as will be seen in our account of such oxidizing disinfectants as potassium perman- ^anat<« and the hvporhloritc of lime. Ozone.— It was formerly supposed that ozone would prove to be a most valuable agent for disinfecting purposes ; but recent experi- ments show that it is not so active a germicide as was anticipated, and that from a practical point of view it has comparatively little value. Lukaschewitsch found that one gramme in the space of a cubic metre failed to kill anthrax spores in twenty-four hours. The cholera >j.ii -ilium in amoist state was killed in this time by the same amount, but fifteen hours' exposure failed to destroy it. Ozone for these ex- periments was developed by means of electricity. ACTION OF GASES AND HALOID ELEMENTS UPON BACTERIA. 169 Wyssokowicz found that the presence of ozone in a culture me- dium restrained the development of the anthrax bacillus, the bacillus of typhoid fever, and others tested, but concludes that this is rather due to the oxidation of bases contained in the nutrient medium than to a direct action upon the pathogenic bacteria. Sonntag, in his carefully conducted experiments, in which a cur- rent of ozonized air was made to pass over silk threads to which were attached anthrax spores, had an entirely negative result. The an- thrax bacillus from the spleen of a mouse, and free from spores, was then tested, also with a negative result, even after exposure to the ozonized air for twenty minutes at a time on four successive days. In another experiment several test organisms (Bacillus anthracis, Bacil- lus pneumonise of Friedlander, Staphylococcus pyogenes aureus, Staphylococcus pyogenes albus, Bacillus murisepticus, Bacillus crassus sputigenus) were exposed on silk threads for twenty-four hours in an atmosphere containing 4. 1 milligrammes of ozone to the litre'of air (0. 19 volumes per cent). The result was entirely negative. When the amount was increased to 13.53 milligrammes per litre the anthrax bacillus and Staphylococcus pyogenes albus failed to grow after twenty-four hours' exposure. The conclusion reached by Ms- sen, from his own experiments and a careful consideration of those previously made by others, is that ozone is of no practical value as a germicide in therapeutics or disinfection. Hydrogen. — This gas has no injurious effect upon bacteria, as is shown by the fact that the anaerobic and facultative anaerobic species grow readily in an atmosphere of pure hydrogen. Hydrogen peroxide in solution in water is a valuable antiseptic and deodorant, but its value as a germicide has been very much overestimated. Miquel, in his experiments to determine the anti- septic value of various agents, places H2O2 third in the list of " sub- stances eminently antiseptic," and states that it prevents the develop- ment of the bacteria of putrefaction in the proportion of 1: 20,000. In the writer's experiments (1885) a solution was used which contained at first 4.8 per cent of H2O2, and five per cent of sulphuric acid which was added by the chemist who prepared the solution, to prevent loss of the hydrogen peroxide. At the end of a month the amount of H20a was again estimated, and found to be 3.98 per cent. Five weeks later the proportion was 2.4 per cent. Tested upon "broken-down" beef tea, this solution was found to destroy the vitality of the bacteria of putrefaction contained in it, in two hours' time, in the proportion of thirty per cent (about 1.2 per cent of H2O2). Anthrax spores were killed in the same time by a twenty-per-cent solution (0.8 per cent H2O,). Tested upon a pure culture of pus cocci, it was active in the proportion of ten per cent (0. 4 per cent of 170 ACTION OF GASES AND OF THE H,O,); a solution containing 0.24 per cent of HaOa failed to kill pus cocci. But the solution used in these experiments contained also five per cent of sulphuric acid, which by itself kills micrococci in the pro- portion of 1 : 200. My conclusion was that, unless the chemists can furnish more concentrated solutions which will keep better than that with which I experimented, we are not likely to derive any practical benefit from the use of hydrogen peroxide as a disinfectant. Altehof er more recently has experimented with a solution contain- ing 9.7 per cent of HaOa, and reports the following results: He added to ninety-eight cubic centimetres of hydrant water two cubic centi- metres of a bouillon culture of the typhoid bacillus, and to this was added sufficient of his aqueous solution of H2O., to make the propor- tion present 1: 1,000. At the end of twenty-four hours the bacillus was proved by culture experiments to be killed. Water containing the cholera spirillum, treated in the same way, was not entirely steril- ized, as a few colonies developed in Esmarch roll tubes ; but the gen- eral result of his experiments was that the ordinary water bacteVia, and the pathogenic bacteria named (cholera, typhoid) when sus- pended in water, required for their destruction exposure for twenty- four hours in a solution containing one part of HaO2 in one thousand of water. Carbon Dioxide. — The experiments of Frankel show that certain bacteria grow in an atmosphere of CO., as well as in the air ; among these are the bacillus of typhoid fever and the pneumonia bacillus of Friedlander. Other species are slightly restricted in their growth, e.g. Bacillus prodigiosus, Proteus vulgaris. Still others grow only when the temperature is elevated, including the pus cocci and the bacillus of swine pest. Most of the saprophytic bacteria failed to grow in an atmosphere of CO,, although their vitality was not de- stroyed by it. Certain pathogenic species were, however, killed by the action of this gas, among others the cholera spirillum, Bacillus anthracis, and Staphylococcus pyogenes aureus. Leone and Hochstetter had previously reported that certain bac- teria are injuriously affected by COa. Frankel also found that the growth of strictly anaerobic species was restricted in an atmosphere of carbon dioxide. The aerobic species which failed to grow in pure CO, grew al.nml.-iiitly when a little atmospheric oxygen was ad- mit i ••• I . In the experiments of Frankla ad the cholera spirillum and the Finkler-Prior spirillum failed to develop in an atmosphere of CO,, and at the end of eight days were no longer capable of growth wh.-n the carbon dioxide was replaced with atmospheric air. < Carbonic Oxide. — Frankland's experiments show that an atmo- sphere of this gas is not favorable to the growth of the cholera spiril- lum or of the Finkl.T-Prior spirillum, although it did not entirely HALOID ELEMENTS UPON BACTERIA. 171 prevent development, and after seven days' exposure the spirilla were not all killed, although a comparatively small number of colonies de- veloped. Bacillus pyocyaneus failed to grow in an atmosphere of CO, but when air was admitted, at the end of seven or eight days, abundant development occurred. Methane, CH4. — We have no exact experiments to determine the action of marsh gas in a pure state on bacteria, but the experi- ments of Kladakis upon illuminating gas maybe taken as repre- senting approximately what might be expected from exposure in pure CH4. An analysis of the gas used in his experiments showed it to contain 37.97 per cent of hydrogen, 39.37 per cent of methane (CH4), 9.99 per cent of nitrogen, 4.29 per cent of ethene (CaH4), 3.97 per cent of carbonic oxide (CO), 0.61 per cent of oxygen, and 0.41 per cent of carbon dioxide. As hydrogen and nitrogen are neutral, and carbonic oxide is shown by the experiments of Frankland not to act as a germicide after several days" exposure to its action, the positive results obtained in the experiments of Kladakis may be ascribed to the presence of CH4 (39.37 per cent) or of C2H4 (4.29 per cent), or of both together. A large number of microorganisms were tested, and among these Proteus vulgaris alone grew in an atmosphere of illuminating gas. The others not only failed to grow in such an atmosphere, but were destroyed by it. Cultures of Bacillus anthracis, Staphylococcus pyo- genes aureus, and Spirillum cholera? Asiatics were sterilized in half an hour by the action of this gas. The gas was also found to be un- suitable for anaerobic cultures. Nitrous Oxide, N2O. — The experiments of Frankland, made upon the cholera spirillum, the spirillum of Finkler-Prior, and the bacillus of green pus, gave results similar to those obtained with CO, viz. , seven days' exposure in an atmosphere of this gas failed to de- stroy the test organisms, but completely restrained the growth of Bacillus pyocyaneus and interfered materially with the development of the two species of spirillum without entirely preventing it. Nitrogen Dioxide, NO. — Frankland found that his test organ- isms were quickly killed by this gas (Bacillus pyocyaneus, Spirillum cholera Asiatics, Spirillum Finkler-Prior). Hydrosulphuric Acid, H2S. — In the experiments of Frankland this gas proved to be quickly fatal to the bacteria tested (Bacillus pyocyaneus, Spirillum cholera? Asiatics, Spirillum Finkler-Prior). On the other hand, Grauer found that this gas did not exercise any injurious influence upon the tubercle bacillus, the bacillus of anthrax, the typhoid bacillus, or the cholera spirillum, after the exposure of these microorganisms in a current of the gas for an hour. It has been shown by the experiments of Holschewnikoff and UTInN OF GASES AND OF THE others that certain species of bacteria cause an abundant evolution of H,S as a result of their development in an albuminous medium (Bacillus sulfureus and Proteus sulfureus). Sulphur Dioxide, SO,.— Very numerous experiments have been made with this gas, owing to the fact that it has been extensively used in various parts of the world for the disinfection of hospitals, ships, apartments, clothing, etc. In the writer's experiments, made in 1880, dry vaccine virus on ivory points was disinfected by exposure for twelve hours in an at- mosphere containing one volume per cent of this gas, and liquid virus, exposed in a watch glass, by one-third of this amount. Sub- sequent experiments (1885) showed that pus micrococci were killed by exposure for eighteen hours in a dry atmosphere containing twenty volumes per cent of S0a, but that four volumes per cent failed. In the presence of moisture this gas has considerably greater germicidal power than this, owing, no doubt, to the formation of the more ac- tive agent, sulphurous acid (H,S03). But in a pure state anhydrous sulphur dioxide does not destroy spores. The writer has shown that the spores of Bacillus anthracis and Bacillus subtilis are not killed by contact for some time with liquid SO2 (liquefied by pressure). Koch exposed various species of spore-bearing bacilli in a disinfection cham- ber for ninety-six hours, the amount of SOa at the outset of the ex- periment being 6.13 volumes per cent, and at the end 3.3 per cent. The result was entirely negative. But in the absence of spores the anthrax bacillus, in a moist con- dition, attached to silk threads, was destroyed in thirty minutes in an atmosphere containing one volume per cent. In another of Koch's experiments the amount of SO, in the disin- fection chamber was at the outset 0.84 per cent, and at the end of twenty-four hours 0. 55 per cent. An exposure of one hour in this at- mosphere killed anthrax bacilli attached to silk threads, in a moist condition ; but four hours' exposure failed to kill Bacillus prodigiosus growing on potato, while twenty-four hours' exposure was successful. A similar result was obtained with Bacillus pyocyaneus. Thinot, as a result of experiments made in 1890, arrives at the conclusion that the specific germs of tuberculosis, glanders, farcy of cattle, typhoid fever, cholera, and diphtheria are destroyed by twenty- four hours' exposure in an atmosphere containing SO, developed by the combustion of sixty grains of sulphur per cubic metre. This amount corresponds closely with that fixed by the Committee on Dis- infoctante of the American Public Health Association on the experi- mental evidence obtained by the writer in 1885. But the committee insisted upon tin- pn-s.-nrr <>f inuistuiv and mado thetimo of exposure twelve hours — "exposure for twelve hours to an. atmosphere con- HALOID ELEMENTS UPON BACTERIA. 173 taining at least four volumes per cent of this gas in the presence of moisture. " Chlorine. — The haloid elements are active germicidal agents, especially chlorine on account of its affinity for hydrogen, and the consequent release of nascent oxygen when it comes in contact with microorganisms in a moist condition. And for the same reason this agent is a much more active germicide in the presence of moisture than in a dry condition. The experiments of Fischer and Proskauer showed that when dried anthrax spores were exposed for an hour in an atmosphere containing 44. 7 per cent of dry chlorine they were not destroyed ; but if the spores were previously moistened and were ex- posed in a moist atmosphere for the same time, four per cent was effective, and when the time was extended to three hours one per cent destroyed their vitality. The anthrax bacillus, in the absence of spores, was killed by exposure in a moist atmosphere containing 1 part to 2,500, the time of exposure being twenty-four hours, and the same amount was effective for Micrococcus tetragenus ; the strepto- coccus of erysipelas and the micrococcus of fowl cholera were killed in three hours by 1 : 2,500, and in twenty-four hours by 1: 25,000. The bacillus of mouse septicaemia and the tubercle bacillus were killed in one hour by 1 : 200. In the writer's experiments (1880) four children were vaccinated with virus from ivory points which had been exposed for six hours in an atmosphere containing one-half per cent of chlorine ; also with four points, from the same lot, not disinfected. Vaccination was un- successful in every case with the disinfected points, and successful with those not disinfected. Koch found that anthrax spores failed to grow after twenty-four hours' exposure in chlorine water. In the experiments of De la Croix to determine the antiseptic power of this agent, it was found that when present in unboiled beef infusion in the proportion of 1 : 15,000 no development of bacteria occurred. Miquel gives the antiseptic value of chlorine as 1 : 4,000. Chloroform. — Immersion for one hundred days in chloroform does not destroy the vitality of anthrax spores (Koch). This agent is without effect on the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). Salkowski found that the anthrax bacillus in the absence of spores, and the cholera spirillum, were killed by being immersed in chloroform water for half an hour. Kirchner reports still more favorable results. In his experiments a one-per- cent solution killed the cholera spirillum in less than a minute, and a one-quarter-per-cent solution in an hour. But the typhoid bacillus required at least one-half per cent acting for an hour. Iodine. — In the writer's experiments (1880) iodine in aqueous solution with potassium iodide was found to be fatal to Micrococcus 174 ACTION OP GASES AND OF THE pneumonia* crouposa^in the proportion of I : 1,000, and to the staphy- lococci of pus in 1 : 500— time of exposure two hours. Iodine water was found by Koch to destroy the vitality of anthrax spores in twenty-four hours, but a two-per-cent solution in alcohol failed to destroy anthrax spores in forty-eight hours. In the experiments of Schill and Fischer twenty hours' contact with a solution of the strength of 1 : 500 failed to destroy the virulence of tuberculous spu- tum, as tested by inoculation experiments. The antiseptic value of iodine is given by Miquel as 1 : 4,000. Bromine.— Fischer and Proskauer have studied the action of bromine vapor upon various microorganisms. They found that ex- posure for three hours in a dry atmosphere to three per cent does not destroy the tubercle bacillus in sputum or the spores of an- thrax. But when the atmosphere is saturated with moisture 1 : 500 is effective ; and when the time of exposure was extended to twenty- four hours, 1 : 3,500. A two-per-cent solution destroys the vitality of anthrax spores in twenty-four hours (Koch). Bromine vapor is an active agent for the destruction of the virus of symptomatic an- thrax (Arloing, Cornevin, and Thomas). Miquel gives the antisep- tic value of bromine as 1 : 1,666, which is considerably below that of chlorine and iodine. Iodine Trichloride. — According to Behriiig, we possess in this agent a disinfectant which possesses the potency of free chlorine and iodine without having their disadvantages. As prepared by O. Rie- del it is a yellowish-red powder of penetrating odor. It remains un- changed for weeks in concentrated aqueous solution (five per cent). A one-per-cent solution destroys anthrax spores suspended in water almost instantly, and a 0.2-per-cent solution within a few minutes. Anthrax spores in blood serum are killed by a one-per-cent solution in forty minutes (Behring). Langenbuch found that a solution of 1 : 1 ,000 kills spores in a short time, and that when added to nutri- ent gelatin in the proportion of 1 : 1,200 it restrains the develop- ment of bacteria. lodoform. — Numerous experiments have been made with this agent, which slm\v that it has little, if any, germicidal power ; but it acts to some extent as an antiseptic. Tilanus reports that the tu- bercle bacillus will not grow in glycerin-agar cultures to which a small quantity of iodoform has been added, and that a pure culture of the tubercle bacillus was not killed in six days by exposure to iodoform vapor, but that after six weeks* exposure it failed to grow. The experiments of Neisser and of Buchner show that while most bacteria are not injuriously affected by exposure to iodoform vapor, the cholera spirillum and the Finkler-Prior spirillum are restrained in their growth by such exposure. When plate cultures of the cholera HALOID ELEMENTS UPON BACTERIA. 175 spirillum were placed under a bell jar beside iodoform powder no development occurred, but when they were removed colonies de- veloped, showing that the spirilla were not killed. Iodoform Ether, according to Yersin, is fatal to the tubercle ba- cillus in one-per-cent solution in five minutes. Cadeac and Meunier found that a saturated solution required thirty-six hours to kill the bacillus of typhoid fever. lodol. — In experiments made by the writer (1885) this agent was found to be without germicidal power. Riedlin found it without any action, even upon the cholera spirillum. Hydrofluoric Acid, HF1. — From a series of experiments made with this gas, Grancher and Chautard arrive at the conclusion that " the direct and prolonged action of hydrofluoric acid upon the tuber- cle bacillus diminishes its virulence but does not kill it." Sozoiodol ^4ctd,according to Draer,is a phenol, in which two atoms of hydrogen are replaced by two of iodine and one atom by the group HSO3. This acid and its salts with soda, potash, zinc, and mercury have been tested by the author named. The acid and its salt with mercury were found to destroy the cholera spirillum in two hours' time in two-per-cent solution. A two-per-cent solution of phenol would have accomplished the same result and in less time. Tribromphenol, according to Draer, is less active than sozoiodol acid ; and it appears from the experimental evidence on record that combinations of iodine, chlorine, or bromine with phenol are less active that the haloid elements alone. According to Karpow (1893) monochlor- phenol, tested upon anthrax spores attached to silk threads, proved to be decidedly more active than phenol. Nosophen (tetraiodphenolphthalein), according to Li even (1895) contains sixty-one per cent of iodine. It is entirely insoluble in water. When added to nutrient gelatin in the proportion of one- quarter per cent it prevented the development of the anthrax bacillus and of Staphylococcus aureus, but failed to prevent the development of Bacillus pyocyaneus (Lieven). IX. ACTION OF ACIDS AND ALKALIES. Sulphuric Acid, H,SO4.— The experiments of Koch (1881) showed that anthrax spores were still capable of growing after ex- posure in a one-per-cent solution of sulphuric acid for twenty days. In the writer's experiments (1885) a four-per-cent solution failed to destroy the spores of Bacillus subtilis in four hours, and an eight- per-cent solution was found to be required for the sterilization of culture fluids containing spores ; but the multiplication of the bacte- ria of putrefaction was prevented by the presence of this acid in a culture solution in the proportion of 1 : 800. Pus micrococci were destroyed by exposure for two hours in a solution containing 1 : 200. The experiments of Boer show that there is a considerable differ- ence in the resisting power of different pathogenic bacteria. The time of exposure being two hours, cultures in bouillon twenty-four hours old gave the following results : Restrains development. Destroys vitality. 1 • 2550 1 : 1300 Diphtheria bacillus 1 : 2050 1 :500 Glanders bacillus 1 : 750 1 : 200 Typhoid bacillus 1 : 1550 1 :500 Cholera spirillum 1 :7000 1 :1300 Leitz, in his studies relating to the bacillus of typhoid fever, reports the following results : The dejections of typhoid patients, i nixed with an equal proportion of the disinfecting solution, were .sterilized by a five-per-cent solution of sulphuric acid in three days. A pure culture was sterilized in fifteen minutes by two per cent, and in liv»> minutes by five per cent. Sulphurous Acid, H.SO,.— In the writer's experiments (1885) micrococci were destroyed in two hours by 1 : 2,000 by weight of SO, added to water. Kitasato found that solutions of sulphurous acid m the proportion of 0.28 per cent killed the typhoid bacillus, and <>. 1 48 per cent the cholera spirillum. De la Croix found that one ACTION OF ACIDS AND ALKALIES. 177 gramme of SO2 added to two thousand of bouillon prevents the de- velopment of putrefactive bacteria and after a time destroys the vitality of these bacteria. The writer found that pus cocci failed to grow in a culture solution containing one part of SO2 in five thousand of water. Nitric Acid, HNO3. — In the writer's experiments an eight-per- cent solution which contained 0.819 gramme of HNO3 in each cubic centimetre sterilized broken-down beef tea containing spores, and five per cent failed to do so. Kitasato, in experiments upon the chol- era spirillum and typhoid bacillus, obtained results corresponding with those obtained with hydrochloric acid — 0. 2 per cent destroyed vitality at the end of four or five hours. In these experiments the acid used contained 0.35 gramme HNO3 in one cubic centimetre. Nitrous Acid. — In the writer's experiments on vaccine virus (1880) exposure for six hours in an atmosphere containing one per cent of nitrous acid destroyed the virulence of dried virus upon ivory points. Hydrochloric Acid, HC1. — Anthrax spores are destroyed in ten days by a two-per-cent solution, but not in five days (Koch). Tested upon broken-down beef tea containing spores of Bacillus subtilis, it was effective in two hours in the proportion of fifteen per cent, but failed in ten per cent (Sternberg). In the experiments of Kitasato this acid destroyed the typhoid bacillus in five hours in the proportion of 0.2 per cent, and the cholera spirillum in 0.132 per cent — the acid used contained 0. 26 gramme HC1 in one cubic centimetre. We give the more recent determinations of Boer in tabular form. Its germi- cidal power was tested upon bouillon cultures which had been kept for twenty-four hours in an incubating oven ; time of exposure to the action of the acid solution, two hours. Restrains development. Destroys vitality. Anthrax bacillus 1 : 3400 1 : 1100 Diphtheria bacillus . . . ... 1 : 3400 1 -700 Glanders bacillus < 1 :700 1-200 Typhoid bacillus 1 : 2100 1 :300 Cholera spirillum 1 : 5500 1 : 1350 Chromic Acid. — In Koch's experiments a one-per-cent solution destroyed anthrax spores in from one to two days. In the propor- tion of 1 : 5,000 it prevents the development of putrefactive bacteria (Miquel). Osmic Acid. — A solution of one per cent kills anthrax spores in twenty-four hours (Koch). It is an antiseptic in the proportion of 1 :6,666 (Miquel). Phosphoric Acid. — Exposure for four or five hours to a solution 12 178 ACTION OF ACIDS AND ALKALIES. containing 0.3 per cent destroys the typhoid bacillus, and 0.183 per cent the cholera spirillum (Kitasato). The acid used contained 0. 1 :>•> gramme H,PO4 in one cubic centimetre. .Acetic Acid.— A five-per-cent solution failed to kill anthrax spores after five days' exposure (Koch). In Abbott's experiments glacial acetic acid in fifty-per-cent solution failed in two hours to kill anthrax spores, but micrococci were killed by two hours' exposure to a one-per-cent solution. A solution of 1 : 300 of glacial acetic acid destroys the cholera spirillum in half an hour (Van Ermengem). In the proportion of 0.25 per cent it restrains the growth of the typhoid bacillus, and 0.3 per cent destroys its vitality after five hours' expo- sure ; the cholera spirillum fails to grow in presence of 0.132 per cent and is destroyed by 0.2 per cent (Kitasato). Lactic Acid. — The bacillus of typhoid fever is killed in five hours by a solution containing 0.4 per cent, the cholera spirillum by 0.3 per cent (Kitasato). Citric Acid. — The bacillus of typhoid fever is killed in five hours by 0.43 per cent, the cholera spirillum by 0.3 percent (Kitasato). The cholera spirillum is killed in half an hour by 1 : 200 (Van Er- mengem). Oxalic Acid. — The typhoid bacillus requires a solution of 0.36 per cent, the cholera spirillum one of 0. 28 per cent, to destroy vitality in five hours (Kitasato). Boracic Acid. — In the writer's experiments (1883) a saturated solution failed to kill pus cocci in two hours. A five-per-cent solu- tion failed to destroy anthrax spores in five days (Koch). The typhoid bacillus is killed in five hours by 2.7 percent, the cholera ^jiirillum by 1.5 per cent (Kitasato). According to Arloing, Corne- vin, and Thomas, the fresh virus of symptomatic anthrax requires exposure to a twenty-per-cent solution for forty-eight hours for the destruction of vitality. Boracic acid acts as an antiseptic in the pro- portion of 1 : 143 (Miquel). Salicylic Acid. — In the writer's experiments this agent was dis- solved by the addition of sodium biborate, which by itself has no germicidal power. A two-per-cent solution was found to destroy pus cocci in two hours. Dissolved in oil or in alcohol a five-per-cent so- 1 ut ion does not destroy anthrax spores (Koch). Micrococci are de- stroyed by solutions containing 1 : 400 (Abbott). The typhoid bacillus is killed in five hours by 1.6 per cent, the cholera spirillum by 1.3 per cent (Kitasato). A one-per-cent solution destroys Micrococcus Pas- teuri in half an hour (Sternberg). It is an antiseptic in the propor- tion of 1 : 1,000 (Miquel). A solution of 2.5 per cent kills the tubercle bacillus in six hours (Yersin). In the proportion of 1 : 300it destroys the cholera spirillum in half an hour (Van Ermengem). ACTION OF ACIDS AND ALKALIES. 179 Benzoic Acid. — According to Miquel, this acid restrains the de- velopment of putrefactive bacteria when present in bouillon in the proportion of 1: 909. In the proportion of 1 : 2,000 it retards the de- velopment of anthrax spores (Koch). Formic Acid. — The typhoid bacillus is restrained in its growth by 0.25 per cent, and is killed in five hours by 0.35 per cent, the cholera spirillum by 0.22 per cent (Kitasato). Tannic Acid. — A solution of one per cent kills Micrococcus Pas- teuri in the blood of a rabbit in half an hour (Sternberg). A five- per-cent solution failed in ten days to destroy anthrax spores (Koch). A twenty-per-cent solution failed in two hours to destroy the vitality of spores of the anthrax bacillus or of Bacillus subtilis (Abbott). Micrococci are destroyed by 1 : 400, and 1 : 800 failed (Abbott). A twenty-per-cent solution has no effect upon the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). A solution of 1.66 per cent kills the typhoid bacillus in five hours, and 1.5 per cent the cholera bacillus in the same time (Kitasato). It restrains the devel- opment of putrefactive bacteria in the proportion of 1 : 207 (Miquel). Tartaric Acid. — A twenty-per-cent solution of this acid fails, after two hours' exposure, to destroy the spores of Bacillus anthracis or Bacillus subtilis. Micrococci are killed by two hours' exposure in a solution containing 1 : 400 (Abbott). Malic Acid. — This was found by Kitasato to correspond with citric acid in its germicidal power. Valerianic Acid. — A five-per-cent solution in ether failed in five days to destroy anthrax spores (Koch). Oleic Acid. — A solution of five percent in ether does not destroy anthrax spores in five days (Koch). Thymic Acid. — In the proportion of 1 : 500 this acid prevents the putrefactive decomposition of beef tea (Miquel). Butyric Acid. — Five days' immersion in this acid failed to de- stroy anthrax spores (Koch). Arsenious Acid. — A one-per-cent solution destroys the vitality of anthrax spores in ten days, but failed to do so in six days (Koch). In the proportion of 1 : 166 it prevents putrefactive changes in bouillon (Miquel). Gallic Acid. — Abbott found this acid to destroy the bacteria in broken-down beef tea in the proportion of 2.37 per cent, but it failed to destroy anthrax spores in two hours in the same proportion. Mi- crococci were killed in two hours by 1 : 142, while 1 : 250 failed. ALKALIES. Potassium Hydroxide, KHO. — In the writer's experiments aten- per-cent solution of caustic potash was fatal to pus cocci, and an ACTION OF ACIDS AND ALKALI Ks. eight-per-cent solution failed — two hours' exposure. Exposure for twenty-four hours to a ten-per-cent solution failed to kill the tubercle bacillus (Schill and Fischer). A solution of one per cent kills the anthrax bacillus, the bacillus of rothlauf, and several others (Jager). The addition of 0. 14 per cent restrains the development of the typhoid Iwcillus, and 0.18 per cent kills this bacillus in four or five hours; the cholera spirillum failed to grow in cultures containing 0. 18 per cent and was killed by 0.237 per cent in the same time (Kitasato). Sodium Hydroxide, NaHO.— The experiments of Jager and of Kitasato show that soda has about the same germicidal power as caustic potash. Boer obtained the following results with bouillon cultures after two hours' exposure: Anthrax bacillus, 1 : 450 ; diph- theria bacillus, 1 : 300 ; glanders bacillus, 1 : 150 ; typhoid bacillus, 1 : 100 ; cholera spirillum, 1 : 150. In about one-half the amount required to destroy vitality the development of the above-named bac- teria was prevented. In the proportion of 1 : 56 it acts as an anti- septic (Miquel). Ammonia, NH3. — In Kitasato 's experiments the typhoid bacillus wan destroyed in five hours by 0. 3 per cent of NH3, and the cholera spirillum by about the same amount. Boer obtained the following results, the time of exposure being two hours : Anthrax bacillus. 1 : :HX) : diphtheria bacillus, 1 : 250 ; glanders bacillus, 1 : 250 ; typhoid I>acillu8, 1 : 200 ; cholera spirillum, 1 : 350. The growth of the an- thrax bacillus and of the diphtheria bacillus in culture solutions was prevented by 1 : 050. Calcium Hydroxide, Ca2HO. — According to Kitasato, the ty- phoid bacillus and the cholera spirillum, in bouillon cultures, are killed in four or five hours by the addition of 0.1 per cent of calcium • »\ ii it his bouillon cultures were largely diluted with distilled water. From a practical point of view the experiments of Pfuhl are more valuable. Calcium hydrate was added to the dejections of typhoid jwitieiits. When added in the proportion of three per cent steriliza- tinii was effected in six hours, and by six per cent in two hours. When milk of lime containing twenty per cent of calcium hydrate w;w used the results were still more favorable, the typhoid bacillus and cholera spirillum being killed in one hour by the addition of two per cent of the disinfectant. The practical value of lime- wash H'"<1 to walls has been determined by Jager. Silk threads soaked in cultures of various pathogenic bacteria were attached to boards .u i.l th<> li mo-wash applied with a camel's-hair brush. Anthrax ba- nlh (without spoivs), th<> glanders bacillus, Staphylococcus pyogene? nnvus. and several other pathogenic bacteria were killed by a single application after twenty-four hours, but the tubercle bacillus was not ACTION OF ACIDS AND ALKALIES. 181 killed by three successive applications. Iii the writer's experiments (1885) the typhoid bacillus and Staphylococcus pyogenes aureus were killed in two hours by a solution containing 1 : 40 of calcium oxide, and 1 : 80 failed. Spores of the anthrax bacillus and of several other spore-forming species were not killed by two hours' exposure to a milk of lime containing twenty per cent of calcium oxide. Potash Soap has been shown by Jolles (1895) to have considerable germicidal value. In experiments with a soap containing 67.44 per cent of fat acids, 10.4 per cent of combined alkali, and 0.041 per cent of free alkali, the following results were obtained: The typhoid bacillus was destroyed at 18° C. by a one-per-cent solution in twenty-four hours; by a six-per-cent solution in thirty minutes. The Bacillus coli communis required somewhat stronger solutions or longer exposure — eight-per-cent solution required thirty minutes. These experiments show that scrubbing with soap and water is a reliable method of disinfecting surfaces. Solutions of potash — com- mon lye — or of soda also are useful for certain purposes in domes- tic disinfection, and scientific researches justify the continued use of the cleansing methods which have heretofore been in use by careful housewives. X. ACTION OF SALTS. WHILE some of the metallic salts, and especially those of mer- cury, silver, and gold, have remarkable germicidal power, others, even in concentrated solutions, do not destroy the vitality of bacteria exposed to their action. For convenience of reference we shall con- sider the agents in this group in alphabetical order, but first we give Miquel's tables of antiseptic value. This author recognizes the im- portance of experiments to determine the restraining power of chem- ical agents for various species of pathogenic bacteria, but says : " As to me, faithful to a plan I adopted at the outset, I will treat the sub- ject in a more general manner by making known simply the mini- mum weight of the substances capable of preventing the evolution of any bacteria or germs. The method adopted is very simple. To a liquid always comparable to itself it is sufficient at first to add a known weight of the antiseptic and some atmospheric germs or adult bacteria, and to vary the quantity of the antiseptic until the amount is ascertained which will preserve indefinitely the liquid from putre- faction. In order to obtain germs of all kinds in a dry state it suf- fices to take them, where they are most abundant, in the dust col- lected in the interior of houses or of hospitals; and to procure a \ triety of adult bacteria we may take the water of sewers." SUBSTANCES EMINENTLY ANTISEPTIC. Efficient in the proportion of — Mercuric iodide, . . . . . . 1 : 40000 Silver iodide, ...... 1:33000 Hydrogen peroxide, . . . . . . 1 : 20000 Mercuric chlorid* ..... l:143lO Silver nitrate, . . . . 1 : 12500 SUBSTANCES VERY STRONGLY ANTISEPTIC. Osmicacid, . . i:6666 (Jnromio acid. ..... l-5iiOO Chlorine, 1:'4000 iodine, ..... 1:4000 Ohloride of gold, . ..... l:40uO Bichloride of platinum. ..... 1:3333 Hydrocyanic acid. . 1:2500 ACTION OF SALTS. 183 Bromine, ....... 1:1666 Cupric chloride, . . . . . . 1 : 1428 Thymol, 1:1340 Cupric sulphate, . . . . . 1:1111 Salicylic acid, . . . . . . 1 : 1000 SUBSTANCES STRONGLY ANTISEPTIC. Benzoic acid, . . . . . . 1 : 909 Potassium bichromate, . . . . . 1 : 909 Potassium cyanide, . . . . . 1 : 909 Aluminum chloride, . . . . . 1 : 714 Ammonia, ...... 1:714 Zinc chloride, . . . . . 1 : 526 Mineral acids, . . . . . 1:500 to 1:333 Thymicacid, . . . . . . .1:500 Lead chloride, . . . . . . 1:500 Nitrate of cobalt, . . . . . .1:476 Sulphate of nickel, . . . . . 1 : 400 Nitrate of uranium, ...... 1:356 Carbolic acid, . . . . . . 1:333 Potassium permanganate, . . . . . 1 : 285 Lead nitrate, . . . . . . 1 : 277 Alum, 1:222 Tannin, 1:207 SUBSTANCES MODERATELY ANTISEPTIC. Bromhydrate of quinine, ..... 1:182 Arsenious acid, . . . . . . 1:166 Boracic acid, . . . . . . 1 : 143 Sulphate of strychnia, . . . . . 1 : 143 Arsenite of soda, . . . . . .1:111 Hydrate of chloral, 1:107 Salicylate of soda, . . . . . .1:100 Ferrous sulphate, . . . . . 1 : 90 Caustic soda, . . . . . . 1 : 56 SUBSTANCES FREELY ANTISEPTIC. Perchloride of manganese, . . . . 1 : 40 Calcium chloride, . . . . . 1 : 25 Sodium borate, . . . . . . 1 : 14 Muriate of morphia, . . . . . . 1 : 13 Strontium chloride, . . . . . 1 : 12 Lithium chloride, . . . . . .1:11 Barium chloride, . . . . . . 1 : 10 Alcohol 1:10 SUBSTANCES VERY FEEBLY ANTISEPTIC. Ammonium chloride, . . . . . 1:9 Potassium arsenite, . . . . . .1:8 Potassium iodide, . . . . . 1:7 Sodium chloride, . . . . . .1:6 Glycerin (sp. gr. 1.25), .... Ammonium sulphate, . . . . .1:4 Sodium hyposulphite, . . . . . 1:3 }g4 ACTION OF SALTS. ANTISEPTIC! AND GERMICIDAL VALUE OF VARIOUS SALTS, ARRANGED ALPHABETICALLY. A lum.— Antiseptic in the proportion of 1 : 222 (Miquel). Aluminum Acetate. — According to De la Croix, this salt is an antiseptic in the proportion of 1 : 6,310. Kuhn found it to be anti- septic in 1 :5,250. Aluminum Chloride. — Antiseptic in the proportion of 1 : 714 (Miquel). Ammonium Carbonate. — When present in the proportion of 1 : 125 it restrains the development of typhoid bacilli, and in five hours' time it kills these bacilli in the proportion of 1 : 100 ; the cholera spirillum is killed in the same time by 1 : 77 (Kitasato). Ammonium Chloride. — Antiseptic in the proportion of 1:9 (Miquel). A five-per-cent solution does not kill anthrax spores in twenty-five days (Koch). Ammonium Fluosilicate. — The bacillus of anthrax and of ty- phoid fever fail to grow in nutrient gelatin containing 1 : 1,000, and a two-per-cent solution kills anthrax spores in one-quarter to three- quarters of an hour (Faktor). Ammonium Sulphate. — Antiseptic in the proportion of 1:4 (Miquel). A five-per-cent solution failed in two days to kill an- thrax spores, but was effective in five days (Koch). Barium Chloride is an antiseptic in the proportion of 1 : 10 (Miquel). Calcium Chloride is an antiseptic in the proportion of 1 : 25 (Miquel). A saturated solution does not destroy anthrax spores (Koch). Calcium Hypochlorite. — This is a powerful germicidal agent and has great value as a practical disinfectant. Good chloride of lime contains from twenty-five to thirty per cent of available chlo- rine as hypochlorite. The experiments made by the Committee on Disinfectants of the American Public Health Association in 1885 showed that a solution containing 0. 25 per cent of chlorine as hypo- chlorite is an effective germicide, even when allowed to act only for one or two minutes. In Bolton's experiments a solution of chlo- ride of lime of 1 : 2,000 (available chlorine 0.015) destroyed the ty- phoid bacillus and the cholera spirillum in two hours. For the de- - traction of anthrax spores a one-per-cent solution was required (available chlorine 0.3 per cent). Nissen found that the typhoid bacillus and the cholera spirillum are destroyed with certainty in live, minutes by a solution containing 0.12 percent, anthrax bacilli in one minute by o.l per cent, Staphylococcus pyogenes aureus in one minute by 0.2 per cent, anthrax spores in thirty minutes by a ACTION OF SALTS. 185 five-per-ceiit solution and in seventy minutes by a one-per-cent solu- tion. Experiments made by the same author upon the sterilization of faeces showed that 0. 5 per cent to one per cent could be relied .upon to destroy the typhoid bacillus or the cholera spirillum in faeces in ten minutes. Chloral Hydrate. — Antiseptic in the proportion of 1 : 107 (Mi- quel). A tvventy-per-cent solution destroys pus cocci in two hours (Sternberg). Cupric Chloride. — Antiseptic in the proportion of 1 • 1,428 (Miquel). Cupric Sulphate. — Antiseptic in the proportion of 1 : 111 (Mi- quel). Kills the cholera spirillum in the proportion of 1 : 3,000 in ten minutes (Nicati and Rietsch). Destroys the cholera spirillum in bouillon cultures in less than half an hour in 1 : 600, and in four hours in 1 : 1,000 ; cultures in blood serum require 1 : 200 (Van Er- mengem). A solution of 1 : 20 kills the typhoid bacillus in ten min- utes (Leitz). This salt failed, in the writer's experiments, to kill the spores of Bacillus anthracis and Bacillus subtilis in two hours' time in a twenty-per-cent solution. In Koch's experiments a five-per-ceiit solution failed to kill anthrax spores in ten days. Kills pus micro- cocci in two hours in the proportion of 1 : 200 (Sternberg). In Bol- ton's experiments made for the Committee on Disinfectants of the American Public Health Association the following results were ob- tained: Recent cultures in bouillon, time of exposure two hours : Ba- cillus of typhoid fever, 1 : 200; cholera spirillum, 1 : 500; Bacillus pyo- cyanus, 1 :200; Brieger's bacillus, 1 :200; Emmerich's bacillus, 1 : 200; Staphylococcus pyogenes aureus, 1 : 100 ; Staphylococcus pyogenes citreus, 1 : 100; Staphylococcus pyogenes albus, 1 : 200; Streptococcus pyogenes, 1 : 500. When ten per cent of dried egg albumin was added to a recent culture in bouillon of the typhoid bacillus the amount required to insure sterilization was 1 : 10. In the report of the Committee on Disinfectants of the American Public Health Association this agent is recommended in "a solu- tion of two to five per cent for the destruction of infectious material not containing spores." The experimental data above given show that this is a liberal allowance for material which does not contain an excessive amount of albumin. In the experiments of Leitz the typhoid bacillus in cultures was destroyed in ten minutes by a five- per-cent solution. Ferric Chloride. — A five-per-cent solution failed in two days to destroy anthrax spores, but was effective in five days (Koch). Ferrous Sulphate. — In the writer's experiments (1883) a solution of twenty per cent failed to destroy micrococci and putrefactive bac- teria. In a more recent experiment ten per cent failed to kill pus 186 ACTION OP SALTS. i, but was fatal to Micrococcus tetragenus— two hours' exposure. K . >ch found that a five-per-cent solution failed to destroy anthrax spores in six days. Exposure to a twenty-per-cent solution for forty- eight hours does not destroy the virus of symptomatic anthrax (Ar- loing, Cornevin, and Thomas). In the experiments of Jager immer- sion in a solution of 1 : 3 destroyed the infective virulence of certain pathogenic bacteria (fowl cholera, rothlauf, glanders), as tested by injection into mice, but failed to kill anthrax spores and tubercle ba- rilli. The antiseptic power of ferrous sulphate is placed by Miquel at 1 : 90. In the writer's experiments 1 : 200 prevented the develop- ment of micrococci and of putrefactive bacteria in bouillon placed in the incubating oven for forty-eight hours. Leitz found that a five-per-cent solution required three days' exposure for the destruc- tion of the typhoid bacillus. Gold Chloride. — Antiseptic in the proportion of 1 : 4,000 (Miquel). Boer has made extended experiments with the chloride of gold and sodium. We give his results below. In his disinfection experi- ments a bouillon culture which had been in the incubating oven for t \\ ••nty-four hours was used, and the time of exposure was two hours. « Restrains development. Destroys vitality. Anthrax bacillus 1 : 40000 1 :8000 Diphtheria bacillus 1:40000 1 :1000 (Jlanders bacillus . .... 1 : 15000 1 -400 Typhoid bacillus 1 : 20000 1 :500 Cholera spirillum 1 • 25000 1 :1000 Lead Chloride. — Antiseptic in the proportion of 1 :500 (Miquel). Lead Nitrate. — Antiseptic in the proportion of 1 : 277 (Miquel). Lithium (1l»l. — Antiseptic in the proportion of 1 : 11 (Mi- quel). Mangam *< rrotochloride. — Antiseptic in the proportion of 1:40 (Miquel). Mercuric Chloride.— Koch's experiments (1881) gave the follow- ing results : A solution of 1 : 1,000 destroys anthrax spores in a few minutes, and 1 : 10,000 is effective after a more prolonged exposure. The writer (1884) obtained similar results— 1 : 10,000 destroyed the Bpo res of Bacillus anthracis and of Bacillus subtilis in two hours. More recent experiments indicate that failure to grow in culture so- 1 nt ions cannot be accepted as evidence of the destruction of vitality in the case of spores exposed to the action of this agent, unless due pi "cautions ;m> t;ik«-n to exclude the restraining influence of the small amount of mercuric chloride which remains attached to the spores. l,:,,i ;l-< .-itainrd that the development of spores is restrained by ACTION OF SALTS. 187 the presence of 1 : 300,000 in a culture medium, and Geppert has re- cently shown that even so small an amount as 1 : 2,000,000 will pre- vent the development of spores the vitality of which has been reduced by the action of a strong solution (1 : 1,000). When this restraining action is entirely neutralized by washing the spores in a solution con- taining ammonium sulphide it requires, according to Geppert, a solu- tion of 1:1,000 acting for one hour to completely destroy the vitality of anthrax spores. Frankel found that a solution of 1 : 1,000 was effective in half an hour. The typhoid bacillus, the bacillus of mouse septicaemia, and the cholera spirillum, in bouillon cultures and in cultures in flesh-peptone-gelatiii, are destroyed in two hours by 1 : 10,000 ; but in a bouillon culture to which ten per cent of dried egg albumin was added a one-per-cent solution was required to de- stroy the typhoid bacillus in the same time (Bolton). According to Van Ermengem, cultures of the cholera spirillum in bouillon are steril- ized in half an hour by 1 : 60,000, but cultures in blood serum require 1 : 800 to 1 : 1,000. In experiments upon tuberculous sputum Schill and Fischer found that exposure of fresh sputum to an equal amount of a 1 : 2,000 solution for twenty -four hours failed to disinfect it, as shown by inoculation experiments in guinea-pigs. The antiseptic power of mercuric chloride is given by Miquel as 1 : 14,300. In the writer's experiments 1 : 33,000 was found to prevent the development of putrefactive bacteria in bouillon, but a minute bacillus contained in broken-do wii beef infusion multiplied, after several days, in 1 : 20,000. The pus cocci were restrained in their development by 1 : 30,000. In Behring's experiments the anthrax bacillus and cholera spiril- lum were killed in one hour by 1 : 100,000 when the temperature was 36° C., but at a temperature of 3° C. the proportion required was 1 : 25,000. The same author states that at 22° C. Staphylo- coccus aureus in bouillon is not always killed in twenty-five minutes by 1 : 1,000. In a recent series (1891) of experiments Abbott has shown that a 1 : 1,000 solution does not always destroy Staphylococcus pyogenes aureus in five minutes. He says: "Frequently all the organisms would be destroyed after five minutes' exposure, but almost as often a certain few would resist for that length of time, and even longer, going in some cases to ten, twenty, and even thirty minutes. " According to Yersin, a solution of 1 : 1,000 kills the tubercle bacil- lus in one minute. We might add considerably to the experimental data given, but the results already recorded are sufficient to show the value of this agent as an antiseptic and germicide, and justify its use for general purposes of disinfection in the proportion of 1 : 500 or 1 : 1,000 for material containing spores, and in the proportion of 1 : 2,000 to Ihtf ACTION OF SALTS. 1 : :>,000 for pathogenic bacteria in the absence of spores; due regard }>eing had to the fact that the presence of albumin very materially reduces its germicidal potency, and that it may be decomposed and neutralized by alkalies and their carbonates, by hydrosulphuric acid, and by many other substances. The albuminate of mercury, as has been shown by Lister, is solu- ble in an excess of albumin, and, according to Behring, is just as effective as an aqueous solution containing the same amount of sub- limate when dissolved in an albuminous liquid like blood serum (?). In practice the addition of a mineral acid to sublimate solutions, or of sodium, potassium, or ammonium chloride, is to be recom- mended, to prevent the precipitation of the mercuric chloride by al- bumin in fluids containing it. Behring recommends the addition of five parts of sodium or potassium chloride to one of the subli- mate. Such a solution is more stable than a simple solution of sub- limate, and no precipitate is formed by the addition of alkalies or by albumin. The same result is obtained, according to La Place, by the addi- tion of five parts of hydrochloric or tartaric acid to one part of sub- limate in aqueous solution. Mercuric Cyanide, Hg(CN)2, and the Oxycyanide of mercury have been tested, with the following results : Staphylococcus aureus is destroyed in five minutes by 1 : 100, in one hour by 1 : 1,000, in two hours by 1 : 1,500 (Chibret). The development of Bacillus an- thracis in culture solutions is prevented by the presence of cyanide of mercury in the proportion of 1 : 25,000, and by the oxy cyanide by 1 : 16,000 (Behring). Boer obtained the following results with the oxycyanide — cul- turos in bouillon, twenty-four hours in incubating oven, time of exposure two hours : Restrained development. Destroyed vitality. Anthrax bacillus • 80000 1 • 40000 Diphtheria bacillus • soooo i . 4.0000 Glanders bacillus • fiOOOO 1 • '^0000 Typhoid bacillus • 60000 1 • SOOOO Cholera spirillum • 'Mini KI 1 • ftoooo M< rcuric Iodide.— The antiseptic value of this salt is placed by Miquel at 1 : 40,000, which is more than double that given by the ^;mie author to the Im-hloride. In the writer's experiments upon the antiseptir value of salts and oxides of mercury the following results were obtained : ACTION OF SALTS. 189 Active. Failed. Biniodide of mercury 1 • 20000 1 • 40000 Bichloride 1 15000 1 • 20000 Protiodide 1 10000 1 • 20000 Yellow oxide 1 : 1000 1 2000 Black oxide 1 -500 1 • 1000 Morphia Htjdrochlorate. — Antiseptic in. the proportion of 1 : 13 (Miquel). Nickel Sulphate. — Antiseptic in the proportion of 1 : 400 (Mi- quel). Platinum Bichloride. — Antiseptic in the proportion of 1 : 3,333 (Miquel). Potassium Acetate. — A saturated solution of this salt failed to kill anthrax spores in ten days (Koch). Potassium Arsenite. — In the writer's experiments Fowler's solu- tion failed to kill micrococci in two hours in the proportion of four per cent. Miquel places the antiseptic value of potassium arsenite at 1 : 8. Potassium Bichromate. — A five-per-cent solution failed in two days to destroy anthrax spores (Koch). Efficient as an antiseptic in the proportion of 1 : 909 (Miquel). Potassium Bromide. — The bacillus of typhoid fever and the cholera spirillum fail to grow in culture solutions containing 9 to 10.6 per cent, and are killed in four or five hours by ten to twelve per cent (Kitasato). Potassium Carbonate. — The development of the typhoid bacil- lus and of the cholera spirillum is prevented by 0.74 to 0.81 per cent, and these bacteria are killed in five hours by 1 per cent (Kita- sato). Potassium Chlorate. — In the writer's experiments a four-per- cent solution failed in two hours to kill Micrococcus Pasteuri. A five-per-cent solution failed in six days to destroy anthrax spores (Koch). Potassium Chromate. — A five-per-cent solution failed to kill anthrax spores in five days (Koch). Potassium Cyanide. — Antiseptic in the proportion of 1 : 909 (Miquel). Potassium Iodide. — A solution of five per cent does not destroy anthrax spores in eighty days (Koch). Putrefactive bacteria in broken-down beef infusion are not destroyed by two hours' exposure in a twenty-per-cent solution (Sternberg). The typhoid bacillus and the cholera spirillum do not grow in culture solutions containing 190 ACTION OF SALTS. eight per cent, and are destroyed by five hours' exposure to 9.23 per cent'(Kitasato). Antiseptic in the proportion of 1 : 7 (Miquel). Potaasium Permanganate.— In the writers experiments (1881) a two-per-cent solution was required to destroy Micrococcus Pasteuri in the blood of a rabbit. In later experiments pus cocci in bouillon were killed by 1 : 833— time of exposure two hours. One per cent was found by Koch not to destroy anthrax spores in two days, but five per cent was effective in one day. The glanders bacillus is de- stroyed in two minutes by a one-per-cent solution (Loflfler). The experiments of Jager show that a one-per-cent solution is not reli- able for the destruction of anthrax bacilli and other pathogenic bac- teria tested, but a five-per-cent solution was effective. The tubercle bacillus was not, however, killed by exposure in a five-per-cent solu- tion. According to Miquel, permanganate of potash is an antiseptic in the proportion of 1 : 285. Quinine Hydrobromate. — Antiseptic in the proportion of 1 : 182 (Miquel). Quinine Hydrochlorate. — Antiseptic in the proportion of 1 : 900 (Ceri). Quinine dissolved with hydrochloric acid destroys anthrax spores in ten days in one-per-cent solution (Koch). Quinine Sulphate. — The writer found that in the proportion of 1 : 800 quinine prevents the development of various micrococci and l»;tcilli. A ten-per-cent solution does not destroy the bacilli of symp- tomatic anthrax (Arloing, Cornevin, and Thomas). Silver Nitrate. — Miquel places nitrate of silver next to mercuric chloride as an antiseptic, effective in the proportion of 1 : 12,500. Behring also places it next to bichloride as an antiseptic and germi- cide, and says that it is even superior to this salt in albuminous fluids. He reports that it prevents the development of anthrax spores when present in a culture liquid in the proportion of 1 : 80,000, and in the proportion of 1 : 10,000 destroys these spores in forty- eight hours. We give below the result of recent experiments by Boer, in which the time of exposure was two hours : Restrains development. Destroys vitality. • 600^0 . 20000 Dipliflicriu liarilius • 60000 • 2500 • 75000 • 4000 Typhoid bacillus • 50000 •4000 ( 'Imlrrii spirillum • 50000 • 4000 Silver Chloride.— A. solution of chloride of silver in hyposulphite of soda is much less effective as an antiseptic than nitrate of silver. ACTION OF SALTS. 191 Behring found that to prevent the development of anthrax spores a solution of 1 : 8,000 was required. Sodium Borate. — In the writer's experiments a saturated solu- tion of borax was found to be without germicidal power. A twenty- per-cent solution does not destroy the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). A five-per-cent solution failed to destroy anthrax spores in fifteen days (Koch). Antiseptic in the proportion of 1 : 14 (Miquel). Sodium Carbonate. — A solution of 2.2 per cent restrains the growth of the typhoid bacillus, and of 2.47 per cent of the cholera spirillum. The first-named bacillus is killed by four or five hours' exposure in a 2. 47-per-cent solution, and the cholera spirillum by 3.45 per cent (Kitasato). Sodium Chloride. — A saturated solution failed in forty-eight hours to destroy the virus of symptomatic anthrax (Arloing, Corne- vin, and Thomas). A saturated solution failed in forty days to de- stroy anthrax spores (Koch). A saturated solution failed in twenty hours to destroy the tubercle bacillus in fresh sputum (Schill and Fischer). In the writer's experiments a five-per-cent solution failed to kill Micrococcus Pasteuri in blood. Antiseptic in the proportion of 1 : 6 (Miquel). According to Forster, the bacillus of typhoid fever, the bacillus of rouget, and the streptococcus of pus are not killed by several weeks' exposure in strong solutions of sodium chlo- ride, but the cholera spirillum is destroyed in a few hours. Cultures of the tubercle bacillus are not sterilized in two months by a satu- rated solution ; and tuberculous organs from an ox, preserved in a solution of salt, did not lose their power of infecting susceptible ani- mals inoculated with material from the diseased tissue. The flesh of swine which died of rothlauf was found by Petri to still contain the bacillus in a living condition after having been preserved in brine for a month. Sodium Hyposulphite. — In the writer's experiments a saturated solution failed in two hours to kill micrococci and bacilli. Exposure for forty-eight hours to a fifty-per-cent solution does not destroy the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). Antiseptic in the proportion of 1 : 3 (Miquel). Sodium Sulphite. — The results with a saturated solution of this salt were, in the writer's experiments, entirely negative. Tin Chloride. — A one-per-cent solution acting for two hours de- stroyed the bacteria in putrefying bouillon, while 0.8 per cent failed (Abbott). Zinc Chloride.— In the writer's experiments 1:200 destroyed Micrococcus Pasteuri in two hours, but a two-per-cent solution was re- quired to kill pus cocci in the same time ; spores of Bacillus anthracis 192 ACTION OF SALTS. were not destroyed by two hours' exposure in a ten-per-cent solution, but a solution of five per cent killed tbe spores of Bacillus subtilis in the same time. Koch found that anthrax spores germinated after being immersed in a five-per-cent solution for thirty days. The de- velopment of Bacillus prodigiosus is only slightly retarded by expo- sure for sixteen hours in a one-per-cent solution. Antiseptic in the proportion of 1 : 526 (Miquel). Zinc Sulphate. — In the writer's first experiments a twenty -per- cent solution failed to destroy in two hours micrococci obtained from the pus of an acute abscess. In later experiments a micrococcus from the same source resisted two hours' exposure to a ten-per-cent solu- tion, but Micrococcus tetragenus was destroyed by this amount. Broken-down beef infusion mixed with an equal quantity of a forty- per-cent solution was not sterilized after two hours' contact. In Koch's experiments anthrax spores were found to germinate after having been immersed for ten days in a five-per-cent solution. XI. ACTION OF COAL-TAR PRODUCTS, ESSENTIAL OILS, ETC. IN the present section we shall consider the action upon bacteria of a variety of organic products, and for convenience will arrange them alphabetically. Acetone. — Anthrax spores grow freely after two days' exposure to the action of this agent; at the end of five days their development is feeble (Koch). Alcohol. — In the writer's experiments ninety-five-per-cent alco- hol did not destroy the bacteria (spores) in broken-down beef tea in forty -eight hours. Micrococcus Pasteuri was destroyed by two hours' exposure in a twenty-four-per-cent solution ; pus cocci required a forty -per-cent solution. Koch found that absolute alcohol had no effect upon anthrax spores exposed to its action for one hundred and ten days. Schill and Fischer found that when tuberculous sputum was mixed with an equal amount of absolute alcohol its infecting power was not destroyed in twenty-four hours, but that in the pro- portion of five parts to one of sputum it was effective in destroying the tubercle bacillus, as proved by inoculation experiments. Yersin found that in pure cultures the tubercle bacillus is killed by five minutes' exposure to the action of absolute alcohol. Aseptol (orthophenol, sulpho-carbolic acid, etc.). — This substance is freely soluble in water. According to Hueppe a three to five-per- cent solution destroys bacteria in the absence of spores, and a ten- per-cent solution destroys anthrax spores in ten minutes. Aniline Dyes. — Recent researches have shown that some of the aniline colors possess very decided germicidal power. Stilling found that solutions of methyl violet containing 1 : 30,000 exercise a re- straining influence upon the development of putrefactive bacteria and pus cocci, and that these microorganisms are destroyed by solu- tions containing 1 : 2,000 to 1 : 1,000. Methyl violet has been placed in the market by Merck under the name of pyoktanin. Janicke re- ports the following results with pyoktanin : Staphylococcus pyogenes aureus was restrained in its development by solutions containing 1 : 2,000,000, Bacillus anthracis by 1 : 1,000,000, Staphylococcus pyo- genes by 1 : 333,300, Spirillum cholerae Asiaticae by 1 : G2,500, Bacil- lus ty phi abdominalis by 1 : 5,000. In blood serum stronger solutions 13 194 ACTION OF COAL-TAR PRODUCTS, were required (1 : 500,000 for Staphylococcus pyogenes aureus). Sta- phylococcus pyogenes aureus, Streptococcus pyogenes, and Bacillus anthracis were killed in thirty seconds by 1 : 1,000, the typhoid bacil- lus by the same amount in thirty minutes. Boer found malachite green to be still more effective than methyl violet. In his experi- ments upon bouillon cultures twenty-four hours old, with two hours' exposure to the action of the disinfectant, he obtained the following results : MALACHITE GREEN. Restrains development. Destroys vitality. 1 : 120000 : 40000 1 : 40000 :8000 1 :5'»00 :300 1 • 5000 :300 1 : 100000 •5000 METHYL VIOLET (PYOKTANIN). Restrains development. Destroys vitality. 1 : 70003 1 • 5000 Diphtheria bacillus 1 : 10000 1 • 2000 1 • 2500 1 • 150 Typhoid bacillus 1 :2500 1 -150 1 * 30000 1 -1000 Aniline Oil. — According to Riedlin, the addition of 1 : 5 of ani- line water prevents the development of all bacteria in nutrient gelatin. Aromatic Products of Decomposition. — Klein has tested the germicidal power of phenylpropionic and phenylacetic acids. He finds that anthrax spores resist both of these acids, in the proportion of 1 : 400, for two days, but in the absence of spores anthrax bacilli are quickly killed by a solution of this strength. Certain non-patho- genic micrococci were not killed by exposure for twenty-five minutes to 1 : 200. The caseous matter of pulmonary tuberculosis infected guinea-pigs after exposure for ninety-six hours to 1 : 200. Aseptol. — A ten-per-cent aqueous solution kills anthrax spores in ten minutes, and a three- to five-per-cent solution is a reliable disin- fectant in the absence of spores (Hueppe). Benzene, C.H,. — Exposure in benzol for twenty days failed to destroy the vitality of anthrax spores (Koch). Camphor.— Alcohol saturated with camphor has no effect upon the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas) The experiments of Cadeac and Meunier show that camphor (oil ESSENTIAL OILS, ETC. 195 of, or tincture?) has but little germicidal power. The typhoid ba- cillus and cholera spirillum were only destroyed after eight to ten days' exposure to the action of camphor ("essence")* Carbolic Acid. — Tested upon anthrax spores, Koch found a one- per-cent solution to be without effect after fifteen days' exposure ; a two-per-cent solution retarded development but did not completely destroy vitality in seven days ; a three-per-cent solution was effec- tive in two days. In the absence of spores Koch found that a one- per-cent solution quickly destroys the vitality of anthrax bacilli. He recommends a five-per-cent solution for the destruction of the "comma bacillus" in the discharges of cholera patients, and a two- per-cent solution for the disinfection of surfaces soiled with such dis- charges. In the writer's experiments 1 : 200 destroyed Micrococcus Pasteuri in two hours ; and pus cocci were destroyed by 1 : 125, while 1 : 200 failed. Davaine showed by inoculation experiments that an- thrax bacilli in fresh blood are destroyed by being exposed to the action of a one-per-cent solution for one hour. A two-per-cent solu- tion destroys the dried virus of symptomatic anthrax in forty-eight hours (Arloing, Cornevin, and Thomas). Solutions in oil or in alco- hol have been shown by Koch to be less effective than aqueous solu- tions. Thus a five-per-cent solution in oil failed to destroy anthrax spores in one hundred and ten days, and the same solution failed to kill the bacilli, in the absence of spores, in less than six days. A five-per-cent solution in alcohol did not destroy anthrax spores in seventy days. Schill and Fischer found that a three-per-cent solu- tion destroyed the infecting power of tuberculous sputum, as shown by inoculation into guinea-pigs, in twenty-four hours, while solutions of one and two per cent failed. Bolton's experiments gave the fol- lowing results, the test organisms being in fresh bouillon cultures and the time of exposure two hours : The cholera spirillum, the bacillus of typhoid fever, the bacillus of schweinerothlauf, Brieger's bacillus, the bacillus of green pus, and the pus cocci (Staphylococcus pyogenes aureus, albus, and citreus, and Streptococcus pyogenes) were all killed by a solution of one per cent, while in a majority of the experiments a one-half -per-cent (1 : 200) solution failed. Cul- tures of the typhoid bacillus in flesh-peptone-gelatin gave the same result (1 : 100 with two hours' exposure), and the addition of ten per cent of dried egg albumin to bouillon cultures did not influence the result. The experiments of La Place show that the addition of hydro- chloric acid to a disinfecting solution containing carbolic acid greatly increases its germicidal power for spores. Thus it is stated that " two per cent of crude carbolic acid with one per cent of pure hydro- chloric acid destroyed anthrax spores in seven days, while two per cent of carbolic acid or one per cent of hydrochloric acid alone did 196 ACTION OF COAL-TAR PRODUCTS, not destroy these spores in thirty days. A f our-per-cent solution of crude carbolic acid with two per cent of hydrochloric acid destroyed spores in less than an hour ; four per cent of carbolic acid alone did not destroy them in twelve days. Van Ermengem reports that in his experiments the cholera spirillum in chicken bouillon was killed in less than half an hour by 1 : 600, and that in blood serum 1 : 400 was effective. Nicati and Rietsch fix the germicidal power for the cholera spirillum as 1 : 200, the time of exposure being ten minutes ; Ramon and Cajal, 1 : 50. Boer gives the following results, the time of exposure being two hours, cultures in bouillon twenty-four hours old: Restrains development. Destroys vitality. 1 :750 1 : 300 Diphtheria bacillus 1 :500 1 : 300 1 :500 1 :300 1 :400 1:200 1 :600 1 :400 Leitz reports the following results : The dejections of patients suffering from typhoid fever, mixed in equal quantity with the disin- fecting solution, were sterilized by a five-per-cent solution of car- l>olic acid in three days. Pure cultures of the typhoid bacillus were sterilized in fifteen minutes by a five-per-cent solution. In the experiments of Nocht upon anthrax spores it was found that while at the room temperature these spores were not destroyed by several days' exposure in a five-per-cent solution, they were de- stroyed in three hours by the same solution at a temperature of 37.5°. Carbolic acid prevents putrefactive changes in bouillon when pre- -'•nt in the proportion of 1 : 333 (Miquel). The tubercle bacillus is killed in thirty seconds by a five-per-cent solution, and in one minute by a one-per-cent solution (Yersin). Coffee Infusion. — Experiments have been made by Heim and by I .mleritz on the antiseptic power of an infusion of coffee. The first- nam.-.l author found that anthrax bacilli no longer developed after tlm>e hours* exposure in a ten-per-cent solution, but spores were not kill.Ml ; it the end of a week. Streptococci in a bouillon culture re- • iu i rod twenty-four hours' exposure, and the staphylococci of pus were 11. >t destroyed in this time. Liideritz found that a three-per-cent iii- fiisi.m restrained the growtli in nutrient gelatin of the typhoid ba- rillu>. ami a five-per-cent infusion killed the bacillus in two days ; the rlinlrra spirillum failed to grow in presence of one per cent, and a solution of this strength killed it in seven hours ; Staphylococcus ESSENTIAL OILS, ETC. 197 pyogenes aureus was prevented from developing by two per cent, and was killed in six days by a five-per-cent solution ; Streptococcus pyogenes was prevented from growing by one per cent, and killed by a ten-per-cent solution in one day ; Proteus vulgaris did not grow in presence of 2. 5 per cent, and was killed in two days by ten per cent. The question as to what constituent of the infusion of roasted coffee was the active germicidal agent was not determined, but the authors referred to agree that it was not caffeine. Creolin. — This is a coal-tar product which resembles crude carbolic acid in appearance, but smells rather like tar than like phenol. It makes a milky emulsion with water, which has been proved by nu- merous experiments to possess very decided germicidal power, being superior to carbolic acid. The first careful test of the germicidal power of this agent was made by Esmarch, who found that a solu- tion of 1 : 200 killed the cholera spirillum in a minute, the typhoid bacillus at the end of several days. Anthrax spores were not de- stroyed in twenty days by a five-per-cent solution, but this solution killed the tubercle bacillus attached to silk threads which were im- mersed in it for a short time, and also disinfected tuberculous sputum. Behring has shown that in albuminous liquids creolin is less effective than carbolic acid. In blood serum 1 : 175 was required to restrain the development of staphylococci, and 1 : 100 to destroy the same in ten minutes. Van Ermengem, as a result of numerous experiments, arrived at the conclusion that creolin is a cheap and useful disinfect- ing agent, in a five-per-cent solution, for various pathogenic organ- isms. Kaupe reports that in his experiments a ten-per-cent solution killed anthrax spores in twenty-four hours. According to Boer, a solution of 1 : 5,000 destroys anthrax bacilli in bouillon cultures in two hours, 1 :2,000 diphtheria bacilli, 1 : 300 the glanders bacillus, 1 : 250 the typhoid bacillus, and 1 : 3,000 the cholera spirillum. Creosote. — This agent was found by the writer to be fatal to micrococci in the proportion of 1 : 200. In the proportion of one per cent it failed, after twenty hours' exposure, to destroy tubercle ba- cilli in sputum (Schill and Fischer). A saturated aqueous solution does not destroy the tubercle bacillus in cultures in twelve hours (Yersin). Guttman, in extended experiments upon various patho- genic organisms, found that development was prevented by 1 : 3,000 to 1 : 4,000. A solution containing 1 : 300 killed Bacillus pyocyarius and Bacillus anthracis in one minute, Bacillus prodigiosus in two minutes, and the Finkler-Prior spirillum in one minute in the pro- portion of 1 : 600. Cresol. — This is a dark, reddish-brown, transparent fluid, some- what thinner than creolin, and, like it, having an odor of tar. It forms an emulsion with water, which is not so stable as that formed 198 ACTION OF COAL-TAR PRODUCTS, by creolin. Of the three cresols, ortho-, meta-, and paracresol, the second was found by Frankel to be most active. This author states that the addition of sulphuric acid adds greatly to its germicidal |M»\ver. A four-per-cent solution, containing equal parts of cresol and H,SO4, killed anthrax spores in less than twenty-four hours. In Behring^s experiments a solution containing ten per cent of each killed anthrax spores in eighty minutes, and five per cent of each in one hundred minutes, while an eighteen-per-cent solution of sulphuric acid alone did not kill them in twenty -four hours. In the experi- ments of Jager a two-percent solution destroyed the tubercle bacillus in cultures and in sputum. As a result of his experiments Behring concludes that cresol has no advantage over carbolic acid as a ger- micide for the destruction of spores. Tested upon Staphylococcus an runs, Streptococcus erysipelatos, and Bacillus pyocyanus, Frankel found that a solution of 0. 3 per cent destroyed these microorganisms in five minutes, while a two-per-cent solution of carbolic acid re- quired fifteen minutes' contact to accomplish the same result. Trikresol (Schering) has been tested, with favorable results, by several bacteriologists. According to Hammerl it is about twice as active a germicide as carbolic acid. Diaphtherin (oxychinaseptol) has considerable antiseptic power, n> shown by the experiments of Rohrer and others. Two to four drops of a one-per-cent solution was found to prevent the develop- ment of test organisms (Staphylococcus pyogenes aureus and Bacillus anthracis) in twelve cubic centimetres of bouillon. Stable (1893) also finds that as an antiseptic it is far superior to carbolic acid or lysol, and that it has the advantage of being non-toxic. Tested upon an- thrax spores it was found to be comparatively inactive as a germicide. A fifteen-per-cent solution destroyed anthrax spores in three days. Disinfektol—This is a coal-tar product similar to creolin which li is been recommended in Germany for disinfecting purposes. It is mi oily, dark-brown fluid having a specific gravity of 1. 086. It forms nn emulsion with water, which has a slightly alkaline reaction. It 1 ms been tested upon typhoid stools by Uffelmann and by Beselin. The last-named author gives the following summary of the results obtained : An emulsion of five per cent of disinfektol equals in value, f'»r the disinfection of the liquid discharges of typhoid patients, 12.5 per cent of creolin, thirty-three percent of hydrochloric acid, five per cent of carbolic acid, 1 : 500 of mercuric chloride. Ether. — Anthrax spores may germinate after being immersed in -il|»huric ether for eight days (Koch). The tubercle bacillus is de- •yed by ten minutes' exposure to the action of ether (Yersin). /•:,s-.s-r nhtil Oils. —Chamberlain has made an extended series of experiments to determine the antiseptic power of the vapor of vola- ESSENTIAL OILS, ETC. 199 tile oils. A large number of essential oils tested were found to pre- vent the development of the anthrax bacillus, while a few did not. At the end of six days the tubes were opened and the oil absorbed by the culture liquid allowed to evaporate. Cultures were now obtained from all except the following, which, it was inferred, had destroyed the vitality of the spores : Angelica, cinnamon of China, cinnamon of Ceylon, geranium of France, geranium of Algeria, origanum. Cadeac and Meunier have also made extended experiments upon the typhoid bacillus and the bacillus of glanders, for the purpose of determining the germicidal power of agents of this class. Their method consisted in the introduction of a sterilized platinum needle into a pure culture of the test organism, in immersing it in the essential oil for a certain time, and then making with it a puncture in a suitable solid culture medium. Their results are given below for the typhoid bacillus. Essences which kill the bacillus after a contact of less than twenty-four hours : At the end of— Cinnamon of Ceylon, . . . . .12 minutes. Cloves, ...... 25 Eugenol, ....... 30 Thyme, ...... 35 Wild thyme, . 35 Verbena of India, ..... 45 Geranium of France, . . . . .50 Origanum, ...... 75 Patchouly, ...... 80 Zedoary, ...... 2 hours. Absinthe 4 u Sandal wood, . . . . . . 12 " The following were effective in from twenty-four to forty-eight hours: Cumin, caraway, juniper, matico, galbanum, valerian, citron, angelica, celery, savin, copaiba, pepper, turpentine, opoponax, rose, chamomile ; the following required from two to four days: Illicium, sassafras, tuberose, coriander; the following from four to eight days: Calamus, sage, fennel, mace, cascarilla, orange of Portugal; the fol- lowing in eight to ten days : Mint, nutmeg, rosemary, carrot, mus- tard, anise, onion, marjoram, bitter almonds, cherry laurel, myrtle, lavender, eucalyptus, cedar, cajuput, wintergreen, camphor. Riedlin reports as the result of his experiments that the essential oils which have the greatest antiseptic value are oil of lavender, eu- calyptus, rosemary, and cloves. Eucalyptol. — Chabaunes and Perret found that a five-per-cent solution of eucalyptol is without effect upon tubercle bacilli in spu- tum. According to Behring, eucalyptol is about four times less ac- tive as a disinfectant than carbolic acid. * 200 ACTION OF COAL-TAR PRODUCTS, Euphorin (Phenylurethan) has been tested by Colasanti (1894), who finds that it has rather feeble germicidal activity. Formaldehyde (formol, formalin) has very decided germicidal power. According to Pottevin (1894) in the absence of spores a solu- tion of 1 : 1,000 kills bacteria, in comparatively small numbers, in from fifteen minutes to several hours. For the destruction of spores a much stronger solution is required — a fifteen-per-cent solution at 15° C. killed anthrax spores in one and one-half hours, and spores of Bacillus subtilis in twenty hours. At higher temperatures the germicidal action is more energetic, and microorganisms exposed to the vapor of formol are very quickly destroyed. Vanderlinden and de Buck (1895) find that solutions of formalin are decidedly inferior to corresponding solutions of carbolic acid, creolin, or solveol, and are, too irritating to be used in surgical practice. They report that a solution of five per cent failed to destroy their test organisms — Bacillus coli communis, Bacillus typhi abdominalis, Staphylococcus pyogenes aureus. Experiments made by Reed, at the Army Medical Museum in Washington, show that the diphtheria bacillus and other test organisms are quickly killed by formalin vapor. Glycerin has no action upon the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas), and is inert as regards the spores of anthrax (Koch). Glycerin prevents putrefactive decomposition in bouillon when present in the proportion of 1 :4 (Miquel). Roux has shown that the addition of five per cent of glycerin to a culture medium is favorable to the growth of the tubercle bacillus ; it is also appropriated as pabulum by various other species. Ouaiacol. — Kuprianow, as a result of extended experiments with this agent (1894), reports that it ranks below cresol and carbolic acid flfefi germicide. In the proportion of 1 : 500 it restrains the develop- ment of the cholera spirillum, and the author named suggests its in- ternal administration in this disease on account of its non-toxic and non-irritant properties. Hydroxylamin.— Heinisch found that the development of the anthrax bacillus is prevented by 1 : 77 of hydroxylamin hydro- chlorate, and of the diphtheria bacillus by 1 : 75. In these experiments a solution of soda was added to release the hydroxylamin. Marp- mann found that 1:100 preserved milk without change for four to six weeks, and that alkaline fermentation of urine was prevented by 1:1,000. Ichthyol.— Latteux (1892) reports that the various pathogenic bacteria used by him as test organisms were killed by a five-per-cent solution (time ?) with exception of Streptococcus pyogenes, which required a six to seven-per-cent solution. The more recent experi- ments of Abel (1893) gave less favorable result, but the agent was ESSENTIAL OILS, ETC. 201 shown to have considerable antiseptic value — 1 : 2,000 restrained the development of streptococci; 1: 500 of the diphtheria bacillus ; 1: 20 of Staph ylococcus pyogenes aureus ; 1 : 33 the bacillus of typhoid fever. Streptococci and diphtheria bacilli were destroyed in twenty- four hours by a solution of 1 : 200 ; Staphylococcus aureus, subjected to the action of pure ichthyol, was destroyed in five hours — in a five- per-cent solution it survived for four days. Cultures of the typhoid bacillus mixed with a fifty-per-cent solution were not completely sterilized in thirty hours; a small number of bacilli in bouillon were, however, destroyed by a three-per-cent solution in forty-eight hours. Anthrax spores on silk threads were not destroyed by a fifty-per-cent solution at the end of one hundred and forty days. Indol. — When added in excess to water this agent failed to de- stroy anthrax spores in eighty days (Koch). Izal is a coal-tar product which has recently been introduced as a disinfectant. Klein (1892) reports that in the strength of ten per cent it kills anthrax spores in fifteen minutes. In the absence of spores various pathogenic bacteria were killed in five minutes by a solution containing 1 : 200. Lanolin. — According to Gottstein, various microorganisms tested by him failed to grow in cultures after having been in contact with pure lanolin for five to seven days. Loretin. — Korff (1895) claims for this agent that a two-per-cent solution is superior to corresponding solutions of lysol, metakresol, or phenol, and that it has the advantage of being non-toxic, odorless, and non-irritating. Lysol. — Weiss (1895) has tested this product and reports that a solution of three-fourths per cent destroyed his test organisms (pus cocci, typhoid bacillus, Bacillus coli communis, etc.) in five minutes. Anthrax spores were destroyed by the same solution in one hour. Naphthol. — In the proportion of 1 : 10,000 naphthol prevents the development of the glanders bacillus, the anthrax bacillus, the typhoid bacillus, the micrococcus of fowl cholera, of Staphylococcus aureus and albus, and of several other microorganisms tested by Maximo- vitch. The same author states that although insoluble in cold water, water at 70° C. dissolves 0.44 in one thousand parts. When urine is shaken up with naphthol in powder it does not undergo fermenta- tion. In the experiments of Foote hydronaphthol was found to show some germicidal power in the proportion of 1 : 2,300, but the conclu- sion is reached that a saturated aqueous solution (1 : 1,150) does not equal a one-per-cent solution of carbolic acid or of creolin. The writer, in 1892, obtained the following results in experiments with naphthols upon the cholera spirillum. 202 ACTION OF COAL-TAR PRODUCTS, Alpha-naphthol and beta-naphthol have about the same antiseptic and germicidal value. In the proportion of 1 : 16,000 both prevent the develop- ment of the cholera spirillum in peptonized beef -tea, while 1 : 24,000 fails to pi-event development. In the proportion of 1 : 3,000 both destroy the vital- ity of the cholera spirillum in bouillon cultures, twenty-four hours old, after two hours' contact, while 1 : 4,000 fails to destroy this microorganism in the time mentioned — two hours. In experiments made with a solution of 1 : 1,000, added to an equal quantity of a twenty-four hours old bouillon culture — making 1 : 2,000 after mixture — and in which the time of contact varied from live to thirty minutes, alpha-, beta-, and hydronaphthol were found to destroy the cholera germ by fifteen minutes' exposure, but to fail after ten minutes' contact, so that the germicidal value of each of these is similar, or nearly so. In all these experiments the line was sharply drawn between success and failure. No development occurred and the bouillon remained transparent in those experiments in which the germicidal action was complete, and a characteristic development occurred within twenty-four hours in those ex- periments in which there was a failure to destroy the spirillum. Benzo-naphthol has 110 germicidal power, probably because it is insoluble in water. At least this is my inference from the experiments made. One gamme was added to one thousand cubic centimetres of distilled water, and after vigorous shaking was placed in the steam sterilizer for half an hour. At the end of this time the greater portion, at least, of the beiizo-naphthol re- mained undissolved at the bottom of the flask. The saturated solution (?) was then filtered and added to recent bouillon cultures of the cholera spiril- lum in the proportion of 1 : 1, 1 : 2, 1 : 4, and 2:1. At the end of two hours sterile bouillon in test tubes was inoculated from each of these and placed in the incubating oven. At the end of forty-eight hours a characteristic devel- opment of the cholera spirillum had occurred in all of the tubes. Olive Oil. — Anthrax spores germinate after having been im- mersed for ninety days in pure olive oil (Koch). Oil of Mustard. — Koch found that the development of anthrax spores is prevented by 1 : 33,000. Oil of Peppermint.— A. five-per-cent solution in alcohol failed in twelve days to destroy anthrax spores, but the development of these spores is restrained by 1 : 33,000 (Koch). Oil of Turpentine destroys anthrax spores in five days, but failed to do so in one day (Koch) . The development of anthrax spores is prevented by 1 : 75,000 (Koch). The addition of 1 : 200 to nutrient gelatin prevents the development of bacteria (Riedlin) . An excess of oil of turpentine added to a liquefied gelatin culture of Staphylo- ooccus aureus does not destroy this micrococcus in five hours (v. < liiistmas-Dirckinck-Holmfeld). Saprol. — Laser (1802) recommends this agent for the disinfection < >t t he excreta of cholera and typhoid patients. He reports that in the proportion of 1 : 100 it sterilizes liquid faeces in twenty-four hours. Skatol in excess in water has no germicidal power, as tested upon anthrax spores (Koch). smofo?. — The researches of Beu show that meats which have been preserved by smoking commonly contain living bacteria capable of growing in culture media; and Petri has shown that pork which has ESSENTIAL OILS, ETC. 203 been salted for a month and then smoked for fourteen days may still contain the bacillus of rothlauf in a living condition, as shown by in- oculation experiments. It was not until about six months after smok- ing that the bacillus failed to give evidence of vitality. Thymol. — A five-per-cent solution in alcohol does not destroy anthrax spores in fifteen days, but the development of these spores is retarded by a solution of 1 : 80,000 (Koch). The anthrax bacillus and staphylococci fail to grow in culture media containing 1 : 3,000 (Samter). The tubercle bacillus is destroyed by contact with thy- mol for three hours (Yersin). Thymol has about four times less germicidal power than carbolic acid (Behring). Antiseptic in the proportion of 1 : 1,340 (Miquel). Tobacco Smoke. — Tassinari found that tobacco smoke restrains the development of bacteria, and that certain species failed to de- velop after exposure for half an hour in an atmosphere of tobacco smoke — spirillum of cholera and Friedlander's bacillus. XII. ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS. Blood Serum.— Bacteriologists have long been aware of the fact that many species of bacteria, when injected into the circulation of a living animal, soon disappear from the blood, and that the blood of such an animal a few hours after an injection of putrefactive bacte- ria, for example, does not contain living bacteria capable of develop- ing in a suitable nutrient medium. Wyssokowitsch, in an extended series of experiments, has shown that non-pathogenic bacteria in- jected into the circulation may be obtained in cultures from the liver, spleen, kidneys, and bone marrow after they have disappeared from the blood, but that, as a rule, those present in these organs have lost their vitality, as shown by culture experiments, in a period varying from a few hours to two or three days. According to the theory of Metschnikoff, this destruction of bacteria in the blood and tissues of a living animal is effected by -the cellular elements, and especially by the leucocytes, which pick up and digest these vegetable cells very much as an amoeba disposes of similar microorganisms which serve it as food. Some such theory seemed necessary to account for the disappearance of bacteria from the blood before the demonstration was made that the serum of the circulating fluid, quite indepen- dently of its cellular elements, possesses very decided germicidal power. Von Fodor first (1887) called attention to the fact that anthrax ba- Hlli maybe destroyed by freshly drawn blood ; and Nuttall (1888), in an extended series of experiments, showed that various bacteria are destroyed within a short time by the fresh blood of warm- hlnndcd animals. Thus tlu> anthrax bacillus in rabbit's blood was usually killed in from two to four hours when the temperature was maintained at 37°-38° C., and the same result was obtained with pigeon's blood at 41° C. But when the blood was allowed to stand for a considerable time, or was heated for forty-five minutes to 45° C., it served as a culture fluid, and an abundant development of anthrax bacilli occurred in it. Bacillus subtilis and Bacillus mega- ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS. 205 therium were also destroyed in two hours by fresh rabbit's blood, but it was without action on Staphylococcus pyogenes aureus, which at a temperature of 37. 5° C. was found to have increased in num- bers at the end of two hours. Further researches by Nissen and Behring show that there is a wide difference in the blood of dif- ferent animals as to germicidal power, and that certain bacteria are promptly destroyed, while other species are simply restrained for a time in their development or are not affected. Thus Nissen found that the cholera spirillum, the bacillus of anthrax, the bacillus of typhoid fever, and Friedlander's pneumococcus were killed, while Staphylococcus pyogenes aureus and albus, the streptococcus of ery- sipelas, the bacillus of fowl cholera, the bacillus of rothlauf, and Proteus hominis were able to multiply in rabbit's blood after having been restrained for a short time in their development. In the case of the cholera spirillum a period of ten to forty minutes sufficed for the complete destruction of a limited number, but when the number exceeded 1,200,000 per cubic centimetre they were no longer de- stroyed with certainty, and after five hours an increase occurred. The anthrax bacillus was commonly destroyed within twenty minutes and the typhoid bacillus at the end of two hours. In the experi- ments of Behriiig and Mssen it was found that the most pronounced germicidal effect upon the anthrax bacillus was obtained from the blood of the rat, an animal which has a natural immunity against anthrax ; while the blood of the guinea-pig, a very susceptible ani- mal, had no restraining effect and served as a favorable culture medium for the anthrax bacillus. And the remarkable fact was de- monstrated that when the blood of a rat was added to the blood of the guinea-pig in the proportion of 1:8, it exercised a decided re- straining influence upon the growth of the anthrax bacillus. Later researches have shown that cultivation in the blood of an immune animal causes an attenuation of the virulence of an anthrax cul- ture (Ogata and Jasuhara) ; and also that the injection of the blood of a frog or rat — naturally immune — into a susceptible animal which has been inoculated with a virulent culture of the anthrax bacillus, will prevent the death of the inoculated animal. Buchner has shown that the germicidal power of the blood of dogs and rabbits does not depend upon the presence of the cellular elements, but is present in clear serum which has been allowed to separate from the clot in a cool place. Exposure for an hour to a temperature of 55° C. destroys the germicidal action of serum as well as of blood ; the same effect is produced by heating to 52° C. for six hours or to 45. C° C. for twenty hours. The germicidal power of blood serum is not destroyed by freezing and thawing, but is lost after it has been kept for some time. Buchner 's experiments led 206 ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS. him to the conclusion that the germicidal power of fresh blood serum depends upon the presence of some albuminous body present in it. This view is sustained by the researches of Ogata, who has obtained from the blood of dogs and other animals a glycerin ex- tract of a " ferment" which is insoluble in alcohol or in ether and which has germicidal properties. According to Emmerich and Tsuboi (1893), when the serum- albumin is precipitated by alcohol, dried in a vacuum at 40° C., and dissolved in water it has no longer any germicidal activity. But if the precipitated and dried albumin is dissolved at 39° C. in a weak solution (0.05-0.08 per cent) of soda or potash it recovers its original germicidal value. It has been demonstrated by several experimenters that other albuminous fluids possess a similar germicidal power. Thus Nuttall found that a pleuritic exudation from man destroyed the anthrax bacillus in an hour, the aqueous humor of a rabbit in two hours. Wurz has experimented with fresh egg albumin, and found that the anthrax bacillus failed to grow after having been exposed for an hour to the action of albumin from a hen's egg ; other bacteria tested were not killed so promptly, but a decided germicidal action was manifested. Prudden has shown that the albuminous fluid obtained from a hydrocele, or from the abdominal cavity in ascites, possesses similar germicidal power ; and Fokker has demonstrated that fresh in ilk destroys the vitality of certain bacteria which induce an acid fermentation of this fluid. The results heretofore referred to induced Hankin to experiment with cell globulin obtained from the spleen or lymphatic glands of a dog or cat. This is extracted by means of a solution of chloride of sodium, the solution is filtered, and the globulin precipitated by the ;i«l. Chloride of zinc. A ten-per-cent solution. lo. Sulphur dioxide.* Exposure for twelve hours to an atmosphere con- taining at least four volumes per cent of this gas in presence of moisture. The committee would make the following recommendations with refe- iv nee to the practical application of these agents for disinfecting purposes: FOR EXCRETA. (a) In the sick-room : 1. Chloride of lime in solution, four per cent. In the absence of spores: 2. Carbolic acid in solution, five per cent. Sulphate of copper in solution, five per cent. 1 Should contain at least twenty-five per cent of available chlorine. • Should contain at least three per cent of available chlorine. 1 This will require the combustion of between three and four pounds of sulphur l»r every thousand cubic feet of air space. PRACTICAL DIRECTIONS FOR DISINFECTION. (b) In privy vaults : 1. Mercuric chloride in solution, 1: 500. * 2. Carbolic acid in solution, five per cent. (c) For the disinfection and deodorization of the surface of masses of or- ganic material in privy vaults, etc. : Chloride of lime in powder. FOR CLOTHING, BEDDING, ETC. (a) Soiled underclothing-, bed linen, etc. : 1. Destruction by fire, if of little value. 2. Boiling- for at least half an hour. 3. Immersion in a solution of mercuric chloride of the strength of 1 : 2,000 for four hours. 4. Immersion in a two-per-cent solution of carbolic acid for four hours. (6) Outer garments of wool or silk, and similar articles, which would be injured by immersion in boiling water or in a disinfecting solution: 1. Exposure in a suitable apparatus to a current of steam for ten min- utes. 2. Exposure to dry heat at a temperature of 110° C. (230° F.) for two hours. (c) Mattresses and blankets soiled by the discharges of the sick : 1. Destruction by fire. 2. Exposure to superheated steam, 105° C. (221° F.), for ten minutes. (Mattresses to have the cover removed or freely opened.) 3. Immersion in boiling water for half an hour. FURNITURE AND ARTICLES OF WOOD, LEATHER, AND PORCELAIN. Washing, several times repeated, with — 1. Solution of carbolic acid, two per cent. FOR THE PERSON. The hands and general surface of the body of attendants of the sick, and of convalescents, should be washed with — 1. Solution of chlorinated soda diluted with nine parts of water, 1: 10. 2. Carbolic acid, two-per-cent solution. 3. Mercuric chloride, 1: 1,000. FOR THE DEAD. Envelop the body in a sheet thoroughly saturated with — 1. Chloride of lime in solution, four per cent. 2. Mercuric chloride in solution, 1 : 500. 3. Carbolic acid in solution, five per cent. FOR THE SICK-ROOM AND HOSPITAL WARDS. (a) While occupied, wash all surfaces with— 1. Mercuric chloride in solution, 1: 1,000. 2. Carbolic acid in solution, two per cent. (6) When vacated, fumigate with sulphur dioxide for twelve hours, burn- ing at least three pounds of sulphur for every thousand cubic feet of air space in the room ; then wash all surfaces with one of the above-mentioned disinfecting solutions, and afterward with soap and hot water; finally throw open doors and windows, and ventilate freely. 1 The addition of an equal quantity of potassium permanganate as a deodorant, and to give color to the solution, is to be recommended. [The writer no longer in- dorses this recommendation. See his paper on " The Disinfection of Excreta," ap- pended.] 14 210 PRACTICAL DIRECTIONS FOR DISINFECTION. FOR MERCHANDISE AND THE MAILS. The disinfection of merchandise and of the mails will only be required under exceptional circumstances ; free aeration will usually be sufficient. If disinfection seems necessary, fumigation with sulphur dioxide will be the only practicable method of accomplishing it without injury. RAGS. (a) Rags which have been usea for wiping away infectious discharges should at once be burned. (b) Rags collected for the paper-makers during the prevalence of an epi- demic should be disinfected, before they are compressed in bales, by— 1. Exposure to superheated steam of 105° C. (221° F.") for ten minutes. 2. Immersion in boiling water for half an hour. SHIPS. (a) Infected ships at sea should be washed in every accessible place, and especially the localities occupied by the sick, with— 1. Solution of mercuric chloride, 1 : 1,000. 2. Solution of carbolic acid, two per cent. The bilge should be disinfected by the liberal use of a strong solution of mercuric chloride. (b) Upon arrival at a quarantine station, an infected ship should at once be fumigated with sulphurous acid gas, using three pounds of sulphur for every thousand cubic feet of air space; the cargo should then be dis- charged on lighters; a liberal supply of the concentrated solution of mercuric chloride (four ounces to the gallon) should be thrown into the bilge, and at the end of twenty- four hours the bilge watei should be pumped out and re- placed with pure sea water; this should be repeated. A second fumigation, after the removal of the cargo, is recommended; all accessible surfaces should be washed with one of the disinfecting solutions heretofore recommended, and subsequently with soap and hot water. FOR RAILWAY CARS. The directions given for the disinfection of dwellings, hospital wards, and ships apply as well to infected railway cars. The treatment of excreta with a disinfectant, before they are scattered along the tracks, seems desirable at all times in view of the fact that they may contain infectious germs. Dur- ing the prevalence of an epidemic of cholera this is imperative. For this purpose the standard solution of chloride of lime is recommended. DISINFECTION BY STEAM. The Committee on Disinfectants, in tne above-quoted "Conclu- sions," recommends the use of " steam under pressure, 105° C. (221° F.), for ten minutes" for the destruction of spore-containing infec- tious material. The spores of all known pathogenic bacteria are d< '- stroyed by a temperature of 100° C. maintained for five minutes, and in view of this fact the temperature fixed by the committee is ample, and to exact a higher temperature or longer exposure would be un- n-asonahle. hut in practical disinfection the temperature required to destroy infectious material is not the only question to be considered. Economy in the construction and operation of the steam disinfecting apparatus must have due attention, and an important point relates PRACTICAL DIRECTIONS FOR DISINFECTION. 211 to the penetration of porous, non-conducting articles, such as rolls of blankets, clothing, etc. These points have been the subject of nu- merous experimental investigations, and the principles involved have been elucidated, especially by the investigations of Esmarch (1887), of Budde (1889), and of Teuschner (1890). It has been shown that streaming steam is more effective than confined steam at the same temperature, because it penetrates porous objects more quickly. Also that superheated, " dry " steam is not as effective as flowing steam at 100° C. ; on the other hand, it corre- sponds in effectiveness with dry air, and the temperature must be raised to 140° to 150° C. in order to quickly destroy the spores of bacilli. Esmarch's investigations show that streaming steam penetrates porous objects, like rolled blankets, more readily than confined steam ; but the later researches of Budde and of Teuschner show that a temperature of 100° C. is more rapidly reached in the interior of such rolls when the flowing steam is under pressure. With the same pressure (fifteen pounds) a temperature of 100° C. was reached in two and one-half minutes when the steam was flowing, and in eleven minutes by steam at rest (Budde). Intermittent pressure was not found by Budde to present any advantages over continuously flowing steam ; on the contrary, the time of penetration was longer. Teuschner, whose investigations are the most recent, arrives at the following conclusions : 1. Strongly superheated steam is not to be recommended for practical disinfection. On the contrary, a slight superheating of the steam, such as occurs in the apparatus of Schimmel, is not objectionable. 2. Those forms of apparatus in which the steam enters from above are much safer and quicker in their disinfecting action than those in which this is not the case. In the construction of such apparatus care must be taken, in order to secure penetration of the objects, that the air and steam have a free escape below. 3. Disinfection is hastened by previously warming the apparatus. 4. The most rapid disinfecting action is secured by the use of streaming steam in a state of tension (under pressure). 5. Objects which have been in contact with fatty or oily substances require a longer time for disinfection than those which have not. 6. To accomplish disinfection it is necessary to expel, as completely as possible, all air from the objects to be disinfected, and also to secure a suffi- cient condensation of the steam. 7. The condensation of the steam advances in a sharply denned line from the periphery to the centre of porous objects. 8. The temperature necessary for disinfection is only found in the zone where condensation has already taken place. 9. Only a few centimetres from the zone in which the temperature is 100° C.— when disinfection is incomplete— there may be places in which the temperature is 40° C. or more below the boiling point. 212 PRACTICAL DIRECTIONS FOR DISINFECTION. DISINFECTION OF THE HANDS. The importance of a reliable method of disinfecting the hands of surgeons, obstetricians, and nurses after they have been in contact with infectious material from wounds, puerperal discharges, etc., is now fully recognized, and some surgeons consider it necessary to completely sterilize the hands before undertaking any surgical opera- tion which will bring them in contact with the freshly-cut tissues. The numerous experiments which have been made with a view to ascertaining the best method of accomplishing such sterilization of the hands show that it is by no means a simple matter to effect it, and especially to insure the destruction of microorganisms con- cealed beneath the finger nails. Fiirbringer, in an extended series of experiments (1888), found that a preliminary cleansing with soap and a brush was even more important than the degree of potency of che disinfecting wash subsequently applied. He recommends the following procedure : 1. Remove all visible dirt from beneath and around the nails. 2. Brush the spaces beneath the nails with soap and hot water for a minute. 3. Wash for a minute in alcohol (not below eighty per cent), and, before this evaporates, in the following solution : 4. Wash thoroughly for a minute in a solution containing 1 : 500 of mercuric chloride or three per cent of carbolic acid. Roux and Reynes tested the above method of Fiirbringer, and found that it gave better results than others previously proposed, al- though not always entirely successful in securing complete steriliza- tinll. Boll has recently (1890) reported favorable results from the fol- lowing method : 1. Cleanse the finder nails from visible dirt with knife or nail scissors. 2. Brush the hands for three minutes with hot water and potash soap. :; Wash for half a minute in a three-per-cent solution of carbolic acid, and subsequently in a 1 : 2,000 solution of mercuric chloride. 4. Rub the spaces beneath the nails and around their margins with iodo- form gauze wet in a ftve-per-cent solution of carbolic acid. Welch, as a result of extended experiments made at the Johns Hopkins Hospital, recommends the following procedure : 1. The nails are kept short and clean. •-' The hands are washed thoroughly for several minutes with soap and water, the water being as warm as can be comfortably borne, and being fre- quently changed. A brush sterilized by steam is used. The excess of soap is washed off with water. :*. The hands are immersed for one or two minutes in a warm saturated solution of permanganate of notash and are rubbed over thoroughly with a sterilized swab. PRACTICAL DIRECTIONS FOR DISINFECTION. 213 4. They are then placed in a warm saturated solution of oxalic acid, where they remain until complete decolorization of the permanganate occurs. 5. They are then washed off with sterilized salt solution or water. 6. They are immersed for two minutes in sublimate solution, 1 : 500. The bacteriological examination of the skin thus treated yields almost uniformly negative results, the material for the cultures being taken from underneath and around the nails. This is the procedure now employed in the gynecological and surgical wards of the hospital. THE DISINFECTION OF EXCRETA. ' The following paper by the present writer was read before the Section on State Medicine at the last (1891) meeting of the American Medical Association : The Committee on Disinfectants appointed by the American Public Health Association in 1884, in its final report submitted in 1887, gives the following general directions : "Disinfection of Excreta, etc. — The infectious character of the dejections of patients suffering from cholera and from typhoid fever is well established, and this is true of mild cases and of the earliest stages of these diseases as well as of severe and fatal cases. It is probable that epidemic dysentery, tuberculosis, and perhaps diphtheria, yellow fever, scarlet fever, and typhus fever, may also be transmitted by means of the alvine discharges of the sick. It is, therefore, of the first importance that these should be disin- fected. In cholera, diphtheria, yellow fever, and scarlet fever all vomited material should also be looked upon as infectious. And in tuberculosis, diphtheria, scarlet fever, and infectious pneumonia the sputa of the sick should be disinfected or destroyed by fire. It seems advisable also to treat the urine of patients sick with an infectious disease with one of the disinfect- ing solutions below recommended. "Chloride of lime, or bleaching powder, is perhaps entitled to the first place for disinfecting excreta, on account of the rapidity of its action. 44 The following standard solution is recommended: "Dissolve chloride of lime of the best quality, ! in pure water, in the pro- portion of six ounces to one gallon. Use one quart of this solution for the disinfection of each discharge in cholera, typhoid fever, etc.3 Mix well and leave in the vessel for at least one hour before throwing into the privy vault or water closet. " The same directions apply to the disinfection of vomited matters. In- fected sputum should be discharged directly into a cup half-full of the solu- tion. A five-per-cent solution of carbolic acid may be used instead of the chloride of lime solution, the time of exposure to the action of the disinfect- ant being four hours" (op. cit., pp. 237, 238). The object of this paper is to inquire whether these recommendations, which were based upon the experimental data available at the time they were made, are sustained by subsequent investigations ; and whether any other agents have been shown to possess superior advantages for the pur- pose in view. But first we desire to call attention to another portion of the report of the 1 Good chloride of lime should contain at least twenty-five per cent of available chlorine (page 92). It may be purchased by the quantity at three and one-half cents per pound. The cost of the standard solution recommended if therefore but little more than one cent a gallon. A clear solution may be obtained by filtration or by decantation, but the insoluble sediment does no harm and this is an unnecessary re- finement. 8 For a very copious discharge use a larger quantity. 214 PRACTICAL DIRECTIONS FOR DISINFECTION. Committee on Disinfectants. On page 236 the following definition of disin fection and disinfectants is given : " The object of disinfection is to prevent the extension of infectious dis- eases by destroying the specific infectious material which gives rise to them. This is accomplished by the use of disinfectants. There can be no partial disinfection of such material; either its infecting power is destroyed or it is not. In the latter case there is a failure to disinfect. Nor can there be any disinfection in the absence of infectious material." I have italicized the last sentence because I wish to call especial attention to it. I am frequently asked, " What is the best disinfectant to put into a water closet? " Now, if a closet or privy vault is resorted to only by healthy pers ons and no infectious material has been thrown into it, there is nothing 111 it to disinfect, and the recommendation of the Committee on Disinfect- ants does not apply to it at all. It may smell badly, and in this case the bad odor may be neutralized by the use of deodorants ; or we may prevent the putrefactive decomposition of its contents, and thus prevent the forma- tion of the offensive gases given off as a result of such decomposition, by the use of antiseptics. But to accomplish this it is not necessary to sterilize the entire contents by the use of active germicidal agents. A solution of sulphate of iron or of chloride of zinc is a useful antiseptic and deodorizing agent, and the Committee on Disinfectants, in making its recommendations, did not intend to discourage the use of such agents. But exact experimental data showed that these agents could not be depended upon for the destruction of infectious disease germs, and the recommenda- tions made related to disinfection in the strict and proper use of the term as above defined. This definition is now accepted by sanitarians in all parts of the world, but many practising physicians still use the term disinfectant as synonymous with deodorant. For example, I find in a recent sanitary periodical, under the heading "Medical Excerpt,'' an item copied from the American Journal of Obstetrics, to which the name of a distinguished gy- necologist is attached, in which the following statement is made with reference to a much-advertised so-called "disinfectant": "Asa disinfectant I have used it in my house for over a year with great satisfaction." Now, the agent referred to has been proved by exact experiments to have comparatively little disinfecting power, although it is a very good deodorant. According to our definition, " the object of disinfection is to prevent the extension of infectious diseases by destroying the specific infectious material which gives rise to them." Are we to suppose that the distinguished gynecologist above quoted had such infectious material in his house "for over a year " at the time he was employing " with great satisfaction " the agent he recommends? If not, the term was improperly employed, for " there can be no disinfec- tion in the absence of infectious material." I wish to emphasize this point, t>ecause I have reason to believe that, in the army at least, the recommen- dation of the Committee on Disinfectants has led to the substitution of chlo- ride of lime for cheaper deodorants and antiseptic agents — and especially for sulphate of iron— in latrines which are frequented only by healthy persons and consequently need no disinfection. The amount of chloride of lime issued from the Medical Purveying Depot at San Francisco during the past si\ months for use at military posts on the Pacific coast is more than double the amount of sulphate of iron ; but there has been no epidemic of an infectious disease, and probably comparatively little call for the use of a disinfecting agent in the sick-room. We quote again from the report of the Committee on Disinfectants : " In the sick-room we have disease germs at an advantage, for we know where to find them as well as how to kill them. Having this knowledge, not to apply it would be criminal negligence, for our efforts to restrict the extension of infectious diseases must depend largely upon the proper use of disinfectants in the sick-room" (op. cit, p. 237). " The injurious consequences which are likely to result from such mis- apprehension and misuse of the word disinfectant will be appreciated when PRACTICAL DIRECTIONS FOR DISINFECTION. 215 it is known that recent researches have demonstrated that many of the agents which have been found useful as deodorizers or as antiseptics are en- tirely without value for the destruction of disease germs. " This is true, for example, as regards the sulphate of iron, or copperas, a salt which has been extensively used with the idea that it is a valuable dis- infectant. As a matter of fact, sulphate of iron in saturated solution does not destroy the vitality of disease germs, or the infecting power of material containing them. This salt is, nevertheless, a very valuable antiseptic, and its low price makes it one of the most available agents for the arrest of putre- factive decomposition" (op. cit., p. 237). Chloride of lime is also a valuable antiseptic and deodorant, and I know of 110 objection to substituting it for sulphate of iron other than the question of cost. The first cost of chloride of lime, by the quantity, is about double that of sulphate of iron, but practically the difference is much greater, be- cause it is necessary to preserve the chloride of lime in air-tight packages. When exposed to the air it deteriorates in value very rapidly. It is, there- fore, necessary to pack it in air-tight receptacles which will not be injured by the corrosive action of free chlorine, and in comparatively small quanti- ties so that the contents of a package may be used soon after it is opened. We now proceed to consider the experimental data relating to the germi- cidal value of chloride of lime. The Committee on Disinfectants gave it "the first place for disinfecting excreta, on account of the rapidity of its action." This recommendation was upon experimental data obtained in the pathological laboratory of the Johns Hopkins University, under the writer's direction, and is sustained by more recent experiments made in Germany. The experiments of Bolton, made for the Committee on Disinfectants in 1886, j»-ave the following results : The time of exposure being two hours, the typhoid bacillus and cholera spirillum in bouillon cultures were killed by a solution containing one part to one thousand parts of water (containing 0.03 per cent of available chlorine). Anthrax spores were killed in the same time by a solution containing 0.3 per cent of available chlorine. Typhoid faeces were sterilized by a two-per-cent solution, and in several instances by a one- lialf-per-cent solution ; but some resistant spores of non-pathogenic bacilli sur- vived in two experiments in which a solution of 1 : 100 was used. In bouillon cultures to which ten per cent of dried egg albumin had been added the typhoid bacillus was destroyed by one-half per cent (1 : 200). Nissen, whose experiments were made in Koch's laboratory in 1890, found that anthrax spores were destroyed in thirty minutes by a five-per-cent solution, and in seventy minutes by a one-per-cent solution. In his experi- ments the typhoid bacillus and the cholera spirillum were destroyed with certainty in five minutes by a solution containing 0.12 per cent (1: 833) ; the anthrax bacillus in one minute by 1 : 1, 000 ; Staphylococcus pyogenes aureus in one minute by 1 : 500. Experiments made by the same author on the sterili- zation of f;eces showed that one per cent could be relied upon to destroy the bacillus of typhoid fever and the spirillum of cholera in faeces in ten min- utes. Carbolic Acid — The Committee on Disinfectants says: " A five-per-cent solution of carbolic acid may be used instead of the chloride of lime solution, the time of exposure to the action of the disinfectant being four hours." This recommendation is made in view of the fact that in those diseases in which it is most important to disinfect the excreta the specific germ does not form spores. This is now believed to be true of the typhoid bacillus, the spirillum of cholera, the bacillus of diphtheria, the bacillus of glanders, and the streptococcus of erysipelas ; and it has been shown by exact experiments that all of these pathogenic bacteria are destroyed in two hours by a one-per- cent solution, or less, of this agent. Spores require for their destruction a stronger solution and a longer time. Koch found a one-per-cent solution to be without effect on anthrax spores after fifteen days' exposure ; a two-per-cent solution retarded their develop- 216 PRACTICAL DIRECTIONS FOR DISINFECTION. ment, but did not destroy their vitality in seven days; a three-per cent olu- tion was effective in two days. According to Nocht, at a temperature of 37.50° C. anthrax spores are killed by a five-per-cent solution in three hours. Carbolic acid possesses the advantage of not being neutralized by the sub- stances found in excreta, or by the presence of albumin. ThusBolton found that the addition of ten per cent of dried albumin to a bouillon culture of the typhoid bacillus did not materially influence the result, the bacillus be- ing destroyed in two hours by a one-per-cent solution. This agent, then, is firmly established as a valuable disinfectant for ex- creta, but we still give the preference to the standard solution of chloride of lime of the Committee on Disinfectants for use in the sick-room, "on account of the rapidity of its action," and also on account of its compara- tive cheapness. At the International Sanitary Conference at Rome (1885) the writer, who was associated with Dr. Koch on the Committee on Disinfectants, presented the claims of chloride of lime, and in the recommendations of the commit- tee it was placed beside carbolic acid with the following directions: " Carbolic acid and chloride of lime are to be used in aqueous solution. "Weak solutions, carbolic acid, two percent; chloride of lime, one per cent. ' ' Strong solutions, carbolic acid, five per cent ; « hloride of lime, four per cent." The strong solutions were to be used for the disinfection of excreta. Creolin, a coal-tar product, which is a syrupy; dark-brown fluid with the odor of tar, has during the past three years received much attention from the German bacteriologists. It is probably the same product which was tested under the writer's direction for the Committee on Disinfectants, in 1885, under the name of "Little's soluble phenyle." It stood at the head of the ** Commercial Disinfectants " tested. The experiments made in Ger- many show that it is not so active for spores as carbolic acid, but that it very promptly kills known pathogenic bacteria, in the absence of spores, in solutions of two per cent or less. Eisenberg found that a solution of two per cent killed all test organisms within fifteen minutes. Esmarch found it especially fatal to the cholera spirillum, which was killed by solutions of 1 : 1,000 in ten minutes. The typhoid bacillus showed much greater resist- ing power— a one-half-per-cent solution failed after ten minutes' exposure. The pus cocci was still more resistant. Behring has shown that the pre- sence of albumin greatly diminishes its germicidal power. As a deodorant it is superior to carbolic acid, and on this account is to be preferred in the sick-room. A recently prepared emulsion may be used to disinfect the liquid excreta of cholera or typhoid patients, in the proportion of four per cent, two hours' time being allowed for the action of the disinfectant. The ex- periments of Jager upon pure cultures of the tubercle bacillus attached to silk threads were successful in destroying the infecting power of these cul- tures, as tested by inoculation into the anterior chamber of the eye of a rabbit, when solutions of two per cent were used. The value of this agent as a disinfectant is then fully established ; as to its cost in comparison with the agents heretofore mentioned I am not in- formed. Quicklime.— Experiments made in Koch's laboratory in 1887 by Libo- rius led him to place a high value upon recently burned quicklime as a dis- infectant. More recent experiments by Jager, Kitasato, Pfuhl, and others buve shown that, this ;i«n-nt has considerable jrermicidal power in the, ab- sence of spores, and that the value which has long been placed upon it for the treatment of excrementitious material in latrines, etc., and as a wash for exposed surfaces, is justified by the results of exact experiments made upon known pathogenic bacteria. The germicidal power of lime is not interfered with by the presence of albuminous materi.il, but is neutralized by phos- ]>h-ites, carbonates, and other bases, and by carbonic acid. In the writer's experiments a saturated aqueous solution of calcium oxide PRACTICAL DIRECTIONS FOR DISINFECTION. 217 failed to kill typhoid bacilli ; but when suspended in water in the proportion of 1 : 40 by weight this bacillus was killed at the end of two hours. Anthrax spores were not killed in the same time by a lime wash containing twenty per cent by weight of pure calcium oxide. According to Kitasato, the typhoid bacillus and the cholera spirillum in bouillon cultures are destroyed by the addition of one-tenth per cent of calcium oxide. Pfuhl experimented upon sterilized faeces to which pure cultures of the typhoid bacillus or cholera spirillum were added. The liquid discharges of patients with typhoid fever or diarrhoea were used for the purpose. He found that sterilization was effected at the end of two hours by adding fragments of calcium hydrate in the proportion of six per cent, and that three per cent was effective in six hours. When a milk of lime was used which could be thoroughly mixed with the dejecta the result was still more favorable. A standard preparation of milk of lime containing twenty per cent of calcium hydrate killed the typhoid bacillus and the cholera spirillum in one hour when added to liquid faeces in the proportion of two per cent. The experiments with this agent show that time is an important factor, and that much longer exposures, as well as stronger solutions, are required to destroy pathogenic bacteria than is the case with chloride of lime. For this reason we still give the last-named agent the preference for the disinfec- tion of excreta in the sick-room. But in latrines the time required to accom- plish disinfection is of less importance, and we are disposed to give recently burned quicklime the first place for the disinfection of excreta in privy vaults or on the surface of the ground. It may be applied in the form of milk of lime, prepared by adding gradually eight parts, by weight, of water to one part of calcium hydrate. This must be freshly prepared, or protected from the air to prevent the formation of the inactive carbonate of lime. According to Behring, lime has about the same germicidal value as the other caustic alkalies, and destroys the cholera spirillum and the bacillus of typhoid fever, of diphtheria, and of glanders after several hours' exposure, in the proportion of fifty cubic centimetres normal-lauge per litre. Wood ashes or lye of the same alkaline strength may therefore be substituted for quicklime. Finally, it must not be forgotten that we have a ready means of disinfect- ing excreta in the sick-room or its vicinity by the application of heat. Exact experiments, made by the writer and others, show that the thermal death-point of the following pathogenic bacteria, and of the kinds of virus mentioned is below 60° C. (140° F.): Spirillum of cholera, bacillus of an- thrax, bacillus of typhoid fever, bacillus of diphtheria, bacillus of glanders, diplococcus of pneumonia (Micrococcus Pasteuri), streptococcus of erysipelas, staphylococci of pus, micrococcus of gonorrhoea, vaccine virus, sheep pox virus, hydrophobia virus. Ten minutes' exposure to the temperature men- tioned may be relied upon for the disinfection of material containing any of these pathogenic organisms, except the anthrax bacillus when in the stage of spore formation. The use, therefore, of boiling water in the proportion of three or four parts to one part of the material to be disinfected may be safely recommended for such material. Or, better still, a ten-per-cent solu- tion of sulphate of iron or of chloride of zinc at the boiling point may be used in the same way (three parts to one). This will have a higher boiling point than water, and will serve at the same time as a deodorant. During an epidemic of cholera or typhoid fever such a solution might be kept boil- ing in a proper receptacle in the vicinity of hospital wards containing patients, and would serve to conveniently, promptly, and cheaply disinfect all excreta. For the disinfection of fseces in privy vaults, etc., Vincent (1895) gives the first place to sulphate of copper, which should be used in the proportion of eight to ten grammes per litre of contents, together with an equal part, by weight, of sulphuric acid. PKACTICAL IHKKCTIONS F()K DISINFECTION. DISINFECTION IN DIPHTHERIA. At the meeting of the Tenth International Medical Congress in Berlin (1890) Loffler made an important communication upon the measures to be taken to prevent the spread of diphtheria. His con- el usions are summarized as follows : 1. The cause of diphtheria is the diphtheria bacillus, which is found in the secretions of the affected mucous membrane. , 2. With this secretion it is distributed outside of the body and may be deposited upon anything in the vicinity of the sick. 3. Those sick with diphtheria carry about bacilli capable of infecting others so long as there is the slightest trace of diphtheritic deposit, and even for several days after such deposit has disappeared. 4. Those sick with diphtheria are to be rigidly isolated so long as the diphtheria bacilli are present in their secretions. Children who have been sick with diphtheria should be kept from school for at least four weeks. 5. The diphtheria bacilli may preserve their vitality in dried fragments of diphtheritic membrane for four or five months. Therefore all objects which may have been exposed to contact with the excretions of those sick with diphtheria, such as linen, bedclothing, utensils, clothing of nurses, etc., should be disinfected by boiling in water or treated with steam at 100° C. In the same way the rooms occupied by diphtheria patients are to be care- fully disinfected. The floors should be repeatedly scrubbed with hot sub- limate solution (1:1,000) and the walls rubbed down with bread. The recommendation made by Loffler with reference to rubbing d«»\vn the walls of an infected apartment with bread is based upon the experiments of Esmarch (1887), as a result of which he arrived at the conclusion that this is the most reliable method of removing 1 i.-tcteria attached to the walls of an apartment. Fresh bread is used, and, after having been used, is destroyed by burning. We judge that this method would be especially applicable to painted surfaces or to walls covered with paper. For plastered walls the liberal ap- plication of lime wash is probably the safest method of disinfection. I ;• •« cntly the use of the vapors of formaldehyde has been proposed for the disinfection of the sick-room, hospital wards, etc. Miquel (1894) does not think favorably of this agent for the purpose indicated, although it is a very active germicide and may bo used with advantage to disinfect certain articles wh i«-li run be exposed to the vapors in a closed receptacle, and which would he injured l.y exposure to steam. Lehmann (1893) has shown that articles of leather, wool, or silks and furs may be disinfected in this way without in- jury, hut the vapor will not penetrate' to the interior of bundles.' Theauthor last named considers it especially well adapted for the disinfection of hair- hnishi-s and combs. The disinfection of the sick-room and its contents by means of ammonia has been proposed by von Rigler (1893). His experiments led him to the conclusion that one kilo of liquid ammonia, poured into shal- low dishes, would sutlice for the disinfection of one hundred cubic metres of space, including hangings, furniture, etc. The carefully conducted experi- ments of de Ims, although the species concerned in its production has not been determined, owing to the fact that modern bacteriologists have had few, if any, opportunities for studying it. The history of the disease, i t s rapid extension in infected surgical wards, the extensive slough- ing which occurs within a few hours in previously healthy wounds, and the effect of deep cauterization by the hot iron, nitric acid, or hromino in arresting the progress of the disease, all support this view of its etiology. Whether it is due to a specific pathogenic micro- MODES OF ACTION. 225 organism, or to exceptional pathogenic power acquired by some one of the common bacteria which infest suppurating wounds, cannot be determined in the absence of exact experiments by modern methods. But the latter view has seemed to the writer the most probably cor- rect. There are many facts which go to show that pathogenic viru- lence may be increased by cultivation in animal fluids, and where wounded men are brought together under unfavorable sanitary con- ditions, as has been the case where hospital gangrene has made its appearance, it may be that some common saprophyte acquires the power of invading the exposed tissues instead of simply feeding upon the secretions which bathe its surface. Koch has described a progressive tissue necrosis in mice, due to a streptococcus, which he first obtained by inoculating a mouse in the ear with putrid material. The morbid process is entirely local and rapidly progressive, causing a fatal termination in about three days, without invasion of the blood. In diphtheritic inflammations of mucous membranes we have a local invasion of the tissues and a characteristic plastic exudation. In true diphtheria the local inflammation and necrotic changes in the invaded tissues are not sufficient to account for the serious gen- eral symptoms, and we now have experimental evidence that the diphtheria bacillus produces a very potent toxic substance to which these symptoms are no doubt largely due. The diphtheria bacillus of Loftier appears to be the cause of the fatal malady which goes by this name, but undoubtedly other microorganisms may be con- cerned in the formation of diphtheritic false membranes. In cer- tain forms of diphtheria, and especially when it occurs as a com- plication of scarlet fever, measles, and other diseases, the Klebs- Loffler bacillus is absent, and a streptococcus, which appears to be identical with Streptococcus pyogenes, is found in considerable num- bers and is probably the cause of the diphtheritic inflammation. An epidemic of diphtheria occurring among calves was studied by Loffler, and is ascribed by him to his Bacillus diphtheria vitulo- rum. The same bacteriologist has shown that the diphtheria of chickens and of pigeons is due to a specific bacillus which differs from that found in human diphtheria, and which he calls Bacillus diphtherias columbrarum. Recently Prof. Welch has studied the histological lesions pro- duced by filtered cultures of the diphtheria bacillus. Cultures in glycerin-bouillon, several weeks old, were filtered through porce- lain, and the sterile filtrate was injected beneath the skin of guinea- pigs. One cubic centimetre of this filtrate was injected into a gui- nea-pig on the 10th of December, and two cubic centimetres more on the 14th of the same month. The animal succumbed at the end of 15 226 MODES OF ACTION. three weeks and five days after the first inoculation. At the autopsy "the lymphatic glands of the inguinal and axillary regions were found to be enlarged and reddened; the cervical glands were swollen and the thyroid gland was greatly congested. There was a consider- able excess of clear fluid in the peritoneal cavity. Both layers of the peritoneum were reddened, the vessels of the visceral layer being es- pecially injected. The spleen was enlarged to double the average size; it was mottled, and the white follicles were distinctly outlined against the red ground. The liver was dark in color and contained much blood. . . . The kidneys were congested and the cut surface was cloudy. . . . The pericardial sac was distended with clear se- rum. Under the epicardium were many ecchymotic spots. The lungs exhibited areas of intense congestion or actual haemorrhage into the tissues. . . . The histological lesions in this case are identi- cal with those observed by us in connection with the inoculation of the living organisms." To what extent non-specific catarrhal inflammations of mucous membranes are caused by the local action of microorganisms has not been determined, but in gonorrhoea the proof is now considered satisfactory that the " gonococcus " of Neisser is the cause of the intense local inflammation and purulent discharge. In this disease the action of the pathogenic microorganism seems to be limited to the tissues invaded by it, as there is no general systemic disturbance indicating the absorption of a toxic ptomaine. Chronic catarrhal inflammations appear, in some cases at least, to be kept up by the presence of microorganisms, which are always found in the discharges from inflamed mucous surfaces. The influence of microorganisms, and especially of the pus cocci, in preventing the prompt healing of wounds, is now well established. An extensive suppurating wound or collection of pus, especially if putrefactive bacteria are present, causes fever and nervous symp toms, due to the absorption of toxic products. More intense general symptoms result from the presence of the streptococcus of pus than from the less pathogenic staphylococci ; this is seen in erysipelatous inflammations and in puerperal metritis due to the presence of this micrococcus. Like the other pus cocci, the Streptococcus pyogenes does not usually invade the blood, but when introduced into the sub- cutaneous tissues it induces a local inflammator}^ process, with a ten- dency to pus formation, and it invades the neighboring lymph chan- nels, in which the conditions appear to be especially favorable for its multiplication. Kinally, certain pathogenic bacteria, when introduced into the bodies of susceptible animals, quickly invade the blood and multiply in it. In so doing they necessarily interfere with its physiological MODES OF ACTION. 227 functions by appropriating for their own use material required for the nutrition of the tissues ; and at the same time toxic substances are formed which play an important part in the production of the morbid phenomena, which in this class of diseases very commonly lead to a fatal result. The pathogenic bacteria which invade the blood may also, in certain cases, give rise to local necrosis and dis- turbance of function in various organs in a mechanical way by blocking up the capillaries. The invasion of the blood which occurs in anthrax and in vari- ous forms of septicaemia in the lower animals, induced by subcuta- neous inoculation with pure cultures of certain pathogenic bacteria, does not generally immediately follow the inoculation. Usually a considerable local development first occurs, which gives rise to more or less inflammation of the invaded tissues, and very commonly to an effusion of bloody serum in which the pathogenic microorganism is found in great numbers. Even in susceptible animals the blood seems to offer a certain resistance to invasion, which is overcome after a time by the vast number of the parasitic host located in the vicinity of the point of inoculation, aided probably by the toxic sub- stances developed as a result of their vital activity. The experiments of Cheyne (1886) seem to show that in the case of very pathogenic species, like the anthrax bacillus or Koch's bacil- lus of mouse septicaemia, a single bacillus introduced subcutaneously may produce a fatal result in the most susceptible animals, while greater numbers are required in those which are less susceptible. Thus a guinea-pig succumbed to general infection after being inocu- lated subcutaneously with anthrax blood diluted to such an extent that, by estimation, only one bacillus was present in the fluid in- jected ; and a similar result in mice was obtained with Bacillus murisepticus. In the case of the microbe of fowl cholera (Bacillus septicaemias haemorrhagicae) Cheyne found that for rabbits the fatal dose is 300,000 or more, that from 10,000 to 300,000 cause a local abscess, and that less than 10,000 produce no appreciable effect. The common saprophyte Proteus vulgaris was found to be patho- genic for rabbits when injected into the dorsal muscles in sufficient numbers. But, according to the estimates made, 225,000,000 were required to cause death, while with doses of from 9,000,000 to 112,- 000,000 a local abscess was produced, and less than 9,000,000 gave an entirely negative result. Secondary infections occurring in the course of specific infec- tious diseases are of common occurrence. Thus a pneumonia may be developed in the course of an attack of measles or of typhoid fever ; or infection by the common pus cocci in the course of scarlet fever, typhoid fever, mumps, etc., may give rise to local abscesses, 228 MODES OF ACTION. to endocarditis, etc. Again, mixed infection may be induced by injecting simultaneously into susceptible animals two species of path- ogenic bacteria. Bumm, Bockhart, and others have reported cases of mixed gonor- rhoeal infection in which the.pyogenic micrococci gave rise to ab- scesses in the glands of Bartholin, to cystitis, parametritis, or to " gonorrhceal inflammation " of the knee joint. Babes gives numer- ous examples of mixed infection in scarlet fever and in other diseases of childhood. Anton and Fiitterer have studied the question of secondary infection in typhoid fever. Karlinski has reported a case of secondary infection with anthrax in a case of typhoid fever, infec- tion occurring by way of the intestine. Many other examples of secondary or mixed infection are recorded in the recent literature of bacteriology and clinical medicine, but enough has been said to call attention to the importance of the subject. The researches of Romer, Kanthack (1892), and others show that the injection of the filtered products of certain bacteria (Bacillus pyocyaneus, Vibrio Metchnikovi, etc.) produces a decided leucocy- tosis in the animals experimented upon. And a similar result, prob- ably from a like cause, has been shown by recent experiments to occur in pneumonia (Billings) and other infectious diseases. Certain bacterial products have been shown by experiment to pro- duce fever when injected into the circulation or beneath the skin of lower animals ; others produce rapid respiration, dilatation of pupils, diarrhoea, and paralysis or convulsions (typhotoxin of Brieger, methyl-guanidin, etc.) ; the toxic effects of some are immediate and of others more or less remote (toxalbumin of diphtheria) ; others have a primary toxic effect which is followed after a time by toxic symp- toms of a different order (Pneumobacillus liquefaciens bovis). II. CHANNELS OF INFECTION. WE have abundant evidence that susceptible animals may be in- fected by the injection of various pathogenic bacteria beneath the skin, and accidental infection through an open tvound or abrasion of the skin is the common mode of infection in tetanus, erysipelas, hospital gangrene, and the " traumatic infectious diseases" generally. Other infectious diseases, like anthrax and glanders, are frequently transmitted in the same way. • We have also satisfactory evidence that tuberculosis may be transmitted to man by the accidental inocu- lation of an open wound ; and in view of the fact that susceptible animals are readily infected in this way, it would be strange if it were otherwise. The question whether infection may occur through the unbroken skin has been studied by several bacteriologists and an affirmative result obtained. Thus Schimmelbusch produced pustules upon the thigh in two young persons suffering from pyaemia by rubbing upon the surface a pure culture of Staphylococcus pyogenes aureus which he had obtained from the pus of a furuncle. The same author also succeeded in infecting rabbits and guinea-pigs with anthrax, and rabbits with rabbit septicaemia, by rubbing pure cultures upon the uninjured skin. Similar results had previously been reported by Roth, who also showed that infection might occur through the uninjured mucous membrane of the nose. Machnoff also suc- ceeded in infecting guinea-pigs with anthrax through the unin- jured skin of the back, and, as a result of subsequent microscop- ical examination of stained sections, arrived at the conclusion that, the principal channel through which infection was accomplished was the hair follicles. Braunschweig, in a series of experiments in which he introduced various pathogenic bacteria into the conjunctival sac of mice, rabbits, and guinea-pigs, obtained a negative result with the anthrax bacillus, the bacillus of mouse septicaemia, the bacillus of chicken cholera, and Micrococcus tetragenus; but the bacillus ob- tained by Ribbert from the intestinal diphtheria of rabbits gave a positive result in five mice, two guinea-pigs, and a rabbit. CHANNELS OP INFECTION. Infection through the mucous membrane of the intestine no doubt occurs in certain diseases. This is believed to be a common mode of the infection of sheep and cattle with anthrax, and probably also in the infectious disease of swine known as hog cholera. The anthrax bacillus would be destroyed by the acid secretions of the stomach, but if spores are present in food ingested they will reach the intestine. The experiments of Korkunoff do not, however, sup- port the view that infection is likely to occur in this way. In a series of experiments upon white mice fed with bread containing a quantity of anthrax spores the result was uniformly negative, but exception- ally infection occurred in rabbits. The same author obtained posi- tive results in rabbits fed with food to which a pure culture of the bacillus of chicken cholera had been added. Buchner, in experiments upon mice and guinea-pigs fed with material containing anthrax spores, obtained a positive result in four out of thirty-three animals. This is no doubt the usual mode of in- fection in typhoid fever in man. Infection may also occur through the mucous membrane of the respiratory organs. This has been demonstrated by several bac- teriologists, and especially by the experiments of Buchner, who mixed dried anthrax spores with ly cop odium powder or pulverized charcoal, and caused mice and guinea-pigs to respire an atmosphere containing this powder in suspension. In a series of sixty- six experi- ments fifty animals died of anthrax, nine of pneumonia, and seven survived. That infection did not occur through the mucous mem- brane of the alimentary canal was proved by comparative experi- ments in which animals were fed with double the quantity of spores used in the inhalation experiments. Out of thirty-three animals fed in this way but four contracted anthrax. That infection occurred through the lungs was also demonstrated by the microscopical ex- amination of sections and by culture experiments, which showed that the lungs were extensively invaded, while in many cases the spleen contained no bacilli. Positive results were also obtained with cul- tures of the anthrax bacillus not containing spores, which the ani- mals were made to inhale in the form of spray. But in this case a considerable quantity was required, and a sero-fibrinous pneumonia was usually produced as well as general infection; the inhalation of > mall quantities gave no result. Positive results in rabbits were also • >l>taim>d by causing them to inhale considerable quantities of a spray containing the bacillus of chicken cholera. The fact that large quantities of a liquid culture of these virulent bacilli were required to infect very susceptible animals by way of the pulmonary mucous membrane, and that Buchner failed to cause tht» infection of tlu»se animals with small quantities of a pure culture CHANNELS OF INFECTION. 231 inhaled in the form of spray, indicates that this is not a common mode of infection in the absence of spores. This view receives further support from the experiments of Hildebrandt, who made tracheal fistulse in three rabbits, and, after the wound had entirely healed, injected into the trachea of each a pure culture of the anthrax bacillus, which was proved to be virulent by inoculation in mice or guinea-pigs. All of the animals remained in good health. On the other hand, three rabbits which received in the same way a pure cul- ture of the bacillus of rabbit septicaemia died as a result of general infection. That man may be infected with anthrax by way of the respira- tory organs seems to be well established. In England the disease known as " wool-sorter's disease" results from infection in this way among workmen engaged in sorting wool, which is liable to contain the spores of the anthrax bacillus when obtained from the skin of an animal which has fallen a victim to this disease. That infection occurs through the lungs is shown by the fact that these organs are first involved, the disease being, in fact, a pulmonic anthrax. While these experiments prove the possibility of infection through the respiratory mucous membrane, other experiments made by Hil- debrandt show that under ordinary circumstances bacteria suspended in the air do not reach the trachea in rabbits, but are deposited upon the mucous membrane of the mouth, nares, and fauces. In healthy rabbits the tracheal mucus was, as a rule, found to be free from bac- teria, while they were very numerous in mucus obtained from the mouth or nares. But when a rabbit was made to inhale for half an hour an atmosphere charged with the spores of Aspergillus f umigatus their presence in the lungs was demonstrated by cultivation, the ani- mal being killed for the purpose half an hour after the inhalation experiment. The rapidity with which infection may occur is shown by the experiments of Nissen, Pfuhl, and others. In mice inoculated with anthrax bacilli at the tip of the tail fatal anthrax has resulted, although the tail was amputated ten minutes after the inoculation. Schimmelbusch inoculated fresh wounds with anthrax cultures (in mice) and immediately after treated the wounds with strong anti- septic solutions, but the animals succumbed to infection. Cultures of the anthrax bacillus have been obtained from the liver, spleen, and kidneys half an hour after the infection of an open wound on the surface of the body (Schimmelbusch and Ricker). The experiments of Sherrington and others show that pathogenic bacteria may escape by way of the kidneys into the bladder, or through the liver into the gall bladder. But his experiments indicate that such escape does not occur through healthy organs. Non-pathogenic bacteria injected 232 CHANNELS OF INFECTION. into the circulation were not found in the urine, and when a consid- erable quantity of a pathogenic species was injected into a vein there was no immediate appearance of bacteria in the urine, but they were found later, probably as a result of lesions in the secreting organ due to their local action or to that of their toxic products. In man the presence of pathogenic bacteria in the urine has been frequently veri- fied, especially in typhoid fever, pneumonia, and streptococcus in- fection. When, as a result of the establishment of foci of infection in the liver, localized necrosis of tissue occurs, the pathogenic bac- teria to which the infection is due escape with the bile and enter the intestine. It is probable that escape through the walls of the intestine does not occur unless there is a local lesion of some kind, as in typhoid fever. The presence of tubercle bacilli in the milk of cows has been repeatedly demonstrated, and in a certain proportion of the cases they have been found in the milk of cows whose udders gave no evidence of being the seat of a tubercular process. Usually, how- ever, when tubercle bacilli are found in the milk the cow's udder is already involved in the disease. The milk of women with puer- peral fever has been found to contain streptococci ; and in mastitis from a localized infection by pyogenic cocci these are found in the milk. It must be remembered, however, that both Staphylococcus albus and aureus have been found in the milk of healthy women. The micrococcus of pneumonia has been found in the milk of women suffering from croupous pneumonia (Foa, and Bordoni-Uffreduzzi) . Various observers (Brunner, Tizzoni, von Eiselsberg) have reported the presence of pus cocci in the sweat of patients suffering from sep- ticaemia, and the experiments of Brunner indicate that they may have escaped through the sweat glands. This, however, does not appear to be definitely established. III. SUSCEPTIBILITY AND IMMUNITY. No questions in general biology are more interesting, or more important from a practical point of view, than those which relate to the susceptibility of certain animals to the pathogenic action of cer- tain species of bacteria, and the immunity, natural or acquired, from such pathogenic action which is possessed by other animals. It has long been known that certain infectious diseases, now demonstrated to be of bacterial origin, prevail only or principally among animals of a single species. Thus typhoid fever, cholera, and relapsing fever are diseases of man, and the lower animals do not suffer from them when they are prevailing as an epidemic. On the other hand, man has a natural immunity from many of the infectious diseases of the lower animals, and diseases of this class which prevail among animals are frequently limited to a single species. Again, several species, including man, may be susceptible to a disease, while other animals have a natural immunity from it. Thus tuberculosis is common to man, to cattle, to afpes, and to the small herbivorous ani- mals, while the carnivora are, as a rule, immune ; anthrax may be communicated by inoculation to man, to cattle, to sheep, to guinea- pigs, rabbits, and mice, but the rat, the dog, carnivorous animals, and birds are generally immune ; glanders, which is essentially a disease of the equine genus, may be communicated to man, to the guinea- pig, and to field mice, while house mice, rabbits, cattle, and swine are to a great extent immune. In addition to this general race immunity or susceptibility we have individual differences in susceptibility or resistance to the ac- tion of pathogenic bacteria, which may be either natural or acquired. As a rule, young animals are more susceptible than older ones. Thus in man the young are especially susceptible to scarlet fever, whooping cough, and other "children's diseases," and after forty years of age the susceptibility to tubercular infection is very much diminished. Among the lower animals it is a matter of common laboratory experience that the very young of a susceptible species may be infected when inoculated with an "attenuated culture" which older animals of the same species are able to resist. 234 SUSCEPTIBILITY AMD IMMUNITY. Considerable differences as to susceptibility may also exist among adults of the same species. In man these differences in individual susceptibility to infectious diseases are frequently manifested. Of a number of persons exposed to infection in the same way, some may . -scape entirely while others have attacks differing in severity and duration. In our experiments upon the lower animals we constantly meet with similar results, some individuals proving to be exception- ally resistant. Exceptional susceptibility or immunity may be to some extent a family characteristic or one of race. Thus the negro race is decidedly less subject to yellow fever than the white race, and this disease is more fatal among the fair-skinned races of the north of Europe than among the Latin races living in tropical or sub- tropical regions. On the other hand, small-pox appears to be excep- tionally fatal among negroes and dark-skinned races generally. A very remarkable instance of race immunity is that of Algerian sheep against anthrax, a disease which is very fatal to other sheep. In the instances mentioned race immunity is probably an ac- quired tolerance due to natural selection and inheritance. If, for example, a susceptible population is exposed to the ravages of small- pox, the least susceptible individuals will survive and may be the pa- rents of children who will be likely to inherit the special bodily char- acters upon which this comparative immunity depends. The ten- dency of continuous or repeated exposure to the same pathogenic agent will evidently be to establish a race tolerance ; and there is n '.ison to believe that such has been the effect in the case of some of the more common infectious diseases of man, which have been noticed to prevail with especial severity when first introduced among a virgin population, as in the islands of the Pacific, etc. In the same way we may explain the immunity which carnivor- ous animals have for anthrax and various forms of septicaemia to which the herbivora are very susceptible when the pathogenic germ is introduced into their bodies by inoculation. From time immemo- rial the carnivora have been in the habit of fighting over the dead bodies of herbivorous animals, some of which may have fallen a prey to these infectious germ diseases, and in their fighting they receive wounds, inoculated with the infectious material from these bodies, which would be fatal to a susceptible animal. If at any time in the j.ast a similar susceptibility existed among the carnivora, with indi- vidual differences as to resisting power, it is evident that there would be a constant tendency for the most susceptible individuals to perish and for the least susceptible to survive. But if we admit this to be a probable explanation of the immu- nity of carnivorous animals from septic infection, we have not yet t-\|.l aiiicd tin- praise reason for the immunity enjoyed by the SUSCEPTIBILITY AND IMMUNITY. 235 selected individuals and their progeny. The essential difference be- tween a susceptible and immune animal depends upon the fact that in one the pathogenic germ, when introduced by accident or ex- perimental inoculation, multiplies and invades the tissues or the blood, where, by reason of its nutritive requirements and toxic pro- ducts, it produces changes in the tissues and fluids of the body incon- sistent with the vital requirements of the infected animal ; while in the immune animal multiplication does not occur or is restricted to a local invasion of limited extent, and in. which after a time the re- sources of nature suffice to destroy the parasitic invader. Now the question is, upon what does this essential difference de- pend ? Evidently upon conditions favorable or unfavorable to the development of the pathogenic germ ; or upon its destruction by some active agent present in the tissues or fluids of the body of the immune animal; or upon a neutralization of its toxic products by some substance present in the body of the animal which survives infec- tion. What, then, are the unfavorable conditions which may be supposed to prevent development in immune animals ? In the first place, the temperature of the body may not be favorable. Certain pathogenic bacteria are only able to develop within very narrow temperature lim- its, and, if all other conditions were favorable, could not be expected to multiply in the bodies of cold-blooded animals. Or the temperature of warm-blooded animals, and especially of fowls, may be above the point favorable for their development. This is the explanation offered by Pasteur of the immunity of fowls, which are usually re- fractory against anthrax ; and in support of this view he showed by experiment that when chickens are refrigerated after inoculation, by being partly immersed in cold water, they are liable to become in- fected and to perish. But, as pointed out by Koch, the sparrow, which has a temperature as high as that of the chicken, may con- tract anthrax without being refrigerated. We must not, therefore, too hastily conclude that the success in Pasteur's experiment de- pended alone upon a reduction of the body heat. Gibier has shown that the anthrax bacillus may multiply in the bodies of frogs or fish, if these are kept in water having a temperature of 35° C. But the anthrax bacillus grows within comparatively wide tempera- ture limits, while other pathogenic bacteria are known to have a more restricted temperature range and would be more decidedly influenced by this factor — e.g., the tubercle bacillus. The composition of the body fluids, and especially their reaction, 13 probably a determining factor in some instances. Thus Behring has ascribed the failure of the anthrax bacillus to develop in the white rat, which possesses a remarkable immunity against anthrax, 236 srsCKI'TIHILITY AND IMMUNITY. to the highly alkaline reaction of the blood and tissue juices of this animal. Behring claims to have obtained experimental proof of the truth of this explanation by feeding white rats on an exclusively vegetable diet or by adding acid phosphate of lime to their food, by which means this excessive alkalinity of the blood is diminished. Rats so treated are said to lose their natural immunity, and to die as a result of inoculation with virulent cultures of the anthrax bacillus. The experiments of Nuttall, Behring, Buchner, and others have established the fact that recently drawn blood of various animals possesses decided germicidal power, and Buchner has shown that this property belongs to the fluid part of the blood and not to its cellular elements. It has also been shown that aqueous humor, the fluid of ascites, and lymph from the dorsal lymph sac of a frog possess the same power. This power to kill bacteria is destroyed by heat, and is lost when the blood has been kept for a considerable time, but it is not neutralized by freezing. Further, this power to destroy bacteria differs greatly for different species, being very de- cided in the case of certain pathogenic bacteria, less so for others, and absent in the case of certain common saprophytes. Behring has also shown that the blood of different animals differs consider- ably in this regard, and that the blood of the rat and of the frog, which animals have a natural immunity against anthrax, is espe- cially fatal to the anthrax bacillus. The experiments made show that this germicidal power is very prompt in its action, but that it is limited as to the number of bacteria which can be destroyed by a given quantity of blood serum. When the number is excessive, de- velopment occurs after an interval during which a limited destruc- tion has taken place. It would appear that the element in the blood to which this germicidal action is due is neutralized in exercising this power ; and as, independently of this, blood serum is an excel- lent culture medium for bacteria, an abundant development takes place when the destruction has been incomplete. Buchner (1880) first proved by experiment that the germicidal power of the blood of dogs and rabbits does not depend upon the presence of the cellular elements, but is present in clear serum which has been allowed to separate from the clot in a cool place. Exposure for an hour to a temperature of 55° C. destroys the germicidal action of serum as well as of blood. The researches of Buchner, of Hankin, and others, show that this germicidal power of fresh blood serum depends upon the presence of proteids, to which the first-named bacteriologist has given the name of " alexins." Hankin, in his paper upon the origin of these "defen- sive proteids" in the animal body (1892), arrives at the conclusion SUSCEPTIBILITY AND IMMUNITY. 237 that while they are present in the cell-free serum they are the prod- uct of certain leucocytes — Ehrlich's eosinophile cells. He believes that the eosinophile granules become dissolved in the serum and con- stitute the germicidal proteid which is shown to be present by ex- periments upon bacteria. According to Hankin the separation of these granules can be witnessed under the microscope. They first accumulate upon one side of the cell and then gradually disappear, and as this occurs a considerable increase in the bactericidal power of the serum can be demonstrated. The germicidal power of the blood serum is also said to be increased when the number of leuco- cytes is considerably augmented, as occurs when a sterilized culture of Vibrio Metschnikovi is injected subcutaneously. Also by treat- ment which favors a separation of the alexin from the leucocytes, i.e., a solution of the eosinophile granules. This may be accom- plished by the injection of an extract of the thymus gland of the calf , or by simpty allowing the drawn blood to stand for several hours at a temperature of 38° to 40° C. Buchner's latest communication upon the subject shows that he also attributes the origin of the germicidal proteid in fresh blood serum to the leucocytes. In his paper on "Immunity," read at the Eighth International Congress on Hygiene and Demography (Buda- pest, 1894), he calls attention in the first place to the fact that a clearly marked distinction must be made between natural immunity and acquired immunity, inasmuch as the " alexins " and " antitoxins " have very different properties. The first-mentioned proteids are de- stroyed by a comparatively low temperature (55° to CO0 C.), while the antitoxins resist a considerably higher temperature, and, unlike the alexins, have no bactericidal or globulicidal action. A very remark- able fact developed in Buchner's experiments is that the blood serum from the dog and from the rabbit, when mixed, neutralize each other so far as their germicidal power is concerned. By injecting sterilized emulsions of wheat-flour paste in the pleural cavity of rabbits and dogs Buchiier succeeded in obtaining an exudate which had more decided germicidal power than the blood or serum of the same animal. This was evidently due to the large number of leucocytes present, but not to their phagocytic action, as was shown by experiment. By freezing the exudate the leucocytes were killed, but the germicidal action of the fluid was rather in- creased than diminished by freezing. While freezing had no effect upon the germicidal action of the pleural exudate, this was always neutralized by exposure to a temperature of 55° C. Emmerich, Tsuboi, Steinmetz, and Low (1892), as a result of ex- tended experiments, arrived at the conclusion that the germicidal action of blood serum " depends upon a specific property of the alkali 238 slSCEPTIBILITY AND IMMUNITY. serumalbuiiiin, and that it is a purely chemical process." They state that when the germicidal power is neutralized by heat it may be restored by the addition of an alkali. Buclmer repeated the ex- periments of Emmerich and his associates and obtained similar re- sults, but interprets them differently. According to him the serum does not regain its germicidal power, but after the addition of an alkali and subsequent dialyzing the nutritive value of the serum is so diminished that the bacteria do not develop in it. Pane (1892) has made experiments which give additional weight to the assumption that the alkalinity of the blood is an important factor in accounting for immunity. He states that carbonate of soda, dissolved in water, in the proportion of 1:3,000, has a de- cided germicidal action upon the anthrax bacillus, equal to that of the blood serum of the rabbit. And that when rabbit serum is com- pletely neutralized it no longer has any injurious action on anthrax bacilli. Zagari and Innocente (1892) also arrived at the conclusion that the diminished resistance to anthrax infection resulting from curare poisoning in frogs, and from chloral or alcohol in dogs (Platania) , in fowls as a result of starvation (Canalis and Morpurgo), in white mice as a result of fatigue (Charm and Roger), is, in fact, due to diminished alkalinity of the blood, which they found to correspond with the increased susceptibilit}' resulting from the causes men- tioned . Buchner (1892) states that several of the ammonium salts, and especially ammonium sulphate, cause an increase in the germicidal action of blood serum, and also increase its resistance to the neutral- izing effects of heat. The experiments of Pansini and Calabrese (1M)4) show, on the contrary, that the addition of uric acid to blood serum diminishes its bactericidal activity, as does also the presence of glucose. That certain infectious diseases are especially virulent in |>ersons suffering from diabetes is a frequently repeated clinical observation. Van Fodor has shown by experiment that the injection of an alkali into the circulation of a rabbit increases its resistance to anthrax infection and the germicidal activity of its blood serum. The same bacteriologist has found that when a rabbit is infected with anthrax, the alkalinity of its blood is notably increased during the first twenty-four hours, when we may suppose that the powers of nature .11-0 brought to bear to resist the invading parasite, and that after this time it rapidly diminishes. Ten hours after infection (by subcutaneous inoculation?) the alkalinity of the blood had increased 51.fi per rent. Shortly before the death of the animal a diminution <>f M.8 j>er cent was noted. This diminution was observed in thirty- SUSCEPTIBILITY AND IMMUNITY. 239 four out of thirty-nine animals experimented upon, and these ani- mals succumbed to the anthrax infection in a shorter time than did the other five in which there was no such diminution. It seems probable that the germicidal property of freshly drawn blood serum is not due to its alkalinity, per se, but to the fact that the germicidal constituent is only soluble in an alkaline fluid. The researches of Vaughn, McClintock, and Novy indicate that this ger- micidal constituent is a nucleiu. Dr. Vaughn, in his last published paper upon "Nucleins and Nuclein Therapy," says: "Kossel, of Berlin, has confirmed our statements concerning the germicidal action of the nucleins. Dr. McClintock and I have also demon- strated that the germicidal constituent of blood serum is a nuclei n. This nuclein is undoubtedly furnished by the polynuclear white corpuscles." Denys has (1894) reported the results of experi- ments made in his laboratory by Van der Velde, which give sup- port to the conclusion reached by Vaughn. In these experiments a sterilized culture of staphylococci was injected into the pleural cavity of rabbits in order to obtain an exudate. At intervals of two hours this exudate was obtained by killing one of the animals in the series experimented upon, and at the same time blood from the animal was secured. Both the exudate and the blood were placed in a centrifugal machine, in order to obtain a serum free from corpuscular elements. The germicidal activity of the serum was then tested. The general result of the experiments was to show that the longer the interval after the injection into the pleural cavity the more potent the ger- micidal activity of the exudate became, and that there was no corre- sponding increase in the activity of the blood serum obtained from the circulation. At the end of ten or twelve hours, the serum from the exudate killed all of the staphylococci in a bouillon culture twenty times as great in quantity as the germicidal serum used in the ex- periment. The absence of any increase in germicidal power in the blood serum taken from the general circulation shows that the nota- ble increase manifested by the exudate was due to local causes; and as a matter of fact it corresponded with an increase in the number of leucocytes as found in the pleural exudate. Thus it will be seen that the independent researches of Hankin, of Buchner, of Vaughn, and of other competent bacteriologists, have led them to the same ultimate result so far as the origin of the ger- micidal constituent of the blood is concerned, and that the leucocytes appear to play an important role in the protection of the animal body from invasion by bacteria (natural immunity). It has been shown by several investigators that the number of leucocytes increases in certain infectious diseases, and this increase, together with an increased alkalinity of the blood, which has here- 240 SUSCEPTIBILITY AND IMMUNITY. tofore been referred to, appears to be a provision of nature for over- coming the infection which has already occurred. It has been demonstrated by experiment that naturally immune animals may be infected by the addition of certain substances to cul- tures of pathogenic bacteria. Thus Arloing was able to induce symp- tomatic anthrax in animals naturally immune for this disease by mixing with his cultures various chemical substances, such as car- bolic acid, pyrogallic acid, and especially lactic acid (twenty per cent). Leo has shown that white mice, which are not subject to the pathogenic action of the glanders bacillus, may be rendered sus- ceptible by feeding them for some time upon phloridzin, which gives rise to an artificial diabetes, and causes the tissues to become im- pregnated with sugar. Bouchard has found that very small doses of a pure culture of Bacillus pyocyaneus are fatal to rabbits when at the same time a considerable quantity of a filtered culture of the same bacillus is in- jected into a vein. The animal could have withstood the filtered culture alone, or the bacillus injected beneath its skin ; but its resist- ing power — natural immunity — is overcome by the combined action of the living bacilli and the toxic substances contained in the filtered culture. The same result may be obtained by injecting sterilized cultures of a different microorganism. Thus Roger has shown that the rabbit, which has a natural immunity against symptomatic anthrax, succumbs to infection when inoculated with a culture of the bacillus of this disease, if at the same time it receives an injection of a sterilized or non-sterilized culture of Bacillus prodigiosus. Monti has succeeded in killing animals with old and attenuated cultures of Streptococcus pyogenes, or of Staphylococcus pyogenes aureus, by in- jecting at the same time a culture of Proteus vulgaris. In a similar way, it seems probable, the normal resistance of man to infection by certain pathogenic bacteria may be overcome. Thus when water contaminated by the presence of the typhoid bacillus is used for drinking by the residents of a certain town or district, not all of those who in this way are exposed to infection contract typhoid fever; and among those who do, there is good reason to believe that, in certain cases at least, the result depends upon an additional factor of the kind suggested by the above-mentioned experiments— e.g., the consumption of food containing putrefactive products, or the respi- r; it inn of an atmosphere containing volatile products of putrefaction. The natural immunity of healthy animals may also be neutralized by other agencies which have a depressing effect upon the vital re- sisting power. Thus Nocard and Roux found by experiment that an attenuated culture of the anthrax bacillus, which was not fatal to guinea-pigs, killed these animals when injected into the muscles of SUSCEPTIBILITY AND IMMUNITY. 241 the thigh after they had been bruised by mechanical violence. Abarrin and Roger found that white rats, which are not susceptible to anthrax, became infected and frequently died if they were ex- hausted, previous to inoculation, by being compelled to turn a revolv- ing wheel for a considerable time. Pasteur found that fowls, which have a natural immunity against anthrax, become infected and perish if they are subjected to artificial refrigeration after inocula- tion. This has been confirmed by the more recent experiments of Wagner (1891). According to Canalis and Morpurgo, pigeons which are enfeebled by inanition eaily contract anthrax as a result of inoculation. Arloing states that sheep which have been freely bled contract anthrax more easily than others ; and Serafini found that when dogs were freely bled the bacillus of Friedlander, injected into the trachea or the pleural cavity, entered, and apparently mul- tiplied to some extent in the blood, whereas without such previous bleeding they were not to be found in the circulating fluid. Certain anesthetic agents have been shown also to produce a similar result. Platania communicated anthrax to immune animals — dogs, frogs, pigeons — by bringing them under the influence of curare, chloral, or alcohol; and Wagner obtained similar results in his experiments upon pigeons to which he had administered chloral. In man, clini- cal experience shows that those who are addicted to the excessive use of alcohol are especially liable to contract certain infectious diseases —pneumonia, erysipelas, yellow fever, etc. The micrococcus of pneumonia is habitually present in the sali- vary secretions of many healthy individuals, and it is evident that an attack of pneumonia does not depend alone upon the presence of this micrococcus, which has, nevertheless, been conclusively shown to be the usual infectious agent in cases of croupous pneumonia. No doubt the introduction of the pathogenic micrococcus to the vulner- able point — the lungs — is an essential factor in the development of a case of pneumonia, but there is reason to believe that there are other factors equally essential. Thus it is well known that an attack of pneumonia often results from exposure to cold, which may act as an exciting cause ; and, also, that a recent attack of an acute febrile disease — especially measles — constitutes a predisposing cause. It is generally recognized that malnutrition, want of exercise, insanitary surroundings, and continued respiration of an atmosphere loaded with dust, as in cotton mills, or a recent attack of pneumonia, con- stitute predisposing causes to tubercular infection by way of the lungs. While natural immunity may be overcome by the various depress- ing agencies referred to, it is also true that it has only a relative value in the absence of these predisposing causes, and may be over- 16 242 SUSCEPTIBILITY AND IMMUNITY. come by unusual virulence of the pathogenic infectious agent, or by the introduction into the body of an excessive amount of a pure cul- ture of the same. The pathogenic potency of known disease germs varies as widely as does the susceptibility of individuals to their specific action. In general it may be said that the more recently the germ comes from a developed case of the disease to which it gives rise the more viru- lent it is, and the longer it has been cultivated outside of the animal body the more attenuated is its pathogenic power. Thus when the discharges of a typhoid fever patient find their way directly to a water-supply of limited amount a large proportion of those who drink the water are likely to be attacked; but when a considerable interval of time has elapsed since the contamination occurred, although the germs may still be present, the liability to attack is much less on account of diminished pathogenic virulence. The development of an attack also depends, to some extent, upon the number of germs introduced into a susceptible individual at one time. The resources of nature may be sufficient to dispose of. a few bacilli, while a large number may overwhelm the resisting power of the individual. The experiments of Cheyne (1886) show that in the case of very pathogenic species a single bacillus, or at least a very small number, introduced beneath the skin, may produce fatal infection in a very susceptible animal, while greater numbers are required in those less susceptible. Thus a guinea-pig succumbed to general infection after being inoculated subcutaneously with anthrax blood diluted to such an extent that, by estimation, only one bacillus was present in the fluid injected ; and a similar result was obtained in mice with Bacillus murisepticus. In the case of the microbe of fowl cholera (Bacillus septica3mia hemorrhagicaB) , Cheyne found that for rabbits the fatal dose was 300,000 or more, that from 100,000 to 30,000 cause a local abscess, and that less than 10,000 produce no appreciable effect. The common saprophyte, Proteus vulgaris, was found to be pathogenic for rabbits when injected into the dorsal muscles in sufficient num- bers. But, according to the estimates made, 225,000,000 were re- quired to cause death, while doses of from 9,000,000 to 112,000,000 produced a local abscess, and less than 9,000,000 gave an entirely negative result. ACQUIRED IMMUNITY. It has long been known that, in a considerable number of infec- tious diseases, a single attack, however mild, affords protection against subsequent attacks of the same disease; that in some cases this protection appears to be permanent, lasting during the life of the SUSCEPTIBILITY AND IMMUNITY. 213 individual; that in others it is more or less temporary, as shown by the occurrence of a subsequent attack. The protection afforded by a single attack not only differs in dif- ferent diseases, but in the same disease varies greatly in different individuals. Thus certain individuals have been known to suffer several attacks of small-pox or of scarlet fever, although, as a rule, a single attack is protective. Exceptional susceptibility or insuscepti- bility may be not only an individual but a family characteristic, or it may belong to a particular race. In those diseases in which second attacks are not infrequent, as, for example, in pneumonia, in influenza, or in Asiatic cholera, it is difficult to judge from clinical experience whether a first attack exerts any protective influence. But from experiments upon the lower ani- mals we are led to believe that a certain degree of immunity, lasting for a longer or shorter time, is afforded by an attack of pneumonia or of cholera, and probably of all infectious diseases due to bacterial parasites. In the malarial fevers, which are due to a parasite of a different class, one attack affords no protection, but rather predis- poses to a subsequent attack. In those diseases in which a single attack is generally recognized as being protective, exceptional cases occur in which subsequent attacks are developed as a result of unusual susceptibility or expo- sure under circumstances especially favorable to infection. Maiselis (1894) has gone through the literature accessible to him for the purpose of determining the frequency with which second attacks occur in the various diseases below mentioned. The result is as follows : Second Third Fourth „, . , Attacks. Attacks. Attacks. Small-pox 505 9 0 514 Scarlet fever 29 4 0 33 Measles 36 1 0 37 Typhoid fever 202 5 1 208 Cholera 29 3 2 34 These figures support the view generally entertained by physi- cians that second attacks of scarlet fever and of measles are compar- atively rare, while second attacks of small-pox are not infrequently observed. Considering the very large number of cases of typhoid fever which occur annually in all parts of Europe and America, the number of second attacks collected does not bear a very large propor- tion to the total number taken sick, although the recorded cases, of course, fall far short of the total number of second attacks of this and the other diseases mentioned. The second attacks of cholera recorded are not numerous, and, no doubt, a carefullly conducted investigation made in the areas of en- 244 SUSCEPTIBILITY AND IMMUNITY. demic prevalence of this disease would show that second attacks are more common than is indicated by these figures. That immunity may result from a comparatively mild attack as well as from a severe one is a matter of common observation in the case of small-pox, scarlet fever, yellow fever, etc. ; and since the dis- covery of Jenner we have in vaccination a simple method of produc- ing immunity in the first-mentioned disease. The acquired immunity resulting from vaccination is not, however, as complete or as per- manent as that which results from an attack of the disease. These general facts relating to acquired immunity from infectious diseases constituted the principal portion of our knowledge with re- ference to this important matter up to the time that Pasteur (1880) demonstrated that in the disease of fowls known as chicken cholera, which he had proved to be due to a specific microorganism, a mild attack followed by immunity may be induced by inoculation with an " attenuated virus" — i.e., by inoculation with a culture of the patho- genic microorganism the virulence of which had been so modified that it gave rise to a comparatively mild attack of the disease in question. Pasteur's original method of obtaining an attenuated virus consisted in exposing his cultures for a considerable time to the ac- tion of atmospheric oxygen. It has since been ascertained that the same result is obtained with greater certainty by exposing cultures for a given time to a temperature slightly below that which would destroy the vitality of the pathogenic microorganism, and also by ex- posure to the action of certain chemical agents (see Part Second, p. 124). Pasteur at once comprehended the importance of his discovery, and inferred that what was true, of one infectious germ disease was likely to be true of others. Subsequent researches, by this savant and by other bacteriologists, have justified this anticipation, and the demonstration has already been made for a considerable number of similar diseases — anthrax, symptomatic anthrax, rouget. A virus which has been attenuated artificially — by heat, for ex- ample— may be cultivated through successive generations without re- gaining its original virulence. As this virulence depends, to a con- siderable extent at least, upon the formation of toxic products during the development of the pathogenic microorganism, we naturally infer that diminished virulence is due to a diminished production of these toxic substances. There is reason to believe that a natural attenuation of virulence may occur in pathogenic bacteria which are able to lead a sapro- phytic existence during their multiplication external to the bodies of living animals, and the comparatively mild character of some epi- demics is probably due to this fact. SUSCEPTIBILITY AND IMMUNITY. 245 Again, cultivation within the body of a living animal may, in certain cases, cause a diminution in the virulence of a pathogenic microorganism. Thus Pasteur and Thuiller have shown that the microbe of rouget when inoculated into a rabbit kills the animal, but that its pathogenic virulence is nevertheless so modified that a cul- ture made from the blood of a rabbit killed by it is a suitable " vac- cine " for the pig. On the other hand, we have experimental evidence that the viru- lence of attenuated cultures may be reestablished by passing them through the bodies of susceptible animals. Thus a culture of the bacillus of rouget, attenuated by having been passed through the body of a rabbit, is restored to its original virulence by passing it through the bodies of pigeons. And a culture of the anthrax bacillus which will not kill an adult guinea-pig may be fatal to a very young animal of the same species or to a mouse, and the bacillus cultivated from the blood of such an animal will be found to have greatly in- creased virulence. In Pasteur's inoculations against anthrax "attenuated" cultures are employed which contain the living pathogenic germ as well as the toxic products developed during its growth. Usually two inocu- lations are made with cultures of different degrees of attenuation — that is to say, with cultures in which the toxic products are formed in less amount than in virus of full power. The most attenuated virus is first injected, and after some time the second vaccine, which if injected first might have caused a considerable mortality. The animal is thus protected from the pathogenic action of the most virulent cultures. Now, it has been shown by recent experiments that a similar im- munity may result from the injection into a susceptible animal of the toxic products contained in a virulent culture, independently of the living bacteria to which they owe their origin. Chauveau, in 1880, ascertained that if pregnant ewes are protected against anthrax by inoculation with an attenuated virus, their lambs, when born, also give evidence of having acquired an immunity from the disease. As the investigations of Davaine seemed to show that the anthrax bacillus cannot pass through the placenta from the mother to the foetus, the inference seemed justified that the acquired immunity of the latter was due to some soluble substance which could pass the placental barrier. More recent researches by Strauss and Chamber- lain, Malvoz and Jacquet, and others, show that the placenta is not such an impassable barrier for bacteria as was generally believed at the time of Chauveau's experiments, so that these cannot be accepted as establishing the inference referred to. But, as stated, we have more recent experimental evidence which shows that immunity may 246 SUSCEPTIBILITY AND IMMUNITY. result from the introduction into the bodies of susceptible animals of the toxic substances produced by certain pathogenic bacteria. The first satisfactory experimental evidence of this important fact was obtained by Salmon and Smith in 1886, who succeeded in making pigeons immune from the pathogenic effects of cultures of the bacil- lus of hog cholera by inoculating them with sterilized cultures of this bacillus. In 1888 Roux reported similar results obtained by in- jecting into susceptible animals sterilized cultures of the anthrax bacillus. Behring and Kitasato, in 1890, reported their success in establishing immunity against virulent cultures of the bacillus of tetanus and the diphtheria bacillus by inoculating susceptible ani- mals with filtered, germ-free cultures of these pathogenic bacteria. In 1892 Behring, Kitasato, and Wassermann published the re- sults of interesting experiments with a bouillon made from the thymus gland of the calf. They found that the tetanus bacillus cul- tivated in this bouillon did not form spores and had comparatively little virulence. Mice or rabbits inoculated with it in small doses — 0.001 to 0.2 cubic centimetre for a mouse — proved to be subsequently immune. And the blood serum of an immune rabbit injected into the peritoneal cavity of a mouse — 0.1 to 0.5 cubic centimetre — was found to give it immunity from the pathogenic action of a virulent culture of the tetanus bacillus. Similar results were obtained with several other pathogenic bacteria cultivated in the thymus bouillon- spirillum of cholera, bacillus of diphtheria, typhoid bacillus. We give here the directions for preparing the thymus bouillon as used by the authors named : Two or three thymus glands are chopped into small pieces immediately after they are taken from the animal. An equal part of distilled water is added to the mass and stirred for some time ; it is then placed in an ice chest for twelve hours. The juices are now expressed through gauze by means of a flesh press. A clouded, slimy fluid is obtained, which constitutes a stock solution. This is diluted with water, and a certain quantity of carbonate of soda is added to the solution before sterilization. By this means coagulation and precipitation of the active substance from the thymus gland are avoided. The exact amount of water and of sodium carbonate required to prevent pre- cipitation must be determined by experiment, as it differs for different glands. Usuall v an eoual portion of water and sufficient soda solution to turn litmus paper feeblv blue will give the desired result. The liquid is now heated in a large flask, which is left for fifteen minutes in the steam sterilizer. The liquid is allowed to cool and then filtered through fine linen to remove any suspended coagula ; the filtrate has a milky opalescence. It is now placed in test tubes and again sterilized. The active principle is precipitated by the addition of a few drops of acetic acid. In Pasteur's inoculations against hydrophobia, made subsequently to infection by the bite of a rabid animal, an attenuated virus is in- SUSCEPTIBILITY AND IMMUNITY. 247 troduced subcutaneously in considerable quantity by daily injections, and immunity is established during the interval — so-called period of incubation— which usually occurs between the date of infection and the development of the disease. That the immunity in this case also depends upon the introduction of a chemical substance present in the desiccated spinal cord of rabbits which have succumbed to rabies, which is used in these inoculations, is extremely probable. But, as the germ of rabies has not been isolated or cultivated artificially, this has not yet been demonstrated. Wooldridge claims to have made susceptible animals immune against anthrax by inoculating them with an aqueous extract of the testicle or of the thymus gland of healthy animals. We may mention also the interesting results obtained by Em- merich, Freudenreich, and others, who have shown that an anthrax infection in a susceptible animal inoculated with a virulent culture may be made to take a modified and non-fatal course by the simul- taneous or subsequent inoculation of certain other non-pathogenic bacteria — streptococcus of erysipelas, Bacillus pyocyaneus. In a series of experiments made by the writer some years ago evidence was obtained that, under certain circumstances, immunity from the effects of one pathogenic bacillus may be obtained by the previous injection of a pure culture of a different species. In the experiments referred to injections into the cavity of the abdomen of a culture of Bacillus pyocyaneus protected rabbits from the lethal effects of Bacillus cuniculicida Havaniensis, when subsequently in- jected into the cavity of the abdomen in such amount (one cubic centimetre of a bouillon culture) as invariably proved fatal in rabbits not protected by such injections. Before considering the theories which have been offered in expla- nation of acquired immunity it is desirable to call attention to certain observations which have been made during the past few years relat- ing to "chemiotaxis." The term chemiotaxis was first used by Pfeffer to designate the property, observed by himself and others, which certain living cells exhibit with reference to non-living organic material, and by virtue of which they approach or recede from certain substances. The chemiotaxis is said to be positive when the living cell approaches, and negative when it recedes from, a chemical substance. As examples of this we may mention the approach of motile bacteria to nutrient material or to the surface of a liquid medium where they find the oxygen required for their vital activities ; and of leucocytes to cer- tain substances when these are introduced beneath the skin of warm- er cold-blooded animals. This subject has recently received much 248 SUSCEPTIBILITY AND IMMUNITY. attention and has been studied especially by Ali-Cohen, Massart and Bordet, Gabritchevski, and others. According to Gabritchevski, the following substances have a neg- ative chemiotaxis for the leucocytes : Sodium chloride in ten-per-cent solution, alcohol in ten-per-cent solution, quinine, lactic acid, gly- cerin, chloroform, bile. On the other hand, a positive chemiotaxis is excited by sterilized or non-sterilized cultures of various bacteria. This is shown by the fact that when a small capillary tube, closed at one end, which contains the substance to be tested, is introduced be- neath the skin of an animal, the leucocytes are repelled from the tube by certain substances, while those which incite positive chemiotaxis cause them to enter the tube in great numbers. The experiments of Buchner seem to show that the positive chemiotaxis induced by sterilized cultures of bacteria introduced beneath the skin of an animal, is due to the proteid contents of the cells rather than to the chemical products elaborated as a result of their vital activity. But that such chemical products may, in some instances at least, produce a positive chemiotaxis independently of the bacteria is shown by the experiments of Gabritchevski with filtered cultures of Bacillus pyocyaneuc — confirmed by Massart and Bordet. An important observation made by Bouchard, and confirmed by Massart and Bordet, is the following : When a tube containing a cul- ture of Bacillus pyocyaneus is introduced beneath the skin of a rabbit it is found, at the end of a few hours, to contain a great number of leucocytes. But if immediately after its introduction ten cubic centi- metres of a sterilized culture of the same bacillus are injected into the circulation through a vein, very few leucocytes enter the tube intro- duced beneath the skin — that is, the chemiotaxis of the leucocytes for the bacilli contained in the tube has been neutralized by injecting a considerable quantity of the soluble products of the same bacillus into the circulation. Buchner, having shown that the bacterial cells contain a proteid substance which attracts the leucocytes, experimented with various other proteids and found that gluten, casein from wheat, and legumin from peas had a similar effect. Starch has no effect, but a mass of flour, made from wheat or from peas, introduced beneath the skin of a rabbit or of a guinea-pig, with antiseptic precautions, in the course nf ;i day or t\v<> is enveloped and penetrated by immense numbers of leucocytes. If, instead of introducing these substances which induce positive chemiotaxis beneath the skin, they are injected into the cir- culation, Buchner has shown that a great increase in the number of leucocytes occurs. SUSCEPTIBILITY AND IMMUNITY. 249 THEORIES OF IMMUNITY. Exhaustion Theory. — For a time Pasteur supported the view that during an attack of an infectious disease the pathogenic micro- organism, in its multiplication in the body of a susceptible animal, exhausts the supply of some substance necessary for its development, that this substance is not subsequently reproduced, and that conse- quently the same pathogenic germ cannot again multiply in the body of the protected animal. This view is sustained in a memoir pub- lished in the Comptes Rendus of the French Academy in 1880, in which Pasteur says : " It is the life of a parasite in the interior of the body which produces the malady commonly called ' cholera des ponies,' and which causes death. From the moment when this culture (i.e., the multiplication of the parasite) is no longer possible in the fowl the sickness cannot appear. The fowls are then in the constitutional state of fowls not subject to be attacked by the disease. These last are as if vaccinated from birth for this malady, because the foetal evolution has riot introduced into their bodies the material neces- sary to support the life of the microbe, or these nutritive materials have disappeared at an early age. "Certainly one should not be surprised that there may be constitutions sometimes susceptible and sometimes rebellious to inoculation — that is to say, to the cultivation of a certain virus — when, as I have announced in my first note, one sees a preparation of beer yeast made, exactly like one from the muscles of fowls (bouillon), to show itself absolutely unsuited for the cul- tivation of the parasite of fowl cholera, while it is admirably adapted to the cultivation of a multitude of microscopic species, notably to the bacteride charbonneuse (Bacillus anthracis). "The explanation to which these facts conduct us, as well of the consti- tutional resistance of some individuals as of the immunity produced by protective inoculations, is only natural when we consider that every culture, in general, modifies the medium in which it is effected— a modification of the soil when it relates to ordinary plants; a modification of plants and ani- mals when it relates to their parasites ; a modification of our culture liquids when it relates to muce'dines, vibrioniens, or ferments. ' ' These modifications are manifested and characterized by the circum- stance that new cultivations of the same species in these media become promptly difficult or impossible. If we sow chicken bouillon with the mi- crobe of fowl cholera, and, after three or four days, filter the liquid in order to remove all trace of the microbe, and subsequently sow anew in the fil- tered liquid this parasite, it will be found quite powerless to resume the most feeble development. The liquid, which is perfectly limpid after being fil- tered, retains its limpidity indefinitely. "How can we fail to believe that by cultivation in the fowl of the atten- uated virus we place its body in the state of this filtered liquid which can no longer cultivate the microbe ? The comparison can be pushed still further; for if we filter the bouillon containing the microbe in full develop- ment, not on the fourth day of culture, but on the second, the filtered liquid will still be able to support the development of the microbe, although with less energy than at the outset. We comprehend, then, that after a cultiva- tion of the modified (attenue) microbe in the body of the fowl we may not have removed from all parts of its body the aliment of the microbe. That which remains will permit, then, a new culture, but in a more restricted measure. "This is the effect of a first inoculation ; subsequent inoculations will 250 SUSCEPTIBILITY AND IMMUNITY. remove progressively all the material necessary for the development of the parasite. " In discussing this theory, in a paper published in the American Journal of the Medical Sciences (April, 1881), the writer says: "Let us see where this hypothesis leads us. In the first place, we must have a material of small-pox, and a material of measles, and a material of scarlet fever, etc., etc. Then we must admit that each of these different materials has been formed in the system and stored up for these emergencies — attacks of the diseases in question — for we can scarcely conceive that they were all packed away in the germ cell of the mother and the sperm cell of the father of each susceptible individual. If, then, these peculiar materials have been formed and stored up during the development of the individual, how are we to account for the fact that no new production takes place after an attack of any one of the diseases in question ? " Again, how shall we account for the fact that the amount of material which would nourish the small-pox germ, to the extent of producing a case of confluent small-pox, may be exhausted by the action of the attenuated virus (germ) introduced by vaccination ? Pasteur's comparison of a fowl protected by inoculation with the microbe of fowl cholera, with a culture fluid in which the growth of a particular organism has exhausted the pabu- lum necessary for the development of additional organisms of the same kind, does not seem to me to be a just one, as in the latter case we have a limited supply of nutriment, while in the former we have new supplies constantly provided of the material — food — from which the whole body, including the hypothetical substance essential to the development of the disease germ, was built up prior to the attack. Besides this we have a constant provision for the elimination of effete and useless products. " This hypothesis, then, requires the formation in the human body, and the retention up to a certain time, of a variety of materials which, so far as we can see, serve no purpose except to nourish the germs of various specific diseases, and which, having served this purpose, are not again formed in the same system, subjected to similar external conditions, and supplied with the same kind of nutriment." It is unnecessary to discuss this hypothesis any further, inasmuch as it is no longer sustained by Pasteur or his pupils, and is evidently untenable. The Retention Theory, proposed by Chauveau (1880), is subject to similar objections. According to this view, certain products formed during the development of a pathogenic microorganism in the body of a susceptible animal accumulate during the attack and are subse- quently retained, and, being prejudicial to the growth of the particu- lar microorganism which produced them, a second infection cannot occur. Support for this theory has been found by its advocates in the fact that various processes of fermentation are arrested after a time by the formation of substances which restrain the development < >f the microorganisms to which they are due. But in the case of a living animal the conditions are very different, and it is hard to con- ceive that adventitious products of this kind could be retained for years, when in the normal processes of nutrition and excretion the tissues and fluids of the body are constantly undergoing change. Certainly the substances which arrest ordinary processes of f ermen- SUSCEPTIBILITY AND IMMUNITY. 251 tation by their accumulation in the fermenting liquid, such as alco- hol, lactic acid, phenol, etc. , would not be so retained. But we can- not speak so positively with reference to the toxic albuminous substances which recent researches have demonstrated to be present in cultures of some of the best known pathogenic bacteria. It is difficult, however, to believe that an individual who has passed through attacks of half a dozen different infectious diseases carries about with him a store of as many different chemical substances pro- duced during these attacks, and sufficient in quantity to prevent the development of the several germs of these diseases. Nor does the experimental evidence relating to the action of germicide and germ- restraining agents justify the view that a substance capable of preventing the development of one microorganism should be with- out effect upon others of the same class ; but if we accept the re- tention hypothesis we must admit that the inhibiting substance produced by each particular pathogenic germ is effective only in restraining the development of the microbe which produced it in the first instance. Pasteur discusses this hypothesis in his paper from which we have already quoted, as follows : 4 ' We may admit the possibility that the development of the microbe, in place of removing or destroying certain matters in the bodies of the fowls, adds, on the contrary, something which is an obstacle to the future develop- ment of this microbe. The history of the life of inferior beings authorizes such a supposition. The excretions resulting from vital processes may arrest vital processes of the same nature. In certain fermentations we see anti- septic products make their appearance during, and as a result of, the fer- mentation, which put an end to the active life of the ferments and arrest the fermentations long before they are completed. In the cultivation of our microbe, products may have been formed the presence of which, possibly, may explain the protection following inoculation. "Our artificial cultures permit us to test the truth of this hypothesis. Let us prepare an artificial culture of the microbe, and after having evapo- rated it, in vacuo, without heat, let us bring it back to its original volume by means of fresh chicken bouillon. If the extract contains a poison for the life of the microbe, and if this is the cause of its failure to multiply in the filtered liquid, the new liquid should remain sterile. Now, this is not the case. We cannot, then, believe that during the life of the parasite certain substances are produced which are capable of arresting its ulterior development." This experiment of Pasteur appears to be conclusive so far as the particular pathogenic microorganism referred to is concerned ; and we may say, in brief, that more recent investigations do not sustain the view that acquired immunity is due to the retention of products such as are formed by pathogenic bacteria in artificial culture media, and which act by destroying these bacteria or restraining their devel- opment when they are introduced into the bodies of immune animals. Moreover, if we suppose that the toxic substances which give pathogenic power to a particular microorganism are retained in the 252 SUSCEPTIBILITY AND IMMUNITY. body of an immune animal, we must admit that the animal has ac- quired a tolerance to the pathogenic action of these toxic substances, for their presence no longer gives rise to any morbid phenomena. And this being the case, we are not restricted to the explanation that immunity depends upon a restraining influence exercised upon the microbe when subsequently introduced. The Vital Resistance Theory. — Another explanation offers itself, viz., that immunity depends upon an acquired tolerance to the toxic products of pathogenic bacteria. This is a view which the writer has advocated in various published papers since 1881. In a paper contributed to the American Journal of the Medical Sci- ences in April, 1881, it is presented in the following language: "The view that I am endeavoring- to elucidate is that, during a non- fatal attack of one of the specific diseases, the cellular elements implicated which do not succumb to the destructive influence of the poison acquire a tolerance to this poison which is transmissible to their progeny, and which is the reason of the exemption which the individual enjoys from future attacks of the same disease. " l In my chapter on "Bacteria in Infectious Diseases," in "Bac- teria," published in the spring of 1884, but placed in the hands of the publishers in 1883, I say: " It may be that the true explanation of the immunity afforded by a mild attack of an infectious germ disease is to be found in an acquired tolerance to the action of a chemical poison produced by the microorganism, and conse- quent ability to bring the resources of nature to bear to restrict invasion by Uie parasite." The "resources of nature" are referred to in the same chapter as follows : 44 The hypothesis of Pasteur would account for the fact that one individual suffers a severe attack and another a mild attack of an infectious disease, after being subjected to the influence of the poison under identical circum- stances, by the supposition that the pabulum required for the development of this particular poison is more abundant in the body of one individual than in the other. The explanation which seems to us more satisfactory is that the vital resistance offered by the cellular elements in the bodies of these two individuals was not the same for this poison. It is well known that in conditions of lowered vitality resulting from starvation, profuse discharges, or any other cause, the power to resist disease poisons is greatly diminished, and, consequently, that the susceptibility of the same individual differs at different times. 44 From our point of view, the blood, as it is found within the vessels of a living animal, is not simply a culture fluid maintained at a fixed tempera- ture, out under these circumstances is a tissue, the histological elements of which present a certain vital resistance to pathogenic organisms which may be introduced into the circulation. 44 If we add a small quantity of a culture fluid containing the bacteria of putrefaction to the blood of an animal, withdrawn from the circulation into a proper receptacle and maintained in a culture oven at blood heat, we will find that these bacteria multiply abundantly, and evidence of putrefactive 1 "What is the Explanation of the Protection from Subsequent Attacks, result- ing from :m Attack of Certain Diseases, etc ?" American Journal of the Medical Sciences, April, 1881, p. 370. SUSCEPTIBILITY AND IMMUNITY. #53 decomposition will soon be perceived. But if we inject a like quantity of the culture fluid with its contained bacteria into the circulation of a living- animal, not only does no increase and no putrefactive change occur, but the bacteria introduced quickly disappear, and at the end of an hour or two the most careful microscopical examination will not reveal the presence of a single bacterium. This difference we ascribe to the vital properties of the fluid as contained in the vessels of a living animal; and it seems probable that the little masses of protoplasm known as white blood corpuscles are the essential histological elements of the blood, so far as any manifestation of vitality is concerned. The ivriter has elsewhere (1881) suggested that the disappearance of the bacteria from the circulation, in the experiment referred to, may be effected by the white corpuscles, which, it is well known, pick up, after the manner of amosbae, any particles, organic or inorganic, which come in their way. And it requires no great stretch of credulity to believe that they may, like an amoeba, digest and assimilate the protoplasm of the captured bacterium, thus putting an end to the possibility of its do- ing any harm. " In the case of a pathogenic organism we may imagine that, when cap- tured in this way, it may share a like fate if the captor is not paralyzed by some potent poison evolved by it, or overwhelmed by its superior vigor and rapid multiplication. In the latter event the active career of our conserva- tive white corpuscle would be quickly terminated and its protoplasm would serve as food for the enemy. It is evident that in a contest of this kind the balance of power would depend upon circumstances relating to the inherited vital characteristics of the invading parasite and of the invaded leucocyte." In the same chapter the writer quotes from his paper on acquired immunity, published in 1881, as follows : " The difficulties into which this hypothesis [the exhaustion theory of Pas- teur] leads us certainly justify us in looking further for an explanation of the phenomena in question. This explanation is, I believe, to be found in the peculiar properties of the protoplasm, which is the essential framework of every living organism. The properties referred to are the tolerance which living protoplasm may acquire to certain agents which, in the first instance, have an injurious or even fatal influence upon its vital activity ; and the property which it possesses of transmitting its peculiar qualities, inherent or acquired, through numerous generations, to its offshoots or progeny. "Protoplasm is the essential living portion of the cellular elements of ani- mal and vegetable tissues ; but as our microscopical analysis of the tissues has not gone beyond the cells of which they are composed, and is not likely to reveal to us the complicated molecular structure of the protoplasm, upon which, possibly, the properties under consideration depend, it will be best, for the present, to limit ourselves to a consideration of the living cells of the body. These cells are the direct descendants of the pre-existent cells, and may all be traced back to the sperm cell and the germ cell of the parents. Now, the view which I am endeavoring to elucidate is that, during a non- fatal attack of one of the specific diseases, the cellular elements implicated, which do not succumb to the destructive influence of the poison, acquire a tolerance to this poison which is transmissible to their progeny, and which is the reason of the exemption which the individual enjoys from future attacks of the same disease. " The known facts in regard to the hereditary transmission by cells of ac- quired properties make it easy to believe in the transmission of such a tolerance as we imagine to be acquired during the attack ; and if it is shown by analogy that there is nothing improbable in the hypothesis that such a tolerance is acquired, we shall have a rational explanation, not of heredity and of the mysterious properties of protoplasm, but of the particular result under consideration. The transmission of acquired properties is shown in the budding and grafting of choice fruits and flowers, produced by cultiva- 254 SUSCEPTIBILITY AND IMMUNITY, tion, upon the wild stock from which they originated. The acquired proper- ties are transmitted indefinitely; and the same sap which on one twig nour- ishes a sour crab apple, on another one of the same branch is elaborated into a delicious pippin. " The tolerance to narcotics — opium and tobacco — and to corrosive poisons — arsenic — which results from a gradual increase of dose, may be cited as an example of acquired tolerance by living protoplasm to poisons which at the outset would have been fatal in much smaller doses. "The immunity which an individual enjoys from any particular disease must be looked upon as a power of resistance possessed by the cellular ele- ments of those tissues of his body which would yield to the poison in the case of an unprotected person." This theory of immunity, advanced by the author in 1881, has received considerable support from investigations made since that date, and especially from the experimental demonstration by Sal- mon, Roux, and others that, as suggested in the paper from which I have quoted, immunity may result from the introduction into the body of a susceptible animal of the soluble products of bacterial growth — filtered cultures. The theory of vital resistance to the toxic products evolved by pathogenic bacteria is also supported by numerous experiments which show that natural or acquired immunity may be overcome when these toxic products are introduced in excess, or when the vital resisting power -of the animal has been reduced by various agencies. More direct experimental evidence in favor of the view under con- sideration is that obtained by Beumer in his experiments with steril- ized cultures of the typhoid bacillus. He found that after the re- peated injection of non-lethal doses mice were able to resist an amount of this toxine which was fatal to animals of the same spe- cies not so treated. But, on the other hand, Gamaleia found, in his experiments upon guinea-pigs which had been made immune against the pathogenic action of a spirillum, called by him Vibrio Metschni- kovi, that these animals have no increased tolerance for the toxic products of this microorganism. Although immune against infec- tion by the living microbe, they were killed by the same quantity of a sterilized culture as was fatal to guinea-pigs which had not been rendered immune. Charrin has obtained similar results in experiments with filtered cultures of Bacillus pyocyaneus. Rabbits which had an artificial im- munity against the pathogenic action of the bacillus were killed by doses of a sterilized culture such as were fatal to other rabbits of the same size not immune. In subsequent experiments by Charrin and Gameleia "vaccinated" rabbits were found to be even more suscepti- ble to the toxic action of filtered cultures than were those not vacci- nated. Metschnikoff (1891) has followed up this line of experiment, and has shown that when considerable amounts of filtered cultures of Bacillus pyocyaneus are injected subcutaneously in rabbits a cer- SUSCEPTIBILITY AND IMMUNITY. 255 tain tolerance to the toxic action of the same cultures is established in some instances. But his results do not give any substantial sup- port to the view that immunity depends upon an acquired tolerance to the toxic action of the chemical products contained in cultures of the pathogenic bacteria with which he experimented — Bacillus pyo- cyaneus and Vibrio Metschnikovi. In view of the results of experimental researches above recorded, and of other recent experiments which show that, in certain cases at least, acquired immunity depends upon the formation of an anti- fcoxine in the body of the immune animal, we are convinced that the theory of immunity under discussion, first proposed by the writer in 1881, cannot be accepted as a sufficient explanation of the facts in general. At the same time we are inclined to attribute considerable importance to acquired tolerance to the toxic products of pathogenic bacteria as one of the factors by which recovery from an infectious disease is made possible and subsequent immunity established. The " vital-resistance theory" of the present writer, as set forth in the above-quoted extracts from his published papers, is essentially the same as that advocated by Buchner at a later date (1883). Buch- ner supposes that during the primary infection, when an animal re- covers, a " reactive change " has been produced in the cells of the body which enables it to protect itself from the pathogenic action of the same microorganism when subsequently introduced. Of course when we ascribe immunity to the " vital resistance" of the cellular elements of the body, we have not explained the modus operandi of this vital resistance or " reactive change," but have simply affirmed that the phenomenon in question depends upon some acquired property residing in the living cellular elements of the body. We have suggested that that which has been acquired is a tolerance to the action of the toxic products produced by patho- genic bacteria. But, as already stated, in the light of recent experi- ments this theory now appears to us to be untenable as a general explanation of acquired immunity. The Theory of Phagocytosis. — The fact that in certain infectious diseases due to bacteria the parasitic invaders, at the point of inocu- lation or in the general blood current, are picked up by the leuco- cytes and in properly stained preparations may be seen in their in- terior, has been known for some years. In mouse septicaemia — an infectious disease described by Koch in his work on "Traumatic Infectious Diseases, " published in 1878 — the slender bacilli which are the cause of the disease are found in large numbers in the interior of the leucocytes. Koch says, in the work referred to : " Their rela- tion to the white blood corpuscles is peculiar ; they penetrate these and multiply in their interior. One often finds that there is 256 SUSCEPTIBILITY AND IMMUNITY. hardly a single white corpuscle in the interior of which bacilli can- not be seen. Many corpuscles contain isolated bacilli only ; others have thick masses in their interior, the nucleus being still recog- nizable ; while in others the nucleus can be no longer distinguished ; and, finally, the corpuscle may become a cluster of bacilli, breaking up at the margin — the origin of which one could not have explained had there been no opportunity of seeing all the intermediate steps between the intact white corpuscle and these masses " (Fig. 78). It will be noted that in the above, quotation Koch affirms that the bacilli penetrate the leucocytes and multiply in their interior. Now, the theory of phagocytosis assumes that the bacilli are picked up by the leucocytes and destroyed in their interior, and that immunity de- pends largely upon the power of these " phagocytes" to capture and destroy living pathogenic bacilli. The writer suggested this as an hypothesis as long ago as 1881, in a paper read before the American Association for the Advance- ment of Science, in the following language: "It has occurred to me that possibly the white corpuscles may have the office of picking up and digesting bacterial organisms which FIG. 78.— Bacillus of mouse septicaemia in leucocytes from blood of mouse (Koch). by any n^eans find their way into the blood. The propensity exhib- ited by the leucocytes for picking up inorganic granules is well known, and that they may be able not only to pick up but to assimi- late, and so dispose of, the bacteria which come in their way, does not seem to me very improbable, in view of the fact that amoebae, which resemble them so closely, feed upon bacteria and similar or- ganisms." ' At a later date (1884) Metschnikoff offered experimental evi- dence in favor of this view, and the explanation suggested in the above quotation is commonly spoken of as the Metschnikoff theory. 1 " A Contribution to the Study of Bacterial Organisms commonly found upon Exposed Mucous Surfaces and in the Alimentary Canal of Healthy Individuals." Il- lustrated by photomicrographs. Proceedings of the American Association for Ad- vancement of Science, 1881, Salem, 1882, xxx., 83-94. Also in Studies from the Biological Laboratory, Johns Hopkins University, Baltimore, vol. ii., No. 2, 1882. SUSCEPTIBILITY AND IMMUNITY. 257 The observations which first led Metschnikoff to adopt this view were made upon a species of daphnia which is subject to fatal infec- tion by a torula resembling the yeast fungus. Entering with the food, this fungus penetrates the walls of the intestine and invades the tissues. In certain cases the infection does not prove fatal, owing, as Metschnikoff asserts, to the fact that the fungus cells are seized upon by the leucocytes, which appear to accumulate around the invading parasite (chemiotaxis) for this special purpose. If they are success- ful in overpowering and destroying the parasite the animal recovers ; if not, it succumbs to the general infection which results. In a simi- lar manner, Metschnikoff supposes, pathogenic bacteria are destroyed when introduced into the body of an immune animal. The colorless blood corpuscles, which he designates phagocytes, accumulate at the point of invasion and pick up the living bacteria, as they are known to pick up inorganic particles injected into the circulation. So far there can be no doubt that Metschnikoff is right. The presence of bacteria in the leucocytes in considerable numbers, both at the point of inoculation and in the general circulation, has been repeatedly demonstrated in animals inoculated with various pathogenic bacteria. The writer observed this in his experiments, made in 1881, in which rabbits were inoculated with cultures of his Micrococcus Pasteuri ; and it was this observation which led him to suggest the theory which has since been so vigorously supported by Metschnikoff. But the presence of a certain number of bacteria within the leucocytes does not prove the destructive power of these cells for living patho- genic organisms. As urged by Weigert, Baumgarten, and others, it may be that the bacteria were already dead when they were picked up, having been destroyed by some agency outside of the blood cells. As heretofore stated, we have now experimental evidence that blood serum, quite independently of the cellular elements contained in it in the circulation, has decided germicidal power for certain patho- genic bacteria, and that the blood serum of the rat and other animals which have a natural immunity against anthrax is especially fatal to the anthrax bacillus. Numerous experiments have been made during the past two or three years with a view to determining whether pathogenic bacteria are, in fact, destroyed within the leucocytes after being picked up, and different experimenters have arrived at different conclusions. In the case of mouse septicaemia, already alluded to, and in gonor- rhoea, one would be disposed to decide, from the appearance and ar- rangement of the pathogenic bacteria in the leucocytes, that they are not destroyed, but that, on the other hand, they multiply in the in- terior of these cells, which in the end succumb to this parasitic in- vasion. In both of the diseases mentioned we find leucocytes so 17 258 SUSCEPTIBILITY AND IMMUNITY. completely filled with the pathogenic microorganisms that it is diffi- cult to believe that they have all been picked up by a voracious pha- gocyte, which has stuffed itself to repletion, while numerous other leucocytes from the same source and in the same microscopic field of view have failed to capture a single bacillus or micrococcus. More- over, the staining of the parasitic invaders, and the characteristic ar- rangement of the ' ' gonococcus " in stained preparations of gonorrhoeal pus, indicate that their vitality has not been destroyed in the interior of the leucocytes or pus cells, and we can scarcely doubt that the large number found in certain cells is due to multiplication in situ rather than to an unusual activity of these particular cells. But in certain infectious diseases, and especially in anthrax, the bacilli in- cluded within the leucocytes often give evidence of degenerative changes, which would support the view that they are destroyed by the leucocytes, unless these changes occurred before they were picked up, as is maintained by Nuttall and others. We cannot consider this question as definitely settled, but, in view of the importance attached to the theory of phagocytosis by many pathologists and bac- teriologists, we reproduce here a paper by Metschnikoff in which his views are fully set forth : LECTURE ON PHAGOCYTOSIS AND IMMUNITY.1 It is not possible to study the bacteriology of disease without noticing that, while ^n many cases the invading microorganisms are to be found solely in the fluids of the body, in not a few affections they present them- selves in the interior of certain cells, and this either partially — some being within the cells, others free in the blood plasma and the lymph that bathes the various tissues — or exclusively, all the bacteria that are visible being intracellular. Many of the facts bearing upon the terms of this relationship between tissue cell and microorganism are now well known, yet it is worth while to recapitulate the more important, in order to show that from them it is possible to gain a general law ; and what is more, that from a study of such facts some insight may be gained into the phenomena of immunity. It may, in the first place, be postulated that whenever a microorganism is discoverable within a cell its passage thither has been by means of proto- plasmic or amoeboid movements, either on the part of the microbe or of the cell itself. The first alternative is the rarer, although it certainly exists, and of this the malarial parasite affords an excellent example ; for here in the amoeboid stage of its existence the hsematozoon makes its way into the in- trrior of a cell that possesses no active movements of its own, namely, the red blood corpuscle, and from the substance of this corpuscle the parasite gains its nourishment. Other sporozoa furnish instances almost equally good. More commonly, however, as in the case of all bacteria, where we have to deal wit h microorganism* which, even when mobile, are destitute of protoplasmic appendages, it is the cells which play the active part ; certain cells include the parasites. Of such the amoebifonn leucocyte of the blood and lymph is the most typical example, capable, as it is, of sending out pseudopodia in all directions, while a closely allied form is the cell of the 1 Delivered at the Institut Pasteur, December 29th, 1890, by Dr. Elias Metschni- koff, Chef de Service do 1' Institut Pasteur, Paris ; late Professor of Zoology in the University of Odessa. SUSCEPTIBILITY AND IMMUNITY. 259 splenic pulp. But there are also cells — as, for instance, those forming the endothelial lining- of the vessels — which are very definitely fixed, which nevertheless can give off protoplasmic processes from their free surface and so capture and include bacteria. All these may be spoken of as phagocytes, and may be divided into the two broad groups affixed phagocytes (endothelial cells, etc.) and free (leu- cocytes). Not that the terms "phagocyte" and "leucocyte" are synonymous, for of the latter three main forms may be distinguished, of which one is practically immobile and never takes up bacteria. This is the lymphocyte, characterized by its relatively small size, its large single nucleus, and the small amount of surrounding protoplasm. The two remaining (phagocytic) forms are, first, the large uninuclear leucocyte, whose prominent nucleus is at times lobed or reniform, which stains well with aniline dyes and possesses much protoplasm and active amoeboid movements — the macrophage — and, second, the microphage, a small form, also staining well, but either multi- nuclear or with one nucleus in the process of breaking up. If now we com- pare the endothelial cells with these, it is evident that their properties con- nect them closely with the macrophage ; and, in fact, there is now little or no doubt that a very large proportion of the macrophages are of endothelial origin. Leaving aside the subject of amoeboid microbes and their life within ani- mal cells, it is to the phagocytes and their relation to the bacteria that I wish specially to draw your attention. Taking as wide a view as possible of this relationship, we can first deter- mine that the more malignant the microorganism the rarer is its presence within the phagocyte. Thus in those which of all diseases are the most rapidly fatal — in chicken cholera affecting birds and rabbits, in hog cholera ("cholera des pores") given to pigeons and rabbits, in the anthrax of mice and other specially sensitive animals, in the "septicemie vibrionienne" of guinea-pigs and birds, and in yet other diseases of peculiarly swift course — the corresponding bacteria are only very exceptionally to be found within the cells, but remain free in the neighborhood of their introduction and thence invade the blood. For all the above-mentioned diseases are not localized, but, on the contrary, present the characters of general acute sep- ticaemia, causing death within twenty to thirty-six hours, or, in certain cases, even within six hours. And when we pass to those diseases in which the bacteria are to be found either in part or almost wholly within the phagocytes, the same law still applies ; for in such cases the disease has lost its suddenness, tending to have a slower course, or, indeed, to be of a chronic nature. Even in those affections in which an acute course is accompanied by considerable phago- cytosis, the fatal termination is far from occurring at the same early period as in the diseases recorded above. Thus mouse septicaemia, characterized as it is by frequent intracellular bacteria, has a duration in the mouse two and a half times as long as that of anthrax in the same animal. But in general a well-marked phagocytosis is associated with diseases presenting an essen- tially chronic development ; it is in affections such as tuberculosis, leprosy, rhinoscleroma, glanders, that the specific bacteria are most readily taken up by the phagocytes ; it is here that, at the seat of the disease, we meet with in- numerable macrophages — epithelioid cells in which lie the individual micro- organisms. Further, if we consider the phenomena associated with the resolution of an infectious disease, this inverse relationship between the malignancy of the malady and the occurrence of phagocytosis is, if possible, yet more clearly demonstrated. Notice, for instance, what obtains during the progress of re- lapsing fever, a malady still fairly common in Russia and other Sclavonic countries, and one which, while presenting many difficulties to the bacteri- ologist, in that the specific spirochaete has so far resisted cultivation, and in that it cannot be communicated to the ordinary animals of the laboratory, is nevertheless in many respects not ill -adapted for our present purpose. Here, 260 SUSCEPTIBILITY AND IMMUNITY. during the sudden access of the fever, the spirilla are present in the blood in, enormous numbers ; they all are free in the plasma, and not a single intra- cellular spirillum is to be met with. During the apyretic stage (and in the monkey this is, at the same time, the stage of resolution) not a single free spiril- lum is discoverable in the blood, while the phagocytes of the spleen contain the microbes. The like phenomena repeat themselves in all those cases where it is possible to follow the fate of the microorganisms of acute disease during the stage of recovery. Thus rats and pigeons very frequently survive an attack of anthrax, and, where this occurs, the bacteria, which ,at the com- mencement of the disease were for the most part free, now, during resolution, are for the most part included within leucocytes and splenic phagocytes. Nor is this all. Analogous phenomena as a rule attend immunity, which most often is but recovery in operation from the very onset of a disease The more closely one studies this condition of immunity the more is one led to the conviction that immunity and recovery are very intimately con- nected ; that one can pass by slight gradations from the resolution of disease to the production of immunity. So it is that, in inoculating refractory ani- mals with the microbe to whose action they have been rendered immune, it is found that the parasite begins to develop, but that from the onset a reac- tion on the part of the organism shows itself, accompanied by a considerable emigration of leucocytes, which soon include the bacteria in great numbers. This relationship of phagocytosis to acquired immunity is in the highest degree instructive. Where a given species of animal is specially sensitive to the onslaught of one or other microorganism, there, during the course of the disease, the phagocvtes are inoperative, including none, or almost none, of the bacteria. On the other hand, when by previous vaccination these animals have been rendered refractory, their phagocytes have acquired the property of including the same bacteria. As an example of this I may cite the action of the bacillus of anthrax and of the Vibrio Metschnikovi. In ordinarv rabbits the development of anthrax is only followed by a very feeble phagocytosis, while in vaccinated rabbits this phagocytosis is very ex- tensive. Corresponding but yet more strongly marked differences are to be made out between the unvaccinated guinea-pig — an animal most readily affected by the vibrionic septicaemia — and the guinea-pig vaccinated against the same; after inoculation with the Vibrio Metschnikovi none of the vibrios are to be found in the cells of the former; in the latter the phagocytes are simply replete with the microbes. The facts enumerated thus far would seem to prove that there exists a certain antagonism between ihe microbes and the phagocytes, and this view is confirmed by the fact that in general the microbes find the interior of the phagocytes an unfavorable medium for their development and continued existence. Very often it is possible to determine absolutely that the parasites are killed within the phagocytes; after inoculating refractory animals with bacteria, an afflux of white corpuscles toward the region of inoculation fol- lowed by the inclusion of the bacteria and by their death, is seen to occur. '•-; >'•'-"•> can be well followed where the anthrax bacilli are taken into te phagocytes of animals that are, or have been rendered, immune They occur also with a long series of other microorganisms studied in thisconnec- tion, and, among others, m the case of the tubercle bacillus invading animals that are more or less resistant. The giant cells of tuberculosis are, in fact huge muUinuclear phagocytes, and here the intracellular destruction of the the more clearly demonstrable, inasmuch as the microorganisms «>x In bit such very evident signs of degeneration; the bacilli swell their en- veloping membrane becomes much thickened and highly refractive and in imio the .content* lose their power of fixing the stain in* material so tint eventually nothing is left Vut slightly yellowish form's rec^Whi pro- !""•!""'- •""! I"-"""', tlm Pillared burilli; ami ihrsr shadowy bodies unite rerbetg^^ SUSCEPTIBILITY AND IMMUNITY. 261 tures or in caseating masses — these changes may well be regarded as due to a specific action upon the part of the giant cells. The broad fact that the invasion of the organism by microbes most often induces, on the one hand, an inflammatory reaction with its associated emi- gration of leucocytes, and that, on the other hand, the phagocytes are capable of including and destroying the invaders, leads us to admit that the afflux of phagocytes to the invaded region, and their bactericidal properties, are mechanisms which serve to ward off bacterial attack and to maintain the integrity of the organism. Where the phagocytes do not, either immediately or eventually, intervene, but leave the field free to the microbes, these last multiply without hindrance and succeed in killing the animal within, it may be, an excessively short period. Thus the microorganism of hog cholera, which is left quite untouched, kills the pigeon in the course of a few hours — often within five hours after inoculation ; chicken cholera kills not only pigeons but also rabbits in an equally short period. In other diseases in which the phagocytes appear upon the scene in relatively large numbers, and even include the microorganisms, the latter gain the day whenever and wherever the phagocytes are incapable of destroying them or of preventing their growth. This manifest bactericidal action is to be compared with the phenomena of intracellular digestion characteristic of amoeboid cells in general, and of leucocytes and other microbic phagocytes in particular. These cells have the power of digesting with ease red corpuscles and other organized ele- ments, just as have the amoebae proper and other protozoa. Among these last are many which have been found to include and transform bacteria in exactly the same way as do the phagocytes of the higher animals. Now, in determining the intervention or non-intervention of the leuco- cytes in this war between the organism and the bacteria, a very great part is played by the sensitiveness of these cells to external influences, and es- pecially to the chemical composition of their environment. The leucocytes are powerfully attracted by many microorganisms and the resultants of their growth, and as powerfully repelled by others and their resultants, or, as it is expressed, they have a positive chemiotaxis for certain microbes, a negative chemiotaxis for others. The existence of these chemiotactic pro- perties has been so clearly proved of late by the researches of Leber, Mas- sart and Bordet, and Gabritschevski that I need not enter into a fuller ex- planation of the subject here. Where negative chemiotaxis manifests itself, there, being shunned by the white corpuscles, the parasites freely propagate themselves and induce the death of their host. Nevertheless this chemio- taxis is not immutable, and the cells can become accustomed to substances from which they shrank at first — a negative may thus be transformed into a positive chemiotactic state. Such obtains in acquired immunity ; the cells which in the unvaccinated animal never included the bacteria, now in the vaccinated take them up readily. . . . There is not a single portion of the theory which I have just expounded but has encountered a lively opposition. Even the fundamental fact that the phagocytes are capable of including the microbes has had doubts thrown upon it ; it has been held that the latter insinuate themselves into the for- mer. Only after successive series of observations upon the phagocytes and the living microbes has it been proved that assuredly it is the phagocytes which, by the aid of their pseudopodia, themselves include the microorgan- isms. The observer can see the whole process in the case of immobile ba- cilli— can see the leucocyte approach, send out pseudopodia, arid gradually include the individual bacillus. Or, conversely, in cases of negative che- miotaxis, one can, in blood taken from the monkey during the access of re- lapsing fever, observe the actively moving spirilla come into contact with a leucocyte, and even become attached by one end to its surface ; yet, how- ever active the movement, one never finds that the spirillum succeeds in piercing the surface and gaining an entrance. If it be suggested that this entry may take place in consequence of the force of active growth and elon- 2G2 SUSCEPTIBILITY AND IMMUNITY. Cation of bacilli, then, apart from the fact that here but one set of cases is embraced, it can be determined that this force is too feeble — it can be seen that, during the active growth of the anthrax organism in the blood, the elongating chains of bacilli curve in and out between the corpuscles, but never penetrate the cells. From another side the objection has been formulated that in many cases the organism fleets rid of its invaders without the aid of the phagocytes. According to those who support this objection, this happens in the anthrax of pigeons (CzaplewskiJ and of refractory rats (Behring, Franck), in symp- tomatic anthrax of various refractory animals (Rogowicz), and in the septi- ca-mia of vaccinated guinea pigs, due to the Vibrio Metschnikovi (R. Pfeif- fer). A reexamination of the cases here adduced has, however, shown that in each a very considerable phagocytosis can be proved, and that the negative results of the above observers have been due to insufficient methods of observation. While accepting that the phagocytes do truly absorb the microorgan- isms, other opponents of the theory have urged that these cells are only capable of including microorganisms already killed by other means, and that living microbes are solely to be found within the cells in those cases where there has been a fatal ending — in tuberculosis, mouse septicaemia, and so on. Against this may be brought the fact determined by Lubarsch, that the phagocytes of several animals, refractory to anthrax, take up living ba- cilli that have been injected, with greater eagerness than they include those which have been killed before injection. But, further, this objection may be disposed of by direct observation of bacteria undergoing development from within the interior of phagocytes after the latter hayel>een destroyed by a substance which is at tne same time a favorable medium for bacterial growth — as, for instance, beef broth. Such observations have been made upon pigeons rendered immune to anthrax. During the last year or two great stress has been laid upon the fact that the body humors themselves possess most marked bactericidal properties, and, in fact, against the theory of phagocytosis has been brought another, baned upon this power of the humors to destroy the microorganisms. Ob- server after observer has remarked that in blood plasma, defibrinated blood, blood serum, and in the blood as a whole, in the removed aqueous humor and other fluids and exudations of the body, many species of bacteria perish after a longer or shorter interval ; and forthwith an endeavor has been made to find in these facts some elucidation of the phenomena of immunity. Yet the more deeply one examines into the question the more one is con- vinced that no relationship exists between the two. Thus it happens often that tho bactericidal property is more developed in susceptible species than in refractory ; so, with regard to the anthrax bacilli, in the very sensitive rabbit the bactericidal properties of the humors are more pronounced than they are m the refractory dog ; and Bearing and Nissen, the two who al- most simultaneously first drew our attention to these phenomena, in their -• combined research, recently published, admit that, as against the bacteria of anthrax, pneumonia, and diphtheria, this bactericidal property exists to iiH-sani.-d.--n-.. m the juices of animals of the same species, whether they be susceptible or -have been rendered immune. Often, again, it has been determined that the blood removed from the organism has a greater power of destroying bacteria than it has within the organism. A small quantity of Wood withdrawn from the body will, in certain instances, kill a mass of I. acilli greater than that which, injected into the circulation, would inevi- tably cause death. Evident ly. therefore, in this bactericidal influence extra- raMUlar phenomena enact an imp,, riant role phenomena, that is which naveno connection with what occurs in the living refractory organism •'"» another point ot view strong ar-nments have been directed against this theory of the tissue fluids. It has been shown, especially by the re- •cnesof M. Hall km.-, that the death of the bacteria transported into or- llui.ls is largely due to the sudden change of medium, and that, in SUSCEPTIBILITY AND IMMUNITY. 263 passing from one medium to another by successive slight modifications in the fluid of growth, it is easy to make bacteria live in fluids which, when the change of environment has been abrupt, swiftly lead to their destruc- tion. In order to gain an idea as to the part played in the refractory animal by the fluids and the phagocytes respectively, the endeavor has been made to separate the two by placing under the skin of frogs (which are naturally immune to anthrax) minute packets formed of filter paper or of animal membrane, and containing the bacilli. The paper, while permitting the passage of fluid, wards off the wandering amoeboid cells for a certain time. Shielded in this way from the phagocytes, though exposed to the action of the juices, the bacilli grow well and produce the characteristic felted mass of anthrax filaments. Baumgarten has not been able to confirm this experi- ment, but Hueppe and Lubarsch have repeatedly verified it. But it is not even necessary to take these precautions in order to assure one's self that anthrax spores germinate in the juices of refractory animals. Recently, for instance, M. Trapeznikoff has found that, when these spores are injected into the dorsal lymph sac of the frog, they constantly tend to develop into bacilli, whose further growth is stopped by the phagocytes, which include them, along with such spores as have not had time to germi- nate. Eventually the bacilli so absorbed are digested by their hosts, while the included spores remain intact, although incapable of giving birth to bacilli for so long a time as the phagocytes remain alive. And I might ad- duce other similar cases. Such a comparative examination proves that in the living body the bactericidal property resides in the phagocytes and not in the fluids. Still, it may be urged that possibly these cells, which can thus devour and destroy the living microbes, are only in a position to attack bacteria whose virulence has already been lessened by other means Were this so, the mi- crobes present in a refractory organism should behave, not like parasites, but as simple, inoffensive saprophytes. Hence these microbes — powerless to produce upon a refractory soil the toxic substances which render them pathogenic and dangerous — should easily be included and destroyed; so that, according to this hypothesis, which "has frequently been brought for- ward, the phagocytes play a purely secondary and dependent part, waiting until the microbes are weakened before they seize upon them. In favor of this view the fact has been cited that certain microorganisms cultivated in the blood, or serum, of vaccinated animals become attenuated, so that they no longer induce a fatal disease. The Bacillus anthracis grown in the blood of vaccinated sheep no longer kills rabbits, and, according to Roger, the Streptococcus erysipelatos grown in the blood of vaccinated rabbits only occasions a slight and passing disturbance in susceptible members of the same species. But here again we are dealing with fluids withdrawn from the body, and so modified in various ways. Let us make an observation more strictly to the point. Take, for instance, a rabbit vaccinated against anthrax and inoculate it with anthrax bacilli, thus allowing these to exist directly within the refractory organism. Such bacilli as are not destroyed preserve their virulence for a sufficiently long period, and it is possible to kill a guinea-pig with a drop of exudation, taken from the region of injection thirty hours after subcutaneous inoculation, eight days after inoculation into the anterior chamber of the eye. A sojourn of so long duration within the vaccinated organism, then, has not deprived the microbes of their viru- lence, although twenty-four hours suffice to completely attenuate the bacilli cultivated in the removed blood of vaccinated sheep. Years ago it was established in M. Pasteur's laboratory that the refrac- tory organism, instead of being an unfavorable soil for the preservation of virulence, tends the rather to reinforce this property. To exalt the viru- lence of an attenuated microorganism, one always employs, not animals very susceptible to the specific disease, but those which are slightly suscep- tible, or it may be, under many circumstances, refractory. In this manner 204 SUSCEPTIBILITY AND IMMUNITY. the most active anthrax virus has usually been obtained by passage through birds, notably fowls ; the greatest virulence of chicken cholera was gained by passage through the vaccinated cock ; and quite recently M. Malm has shown that passage of the anthrax bacillus through the organisms of dogs, which of all mammals are the most refractory in this respect, increases its virulence in a most remarkable manner, so that the general law may be laid down that an organism which is but slightly susceptible or is refractory is able not only to preserve, but even to exalt, the virulence of bacteria. The principal argument in favor of the hypothesis that pathogenic microor- ganisms become simple inoffensive saprophytes when they find themselves in a refractory region, loses therefore its raison d'etre. M. Bouchard, in his objection to the theory of phagocytosis, may be re- garded as introducing but a modification of this hypothesis. He holds that pathogenic bacteria placed under favorable conditions give rise to substances which hinder the inflammatory process, and that only when these inhibi- tory substances are inadequately represented do the cells intervene. When, therefore, the organism rendered refractory by vaccination becomes an un- favorable soil for the production of these inhibitory bodies, the bacteria can no longer prevent the inflammatory reaction ; free emigration of the leuco- cytes ensues, these cells seize upon the impotent microbes and put a stop to their further growth. In this theory the part played by the phagocytes is again secondary, depending upon a dearth of anti-inflammatory substance. If the theory could be accepted in certain cases, it is nevertheless inap- plicable as a general rule. In all those affections which are characterized by the absence of leucocytes upon the field of battle there is certainly no lack of inflammation. The very reverse obtains. In anthrax affecting small mammals, just as in the vibrionic septicaemia of pigeons and guinea-pigs, and other analogous diseases, we find that there is a very distinct dilatation of the vessels, accompanied by great exudation ; the inflammatory reaction is well marked ; nothing is wanting save the determination of the white cor- puscles. Or, employing yet further that affection which is, as it were, the touchstone of the bacteriologist, a still clearer proof of our contention is to be gained if we inoculate a rabbit on the one ear with a small quantity of virulent, on the other with a like quantity of attenuated, anthrax virus. In the course of a few hours the external signs of inflammation are far more conspicuous in the former ; the vessels are greatly enlarged and there is literally a huge exudation of clear serous fluid into the part ; in the latter the external signs are less prominent, but examination of the seat of inocu- lation shows it to be packed with leucocytes. Consequently, the phenome- non we are discussing is to be explained, not by an absence of the inflamma- tory process, but much more satisfactorily by a negative chemiotaxis of the leucocytes, which, instead of being attracted by the bacterial products, are repelled ; where the animal is vaccinated or refractory a much slighter in- flammation is sufficient to produce an abundant emigration of the leu- cocytes. Recently Behring has brought forward another view which would ex- plain immunity in a wholly different way. According to him, the bac- teria can live, and even preserve their virulence, in the refractory organism, I Mit the toxines excreted by them now undergo a modification so as to be rendered completely inoffensive for the animal. And to this "toxicide property " of the organism is to be attributed the essential quality of the immune state. It is impossible to pronounce upon the arguments that have led up to this theory, for as vet they have not been circumstantially set forth ; but already one can declare that such a theory is in no wise applicable to the phenomena of immunity in general. In three diseases remarkable for their u renounced toxic character — vibrionic septicaemia, pyocyanic dis- MM, a nd hog cholera affecting the rabbit- as shown by the experiments of Charrm, Gamalem, and Selander, the toxines are so little attacked by the re- fractory organism that the same quant ity of those substances (freed from bacteria) suffices to kill an animal very susceptible to one or other disease, SUSCEPTIBILITY AND IMMUNITY. 265 and an animal vaccinated against it and thus completely immune. So, too, non-fatal doses of these toxines produce in animals of the two categories the same febrile and inflammatory reactions. The proof is clear that there is no special destruction of toxines in the refractory animal, and that the ' ' toxicide property," if it exists, is not one whit more developed after vaccination than before. Passing- in review all these counter theories, we see that each of them can only be applied to a certain number of facts ; in some an attenu- ating or even bactericidal influence of the juices is relied upon, in others an anti-inflammatory action, in yet others a toxicide property. Still the pha- gocytic reaction is the only constant in all those cases of immunity and recovery that have as yet been sufficiently studied, and while certain of the factors mentioned (the attenuating and toxicide properties) do not in the least touch upon the continued existence or otherwise of the microorganism, the bactericidal power of the phagocyte puts an end to the parasite itself, and thus at a given moment prevents further manifestation of its virulence, or preserves the animal attacked at a time when the toxicide properties would be found wanting, and the microbe remaining alive would consequently gain the upper hand. But while thus placing before you the important part played by the pha- gocytes, I do not wish it to be thought that these cells are unaided in their contest by other defensive means possessed by the organism. This is far from being my view. Thus, in the febrile reaction, we see a puissant auxil- iary very definitely favoring the work of the phagocytes. This febrile re- action has only to be inhibited — as was done by M. Pasteur in the anthrax of fowls — and animals naturally refractory to the affection succumb to the ravages of the bacilli. It is not possible at the present time to state fully and accurately all these influences which are associated in aiding phago- cytic action, but already we have the right to maintain that, in the prop- erty of its amoeboid cells to include and to destroy microorganisms, the animal body possesses a formidable means of resistance and defence against these infectious agents.1 We are disposed to agree with Metschnikoff in his final conclu- sion,,as above stated in italics. But in view of experimental evi- dence, to be referred to later, we cannot accept the so-called Metsch- nikoff theory as a sufficient explanation for the facts relating to natural and acquired immunity in general, and must regard phago- cytosis simply as a factor which, in certain infectious diseases, ap- pears to play an important part in enabling immune animals to resist invasion by pathogenic bacteria. Going back to the demonstrated fact that susceptible animals may be made immune by inoculating them with the toxic products pro- duced during the growth of certain pathogenic bacteria, we may suppose either that immunity results from the continued presence of these toxic products in the body of the inoculated animal, or from a tolerance acquired at the time of the inoculation and subsequently retained — by transmission from cell to cell, as heretofore suggested. Under the first hypothesis — retention theory — immunity may be ex- plained as due to a continued tolerance on the part of the cellular ele- ments of the body to the toxic substances introduced and retained ; or to the effect of these retained toxic products in destroying the pathogenic bacteria, or in neutralizing their products when these are 1 From the British Medical Journal. SUSCEPTIBILITY AND IMMUNITY. subsequently introduced into the body of the immune animal. We <3annot understand how toxic substances introduced in the first in- stance can neutralize substances of the same kind introduced at a later date. There is something in the blood of the rat which, accord- ing to Behring, neutralizes the toxic substances present in a filtered culture of the tetanus bacillus ; but whatever this substance may be, it is evidently different from the toxic substance which it destroys, and there is nothing in chemistry to justify the supposition last made. Is it, then, by destroying the pathogenic microorganism that these inoculated and retained toxic products preserve the animal from future infection ? Opposed to this supposition is the fact that the blood of an animal made immune in this way, when removed from the body, does not prove to have increased germicidal power as compared with that of a susceptible animal of the same species. Again, these same toxic substances in cultures of the anthrax bacillus, the tetanus bacillus, the diphtheria bacillus, etc. , do not destroy the pathogenic germ after weeks or months of exposure. And when we inoculate a susceptible animal with a virulent culture of one of these microorganisms, the toxic substances present do not prevent the rapid development of the bacillus ; indeed, instead of proving a germicide, they favor its development, which is more abundant and rapid than when attenuated cultures containing less of the toxic material are used for the inoculation. In view of these facts we are unable to adopt the view that acquired immunity results from the direct action of the products of bacterial growth, introduced and retained in the body of the immune animal, upon the pathogenic microorganism when subsequently introduced or upon its toxic products. But there is another explanation which, although it may appear a priori to be quite improbable, has the support of recent experimen- tal evidence. This is the supposition that some substance is formed i n the bod i i <>f I lie immune animal which neutralizes the toxic products of the pathogenic microorganism. How the presence of these toxic products in the first instance brings about the formation of an "antitoxin" by which they are neutralized is still a mystery; but that such a substance is formed appears to be proved by the ex- peri ni« nu of Ogata, Behring and Kitasato, Tizzoni and Cattani, G. and F. Klemperer, and others. Ogata and Jasuhara, in a series of experiments made in the Hy- gienic Institute at Tokio (1890), discovered the important fact that the blood of an animal immune against anthrax contains some sub- stance which neutralizes the toxic products of the anthrax bacillus. When cultures were made in the blood of dogs, frogs, or of white rats, which animals have a natural immunity against anthrax, they were found not to kill mice inoculated with them. Further experi- SUSCEPTIBILITY AND IMMUNITY. 267 ments showed that mice inoculated with virulent anthrax cultures did not succumb to anthrax septicaemia if they received at the same time a subcutaneous injection of a small quantity of the blood of an immune animal. So small a dose as one drop of frog's blood or one- half drop of dog's blood proved to be sufficient to protect a mouse from the fatal effect of an anthrax inoculation. And the protective inoculation was effective when made as long as seventy-two hours before or five hours after infection with an anthrax culture. Fur- ther, it was found that mice which had survived anthrax infection as a result of this treatment were immune at a later date (after several weeks) when inoculated with a virulent culture of the anthrax bacillus. Behring and Kitasato have obtained similar results in their ex- periments upon tetanus and diphtheria, and have shown that the blood of an immune animal, added to virulent cultures before in- oculation into susceptible animals, neutralizes the pathogenic power of these cultures. . They have shown by experiment that the blood of a rabbit which has an acquired immunity against tetanus, mixed with the virulent filtrate from a culture of the tetanus bacillus, neutralizes its toxic power. One cubic centimetre of this filtrate was mixed with five cubic centimetres of serum from the blood of an immune rabbit and allowed to stand for twenty-four hours ; 0. 2 cubic centimetre of this injected into a mouse was without effect, while 0.0001 cubic centi- metre of the filtrate without such admixture was infallibly fatal to mice. The mice inoculated with this mixture remained immune for forty to fifty days, after which they gradually lost their immunity. The blood or serum from an immune rabbit, when preserved in a dark, cool place, retained its power of neutralizing the tetanus tox- albumin for about a week, after which time it gradually lost this power. The blood of chickens, which have a natural immunity against tetanus, was found not to have a similar power. Behring and Kitasato have also shown that the serum of a diphtheria-immune rabbit destroys the potent toxalbumin in diphtheria cultures. It does not, however, possess any germicidal power against the diph- theria bacillus. Ogata, in 1891, reported that he had succeeded in isolating from the blood of dogs and of chickens a substance to which he ascribes the nat- ural immunity of these animals from certain infectious diseases, and the power of their blood to protect susceptible animals from the same diseases. This substance is soluble in water and in glycerin, but in- soluble in alcohol or ether, by which it is precipitated without being destroyed. Its activity is neutralized by acids, but not by weak alkaline solutions. Ogata supposes the substance isolated by him to SUSCEPTIBILITY AND IMMUNITY. be the active agent in blood serum by which certain pathogenic bac- teria are destroyed, as shown by the experiments of Nuttall, Buchner, and others. Hankin had previously isolated an albuminoid sub- stance from the spleen and blood of the rat, to which he ascribed the immunity of this animal from anthrax. This substance, according to the author named, is a globulin; it is insoluble in alcohol and in distilled water, and does not dialyze. Tizzoni and Cattani ascribe the protection of animals which have acquired an immunity against tetanus to the presence of an albumi- nous substance which they call the tetanus-antitoxin. This they have isolated from the blood of immune animals. They arrive at the conclusion that it is a globulin, or a substance which is carried down with the globulin precipitate, and that it is different from the globulin, above referred to, obtained by Hankin from animals im- mune against anthrax. G. and F. Klemperer, in 1891, published an important memoir in which they gave an account of their researches relating to the ques- tion of immunity, etc., in animals subject to the form of septicaemia produced by the Micrococcus pneumonia crouposaB. They were able to produce immunity in susceptible animals by introducing into their bodies filtered cultures of this micrococcus, and proved by experiment that this immunity had a duration of at least six months. They arrived at the conclusion that the immunity induced by injecting fil- tered cultures is not directly due to the toxic substances present in these cultures, but that they cause the production in the tissues of an antitoxin which has the power of neutralizing their pathogenic action. The toxic substance present in cultures of the "diplococcus of pneumonia" they call " pneumotoxin" ; the substance produced in the body of an artificially immune animal, by which this pneumo- toxin is destroyed if subsequently introduced, they call " anti -pneumo- toxin." Emmerich, in a communication made at the meeting of the In- ternational Congress for Hygiene and Demography, in London, re- ported results which correspond with those of G. and F. Klemperer so far as the production of immunity is concerned, and also gave an account of experiments made by Donissen in which the injection of twenty to twenty-five cubic centimetres of blood or expressed tissue juices, filtered through porcelain, from an immune rabbit into an unprotected rabbit, subsequently to infection with a bouillon culture of "diplococcus pneumonia," prevented the development of fatal septicaemia. Even when the injection was made twelve to fifteen hours after infection, by inhalation, the animal recovered. Emmerich and Mastraum had previously reported similar results in experiments made upon mice with the Bacillus erysipelatos suis SUSCEPTIBILITY AND IMMUNITY. 269 (rothlauf bacillus). White mice are very susceptible to the patho- genic action of this bacillus. But mice which, subsequently to in- fection, were injected with the expressed and filtered tissue juices of an immune rabbit, recovered, while the control animals succumbed. According to Emmerich, the result in these experiments was due to a destruction of the pathogenic bacilli in the bodies of the infected animals ; and the statement is made that at the end of eight hours after the injection of the expressed tissue juices all bacilli in the body of the infected animal were dead. The same liquid did not, however, kill the bacilli when added to cultures external to the body of an animal. The inference, therefore, seems justified that the result de- pends, not upon a substance present in the expressed juices of an immune animal, but upon a substance formed in the body of the animal into which these juices are injected. We have, however, an example of induced immunity in which the result appears to depend directly upon the destruction of the pathogenic microorganism in the body of the immune animal. In guinea-pigs which have an acquired immunity against Vibrio Metsch- nikovi the blood serum has been proved to possess decided germicidal power for this "vibrio," whereas it multiplies readily in the blood serum of non-immune guinea-pigs (Behring and Nissen). There is experimental evidence that animals may acquire an arti- ficial immunity against the toxic action of certain toxalbumins from other sources than bacterial cultures. Thus Sewell (1887) has shown that a certain degree of tolerance to the action of rattlesnake venom may be established by inoculating susceptible animals with small doses of the " hemialbumose " to which it owes its toxic potency. These results have been confirmed by the more recent experiments Of Calmette (1894) and of Fraser (1895). In his paper detailing the results of his experiments the first-named author says : ' ' Animals may be immunized against the venom of serpents either by means of repeated injections of doses at first feeble and progressively stronger, or by means of successive injections of venom mixed with certain chemical substances, among which I mention especially chloride of gold and the hypo- chlorites of lime or of soda. "The serum of animals thus treated is at the same time preventive, anti- toxic, and therapeutic, exactly as is that of animals immunized against diphtheria or tetanus. "If we inoculate ascertain number of rabbits, under the skin of the thigh, with the same dose, one milligramme of cobra venom for example, and if we treat all of these animals with the exception of some for control, by subcutaneous or intraperitoneal injections of the serum of rabbits im- munized against four milligrammes of the same venom, all of the control animals not treated will die within three or four hours, while all of the animals will recover which receive five cubic centimetres of the therapeutic serum within an hour after receiving the venom." In this connection we may remark that there is some evidence to 270 SUSCEPTIBILITY AND IMMUNITY. show that persons who are repeatedly stung by certain poisonous in- sects—mosquitoes, bees — acquire a greater or less degree of immu- nity from the distressing local effects of their stings. Ehrlich, of Berlin, in 1891, reported his success in establishing immunity in guinea-pigs against two toxalbumins of vegetable origin: one — ricin— from the castor-oil bean (Ricinus communis), the other— abrin— from the jequirity bean. The toxic potency of ricin is somewhat greater than that of abrin, and it is esti- mated by Ehrlich that one gramme of this substance would suffice to kill one and a half millions of guinea-pigs. When injected be- neath the skin, in dilute solution, it produces intense local inflamma- tion, resulting in necrosis of the tissues. Mice are less susceptible than guinea-pigs and are more easily made immune. This is most readily effected by giving them small and gradually increasing doses with their food. As a result of this treatment the animal resists subcutaneous injections of two hundred to four hundred times the fatal dose for animals not having this artificial immunity. The fatal dose of abrin is about double that of ricin. When injected into mice in the proportion of one cubic centimetre to twenty grammes of body weight a solution of one part in one hundred thousand of water proved to be a fatal dose. The local effects are also less pronounced when solutions of abrin are used ; they consist principally in an ex- tensive induration of the tissues around the point of injection and a subsequent falling off of the hair over this indurated area. When introduced into the conjunctival sac, however, abrin produces a local inflammation in smaller amounts than ricin, a solution of 1 : 800 being sufficient to cause a decided but temporary conjunctivitis. Solutions of 1 : 50 or 1 : 100 of either of these toxalbumins, introduced into the eye of a mouse, give rise to a panophthalmitis which com- monly results in destruction of the eye. But in mice which have been rendered immune by feeding them for several weeks with food containing one of these toxalbumins, no reaction follows the intro- duction into the eye of the strongest possible solution, or of a paste made by adding abrin to a little ten-per-cent salt solution. Ehrlich gives the following explanation of the remarkable degree of im- munity established in his experiments by the method mentioned: " All of these phenomena depend, as may be easily shown, upon the fact that the blood contains a body — antiabrin — which completely neutralizes the action of the abrin, probably by destroying this body." In a more recent paper Ehrlich has given an account of subse- quent experiments which show that the young of mice which have an acquired immunity for these vegetable toxalbumins may acquire immunity from the ingestion of the mother's milk ; and also that immunity against tetanus may be acquired in a very brief time by SUSCEPTIBILITY AND IMMUNITY. 271 young mice through their mother's milk. In his tetanus experi- ments Ehrlich used blood serum from an immune horse to give im- munity to the mother mouse when her young were already seven- teen days old. Of this blood serum two cubic centimetres were injected at a time on two successive days. The day after the first injection one of the sucklings received a tetanus inoculation by means of a splinter of wood to which spores were attached. The animal remained in good health, while a much larger control mouse inoculated in the same way died of tetanus at the end of twenty-six hours. Other sucklings, inoculated at the end of forty-eight and of seventy-two hours after the mother had received the injection of blood serum, likewise remained in good health, while other control mice died. The possibility of conferring immunity by means of the milk of an immune animal is further shown by the experiments of Brieger and Ehrlich (1892). A female goat was immunized against tetanus by the daily injection of " thymus-tetanus bouillon." The dose was gradually increased from 0.2 cubic centimetre to 10 cubic centimetres. At the end of thirty-seven days a mouse, which received 0.1 cubic centimetre of the milk of this goat in the cavity of the abdomen, proved to be immune against tetanus. Further experiments gave a similar result, even when the milk of the goat was not injected into the peritoneal cavity of the mouse until several hours after inocu- lation with a virulent culture of the tetanus bacillus. When the casein was separated the milk retained its full im- munizing activity, and by concentration in vacuo a thick milk was obtained which had a very high immunization value — 0.2 cubic centimetre of this milk protected a mouse against forty-eight times the lethal dose of a tetanus culture. In a subsequent communication (1893) Brieger and Ehrlich de- scribe their method of obtaining the antitoxin of tetanus from milk in a more concentrated form. They found by experiment that it was precipitated by ammonium sulphate and magnesium sulphate. From twenty-seven to thirty per cent of ammonium sulphate added to milk caused a precipitation of the greater part of the antitoxin. This pre- cipitate was dissolved in water, dialyzed in running water, then filtered and evaporated in shallow dishes at 35° C. in a vacuum. One litre of milk from an immune goat gave about one gramme of a transparent, yellowish-white precipitate, which contained fourteen per cent of ammonium sulphate. This precipitate had from four hundred to six hundred times the potency of the milk from which it was obtained in neutralizing the tetanus toxin. In a still later communication (1893) Brieger and Cohn give an improved method of separating the antitoxin from the precipitate 272 SUSCEPTIBILITY AND IMMUNITY. thrown down with ammonium sulphate. The finely pulverized pre- cipitate is shaken up with pure chloroform, and when this is allowed to stand the antitoxin rises to the surface while the ammonium salt sinks to the bottom. By filling the vessel to the margin with chloro- form, the antitoxin floating on the surface can be skimmed off, after which it quickly dries. By this method the considerable loss which occurred in the dialyzer, used in the previously described method, is avoided. A most interesting question presents itself in connection with the discovery of the antitoxins. Does the animal which is immune from the toxic action of any particular toxalbumin also have an im- munity for other toxic proteids of the same class? The experimental evidence on record indicates that it does not. In Ehrlich's experi- ments with ricin and abrin he ascertained that an animal which had been made immune against one of these subtances was quite as sus- ceptible to the toxic action of the other as if it did not possess this immunity, i.e., the antitoxin of ricin does not destroy abrin, and vice versa. As an illustration of the fact, he states that in one ex- periment a rabbit was made immune for ricin to such an extent that the introduction into its eye of this substance in powder produced no inflammatory reaction ; but the subsequent introduction of a solution of abrin, of 1 to 10,000, caused a violent inflammation. Evidently these facts are of the same order as those relating to immunity from infectious diseases, and, taken in connection with the experimental data previously referred to, give strong support to the view that the morbid phenomena in all diseases of this class are due to the specific toxic action of substances resembling the toxalbumins already discovered ; and that acquired immunity from any one of these diseases results from the formation of an antitoxin in the body of the immune animal. Hankin calls these substances produced in the bodies of immune animals " defensive proteids," and proposes to classify them as fol- lows : First, those occurring naturally in normal animals, which he calls sozins ; second, those occurring in animals that have acquired an artificial immunity — these he calls phylaxins. Each of these classes of defensive proteids is further subdivided into those which act upon the pathogenic microorganism itself and those which act upon its toxic products. These subclasses are distinguished by the prefixes myco and t oxo attached to the class name. In accordance with this classification a mycosozin is a defensive proteid, found in the body of a normal animal, which has the power of destroying bacteria. A toxosozin is a defensive proteid, found in the body of a normal SUSCEPTIBILITY AND IMMUNITY. 273 animal, which has the power of destroying the toxic products of bac- terial growth. A mycophylaxin is a defensive proteid produced in the body of an animal which has an acquired immunity for a given infectious disease, which has the power of destroying the pathogenic bacteria to which the disease is due. A toxophylaxin is a defensive proteid produced in the body of an animal which has an acquired immunity for a given infectious disease, which has the power of destroying the toxic products of the pathogenic bacteria to which the disease is due. Buchner had previously proposed the name " alexines " for these defensive proteids. The importance of the experimental evidence above referred to in explaining the phenomena of natural and acquired immunity is ap- parent. The facts stated also suggest a rational explanation of re- covery from an attack of an acute infectious disease. But the idea that during such an attack an antidote to the disease poison is de- veloped in the tissues is yet so novel, and the experimental evidence in support of this view is of such recent date, that it would be pre- mature to accept this explanation as applying to immunity in gene- ral. It seems difficult to believe that an individual who has passed through attacks of measles, mumps, whooping cough, scarlet fever, small-pox, etc. , has in his blood or tissues a store of the antitoxine of each of these diseases, formed during the attack and retained during the remainder of his life, or continuously produced so long as the immunity lasts. Moreover, in those diseases to which the experi- mental evidence above recorded relates — diphtheria, tetanus, pneu- monia— as they occur in man, no lasting immunity has been shown to result from a single attack, and in this regard they do not come into the same class with the eruptive fevers and other diseases in which a single attack usually protects during the lifetime of the in- dividual. In those instances in which acquired immunity has been shown to be due to the production in the body of the immune animal of an antitoxin, it is still uncertain whether there is a continuous produc- tion of the protective proteid, or whether that formed during the attack remains in the body during the subsequent immunity. The latter supposition appears at first thought improbable ; but when we remember that the protective proteids which have been isolated by Hankin from the blood and spleen of rats, and by Tizzoni and Cat- tani from the blood of animals made immune against tetanus, do not dialyze, it does not seem impossible that these substances might be retained indefinitely within the blood-vessels. On the other hand, the passage of the tetanus antitoxin into the mother's milk, as 18 274 SUSCEPTIBILITY AND IMMUNITY. shown by Ehrlich's experiments upon mice, indicates a continuous supply, otherwise the immunity of the mother would soon be lost. The writer has obtained (May, 1892) experimental evidence that the blood of vaccinated, and consequently immune, calves contains something which neutralizes the specific virulence of vaccine virus, both bovine and humanized. Four drops of blood serum from a calf which had been vaccinated two weeks previously, mixed with one drop of liquid lymph recently collected in a capillary tube, after con- tact for one hour was used to vaccinate a calf ; the same animal was also vaccinated with lymph, preserved on three quills, which was mixed with four drops of serum from the immune calf and left for one hour. The result of these vaccinations was entirely negative, while vaccinations upon the same calf made with virus from the same source, and mixed with the same amount of blood serum from a non-immune calf, gave a completely successful and typical result, The experimental evidence detailed shows that in certain dis- eases acquired immunity depends upon the formation of anti- toxins in the bodies of immune animals- As secondary fac- tors it is probable that tolerance to the toxic products of pathogenic bacteria and phagocytosis have considerable importance, but it is evident that the principal role cannot be assigned to these agencies. As a rule the antitoxins have no bactericidal action; but it has been shown by the experiments of Gamaleia, Pfeiffer, and others, that in animals which have an acquired immunity against the spiril- lum of Asiatic cholera and against spirillum Metschnikovi, there is a decided increase in the bactericidal power of the blood serum, and that immunity probably depends upon this fact. The researches of Metschnikoff upon hog cholera, of Issaef upon pneumonia, and of Sanarelli upon typhoid fever indicate that the immunity conferred upon susceptible animals by protective inocula- tions is not due to an antitoxin but to a substance present in the blood of immune individuals which acts directly upon the pathogenic microorganism, as is the case in cholera-immune animals. The ani- mals immunized are said to be quite as sensitive to the action of the bacterial poisons as are those which have not received protective inoculations. "Their serum does not protect against the toxin, but against the microbe" (Roux). PLATE IV. FIGS. 1, 2, and 3. — Leucocytes from the spleen of an inoculated monkey, containing Spirillum Obermeieri. (Soudake witch.) Fias. 4 and 5. —Leucocytes (" macrophages ") from a preparation of muscle from a pigeon which succumbed to an anthrax inoculation. In Fig. 4 the bacilli are deeply stained ; in Fig. 5 they are pale. (Metschnikoff.) FIG. 6. — Leucocyte from a frog seventy-two hours after the injection of anthrax spores. (Trapeznikoff.) FIGS. 7 and 8. — Leucocytes from a chicken four hours after the injection of anthrax spores. (Trapeznikoff.) Brt. pp. 174- 71S. STEKNBERG'S BACTERlOIflGT. P 1 at e TV. Fig. I. Fig. 2. 3. Fig. 5. Fig .6. Fio 7. PHAGOCYTES IV. PYOGEKEC BACTERIA. THE demonstration made by Ogston, Rosenbach, Passet, and others that micrococci are constantly present in the pus of acute abscesses, led to the inference that there can be no pus formation in the absence of microorganisms of this class. But it is now well established, by the experiments of Grawitz, De Bary, Steinhaus, Scheurlen, Kaufmann, and others, that this inference was a mis- taken one, and that certain chemical substances introduced beneath the skin give rise to pus formation quite independently of bacteria. Among the substances tested which have given a positive result are nitrate of silver, oil of turpentine, strong liquor ammonias, cada- verin, etc. The demonstration has also been made by numerous in- vestigators that cultures of pus cocci, when sterilized by heat, still give rise to pus formation when injected subcutaneously. This was first established by Pasteur in 1878, who found that sterilized cul- tures of his " microbe generateur du pus " induced suppuration as well as cultures containing the living microbe. This fact lias since been confirmed, as regards the pus staphylococci and various bacilli, by a number of bacteriologists. Wyssokowitsch produced abscesses containing sterile pus by injecting subcutaneously agar cultures of the anthrax bacillus sterilized by heat. Buchner obtained similar results in a series of forty experiments from the injection of steril- ized cultures of Friedlander's bacillus (" pneumococcus ")> and has shown that the pus-forming property belongs to the bacterial cells and not to a soluble chemical substance produced by them. When cultures were filtered by means of a Chamberlain filter the clear fluid which passed through the porous porcelain was without effect, while the dead bacteria retained by the filter produced aseptic pus infiltration in the subcutaneous tissues within forty-eight hours after having been injected. Subsequent experiments gave similar results with seventeen different species tested, including Staphylo- coccus pyogenes aureus, Staphylococcus cereus flavus, Sarcina auran- tiaca, Bacillus prodigiosus, Bacillus Fitzianus, Bacillus subtilis, Bacillus coli communis, Bacillus acidi lactici, etc. From the experi- 276 PYOGENIC BACTERIA. ments made to determine the exact cause of pus formation following the injection of sterilized cultures Buchner arrives at the conclusion that it is due to the albuminous contents of the bacterial cells. While it is demonstrated that a large number of microorganisms, either living or in sterilized cultures, may give rise to the formation *• "fr^of pus, the extended researches of Rosenbach, Passet, and other bacteriologists show that few species are usually concerned in the formation of acute abscesses, furuncles, etc., in man. Of these the two most important, by reason of their frequent occurrence and path- ogenic power, are Staphylococcus pyogenes aureus and Strepto- coccus pyogenes ; next to these comes Staphylococcus pyogenes albus, and the following species are occasionally found : Staphylo- coccus pyogenes citreus, Staphylococcus cereus flavus, Staphylococcus cereus albus, Micrococcus tenuis, Bacillus pyogenes f cetidus, Micro- coccus tetragenus, Micrococcus pneumonias crouposae. Two or more species are often found in the same abscess ; thus Passet, in thirty- three cases of acute abscess, found Staphylococcus aureus and albus associated in eleven, albus alone in four, albus and citreus in two, Streptococcus pyogenes alone in eight, albus and streptococcus in one, and albus, citreus, and streptococcus in one. Hoff a found, in twenty-two cases of inguinal bubo, aureus in ten, albus in nine, and citreus in three. Bumm, in ten cases of puerperal mastitis, found aureus in seven and Streptococcus pyogenes in three. Rosenbach found staphylococci alone sixteen times, Streptococcus pyogenes alone fifteen times, staphylococci and streptococci associated five times, and Micrococcus tenuis three times in thirty-nine acute abscesses and phlegmons examined by him. . ,oj Robb and Ghrisky have shown that under the most rigid antisep- ^ tic treatment microorganisms are constantly found attached to su- tures when these are removed from wounds made by the surgeon, and that a skin abscess frequently results from the presence of the most common of these microorganisms — Staphylococcus epidermidis albus. The authors named state their conclusions as follows : *' A wound, at some time of its existence, always contains organisms. They occur either on the stitches or in the secretions. 44 The number of bacteria is influenced by the constricting action of the ligatures or drainage tube, or anything interfering with the circulation of the^tissues. 4 'The virulence of the organisms present will influence the progress of the wound. "The bpdv temperature is invariably elevated if the bacteria are viru- lent; and, indeed, in cases where many of the less virulent organisms are found, almost without exception there is some rise of temperature." The organism most frequently found — Staphylococcus epidermi- PYOGENIC BACTERIA. 277 dis albus — has but slight virulence. Out of forty-five cases in which a bacteriological examination was made this micrococcus was ob- tained in pure cultures in thirty -three ; in five cases it was associated with Staphylococcus pyogenes aureus, in one case with Streptococ- cus pyogenes, in three cases Streptococcus pyogenes was obtained alone. In abscesses resulting from inflammation of the middle ear the micrococcus commonly known under the name of " diplococcus pneumonias " — Micrococcus pneumoniae crouposae — has been obtained in pure cultures in a considerable number of cases when the pus has been examined immediately after paracentesis of the tympanic mem- brane. We shall not, however, describe this among the pyogenic bacteria, but will give an account of it in the following section (Bac- teria in Croupous Pneumonia, etc.). Bacillus pyocyaneus, which is described by some authors among the pyogenic bacteria, is found only in the pus of open wounds, where its presence is evidently acci- dental. We shall describe it among the chromogenic saprophytes. 1. STAPHYLOCOCCUS PYOGENES AUREUS. Synonym. — Micrococcus of infectious osteomyelitis (Becker). Observed by Ogston (1881) in the pus of acute abscesses, but not differentiated from the associated staphylococci and the streptococ- cus of pus. Obtained by Becker from the pus of osteomyelitis (1883). Isolated from the pus of acute abscesses and accurately described by Rosenbach (1884) and by Passet (1885). The Staphylococcus pyogenes aureus is a facultative parasite, and / is the most common pyogenic micrococcus found in suppurative pro- cesses generally. But it is also a common and widely distributed saprophyte, which finds the conditions necessary for its existence on the external surface of the human body and of moist mucous mem- branes. This is shown by the researches of numerous bacteriolo- gists. Thus Ullmann found it upon the skin and in the secretions of the mouth of healthy persons, and also in the dust of occupied apart- ments, in water, etc.; Bockhart obtained it in cultures from the surface of the body and from the dirt beneath the finger nails of healthy persons ; Biondi, Vignal, and others in the salivary secre- tions ; B. Frankel in mucus from the pharynx ; Von Besser and Wright in nasal mucus ; Escherich in the alvine discharges of healthy infants ; C. Frankel in the air ; and Liibbert in the soil. Its presence in the air, in water, or in the soil is, however, quite excep- tional, and is probably to be considered the result of accident, its normal habitat as a saprophyte appearing to be rather upon the sur- face of the b9dy and of mucous membranes. v.o*^ ^ , gono- varies from 0.8 to- 1.6 w in the long dia- cocci in PUS cells and epithelial c*n , _ from case of gonorrhoeal ophthal- meter — average about 1.25 jw— and from mia; c, form and mode of division 0.6 to 0.8 >u in the line of the interspace of gonococci-schematic. between the biscuit-shaped elements, which sometimes present a slight concavity of the flattened surfaces. Mul- tiplication occurs alternately in two planes, and as a result of this groups of four are frequently observed. But diplococci are more numerous and are considered as the characteristic mode of grouping. Single, spherical, undivided cells are rarely seen. It must be remembered that the morphology of this micrococcus as above described does not suffice to distinguish it, for Bumm has shown that " the biscuit form is not at all specific for the gonococcus, but is shared with it by a number of microorganisms, which consist of two hemispherical elements with the flattened surfaces facing each 296 PYOGENIC BACTERIA. other and separated by a cleft, and some of these correspond in their morphology, in every detail, with the gonococcus." Stains quickly with the basic aniline colors, especially with methyl violet, gentian violet, and f uchsin ; not so quickly with methylene blue, which is, however, one of the most satisfactory staining agents for demonstrating its presence in pus. Beautiful double-stained preparations may be made irom gonorrhoeal pus, spread upon a cover glass and " fixed/' secundum artem, by the use of methylene blue and eosin. Does not stain by Gram's method— i.e., the cocci are decolorized, after having been stained with an ani- line color, by being immersed in the iodine solution employed in Gram's method of staining. But this character cannot be depended upon alone for establishing the diagnosis, for Bumm has shown that Fia. 86.—" Gonococcus " in gonorrhoeal pus. From a photomicrograph by Frankel and Pfeiffer. X 1,000. other diplococci are occasionally found in gonorrhoeal pus which do not stain by this method. It serves to distinguish them, however, from the common pus cocci heretofore described — Staphylococcus aureus, Staphylococcus albus, Staphylococcus citreus — which retain their color when treated in the same way. A more trustworthy diagnostic character is that these biscuit-shaped diplococci are found within the pus cells, sometimes one or two pairs only, but more frequently in considerable numbers, and occasionally iii such numbers as to com- pletely fill the cell. No similar picture is presented by pus from any other source, with the exception of that from a form of " puerperal cystitis " which Bumm has described. But in this the diplococci contained in the pus cells were to be distinguished by the fact that they retained their color when treated by Gram's method. Owing PYOGENIC BACTERIA. 297 to the difficulty of cultivating this micrococcus, and the importance, under certain circumstances, of not making a mistake in its diag- nosis, these characters are of exceptional value. Biological Characters. — Bumm (1885) first succeeded in culti- vating the " gonococcus " upon human blood serum, obtained from the placenta of a recently delivered woman. He found that the cul- tures thrive best in a moist atmosphere at 30° to 34° C. The growth under the most favorable conditions is slow, and frequently no devel- opment occurs when pus containing numerous gonococci is placed upon blood serum in an incubating oven; or after a slight multi- plication development ceases and the cocci undergo degenerative changes and quickly disappear. Cultures upon the surface of blood serum form a very thin, often scarcely visible layer, with a smooth, moist, shining surface, and by reflected light a grayish-yellow color. The growth at the end of twenty-four hours may extend for a distance of a millimetre along the line of inoculation, but at the end of two or three days no fur- ther development occurs and the cocci soon lose their vitality. This micrococcus, then, is aerobic. Whether it may also be a facultative anaerobic has not been definitely determined, but it does not grow along the line of puncture when stick cultures are made in blood se- rum. Its rapid and abundant multiplication in gonorrhoeal infection of mucous membranes, and the difficulties attending its cultivation in artificial media, show that the gonococcus is a strict parasite. Lestikow and Loffler, prior to the publication of Bumm's impor- tant monograph, had reported successful results in cultivating the gonococcus upon a mixture of blood serum and gelatin. Bockhart has since recommended a mixture of nutrient agar (two parts), lique- fied at a temperature of 50° C., with blood serum (two to three parts) at 20° C. By quickly mixing with this a little pus containing the gonococcus he was able to obtain colonies upon plate cultures, made by pouring the liquid medium upon sterile glass plates in the usual manner. Ghon and Schlagenhaufer in 1893 reported that they obtained good results by adding phosphate of soda to blood-serum agar, made according to the method of Wertheim — one part of human blood serum from the placenta to two or three parts of nutrient agar. Also that they were successful in cultivating the gonococcus in an acid medium made by adding one part of urine to two of nutrient agar (two per cent). Turro (1894) has since published the results of his experiments relating to the cultivation of this micrococcus in acid media. According to him it grows in normal urine, either with or without the addition of peptone (one per cent) ; also in acid gelatin, prepared in the usual way but without neutralization (?). 298 PYOGENIC BACTERIA. Turro also claims to have produced specific urethritis in dogs by inoculation with his cultures. Heiman (1895) as a result of an ex- tended experimental research, arrives at the conclusion that "the diplococcus described by Turro in connection with his acid media is not the gonococcus." His inoculation experiments in dogs, made with pure cultures of the gonococcus, gave an entirely negative result. For the cultivation of the gonococcus, Heiman recommends a medium made from "chest serum" obtained from a patient suffering with hydrothorax or acute pleurisy. This was found to be superior to placenta serum, sheep-blood serum, or peritoneum serum, because of the great amount of serum albumin which it contains. Two per cent of agar, one per cent of peptone, and one-half per cent of sodium chloride were added to the chest serum, and the medium was sterilized by "fractional sterilization." Fio. 87.— Gonorrhoeal conjunctivitis, second day of sickness; section through the mucous mem- brane of upper eyelid; invasion of the epithelial layer by gonococci. (Bumm.) Schrotter and Winkler (1890) report their success in cultivating the gonococcus upon albumin from the egg of the pewit — "Kibitz." In the culture oven at 38° C. a thin, transparent, whitish layer was already visible at the end of six hours and rapidly extended ; the growth was less abundant at the end of three days, and had entirely ceased by the fifth day. Attempts to cultivate the same microor- ganism in albumin from hens' eggs gave a negative result. Aufuso (1891) has cultivated the gonococcus in fluid obtained from the knee joint in a case of chronic synovitis, but failed to culti- vate it in the fluid of ascites. A culture of the twelfth generation made upon the culture medium mentioned, solidified by heat, was introduced into the urethra of a healthy man and gave rise to a characteristic attack of gonorrhoea. Development does not occur below 25° or above 38° C. The writer has shown that a temperature of 60° C. maintained for ten minutes destroys the infective virulence of gonorrhceal pus. Pathogenesis.— That the gonococcus is the cause of the specific inflammation and purulent discharge characteristic of gonorrhoea is now generally admitted upon the experimental evidence obtained by PYOGENIC BACTERIA. 299 Bumm. Having succeeded in obtaining it in pure cultures from gonorrhoeal pus, he made successful inoculations in the healthy ure- thra in two cases— once with a third culture and once with one which had been transferred through twenty successive generations. In both cases a typical gonorrhoea developed as a result of the inocu- lation. The mucous membranes in man which are subject to gonorrhoeal infection are those of the urethra, the conjunctiva, the cervix uteri, and the vagina in children — the vagina in adults is not involved. Inoculations of gonorrhoeal pus into the vagina or conjunctival sac of the lower animals — dogs, rabbits, horses, apes — are without result. The very numerous researches which have been made by compe- tent bacteriologists show that the gonococcus is constantly present in gonorrhoeal discharges, and in view of the facts above stated its etio- logical import appears to be fully established. Bumm has studied the development of blennorrhoea neonatorum, and has shown that soon after infection the presence of gonococci may be demonstrated in the superficial epithelial cells of the mucous membrane and be- tween them ; that they soon penetrate to the deeper layers, and that by the end of forty-eight hours the entire epithelial layer is invaded by the diplococci, which penetrate by way of the connecting mate- rial— " Kittsubstance " — between the cells. They also multiply in the superficial layers of connective tissue and give rise to an inflam- matory reaction, which is shown by an abundant escape of leuco- cytes from the dilated capillary network. The penetration of the gonococci to the deeper layers of the mucous membrane of the ure- thra, and even to the corpus cavernosum, was observed by Bockhart in a case studied by him in which death occurred during an acute attack of gonorrhoea. But Bumm concludes from his researches that this is not usual, and that the invasion is commonly limited to the superficial layers of the mucous membrane. Staphylococcus pyogenes aureus is not infrequently associated with the gonococcus in late gonorrhoeal discharges, and the abscesses which occasionally develop as a complication of gonorrhoea, in the prostate, the inguinal glands, or around the urethra, are probably due to its presence, which has been demonstrated in the pus from such abscesses in a number of cases. The same is true of the joint affections and endocarditis which sometimes occur in the course of an attack of gonorrhoea. Although some authors have claimed to find the gonococcus in these so-called metastatic gonorrhoeal inflam- mations, the evidence is not satisfactory, and it seems probable that the Staphylococcus aureus is the usual microorganism concerned in these affections. V. BACTERIA IN CROUPOUS PNEUMONIA. THE following account of " The Etiology of Croupous Pneumo- nia " is from a paper read by the writer at the annual meeting of the Medical Society of the State of New York, at Albany, N. Y., Feb- ruary 6th, 1889 : Quain's "Dictionary of Medicine," says: servers that, like the specific fevers, it is due to a specific cause. Pneumonia, whilst differing from these fevers in not being- contagious, resembles them in the typical character of its clinical phenomena and, to a less extent, of its local lesion. The changes in the lung occurring in pneumonia cannot be induced by artificial injury of the organ, and it must therefore be admitted that there is something special in the inflammatory process." This "something special "has been demonstrated by recent researches, and it is the object of the present paper to give a historical account of the de- velopment of our knowledge with reference to this specific infectious agent, and of the experimental evidence upon which the claim is founded that the microorganism referred to bears an etiological relation to the disease in question. Evidently, if pneumonia is a specific infectious disease, the microorgan- ism which causes it is widely distributed, and the development of an attack depends rather upon secondary predisposing and exciting causes than upon the accidental introduction of the specific agent. It cannot be maintained that the disease, as a general rule, is transmitted from individual to individual — i.e., by personal contagion. Clinical expe- rience is entirely opposed to this view, although we have ample evidence that it may occur as an epidemic among individuals who are exposed to the same conditions of environment — as in jails, barracks, etc. Thus at Chris- tiania, Sweden, an epidemic of pneumonia occurred in 1847 in the prison, during which sixty-nine of the prisoners were attacked. And again in 1866 and 1867, during a period of six months (December, 1866, to May, 1867), a similar epidemic was observed in the same prison — sixty-two cases. Other prison epidemics recorded are those at Frankfort in 1875 (seventy -five cases) and in 1876 (ninety-eight cases) ; at Maringen in 1875 (eighty-three cases) and in 1878 (fifty-eight cases) ; at the prison of DAnsberg in 1880 (one hun- dred and sixty-one cases, with forty-six deaths, in a period of five months). Ajrain, we have numerous records of village epidemics and of epidemics confined to single houses. In outbreaks of this character, as in epidemics of typhoid fever, of cholera, and of yellow fever, there is a succession of cases occurring at different intervals, but it does not follow that these cases bear any direct relation to each other. On the contrary, everything indi- cates that, as in the diseases mentioned, in the presence of the infectious BACTERIA IN CROUPOUS PNEUMONIA. 301 agent common predisposing causes relating to the environment, acting upon persons having various degrees of resisting power, induce attacks at various intervals ; or it may be that in the presence of the specific cause and predis- posing influences an exciting cause, such as exposure to cold, alcoholic ex- cess, etc., is the immediate factor in the development of an attack. Without stopping to discuss further the facts relating to the epidemic prevalence of the disease under consideration, I call attention to the well- established fact that pneumonia prevails over a wide area of the inhabited surface of the earth, and that by far the larger number of cases occur inde- pendently of any recognized connection with previous cases, and often un- der circumstances in which such connection can be very positively excluded. And, on the other hand, the direct transmission of the disease by the sick to those most closely associated with them, as nurses, etc., if it occurs at all, is evidently a rare exception to the general rule. We must then conclude, as stated at the outset, that if pneumonia is a specific infectious disease the microorganism which causes it is widely dis- tributed. As a matter of fact, the pathogenic micrococcus which, from the evidence now at hand, appears to be the specific etiological agent in acute pneumonia has been found in the buccal secretions of healthy individuals in various parts of the world — in America, in France, in Italy, and in Ger- many, and no doubt more extended researches will show that it is extremely common. This statement may appear at the outset to make the view that the micro- coccus in question is the cause of croupous pneumonia quite untenable. For, it may be asked, how is it that the individuals who have this microor- ganism in their buccal secretions escape an attack of pneumonia ? In the present state of our knowledge this question no longer presents any serious difficulties. We know, for example, that the pus organisms— Staphylococcus pyogenes aureus, albus, and citreus — are very frequently found in the buc- cal secretions and on the surface of the body of healthy individuals, and that, although these micrococci are recognized as the cause of furuncles and of all sorts of acute abscesses, they only give rise to the formation of such abscesses under certain special conditions relating to the general health of the individual, or to a traumatism by which their introduction to vulnerable parts is effected. Again, the tetanus bacillus is a widely distributed micro- organism which has been found in the earth, and especially in rich loam, in various parts of the globe. But the hands of farmers and gardeners are con- stantly soiled with such earth without their contracting tetanus. In this instance it has long been recognized that a traumatism is an essential factor in the chain of events which leads to the development of tetanus, and now we believe, on satisfactory experimental evidence, that it is not the trauma- tism per se, or the injury to the nerves, or exposure to cold, which in certain cases gives rise to this infectious malady, but that the result depends upon the introduction of a specific infectious agent at the time the wound was re- ceived or subsequently — the tetanus bacillus of Nicolaier. In the case of the tubercle bacillus, also, it is extremely probable, in the light of our present knowledge, that this bacillus, in a living condition, not infrequently finds a lodgment in the mouth, upon the Schneiderian mucous membrane, or in the larger bronchial tubes of most individuals who live in populous communities. Here also the infectious agent is only one factor, although an essential one, in the production of the infectious disease. It must be introduced to the vulnerable location, and must find a favorable nidus in the tissues invaded. We have good reason to believe that in this, as well as in other infectious diseases, there are wide differences, inhe- rited or acquired, in the susceptibility of the tissues to invasion when the infectious agent has been introduced to a favorable location. In a paper read before the Pathological Society of Philadelphia in April, 1885, in discussing the relation of my Micrococcus Pasteuri to croupous pneumonia, I say: "It seems extremely probable that this micrococcus is concerned in the etiology of croupous pneumonia, and that the infectious 302 BACTERIA IN CROUPOUS PNEUMONIA. nature of this disease is due to its presence in the fibrinous exudate into the pulmonary alveoli. "But this cannot be considered as definitely established by the experi- ments which have thus far been made upon the lower animals. The con- stant ' presence of this micrococcus in the buccal secretions of healthy per- sons indicates that some other factor is required for the development of an attack of pneumonia; and it seems probable that this other factor acts by re- ducing the vital resisting power of the pulmonary tissues, and thus making them vulnerable to the attacks of the microbe. This supposition enables us to account for the development of the numerous cases of pneumonia which cannot be traced to infection from without. The germ being always pre- sent, auto-infection is liable to occur when, from alcoholism, sewer-gas poisoning, crowd poisoning, or any other depressing agency, the vitality of the tissues is reduced below the resisting point. We may suppose, also, that a reflex vaso- motor paralysis, affecting a single lobe of the lung, for exam- ple, and induced by exposure to cold, may so reduce the resisting power of the pulmonary tissue as to permit this micrococcus to produce its character- istic effects. " Again, we may suppose that a person whose vital resisting power is reduced by any of the causes mentioned may be attacked by pneumonia from external infection with material containing a pathogenic variety of this micrococcus having a potency, permanent or acquired, greater than that possessed by the same organism in normal buccal secretions." This is the theory by which I have attempted to explain the etiological role of this micrococcus in croupous pneumonia. Let us now consider the principal facts which have led to a belief in its causal relation to this disease. Friedlander, in 1882, observed, in eight fatal cases of pneumonia in which he made autopsies, microorganisms, having an oval form, in the exudate into the pulmonary alveoli ; they were in pairs or in short chains. Without af- firming that this microorganism is the cause of pneumonia, Friedlander seems to have considered it extremely probable that it bore an etiological re- lation to this disease. During the same year Leyden and Gunther announced at a meeting of the Medical Society of Berlin (November 20th, 1882) that they had found the same micrococcus in the fibrinous exudate of pneumonia, obtained through the thoracic walls by means of a Pravaz syringe. At the same time Gunther stated that the elliptical cocci, in specimens stained with gentian violet, were surrounded with a colorless capsule. The following year Matruy published his observations. In sixteen cases he had found an elongated coccus in the fibrinous exudate of pneumonia, and always having a very transparent capsule. He had also encountered the same microorganism in the sputa of patients with other diseases, but not so abundantly as in pneumonia. On November 19th, 1883, Friedlander communicated to the Medical Soci- ety of Berlin the results of his culture and inoculation experiments. His "pneumococcus" was characterized by the presence of a capsule which, as he says, " always takes the form of the microorganism; if this is round the capsule is round; if it is elliptical the capsule is an ellipse." This capsule, however, was only found in preparations made from the blood of an inocu- lated animal or from the fibrinous exudate into the alveoli ; in cultures it was no longer seen. The cultures in flesh-peptone gelatin presented a nail- shaped growth which was believed to be characteristic. Growth was rapid in a variety of culture media at the ordinary room temperature (65° to 75° F.), and in a gelatin culture medium no liquefaction occurred. The following results were obtained by Friedlander in his inoculation ex- periments: In one series of experiments the "pneumococci," mixed with distilled water, were injected through the thoracic walls into the lungs. Nine rabbits inoculated in this way gave an entirely negative result. Six 1 1 should have said frequent instead of " constant presence." BACTERIA IN CROUPOUS PNEUMONIA. 303 out of eleven guinea-pigs are said to have succumbed and to have presented the lesions of pneumonia. All of the mice injected died within twenty -four hours, and at the autopsy the lungs were found to be congested and to pre- sent foci of red hepatization. In a second series of experiments upon mice they were made to inhale a spray containing the pneumococci in suspension. Several of these animals died and are said to have presented a typical pneu- monia. The " pneumococcus, " surrounded by its characteristic capsule, was found in the lungs, the spleen, the blood, and the liquid contained in the pleural cavity. Upon this evidence Friedlander's "pneumococcus," which is now usually described as a bacillus, was very generally accepted as the specific cause of flbrinous pneumonia, and cultures were distributed throughout the labora- tories of Europe bearing- the label, " Pneumococcus of Friedlander." For some time after the publication of Friedlander's paper all observations made with reference to the presence of oval cocci or of encapsulated cocci in the fibrinous exudate of pneumonia were supposed to confirm his discovery. But now we know that there is another oval coccus which is far more fre- quently present in the exudate of acute pneumonia, which also presents the appearance of being surrounded by a transparent capsule — less pronounced, however, than that of Friedlander's bacillus — but which is entirely distinct from that of Friedlander and is probably the true pneumococcus. I shall give the distinctive characters of this microorganism later. At the same time that Friedlander was pursuing his researches in Berlin, Talamon, a French physician, was engaged in similar researches in the lab- oratory of the Hotel-Dieu. His results were communicated to the Anato- mical Society of Paris on November 30th, 1883, a few days after Friedlan- der's communication to the Medical Society of Berlin (Germain See). " Talamon did not describe his microbe as having a capsule; according to him, the pneumonia-coccus is characterized by its form. When seen in the fibrinous exudate the microbe has an elliptical form, like a grain of wheat. Cultivated in a liquid medium — an alkaline solution of extract of beef — it is elongated and attenuated, and presents the appearance of a grain of barley. On account of this appearance Talamon has proposed to call it the lanceolate coccus. This organism is encountered in the pneumonic exudate obtained after death, or drawn during life by means of a Pravaz syringe from the hepatized portions of the lung. Once only, out of twenty-five cases, it was found in the blood of a patient at the moment of death." Talamon's inoculation experiments in dogs and guinea-pigs gave a nega- tive result, but out of twenty rabbits injected through the walls of the thorax into the lungs eight showed the characteristic lesions of fibrinous pneumonia. Prof. See says, with reference to the evidence in the case of these rabbits as compared with that obtained by Friedlander in his mice: 44 The rather brief description of the lesions obtained by Friedlander in the mice inoculated by him leaves some doubt in the mind; for the presence of foci (noyaux) of induration in congested lungs is not sufficient to character- ize fibrinous pneumonia. But the lungs of the rabbits presented by Tala- mon to the Anatomical Society in support of his communication leave no room for discussion. As he observed, it was not at all a question of foci of congestion, or of broncho-pneumonia, such as one observes habitually in rabbits which die of septicaemia, but a veritable lobar fibrinous pneumonia with pleurisy and pericarditis of the same nature. The naked-eye examina- tion, as well as the microscope, showed no difference in the lesions produced in the rabbit and the pneumonia of man." On another page Prof. See says : 4 ' Afanassiew repeated in the laboratory of Prof. Cornil the experiments of Friedlander and of Talamon ; by the cul- ture in peptonized gelatin of the pneumonic exudate taken from the cadaver he obtained two species of organisms, round micrococci of large and small dimensions, and oval cocci which corresponded to the microbes described by the two authors " (Friedlander and Talamon) '4 whose researches we have just reviewed." This quotation indicates that Prof. See did not question the 304 BACTERIA IN CROUPOUS PNEUMONIA. identity of the oval or " lanceolate " coccus found by Talamon in pneumonic exudate, and which in his experiments produced typical pneumonia in rab- bits, and the so-called " pneumococcus " of Friedlander, which, according to his account, gave a negative result when injected into rabbits, but caused pneumonia in mice when injected directly into the lungs. Prof. See was not alone in making this inference, which has turned out to be a mistaken one. The identity of the oval cocci, which had now been seen in the pul- monary exudate by numerous observers, with the microorganism which Friedlander had isolated and cultivated from material obtained post mortem from hepatized lungs, was generally admitted ; and all of the observations relating to the presence of oval cocci, having a more or less distinct capsule, in the exudate of fibrinpus pneumonia, were supposed to give support to the alleged discovery of Friedlander. Now we know that the oval coccus most frequently found in such material is not that of Friedlantier, but that it is identical with a coccus first observed by the writer in September, 1880, in the blood of rabbits injected with his own saliva and subsequently (1885) named by him Micrococcus Pasteuri. This was, without doubt, the coccus which produced pneumonia in Tala- mon's experiments upon rabbits ; and we must give him the credit of having first experimentally aempnstrated the fact that fibrinous pneumonia may be induced by the introduction of this microorganism into the parenchyma of the lung in these animals. Salvioli, whose experiments were also made in 1884, had a uniformly fatal result from the injection of pneumonic sputum into rabbits (four). He also observed the oval coccus in the material injected, and in the blood of the animals which succumbed to his injections, but did not recognize the identity of this coccus with that which my own experiments and those of Pasteur, Vulpian, and others had shown to be present in normal human saliva and to induce a fatal form of septicaemia in rabbits. On the other hand, he also appears to have taken it for granted that the oval micrococcus encountered by him, and which, under certain circumstances, was sur- rounded by a transparent capsule, was the '* pneumococcus " of Friedlander. Klein appears to have made the same mistaken inference. This is shown by the following quotation from his paper published in 1885: 44 In seeking to ascertain what might be the relation between the so-called pneumococci and croupous pneumonia, I have made extensive examination of the lungs and blood of persons dead of the disease, and also of the sputum of living patients at various stages of their illness. ... In some of the air vesicles, tnough few and far between, there were present undoubtedly the capsulated cocci spoken of by Friedlander and others as pneumococci. . . . As regards the living patients, if we examine tvpical sputum of croupous pneumonia we find, besides numerous red blood discs and white blood cor- puscles, also a few epithelial cells, and in the general gelatinous matrix numbers of microorganisms, chiefly belonging to the species micrococci. . . . 4 'These are, as far as size and arrangement go, of two principal types: (a) Oval micrococci about 0.001 millimetre in length, occurring isolated, but more commonly as dumbbells and slightly curved chains of four, six, and even eight elements. . . . But in all tnese micrococci the elements are dis- tinctly surrounded by a hyaline zone which, in stained preparations, can be made out as an unstained halo, though in some stained specimens it as- sumes a tint that is fainter than that of the micrococcus itself; this corre- sponds to the capsule of Friedlander, and for this reason he called them capsule micrococci." In a footnote to the paper from which I have quoted Klein says: 44 While this paper is passing through the press I receive from Dr. Stern- berg, of Baltimore, a paper in which he conclusively proves that the mi- crococci of human saliva, which produce in some instances septicaemia on inoculation into rabbits, are identical with the pneumococci of Friedlander, Salvioli, and others." My own experiments with pneumonic sputum were made in January, BACTERIA IN CROUPOUS PNEUMONIA. 395 1885, and led me to the identification of the oval coccus found in this ma- terial with the coccus found in my own saliva (by inoculations into rabbits) in September, 1880, and subsequently studied by me in an extended series of experiments made during the following years, 1880-84. But, at the same time, I fell into the error of inference, previously made by Prof. See, by Salvioli, and others, and assumed that the "pneumo- coccus " which Friedlander had obtained from the same source was the same, although I found it difficult to reconcile the experimental data, inasmuch as he had obtained uniformly negative results in his inoculations into rabbits. To explain this discrepancy I suggested that Friedlander's pneu- mococcus was probably a variety having a different degree of pathogenic power. This supposition seemed to find support in the fact, which I had previ- ously observed, that my Micrococcus Pasteuri became attenuated, as to its pathogenic power, when the cultures were kept for some time ; and that there seemed, from the experimental evidence before me, to be different recognized my mistake and hastened to correct the error.1 For a detailed account of my experiments with pneumonic exudate I must refer to my paper published in the ' ' Transactions of the Pathological Society of Philadelphia" (vol. xii.) and in the American Journal of the Medical Sciences (July, 1885). With reference to my conclusion that the oval coccus of Talamon and of Salvioli was identical with my Micrococcus Pasteuri, I may say that this conclusion has been sustained by the subsequent investigations of Frankel, Weichselbaum, Bordoni-Uffreduzzi, Netter, Gameleia, and others. Frankel's first paper relating to the presence of this microorganism in pneumonic exudate was published in 1885. Having ascertained that his own saliva contained this oval micrococcus, he was induced to make an extended and interesting series of experiments which led him to the conclusion that this microorganism is far more con- stantly present in. the exudate of fibrinous pneumonia than is the so-called " pneumococcus " of Friedlander. He says: "Finally, as regards the relative frequency of the two hitherto investi- gated microorganisms in cases of pneumonia, no positive statement can yet be made. Nevertheless I am inclined to regard the lancet-shaped pneu- mococcus, which is identical with the microbe of sputum septicaemia, as the more frequent, and the usual infectious agent of pneumonia, on the ground that this organism is so much more frequently found in the sputum of pneu- monic patients than in that of healthy individuals. This conclusion is further supported by the fact that it has not hitherto been possible to isolate, directly from the rusty sputum, Friedlander's bacillus." The extended researches of Weichselbaum, published in 1886, give strong support to the view that this coccus is the usual infectious a,gent in croupous pneumonia. He examined, in all, the exudate in one hundred and twenty- nine cases of pneumonia. In ninety-four of these cases the micrococcus in question, called by Weichselbaum " diplococcus pneumonias, " was obtained (fifty-four times in cultures); in twenty-one cases he obtained a streptococcus, and in nine only was the bacillus of Friedlander encountered. Wolf, whose studies were made in Weichselbaum's laboratory, reported the result of his researches in 1887. He found the "diplpcoccus pneumoniae" in sixty-six out of seventy cases of croupous pneumonia examined, and the " pneumococcus of Friedlander " in three cases. Netter, whose paper was published in November, 1887, found Micrococcus 1 See my paper published in the American Journal of the Medical Sciences for July, 1886. 20 300 BACTERIA IN CROUPOUS PNEUMONIA. Pasteuri in seventy-five per cent of his cases of pneumonia, and in the sputum of convalescents from this disease its presence was verified in sixty per cent of the cases by inoculation experiments in rabbits. He makes the interest- ing observation that the sputum of recent convalescents is less virulent for rabbits than that collected at a later period. Gameleia, who has recently published in the Annales of the Pasteur Institute an important paper upon the etiology of fibrinous pneumonia, veri- fied the presence of Micrococcus Pasteuri in twelve fatal cases in which he collected material post mortem. He states that in a series of forty con- secutive cases Dr. Goldenberg, whose experiments were made in his laboratory, found this micrococcus in everv case by inoculation experiments in rabbits or in mice. According to Gameleia, inoculations in mice are more reliable than those made in rabbits, as the mouse is the more susceptible animal. He says: "The author, Weichselbaum, who has made the most extended research upon the etiology of pneumonia, used in his researches the method of culti- vation upon gelatin. We must adopt the opinion of Baumgarten, who does not accord any decided value to the negative results of Weichselbaum with reference to the constant presence of Streptococcus Paste.uri. Netter, who adopted the method of inoculating the pneumonic sputum into rabbits, and who only found the microbe of Pasteur in seventy-five per cent of his cases, was wrong, in our opinion, in making use of an animal which is too resist- ant to determine the presence of small quantities of virus. This opinion is confirmed by the fact that Netter rendered some rabbits refractory by his inoculations with material in which he had not found the specific microbe. " En rteumt, taking our stand upon the positive results which we have always obtained, as well as upon the superiority of the method of research (inoculations in mice) which wre have adopted, we believe ourselves au- thorized to conclude that fibrinous pneumonia is always dependent upon the microbe of Pasteur." Frankel, Weichselbaum, and other recent authors, while maintaining that Micrococcus Pasteuri is the most frequent etiological agent in the pro- duction of pneumonia, have been disposed to admit that in a certain propor- tion of the cases the bacillus of Friedlander, and possibly other microorgan- isms, may bear the same relation to the pneumonic process. Gameleia, on the other hand, believes that the bacillus of Friedlander is a simple sapro- phyte, the occasional presence of which in pneumonic exudate is without etiological import. He remarks as follows : " We may be brief as regards the second objection made against the etio- logical unity of fibrinous pneumonia, viz., with reference to the etiologicai rights of the microbe of Friedlander. This microbe is found in normal sali- va, it is a true saprophyte, and may at times invade the diseased or dead lung. Weichselbaum only found it in seven per cent of his cases, and al- most always associated with other microbes, for he only encountered it pure in three cases. As to the researches of the authors who preceded Frankel, it is sure that the microbe which they often found in sections of diseased lungs, and which they called the microbe of Friedlander, was in fact the mi- crobe of Pasteur, since it was colored by the method of Gram, which decol- orizes the bacillus of Friedlander. Many of the positive results, then, which have been reported relative to the last-mentioned microorganism, oufHht to be put to the account of the other." This opinion the present writer has entertained since his researches made in 1885. The experimrntal evidence oflVred l>y (Jameleia in favor of the etiologi- cal role of this micrococcus is most important. It will be remembered that Talamon produced typical pneumonia in eight rabbits, in 1883, by inoculating them through the thoracic walls with pneumonic exudate. Gameleia says : 4 'The number of ray rabbits iu which pneumonia was induced is about two hundred," BACTERIA IN CROUPOUS PNEUMONIA. 307 The writer found in his experiments, made in 1881, that in making- a series of inoculations in rabbits the virus increased in virulence, and that, on the other hand, the micrococcus lost its virulence when the cultures were kept for some time. This fact has been verified by the subsequent re- searches of Frankel and of Gameleia. The last-named author has shown that to induce pneumonia in very susceptible animals, like the rabbit, an attenu- ated variety of the microbe should be injected, for the most virulent cul- tures quickly cause death by septicaemia. As he expresses it : "Animals which are too susceptible, like the rabbit and the mouse, do not have pneu- monia, because they do not offer a local reaction ; the virus is generalized in their bodies and they die of an acute septicaemia " On the other hand, Gameleia has shown that " animals which are but little susceptible to the pneumonic virus offer a local resistance which gives rise to very pronounced reactionary phenomena (extended fibrino-granular oedema), and consequently they present, as a result of intrapulmonary infec- tion, a typical fibrinous pneumonia. Such animals are the dog and the sheep." In his experiments upon these animals Gameleia obtained the following results: The sheep was found to survive subcutaneous inoculations, unless very large doses (five cubic centimetres) of the most potent virus were ad- ministered. But intrapulmonary inoculation was always followed by typi- cal fibrinous pneumonia, which in the majority of cases proved fatal. The microbe was rarely found in the blood, and successive inoculations from one sheep to another were not successful. Death occurred, after an intrapulmonary inoculation, on the third, fourth, or fifth day. The pneu- monia produced was lobar, and was attended with an extensive fibrinous exudation in which the coccus was found in great abundance. In all, fifty sheep were experimented upon. The writer found in his experiments, made in 1881, that the dog resists inoculations with this coccus. Gameleia also obtained negative results when moderate doses were injected beneath the skin, but states that ' ' intrathoracic infection always causes a frank, fibrinous pneumonia which is rarely fatal ; recovery usually occurs in from ten to fifteen days, after the animal has passed through all the stages of red and gray hepatization which character- izes this affection in man." Twelve dogs were experimented upon. This micrococcus, then, which in very susceptible animals (mouse, rabbit) invades the blood and quickly causes aeath by septicaemia, when injected through the thoracic walls in less susceptible animals (dog, sheep), or in an attenuated form in the rabbit, gives rise to the local lesions which character- ize fibrinous pneumonia. Man comes in the category of slightly susceptible animals, as is shown by the comparatively small mortality from pneumonia, and the fact that the micrococcus found in the exudate into the pulmonary alveoli does not invade the blood, unless in rare instances. We may therefore agree with Gameleia in the following statement : "It is clear that the results obtained in the dog and the sheep, animals which have but a slight susceptibility, are most applicable to human patho- logy." In my paper read before the Pathological Society of Philadelphia in April, 1885, from which I have already quoted, I say: " It seems extremely probable that this micrococcus is concerned in the etiology of croupous pneu- monia. . . . But this cannot be considered as definitely established by the experiments which have thus far been made upon the lower animals.1' The experiments of Gameleia go far toward settling this question in a definite manner, and, considered in connection with those of Talamon and Salvioli, and the extended researches of Frankel, Weichselbaum, and Netter, leave but little doubt that this is the true infectious agent in acute lobar pneumonia. BACTERIA IN CROUPOUS PNEUMONIA. 7. BACILLUS OF FRIEDLANDER. Synonyms. — Pneumococcus (Friedlander) ; Bacillus pneumoniae (Flugge). Obtained by Friedlander and Frobenius in pure cultures (1883) from the exudate into the pulmonary alveoli in cases of croup- ous pneumonia. Subsequent researches show that it is only present in a small proportion of the cases — nine times in one hundred and twenty-nine cases examined by Weichselbaum, three times in seventy cases examined by Wolf. Morphology. — Short rods with rounded ends, often so short as to resemble micrococci, especially in very (^m recent cultures ; commonly united in pairs °& ^W ^@(§\/^ or chains of four, and under certain cir- ^ifo Q° ^Qs^&r cumstances surrounded by a transparent Ji j capsule. The gelatinous envelope — so- Fio.88.-Baciiius of Friedlander; called capsule — is not seen in preparations o, from a culture; 6, from blood of made from cultures in artificial media, but mouse, showing capsule. (Flugge.) . . . .. is very prominent in properly stained prepa- rations from the blood of an inoculated animal. It often has a diame- ter equal to or greater than that of the enclosed cell, and appears to consist of a substance resembling mucin, which is soluble in water or dilute alcohol. Where several cells are united in a chain they may all be enclosed in a common envelope, or each may have its own cap- sule. This capsule is not peculiar to Friedlander's bacillus, as he at first supposed, but is found in other bacilli and also in the writer's Micrococcus Pasteuri. Friedlander's bacillus stains readily with the aniline colors, but is decolorized by the iodine solution used in Gram's method. In preparations from the blood of an inoculated animal, stained by an aniline color, the capsule appears as an unstained envelope surround- ing the stained cell, but by special treatment the capsule may also be stained. Friedlander's method is as follows : The section or cover- glass preparation is placed for twenty-four hours in a solution of gentian violet and acetic acid, containing fifty parts of a concentrated alcoholic solution of gentian violet, one hundred parts of distilled water, and ten parts of acetic acid. The stained preparation is washed for a minute or two in a one-per-cent solution of acetic acid, dehydrated with alcohol, cleared up with oil of cloves or cedar, and mount.Ml in balsam. The bacillus is quickly stained in dried cover- glass preparations by immersion in aniline- water-gentian-violet solu- tion (two or three minutes). The stained preparation should be de- colorized by placing it in absolute alcohol for half a minute, and then washed in distilled water. BACTERIA IN CROUPOUS PNEUMONIA. 309 Biological Characters. — This bacillus does not, so far as is known, form reproductive spores ; it is non-motile and does not liquefy gelatin. It is aerobic and a facultative anaerobic. In gelatin stick cultures it presents the "nail-shaped" growth first described by Friedlander, which is not, however, peculiar to this bacillus. The head of the nail is formed by the development around the point of entrance of the inoculating needle of a rounded, white mass hav- ing a smooth, shining surface, and its stem by the growth along the line of puncture. This consists of closely crowded, opaque, white, spherical colo- nies. Gas bubbles sometimes develop in gelatin cultures, and in old cultures the gelatin about the line of growth acquires a yellowish-brown color. The growth in nutrient agar resembles that in gelatin. Upon the surface of blood serum abun- dant grayish- white, viscid masses are developed. Upon potato the growth is abundant, quickly cov- ering the entire surface with a thick, yellowish- white, glistening layer which often contains gas bubbles when the temperature is favorable. Col- onies in gelatin plates appear at the end of twenty- four hours as small, white spheres, which increase rapidly in size, and upon the surface form round- ed, smooth, glistening, white masses of consider- able size. Under the microscope the colonies pre- sent a somewhat irregular outline and a slightly & J bacillus; stick culture in granular appearance. Growth occurs at compara- gelatin; end of four days tively low temperatures— 16° to 20° C.— but is more *etn16°-18° c' rapid in the incubating oven. The thermal death- point, as determined by the writer, is about 56° C. In the ordinary culture media it retains its vitality for a long time, and may grow when transplanted to fresh culture material after having been pre- served for a year or more. At a temperature of 40° C. development ceases. Pathogenesis. — In Friedlander's experiments the bacillus from pure cultures, suspended in water, was injected through the thoracic wall 4nto the right lung of dogs, rabbits, guinea-pigs, and mice. Rabbits proved to be immune ; one dog out of five, six guinea-pigs out of eleven, and all of the mice (thirty-two) succumbed to the inoculation. At the autopsy the pleural cavities were found to con- tain a sero-purulent fluid ; the lungs were intensely congested, con- tained but little air, and in places showed limited areas of red infil- tration ; the spleen was considerably enlarged ; the bacillus was 310 BACTERIA IN CROUPOUS PNEUMONIA. found in great numbers in the lungs, the fluid in the pleural cavi- ties, and in the blood obtained from the general circulation or from the various organs of the body. Similar appearances presented them- selves in the case of the guinea-pigs which succumbed to the inocu- lation. These results show that the bacillus under consideration is path- ogenic for mice and for guinea-pigs, but they are by no means sufficient to prove that it is capable of producing a genuine croupous pneumonia in man, and it is still uncertain whether its occasional presence in the exudate into the pulmonary alveoli in cases of this disease has any etiological importance. 8. MICROCOCCUS PNEUMONIA CROUPOS^E. Synonyms. — Micrococcus Pasteuri (Sternberg) ; Micrococcus of sputum septicaemia (Frankel) ; Diplococcus pneumonia (Weichsel- baum) ; Bacillus septicus sputigenus (Fliigge) ; Bacillus salivarius septicus (Biondi) ; Lancet-shaped micrococcus (Talamon) ; Strepto- coccus lanceolatus Pasteuri (Gameleia). Discovered by the present writer in the blood of rabbits inocu- lated subcutaneously with his own saliva in September, 1880 ; by Pasteur in the blood of rabbits inoculated with the saliva of a child which died of hydrophobia in one of the hospitals of Paris in De- cember, 1880 ; identified with the micrococcus in the rusty sputum of pneumonia, by comparative inoculation and culture experiments, by the writer in 1885 (paper published in the American Journal of the Medical Sciences, July 1st, 1885). Proved to be the cause of croup- ous pneumonia in man by the researches of Talamon, Salvioli, Stern- berg, Frankel, Weichselbaum, Netter, Gameleia, and others. The Presence of Micrococcus Pasteuri in the Salivary Secre- tions of Healthy Individuals. — In September, 1880, while engaged in investigations relating to the etiology of the malarial fevers, I in- jected a little of my own saliva beneath the skin of two rabbits as a control experiment. To my surprise the animals died, and I found in their blood a multitude of oval microorganisms, united for the most part in pairs, or in chains of three or four elements. These experiments are recorded in my paper entitled " Experimental Inves- tigations Relating to the Etiology of the Malarial Fevers/' published in the Report of the National Board of Health for 1881, pp. 74, 75. Following up my experiments made in New Orleans (in Septem- ber, 1880), in Philadelphia (January, 1881), and in Baltimore (March, 1881), I obtained the following results : " The saliva of four students, residents of Baltimore (in March), gave negative results ; eleven rabbits injected with the saliva of six individuals in Philadelphia (in January) gave eight deaths and three BACTERIA IN CROUPOUS PNEUMONIA. 311 negative results; but in the fatal cases a less degree of virulence was shown in six by a more prolonged period between the date of injec- tion and the date of death. This was three days in one, four days in four, and seven days in one." In a paper published in the Journal of the Royal Microscopical Society (June, 1886) I say : " My own earlier experiments showed that there is a difference in the pathogenic potency of the saliva of different individuals, and I have since learned that the saliva of the same individual may differ in this respect at different times. Thus during the past three years injections of my own saliva have not infrequently failed to cause a fatal result, and in fatal cases death is apt to occur after a some- what longer interval, seventy-two hours or more ; whereas in my earlier experiments the animals infallibly died within forty-eight hours." The presence of my Micrococcus Pasteuri was demonstrated in the blood of the rabbits which succumbed to the inoculations. Claxton, in a series of experiments made in Philadelphia in 1882, injected the saliva of seven individuals into eighteen rabbits. Five of these died within five days, and nine at a later period. Frankel, whose first publication was made in 1885, discovered the presence of this micrococcus in his own salivary secretions in 1883, and has since made extended and important researches with refe- rence to it. The saliva of five healthy individuals and the sputa of patients suffering from other diseases than pneumonia, injected into eighteen rabbits, induced fatal " sputum septicaemia " in three only. When he commenced his experiments his saliva was uni- formly fatal to rabbits, but a year later it was without effect. Wolf injected the saliva of twelve healthy individuals, and of three patients with catarrhal bronchitis, into rabbits, and induced " sputum septicaemia " in three. Netter examined the saliva of one hundred and sixty-five healthy persons, by inoculation experiments in rabbits, and demonstrated the presence of this micrococcus in fifteen per cent of the number. Vignal, in his recent elaborate paper upon the microorganisms of the mouth, says : " Last year I encountered this microbe continually in my mouth during a period of two months, then it disappeared, and I did not find it again until April of this year, and then only for fifteen days, when it again disappeared without appreciable cause. " The Presence of Micrococcus Pneumonice Crouposce in Pneu- monic Sputum. — Talamon, in 1883, demonstrated the presence of this micrococcus in pneumonic sputum, described its morphological char- acters, and produced typical croupous pneumonia in rabbits by in- 312 BACTERIA IN CROUPOUS PNEUMONIA. jecting material containing it into the lungs through the thoracic walls. Salvioli, in 1884, demonstrated its presence in pneumonic sputum by injections into rabbits. In 1885 the writer made a similar demonstration, and by compara- tive experiments showed that the micrococcus present in the blood of rabbits inoculated with the rusty sputum of pneumonia was iden- tical with that which he had discovered in 1880 in rabbits inoculated with his own saliva. The same year (1885) A. Frankel made a similar demonstration, and published a paper containing valuable additions to our knowl- edge relating to the biological characters of this microorganism (first publication appeared July 13th, 1885). In 1886 Weichselbaum published the results gf his extended re- searches relating to the presence of this micrococcus in the fibrinous exudate of croupous pneumonia. He obtained it in ninety-four cases (fifty-four times in cultures) out of one hundred and twenty-nine cases examined. Wolf (1887) found it in sixty-six cases out of seventy examined. Netter (1887) in seventy-five per cent of his cases, and in the sputum of convalescents from pneumonia in sixty per cent of the cases ex- amined, by inoculations into rabbits. Gameleia (1887) in twelve fatal cases of pneumonia in which he collected material from the lungs at the post-mortem examination. Goldenberg, whose researches were made in Gameleia's labora- tory, found it in pneumonic sputum in forty consecutive cases, by inoculations into rabbits and mice. The Presence of Micrococcus Pneumonice Crouposce in Menin- gitis.— Numerous bacteriologists have reported finding diplococci in the pus of meningitis, and frequently the microorganisms have been fully identified as " diplococcus pneumoniae." Thus Netter (1889), in a resume of the results of researches made by him in twenty-five cases of purulent meningitis, reports as follows : Thirteen cases were examined microscopically, by cultures, and by inoculations into susceptible animals ; six cases by microscopical examination and experiments on animals; and the remainder only by microscopical examination. Four of the cases were complicated with purulent otitis, six with pneumonia, three with ulcerative endo- carditis. The " pneumococcus" was found in sixteen of the twenty- five cases ; in four Streptococcus pyogenes was present ; in two Diplococcus intracellularis meningitidis of Weichselbaum ; in one Friedlander's bacillus ; in one Newmann and Schaffer's motile ba- cillus ; in one a small curved bacillus. In forty-five cases collected from the literature of the subject by BACTERIA IN CROUPOUS PNEUMONIA. 313 Netter this micrococcus was present in twenty-seven, Streptococcus pyogenes in six, and the Diplococcus intracellularis meningitidis of Weichselbaum in ten. Monti (1889), in four cases of cerebro-spinal meningitis, demon- strated the presence of the same micrococcus. In three of his cases pneumonia was also present. In two Staphylococcus pyogenes aureus was associated with the " diplococcus pneumonias." Micrococcus Pneumonice Crouposce in Ulcerative Endocar- ditis.— Weichselbaum, in a series of twenty-nine cases examined (1888), found " diplococcus pneumonise" in seven. Micrococcus Pneumonice Crouposce in Acute Abscesses. — In a case of parotitis occurring as a complication of croupous pneumonia this micrococcus was obtained from the pus in pure cultures by Testi (1889); and in another case in which, as a complication of pneumonia, there developed a purulent pleuritis, abscess of the parotid on both sides, and multiple subcutaneous abscesses, the pus from all of the sources named contained the "diplococcus" in great numbers, as FIG. 90. FIG. 91. FIG. 98. FIG. 90.— Micrococcus pneumonias crouposse from blood of rabbit inoculated with normal human saliva (Dr. S.). X 1,000. FIG. 91. — Micrococcus pneumonias crouposse from blood of rabbit inoculated subcutaneously with fresh pneumonic sputum from a patient in the seventh day of the disease. X 1,000. FIG. 92.— Surface culture of Micrococcus pneumoniae crouposse, on nutrient agar, showing the development of long chains. X 1,000. 1 shown not only by microscopical examination but by inoculation into rabbits. In a case of tonsillitis resulting in the formation of an abscess Gabbi (1889) obtained the same coccus in pure cultures. In otitis media this micrococcus has been found in a consider- able number of cases in the pus obtained by paracentesis of the tympanic membrane, and quite frequently in pure cultures — by Zau- fal (1889) in six cases; Levy and Schrader (1889) in three out of ten cases in which paracentesis was performed; by Netter (1889) in five out of eighteen cases occurring in children. Monti (1889) and Belfanti (1889) report cases of arthritis of the wrist joint, occurring as a complication of pneumonia, in which this micrococcus was obtained in pure cultures. Ortmann and Samter 1 The above figures are from Dr. Sternberg's paper published in the American Journal of the Medical Sciences for July and October, 1885. 314 BACTERIA IN CROUPOUS PNEUMONIA. (1889), in a case of purulent inflammation of the shoulder joint fol- lowing pneumonia and pleurisy, obtained the "diplococcus pneu- monise " in pure cultures. Morphology. — Spherical or oval cocci, usually united in pairs, or in chains consisting of three or four elements. Longer chains, con- taining ten or more elements, are frequently formed, especially in cultures upon the surface of nutrient agar, and in liquid media; it may therefore be regarded as a streptococcus. As observed in the blood of inoculated animals it is usually in pairs consisting of oval or lance-oval elements, which are surrounded by a transparent cap- sule. Owing to the elongated form of the cocci when in active growth, it has been regarded by some authors as a bacillus; but in cultures in liquid media, when development by binary division has ceased, the cells are spherical, or nearly so, and in cultures on the surface of nutrient agar the individual cells more nearly approach a spherical form than in the blood of an inoculated animal. The " lan- ceolate " form was first referred to by Talamon, who described it as having the form of a grain of wheat, or even still more elongated like a grain of barley, as seen in the fibrinous exudate of croupous pneumonia. * The transparent material surrounding the cells — so- called capsule — is best seen in stained preparations from the fibrinous exudate of croupous pneumonia or from the blood of an inoculated animal. It appears as an unstained marginal band surrounding the elliptical cells, and varies greatly as to its extent in different prepara- tions. This capsule probably consists of a substance resembling mucin, and, being solu- ble in water, its extent depends partly upon the methods employed in preparing speci- mens for microscopical examination. It is occasionally seen in stained preparations from the surface of cultures on blood serum ; and in drop cultures examined under the micro- ^P6' by usi"g a sma11 di^^agm it may suie, attached to pus cells from be seen to surround the cocci as a scarcely exudate in pleura! cavity of vidihln Vialn inoculated rabbit. (Salvioli.) V1S1^e. nal°' This micrococcus stains readily with the aniline colors; and also by Gram's method, which constitutes an important character for distinguishing it from Friedlander's ba- cillus. Biological Characters. — Grows in the presence of oxygen — aerobic — but is also a facultative anaerobic. Like other micro- cocci, it has no spontaneous movements. It grows in a variety of culture media when they have a slightly alkaline reaction, but will not develop in a medium which contains the slightest trace of free . BACTERIA IN CROUPOUS PNEUMONIA. 315 acid. Nor will it grow at the ordinary room temperature. Scanty development may occur at a temperature of 22° to 24° C., but a temperature of 35° to 37° C. is most favorable for its growth, which is very rapid in a suitable liquid medium. In an infusion made from the flesh of a chicken or a rabbit it multiplies, in the incubating oven, with remarkable rapidity ; at the end of six to twelve hours after inoculation the previously transparent fluid will be found to present a slight cloudiness and to be filled throughout with the cocci in pairs and short chains. It does not produce a milky opacity in liquid media, like the pus cocci, for example, but the fluid becomes slightly clouded ; multiplication ceases at the end of about forty- eight hours or less, and the liquid medium again becomes transpa- rent as a result of the subsidence of the cocci to the bottom of the receptacle. It may be cultivated in flesh-peptone-gelatin, containing fifteen per cent of gelatin, at a temperature of 24° C. , or in liquefied gela- tin (ten per cent) in the incubating oven. In gelatin (fifteen per cent) stick cultures small white colonies develop all along the line of puncture, and in gelatin plates small, spherical, slightly granular, whitish colonies are formed : the gelatin is not liquefied. In agar plates extremely mi- nute colonies are developed in the course of forty-eight hours, which resemble little, transparent drops of fluid, and under the microscope some of these are observed to FIG. have a compact, finely granular central coccus pneumonise crouposse upon portion surrounded by a paler, transparent, finely granular marginal zone. Upon the surface of nutrient agar or coagulated blood serum development occurs in the form of minute, transparent, jelly-like drops, which form a thin layer along the line of inoculation in "streak cultures" ; and in agar stick cultures the growth along the line of puncture is rather scanty, almost homogeneous, and semi-transparent. Upon potato no development occurs, even in the incubating oven. Milk is a favorable culture medium, and the casein is coagulated as a result of its presence. It ceases to grow on solid media at about 40° C., and in favorable liquid media at 42° C. Its thermal death-point, as determined by the writer, is 52° C., the time of exposure being ten minutes. It loses its vitality in cultures in a comparatively short time — four or five days on agar — and is very sensitive to the action of germicidal agents. Its pathogenic power also undergoes attenuation very 316 BACTERIA IN CROUPOUS PNEUMONIA. quickly when it is cultivated in artificial media, but may be restored by passing it through the bodies of susceptible animals. Attenua- tion of virulence may also be effected by exposing bouillon cultures to a temperature of 42° C. for twenty-four hours, or by five days' exposure to a temperature of 41° C. Emmerich reported in 1891 to the Congress of Hygiene and Demography in London the results of experiments made by him relating to immunity in rabbits and mice. Rabbits were rendered immune by the intravenous injection of a very much diluted but virulent culture of the micrococcus. The flesh of these immune rabbits was rubbed up into a fine paste, and the juices obtained by compressing it in a clean, sterilized cloth. This bloody juice was kept for twelve hours at a temperature of 10° C., and then sterilized by passing it through a Pasteur filter. Some of this juice was injected into a rabbit, which with twenty-five others was then made to re- spire an atmosphere charged with a spray of a bouillon culture of the micrococcus. As a result of this all of the rabbits died except the one which had previously been injected with the immunizing juice. In a similar experiment upon mice six of these animals, which had previously been injected with the immunizing juice, survived the in- jection of a full dose of a virulent culture, while a control mouse, not previously injected with the juice, promptly died after receiving the same quantity of the virulent culture. The writer in 1881, in experiments made to determine the value of various disinfectants, as tested upon this micrococcus, obtained experimental evidence that its virulence is attenuated by the action of certain antiseptic agents. Commenting upon the results of these experiments in my chapter on " Attenuation of Virus/' in " Bacte- ria "(1884), I say: "Sodium hyposulphite and alcohol were the chemical reagents which produced the result noted in these experiments ; but it seems probable that a variety of antiseptic substances will be found to be equally effective when used in the proper proportion. Subsequent experiments have shown that neither of these agents is capable of destroying the vitality of this septic micrococcus in the proportion used (one per cent of sodium hyposulphite or one part of ninety-ftve-per-cent alcohol to three parts of virus), and that both have a restraining influence upon the development of this microorgan- ism in culture fluids." The following results were obtained by the writer in his experi- ments (1881 and 1883) to determine the germicidal and antiseptic value of the agents named, as tested upon this micrococcus. Alcohol. — A twenty-four-per-cent solution was effective upon bouillon cultures in two hours. Boric Acid. — A saturated solution failed to destroy vitality after two hours' exposure, but 1 : 400 restrained development. BACTERIA IN CROUPOUS PNEUMONIA. 317 Carbolic Acid. — A one-per-cent solution destroys vitality in two hours, and 1 : 500 restrains development. Cupric Sulphate destroys the virulence of the coccus in the blood of a rabbit in the proportion of 1 : 400 in half an hour. Ferric Sulphate failed to destroy vitality in a saturated solution, but restrained development in the proportion of 1 : 200. Hydrochloric Acid destroys the virulence of the blood of a rab- bit containing this micrococcus in the proportion of 1 : 200. Iodine, in aqueous solution with potassium iodide, destroys vital- ity in the proportion of 1: 1,000 and prevents development in 1: 4,000. Mercuric Chloride. — One part in forty thousand prevents the development of this micrococcus, and 1 : 20,000 was found to destroy vitality in two hours. Nitric Acid. — One part in four hundred destroyed the virulence of rabbit's blood containing this micrococcus. Caustic Potash. — A two-per-cent solution destroyed vitality in two hours. Potassium Permanganate. — A two-per-cent solution destroyed the virulence of rabbit's blood containing this coccus. Salicylic Acid, dissolved by the addition of sodium biborate. — A solution of 1 : 400 prevented development. Sulphuric Acid. — One part in two hundred destroys vitality, and 1 : 800 prevents development. In a paper by Bordoni-Uffreduzzi relating to the resisting power of pneumonic virus for desiccation and light, the following results are given : Pneumonic sputum attached to cloths, when dried in the air and exposed to diffuse daylight, retained its virulence, as shown by injection in rabbits, for a period of nineteen days in one series of ex- periments and for fifty-five days in another. Exposed to direct sun- light the same material retained its virulence after twelve hours' exposure. Cultures have far less resistance, and the protection afforded by the dried albuminous material in which the micrococci were embedded, in the experiments referred to, probably accounts for the virulence being retained so long a time. Kruse and Pansini (1892) have published an elaborate paper giv- ing an account of their researches relating to "diplococcus pneumo- niae " and allied streptococci. We give below a summary statement of their results : Many varieties were obtained by the observers named in their cultures from various sources — from the lungs of individuals dead from pneumonia, from pleuritic exudate, from pneumonic sputa, from bronchitic sputa, from the saliva of healthy persons, from the secretion in a case of subacute nasal catarrh , from the urine of a patient with nephritis. Pure cultures were obtained by the use of agar plates or by inoculations into rabbits. In all about thirty varieties were obtained and cultivated 318 BACTERIA IN CROUPOUS PNEUMONIA. through many successive generations. As a rule, the different varieties, which* at first were seen to have the form of diplococci, when cultivated for a length of time in artificial media presented the form of streptococci ; and the elements which at first were lancet-shaped showed a tendency to become spherical. The more virulent varieties usually presented the form of diplococci with lancet-shaped elements, or of short chains. A variety which formed long chains could be pronounced, in advance of the experiments on animals, to possess comparatively little virulence. When by inoculations in animals the virulence of such a variety was restored, the tendency to form chains was less pronounced. Although, as a rule, no development occurs at 20° C., certain varieties were obtained which, after long cultivation in artificial media, showed a de- cided growth at 18° C. Decided differences were shown by the cultures from various sources as regards their growth in rnilk. Out of eighty-four cultures from various sources eleven did not produce coagulation. As a rule, cultures which caused coagulation of milk were virulent for rabbits, and when such cultures lost their virulence they usually lost at the same time the power of coagulating milk. Virulent cultures die out sooner than those which have become at- tenuated by continuous cultivation in artificial media; the first, on the sur- face of agar, usually fail to grow at the end of a week, while the attenuated cultures may survive for three weeks or more. Pathogenesis. — This micrococcus is very pathogenic for mice and for rabbits, less so for guinea-pigs. The injection of a minute quan- tity— 0.2 cubic centimetre or less — of a virulent culture beneath the skin of a rabbit or a mouse usually results in the death of the animal in from twenty-four to forty-eight hours. The following is from the writer's first published paper (1881), and refers to the pathological appearances in rabbits : " The course of the disease and the post-mortem appearances indicate that it is a form of septicaemia. Immediately after the injection there is a rise of temperature, which in a few hours may reach 2° to 3° C. (3.6° to 5.4° F.); the temperature subsequently falls, and shortly before death is often several degrees below the normal. There is loss of appetite and marked debility after twenty -four hours, and the animal commonly dies during the second night or early in the morning of the second day after the injection. Death occurs still more quickly when the blood from a rabbit recently dead is in- jected. Not infrequently convulsions immediately precede death. 4 'The most marked pathological appearance is a diffuse inflammatory oadema or cellulitis, extending in all directions from the point of injection, but especially to the dependent portions of the body. Occasionally there is a little pus near the puncture, but usually death occurs before the cellulitis reaches the point of producing pus. The subcutaneous connective tissue contains a quantity of bloody serum, which possesses virulent properties and which contains a multitude of micrococci. There is usui lly more or less in- flammatory adhesion of the integument to the subjacent tissues. The liver is sometimes dark-colored and gorged with blood, but more frequently it is of a lighter color than normal and contains much fat. The spleen is either normal in appearance or enlarged and dark-colored. Changes in this organ are more marked in those cus.-s which are of the longest duration. The blood commonly contains an immense number of micrococci, usually •joined in pairs and having a diameter of about 0.5 //. These are found in blood drawn tmm superficial veins, from arteries, and from the cavities of the heart immediately after death, and in a few cases their presence has been BACTERIA IN CROUPOUS PNEUMONIA. 319 verified during life. Observations thus far made, however, indicate that it is only during the last hours of life that these parasites multiply in the cir- culating fluid, and in a certain proportion of the cases a careful search has failed to reveal their presence in the hlood in post-mortem examinations made immediately after the death of the animal." In animals which are not examined until some hours after death a considerable increase in the number of micrococci occurs post mor- tem. The fact that this micrococcus varies very much as to its pathogenic power, as a result of conditions relating to the medium in which it develops, was insisted upon in my first published paper, and has been fully established by later researches (Frankel, Gameleia). Susceptible animals inoculated with attenuated cultures acquire an immunity against virulent cultures. FIG. 9).— Micrococcus pneumonias crouposae in blood of rabbit inoculated with pneumonic spu- tum, x 1,000. In dogs subcutaneous injections usually give a negative result, or at most a small abscess forms at the point of inoculation. In a single experiment, however, the writer has seen a fatal result in a dog from the injection of one cubic centimetre of bloody serum from the subcutaneous connective tissue of a rabbit recently dead. This shows the intense virulence of the micrococcus when cultivated in the body of this animal. Pneumonia never results from subcutane- ous injections into susceptible animals, but injections made through the thoracic walls into the substance of the lung may induce a typi- cal fibrinous pneumonia. This was first demonstrated by Talamon (1883), who injected the fibrinous exudate of croupous pneumonia, obtained after death, or drawn during life by means of a Pravaz syringe from the hepatized portions of the lung, into the lungs of 320 BACTERIA IN CROUPOUS PNEUMONIA. rabbits. According to See, eight out of twenty animals experi- mented upon exhibited "a veritable lobar, fibrinous pneumonia, with pleurisy and pericarditis of the same nature." Gameleia has also induced pneumonia in a large number of rabbits, and also in the dog and the sheep, by injections directly into the pulmonary tissue. Sheep were found to survive subcutaneous inoculations, unless very large doses (five cubic centimetres) of the most potent virus were in- jected. But intrapuhnonary inoculations invariably induced a typi- cal fibrinous pneumonia which usually proved fatal. In dogs simi- lar injections gave rise to a "frank, fibrinous pneumonia which rarely proved fatal, recovery usually occurring in from ten to fifteen days, after the animal had passed through the stages of red and gray hepatization characteristic of this affection in man." Monti claims to have produced typical pneumonia in rabbits by injecting cultures of this micrococcus into the trachea. From the evidence obtained in these experimental inoculations, and that recorded relating to the presence of this micrococcus in the fibrinous exudate of croupous pneumonia, we are justified in con- cluding that it is the usual cause of this disease, and consequently have described it under the name Micrococcous pneumonise crou- posae. We prefer this to the name commonly employed by German authors — " diplococcus pneumonise" — because this micrococcus, al- though commonly seen in pairs, forms numerous short chains of three or four elements in cultures in liquid media, and upon the sur- face of nutrient agar may grow out into long chains. It would, therefore, more properly be called a streptococcus than a diplococcus. G. and F. Klemperer, in 1891, published an important memoir relating to the pathogenic action of this micrococcus. They suc- ceeded in conferring immunity upon susceptible animals by inocu- lating them with filtered cultures of the micrococcus, and in some instances this immunity had a duration of six months. A curi- ous fact developed in their researches was that the potency of the substance contained in the filtered cultures was increased by subjecting these to a temperature of 41° to 42° C. for three or four days, or to a higher temperature (60° C.) for an hour or two. When injected into a vein after being subjected to such a temperature im- 111 unity was complete at the end of three or four days ; but the same material not so heated required larger doses and a considerably longer time (fourteen days) to confer immunity upon a susceptible animal. Tlir nmvarinrd material caused a considerable elevation of temperature, lasting for some days. The authors mentioned con- clude from their investigations that the toxic substance present in cultures of Micrococcus pneumonise crouposse is a proteid substance, which they propose to call pneumotoxin. The substance produced BACTERIA IN CROUPOUS PNEUMONIA. 321 in the body of an immune animal, as a result of protective inocula- tions, upon which the immunity of these animals depends, is also a proteid, which they call anti-pneumotoxin. This they isolated from the blood serum of immune animals. By experiment they were able to demonstrate that the blood serum containing this protective pro- teid, when injected into other animals, rendered them immune ; and also that it arrested the progress of the infectious malady induced by inoculating susceptible animals with virulent cultures of the micro- coccus. When injected into the circulation of an infected animal its curative action was manifested by a considerable reduction of the body temperature. While the micrococcus of pneumonia is not usually seen in the blood in cases of pneumonia it is probably present in small numbers, and secondary infection of the kidneys appears to be a common occur- rence. Thus Frankel and Reiche (1894) report that in twenty-two cases out of twenty-four in which they had an opportunity to exam- ine the kidneys, this micrococcus was present. It was found espe- cially in the larger branches of the veins and arteries, but also in the intertubular vessels and the glomeruli. The kidneys gave evidence of degenerative changes, and it is probable that the " pneumococcus " would have been found in the urine of some of these cases if a bac- teriological examination had been made during life. 21 VI. PATHOGENIC MICROCOCCI NOT DESCRIBED IN SECTIONS IV. AND V. 9. DIPLOCOCCUS INTRACELLULARIS MENINGITIDIS. DISCOVERED by Weichselbaum (1887) in the exudate of cerebro spinal meningitis (six cases), for the most part within the cells. Morphology. — Micrococci, usually united in pairs, in groups of four, or in little masses ; sometimes solitary and larger (probably being upon the point of dividing). Distinguished by their presence in the interior of pus cells in the exudate, in this respect resembling the gonococcus. Stain best with Loffler's alkaline solution of methylene blue. Do not retain their color when treated with iodine solution (Gram's method). Biological Characters. — This micrococcus does not grow at the room temperature, but upon nutrient agar an abundant development occurs in the incubating oven. Upon the surface of agar a tolerably luxuriant, viscid growth, which by reflected light is gray and by transmitted light grayish- white ; along the line of puncture growth occurs only near the surface, indicating that this micrococcus will not grow in the absence of oxygen. Upon plates made from agar- agar (one per cent) and gelatin (two per cent) very small colonies are formed in the interior of the mass, and larger ones, of a grayish color, on the surface. The former, under the microscope, are seen to be round or slightly irregular, finely granular, and of a yellowish- brown color. The superficial colonies have a yellowish-brown nu- cleus, surrounded by a more transparent zone. The growth upon coagulated blood serum is very scanty, as is that in bouillon ; no growth occurs upon potato. This micrococcus quickly loses its power of reproduction in artificial cultures — within six days — and should U> transj.lanltMl to {'roll material at short intervals— t\vo days. Pathogenesis. — Mice are especially susceptible, and usually die within forty-eight hours after inoculation. Also pathogenic for guinea-pigs, rabbits, and dogs. PATHOGENIC MICROCOCCI NOT HERETOFORE DESCRIBED. 323 10. STAPHYLOCOCCUS SALIVARIUS PYOGENES. Obtained by Biondi (1887) from an. inoculation abscess in a guinea-pig injected subcutaneously with, saliva from a child suffering from scarlatina anginosa. Morphology. — Spherical cocci, 0.3 to 0.5. u in diameter, usually solitary in the pus of abscesses or in irregular agglomerations. Stains best by Gram's method. Biological Characters. — Grows at a comparatively low temperature (12° to 14° C.), and more rapidly in the incubating oven. In gelatin stick cultures, at the room temperature, growth occurs along the line of punc- ture, and at the end of eight days liquefaction commences in the form of a funnel, at the bottom of which little, white, shining masses accumu- late, while at the surface of the liquefied gelatin a white, viscid layer forms. In gelatin plates spherical, well-defined, opalescent, whitish colonies are formed, which cause a tardy liquefaction of the surrounding gelatin. Upon agar-agar the growth is rapid, in the form of a thick layer along the line of inoculation in streak cultures, which has a breadth of about one millimetre at the end of twenty-four hours in the incubating oven, and presents an orange-yellow color at the centre, fading out to white at the margins. The yellow color is not by any means as pronounced as in similar cultures of Staphylococcus pyogenes aureus, and liquefaction of gelatin is much slower. Pathogenesis. — Produces a local abscess when inoculated into dogs, rab- bits, guinea-pigs, or mice. When injected into the anterior chamber of the eye of rabbits, hypopyon, followed by spontaneous perforation of the cor- nea, resulted. Injected into the circulation of a guinea-pig (0.2 to 0.4 cubic centimetre) it gave rise to general infection, and death at the end of eight to ten days. 11. MICROCOCCUS OF PROGRESSIVE TISSUE NECROSIS IN MICE. Obtained by Koch (1879) from mice inoculated subcutaneously with putrid blood. Morphology. — Round cells, 0.5// in diameter, united in chains, or at times in crowded masses. Biological Characters not given. Pathogenesis. — Causes necrosis of the tissues in the vicinity of the point of inoculation in mice, which extends rapidly and causes the death of the animal in about three days. The blood and internal organs remain free from micrococci. (Possibly a very pathogenic variety of Streptococcus pyogenes?) 12. MICROCOCCUS OF PROGRESSIVE ABSCESS FORMATION IN RABBITS. Obtained by Koch (1879) from rabbits inoculated with putrid blood. Morphology.— Minute cocci, about 0.15 u in diameter, usually associated in thick, cloud-like zooglcea masses. Biological Characters not given, Pathogenesis. — In rabbits an extensive abscess forms inthe vicinity of the point of in- oculation, and the animal dies in about twelve days. The walls of the abscess are formed of a thin layer of micrococci associated in zoog- loea masses; the interior contains finely gran- ular, cheesy material, in which the cocci ap- pear to have degenerated and perished. The contents of the abscess injected into other Fro. 96.— Micrococcus of progressive rahhifq rirnr1iir>f> a «iTYiilaT« i^snlt Thp mWn- tissue necrosis in mice; section of the 6 a SimUar result, ine miCl cartilage cells; 5, streptococci. coccus does not invade the blood. CKoch.) PATHOGENIC MICKOCOCCI Fio. 97. — Micrococcus of pyaemia in rabbits, in capil- lary from the cortical portion of the kidney. X700. (Koch.) 13. MICROCOCCUS OF PY^MIA IN RABBITS. Obtained by Koch (1879) in rabbits inoculated subcutaneously with putrefying flesh infusion. Morphology. — Round cells, 0.25 u in diameter, solitary or in pairs, which usually surround tHe blood corpuscles in a characteristic manner. Biological Characters not given. Pathogenesis. — When injected subcutaneously in rabbits the blood is invaded and death occurs from general infection. At the autopsv a puru- lent infiltration is found at the point of injection, there is peritonitis, and metastatic abscesses are found in the lungs and liver. Numerous micro- cocci, closely surrounding the blood corpuscles, are found in the capillaries of the various organs, the blood of the heart, etc. Two or three drops of blood from the heart of a rabbit recently dead, in- jected into another animal of the same species, cause its death in about forty hours. 14. MICROCOCCUS OF SEPTICAEMIA IN RABBITS. Obtained by Koch (1879) from rabbits inoculated subcutaneously with putrefying flesh infusion. Morphology. — Oval cells, haying a long diameter of 0.8 to 1.0 /*. Biological Characters not given. Pathogenesis. — Produces general infection and death in rabbits and mice. At the autopsy slight oedema is observed at the point of inoculation ; the spleen is greatly enlarged ; no peritonitis and no embolic processes are found, such as characterize the pathogenic action of the last-described species (No. 13) ; nor do the cocci accumulate around the red blood corpuscles. They are found in the capillaries of the various organs in masses, and especially in the glomeruli of the kidneys. 15. MICROCOCCUS SALIVARIUS SEPTICUS. Obtained by Biondi (1887) from the saliva of a case of puerperal septicae- mia, by inoculations into animals. Morphology.— Spherical or slightly oval cocci, which, when in rapid mul- tiplication, show slight lateral protrusions. Biological Characters. — Grows in nutrient gelatin or agar at a tem- perature of 18° to 20° C., and more rapidly in the incubating oven. Does not liquefy gelatin. In gelatin plates forms spherical, grayish- white colonies, which may acquire a dark color. In gelatin stick cultures grows along the .line of puncture in the form of a column made up of crowded white colo- nies. Very scanty growth on potato. Stains with all the aniline colors and by Gram's method. Pathogenesis.— Produces general infection and death in from four to six days when inoculated into mice, guinea-pigs, or rabbits. The cocci are found in great numbers, often assembled in masses, in the capillaries of the various organs, but no evidence of inflammatory reaction of the tissues is to be observed. 16. MICROCOCCUS SUBFLAVUS (Flligge). Synonym.— Yellowish-white diplococcus (Bumm). Obtained by Bumm (1885) from the lochial discharge of puerperal women and from vaginal mucus. Has also been obtained from the urine in cases NOT DESCRIBED IN SECTIONS IV. AND V. 325 of vesical catarrh, and in the vesicles of pemphigus ; also by Frankel in the vaginal secretion of children suffering- from colpitis not of gonorrhceal origin. Morphology.— Diplococci, associated in biscuit-shaped pairs, separated by a cleft, and closely resembling the gonococcus of Neisser. Cells from 0.5 to 1.5 n in diameter. Stains with the aniline colors and by Gram's method — by which char- acter it may be distinguished from the micrococcus of gonorrhoea. Biological Characters. — Grows at the room temperature upon the surface of nutrient gelatin; small, grayish- white colonies appear along the line of inoculation at the end of twenty-four hours, which later form a confluent layer, first of a pale yellow and finally of an ocherous color. In the course of a few days liquefaction of the gelatin commences in the vicinity of the growth. Coagulated blood serum is also liquefied by this micrococcus. Pathogenesis. — Inoculations upon mucous membranes susceptible to gon- orrhceal infection are without result. But by injecting the diplococcus from pure cultures, in suspension in distilled water, beneath the skin in man, Bumm obtained as a result local abscess formation — abscesses varying in size from that of a pigeon's egg to that of a man's fist. The diplococcus was present in great numbers in the pus of these abscesses. 17. MICROCOCCUS OF TRACHOMA (?). Obtained by Sattler (1885) from the contents of the tuachomatous follicles in cases of Egyptian ophthalmia; also by Michel (1886), who has given a more exact description of this micrococcus, and has made inoculation experi- ments which he believes establish its etiological relation to the form of oph- thalmia with which it is associated (?). Morphology. — Very small, biscuit-shaped micro-cocci, in pairs — diplococci — separated by a very narrow dividing line. (This description would apply to some of the more common pus cocci, e.g., Stapbylococcus pyogenes aureus, which have also been shown to consist of two hemispherical halves separated by a narrow line of division.) Biological Characters. — Grows slowly upon nutrient gelatin at the room temperature, and does not liquefy this medium, upon the surface of which a grayish-white, broadly extended, glistening layer is formed, which later has a yellowish tint and tulip-shaped margins. Spherical colonies are formed along the line of puncture, which are arranged in a linear series, like a chaplet. In blood serum it grows along the line of puncture as a white, band-like stripe, which subsequently spreads out in the form of white clouds. The growth is more rapid upon nutrient agar or blood serum in the incur bating oven. The development upon potato is very scanty. The cultures are viscid, drawing out into long threads when touched with a platinum needle. This micrococcus does not grow in the absence of oxygen — aerobic. Stains by the aniline colors and by Gram's method. Pathogenesis. — Not pathogenic for rabbits when injected subcutaneously or into the anterior chamber of the eye ; but, according to Sattler and to Michel, when inoculated by puncture into the conjunctivse in man it causes a follicular inflammation resulting in typical trachoma. But Michel was not able to demonstrate the presence of this micrococcus in all of his cases, and extensive researches made since by Baumgarten and by Kartulis (1887) show that in many cases of trachoma, and even in Egyptian ophthalmia (Kartulis) , it cannot be found. According to the last-named author, the viru- lent ophthalmia which prevails in Egypt is gonorrhceal in its origin, and he has demonstrated the presence of the gonococcus in a large series of cases. A milder, but infectious, acute catarrhal conjunctivitis is characterized by the presence of a minute bacillus, resembling the bacillus of mouse septi- caemia, and found in the pus cells. A third group of chronic cases with trachoma, in the researches of Kartulis, failed to show the presence of Sat- tler's trachoma coccus or any other microorganisms in the contents of the diseased follicles. 326 PATHOGENIC MICROCOCCI 18. MICROCOCCUS TETRAGENUS. First described by Gaffky (Fliigge). Obtained by Koch and Gaffky (1881) from a cavity in the lung in a case of pulmonary phthisis. Since found occasionally in normal saliva (three times in fifty persons examined by Biondi), and in the pus of acute abscesses (Steinhaus, Park, Vangel). Rather common in the sputum of phthi- sical cases. Morphology. — Micrococci, having a diameter of about one ^, which divide in two directions, forming tetrads, which are enclosed in a transparent, jelly-like envelope— especially well developed as seen in the blood and tissues of inoculated animals. In cultures the cocci are seen in the various stages of division, as large single cells, Fio. 98 — Micrococcus tetragenus; section of lung of mouse. X 800. (Flugge.) pairs of oval elements, or groups of four resulting from the trans- verse division of these latter. Stains quickly with aniline colors, and in preparations from the blood of an inoculated animal the transparent envelope may also be feebly stained. Stains also by Gram's method. Biological Characters. — This micrococcus grows, rather slowly, in nutrient gelatin at the ordinary room temperature, without lique- faction of the gelatin. Upon gelatin plates small white colonies are developed in from twenty-four to forty-eight hours, which under the microscope, with a low power, are seen to be spherical or lemon- shapnK finely -rantilar. and with a inulhcrry-likc surface. When they come to the surface they form white, elevated, and rather thick masses having a diameter of one to two millimetres. In gelatin Stick cultures a broad and thick white mass forms upon the surface, NOT DESCRIBED IN SECTIONS IV. AND V. 327 and along the line of puncture a series of round, milk-white or yel- lowish masses form, which usually remain distinct, but may become confluent. Upon the surface of agar the growth is similar to that upon gelatin, or in streak inoculations may consist of a series of spherical, white colonies. Upon cooked potato a thick, viscous layer is formed of milk-white color ; the growth upon blood serum is also abundant, especially in the incubating oven. This micrococcus is a facultative anaerobic. Pathogenesis. — Subcutaneous inoculation of a culture of this micrococcus in minute quantity is fatal to white mice in from two to six days. The animals remain apparently well for the first day or two, then remain quiet and somnolent until death occurs. The cocci are found in comparatively small numbers in the blood of the heart, but are more numerous in the spleen, lungs, liver, and kidneys, from which organs beautiful stained preparations may be made show- ing the tetrads surrounded by their transparent capsule. Common house mice and field mice are, for the most part, immune, as are the rabbit and the dog. Guinea-pigs sometimes die from general infec- tion, and sometimes a local abscess is the only result of a subcutane- ous inoculation. 19. MICROCOCCUS BOTRYOGENUS (Rabe). Synonyms. — Micrococcus of " myko-desmoids " of the horse; Mi- crococcus askoformans (Johne) ; Ascococcus Johnei (Cohn). First described by Bellinger (1870) ; morphological characters and location in the diseased tissues described by Johne (1884) ; biological characters determined by Rabe (1886). Is found in certain diffused or circumscribed growths in the con- nective tissue of horses — " myko-desmoids." Morphology. — Micrococci, having a diameter of 1 to 1.5 /*, usu- ally united in pairs. In the tissues the cocci are united in colonies of fifty to one hun- dred /* in diameter, and these are associated in mulberry-like masses visible to the naked eye. The separate colonies are enclosed in a homogeneous, transparent envelope — as in Ascococcus Billrothii. This is not the case, however, in cultures in artificial media. Stains with the aniline colors. Biological Characters. — In gelatin plate cultures spherical, sharply defined, silver-gray colonies are developed ; later these have a yellowish color and a metallic lustre, and the plate presents the ap- pearance of being powdered with grains of pollen. It gives off a peculiar fruit-like odor, reminding one of the odor of strawberries. In gelatin stick cultures growth occurs along the line of puncture as a pale grayish- white line, which later becomes milk-white ; an air 328 PATHOGENIC MICROCOCCI bubble forms near the surface of the gelatin ; very slight liquefac- tion occurs in the immediate vicinity of the line of growth, and after a time the grayish-white thread sinks into an irregular mass, lying at the bottom of the puncture. Upon nutrient agar scarcely any de- velopment occurs. Upon potato the growth is abundant, in the form of a pale-yellow, circular layer, and the culture gives off the peculiar odor above described. Pathogenesis. — When inoculated into guinea-pigs general infec- tion and death result. In sheep and goats it produces a local in- flammatory oedema and sometimes necrosis of the tissues. In horses inoculated subcutaneously an inflammatory oedema first occurs, fol- lowed at the end of from four to six weeks by the development of new growths in the connective tissue, resembling the tumors found in cases of the disease in the animal from which the micrococcus in question was first cultivated. These tumors contain characteristic mulberry-like conglomerations of colonies made up of the coccus. 20. MICROCOCCUS OF MANFREDI. Synonym. — Micrococcus of progressive granuloma formation. Obtained by Manfredi (1886) from the sputum of two cases of croupous pneumonia following measles. Morphology. — Oval micrococci, having a diameter of 0.6 to 1.0 /* and from 1.0 to 1.5 /* in length ; usually associated in pairs, and oc- casionally in short chains containing three or four elements. Stains with the aniline colors and by Gram's method. Biological Characters. — Aerobic ; does not liquefy gelatin. Upon gelatin plates forms small, spherical colonies, at first grayish- white, which spread out upon the surface as thin, transparent plates, which by transmitted light have a bluish, by reflected light a pearl- gray color. Later these become thicker and have a pearly lustre. Under the microscope (forty to fifty diameters) the colonies are seen to be slightly granular and the margins have an irregular outline. In gelatin stick cultures a scanty growth occurs along the line of puncture, and a rather thin and limited growth about the point of inoculation. Upon blood serum a thin, greenish-yellow layer, which has irregular margins and a slightly granular, shining surface, is developed. The growth upon potato, at 37° C., is scanty, and con- sists of a very thin, moist layer, which has a yellowish color and is slightly granular. Growth occurs in favorable media — bouillon, gelatin— at temperatures of 18° to 48° C., but ceases at a temperature of 48° to 50° C. Pathogenesis. — Pathogenic for dogs, rabbits, guinea-pigs, mice, and l.inls. In mammals the principal pathological appearance re- sulting from infection consists in the formation of " granulation tu- NOT DESCRIBED IN SECTIONS IV. AND V. 329 mors " in the parenchymatous organs. These vary in size from that of a millet seed to that of a pea, and undergo caseation. They con- tain the micrococcus and are infectious. Mammals die in from nine to fifteen days ; birds in from one to three or four, and without the formation of the characteristic granuloma, but with general infec- tion of the blood. Cultures which have been kept for several months retain their pathogenic power. 21. MICROCOCCUS OF BOVINE MASTITIS (Kitt). Obtained by Kitt (1885) from the udder of cows suffering from mastitis and giving milk mixed with pus. Morphology. — Micrococci, having a diameter of 0.2 to 0.5 ju, solitary, united in pairs, in irregular groups, and occasionally in chains. Stains with the aniline colors. Biological Characters. — Does not liquefy gelatin. Upon gelatin plates forms spherical, translucent, glistening colonies, the size of a hemp seed to that of a pin's head ; in gelatin stick cultures a nail-shaped growth occurs, the mass at the point of puncture being opaque and of a white color. Upon potato, colonies are quickly developed which have a grayish -white or dirty yellow color, and after a few days have a shining, wax-like appearance. Grows rapidly in milk, causing an acid reaction ; in six hours in the incu- bating oven the milk is pervaded by the micrococcus, or in twelve hours at 20° C. Pathogenesis. — Injection of pure cultures, suspended in distilled water, into the mammary glands of cows, produces typical, acute, purulent mas- titis (Kitt). The micrococcus produced the same result after having been cultivated in artificial media for a year. Subcutaneous inoculations in cows, pigs, guinea-pigs, rabbits, and mice were without result. Injections into the mammary gland of goats were also without effect. 22. MICROCOCCUS OF BOVINE PNEUMONIA (?). Isolated by Poels and Nolen (1886) from the lungs of cattle suffering from '"Limgenseuche" (infectious pleuro-pneumonia of cattle). Morphology. — Micrococci, varying considerably in size — average dia- meter 0.9 #; solitary, in pairs, or in chains containing several elements; sur- rounded by a transparent capsule, which stains with difficulty. Stains with all the aniline colors, and with difficulty by Gram's method. Biological Characters. — Does not liquefy gelatin, and grows like the ba- cillus of Friedlander in gelatin stick cultures (nail-shaped growth). In gela- tin plates the colonies are spherical, white, and have a very faint yellowish tinge. Grows more rapidly on agar in the incubating oven, and upon po- tato in the form of a very pale-yellowish layer. Is destroyed by a tempera- ture of 66° C. maintained for fifteen minutes. Pathogenesis — Pure cultures injected into the lungs of dogs, rabbits, and guinea-pigs are said to give rise to pneumonic inflammation, and simi- lar results were obtained by injection into the trachea of dogs and by in- halation experiments. Injection of a pure culture into the lungs of a cow caused extensive pneumonic changes; but these did not entirely correspond with the appearances found in the lungs of cattle suffering from infectious pneumonia. Cattle inoculated with a pure culture, by means of a sterilized lancet, did not fall sick, but are believed by Poels and Nolen to have been protected from the disease by such inoculations. The specific relation of the micrococcus above described to the disease with which it was associated, in the researches of the authors mentioned, has not been established by subsequent investigations. 22 330 PATHOGENIC MICROCOCCI 23. STREPTOCOCCUS SEPTICUS (Fliigge). Found by Nicolaier and by Guarneri in unclean soil during researches made in Fliigge's laboratory in Gottingen. Morphology. — Cannot be distinguished from Streptococcus pyogenes, but does not so constantly form chains, being found in the tissues of inoculated animals, for the most part in pairs. Biological Characters.— Grows more slowly than Streptococcus pyogenes ; in gelatin plates very minute colonies first appear at the end of three or four days, or along the line of puncture in gelatin stick cultures after five or six days. Does not liquefy gelatin. Pathogenesis. — Is very pathogenic for mice and for rabbits, causing death from general infection in two or three days. 24. STREPTOCOCCUS BOMBYCIS. Synonym. — Microzyma bombycis (Bechamp). Found in the bodies of infected silkworms suffering from la flacherie (maladie des morts-plats). Etiological relation established by Pasteur. Morphology. — Oval cells, not exceeding 1.5 ju, in diameter, in pairs or in chains. Biological Characters. — Not determined with precision. Pathogenesis. — The infected silkworm ceases to eat, becomes weak, and dies. Its body is soft and diffluent, and at the end of twenty-four to forty- eight hours is filled with a dark-brown fluid and with gas. 25. NOSEMA BOMBYCIS. Synonyms. — Micrococcus ovatus; Panhistophyton ovatum. Found in the blood and all of the organs of silkworms infected with p4brine (Fleckenkrankheit). First observed by Cornalia. Etiological relation established by Pasteur. Morphology. — Shining, oval cells, three to four # long and two/* broad; solitary, in pairs, or in irregular groups. Biological Characters. — Not determined with precision. Pathogenesis. — Dark spots appear upon the skin of infected silkworms, which lose their appetite, become slender and feeble, and soon die. The oval corpuscles are found in all of the organs, and also in the eggs of butterflies hatched from infected larvae. Some authors are of the opinion that the oval corpuscles found in this disease do not belong to the bacte- ria, but to an entirely different class of microorganisms — the Psorospermia (Metschnikoff). 26. MICROCOCCUS OF HEYDENREICH. Synonyms.— Micrococcus of Biskra button— Fr. " clou de Biskra "; Ger. "Pendesche Geschwur." Found by Heydenreich (1888) in pus and serous fluid obtained from the tumors and ulcers in the Oriental skin affection known as Biskra button. Morphology.— Diplococci, from 0.86 to 1 /* in length, surrounded by a capsule ; sometimes associated to form tetrads. ,s'/a///.s with the usual aniline colors. Biological Characters .— An aerobic, liquefying micrococcus Grows in the usual culture media at the room temperature. In gelatin stick cultures, at 20° C., at the end of fortv-eight hours growth occurs along the line of puncture in the form of small, crowded colonies, which produce a grayish- white line; upon the surface a thin, circular layer of a yellowish-white color is developed. At the end of three to four days liquefaction commences near the surface, where a funnel is formed which extends until about the fourteenth day, when the gelatin is completely liquefied. Upon the surface NOT DESCRIBED IN SECTIONS IV. AND V. 331 of agar, at 37° C., a grayish- white or yellowish layer is formed at the end of twenty-four hours, which has a varnish-like lustre. Upon potato, at 30° to 35° C. , at the end of forty-eight hours a white or yellow layer has de- veloped. Pathogenesis. — According to Heydenreich, inoculations in rabbits, dogs, chickens, horses, and sheep cause a skin affection which is identical with that which characterizes Biskra button in man. When rubbed into the healthy skin of man it also produces the development of abscesses. 27. MICROCOCCUS OP DEMME. Synonym. — Diplococcus of pemphigus acutus (Demme). Obtained by Demme (1886) from the contents of the bullae in a case of pemphigus. Morphology.— Micrococci of from 0.8 to 1.4 // in diameter; usually united in pairs resembling the " gonococcus " and having a length of 1.8to3/*, very opaque and not surrounded by a capsule ; usually associated in irregu- lar masses. Biological Characters. — Aerobic micrococci. Do not grow at the room temperature. Upon agar plates, at 37° C., at the end of thirty-six to forty- eight hours milk-white, spherical colonies, which project above the surface, are developed ; later club-shaped outgrowths form around the periphery of the colony, giving it the appearance of a rosette, or sometimes of a bunch of grapes. At the end of two weeks the surface is covered with smooih projec- tions and has a cream-like color. In streak cultures upon agar a similar growth occurs along the impfstrich, having club-like projections and stalac- tite-like outgrowths. Growth also occurs upon potato at a temperature of 37° C. This micrococcus develops slowly in the incubating oven, and scarcely any growth occurs at a temperature below 32° C. Pathogenesis. — The injection of a pure culture into the lungs of guinea- pigs gave rise to emaciation and debility and to the formation of foci of broncho-pneumonia, the size of a pea, in the lungs. 28. STREPTOCOCCUS OF MANNEBERG. Obtained by Manneberg (1888) from the urine in acute cases of Bright's disease. Morphology. — Micrococci, aboutO.9// in diameter, solitary, in pairs, or in chains of six to ten elements. Does not differ in morphology from Strep- tococcus pyogenes. Stains with the usual aniline colors and also by Gram's method. Biological Characters. — An aerobic and facultative anaerobic micro- coccus, which slowly produces a viscid softening of nutrient gelatin. Grows in the usual culture media at the room temperature. In gelatin stick cul- tures forms a white stripe along the line of puncture, which consists of small colonies. At the end of three or four weeks a funnel is formed containing very viscid liquefied gelatin, and at the same time brush-like outgrowths are seen along the line of development. Upon the surf ace of agar the growth resembles that of Streptococcus pyogenes, but is somewhat more abundant. IJpon potato, at 37° C., at the end of four or five days white, drop-like colo- nies are developed of about 0.5 millimetre in diameter; these become con- fluent and form a slimy layer. Milk becomes strongly acid and coagulates within twelve hours when inoculated with this micrococcus. Pathoge nesis.— Subcutaneous injection of 0.75 to 1 cubic centimetre causes the formation of a local abscess in dogs and rabbits. Intravenous injections produce inflammatory changes in the kidneys ; at the end of three or four days the urine contains red blood corpuscles, renal epithelium, blood casts, albumin, and streptococci. 332 PATHOGENIC MICROCOCCI 29. MICROCOCCUS ENDOCARDITIDIS RUGATUS (Weichselbaum). Obtained by Weichselbaum (1890) from the affected cardiac valves in a fatal case of ulcerative endocarditis. Morphology. — Micrococci, resembling- the staphylococci of pus in dimen- sions and mode of grouping; solitary, in pairs, in groups of four, or in ir- regular masses. Biological Characters. — An aerobic micrococcus. Does not grow at the room temperature. Upon agar plates, at 37° C. , at the end of three or four days the superficial colonies consist of a small, brown, central mass sur- rounded by a granular, semi transparent, grayish marginal zone; gradually they attain a characteristic wrinkled appearance; the deep colonies, under a low power, are irregular, finely granular, and contain a large central, yel- lowish-brown nucleus surrounded by a narrow, grayish -brown peripheral zone. In agar stick cultures small, spherical colonies are formed upon the surface, which become confluent, forming a grayish-white, wrinkled la}rer which has a stearin-like lustre and is very viscid ; a scanty growth occurs along the line of puncture. Upon potato, at 37° C , a scanty development occurs in the form of a small, dry, pale-brown mass. Upon blood serum isolated or confluent, colorless colonies are formed the size of a poppy seed ; these are closely adherent to the surface of the culture medium. Pathogenesis. — When injected subcutaneously into the ear of a rabbit it produces tumefaction and redness; in guinea-pigs, formation of pus. When injected into the circulation of dogs, after injury to the aortic valves, an en- docarditis is developed. 30. MICROCOCCUS OF GANGRENOUS MASTITIS IN SHEEP. Obtained bjr Nocard (1887) from the milk of sheep suffering from gan- grenous mastitis (rnal de pis or d'araignee), a fatal disease which attacks especially sheep which are being milked for the manufacture of cheese, at Roauefort and elsewhere in France. Morphology.— Micrococci, solitary, in pairs, or in irregular groups, resem- bling the staphylococci of pus in dimensions and arrangement. Stains with the usual aniline colors and also by Gram's method. Biological Characters.— An aerobic and facultative anaerobic, liquefy- ing micrococcus. Grows at the room temperature in the usual culture me- dia. Upon gelatin plates, at the end of forty-eight hours, the colonies are spherical and white in color; under a low power the superficial colonies are circular in outline, homogeneous, and brown in color; they are surrounded by a semi -transparent aureole ; liquefaction around the superficial colonies occurs sooner than around those beneath the surface of the gelatin. In gelatin stick cultures, at 18° to 20° C., on the second day liquefaction of the gelatin commences near the surface ; by the fifth day a pouch of liquefied gelatin has formed, which has the shape of an inverted cone; at the bottom of this an abundant deposit of micrococci is seen, while the liquefied gela- tin above is clouded throughout. In agar stick cultures development oc- curs upon the surface as a thick white layer, which gradually extends over the entire surface, and after a time acquires a yellowish tint; develop- ment also occurs along the line of puncture. Upon potato a thin, viscid, grayish layer is slowlv developed; the outline is irregular and the edges Incker than the central portion ; the central portion of this la yc-r gradually acquires a yellow color, while the periphery remains of a dirty-white or grayish color. Blood serum is liquefied by this micrococcus. / ''itliogenem—A. few drops of a pure culture injected subcutaneously or ito the mammary gland of sheep cause an extensive inflammatory oedema and the death of the animal in from twenty-four to forty-eight hours. A - globinuria. The cocci are found in the blood in great numbers, for the most part enclosed in the red corpuscles. Morphology. — Biscuit-shaped cocci united in pairs; sometimes oblong in form, isolated 01- united in groups; the free cocci are surrounded by a pale- yellowish, shining aureole of 0.5 to 1 >u in diameter. NOT DESCRIBED IN SECTIONS IV. AND V. 335 Stains best with Loffler's solution of methylene blue ; does not stain by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying micrococcus. Grows very slowly at the room temperature— not below 20° C. In the incubating* oven grows in the usual culture media. In gelatin stick cultures a scanty development of small, white colonies occurs along the line of puncture. Upon the surface of agar small, transparent drops are developed along the impfstrich. Upon potato, at 37° C.. a thin, broad, yellowish, shining layer is developed in the course of a few days— scarcely visible. Upon blood serum small, moist, transparent colonies are developed. Pathogenesis. — Pathogenic for rabbits and rats, which die in from six to ten days after inoculation with a pure culture; the spleen is found to be en- larged, the lungs hyperasmic, and a bloody serum is found in the cavity of the abdomen ; the cocci are present in the blood in considerable numbers, but are rarely seen in the red corpuscles. Inoculations in oxen, horses, goats, sheep, guinea-pigs, and birds were without effect. 35. MICROCOCCUS GINGIV.E PYOGENES. Obtained by Miller (1889) from the mouth of a man suffering from alveo- lar abscess. Morphology. — Large cocci of irregular dimensions, solitary or in pairs. Biological Characters. — An aerobic and facultative anaerobic, non-lique- fying micrococcus. Grows at the room temperature in the usual media. Upon gelatin plates it forms spherical, well-defined colonies, which under a low power are at first slightly colored and later opaque. In gelatin stick cultures an abundant development occurs both upon the surface and along the line of puncture. Upon the surface of agar a tolerably thick, grayish growth occurs along the impfstrich, which has a purplish tint by transmitted light. Pathogenesis. — Subcutaneous injections in mice produce a local abscess and necrosis of the skin, followed sometimes by death. Injections into the cavity of the abdomen produced peritonitis and death in from twelve to twenty-four hours. 36. PSEUDODIPLOCOCCUS PNEUMONIJE. Obtained by Bonome (1888) from the sero-fibrinous exudate in an autopsy of an individual who died of cerebro-spinal meningitis. Morphology.— Oval cocci, in pairs or in chains of five or six elements, often surrounded by a transparent capsule; not to be distinguished troi Micrococcus pneumonias crouposas. Stains with the usual aniline colors and by Gram s method. Biological Characters.— An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature (Micrococcus pneumonias crouposaedoes not grow at the room temperature). In gelatin stick cultures very small colonies are developed along the line of puncture at the end o. twenty-four to twenty-eight hours. Upon the surface of agar a rattier scanty, moist layer is developed along the impfstrich. Upon potato a tnm, scarcely visible layer is developed. In bouillon the development is abun- dant; the culture medium acquires a very acid reaction and gives ott a st odor like that of perspiration. . , . , Pathoqenesis.— Pathogenic for mice, guinea-pigs, and rabbits, in WHICH animals it produces fatal septicaemia; the spleen is not enlarged, as is the case in animals inoculated with Micrococcus pneumonias crouposae. 37. STREPTOCOCCUS SEPTICUS LIQUEFACIENS. Obtained by Babes (1889) from the blood and various organs of a child which died of sapticsernia following scarlatina. 336 PATHOGENIC MICROCOCCI Morphology. — Micrococci, about 0.3 to 0.4 /* in diameter, in pairs or in short chains in which the elements are loosely connected. Stains with the usual aniline colors and by Gram's method. Biological Characters. — An aerobic, liquefying micrococcus. Grows in the usual culture media at the room temperature. In gelatin stick cultures at the end of twenty-four hours a thin, granular, whitish stripe is seen along the line of puncture, while the surface seems somewhat depressed; later liquefaction of the gelatin occurs in funnel form ; the liquefied gelatin is but slightly clouded, and upon the walls of the funnel peculiar, flat, white, leaf- shaped, jagged colonies are seen. Upon the surface of agar, at 36° C. , small, white, thin, shining, transparent colonies are developed, which may attain a diameter of two to three millimetres. Upon blood serum a scarcely visible granular layer is developed. Pathogenesis. — Subcutaneous injections in mice and rabbits produce local inflammation with oedema, and death occurs in about six days ; the streptococci are found in large numbers in the effused serum, in the blood, and in the spleen. After being cultivated for some time in artificial media the cultures lose their pathogenic power. 38. MICROCOCCUS OF KIRCHNER. Obtained by Kirchner (1890) from the bronchial secretions (in sputum) of patients suffering from epidemic influenza — soldiers in garrison at Hanover. Morphology. — Spherical cocci, usually associated in pairs, and surrounded by a capsule. Distinguished from Micrococcus pneumoniae crouposse by be- ing smaller, quite spherical, and the elements in a pair being more widely separated from each other. Found in the bronchial secretion in scattered pairs, or associated in groups ; occasionally seen in chains. Stains with the usual aniline colors, but not by Gram's method. Biological Characters.— An aerobic micrococcus; does not grow in flesh- peptone-gelatin at the room temperature. Upon agar plates, at 36° C., small, grayish- white, transparent, spherical colonies are developed, which later form round, grayish-white plaques. In agar stick cultures an abun- dant development occurs upon the surface, extending to the walls of the test tube ; growth also occurs along the line of puncture. Pathogenesis. — Not pathogenic for rabbits or for white mice. A guinea- pig which received one cubic centimetre of a bouillon culture in the pleural cavity died at the end of twenty -four hours ; the spleen was not enlarged ; lungs hyperaemic ; the micrococci were found in the blood and in the vari- ous organs. Another guinea-pig, which received one cubic centimetre of a bouillon culture in the cavity of the abdomen, recovered after a slight indis- position. 39. MICKOCOCCUS NO. II. OF FISCHEL. Obtained by Fischel (1891) from the blood of two cases of influenza. Morphology.— Micrococci of from 1 to 1.25 n in diameter, mostly in pairs, sometimes in chains. Stains with the usual aniline colors and by Gram's method. Biological Characters.— An aerobic and facultative anaerobic, liquefy- ing micrococcus. Grows in the usual culture media at the room tempera ture. Upon gelatin plates minute colonies, visible only under the micro- scope, are developed at the end of three days. In gelatin stick cultures an abundant milk-white growth occurs along the line of puncture, and lique- - . - - potato, at 37° C., at the end of eight days „ thin, shining layer of a yellowish-white color, and about one centimetre broad is developed; no growth upon potato at the room temperature. No growth occurs in liquid blood serum or in milk. In sterilized water this micrococcus is said by Fischel to lose its vitality in eight hours NOT DESCRIBED IN SECTIONS IV. AND V. 337 Pathogenesis. — Pathogenic for dogs and for horses. Intravenous injec- tion of three to four cubic centimetres in dogs is said to produce symptoms resembling those of distemper in this animal, viz., increased temperature, catarrhal conjunctivitis, in some cases keratitis, and in some a mucous dis- charge from the preputial sac. The micrococcus was not found «in the blood of the dogs inoculated by intravenous injection, later than the fourth day. 40. STREPTOCOCCUS OF BONOME. Obtained by Bonome (1890) from the exudations of the cerebro-spinal meninges and from haemorrhagic extravasations in the lungs in cases of epidemic cerebro-spinal meningitis. This streptococcus is said by Bonome to be distinguished from previously known streptococci by the following characters: It does riot grow readily in artificial culture media, and soon loses its pathogenic power when pre- served in a desiccated condition or cultivated through a few successive gene- rations. It differs from the " pneumococcus " and " meningococcus " by the ball- shaped appearance of its colonies on agar plates, and in the fact that it does not grow upon, blood serum ; also by the difficulty experienced in carrying it through five or six generations in artificial media. Pathogenesis.— In white mice and in rabbits a fibrinous inflammation and death result from inoculations with a pure culture, the symptoms re- sembling those produced by similar inoculations with Micrococcus pneumo- niae crouposse. It does not produce septicaemia in white mice, but in rabbits the cocci are found in the blood in chains surrounded by a capsule. In guinea-pigs and dogs a local fibrinous inflammation results from inocula- tions, and the streptococcus is found in the gelatinous exudate at the point of inoculation. It is distinguished from the streptococcus of erysipelas by its failure to grow in gelatin or in blood serum, and by the appearance of its colonies on agar plates. 41. MICROCOCCUS OF ALMQUIST. Obtained by Almquist (1891) from the bullse of pemphigus neonatorum, in nine children suffering from this disease during an epidemic which oc- curred at Goteborg. Morphology. — Micrococci from 0.5 to 1 ju. in diameter, usually in pairs. Stains readily with the aniline colors. Biological Characters. — An aerobic, liquefying, chromogenic micro- coccus. Closely resembles Staphylococcus pyogenes aureus in its morpho- logy and growth in culture media. Produces a similar golden-yellow pig- ment. Pathogenesis. — According to Almquist, this micrococcus is distinguished from Staphylococcus pyogenes aureus by its specific pathogenic power. Two inoculations made from a pure culture, by means of a lancet, upon his own arm gave rise to a development of bullse like those of pemphigus. The process showed no disposition to extend deeper, but the epidermis was raised by a collection of fluid which was at first transparent and later had a milky opacity. From the contents of these bullae the same coccus was obtained in pure cultures. 42. STAPHYLOCOCCUS PYOSEPTICUS. Obtained by Hericourt and Richet (1888) from an abscess in the skin of a dog. In its morphology and biological characters this micrococcus closely re- sembles Staphylococcus pyogenes albus, and it is probably a pathogenic va- riety of this common species. But the experiments made by the authors referred to show it to be decidedly more pathogenic for rabbits. Subcutane- ous injections of a drop or two of a pure culture caused an extensive inflam- matory oedema, and death in from twelve to twenty-four hours. 338 PATHOGENIC MICROCOCCI NOT HERETOFORE DESCRIBED. 43. STREPTOCOCCUS PERNICIOSUS PSITTACORUM. Micrococcus of gray parrot disease. Eberth and Wolff have described an infectious disease of gray parrots, which is said to be extremely fatal among the imported birds. The disease is characterized by the formation of nodules upon the surface and in the interior of various organs, and especially in the liver. Micrococci of medium size are found in these nodules and in blood from the heart; these are sometimes in chains. Microscopic examina- tion of stained sections shows that these cocci are directly related to the tis- sue necrosis which characterizes the disease. But the micrococcus has not been cultivated and its biological characters are undetermined. 44. MICROCOCCUS OF FORBES. Forbes (1886) has studied an infectious disease of cabbage caterpillars (Pieris rapse), which appears to be due to a micrococcus found by him in large numbers in the bodies of the infected larvae. This micrococcus, which resembles the common staphylococci in form, was cultivated in liquid media and successful inoculation experiments were made. 44A. STREPTOCOCCUS AGALACTI^E CONTAGIOS^. Obtained by Adametz (1894) from the milk of cows suffering from mas- titis (Gelben Gait). According to Adametz all of the streptococci which have been described by different investigators (Kitt, Nocard and Mollereau, Guillebeau, and others) are probably varieties of a single species. Morphology. — Spherical cocci in short chains — 1 p in diameter. Biological Characters. — An aerobic and facultative anaerobic, non- efying streptococcus. Jpon gelatin plates forms flat, transparent, white or bluish-white, slimy colonies, having a slight pearly lustre and an irregular outline. In nutrient gelatin containing five per cent of milk sugar the colonies, at the end of eight days, have a diameter of 0.85 to 1 millimetre; they are milk- white and of a semi-fluid, slimy consistence. Upon agar plates the deep colonies are jmnctiform and white in color- under a low power they are seen to have an irregular dentate contour and a brownish color; the superficial colonies gradually assume the appearance of transparent, flat drops having a diameter of 0.5 to 0.7 millimetre. In sterilized milk fermentation occurs, at 37° C., in from twenty to twenty-four hours; some hours later the casein is precipitated, fine gas bubbles are seen in the lower part of the fluid and a foam upon the surface; the reaction is acid and the casein is not peptonized. The power of producing acid and gas is diminished or lost after a few successive cultures have been made. Streptococcus mastitis sporadice (Guillebeau) is said by Adametz to be distinguished from the streptococcus above described (No. 44A) by being smaller — 0.5 p. in diameter — and by the fact that the cultures do not lose the power of producing fermentation in milk. liqueh Upt VII. THE BACILLUS OF ANTHRAX. [Fr., CHARBON; Ger., MILZBRAND.] ANTHRAX is a fatal infectious disease which prevails extensively among sheep and cattle in various parts of the world, causing heavy losses. In Siberia it constitutes a veritable scourge and is known there as the Siberian plague ; it also prevails to a considerable extent in portions of France, Hungary, Germany, Persia, and India, and local epidemics have occasionally occurred in England, where it is known under the name of splenic fever. It does not prevail in the United States. In infected districts the greatest losses are incurred during the summer season. In man accidental inoculation may occur among those who come in contact with infected animals, and especially during the removal of the skin and cutting -up of dead animals, when there is any cut or abrasion upon the hands. A malignant pustule is developed as the result of such inoculation, but, as a rule, general infection does not occur, as is the case when inoculations are made into the more susceptible lower animals — rabbit, guinea-pig, mouse. Those who handle the hair, hides, or wool of infected animals are also liable to contract the disease by inoculation through opsn wounds, or by the inhalation of dust containing spores of the anthrax bacillus. Cases of pulmonic anthrax, known formerly in England as "wool-sorters' disease/' have been occasionally observed in England and in Ger- many, and are now recognized as being due to infection through the lungs in the manner indicated. The French physician Davaine, who had observed the anthrax bacillus in the blood of infected animals in 1850, communicated to the French Academy of Sciences the results of his inoculation experi- ments in 1863 and 1864, and asserted the etiological relation of the bacillus to the disease with which his investigations showed it to be constantly associated. This conclusion was vigorously contested by conservative opponents, but has been fully established by subsequent investigations, which show that the bacillus, in pure cultures, induces 840 THE BACILLUS OF ANTHRAX. anthrax in susceptible animals as certainly as does the blood of an animal recently dead from the disease. Owing to the fact that this was the first pathogenic bacillus cul- tivated in artificial media, and to the facility with which it grows in various media, it has served more than any other microorganism for researches relating to a variety of questions in pathology, general biology, and public hygiene, some of which are discussed in other sections of this volume. 45. BACILLUS ANTHRACIS. Synonyms. — Milzbrandbacillus, Ger.; Bacteridie du charbon, Fr. First observed in the blood of infected animals by Pollender (1849) and by Davaine (1850). Etiological relation affirmed by Davaine PIG. lOO.-Bacillus anthracis, from a culture, showing development of long threads in convo- luted bundles, x 300. (Klein.) (1863), and established by the inoculation of pure cultures by Pasteur (1879) and by many other investigators. Morphology.— Rod-shaped bacteria having a breadth of 1 to 1.25 /*, and 5 to 20 ^ in length; or, in suitable culture media, growing out into long, flexible filaments, which are frequently united in twisted, cord-like bundles. These filaments in hanging-drop cul- tures, before the development of spores, appear to be homogeneous ; or the protoplasm is clouded and granular, but without distinct seg- mentation. But in stained preparations the filaments are seen to be made up of a series of rectangular, deeply stained segments. In hanging-drop cultures the ends of the rods appear rounded, but in stained preparations from the blood of an infected animal they are seen to present a slight concavity, and a lenticular interspace is formed where two rods come together. The diameter of the rods THE BACILLUS OF ANTHRAX. 341 varies considerably in different culture media ; and in old cultures irregular forms are frequently seen — " involution forms." Under favorable conditions endogenous spores are developed in the long filaments which grow out in artificial culture media. These first appear as refractive granules distributed at regular inter- vals in the segments of the protoplasm, which gradually disappear as the spores are developed ; and these are left as oval, highly re- fractive bodies, held together in a linear series by the cellular enve- lope, and subsequently set free by its dissolution. The germination of these reproductive bodies results in the formation of rods and spore-bearing filaments like those heretofore described. In this pro- cess the spore is first observed to lose its brilliancy, from the ab- sorption of moisture, a promi- nence occurs at one end of the oval body, and soon the external envelope — exosporium — is rup- tured, permitting the softened protoplasmic contents enclosed in the internal spore membrane — endosporium — to escape as a short rod, to which the empty exosporium sometimes remains attached. The anthrax bacillus stains readily with the aniline colors and also by Gram's method, when not left too long in the decolorizing iodine solution. Loffler's solution of methylene blue is an especially good stain- ing fluid for this as well as for many other bacilli. Bismarck brown is well adapted for specimens which are to be photographed, and also for permanent preparations, as it is less liable to fade than the blue and some other aniline colors. Biological Characters. — The anthrax bacillus is aerobic, but not strictly so, as is shown by the fact that it grows to the bottom of the line of puncture in stick cultures in solid media. It is non-mo- tile, and is distinguished by this character from certain common bacilli resembling it in morphology — Bacillus subtilis — which were frequently confounded with it in the earlier days of bacteriological investigation. The anthrax bacillus grows in a variety of nutrient media at a FIG. 101.— Bacillus anthracis, from a culture, showing formation of spores, x 1,000. (Klein.) 342 THE BACILLUS OF ANTHRAX. temperature of 20° to 38° C. Development ceases at temperatures below 12° C. or above 45° C. This bacillus grows best in neutral or slightly alkaline media, and its development is arrested by a decidedly acid reaction of the cul- ture medium. It may be cultivated in infusions of flesh or of vari- ous vegetables, in diluted urine, in milk, etc. In gelatin plate cultures small, white, opaque colonies are devel- oped in from twenty-four to thirty-six hours, which under the micro- scope are seen to be somewhat irregular in outline and of a greenish tint ; later the colonies spread out upon the surface of the gelatin, and the darker central portion is surrounded by a brownish mass of wavy filaments, which are associated in tangled bundles. Mycelial- like outgrowths from the periphery of the colony may often be seen extending into the surrounding gelatin. At the end of two or three days liquefaction of the gelatin commences, and the colony is soon surrounded by the liquefied me- dium, upon the surface of which it floats as an irregular white pellicle. In gela- tin stick cultures growth occurs all along the line of puncture as a white cen- tral thread, from which lateral thread- like ramifications extend into the culture medium. At the end of two or three days liquefaction of the culture medium commences near the surface, where the development has been most abundant. At first a pasty, white mass is formed, but as liquefaction progresses the upper part of the liquefied gelatin becomes transparent from the subsidence of the motionless bacilli, and these are seen upon the surface of the non-liquefied portion of the medium in the form of cloudy, white masses, while below the line of liquefaction the charac- teristic branching growth may still be seen along the line of puncture. In agar plate cultures, in the incubating oven at 35° to 37° C., colonies are developed within twenty-four hours, which under the microscope are seen to be made up of interlaced filaments and are very characteristic and beautiful. Upon the surface of nutrient agar a grayish-white layer is formed, which may be removed in ribbon-like strips ; and in stick cultures in this medium a branching growth is seen, like that in gelatin, but without liquefaction. The addition of Fia. 102.— Culture of Bacillus an- thracis In nutrient gelatin : a, end of four days ; 6, end of eight days. (Baumgarten.) THE BACILLUS OP ANTHRAX. 343 a small quantity of agar to a gelatin medium prevents liquefaction of the gelatin (Fliigge). Upon blood serum a rather thick, white layer is formed and liquefaction slowly occurs. Upon potato the growth is abundant as a rather dry, grayish- white layer, of limited extent, having a somewhat rough surface and irregular margins. Spores are formed only in the free presence of oxygen, as in sur- face cultures upon potato or nutrient agar, or in shallow cultures in liquid media, and at a temperature of 20° to 35° C. They are not formed during the development of the bacilli in the bodies of living FIG. 103.— Colonies of Bacillus anthracis upon gelatin plates : a, at end of twenty-four hours; 6, at end of forty -eight hours, x 80. (Flugge.) animals, but after the death of the animal the bacillus continues to multiply for a time, and spores may be formed where the fluids containing it come in contact with the air — as, for example, in bloody discharges from the nostrils or from the bowels of the dead animal. Varieties incapable of spore production have been produced arti- ficially, by several bacteriologists, by cultivating the bacillus under unfavorable conditions. Roux was able to produce a sporeless va- riety by successive cultivation in media containing a small quantity of carbolic acid — 1 : 1,000. Varieties differing in their pathogenic power may also be pro- duced by cultivation under unfavorable conditions. Thus Pasteur 344 THE BACILLUS OF ANTHRAX. produced an " attenuated virus" by keeping his cultures for a con- siderable time before replanting them upon fresh soil, and supposed the effect was due to the action of atmospheric oxygen. It seems probable that it was rather due to the deleterious action of its own products of growth present in the culture media. It has been shown by Chamberlain and Roux that cultivation in the presence of certain chemical substances added to the culture medium— e. g. , bichromate of potassium 0.01 per cent — causes an attenuation of virulence. The same result occurs when cultures are subjected to a temperature a little below that which is fatal to the bacillus — 50° C. for eighteen minutes (Chauveau); 42.5° C. for two or three weeks (Koch). Attenuation of pathogenic virulence is also effected by cul- tivation in the body of a non-susceptible animal, like the frog (Lu- barsch, Petruschky); or in the blood of a rat (Behring); by exposure to sunlight (Arloing); and by compressed air (Chauveau). Anthrax spores may be preserved in a desiccated condition for years without losing their vitality or pathogenic virulence when in- oculated into susceptible animals. They also resist a comparatively high temperature. Thus Koch and Wolffhiigel found that dry spores exposed in dry air required a temperature of 140° C., maintained for three hours, to insure their destruction. But spores suspended in a liquid are destroyed in four minutes by the boiling temperature, 100° C. (writer's determination). The bacilli, in the absence of spores, according to Chauveau, are destroyed in ten minutes by a temperature of 54° C. For the action of various antiseptic and germicidal agents upon this bacillus we must refer to the sections especially devoted to this subject (Part Second). Toussaint, by injecting filtered anthrax blood into animals, obtained evidence that it contained some toxic substance which in his experi- ments gave rise to local inflammation without any noticeable general symptoms. More recent investigations show that a poisonous sub- stance is formed during the growth of the anthrax bacillus, and that cultures containing this toxin, from which the bacilli have been re- moved by filtration through porcelain, produce immunity when in- jected into susceptible animals, similar to that resulting from inocu- lations with an attenuated virus. It is probable that the pathogenic power of the anthrax bacillus depends largely upon the presence of tliis toxin, and that the essential difference between virulent and attenuated varieties depends upon the more abundant production of this toxic substance by the former. It has also been shown that virulent cultures produce a larger quantity of acid than those which have been attenuated by any of the agencies above mentioned (IVhring). THE BACILLUS OF ANTHRAX. 345 Martin (1890) has studied the chemical products in filtered cul- tures of the anthrax bacillus and obtained the following results: 1. Protoalbumose, deuteroalbumose, and a trace of peptone. The mixed albumoses were found not to be poisonous except in consider- able doses — 0.3 gramme injected subcutaneously killed a mouse weighing twenty-two grammes; smaller doses produced a local oedema. A fatal dose caused extensive cedema, coma, and death in twenty-four hours ; the spleen was sometimes enlarged. Boiling neutralizes to a considerable extent the toxic power. 2. An alkaloid, soluble in water and in alcohol, but insoluble in benzol, chloroform, or ether. The solutions have a strongly alkaline reaction, and crystalline salts are formed with various acids. This alkaloid is somewhat volatile, and when exposed to light loses to a considerable extent its toxic properties. It produces symptoms simi- lar to those resulting from inoculations with the albumoses, but is more toxic and more prompt in its action. The animal quickly falls into a state of coma ; there is extensive oedema about the point of inoculation, and the spleen is usually enlarged. The fatal dose for a mouse weighing twenty- two grammes is from 0.1 to 0.15 gramme ; death occurs within two or three hours. 3. In addition to these toxic substances small quantities of leucin and of tyrosin were found in the filtered cultures. Petermann (1892) has made a series of experiments with filtered cultures of the anthrax bacillus which led him to the conclusion that " large quantities of a culture in serum from the ox, filtered through porcelain, injected into the veins of a susceptible animal, have a pre- ventive action ; but the immunity thus conferred is transitory, not lasting longer than a month or two." Pathogenesis. — The anthrax bacillus is pathogenic for cattle, sheep, horses, rabbits, guinea-pigs, and mice. White rats, dogs, and frogs are immune, as is also the Algerian race of sheep. The spar- row is susceptible to general infection, but chickens, under normal conditions, are not. Young animals are, as a rule, more susceptible than adults of the same species. Man does not belong among the most susceptible animals, but is subject to local infection as a result of accidental inoculation — malignant pustule — and to pulmonic an- thrax from breathing air, containing spores of the anthrax bacillus, during the sorting of wool or hair from infected animals. In animals which have a partial immunity, natural or acquired, as a result of inoculations with attenuated virus, the subcutaneous introduction of virulent cultures may give rise to a limited local inflammatory pro- cess, with effusion of bloody serum in which the bacillus is found in considerable numbers ; but the blood is not invaded, and the animal, after some slight symptoms of indisposition, recovers. In susceptible 23 346 THE BACILLUS OF ANTHRAX. animals injections beneath the skin or into a vein give rise to general infection, and the bacilli multiply rapidly in the circulating fluid. Death occurs in mice within twenty-four hours, and in rabbits, as a rule, in less than forty-eight hours. The blood of the heart and large vessels may be found, in an autopsy made immediately after death, to contain comparatively few bacilli ; but in the capillaries of the various organs, and especially in the greatly enlarged spleen, in the liver, the kidneys, and the lungs, they will be found in great numbers, and well-stained sections of these organs will give an as- tonishing picture under the microscope, which the student should not fail to see in preparations made by himself. The capillaries in many places will be found stuffed full of bacilli ; or they may even be rup- tured as a result of the distention, and the bacilli, together with Fio. 104.— Bacillus anthracis in liver of mouse, x 700. (Flugge.) escaped blood corpuscles, will be seen in the surrounding tissues. In the kidneys the glomeruli, especially, appear as if injected with col- ored threads, and by rupture these may find their way into the urini- ferous tubules. These appearances and the general symptoms indicate that the disease produced by the introduction of this bacillus into the bodies of susceptible animals is a genuine septicaemia. As in other forms of septicaemia, the spleen is found to be greatly enlarged ; it has a dark color and is soft and friable. With this exception the organs pre- sent no notable changes, although the liver is apt to be somewhat enlarged. In the guinea-pig an extensive inflammatory oedema, ex- tending from the point of inoculation to the most dependent parts of the body, is developed ; the subcutaneous connective tissue is infil- trated with bloody serum and has a gelatinous appearance. This animal comes next to the mouse in susceptibility, and cultures which THE BACILLUS OF ANTHRAX. 347 are attenuated to such an extent that they will not kill a rabbit or a sheep may still kill a guinea-pig ; or, if not, may kill a mouse. Pasteur has shown that the pathogenic power of the bacillus may be reestab- lished by inoculations into susceptible animals, and that an attenu- ated culture which will not kill an adult guinea-pig may be fatal to a very young animal of this species, and that cultures from the blood of this will have an increased pathogenic virulence. Very minute quantities of a virulent culture are infallibly fatal to these most susceptible animals, but for rabbits and other less sus- ceptible animals the quantity injected influences the result, and re- covery may occur after subcutaneous or intravenous injection of a very small number of bacilli. FIG. 105.— Bacillus anthracis in kidney of rabbit. X 400. (Baumgarten.) Infection in cattle and sheep commonly results from the ingestion of spores while grazing in infected pastures. The bacillus itself, in the absence of spores, is destroyed in the stomach. While spores are not formed in the bodies of living animals, their discharges contain the bacillus, and this is able to multiply in them and to form spores upon the surface of the ground when temperature conditions are favorable. It is probable that this is the usual way in which pastures become infected, and that the bloody discharges from the bladder and bowels of animals suffering from the disease furnish a nidus for the external development of these reproductive elements ; as also do the fluids escaping from the bodies of dead animals. And possibly, under specially favorable conditions, the bacillus may lead a sapro- phytic existence for a considerable time in the superficial layers of the soil. 348 THE BACILLUS OF ANTHRAX. Buchner has shown by experiment that infection in animals may result from respiring air in which anthrax spores are in suspension in the form of dust ; and in man this mode of infection occurs in the so-called wool-sorters' disease. The question of the passage of the anthrax bacillus from the mother to the foetus in pregnant females has received considerable attention. That this may occur is now generally admitted, and ap- pears to be established by the investigations of Strauss and Chamber- lain, Morisani, and others. That it does not always occur is shown, however, by the researches of other bacteriologists, and especially by those of Wolff. Sirena and Scagliosi (1894) report, as the result of extended experi- ments made by them, that anthrax spores may survive in distilled water for twenty months; in moist or dry earth for two years and nine months ; in sea- water for one year and seven months ; in sewage nearly sixteen months. Marmier (1895) has made an extended experimental research to determine the nature of the specific toxin of the anthrax bacillus. This he obtains from cultures, at a low temperature, in media con- taining peptone and glycerin. It has not the reactions of an albu- minoid body and is not destroyed by a temperature of 100° C. In comparatively large doses it kills animals susceptible to anthrax, and by the administration of smaller doses immunity may be established in such animals. This toxin is contained in the bacterial cells, and is obtained by subjecting these to the action of alcohol, or from the filtrate when cultures are made at a low temperature in a medium containing peptone. It has not, however, been obtained in a pure form, and its exact nature has not been determined. VIII. THE BACILLUS OF TYPHOID FEVER. RECENT researches support the view that the bacillus described by Eberth in 1880 bears an etiological relation to typhoid fever — typhus abdominalis of German authors ; and pathologists are dis- posed to accept this bacillus as the veritable "germ" of typhoid fever, notwithstanding the fact that the final proof that such is the case is still wanting. This final proof would consist in the production in man or in one of the lower animals of the specific morbid phenomena which char- acterize the disease in question, by the introduction of pure cultures of the bacillus into the body of a healthy individual. Evidently it is impracticable to make the test upon man, and thus far we have no satisfactory evidence that any one of the lower animals is subject to the disease as it manifests itself in man. The experiments of Frankel and Simmonds show, however, that this bacillus is patho- genic for the mouse and the rabbit. We shall refer to the experi- ments of these authors later. Before the publication of Eberth's first paper Koch had observed this bacillus in sections made from the spleen and liver of typhoid cases, and had made photomicrographs from these sections. His name is, therefore, frequently associated with that of Eberth as one of the discoverers of the typhoid bacillus. Other investigators had no doubt previously observed the same organism, but some of them had improperly described it as a micrococcus. Such a mistake is easily made when the examination is made with a low powBr ; even with a moderately high power the closely crowded colonies look like masses of micrococci, and it is only by focussing carefully upon the scattered organisms on the outer margin of a colony that the oval or rod-like form can be recognized. Several observers had noted the presence of microorganisms in the lesions of typhoid fever prior to the publication of Eberth 's pa- per, and Browicz in 1875, and Fischel in 1878, had recognized the presence of oval organisms in the spleen which were probably identi- cal with the bacillus of Eberth. The researches of Gaffky (1884) strongly support the view that 350 THE BACILLUS OF TYPHOID FEVER. the bacillus under consideration bears a causal relation to typhoid fever. Eberth was only successful in finding the bacillus in the lymphatic glands or in the spleen in eighteen cases out of forty in which he searched for it. On the other hand, he failed to find it in eleven cases of various nature — partly infectious processes — and in thirteen cases of tuberculosis in which the lymphatic glands were involved, and in several of which there was ulceration of the mucous membrane of the intestine. Koch, independently of Eberth and before the publication of his first paper, had found the same bacillus in about half of the cases examined by him, and had pointed out the fact that they were lo- cated in the deeper parts of the intestinal mucous membrane, beyond the limits of necrotic changes, and also in the spleen, whereas the long, slender bacillus of Klebs was found only in the necrosed por- tions of the intestinal mucous membrane. The researches of W. Meyer (1881) gave a larger proportion of successful results. This author confined his attention chiefly to the swollen plaques of Peyer and follicles of the intestine which had not yet undergone ulceration. The short bacillus which had been de- scribed by Eberth and Koch was found in sixteen out of twenty cases examined. The observations of this author are in accord with those of Eberth as to the presence of the bacillus in greater abundance in cases of typhoid which had proved fatal at an early date. The fact that in these earlier researches the bacilli were not found in a considerable proportion of the cases examined is by no means fatal to the view that they bear an etiological relation to the disease. As Gaffky says in his paper referred to : :< This circumstance admits of two explanations. Either in those cases in which the bacillus has been sought with negative results they may have perished collectively, before the disease process which thev had induced had run its course ; or the proof of the presence of bacilli was wanting only on account of the technical difficulties which attend the finding of isolated colonies." Gaffky's own researches indicate that the latter explanation is the correct one. In twenty-eight cases examined by this author characteristic colonies of the bacillus were found in all but two. In one of these, one hundred and forty-six sections from the spleen, liver, and kid- neys were examined without finding a single colony, and in the other a like result attended the examination of sixty-two sections from the spleen and twnty-one sections from the liver. In the first of these cases, however, numerous colonies were found in recent ulcers of the intestinal mucous membrane, deeply located in that portion of the tissue which was still intact. These recent ulcers were in the neigh- THE BACILLUS OF TYPHOID FEVER. 351 borhood of old ulcers and are supposed to have indicated a relapse of the specific process. In the second case the negative result is thought by Gaffky to have been not at all surprising, as the patient died at the end of the fourth week of sickness, not directly from the typhoid process, but as a result of perforation of the intestine. Gaffky has further shown that in those cases in which colonies are not found in the spleen, or in which they are extremely rare, the presence of the bacillus may be demonstrated by cultivation ; and that, when proper precautions are taken, pure cultures of the bacil- lus may always be obtained from the spleen of a typhoid case. Hein has been able to demonstrate the presence of the bacillus and to start pure cultures from material drawn from the spleen of a living patient by means of a hypodermatic syringe. Philopowicz has re- ported his success in obtaining cultures of the bacillus by the same method. The fact that a failure to demonstrate the presence of microor- ganisms by a microscopic examination cannot be taken as proof of their absence from an organ, is well illustrated by a case (No. 18) in which the bacillus was obtained by Gaffky from the spleen and also from the liver, in pure cultures ; whereas in cover-glass preparations made from the same spleen he failed to find a single rod, and more than one hundred sections of the spleen were examined before he found a colony. To obtain pure cultures from the spleen Gaffky first carefully washes the organ with a solution of mercuric chloride, 1 : 1,000. A long incision is then made through the capsule with a knife sterilized by heat. A second incision is made in this with a second sterilized knife, and a third knife is used to make a still deeper incision in the same track. By this means the danger of conveying organisms from the surface to the interior of the organ is avoided. From the bottom of this incision a little of the soft splenic tissue is taken up on a ster- ilized platinum needle, and this is plunged into the solid culture medium, or drawn along the surface of the same, or added to lique- fied gelatin and poured upon a glass plate. The colonies develop, in an incubating oven, in the course of twenty-four to forty-eight hours. Gaffky has also shown that the bacillus is present in the liver, in the mesenteric glands, and, in a certain proportion of cases at least, in the kidneys, in which it was found in three cases out of seven. The appearance of the colonies in stained sections of the spleen is shown in Figs. 106 and 107. Two colonies are seen in Fig. 106 (at a, a) as they appear under a low power — about sixty diameters. In Fig. 107 one of the colonies is seen more highly magnified — about five hundred diameters. Fraiikel and Simmonds have demonstrated that the bacilli multi- 352 THE BACILLUS OF TYPHOID FEVER. ply in the spleen after death, and that numerous colonies may be found in portions of the organ which have been kept for twenty- four to forty-eight hours before they were placed in alcohol, when other pieces from the same spleen placed in alcohol soon after the death of the patient show but few colonies or none at all. This observation does not in any way weaken the evidence as to the etiological role of the bacillus, but simply shows that dead ani- mal matter is a suitable nidus for the typhoid germ — a fact which has been repeatedly demonstrated by epidemiologists and insisted upon by sanitarians. The authors last referred to confirm Gaff ky as regards the con- stant presence of the bacillus in the spleen. In twenty-nine cases they obtained it by plate cultures twenty-five times, and remark that in the four cases attended with a negative result this result is Fio. 106. FIG. 107. not at all surprising, inasmuch as the typhoid process had termi- nated and death resulted from complications. Gaffky did not succeed in obtaining cultures from the blood of typhoid-fever patients, and concludes from his researches that if the bacilli are present in the circulating fluid it must be in very small numbers. He remarks that possibly the result would be different if the blood were -drawn directly from a vein instead of from the capil- laries of the skin. Friinkel and Simmonds also report that gelatin, to which blood drawn from the forefinger of typical cases had been added, remained sterile when poured upon plates in the usual man- ner— Koch's method. The blood was obtained from six different in- dividuals, all in an early stage of the disease— the second to the third week. A similar experiment made with blood obtained, post mortem, from the large veins or from the heart, also gave a negative result in every instance save one. In the exceptional case a single THE BACILLUS OF TYPHOID FEVER. 353 colony developed upon the plate. In view of these results we are inclined to attribute the successful attempts reported by some of the earlier experimenters (Letzerich, Almquist, Maragliano) to accidental contamination and imperfect methods of research. The more recent work of Tayon does not inspire any greater confidence. This author obtained cultures in bouillon by inoculating it with blood drawn from a typhoid patient, and found that these were fatal, in a few hours, to guinea-pigs, when injected into the peritoneal cavity. The lesions observed are said to have resembled those of typhoid fever — congestion and tumefaction of Peyer's plaques and of the mesenteric glands, congestion of the liver, the kidneys, etc. The presence of the bacillus of Eberth in the alvine evacuations of typhoid patients has been demonstrated by Pfeiffer and by Frankel and Simmonds. This demonstration is evidently not an easy mat- ter, for while the bacilli are probably always present in some portion of the intestine during the progress of the disease, it does not follow that they are present in every portion of the intestinal contents. As only a very small amount of material is used in making plate cul- tures, and as there are at all times a multitude of bacteria of various species in the smallest portion of faecal matter, it is not to be ex- pected that the typhoid bacillus will be found upon every plate. Frankel and Simmonds made eleven attempts to obtain the bacillus by the plate method, using three plates each time, as is customary with those who adhere strictly to the directions of the master, and were successful in obtaining the bacillus in three instances — in two in great numbers and in the third in a very limited number of colo- nies. The numerous attempts which have been made to communicate typhoid fever to the lower animals have given a negative result in every instance. Murchison, in 1867, fed typhoid-fever discharges to swine, and Klein has made numerous experiments of the same kind upon apes, dogs, cats, guinea-pigs, rabbits, and white mice, without result. Birch-Hirschfeld, in 1874, by feeding large quantities of typhoid stools to rabbits, produced in some of them symptoms which in some respects resembled those of typhoid ; but these experiments were repeated by Bahrdt upon ten rabbits with an entirely negative result. Von Motschukoff sky met with no better success in his at- tempts to induce the disease by injecting blood from typhoid patients into apes, rabbits, dogs, and cats. Walder also experimented with fresh and with putrid discharges from typhoid patients, and with blood taken from the body after death, feeding this material to calves, dogs, cats, rabbits, and fowls, without obtaining any posi- tive results. Klebs has also made numerous experiments of a simi- lar nature, and in a single instance found in a rabbit, which died 24 354 THE BACILLUS OF TYPHOID FEVER. forty-seven*hours after receiving a subcutaneous injection of a cul- ture fluid containing his " typhoid bacillus," pathological lesions re- sembling those of typhoid. Eberth and Gaffky very properly decline to attach any import- ance to this solitary case, in which, as the first-named writer re- i narks, a different explanation is possible, and the possibility of an intestinal mycosis not typhoid in its nature must be considered. Gaffky has also made numerous attempts to induce typhoid symptoms in animals by means of pure cultures of Eberth's bacillus, given with their food or injected into the peritoneal cavity or subcu- taneously. The first experiments were made upon five Java apes. For a considerable time these animals were fed daily with pure cul- tures containing spores. The temperature of the animals was taken twice daily. The result was entirely negative. No better success attended the experiments upon rabbits (16), guinea-pigs (13), wm'te rats (7), house mice (11), field mice (4), pigeons (2), one hen and a calf. Cornil and Babes report a similar negative result from pure cul- tures of the typhoid bacillus injected into the peritoneal cavity and into the duodenum in rabbits and guinea-pigs. Frankel and Simmonds have made an extended series of experi- ments upon guinea-pigs, rabbits, and mice, and have shown that pure cultures of the bacillus of Eberth injected into the last-men- tioned animals — mice and rabbits— may induce death, and that the bacillus may again be obtained in pure cultures from their organs. It is not claimed that the animals suffer an attack of typhoid fever as the result of these injections, but that their death is due to the i ! 1 1 reduction into their bodies of the typhoid bacillus, and that this bacillus is thereby proved to be pathogenic. The failure to produce the characteristic lesions of typhoid in the lower animals is evidently not opposed to the view that this bacillus is the specific cause of such lesions in man. Frankel and Simmonds quote from Koch in support of this statement, as follows : in animals ana in man; tuberculosis does not present itself in precisely the same manner in one species of animals as in another. Phthisis, as it occurs in man. \\c cannot, in general, produce in animals; and, nevertheless, we • •annul assrrt tli.it tin- animals ••xp.-i-inn-iitrtl upon do not .sutler from tubrr- culoeis. and that the conclusions which we draw from such experiments are not perfectly correct." In Frankel and Simmonds' experiments a considerable quantity ai material was used, ,m|MM-ially in potato cultures kept in the incubating oven; these are not reproductive' spores, as was at first supposed. The bacilli have numerous flagella arranged around the periphery of the cells — usually from five to twenty, but many short rods have but a single a paper read by t lie writer :it the annual meeting of the Association of American Physicians, Washington, D. C., June 18th, 1886. THE BACILLUS OF TYPHOID FEVER. 359 terminal flagellum. These flagella are spiral in form, about 0. 1 p. in thickness, and from three to five times as long as the rods (Babes). In stained preparations unstained " vacuoles" may often be seen at the margins of the rods, either along the sides or at the ends ; these appear to be due to a retraction of the protoplasm from the cell membrane. The typhoid bacillus stains with the aniline colors, but more slowly than many other bacteria, and easily parts with its color when treated with decolorizing agents — e.g., iodine solution as employed in Gram's method. Loffler's solution of methylene blue is an excellent staining agent for this bacillus, but permanent preparations fade out after a time ; f uchsin, gentian violet, or Bismarck brown, in aqueous solution, may also be used. The flagella may be demonstrated by Lomer's method of staining (p. 32). 3£* \ -Sx.'^ FIG. 110.— Bacillus typhi abdominalis. stained by Loffler's method, showing flagella. x 1,000. From a photomicrograph by Frankel and Pfeiffer. To stain the bacillus in sections of the spleen, etc.,, it is best to leave these in Loffler's methylene blue solution or in the carbol- fuchsin solution of Ziehl for twelve hours or more ; or the aniline- fuchsin solution may be used. The sections should be washed in distilled water only, when ZiehFs solution is used, or with a very di- lute solution of acetic acid when Ehrlich's tubercle stain is employed (Baumgarten). Biological Characters. — The typhoid bacillus is a motile, aero- bic, non-liquefying bacillus, which grows readily in a variety of culture media at the " room temperature." Although it grows most abundantly in the presence of free oxygen, it may also develop in its absence, and is consequently a facultative anaerobic. 360 THE BACILLUS OF TYPHOID FEVER. FIG. 111.— Single colony of Bacillus typhi abdominalis, in nutrient gela- tin, (x?) From a photograph by ROUT. In gelatin plate cultures small, white colonies are developed at the end of thirty-six to forty-eight hours, which under the microscope are seen to be somewhat irregular in outline and of a spherical, oval, or long- oval form ; these have by transmitted light a slightly granular appearance and a yellowish-brown color. At the end of three or four days the colonies upon the surface of the gelatin form a grayish- white layer of one to two millimetres in diameter, with more or less irregular margins, and, when developed from deep colonies, with an opaque central nucleus. These colonies, by transmitted light, have a yellowish-brown color towards the centre, where they are thickest, while the margins are colorless and transparent ; the surface is com- monly marked with a network of lines and furrows. Stick cultures in ten-per-cent gelatin, at 18° to 20° C., at the end of three days show upon the surface a whitish, semi-transparent layer, with sharply defined margins and irregular outline, which has a shining, pearly lustre ; and along the line of puncture a grayish- white growth, made up of crowded colonies, which are larger and more distinct at the bottom of the line of growth. Upon nutrient agar, at a temperature of 35° to 37° C., the growth is more rapid and forms a whitish, semi-transparent layer. The cul- tures give off a faint putrefactive odor. The growth upon blood serum is rather scanty, in the form of transparent, shining patches along the line of inoculation. The typhoid bacillus develops abundantly in milky in which fluid it produces an acid re- action ; it also grows in various vegetable in- fusions and in bouillon. None of the above characters of growth are distinctive, as certain common bacilli found in normal faeces present a very similar appear- anre U'hrri cultivated in tho same media. The growth of this bacillus upon potato is an important character, as was first pointed out by Gaffky, In the incubating oven at the end of forty-eight hours, FIG m._Bacillua typhi abdominalis; stick culture THE BACILLUS OF TYPHOID FEVER. 361 or at the room temperature in three or four days, the surface of the potato has a moist, shining appearance, but there is no visible growth such as is produced by many other bacteria upon this me- dium. A simple inspection would lead to the belief that no growth had occurred; but if with a platinum needle a little material is scraped from any portion of the shining surface and a stained pre- paration is made from it, numerous bacilli will be seen, some of which are likely to be in the form of quite long threads, while others are short and have rounded extremities. This "invisible growth" has been shown by the researches of Buchner and others to be most characteristic upon potatoes having a decidedly acid reaction, as is usually the case. When cultivated upon potatoes having an alkaline reaction a thin, visible film of a yellowish-brown color and of limited extent may be developed. Inasmuch as several common and widely distributed bacteria closely resemble the typhoid bacillus in form and in their growth in nutrient gelatin, this character of invisible growth upon potato is very important for its differentiation, especially as the common bacilli referred to — Bacillus coli communis, bacillus of Em- merich— produce a very distinct and rather thick, yellowish- white mass upon the surface of potato. But recent researches show that this invisible growth, although not a common character, does not belong exclusively to the typhoid bacillus (Babes). This bacillus in its development in culture media produces acids — according to Brieger small quantities of volatile fat acids, and, in presence of grape sugar, lactic acid. It also grows readily in a de- cidedly acid medium, and this character has been employed as a test for differentiating it from other similar bacilli ; but some of these also grow in a decidedly acid medium, and too much reliance cannot be placed upon this test. Brieger has shown that indol is not produced in cultures of the typhoid bacillus, and Kitasato has proposed to use the indol test for differentiating this from other similar bacilli which are said, as a rule, to give the indol reaction. This test consists in the addition to ten cubic centimetres of a bouillon culture which has been in the in- cubating oven for twenty-four hours, of one cubic centimetre of a solution of sodium nitrite (0. 02 gramme to one hundred cubic centi- metres of distilled water), together with a few drops of concentrated sulphuric acid. If indol is present a red color is developed. None of the above-mentioned tests are entirely reliable, but, taken together with the morphological and biological characters above de- scribed, they may enable the bacteriological expert to give a tolerably confident opinion as to the presence of this bacillus in a water supply suspected of contamination, etc. And when a bacillus having these characters is obtained in a pure culture from the spleen of a typhoid THE BACILLUS OF TYPHOID FEVER. cadaver the student may be very sure that he has the typhoid bacillus. But in the presence of various similar bacilli, as in faeces, very careful comparative researches will be required to determine in a definite manner that a non-liquefying bacillus obtained in pure cultures by the plate method is really the one now under consideration— espe- cially so as the cultures of the typhoid bacillus in the same medium may differ considerably at different times, and a number of bacilli are known which resemble it so closely that it is still uncertain whether they are to be considered as varieties of the typhoid bacillus or as distinct species. Thus Babes, in an extended research, found in the organs of typhoid cases, associated with the true typhoid bacillus, other bacilli or varieties very closely resembling it. He has also described three varieties (?), obtained by him from other sources, which could only be differentiated from the true typhoid bacillus by very careful comparison of cultures made side by side in various media. Cassedebat, also, in an extended examination of the river water at Marseilles with reference to the presence of the typhoid bacillus, found three species which very closely resembled it, but which by careful comparison were shown to present slight but constant dif- ferences in their biological characters. He was not able to find the true typhoid bacillus, and his researches, together with those of Babes and other recent investigators, make it appear probable that numerous mistakes have been made by bacteriologists who have reported the finding of the typhoid bacillus in river and well water, in faeces, etc., and who have depended mainly upon the character of invisible growth upon potato in making their diagnosis. Cassedebat states that all three of his pseudo-typhoid bacilli corresponded in their growth upon potato with the bacillus of Eberth. They also corre- sponded in their growth on gelatin, agar-agar, and blood serum, which, as heretofore remarked, has no characteristic features. They all gave a negative indol reaction. Like the typhoid bacillus, they grew in milk without causing coagulation of the casein, but two of them produced an alkaline reaction in this fluid, while the third cor- responded with the typhoid bacillus in producing a decided acid re- action. Differences were also observed in bouillon cultures, and in bouillon and milk to which various aniline colors had been added, as recommended by Holz. Whether the typhoid bacillus, as obtained from the spleen of a typhoid cadaver, is in truth specifically distinct from these similar bacilli, or whether they are all varieties of the same species, result- ing from modifications in their biological characters acquired during their continuous development under different conditions, is an un- si-ttl.-.l qu.-Miuii. Jiut, in view of the experimental evidence now THE BACILLUS OF TYPHOID FEVER. 363 available, there is nothing improbable in the supposition that they are simply varieties, and that, as the result of a saprophytic mode of life, this bacillus may undergo more or less permanent modifications. In the writer's experiments (1887) the thermal death-point of the typhoid bacillus was found to be 56° C., the time of exposure being ten minutes ; and potato cultures containing the refractive granules described by Gaffky as spores were found to be infallibly destroyed by a temperature of 60° C. This result has been confirmed by Buch- ner (1888) and by Janowsky (1890), and the inference seems justified that these granules are not reproductive bodies, as was at first be- lieved ; for spores are distinguished by their great resistance to heat and other destructive agencies. According to Buchner, the bacilli containing these refractive granules are even less resistant than fresh cultures in which they are not present, and he is disposed to look upon them as representing a degeneration of the protoplasm of the cells. They do not stain by the methods which are successful in staining the spores of other bacilli, and, in short, present none of the characters which distinguish spores, except the form and high re- fractive power. The typhoid bacillus retains its vitality for many months in cul- tures; the writer has preserved bouillon cultures for more than a year in hermetically sealed tubes, and has found that development promptly occurred in nutrient gelatin inoculated from these. Dried upon a cover glass, it may grow in a suitable medium after having been preserved for eight to ten weeks (Pfuhl). When added to sterilized distilled water it may retain its vitality for more than four weeks (Bolton), (forty days Cassedebat), and in sterilized sea- water for ten days (De Giaxa). Added to putrefying faeces it may preserve its vitality for several months (Ufflemann), in typhoid stools for three months (Karlinski), and in earth upon which bouillon cultures had been poured for five and one-half months (Grancher and Deschamps). In hanging-drop cultures this bacillus may be seen to exhibit very active movements, the shorter rods rapidly crossing the field with a darting or to-and-fro, progressive motion, while longer filaments move in a serpentine manner. In addition to the volatile fat acids which, according to Brieger, are formed in small amounts in cultures of the typhoid bacillus, and to lactic acid formed in solutions containing grape sugar, a basic substance possessing toxic properties has been isolated by the chemist named — his typhotoxine (C,Hl7N"Oa). Brieger supposes that other basic substances are likewise formed, but believes this to be the speci- fic product to which the pathogenic action of the bacillus is due. It is a strongly alkaline base, which produces in mice and guinea-pigs salivation, paralysis, dilated pupils, diarrhoea, and death. 364 THE BACILLUS OF TYPHOID FEVER. Numerous experiments have been made to determine the amounts of various germicidal agents required to destroy the vitality of this bacillus, and the action of antiseptics in restraining its development. For the results of these experiments the reader is referred to the sections in Part Second relating to the action of antiseptics and disin- fectants. Pathogenesis.— The very numerous experiments which have been made on the lower animals have not been successful in producing in any one of them a typical typhoid process. Nor is this surprising, in view of the fact that, so far as is known, no one of them is liable to contract the disease, as man does, by the use of infected food or water. The experiments of Frankel and Simmonds show that when con- siderable quantities of a pure culture of this bacillus are injected into Fia 118— Section through wall of intestine, showing invasion by typhoid bacilli. X 950. (Baumgarten.) the circulation of rabbits through the ear vein, or into the peritoneal cavity of mice, a certain proportion of the' inoculated animals die, usually within forty-eight hours, and that the bacillus may be re- covered from the various organs, although it is not present in the blood. But death does not always occur from intravenous injections, and subcutaneous or intraperitoneal injections in rabbits are usually without result. Subcutaneous injections in mice proved to be fatal in ten cases out of sixteen inoculated by A. Frankel. Seitz, by following Koch's method — i.e., by rendering the contents of the stomach alka- line, and arresting intestinal peristalsis by the administration of opium — obtained a fatal result, in a majority of the guinea-pigs experi- mented upon, from the introduction of ten cubic centimetres of a bouillon culture into the stomach through a pharyngeal catheter. We may remark, with reference to these results, that while they show that cultures of the typhoid bacillus have a certain pathogenic power, THE BACILLUS OF TYPHOID FEVER. 365 they also show that the animals experimented upon frequently re- covered after comparatively large doses, and that the typhoid bacil- lus is not pathogenic in the same sense as are those microorganisms which, when introduced into the body of a susceptible animal in very minute amount, give rise to general infection and death. On the other hand, a fatal result depends upon the quantity of the culture introduced in the first instance, rather than upon the multiplication of the bacillus in the body of the inoculated animal. This view is confirmed by the experiments of Sirotinin, which show not only that a fatal result depends upon the quantity injected, but also that a similar result follows the injection of cultures which have been ster- ilized by heat or filtration. The pathogenic action, then, depends upon the presence of toxic substances produced during he growth of the bacillus in artificial culture media. The researches of Brieger, heretofore referred to, show the presence in such cultures of a toxic ptomaine — his typhotoxine — to which the pathogenic potency of these cultures appears to be due. White mice and guinea-pigs usually die in from twenty-four to forty-eight hours when inoculated in the cavity of the abdomen with a virulent culture of the typhoid bacillus — 0. 1 cubic centimetre to 0. 5 cubic centimetre of a bouillon culture three days old. According to Kitasato, the virulence of cultures from different cases of typhoid fever varies considerably. Detection of the Typhoid Bacillus in Water. — The generally recognized fact that typhoid fever is usually contracted by drink- ing water contaminated by the typhoid bacillus has led to numer- ous researches having for their object the discovery of a reliable method of detecting this bacillus when present in water in compara- tively small numbers in association with the ordinary water bacilli. The use of Koch's plate method, as commonly employed, will not suffice, because the water bacilli present grow more rapidly and cause liquefaction of the gelatin before visible colonies of the typhoid bacillus are formed ; and, owing to the relatively small number of typhoid bacilli, these are likely to escape detection. The aim of bacteriologists has, therefore, been to restrain the growth of these common water bacilli by some agent which does not at the same time prevent the development of the typhoid bacillus. Chan- temesse and Widal were the first to propose the use of carbolic acid for this purpose. They recommended the addition of 0. 25 per cent of this agent to nutrient gelatin ; but, according to Kitasato, the de- velopment of the typhoid bacillus is restrained by an amount exceed- ing 0. 20 per cent. Holz prepares an acid medium by adding gelatin (ten per cent) to the juice of raw potatoes, and asserts that while the typhoid bacillus grows luxuriantly in this medium, many other bacilli fail to develop ;},;,; THE BACILLUS OF TYPHOID FEVER. in it. The test is said to be still more reliable if 0.05 per cent of car- bolic acid is added to the " potato-gelatin." According to Holz, the addition of more than 0.1 percent of carbolic acid to nutrient gelatin prevents the free development of the typhoid bacillus. Thoinothas claimed to be able to obtain the typhoid bacillus from mixed cultures— as, for example, from faeces— by suspending a small amount of material containing it for several hours in a solution con- taining 0.25 per cent of carbolic acid. While other bacilli are destroyed, the typhoid bacillus is said to survive such exposure. The method of Parietti has recently been tested in a practical way by Kamen, and proved to be satisfactory for the detection of the typhoid bacillus in water which was supposed to be the source of a local epidemic of the disease. The following solution is used : Carbolic acid, 5 grammes. Hydrochloric acid (pure), 4 Distilled water, 100 Several test tubes, each of which contains ten cubic centimetres of neutral, sterilized bouillon, are used in the experiment. From three to nine drops of the acid solution are added to each of these, and the tubes are then placed in an incubating oven for twenty-four hours to ascertain whether they are still sterile after this addition. If the bouillon remains clear, from one to ten drops of the suspected water are added to each tube and they are returned to the incubating oven. If at the end of twenty-four hours the bouillon becomes clouded, this is due, according to Parietti, to the presence of the typhoid bacillus, which is then to be obtained in pure cultures by the plate method. The following method, suggested by Hazen and White, has been tested with favorable results by Foote. This method depends upon the fact that most of the common water bacilli do not grow at a tem- perature of 40° C., whereas this is a favorable temperature for the development of the typhoid bacillus. A small quantity of the sus- pected water is added to liquefied nutrient agar in test tubes, and plates are made. These are placed in an incubating oven at 40° C., and the typhoid bacillus, if present, will develop colonies within two or three days. At the ordinary room temperature the more numerous water bacilli would develop upon the same plates so abundantly that it would be difficult to recognize colonies of the typhoid bacillus. Theobald Smith (Centralb. /. Bakteriol., Bd. xii., page 367), has shown that the typhoid bacillus may be differentiated from other similar bacilli (Bacillus coli communis, bacillus of hog cholera, etc.) by the fact that it does not produce gas in culture media containing Hiigar— grape sugar, cane sugar, or milk sugar. The medium recom- THE BACILLUS OF TYPHOID FEVEK. 367 mended by Smith for making this test is a peptone-bouillon contain- ing two per cent of grape sugar and made slightly alkaline with carbonate of soda. The liquid becomes clouded throughout at the end of twenty-four hours, but not a trace of gas is developed even after several days. On the other hand, the colon bacillus and other bacilli which closely resemble the typhoid bacillus cause an abundant development of gas in this medium. The method of Wurtz will be found useful in the detection of colonies of the typhoid bacillus in plate cultures from contaminated water, etc. This consists in the addition to the nutrient medium of lactose (two per cent) and a solution of litmus. When the colonies develop in plates made from this medium the typhoid colonies re- main blue, while colonies of the " colon bacillus " have a red color, on account of the development of lactic acid. Schild (1894) uses a bouillon containing formalin (1:7,000) and claims that the typhoid bacillus fails to grow in this medium, while the bacilli of the colon group multiply in it and cause the me- dium to become clouded within twenty-four hours. Abel (1894), as a result of extended experiments, arrives at the conclusion that the formalin test cannot be relied upon for distinguishing the typhoid bacillus from certain similar bacilli, which also fail to grow in for- malin solution. But, on the other hand, a bacillus which grows in bouillon containing 1 : 7,000 of formalin can be definitely pronounced to be not the typhoid bacillus. Eisner (1895) recommends the following method for the detection of the typhoid bacillus in water or in f aaces : To potato gelatin, pre- pared by the method of Holz, he added one per cent of potassium iodide. But few species of bacteria will grow in this medium, but Bacillus coli communis grows in it luxuriantly, forming fully de- veloped colonies at the end of twenty-four hours. The typhoid col- onies, on the contrary, are only just visible under a low power at the end of twenty-four hours, and at the end of forty-eight hours are seen as small, shining, drop-like, very finely granular colonies. At the same time the colonies of the colon bacillus are much larger, coarsely granular, and of a brownish color. By this method Eisner succeeded in obtaining pure cultures of the typhoid bacillus from the faeces in fifteen out of seventeen cases of typhoid fever, in various stages of the disease. Lazarus (1895) has tested this method and re- ports that he succeeded without any difficulty in obtaining pure cul- tures of the typhoid bacillus from the alvine discharges of typhoid patients. When the typhoid bacillus and the colon bacillus are planted to- gether, in the same liquid medium, the first-mentioned bacillus, even when in excess at the outset of the experiment, soon disappears and 368 THE BACILLUS OF TYPHOID FEVER. the Bacillus coli communis remains in full possession. According to Wathelet (1895) the colon bacillus will grow in bouillon which has served as a culture medium for the typhoid bacillus, or on the surface of an agar plate from which a typhoid culture has been re- moved; but the typhoid bacillus fails to develop in culture media which have served for the development of the colon bacillus. The various diagnostic tests which have been proposed, and the extensive literature of the subject, show that the recognition of the typhoid bacillus in water, faeces, etc., is attended with serious diffi- culties. This is chiefly due to the fact that bacilli have been ob- tained from various sources which resemble more or less closely the typical typhoid bacillus as obtained from the spleen of a typhoid patient (or cadaver) and the " colon bacillus " as found in the alimen- tary canal of healthy men and animals; and also from the fact that the bacillus, as obtained from typhoid cases, varies to some extent in its biological characters, and that varieties may be produced in the bacillus as obtained, from a single colony, by special modes of culti- vation. From a consideration of these facts certain authors have been led to the conclusion that Bacillus typhi abdominalis and Bacillus coli communis are simply varieties of the same species. This view, however, is not generally accepted, and the characters which serve to differentiate the two bacilli are sufficiently well defined when typical cultures are compared. These characters, briefly stated, are: The invisible growth of the typhoid bacillus on potato ; its failure to give the indol reaction; its failure to coagulate milk, or to produce a change of color in litmus milk ; its failure to produce gas in culture media containing glucose or lactose ; its failure to grow in formalin bouillon (1 : 7,000) ; and its active motility. Whether the closely re- lated bacilli which present some of the characters above indicated, without corresponding in all particulars with typical cultures of the typhoid bacillus, are varieties of this bacillus, which under favorable circumstances could give rise to typhoid infection, has not been defi- nitely determined, but appears to be quite probable. It may be that such varieties are developed when the typhoid bacillus in faeces finds its way into surface waters, under conditions which are favorable for its continued development as a saprophyte. On the other hand, it may be that one or more of the saprophytic bacilli, which are found in water and which closely resemble the typhoid bacillus, may give rise to the infectious disease which we know as typhoid fever when in- t induced into the alimentary canal of a particularly susceptible indi- vidual, and that the special conditions attending its development as a parasite give rise to certain modifications in its biological charac- ters of a nu.re or less permanent kind. Frankland (18U5), as a result of extended experiments, has arrived THE BACILLUS OF TYPHOID FEVER. 369 at the conclusion that when the typhoid bacillus is cultivated for a long time in media which are more and more largely diluted with water, it acquires an increased ability to survive in river water. A predisposition to typhoid infection is established by various -depressing agencies, such as inanition, overwork, mental worry, in- sanitary surroundings, etc. And there is considerable evidence in support of the supposition that exposure to the offensive gases given off from ill-ventilated sewers constitutes a predisposition to the disease. Experiments made by Alessi (1894), in the Hygienic Institute of the University of Rome, give support to this view. The ex- periments were made upon rats, guinea-pigs, and rabbits. The rats were confined in a close cage with perforated bottom, which was placed over the opening of a privy ; the guinea-pigs and rabbits in similar cages having a receptacle below in which their own excreta was allowed to accumulate. The animals which breathed an atmo- sphere vitiated in this way lost, after a time, their usual activity and became emaciated, although they continued to eat greedily. When these animals were inoculated with a small quantity of a culture of the typhoid bacillus (0.25 to 0.5 cubic centimetre) they died within from twelve to thirty-six hours. The same amount of the typhoid culture injected into control animals produced no injurious effect. In the animals which succumbed to typhoid infection there was found a hemorrhagic enteritis, increase in volume of Peyer's glands and of the spleen, and typhoid bacilli in the blood, liver, and spleen. The char- acteristic appearances of typhoid infection were more pronounced in the rabbits and guinea-pigs than in rats. Similar experiments with Bacillus coli communis gave similar results. The time required to induce this predisposition for typhoid infection was from five to seventy-two days for the rats, seven to fifty-eight for the guinea- pigs, and three to eighteen for the rabbits. Alessi found that the susceptibility to infection diminished after a certain time, and sug- gests that in a similar way man may become habituated to breathing an atmosphere containing sewer gases. Pus- Product ion by Typhoid Bacilli. — The recent literature re- lating to the typhoid bacillus includes many observations as to its presence in accumulations of pus in various parts of the body — often in a pure culture. It has been found in a considerable number of cases of periostitis secondary to typhoid fever, in purulent syno- vitis, and in abscesses in various parts of the body. Dmochowski and Janowski (1895), as the result of a review of the literature and a painstaking experimental research, arrive at the con- clusion that ev^n in abscesses, occurring in typhoid fever cases, in which only the pus cocci are found, it is probable that the typhoid • % <. A 370 THE BACILLUS OP TYPHOID FEVER. bacillus originated the process resulting in abscess formation. They assert that the typhoid bacillus dies out in a comparatively short time in abscesses which are directly due to its presence, and that often it may be found in the abscess walls when its presence can no longer be demonstrated in the purulent contents of the abscess cavity. PLATE V. PATHOGENIC BACTERIA. Fia. 1. — Bacillus anthracis from cellular tissue of inoculated mouse. Stained with gentian violet, x 1,000. Photomicrograph by Frankel and Pfeiffer. FIG. 2. — Bacillus anthracis in section of liver of inoculated rabbit. Stained with Bismarck brown, x 250. Photomicrograph by Sternberg. Fia. 3. — Micrococcus gonorrhoeas in gonorrhoeal pus. Stained with gen- tian violet, x 1,000. Photomicrograph by gaslight (Sternberg). FlG. 4. — Anthrax spores from a bouillon culture. Double-stained prepara- tion— with carbol-fuchsin and methylene blue, x 1,000. Photomicrograph by Frankel and Pfeiffer. FIG. 5. — Spirillum choleras Asiaticae from a culture upon starched linen at end of twenty-four hours Stained with fuchsin. x 1,000. Photomi- crograph by Frankel and Pfeiffer. FIG. 6.— Bacillus diphtherias from colony upon -an agar plate, twenty- four hours old. Stained with Ltf filer's solution of methylene blue, x 1,000. Photomicrograph by Friinkel and Pfeiffer. Mi II-. -•• ,:,. PLATH V. STERNBERG'S BACTERIOLOGY , ,. » . I'1 IK. PATHOGENIC BACTERIA IX. BACTERIA IN DIPHTHERIA. DIPHTHERIA is generally recognized by physicians as a specific infectious disease, and, owing to its wide prevalence and fatal char- acter, a precise knowledge of its etiology is of the greatest import- ance. Until, as a result of recent researches, this was determined, pathologists were in doubt as to whether diphtheria should be con- sidered as primarily a local infection, or whether the local manifesta- tions were secondary to a general systemic infection. But this question appears now to be definitely settled in favor of the former view. We have to-day a very precise knowledge of the specific infecting agent, and have evidence that it produces during its growth a very potent toxic substance, the absorption of which from the seat of local infec- tion accounts in a satisfactory manner for the general symptoms of the disease, which are due to toxaemia and not to an invasion of the blood and tissues by the pathogenic microorganism producing it. Numerous researches by competent bacteriologists have failed to demonstrate the presence of bacteria in the blood of patients suffer- ing from diphtheria, but a variety of microorganisms have been ob- tained in cultures from diphtheritic pseudo-membranes, and may be demonstrated by the microscopical examination of stained prepara- tions. Among these are the well-known pus organisms, and espe- cially the Streptococcus pyogenes, which appears to be very commonly present, and is perhaps the active agent in the production of certain forms of pseudo-diphtheria. But the malignant, specific diphtheria, so well known in this country and in Europe, has been demonstrated by the recent researches of bacteriologists to be due to a bacillus first recognized by Klebs in stained preparations of diphtheritic false membranes (1883), and cultivated and described by Loffler in 1884. In his first publication Loffler did not claim to have fully demon- strated the etiological relation of this bacillus, but this appears to be fully established by subsequent researches. In his first research Loftier studied twenty-five cases, and in the greater number of them found in stained preparations the bacil- lus previously described by Klebs. From six of these cases he 372 BACTERIA IN DIPHTHERIA. obtained it in pure cultures, and by inoculations in pigeons, chickens, rabbits, and guinea-pigs proved that it gave rise to a diphtheritic inflammation when inoculated into the mucous membrane of the trachea, conjunctiva, pharynx, or vagina. In a second communica- tion Loffler reported his success in finding the same bacillus in ten additional cases, and also that he had isolated from the same source a non-pathogenic bacillus which resembled it very closely. This pseudo-diphtheria bacillus has since been found by other bacteri- ologists (Von Hoffmann, Roux and Yersin), and it is uncertain whether it is to be considered a distinct species, or a non-pathogenic variety of the diphtheria bacillus as maintained by Roux and Yersin. But its occasional presence does not invalidate the very positive ex- perimental evidence relating to the specific pathogenic power of the true diphtheria bacillus. Loffler, in 1890, reviewed the evidence upon which this bacillus is now generally conceded by bacteriologists to be the specific infectious agent in true diphtheria. The following are the principal points in the demonstration : FIRST. — It is found in all undoubted cases of diphtheria. In support of this we have the results of researches made by Loffler, Wyssokowitsch, D'Espine, Yon Hoffmann, Ortmann, Roux and Yersin, Kolisko and Paltauf, Zarinko and Sorensen, who in nearly every case have demonstrated without difficulty the presence of this bacillus. On the other hand, Prudden failed to find it in a series of twenty-four cases studied by him ; but his own account of these cases indicates that they were not cases of true diphtheria. He says in a subsequent communication : " In view of the doubt existing among practitioners as to whether all forms of pseudo-membranous inflammation should be called diphtheria or not, and with the purpose of making a wholly objective study, the writer distinctly stated at the outset of that paper that all the fatal cases of exten- sive pseudo-membranous laryngitis, as well as pharyngitis, should in his study be considered as cases of diphtheria. This left the question as to the propriety of establishing separate groups of pseudo-membranous inflamma- tion open and free from bias. It was distinctly stated, however, that six- teen out of the twenty-four cases occurred in a large asylum, in which measles and scarlet fever were prevalent during the period in which these studies were under way. Five other cases in another asylum were ex- posed to similar conditions." In a subsequent series of "twelve cases of fatal pseudo-mem- branous inflammation occurring in two children's asylums, in which t T many months there had been no scarlatina and no measles, and in which there was no complicating suppurative inflammation and ii" < i \ -sipelas," Prudden (1890) obtained Loffler's bacillus in cultures t i"in Cloven, and he says : " We are now, it would seem, justified, as it did not appear to the writer BACTERIA IN DIPHTHERIA. 373 that we were two years ago, owing to the large number of important re- searches which have been made in the interim, in saying that the name diphtheria, or at least primary diphtheria, should be applied, and exclusively applied, to that acute infectious disease, usually associated with a pseudo- membranous inflammation of the mucous membranes, which is primarily caused by the bacillus called Bacillus diphtherias of Loftier." With reference to the question as to how long after convalescence is established the diphtheria bacillus may be present in the throat of an infected person, Loffler has made the following research (1890). In a typical case a bacteriological examination was made daily from the commencement until fourteen days after its termination. Fever disappeared on the fifth day, and the exudation had all disappeared on the sixteenth day. Up to this time the bacillus was daily ob- tained in cultures, and subsequently nearly every day up to the twenty-fifth — that is, for three weeks after the febrile symptoms had disappeared. Roux and Yersin have also obtained the bacillus in cultures from mucus scraped from the throats of convalescents sev- eral days after the disappearance of all evidence of the disease. SECOND. The Klebs-Loffler bacillus is found only in diph- theria.— In his earlier researches Loffler obtained the bacillus in a single instance from the mouth of a healthy child, and this fact led him to hesitate in announcing it as his conviction that it was the true cause of diphtheria. But in extended researches made subse- quently he has not again succeeded in finding it, except in associa- tion with diphtheria, and admits now that he may have been mis- taken as to the identity of the bacillus found. This seems not improbable in view of the fact that very similar bacilli have been found by various bacteriologists. Thus Von Hoffmann obtained a very similar but non-pathogenic bacillus from the mucus of chronic nasal catarrh and from healthy mucous membranes ; Babes from cases of trachoma, Neisser from ulcers, Zarinko rrom the surface of various mucous membranes. But all of these were shown to present certain differences in their biological characters by which they could be differentiated from the true diphtheria bacillus. Welch and Abbott in their comparative studies did not find the Loffler bacillus, "or any bacillus that an experienced bacteriologist would be likely to confound with it." They examined mucus from the throats of healthy children, from those suffering from simple in- flammation of the tonsils and pharynx, and from four cases of so- called follicular tonsillitis. As a result of their investigations they agree with Loffler, and with Roux and Yersin, as to " the great prac- tical value, for diagnostic purposes, of a bacteriological examination of cover-glass specimens and by cultures " of cases in which there is any doubt of the true character of the disease. They say further : 374 BACTERIA IN DIPHTHERIA. "The only species of bacteria which we have found constantly in the cases of diphtheria has been the Loftier bacillus. Two other species have been present in many cases, viz., the well-known streptococcus, which grows in much smaller colonies and less rapidly than the Loftier bacillus, and a TlSe colonies of this bacillus are grayish-white, moist, larger than those of the streptococcus, but smaller than those of the Loftier bacillus." THIRD. As shown by Ldffler>s earlier researches, pure cultures of this bacillus induce characteristic diphtheritic inflammation when inoculated into the mucous membranes of certain lower ani- mals. Roux and Yersin have also shown that local paralysis is likely to occur in inoculated animals, as is the case in diphtheria in man. In speaking of their inoculations into the trachea in rabbits those investigators say : "The affection which is thus induced in the rabbit resembles croup in man. The difficulty which the animal experiences in breathing ; the noise made by the air in passing through the obstructed trachea- the aspect of the trachea, which is congested and covered with false membranes ; the cedema- tous swelling of the tissues and glands of the neck, make the resemblance absolutely remarkable." Welch and Abbott give the following account of the results of inoculations into the trachea in kittens : "A half-grown kitten is inoculated into the trachea with one platinum loop from a pure culture of the Loftier bacillus on glycerin-a^ar, eleven days old, derived from Case IV. For the inoculation a small median incision was made over the trachea, in which a hole just large enough to admit the plati- num loop was made. The culture was rubbed over the mucosa of the trachea for an extent about three centimetres in length, and in this process sufficient force was used to abrade the mucous membrane. On the day following the inoculation no special alteration in the animal was observed, but on the morning of the second day it was found very weak. In the course of this day it became so weak as to lie completely motionless, apparently uncon- scious, with very feeble, shallow respiration; several times it was thought to be dead, but on careful examination proved still to be breathing feebly. It was found dead on the morning of the third day. At the autopsy the wound w.ts found gaping and covered with a grayish, adherent, necrotic, distinctly diphtheritic layer. For a considerable distance around the wound the sub- cutaneous tissues were very cedematous, the oedema extending from the lower jaw down over the sternum, and to the sides of the neck, and along the anterior extremities. The lymphatic glands at the angle of the jaw were markedly swollen and reddened. The mucous membrane of the trachea, Ix'^mmiitfat th<- l.-irynxand extending down forsix centimetres, wascovered with a tolerably firm, grayish-white, loosely attached pseudo-membrane, in ;ill respects identical with the croupous membranes observed in the same situation in cases of human diphtheria." BACTERIA IN DIPHTHERIA. 375 47. BACILLUS DIPHTHERIA. First observed by Klebs (1883) in diphtheritic false membranes. Isolated in pure cultures and pathogenic power demonstrated by Loffler (1884). Found in diphtheritic pseudo-membranes, and especially in the deeper portions, intermingled with numerous cellular elements; while the superficial layers of the membrane commonly contain but few cells or bacilli, or are invaded by other species, especially by Strep- tococcus pyogenes. The bacilli are not found in the affected mucous membrane, or in sections from the internal organs in fatal cases of this disease. Morphology. — Rods, straight or slightly curved, with rounded ends, having a diameter of 0.5 to 0.8 jt, and from 2 to 3 IJL in length. Ir- regular forms are very common, and, indeed, are characteristic of this bacil- lus. In the same culture, and especially in an unfavorable culture medium, very great differences in form and dimen- sions may be observed ; one or both ends may appear swollen, or the central por- tion may be notably thicker than the extremities, or the rod may be made up of irregular spherical or oval segments. FIG. 114. — Bacillus diphtheria*, Multiplication occurs by fission only, SLtJSlS aild the bacilli do not grOW Out into fila- (Frankel and Pfeiffer.) ments. In unstained preparations certain portions of the rod, and espe- cially the extremities, are observed to be more highly refractive than the remaining portion ; and in stained preparations these portions are seen to be most deeply colored. The diphtheria bacillus may be stained by the use of Loffler's alkaline solution of methylene blue, but is not so readily stained with some of the other aniline colors commonly employed. It stains also by Gram's method. For the demonstration of the bacillus in sections of diphtheritic membrane " nothing can surpass in brilliancy and sharp differentiation sections stained doubly by the modified Weigert's fibrin stain and picro-car- mine" (Welch and Abbott). Biological Characters. — The diphtheria bacillus is aerobic, non- motile, and non-liquefying; it does not form spores. It grows most freely in the presence of oxygen, but is also a facultative anaerobic. Development occurs in various culture media at a temperature of from 20° to 42° C., the most favorable temperature being about 35° C. BACTERIA IX DIPHTHERIA. It grows readily in nutrient gelatin having a slightly alkaline reac- tion, in nutrient agar, glycerin-agar, or in alkaline bouillon, but the most favorable medium appears to be that first recommended by Loffler— viz., a mixture of three parts of blood serum with one part of bouillon, containing one per cent of peptone, one per cent of grape sugar, and 0.5 per cent of sodium chloride. This mixture is steril- ized and solidified at a low tem- perature, as is usual with blood serum. Upon this the develop- FIO. ns.-coionies of Bacillus diphtheria inent is so rapid in the incubating in nutrient a&ar, end of twenty-four hours. oven ^hat, ^ ^he end of twentv- X 10. (Frankel and Pfeiffer.) ,11 j i four hours, the large, round, ele- vated colonies, of a grayish- white color and moist appearance, may be easily recognized, while other associated bacteria will, as a rule, not yet have developed colonies large enough to interfere with the- recognition of these. Upon nutrient agar plates the deep-lying colonies, when magni- fied about eighty diameters, appear as round or oval, coarsely granu- lar discs, with rather ill-defined margins, or, when several colonies are in juxtaposition, as figures of irregular form. The superficial col- onies are grayish-yellow in color, have an irregular, not well-defined outline and a rough, almost reticulated surface. The growth upon glycerin-agar is very similar. The first inoculations in a plain nu- trient agar tube often give a comparatively feeble growth, which be- comes more abundant in subsequent inoculations in the same medium. In stick cultures in glycerin — or plain — agar, growth occurs to the bottom of the line of inoculation, and also upon the surface, but is not at all characteristic. The same may be said with reference to cultures in nutrient gelatin. Plate cultures in this medium contain- ing fifteen per cent of gelatin, at 24° C., give rather small colonies, which are white by reflected light and under the microscope are seen as yellowish-brown, opaque discs, having a more or less irregular outline and a granular structure. In alkaline bouillon the growth is sometimes in the form of small, whitish masses along the sides and bottom of the tube, but at others a diffusely clouded growth occurs in this medium ; after standing for some time in the incubating oven a thin, white pellicle may form upon the surface of the bouillon. 'I' 1 10 reaction of the bouillon becomes at first acid, but later it has an alkaline reaction (Welch). With reference to the growth onpotato? authors have differed, probably because the growth is scarcely vis- ible ; upon this point we quote from Welch and Abbott : BACTERIA IN DIPHTHERIA. 377 " Our experience lias been that the Bacillus diphtherias grows on ordinary steamed potato without any preliminary treatment, but that the growth is usually entirely invisible or is indicated by a dry, thin glaze after several days. Doubtless the invisible character of the growth has led most observers into the error of supposing that no growth existed, whereas the microscopi- cal examination reveals a tolerably abundant growth, which on the first po- tato is often feebler than on succeeding ones. Irregular forms are par- ticularly numerous in potato cultures, and in general the rods are thicker than on other media. In twenty-four hours, at a temperature of 35° C., microscopical examination shows distinct growth. We have cultivated the bacillus for many generations on potato." Milk is a favorable medium for the growth of this bacillus, and, as it grows at a comparatively low temperature (20° C.), it is evi- dent that this fluid may become a medium for conveying the bacillus from an infected source to the throats of previously healthy children. Cultures of the diphtheria bacillus may retain their vitality for several months, and when dried upon silk threads for several weeks colonies are still developed in a suitable medium — in the room from three to four weeks, in an exsiccator five to ten, and in one instance fourteen weeks. In dried diphtheritic membrane, preserved in small fragments, the bacillus retained its vitality for nine weeks, and in larger fragments for twelve to fourteen weeks. The thermal death-point, as determined by Welch and Abbott, is 58° C. , the time of exposure being ten minutes. Loffler had previ- ously found that it did not survive exposure for half an hour to 60° C. With reference to the action of germicidal and antiseptic agents, we refer to the sections in Part Second relating to this subject. Pathogenesis. — In view of the evidence heretofore recorded, it may be considered as demonstrated that this bacillus gives rise to the morbid phenomena which characterize the fatal disease in man known as diphtheria. We have already referred to the effects of inoculations into the trachea in rabbits and cats, which give rise to a characteristic diph- theritic inflammation, with general toxaemia and death from the absorption of soluble toxic products formed at the seat of local in- fection. This inference as to the cause of death seems justified by the fact that the pathogenic bacillus does not invade the blood and tissues, and is supported by additional experimental evidence which we give below. Subcutaneous inoculations in guinea-pigs of a small quantity of a pure culture of the bacillus (0. 1 to 0. 5 cubic centime- tre of a bouillon culture) cause death in from one to four or five days. The usual changes observed at the autopsy are " an exten- sive local oedema with more or less hypersemia and ecchymosis at the site of inoculation, frequently swollen and reddened lymphatic glands, increased serous fluid in the peritoneum, pleura, and pericar- dium, enlarged and hsBmorrhagic suprarenal capsules, occasionally 378 BACTERIA IN DIPHTHERIA. slightly swollen spleen, sometimes fatty degenerations in the liver, kidney, and myocardium. We have always found the Loffler ba- cilli at the seat of inoculation, most abundant in a grayish-white, fibrino-purulent exudate present at the point of inoculation, and be- coming fewer at a distance from this, so that the more remote parts of the oedematous fluid do not contain any bacilli " (Welch and Ab- bott). The authors quoted agree with Loffler and others in stating that the bacillus is only found at the point of inoculation. In all cases their cultures from the blood and from the various organs gave a negative result. Rabbits are not so susceptible and may recover after the subcu- taneous inoculation of very small doses, but usually die in from four to twenty days when two to four cubic centimetres of a bouillon culture have been introduced beneath the skin. In these animals also there is an extensive local oedema, enlargement of the neigh- boring lymphatic glands, and a fatty degeneration of the liver. Roux and Yersin have shown that in these animals, when death does not ensue too quickly, paralysis of the posterior extremities fre- quently occurs, thus completing the experimental proof of the spe- cific pathogenic power of pure cultures of this bacillus. Similar symptoms are produced in pigeons by the subcutaneous inoculation of 0. 5 cubic centimetre or more, but they commonly re- cover when the quantity is reduced to 0.2 cubic centimetre (Roux and Yersin). The rat and the mouse have a remarkable immunity from the effects of this poison. Thus, according to Roux and Yersin, a dose of two cubic centimetres, which would kill in sixty hours a rabbit weighing three kilogrammes, is without effect upon a mouse which weighs only ten grammes. Old cultures are somewhat less virulent than fresh ones, but when replanted ia a fresh culture medium they manifest their original virulence. Thus a culture upon blood serum which was five months old was found by Roux and Yersin to kill a guinea-pig in five days, but when replanted it killed a second animal of the same species in twenty-four hour-. Evidently a microorganism which destroys the life of a suscepti- ble animal when injected beneath its skin in small quantity, and which nevertheless is only found in the vicinity of the point of in- oculation, must owe its pathogenic power to the formation of some pot i -nt toxic substance, which being absorbed gives rise to toxaemia • ni.i death. This inference in the case of the diphtheria bacillus is fully sustained by the results of recent experimental investigations. Roux and Yersin (1888) first demonstrated the pathogenic power of cultuivs which had IHVM filtered through porous porcelain. Old BACTERIA IN DIPHTHERIA. 379 cultures were found by these experimenters to contain more of the toxic substance than recent ones, and to cause the death of a guinea- pig in the dose of two cubic centimetres in less than twenty-four hours. The filtered cultures produced in these animals the same effects as those containing the bacilli — local oedema, hsemorrhagic congestion of the organs, effusion into the pleural cavity. Some- what larger doses were fatal to rabbits, and a few drops injected subcutaneously sufficed to kill a small bird within a few hours. In their second paper (1889) the authors mentioned state that so long as the reaction of a culture in bouillon is acid its toxic power is com- paratively slight, but that in old cultures the reaction is alkaline, and in these the toxic potency is greatly augmented. With such a culture, filtered after having been kept for thirty days, a dose of one-eighth of a cubic centimetre, injected subcutaneously, sufficed to kill a guinea-pig ; and in larger amounts it proved to be fatal to dogs when injected directly into the circulation through a vein. The same authors, in discussing the nature of the poison in their filtered cultures, infer that it is related to the diastases, and state that its toxic potency is very much reduced by exposure to a com- paratively low temperature — 58° C. for two hours — and completely destroyed by the boiling temperature — 100° for twenty minutes. It was found to be insoluble in alcohol, arid the precipitate obtained by adding alcohol to an old culture proved to contain the toxic sub- stance. Loffler also has obtained, by adding five volumes of alco- hol to one of a pure culture, a white precipitate, soluble in water, which killed rabbits in the dose of 0. 1 to 0. 2 gramme when injected beneath the skin of these animals. It gave rise to a local oedema and necrosis of the skin in the vicinity of the point of inoculation, and to hypersemia of the internal organs. This deadly toxin appears to be an albuminoid substance, but its exact chemical composition has not yet been determined. * Brieger and Frankel have succeeded in rendering guinea-pigs immune against virulent cultures of the diphtheria bacillus by inject- ing bouillon cultures three weeks old, which had been sterilized by exposure for an hour to 60° to 70° C., into the subcutaneous tissues (ten to twenty cubic centimetres). At first the susceptibility of the animal is rather increased than diminished, but at the end of two weeks immunity is said to be complete. Frankel is of the opinion that immunity results from the introduction of a substance which is not identical with the toxic product to which the cultures owe their pathogenic power. This latter is destroyed by a temperature of 55° to 60° C., while the substance which gives immunity is still present in the cultures after exposure to a temperature of 60° to 70°, as shown by the protective results of inoculations made with such cultures. 380 BACTERIA IN DIPHTHERIA. The researches of Behring show that the blood of immune ani- mals contains a substance which neutralizes the toxic product con- tained in virulent cultures of the diphtheria bacillus. This effect is said to be produced when blood from such an animal is added to a filtered culture without the body, as well as when the culture is in- jected into the living animal. This remarkable discovery has al- ready been utilized for the treatment of diphtheria in man with most brilliant results. The method of preparing the diphtheria "anti- toxin " is given in the writer's recent work on " Immunity, Pro- tective Inoculations, and Serum-Therapy." According to Roux and Yersin, " attenuated varieties " of the diphtheria bacillus may be obtained by cultivating it at a temperature of 30.5° to 40° C. in a current of air ; and these authors suggest that a similar attenuation of pathogenic power may occur in the fauces of convalescents from the disease, and that possibly the similar non- pathogenic bacilli which have been described by various investiga- tors have originated in this way from the true diphtheria bacillus. These authors further state, in favor of this view, that from diphtheri- tic false membrane, preserved by them in a desiccated condition for five months, they obtained numerous colonies of the bacillus in ques- tion, but that the cultures were destitute of pathogenic virulence. They say: " It is then possible, by commencing with a virulent bacillus of diphtheria, to obtain artificially a bacillus without virulence, quite similar to the attenuated bacilli which may be obtained from a benign diphtheritic angina, or even- from the mouth of certain persons in good health. This microbe, obtained artificially, resembles com- pletely the pseudo-diphtheritic bacillus ; like it, it grows more abun- dantly at a low temperature; it renders bouillon more rapidly alkaline; it grows with difficulty in the absence of oxygen." % 48. PSEUDO-DIPHTHERITIC BACILLUS. Loffler, Von Hoffmann, and others have reported finding bacilli which closely resemble the Bacillus diphtherias, but which differ IVoiu it chiefly in being non-pathogenic. The following account we take from the latest paper upon the s-ubject by Roux and Yersin 1 1 roisieme memoire, 1800). Found by Roux and Yersin in mucus from the pharynx and ton- sils of children— from forty-five children in Paris hospitals, suffering I'n.m various affections, not diphtheritic, fifteen times; from fifty- nine healthy children in a villa-o school on the seaboard, twenty-six times. Of six children with a simple angina but two furnished cul- farea of this bacillus, while it was obtained in fiveoutof seven casea of measles. BACTERIA IN DIPHTHERIA. 33^ Its characters are given as follows : "The colonies of the pseudo -diphtheritic bacillus, cultivated upon blood serum, are identical with the true diphtheria bacillus. At a temperature of 33° to 35° multiplication is rapid, and it continues at the ordinary tempera- ture, although slowly. Under the microscope the appearance of the bacillus which forms these colonies is the same as that of Bacillus diphtherise. It stains readily with Loffler's solution of methylene blue, and intensely by Gram's method. Sometimes it colors uniformly, at others it appears granu- lar. It grows in alkaline bouillon, giving a deposit upon the walls of the vessel containing the culture, and in this medium often presents the inflated forms, pear-shaped, or club-shaped. It is destroyed in a liquid medium by a temperature of 58° C. maintained for ten minutes. All of these characters are common to the pseudo-diphtheritic bacillus and the true Bacillus diphthe- rias. As a difference between them we may note that the pseudo diphtheritic bacillus is of ten shorter in colonies grown upon blood serum; that its cultures in bouillon are more abundant ; that they continue at a temperature of 20° to 22, at which the true bacillus grows very slowly. When we make a com- parison of cultures in bouillon they become acid and then alkaline, but the change occurs much sooner in the case of the pseudo-diphtheritic bacillus. Like the true bacillus, the pseudo- diphtheritic grows in a vacuum, but less abundantly than the other. k 4 Inoculations into animals of cultures of this bacillus have never caused their death ; but we may remark that in some experiments a notable oedema has been produced in guinea-pigs at the point of inoculation, while in others there has JDeen no local lesion. The most marked oedema resulted from cul- tures obtained from cases of measles. 4 ' Do the facts which we have reported explain the question which occupies us ? Can we conclude that there is a relation between the two bacilli ? On the one side, the presence of the pseudo diphtheritic bacillus in the mouths of healthy persons, and of those who have anginas manifestly not diphtheritic, seems to be opposed to the idea of a relationship between them. On the other hand, when we consider that the non- virulent bacillus is very rare in fatal diphtheria, that it is more abundant in benign diphtheria, that it be- comes more common in severe cases as they progress towards recovery, and, finally, that they are more numerous in persons who have recently had diphtheria than 111 healthy persons, it is difficult to accept the idea that the two microbes are entirely distinct. The morphological differences which have been referred to are so slight that they prove nothing. The two micro- organisms can only be distinguished by their action upon animals, but the difference of virulence does not at all correspond with the difference of ori- gin. As regards the form and the aspect of cultures, the true and false diphtheria bacilli differ less than virulent anthrax differs from a very attenu- ated anthrax bacillus, which, however, originate from the same source. Besides, the sharp distinction which we make between the virulent and non- virulent bacilli is arbitrary; it depends upon the susceptibility of guinea- pigs. If we inoculate animals still more susceptible, there are pseudo diph- theritic bacilli which we must class as virulent; and if, on the contrary, we substitute rabbits for guinea pigs in our experiments, there are diphtheritic bacilli which we must call pseudo-diphtheritic. In our experiments we do not simply encounter bacilli which are very virulent and bacilli which are non- virulent; between these two extremes there are bacilli of every degree of virulence." Abbott, in 1801, published the result of his researches with reference to the presence of the pseudo-diphtheritic bacillus in benign throat affections. He made a bacteriological study of fifty- three patients, nine of whom were suffering from acute pharyngitis, fourteen from acute follicular tonsillitis, eight from ordinary post- 382 BACTERIA INT DIPHTHERIA. nasal catarrh, two from simple enlarged tonsils, fifteen from chronic pharyngitis, one from subacute laryngitis, one from chronic laryngi- tis, one from rhinitis, and two from an affection of the tonsils and pharynx. In forty-nine cases nothing of particular interest was ob- served. A variety of microorganisms were isolated, and of these the pyogenic micrococci were the most common. In four cases microorganisms were found which resembled the Bacillus diphtheria of Loffler in their morphology and growth in cul- ture media, but which proved not to be pathogenic. Abbott says : ' * The single point of distinction that can be made out between the organisms obtained from Cases L, III., and IV. and the true bacil- lus of diphtheria is in the absence of pathogenic properties from the former, whereas in addition to this point of distinction the organism from Case II. gives, as has been stated, a decided and distinct growth upon the surface of sterilized potato. " . 40. BACILLUS DIPHTHERIA COLUMBRARUM. Described by Loffler (1884), who obtained it from diphtheritic pseudo-mem- branes in the mouths of pigeons dead from an infectious form of .diphtheria which prevails in some parts of Germany among these birds and among chickens. Reddened patches first appear upon the mucous membrane of the mouth and fauces, and these are covered later with a rather thick, yellowish layer of fihrinous exudaie. In pigeons the back part of the tongue, the fauces, and tin* corners of the mouth are especially affected; in chickens the tongue, the £iiiiis, the nares, the larynx, and the conjunct! val mucous membrane. The disease is especially fatal among chickens, the voung fowls and those of choice varieties oeing most susceptible. It is attended at the outset by fever, and usually proves fatal within two or three weeks, but may last for several months. Morphology. — Short bacilli with rounded ends, usually associated in ir- regular masses, and resembling the bacilli of rabbit septicaemia (fowl cholera), but a little longer and not quite so broad. In sections from the liver they are seen in irregular groups in the interior of the vessels. Biological Characters. — An aerobic, non-motile, non-liquefying bacillus. (i rows in nutrient gelatin in the form of spherical, white colonies along the line of puncture, and upon the surface as a whitish layer. Under the microscope the colonies in gelatin plates have a yellowish-brown color and a slightly granular surface. Upon blood serum the growth consists of a -• mi-transparent, grayish-white layer. Upon potato a thin layer is formed 1 laving a grayish tint. ruthogenesift. — Pigeons inoculated with a pure culture in the mucous membrane of the mouth are affected exactly as are those which acquire the naturally. Subcutaneous inoculations in pigeons ^ive rise to an in- tlammation resulting in local necrotic changes. Pathogenic for rabbits and for mice. Suhcutaneous injections in mice give rise toa fatal result in about live r sum.- time, but ceases to be virulent— in this case it seems to have undergone modifications and presents tbe form of short bacilli. " Second. In another series of cases, less numerous but neverthe- BACTERIA IN DIPHTHERIA. 385 less considerable, the bacillus persists in a virulent condition for a longer or shorter time after the apparent cure of the malady. . . " Third. The observations collected up to the present time do not enable us to fix precisely the limits of persistence, but it is not far out of the way if we place it at several weeks to a month for the throat. In the nasal fossa3 the bacillus often persists for a still longer time, and its presence commonly coincides with a more or less abundant discharge from the nose." Park and Beebe (1894), in an extended research made for the pur- pose of determining the persistence of the diphtheria bacillus in the throats of convalescents (2,560 cultures made), found that in 304 out of 605 consecutive cases the bacillus disappeared within 3 days after the disappearance of the exudate; in 176 cases it persisted for 7 days; in 64 cases for 12 days; in 36 cases for 15 days; in 12 cases for 3 weeks; in 4 cases for 4 weeks; in 2 cases for 9 weeks. Park and Beebe arrive at the following conclusion with reference to pseudo- diphtheria bacilli : " The name pseudo-diphtheria bacillus should be regarded as ap- plying to those bacilli found in the throat which, though resembling the diphtheria bacilli in many respects, yet differ in others equally im- portant. These bacilli are rather short, and more uniform in size and shape than the typical Loffler bacillus. They stain equally throughout with the alkaline methyl-blue solution, and produce alkali in their growths in bouillon. They are found in about one per cent of the healthy throats in New York City, and seem to have no connection with diphtheria. They are never virulent." Park (1894) has shown that virulent diphtheria bacilli are fre- quently found in the throats of persons who have been associated with diphtheria patients, although no manifestations of the disease were visible. It is therefore apparent that infection requires not only the presence of virulent bacilli, but also of a predisposition to the disease. This corresponds with the facts relating to other in- fectious diseases — e.g.^ tuberculosis, typhoid fever — and among the probable predisposing causes we may mention " sewer-gas poisoning, " catarrhal inflammations of the mucous membranes most commonly involved, inanition, "crowd poisoning," and depressing agencies generally. Bacteriologists have recently given much attention to the question of mixed infection in diphtheria. Funck (1894) accepts the gener- ally received view that mixed infections with the diphtheria bacillus and Streptococcus pyogenes are more serious than an uncomplicated diphtheria, and in an experimental research has attempted to deter- mine whether this is due to an increased production of the diphtheria the presence of the streptococcus. His experiments on guinea-pigs 27 386 BACTERIA IN DIPHTHERIA. showed that when infected with streptococci these animals did not prove to be more sensitive to the action of the diphtheria poison (without living bacilli), and he concludes that the unfavorable influ- ence of the streptococcus in mixed infections is due to increased patho- genic activity on the part of the diphtheria bacillus. Bernheim (1894) found, in his experiments on guinea-pigs, that they suc- cumbed more rapidly to diphtheria infection when they previously or simultaneously received an injection of a streptococcus culture- filtered or unfiltered. Results of Treatment ivith the Antitoxin. — While questions re- lating to therapeutics are not considered in this manual, a brief note upon the results of treatment by the serum of immunized animals may not be out of place. A recent (1895) collective investigation undertaken by the Deutsche medicinische Wochenschrift gave the following results : The number of cases collected was 10,312; all of these occurred between the 1st of October, 1894, and the 1st of April, 1895; 5,883 of these cases were treated with the antitoxin and 4,479 without it. In the first group the mortality was 9.6 per cent, and in the second group 14.7 per cent. Two thousand five hundred and fifty six children treated with the antitoxin were between two and ten years of age; among these the mortality was 4 per cent, while among children of the same age not treated with the antitoxin the mortality was 15.2 per cent. Six hundred and ninety-six patients above ten years of age were treated with a mortality of 1 per cent. Monod (1895), at a meeting of the Paris Academy of Medicine, presented the following statistics demonstrating the influence upon the mortality from diphtheria in France exerted by the antitoxin since its employment from November, 1894. The following figures represent the number of deaths from diphtheria during the first six months in eight years in 108 French cities having a population of more than 20,000: 1805, Average. Average. January 469 205 February 466 187 March 499 155 April 442 160 . May 417 113 June 333 84 2,656 904 It will be seen from the above statement that during the first six months in the year 1895 after the introduction of the antitoxin treat- ment, the number of deaths from diphtheria in the 108 French cities referred to was 1,552 less than the average for the preceding ten years, and we are justified in concluding that a considerable propor- tion of this saving at least is due to this new method of treatment. X. BACTERIA IN INFLUENZA. A NUMBER of bacteriologists have made careful researches during the recent extended epidemic of influenza, and in 1892 a bacillus was discovered, both by Pfeiffer and by Canon, of Berlin, which there is good reason to believe is the specific cause of this dis- ease. Before describing this we shall refer briefly to previous re- searches. Babes has described no less than seventeen distinct species or varieties isolated by him, principally from nasal or bronchial mucus. Among these a considerable number closely resemble Streptococcus pyogenes or Micro - coccus pneumonias crouposae. No one form was found with sufficient con- stancy to justify the inference that it was the specific cause of the disease. Klebs, in examining blood drawn from the fingers of patients with influ- enza, observed an enormous number of small, actively motile, highly refrac- tive bodies, which in their size, form, and movements corresponded entirely with similar bodies previously observed by him in the blood of patients with pernicious anaemia, but whicn were far more numerous. These bodies are believed by Klebs to be flagellate infusoria ("flagellata"). The investiga- tions of other bacteriologists have not thus far confirmed those of Klebs as regards the presence of microorganisms of this class in the blood of patients with influenza. Kowalski, who made bacteriological researches in sixteen cases, was not able to find microorganisms of any kind in the blood, examined both fresh and in dried preparations. In his cultures from the nasal, buccal, and bronchial secretions of the sick he obtained in five cases Staphylococcus pyogenes aureus, in four Staphylococcus pyogenes albus, in two "diplococ- cus pneumonise, " in two Streptococcus pyogenes, in two Staphylococcus pyogenes citreus, in one Friedlander's bacillus, in one Staphylococcus cereus albus, in one Staphylococcus cereus flavus. In addition to these he isolated three species not previously described. One of these he found in seven cases ; this grew upon the surface of agar as small, transparent drops, but did not grow upon potato, in sterilized milk, or in bouillon ; it was a coccus arranged in pairs or in chains, and is designated by Kowalski ' ' Gallertstrep- tococcus." Prior, in a bacteriological study of fifty- three cases, twenty-nine of which were without complication and twenty-four complicated by pneumonia, found in the sputum of uncomplicated cases, as the most abundant and com- mon microorganism at the outset of the attack, Micrococcus pneumonias crouposse ; next to this came Staphylococcus pyogenes aureus and Strepto- coccus pyogenes ; when the acme of the attack was past the two species first named quickly diminished in numbers, while streptococci were found for a longer time. In cases of croupous pneumonia following influenza " diplo- coccus pneumonias " was constantly found in great numbers. Fischel (1891) obtained in cultures from the blood of two cases two dif- 388 BACTERIA IN INFLUENZA. ferentmicrococci, one of which was pathogenic for dogs and horses and gave rise to symptoms in these animals resembling those of influenza (see Micro- coccus No. II. of Fischel, No. 39, nage 324). Kirchner (1891) found constantly in the sputum of recent cases a diplo- coccus enclosed in a jellv-like capsule, which differed in its biological and pathological characters from Micrococcus pneumonioe crouposae (see Micro- coccus of Kirchner, No. 38, page 324). 52. BACILLUS OF INFLUENZA. Discovered by Pfeiffer (1892) in the purulent bronchial secretion, and by Canon in the blood of patients suffering from epidemic in- fluenza. Pfeiffer found the bacillus in thirty-one cases examined by him, and in uncomplicated cases it was present in the purulent bron- chial secretion in immense numbers and in a pure culture. Canon, whose independent observations were published at the same time, examined the blood of twenty influenza patients in stained prepara- tions, and found the same bacillus in nearly all of them. His method of demonstrating it is as follows : The blood is spread upon clean glass covers in the usual way. After the preparations are thoroughly dry they are placed in abso- lute alcohol for five minutes. They are then transferred to the fol- lowing staining solution (Czenzynke's) : concentrated aqueous solu- tion of methylene blue, forty grammes ; one-half -per-cent solution of eosin (dissolved in seventy-per-cent alcohol), twenty grammes ; dis- tilled water, forty grammes. The cover glasses immersed in this staining solution are placed in an incubating oven at 37° C. for from three to six hours, after which they are washed with water, dried, and mounted in balsam. In successful preparations the red blood corpuscles are stained red by the eosin, and the leucocytes blue. The bacillus is seen in these as a short rod, often resembling a diplococcus. It is sometimes seen in large numbers, but usually only a few rods are seen after a long search — four to twenty in a single preparation. In six cases it was found in numerous aggregations containing from five to fifty bacilli each. In these cases the blood was drawn during a fall of temperature or shortly after. Morphology.— Very small bacilli, having about the same diameter as the bacillus of mouse septicaemia, but only half as long. Solitary or united in chains of three or four elements. Stains with difficulty with the basic aniline dyes— best with dilute Ziehl's solution, or Loffler's methylene blue solution, with heat. The two ends of the bacilli are most deeply stained, causing them to resemble diplococci. Pfeiffer says: "I am inclined to believe that some of the earlier observers also saw the bacilli described by me, but that, misled by their peculiar behavior with regard to staining agents, they described them as diplococci or streptococci." Do not stain hy (iram's method. BACTERIA IN INFLUENZA. 389 Biological Characters. — An aerobic, non-motile bacillus. Does not grow in nutrient gelatin at the room temperature. Spore forma- tion not observed . Upon the surface of glycerin-agar in the incubat- ing oven very small, transparent, drop-like colonies are developed at the end of twenty-four hours. These can only be recognized by the aid of a lens. " A remarkable point about them is that the colonies always remain separate from each other, and do not, as all other species known to me do, join together and form a continuous row. This feature is so characteristic that the influenza bacilli can be thereby with certainty distinguished from other bacteria" (Kitasato). On 1.5 per cent sugar-agar the colonies appear as extremely small droplets, clear as water, often only recognizable with a lens (Pfeiffer). In bouillon a scanty development occurs, and at the end of twen- ty-four hours small, white particles are seen upon the surface, which subsequently sink to the bottom, forming a white, woolly deposit, while the bouillon above remains transparent. This bacillus does not grow at temperatures below 28° C. Canon has obtained colonies, resembling those described by Kita- sato, in cultures from the blood of influenza patients. His cultures were made upon glycerin-agar in Petri's dishes. Ten or twelve drops of blood from a puncture made in the finger of the patient, after sterilization of the surface, were allowed to fall upon the agar medium, and this was placed in the incubating oven. As the number of ba- cilli in the blood is small, a considerable quantity is used. The colonies are visible at the end of twenty-four to forty-eight hours. The influenza bacillus is quickly destroyed by desiccation ; a pure culture diluted with water and dried is destroyed with cer- tainty in twenty hours ; in dried sputum the vitality is retained somewhat longer, but no growth occurs after forty hours. The thermal death-point is 60° C. with five minutes' exposure (Pfeiffer and Beck). Patliogenesis. — Pfeiffer infers that this is the specific cause of influenza in man for the following reasons : 1. They were found in all uncomplicated cases of influenza ex- amined, in the characteristic purulent bronchial secretion, often in absolutely pure cultures. They were frequently situated in the pro- toplasm of the pus corpuscles ; in fatal cases they were found to have penetrated from the bronchial tubes into the peribronchitic tis- sue, and even to the surface of the pleura, where in two cases they were found in pure cultures in the purulent exudation. 2. They were only found in cases of influenza. Numerous con- trol experiments proved their absence in ordinary bronchial ca tarrh, etc. 390 BACTERIA IN INFLUENZA. 3. The presence of the bacilli corresponded with the course of the disease, and they disappeared with the cessation of the purulent bronchial secretion. In his preliminary report of his investigations Pfeiffer says : " Numerous inoculation experiments were made on apes, rabbits, guinea-pigs, rats, pigeons, and mice. Only in apes and rabbits could positive results be obtained. The other species of animals showed themselves refractory to influenza." Kruse (1894) reports that he found the bacillus of Pfeiffer in eighteen influenza patients examined by him in the hospital at Bonn. On the other hand, he failed to find it in a considerable number of pa- tients suffering from other diseases of the respiratory passages. His evidence is the more valuable as he had previously (1890) reported his failure to find the bacillus in typical cases of influenza. He now ascribes his failure at that time to imperfect technique. Huber (1893), Richter (1894), Borchardt (1894), and other com- petent bacteriologists, have also confirmed the results reported by Pfeiffer as regards the presence of this bacillus in the bronchial secretions of persons suffering from epidemic influenza, and as to its biological characters. Bujwid (1893) recognizes the bacillus of Pfeiffer as identical with a bacillus which he cultivated from the spleen of an influenza patient in 1890. The researches of Weichselbaum, Kowalski, Friedrich, Kruse, Bouchard, and others have given a negative result as regards the presence of the influenza bacillus in the blood. They were not able to demonstrate its presence either in stained preparations or by cul- ture methods. Pfeiffer, also, during the last epidemic, has made special researches upon this point and has never succeeded in finding the bacillus. Day after day, both in mild and severe cases, he placed from ten to twenty drops of blood from influenza patients on blood- agar — a most favorable medium — but his cultures always remained sterile. In his experiments upon rabbits, Pfeiffer (1893) found that the intravenous injection of a small quantity of culture on blood-agar, twenty-four hours old, suspended in one cubic centimetre of bouillon, caused a characteristic pathogenic effect. The first symptoms were developed within one and a half to two hours after the injection. The animals became extremely feeble, lying flat upon the floor with their limbs extended, and suffered from extreme dyspnoea. The tem- perature mounted to 41° C. or above. At the end of five or six hours they were able to sit upon their haunches again, and in twenty-four hours had nearly recovered from all indications of ill-health. Larger doses caused the death of the inoculated animals. These results are due to toxic products present in the cultures, and Pfeiffer has never PLATE VI. PATHOGENIC BACTERIA. FIG. 1.— Bacillus of influenza in bronchial mucus. X 1,000. Photo- micrograph by Frankel. FIG. 2.— Bacillus of influenza in bronchial mucus, after the termination of the febrile period. The bacilli are for the most part in pus cells. X 1,000. Photomicrograph by Frankel. FIG. «3. — Bacillus tetani from an agar culture. X 1,000. Photomicro- graph by Frankel and Pfeiffer. FIG. 4. — Micrococcus pneumoniae crouposae in sputum of a patient with pneumonia. X 1,000. Stained by Gram's method. Photomicrograph by Frankel and Pfeiffer. FIG. 5. — Micrococcus pneumoniae crouposae in blood of rabbit. X 1,000. Photomicrograph made at the Army Medical Museum, Washington, by Gray. FIG. 6.— Bacillus of hog cholera, showing flagella. Stained by Loffler's method. X 1,000. Photomicrograph made at the Army Medical Museum, Washington, by Gray. TLAlh VI. STERNBERG'S BACTERIOLOGY.' Fig. 3. Fie:. 4. Fig. 5, '""••.•>:• Fig. 0. PATHOGENIC BACTERIA. BACTERIA IN INFLUENZA. 391 observed a septicsemic infection as a result of his inoculation ex- periments. Pfeiffer has found in three cases of bronchopneumonia a pseudo- influenza bacillus which closely resembles the bacillus previously de- scribed by him as peculiar to that disease. This pseudo-influenza bacillus resembles the genuine one in its growth in culture media, but is larger and shows a decided inclination to grow out into long threads. By these morphological characters, which are said to be constant, it may, according to Pfeiffer, be readily distinguished. XL BACILLI IN CHRONIC INFECTIOUS DISEASES. IN tuberculosis, leprosy, glanders, and syphilis we have a group of infectious diseases which present many points of resemblance. All run a chronic course ; all may be communicated to susceptible animals by inoculation ; in all, the lymphatic glands in the vicinity of the point of inoculation become enlarged, and new growths, con- sisting of various cellular elements of a low grade of vitality, are de- veloped in the tissues which are the point of predilection for each ; in all, these new growths show a tendency to degenerative changes, as a result of which abscesses, caseous masses, or open ulcers are formed In two of the diseases in this group — tuberculosis and glan- ders— the infectious agent has been obtained in pure cultures and its specific pathogenic power demonstrated by inoculations in susceptible animals; in one — leprosy — there is but little doubt that the bacillus con- stantly found in the new growths characteristic of the disease bears an etiological relation to it, although this has not been demonstrated, the bacillus not having as yet been cultivated in artificial media. The evidence with reference to the parasitic nature of the fourth dis- ease mentioned as belonging to this group — syphilis — is still unsatis- t.K-tory, but there is every reason to believe that it will also eventu- ally be, proved to be due to a parasitic microorganism. The announcement of the discovery of the tubercle bacillus was made by Koch, in March, 1882, at a meeting of the Physiological S. K-ioty of Berlin. At the same time satisfactory experimental evi- dence was presented as to its etiological relation to tuberculosis in man and in the susceptible lower animals, and its principal biologi- cal i-liaracters were given. Baumgarten independently demonstrated the presence of the tu- bercle bacillus in tuberculous tissues and published the fact soon at't.T tlu> appearance of Koch's first paper. The previous demonstra- tion by VilK'inin (lsr,r>)— confirmed by Cohnheim (1877) and others— that tuberculosis might be induced in healthy animals by inocula- tions of tuberculous material, had paved the way for his discovery, BACILLI IN CHRONIC INFECTIOUS DISEASES. and advanced pathologists were quite prepared to accept it. The more conservative have since been obliged to yield to the experi- mental evidence, which has received confirmation in all parts of the world. To-day it is generally recognized that tuberculosis is a spe- cific infectious disease due to the tubercle bacillus. As evidence of the thorough nature of Koch's personal researches in advance of his first public announcement, we give the following resume of his investigations : In nineteen cases of miliary tuberculosis the bacilli were found in the tubercular nodules in every instance ; also in twenty-nine cases of pulmonary phthisis, in the sputum, in fresh cheesy masses, and in the interior of recently formed cavities ; in tuberculous ulcers of the tongue, tuberculosis of the uterus, testicles, etc. ; in twenty-one cases of tuberculous — scrofulous — lymphatic glands ; in thirteen cases of tuberculous joints ; in ten cases of tubercular bone affections ; in four cases of lupus ; in seventeen cases of Perlsucht in cattle. His ex- perimental inoculations were made upon two hundred and seventy- three guinea-pigs, one hundred and five rabbits, forty-four field mice, twenty-eight white mice, nineteen rats, thirteen cats, and upon dogs, pigeons, chickens, etc. Very extensive comparative researches were also made, which convinced him that the bacillus which he had been able to demonstrate in tuberculous sputum and tissues by a spe- cial mode of staining was not to be found in the sputa of healthy persons, or of those suffering from non-tubercular pulmonary affec- tions, or in organs and tissues involved in morbid processes of a different nature. 53. BACILLUS TUBERCULOSIS. Discovered by Koch (first public announcement of discovery March 24th, 1882). The bacilli are found in the sputum of persons suffering from pulmonary or laryngeal tuberculosis, either free or in the interior of pus cells ; in miliary tubercles and fresh caseous masses, in the lungs or elsewhere ; in recent tuberculous cavities in the lungs ; in tuberculous glands, joints, bones, and skin affections (lupus) ; in the lungs of cattle suffering from pulmonary tubercu- losis— Perlsucht ; and in tubercular nodules generally in animals which are infected naturally or by experimental inoculations. In the giant cells of tubercular growths they have a peculiar and characteristic position, being found, as a rule, upon the side of the cell opposite to the nuclei, which are crowded together in a crescentic arrangement at the opposite pole of the cell. Sometimes a single bacillus will be found in this position, or there may be several. Again, numerous bacilli may be found in giant cells in which the nuclei are distributed around the periphery. They are more numer- 28 394 BACILLI IN CHRONIC INFECTIOUS DISEASES. ous in tuberculous growths of recent origin, and often cannot be demonstrated, by microscopical examination, in caseous material from the centre of older nodules. But such material, when inocu- lated into susceptible animals, gives rise to tuberculosis, and the usual inference is that it contains spores of the tubercle bacillus. Morphology. — The tubercle bacilli are rods with rounded ends, of from 1.5 to 3.5 // in length, and are commonly slightly curved or bent at an angle ; the diameter is about 0.2 p. In stained preparations unstained portions are frequently seen, which are generally believed to be spores, but this is by no means certain. From two to six of these unstained spaces may often be seen in a single rod, and owing to this al- ternation of stained and unstained portions the bacilli may, under a low power, be mistaken for chains of mi- crococci The rods are usually soli- F,o. US. - BaclHus tuberculosis. ^ but "^ ^ United in PairS> °r x i ,000. From a photomicrograph. in short chains containing three or four elements. In old cultures irregular forms may be observed, the rods being sometimes swollen at one extremity, or presenting the appearance of having a lateral bud-like projection — involution forms. The staining characters of this bacillus are extremely important for its differentiation and recognition in preparations of sputum, etc. Unlike most microorganisms of the same class, it does not readily take up the aniline colors, and when stained it is not easily decolorized, even by the use of strong acids. The failure to observe it in tuber- culous material, prior to Koch's discovery, was no doubt due to the fact that it does not stain in the usual aqueous solutions of the aniline dyes. Koch first recognized it in preparations placed in a staining fluid to which an alkali had been added — solution of methylene blue with caustic potash ; but this method was not very satisfactory, and he promptly adopted the method devised by Ehrlich, which consists essentially in the use of a solution of an aniline color — fuchsin or methyl violet — in a saturated aqueous solution of aniline oil, and de- colorization with a solution of a mineral acid — nitric acid one part to three parts of water. The original method of Ehrlich gives very satisfactory results, but various modifications have since been proposed, some of which are advantageous. The carbol-fuchsin solution of Ziehl is now largely employed ; it has the advantage of prompt action and of BACILLI IN CHRONIC INFECTIOUS DISEASES. 395 keeping well. The staining is effected more quickly if heat is ap- plied. The tubercle bacilli stain by Gram's method, but this is not to be recommended for general use, owing to the fact that the pro- toplasm of the rods is frequently contracted into a series of spheri- cal, stained bodies, which might easily be mistaken for micrococci. The examination of sputum for the presence of the tubercle ba- cillus is recognized as a most important procedure for the early diag- nosis of pulmonary tuberculosis. It is at- tended with no special difficulties, and every physician should be acquainted with the technique. The patient ehould be directed to expec- torate into a clean, wide-mouthed bottle or glass-covered jar the material coughed up from the lungs, and especially, in recent cases, that which is coughed up upon first rising in the morning. This should be placed in the physician's hands as promptly FIG. 117.— Bacillus tubercuio- as possible ; although a delay of some days f&^tum' x 1'ooa> (Baum' does not vitiate the result, and the tubercle bacilli may still be demonstrated after the sputum has undergone pu- trefaction. It is well to pour the specimen into a clean, shallow vessel having a blackened bottom — a Petri's dish placed upon a piece of dead- black paper will answer very well. In tuberculous sputum small, len- ticular masses of a yellowish color may usually be observed, and one of these should be selected for microscopical examination, by picking it up with a platinum needle and freeing it as far as possible from the tenacious mucus in which it is embedded. If such masses are not recognized take any purulent-looking material present in the specimen, whether it be in small specks distributed through the mu- cus, or in larger masses. A little of the selected material should be placed in the centre of a clean cover glass and another thin glass cover placed over it. By pressure and a to-and-fro motion the mate- rial is crushed and distributed as evenly as possible ; the glasses are then separated by a sliding motion. The film is permitted to dry by exposure in the air. When dry the cover glass, held in forceps, is passed three times through the flame of an alcohol lamp or Bunsen burner to fix the albuminous coating. Too much heat causes the film to turn brown and ruins the preparation. The staining fluid (ZiehFs carbol-f uchsin) may then be poured upon the cover glass, or this may be floated upon the surface of the fluid contained in a shallow watch glass. Heat is now applied by bringing the cover glass over a flame and holding it there until steam begins to be given off from the surface of the staining fluid ; it is then withdrawn and again 396 BACILLI IN CHRONIC INFECTIOUS DISEASES. gently heated at intervals for a minute or two. The cover glass is then washed in water, and the film will be seen to have a uniform deep-red color. The next step consists in decolorization in the acid solution (twenty-five-per-cent solution of nitric or of sulphuric acid). The cover glass is gently moved about in this solution for a few seconds, and the color will be seen to quickly fade to a greenish tint. The object is to remove all color from the cells and the al- buminous background, so that the bacilli, which retain their color in presence of the acid, may be clearly seen. The preparation is next washed in dilute alcohol (sixty per cent) to remove the fuchsin which has been set free by the acid. If decolorization was not car- ried far enough the film will be seen to still have a red color, espe- cially in places where it is thickest, when it is removed from the dilute alcohol and washed out in water. In this case it will be necessary to return it to the acid solution and again wash it in the dilute alcohol and in water. It may now be placed in a solution of methylene blue or of vesuvin for a contrast stain. The tubercle bacilli are distinguished by the fact that they retain the red color imparted to them in the fuchsin solution, while other bacteria pre- sent, having been decolorized in the acid solution, take the contrast stain and appear blue or brown, according to the color used. The double-stained preparation, after a final washing in water, may be examined at once, or dried and mounted in balsam for permanent preservation. Of the various other methods which have been proposed, that of Frankel, as modified by Gabbett, appears to be the most useful. This consists in staining as above directed with ZiehPs carbol-f uchsin solu- tion, and in then placing the cover glass directly in a second solution which contains both the acid for decolorizing and the contrast stain. This second solution contains twenty parts of nitric acid, thirty parts of alcohol, fifty parts of water, and sufficient methylene blue to make a saturated solution (one to two parts in one hundred). After re- maining in this solution for a minute or two the cover glass is washed in water, and upon microscopical examination the tubercle bacilli, if present, will be seen as red rods which strongly contrast with the blue background. The methods recommended for cover-glass preparations may also be used for staining the tubercle bacillus in thin sections of tuber- culous tissues, except that it is best not to employ heat. The sec- tions may be left for an hour in the carbol-f uchsin solution, or for twelve hours in the Ehrlich-Weigert tubercle stain— eleven cubic centimetres of saturated alcoholic solution of methyl violet, ten cubic (vntimi'tivsof absolute almlml, <>n<> hundred cubictvntimctivs of ani- line water. They should then be decolorized by placing them for BACILLI IN CHRONIC INFECTIOUS DISEASES. 397 about half a minute in dilute nitric acid (ten per cent) ; then wash out color in sixty-per-cent alcohol ; counter-stain for two or three minutes in a saturated aqueous solution of methylene blue ; dehydrate with absolute alcohol or with aniline oil ; clear up in oil of cedar, and mount in xylol balsam. If the aniline-water-methyl-violet solu- tion has been used for staining the bacilli a saturated solution of vesuvin may be used as a contrast stain. Biological Characters. — A parasitic, aerobic, non-motile ba- cillus, which grows only at a temperature of about 37° C. Is also a facultative anaerobic (Frankel). The question as to spore formation has not been definitely deter- mined. It has been generally assumed that the unstained spaces which are frequently seen in the bacilli are spores ; and the fact that FIG. 118.— Section through a tuberculous nodule in the lung of a cow, showing two giant cells containing tubercle bacilli, "x 950. (Baftungarten.) caseous material in which a microscopical examination has failed to demonstrate the presence of bacilli may produce tuberculosis, with bacilli, when inoculated into guinea-pigs, has been explained upon the supposition that this material contained spores. But a few bacilli present in such caseous material might easily escape detection. As pointed out by Frankel, the oval spaces in stained specimens have not the sharply denned outlines of spores. Moreover, the bacilli, when examined in unstained preparations, do not contain corresponding re- fractive bodies, recognizable as spores. And when the bacilli are stained by Gram's method the protoplasm is often contracted in the form of little, spherical stained masses, while the unstained spaces are larger and no longer have the oval form presented in rods stained by Ehrlich's method. The great resisting power of the bacillus to heat and to desiccation has been supposed to be due to the presence 398 BACILLI IN CHRONIC INFECTIOUS DISEASES. of spores. But, so far as resistance to heat is concerned, this is not so great as was at one time believed. Schill and Fischer (1884), as- suming that the tubercle bacillus forms spores, made quite a number of experiments to determine its thermal death-point. They sub- jected sputum containing the bacillus to a temperature of 100° C., and tested the destruction of vitality by inoculations into guinea-pigs. Exposure to steam at a temperature of 100° C. for two to five min- utes was effective in every experiment, with one exception. One guinea-pig died tuberculous after having been inoculated with sputum exposed to this temperature for two minutes. This result was assumed to show that the bacillus would survive lower tempera- tures, but it is evident that additional experiments were required to establish this fact. In 1887 the writer made a few similar experi- ments at a lower temperature, and guinea-pigs inoculated with tuber- culous sputum exposed for ten minutes to a temperature of 90°, 80°, and 60° C. failed to become tuberculous, while another guinea-pig, inoculated with the same material after exposure to a temperature of 50° C. for ten minutes, died tuberculous. These results correspond with those subsequently (1888) reported by Yersin, who tested the thermal death-point of this bacillus by the culture method. This author assumes that the bacilli form spores, but states as a result of his experiments that "at the end of ten days bacilli heated for ten minutes at 55° C. gave a culture in glycerin-bouillon ; those heated to 60°, at the end of twenty-two days; while those heated to 70° and above failed to grow in every instance. This experiment, repeated a great number of times, always gave the same result. The tubercle bacilli then resist a temperature of 60° C. for ten minutes, and it is to be remarked that the resistance of spores to heat appears to be no greater than that of the bacilli themselves/' Yersin remarks in a footnote that "the spores which served for these experiments did not appear as more or less irregular granules taking the coloring matter strongly, but as veritable spores with sharply defined outlines, to the number of one or two in a bacillus, or three at the outside. These spores are particularly clear in cultures upon glycerin-agar several weeks old." It may be that bacteriologists have been mistaken in the infer- ence that all spores possess a greater resisting power for heat than that exhibited by bacilli in the absence of spores. That this is true as iv^anls anthrax spores ami many others, tlio thermal death-point of which has been determined by exact experiments, does not prove that it is true for all. And it is known that there are wide differ- ences in the resisting power both of the spores of different species and in the vegetating cells. To admit that the tubercle bacillus or the typhoid bacillus, etc., may form spores which have no greater BACILLI IN CHRONIC INFECTIOUS DISEASES. 399 resisting power against heat than the bacilli themselves, would there- fore simply be an admission that some bacteriologists had made a mistaken inference based upon incomplete data. In view of the facts stated we can simply repeat what was said at the outset, viz. , the question as to spore formation has not been definitely deter- mined. The tubercle bacillus is a strict parasite, and its biological char- acters are such that it could scarcely find natural conditions, outside of the bodies of living animals, favorable for its multiplication. It therefore does not grow as a saprophyte under ordinary circum- stances. But it has been noted by Roux and Nocard that when it has been cultivated for a time in artificial media containing glycerin it may grow in a plain bouillon of veal or chicken, in which media it fails to develop when introduced directly from a culture originating from the body of an infected animal. This would indicate the pos- sibility of its acquiring the ability to grow as a saprophyte ; and we can scarcely doubt that at some time in the past it was a true sapro- phyte. The experiments of Nuttall indicate that the bacillus may multiply, under favorable temperature conditions, in tuberculous sputum outside of the body. And it is extremely probable that mul- tiplication occurs in the muco-purulent secretion which accumulates in pulmonary cavities in phthisical patients. In these cavities its de- velopment may, in a certain sense, be regarded as saprophytic, as it feeds upon non-living organic material. Koch first succeeded in cultivating this bacillus upon coagulated blood serum, prepared as directed in Section VIII., Part First, of the present volume. Roux and Nocard have since shown (1888) that it grows very well on nutrient agar to which glycerin has been added (six to eight per cent), and also in veal broth containing five per cent of glycerin. It is difficult to obtain pure cultures from tuberculous sputum, on account of the presence of other bacteria which grow much more rapidly and take full possession of the medium before the tubercle bacillus has had time to form visible colonies, l^or this rea- son it is best to first inoculate a guinea-pig with the tuberculous spu- tum and to obtain cultures from it after tuberculous infection has fully developed. The inoculated animals usually die at the end of three or four weeks. It is best to kill one which gives evidence of being tuberculous, and to remove one or more nodules from the lungs through an opening made in the chest walls. The greatest care will be required to prevent contamination by other common microorganisms. The instruments used must be sterilized by heat, and the skin over the anterior thoracic wall carefully turned back ; then, after again sterilizing knives and scissors, cut an opening into the chest cavity, draw out the root of the lung, and take up with 400 BACILLI IN CHRONIC INFECTIOUS DISEASES. slender sterilized forceps, or with a strong platinum loop, one or more well-defined tubercular nodules. These may be conveyed di- rectly to the surface of the solid culture medium and then broken up and rubbed over the surface as thoroughly as possible ; or they may first be crushed between two sterilized glass slides, and then transferred with the platinum loop and thoroughly rubbed into the surface of the culture medium. This breaking-up of the tuberculous nodules and distribution of the bacilli upon the surface of the culture medium is essential for the success of the experiment. Instead of using the tubercular nodules in the lungs, an enlarged lymphatic gland from the axilla or elsewhere may be used, as first recommended by Koch. This is to be crushed in the same way ; and it will be best to inoculate a num~ her of tubes at the same time, as accidental contamination or failure to develop is very liable to occur in a certain number. Owing to the liability of the blood serum to become too dry for the development of the bacillus, it is best to keep the cultures in a moist atmosphere, or to prevent evaporation by applying a rubber cap over the open end of the test tube. This should be sterilized in a solution of mercuric chloride (1 : 1,000) ; and the end of the cotton plug should be burned off just before applying it, for the purpose of destroying the spores of mould fungi, which in a dry atmosphere would be harmless, but under the rubber cap are likely to sprout and to send their mycelium through the cotton plug to the interior of the tube, thus destroying the culture. Upon coagulated blood serum the growth first becomes visible at the end of ten to fourteen days (at 37° 0.), and at the end of three weeks a very distinct and characteristic develop- ment has occurred. The first appearance is that of dry-looking, grayish-white points and scales, which are without lustre, and are sometimes united to form a thin, irregular, membranous-looking layer. Under the microscope, with an amplification of eighty diameters, the early, thin surface growth upon blood serum presents a characteristic appear- ance. The bacilli, arranged in parallel rows, form variously curved figures, of which we may obtain impressions by carefully applying a dry cover glass < mi ureupon blood se- to the surface. Upon staining the preparation in the usual way the same arrangement of the bacilli which adhered to the thin glass cover will be pre- served. The growth is more abundant in subsequent cultures, which have Ix-m kept up in Koch's laboratory from his original pure cultures up to the present time ; in these the bacillus still pre- BACILLI IN CHRONIC INFECTIOUS DISEASES. 401 serves its characters of form and growth, and its specific pathogenic power. Pastor (1892) has succeeded in obtaining pure cultures of the tubercle bacillus from sputum by the following ingenious method : After proving by microscopic examination that the sputum of a tuberculous individual contains numerous bacilli, he has the patient cleanse his mouth as thoroughly as possible with sterilized water, and then expectorate some material, coughed up from the lungs, into a sterilized test tube. By shaking with sterilized water a fine emul- sion is made, and this is filtered through fine gauze. The filtrate, which is nearly transparent, contains numerous tubercle bacilli. A few drops of the emulsion are now added to liquefied gelatin in a test tube, and a plate is made in the usual way. This is kept for three or four days at the room temperature, during which time the com- mon mouth bacteria capable of growth form visible colonies. By means of a hand lens a place is now selected in which no colonies are seen, and a bit of gelatin is excised with a sterilized knife. This piece is transferred to the surface of blood serum or glycerin-agar, and placed in the incubating oven, where in due time colonies of the tubercle bacillus will usually be found to develop. Another method of accomplishing the same result has recently been described by Kitasato. This is a method devised by Koch some time since and successfully employed in his laboratory. The morn- ing expectoration of a tuberculous patient, raised from the lungs by coughing, is received in a Petri/s dish. A bit of sputum, such as comes from the tuberculous cavity in the lungs of such a patient, is now isolated with sterilized instruments and carefully washed in at least ten successive portions of sterilized water. By this procedure the bacteria accidentally attached to the viscid mass of sputum dur- ing its passage through the mouth are washed away. In the last bath the mass is torn apart and a small portion from the interior is used to make a microscopic preparation, the examination of which shows whether only tubercle bacilli are present. If this be the case cultures upon glycerin-agar are started from material obtained from the interior of the same mass. The colonies obtained in this way appear in about two weeks as round, white, opaque, moist, and shin- ing masses. Kitasato's researches show that the greater portion of the tubercle bacilli in sputum obtained in this way, and in the con- tents of lung cavities, are incapable of development, although this fact cannot be recognized by a microscopic examination of stained specimens. On account of the greater facility of preparing and sterilizing glycerin-agar, and the more rapid and abundant development upon this medium, it is now usually employed in preference to blood 402 BACILLI IN CHRONIC INFECTIOUS DISEASES. Serum. The growth at the end of fourteen days is more abundant than upon blood serum at the end of several weeks. When numerous bacilli have been distributed over the surface of the culture medium a rather uniform, thick, white layer, which subsequently acquires a yellowish tint, is developed ; when the bacilli are few in number or are associated in scattered groups separate colonies are developed, which acquire considerable thickness and have more or less irregular outlines ; they are white at first, then yellowish-white. Frankel describes the tubercle bacillus as a facultative anaerobic, and it would appear that it must be able to grow in situations where it can obtain very little oxy- gen from its development in the interior of tu- berculous nodules, lymphatic glands, etc. But in stick cultures in glycerin-agar development only occurs near the surface, and not at all in the deeper portion of the medium. In view of its abundant growth on the surface it is diffi- cult to understand this failure to grow along the line of puncture, if it is in truth a faculta- tive anaerobic. In peptonized veal broth containing five per cent of glycerin the bacillus develops at first in the form of little flocculi, which accumulate at the bottom of the flask and which by agitation are easily broken up. At the end of two or three weeks the bottom of the flask is covered with similar flocculi, which form an abundant deposit. Pawlowski and others report success in cul- tivating the tubercle bacillus upon the surface of cooked potato enclosed in a test tube after the method of Bolton and Koux. The open end of the tube is hermetically sealed in a flame after the bacilli have been planted upon the obliquely-cut surface of the potato; this prevents drying. Ac- cording to Pawlowski, better results are obtained if the surface of the potato is moistened with a five-per-cent solution of glycerin. The growth is said to be seen at the end of about twelve days as grayish, diy-looking flakes ; at the end of three or four weeks it forms a dry, smooth, whitish layer, and no further development occurs. The range of temperature at which this bacillus will grow is very restricted ; 37° C. is usually given as the most favorable point, Fio. 120.— Culture of tu- bercle bacillus upon glyce- rin-agon Photograph by Roux. BACILLI IN CHRONIC INFECTIOUS DISEASES. 403 but Roux and Nocard say that the most favorable temperature ap- pears to be 39°, and that development is slower at 37°. The experiments of Koch, Schill and Fischer, and others show that the bacilli retain their vitality in desiccated sputum for several months (nine to ten months — De Toma) ; but they are said to undergo a gradual diminution in pathogenic virulence, which is more rapid when the desiccated material is kept at a temperature of 30° to 40° C. In the experiments of Cadeac and Malet portions of the lung from a tuberculous cow, dried and pulverized, produced tuberculosis in guinea-pigs at the end of one hundred and two days. They retain their vitality for a considerable time in putrefying material (forty- three days — Schill and Fischer ; one hundred and twenty days — Ca- deac and Malet). The resisting power of this bacillus against ger- micidal agents is also greater than that of certain other pathogenic microorganisms, but not so great as to justify the inference that it forms spores. It is not destroyed by the gastric juice in the sto- mach, as is shown by successful infection experiments in suscep- tible animals, by mixing cultures of the bacillus with their food (Baumgarten, Fischer), and also by experiments with an artificially prepared gastric juice (Falk). They are destroyed, in sputum, in twenty hours by a three-per-cent solution of carbolic acid, even when they present the appearance usually ascribed to the presence of spores (Cavagnis) ; also by absolute alcohol, a saturated aqueous solution of salicylic acid, saturated aniline water, etc. (Schill and Fischer). The more recent experiments of Yersin upon pure cul- tures of the bacillus gave the following results : " Tubercle bacilli, containing spores, were killed by a five-per-cent solution of carbolic acid in thirty seconds, by one-per-cent in one minute ; absolute alco- hol, five minutes ; iodof orm-ether, one per cent, five minutes ; ether, ten minutes ; mercuric chloride, 1 : 1,000 solution, ten minutes ; thymol, three hours ; salicylic acid, 2. 5 per cent, six hours. The tubercle bacillus appears to be especially susceptible to the action of light. In his address before the Tenth International Medi- cal Congress (Berlin, 1890) Koch says that when exposed to direct sunlight the tubercle bacillus is killed in from a few minutes to sev- eral hours, according to the thickness of the layer ; it is also de- stroyed by diffuse daylight in from five to seven days when placed near a window. This fact has an important hygienic bearing, espe- cially in view of the fact that the tubercle bacillus is not readily killed by desiccation, putrefaction of the material containing it, etc. Tuberculous sputum expectorated upon sidewalks, etc., being ex- posed to the action of direct sunlight, will in many cases be disin- fected by this agent by the time complete desiccation has occurred — i. e. , before it is in a condition to be carried into the air as dust. 404 BACILLI IN CHRONIC INFECTIOUS DISEASES. Sawizky in 1891 made a series of experiments to determine the length of time during which d ried tuberculous sputum retains its virulence. He arrived at the conclusion that virulence is not sud- denly but gradually lost, and that in an ordinary dwelling room dried sputum retains its specific infectious power for two and one- half months. Metschnikoff states that when kept at a temperature of 42° C. for some time the tubercle bacillus undergoes a notable diminution in its pathogenic power, and that when kept at a temperature of 43° to 44° it after a time only induces a local abscess when injected subcu- taneously into guinea-pigs. The experiments of Lote also indicate that an " attenuation of virulence " has occurred in the cultures pre- served in Koch's laboratory, originating in 1882 from the lungs of a tuberculous ape. The author named made experiments with cul- tures from this source (ninetieth to ninety-fifth successive culture), and at the same time with a culture obtained from Roux, of Pasteur's laboratory. Rabbits inoculated with cultures from the last-mentioned source developed a hectic fever at the end of two weeks, and died tuberculous at the end of twenty-one to thirty-nine days. Twelve rabbits were inoculated with the cultures from Koch's laboratory ; the injections were made either subcutaneously, into a vein, into the pleural cavity, or into the cavity of the abdo- men. No elevation of temperature occurred in any of the animals, and they were found at the end of a month to have increased in weight. At the end of six weeks one of them was killed and tuber- cular nodules were found in various organs. The remaining animals were killed at the end of one hundred and forty-four to one hundred and forty-eight days. The two inoculated subcutaneously presented no sign of general tuberculosis, but a small yellow nodule contain- ing bacilli was found at the point of inoculation. Those inoculated by injection into a vein showed one or two nodules in the lungs con- taining a few bacilli. In Koch's original experiments rabbits were killed by intravenous inoculation of his cultures in from thirteen to thirty-one days. That this attenuation of virulence depends upon a \v ditliculty in breathing jind usually succumb to general tuberculosis, especially involving the lungs, within four to eight weeks, Injections of tuberculous sputum, or of pure cultures of the BACILLI IN CHRONIC INFECTIOUS DISEASES. 409 bacillus, into the peritoneal cavity give rise to extensive tuberculo- sis of the liver, spleen, and lungs, and to death, as a rule, within three or four weeks. Rabbits are less susceptible to subcutaneous injections, but die within seventeen to twenty days when virulent — recent — cultures are injected into the circulation. As a result of such an inoculation the animal rapidly loses flesh and has a decided elevation of temperature, commencing at the end of the first week and increasing considerably during the last days of life. At the autopsy the spleen and liver are found to be greatly enlarged, but they do not contain any tubercles that can be recognized by the naked eye (Yersin). They contain, however, great numbers of tubercle bacilli, both free and in the cells. Injections of a small quantity of a pure culture into the anterior chamber of the rabbit's eye cause first iris-tuberculosis, followed by swelling and caseation of the near- est lymph glands, and finally general infection and death ; when larger quantities are injected general tuberculosis is quickly devel- oped. The influence of quantity — number of bacilli — is also shown in subcutaneous, intravenous, or intraperitoneal injections into guinea- pigs and rabbits (Hirschberger, Gebhardt, Wyssokowitsch). Thus rabbits which received less than one hundred and fifty bacilli, in sputum, in the experiments of Wyssokowitsch, did not develop tuber- culosis ; and in guinea-pigs the smaller the number injected the more protracted the course of the disease was found to be. Tuberculosis in man no doubt results, in a large proportion of the cases, from the respiration, by a susceptible individual, of air con- taining the tubercle bacillus in suspension in a desiccated condition. As already stated, it has been demonstrated by experiment that the bacillus retains its vitality in desiccated sputum for several months. The experiments of Cornet have demonstrated that in the dust of apartments occupied by tuberculous patients tubercle bacilli are very commonly present in sufficient numbers to induce tuberculosis in guinea-pigs inoculated in the peritoneal cavity with such dust, while negative results were obtained from inoculations with dust from other localities. In view of these facts the usual mode of infection is apparent. Infection may also occur through an open wound or abrasion of the skin, as in the small, circumscribed tumors which sometimes develop upon the hands of pathologists as a result of handling tuberculous tissues. A few instances of accidental inocu- lation through wounds made by glass or earthen vessels containing tuberculous sputum have also been recorded. A more common mode of infection, especially in children, is probably by way of the intesti- nal glands, from the ingestion of the milk of tuberculous cows. That infection may occur by way of the intestine has been proved by ex- periments upon rabbits, which develop tuberculosis when fed upon 29 410 BACILLI IN CHRONIC INFECTIOUS DISEASES. tuberculous sputum. And that the tubercle bacillus is frequently, if not usually, present in the milk of tuberculous cows has been proved by the experiments of Bollinger, Hirschberger, Ernst, and others. In Hirschberger's investigations milk from tuberculous cows induced tuberculosis in guinea-pigs, when injected subcutaneously or into the peritoneal cavity, in fifty-five per cent of the cases studied (twenty). The conclusion is reached that the milk may contain tu- bercle bacilli even when the udder of the cow is not involved. Ernst also, from an examination of the milk from thirty-six tuberculous cows in which the udder was apparently not involved, found the tubercle bacillus by microscopical examination in five per cent of the samples examined (one hundred and fourteen). The prevalence of tuberculosis among cattle is shown by numer- ous investigations, and especially by the official inspections of slaughtered animals made in Germany. Thus in Saxony, in the year 1889, of 611,511 cattle examined 6,135 were found to be tubercu- lous (about one per cent) ; in Berlin, 1887-1888, out of 130,733 ani- mals slaughtered 4,300 were found to be tuberculous (3.2 per cent). In view of the facts stated the great mortality from tubercular dis- eases among children, many of whom are removed from other prob- able sources of infection, is not difficult to understand, and the practical and simple method of preventing infection in this way, af- forded by the sterilization (by heat) of milk used as food for infants, must commend itself to all. 54. BACILLUS TUBERCULOSIS GALLINARUM. The researches of Maffucci (1889) and of Cadiot, Gilbert, and Roger (1890) show that the bacillus obtained from spontaneous tu- berculosis in chickens, although closely resembling the bacillus of human tuberculosis, is not identical with it, varying especially in its pathogenic power. This view is sustained by the observations of Koch, who says in his address before the Tenth International Medi- cal Congress (Berlin, 1890) : " The care which it is necessary to exercise in judging of the characters which serve to differentiate bacteria, even those which are well known, I have learned in the case of the tubercle bacillus This species is so definitely characterized by its staining reactions, its growth in pure cultures, and its pathogenic qualities, and indeed by each of these characters, that it seems iiiij).»>il>le to con found it with other species. Nevertheless in this case also one should not rely upon a single one of the characters mentioned for de- termining the species, but should follow the safe rule that all available characters should he considered, and the identity of a certain bacterium should only be regarded as demonstrated when it has been shown to corre- spond in all of these particulars When I made my first researches with reference to the tubercle bacillus I was controlled by this rule, and tested tubercle bacilli from various sources, not only with reference to their stain- ing reactions, but also with reference to their growth in culture media and BACILLI IN CHRONIC INFECTIOUS DISEASES. 411 pathogenic characters. Only in the tuberculosis of chickens I was not able to apply this rule, as at that time it was not possible for me to obtain fresh material from which to make pure cultures. As, however, all other forms of tuberculosis had given identical bacilli, and the bacilli of chicken tuber- culosis in their appearance and behavior towards the aniline colors entirely corresponded with these, I believed myself justified in assuming their iden- tity, notwithstanding the incompleteness of the research. Later I received pure cultures from various sources, which apparently originated from tuber- cle bacilli, but in several regards differed from these ; especially in the fact that inoculation experiments, made by experienced and reliable investigators, led to dissimilar results, which it was necessary to regard as unexplained con- tradictions. At first I believed that these differences depended upon changes such as are frequently o >served in pathogenic bacteria, when these are culti- vated in pure cultures outside of the body fora long time under more or less unfavorable conditions. In order to solve the riddle I attempted by various influences to change the common tubercle bacilli into the presumed variety referred to. They were cultivated for several months at so high a tempera- ture that only a scanty growth was obtained; in other experiments still higher temperatures were allowed to act repeatedly for so long a time that the cultures were brought as nearly as possible to the point of killing the bacilli. In a similar way I subjected the cultures to the action of chemical agents, of light, or absence of moisture ; they were cultivated for many gen- erations in association with other bacteria ; inoculated successively in ani- mals having but a slight susceptibility. But, in spite of all these attempts, only slight variations were obtained in their characters — far less than other pathogenic bacteria undergo under similar circumstances. Itappears, there- fore, that the tubercle bacilli retain their characters with special obstinacy ; this is in accord with the fact that pure cultures which have now been cul- tivated by me in test tubes for more than nine years, without in the mean- time having been in a living body, are still entirely unchanged with the ex- ception of a slight diminution of virulence. ... It happened about a year ago that I received a living chicken which was suffering from tuberculosis, and I used this opportunity to make cultures directly from the diseased or- gans of this animal, which previously I had not been able to do. When the cultures grew I saw to my surprise that they had precisely the appearance and all of the characters possessed by the enigmatical cultures resembling those of the genuine tubercle bacillus. Later I learned that these also ori- ginated from tuberculosis in fowls, but, upon the assumption that all forms of tuberculosis are identical, had been considered genuine tubercle bacilli. A verification of my observations I find in the recently published researches of Prof. Maffucci with reference to tuberculosis of fowls." According to Maffucci, adult chickens are refractory against the action of the Bacillus tuberculosis from man, and there are slight morphological and biological differences in the bacilli from the two sources. Cadiot, Gilbert, and Roger (1891) have made a series of experi- ments with the bacillus of tuberculosis in fowls. They found the bacilli to be very numerous in the livers of chickens suffering from spontaneous tuberculosis, and inoculated with material from this source six chickens, five rabbits, and twelve guinea-pigs. The chickens, when inoculated in the cavity of the abdomen or by injec- tion into a vein, died in from forty-one to ninety-three days from general tuberculosis. Four of the rabbits died of general tuberculosis, presenting the same appearance as that following inoculation with 412 BACILLI IN CHRONIC INFECTIOUS DISEASES. bacilli from human tuberculosis. Of the guinea-pigs, which were inoculated in the cavity of the abdomen, eleven remained in good health and one only died of general tuberculosis. These experi- ments show a decided difference in the pathogenic properties of tubercle bacilli from the two sources, for the guinea-pig is especially susceptible to tuberculosis as a result of similar inoculations with bacilli from human tuberculosis. We must therefore conclude that the bacillus found in spontaneous tuberculosis in fowls is a distinct variety of Bacillus tuberculosis. Whether this variety would cause tuberculosis in man, if introduced into susceptible subjects, has not been determined ; and, as pointed out by Koch, this question can only be answered in the affirmative if it should be obtained in pure cultures from cases of human tuberculosis. Since the above was written Maffucci has published (1892) an elaborate memoir upon tuberculosis of fowls. His conclusions are stated as follows : " The bacillus of tuberculosis in fowls is distinguished from that of tuber- culosis in mammals by the following points of difference : **1. It does not induce tuberculosis in guinea-pigs, and seldom causes general tuberculosis in rabbits. 4 * 2. The cultures in various media have a different appearance from those of the Bacillus tuberculosis of mammals. " 3. The temperature at which it develops varies between 35° and 45° C., and the thermal death-point is 70° C. "4. At 45° to 50° C. the cultures show long, thick, and branched forms. "5. The bacillus retains its vegetative and pathogenic power at the end of two years. " 6. This bacillus produces a substance which is toxic for guinea-pigs and is but slightly toxic for grown fowls. " 7. The tuberculosis produced in fowls by this bacillus is without giant cells." Additional Notes upon the Tubercle Bacillus (1895).— Several authors (Metschnikoff, Czaplewski, Fischel) have described branch- ing forms of the tubercle bacillus, and Lubinsky (1895) reports that in certain media it grows out into long threads, which, however, he has never observed to be branched. The media used by him are said to give a more abundant growth than occurs upon glycerin-agar ; the most favorable being made of flesh-peptone agar, or flesh -peptone bouillon, containing four per cent of glycerin and mashed potato, one kilo of finely chopped and washed potato to fifteen hundred cubic centimetres of water ; this is cooked for three or four hours and filtered —to the filtrate is added four per cent of glycerin ; one and a half per cent of agar is now added and the mixture is again cooked and filtered. Jones (1895) has observed the branching forms previously de- scribed by several authors, and states that they are only found upon the surface of culture media where there is free access of oxygen. BACILLI IN CHRONIC INFECTIOUS DISEASES. 413 He concludes that the tubercle bacillus does not form endogenous spores, such as are found in various other bacilli, but that in the rods and branched filaments certain objects are seen which are probably reproductive elements, and which closely resemble similar bodies (" Kolben ") seen in the actinomyces fungus, to which Jones believes the tubercle bacillus is closely related. Prudden and Hodenpyl (1891) have shown that the injection of dead tubercle bacilli in rabbits gives rise to the development of nod- ules in the lung containing epithelioid and giant cells, but that these never undergo caseation. This fact is supposed to justify the infer- ence that caseation is due to the products elaborated during the growth of living tubercle bacilli. The results reported by Vissmann (1892) correspond with those reported by Prudden and Hodenpyl. Gamaleia (1892) has also obtained nodules with epithelioid and giant cells from the injection of dead tubercle bacilli, but in his ex- periments he also found caseation of the nodules. Baumgarten sug- gests that this was probably due to the fact that there were some liv- ing tubercle bacilli remaining in the cultures which he injected. Loomis (1890) and Pizzini (1892) have shown that living tubercle bacilli are not infrequently found in the bronchial glands of individ- uals who present no evidence of tubercular disease of the lungs or else- where. The author last mentioned inoculated thirty guinea-pigs with the bronchial, mesenteric, and cervical glands of thirty in- dividuals in whom death was due to accident or acute disease, and who were free from tuberculosis. Twelve of these thirty guinea- pigs developed tuberculosis as a result of the inoculation. Straus (1894) has found tubercle bacilli in the nasal cavities of healthy individuals. Ernst (1895), as the result of extended researches made under the auspices of the Massachusetts Society for Promoting Agriculture, has arrived at the following conclusions with reference to the pres- ence of the tubercle bacillus in the milk of tuberculous cows : " The possibility of milk from tuberculous udders containing the infectious element is undeniable. " With the evidence here presented, it is equally undeniable that milk from diseased cows with no appreciable lesion of the udder may, and not infrequently does, contain the bacillus of the disease." De Schweinitz (1894) has found that by continued cultivation in an artificial medium the tubercle bacillus becomes attenuated, so that when inoculated into guinea-pigs these animals give no evidence of tubercular infection for six months or more. And his experiments indicate that animals which have survived an inoculation with the attenuated tubercle bacillus acquire an immunity against the patho- genic action of virulent cultures. 414 BACILLI IN CHRONIC INFECTIOUS DISEASES. Amann (1895) has given in the Centralblatt fur Bakteriologie (Bd. xvii., page 513) a detailed account of his method for demon- strating the presence of tubercle bacilli in sputum by sedimentation. He mixes the sputum with two to four volumes of cold distilled water, in a glass cylinder which should not be more than half full. He adds one cubic centimetre of chloroform and a small quantity of shot; the glass cylinder is then closed with a rubber cork and vio- lently shaken for some minutes. From four to six volumes of dis- Fio. 122.-Section of a recent lepra nodule of the Bkin. X 950. (Baumgarten.) tilled water are then added and the mixture is placed in a V-formed glass tube for sedimentation ; two cubic centimetres of carbol-f uchsin solution are added and distributed by gentle agitation of the tube. At the end of two days the sedimentation is complete and the stained bacilli, cells, connective-tissue fibres, etc., are taken up with a pipette for examination under the microscope. 55. BACILLUS LEPR^E. Discovered by Hansen (1879), chiefly in the interior of the peculiar round or oval cells found in leprous tubercles. Discovery confirmed by Neisser (1879) and by many subsequent observers. While found chiefly in the leprous tubercles of the skin and mucous membranes, the bacilli have also been found in the lymphatic glands, the liver, the spleen, the testicles, and, in the anesthetic form of the 29 Bacinus (Baumgarten). Septica!m.i89^b8tf^." With reference to the American swine plague described by Salmon and Smith, we are informed by Smith, in his most recent publication upon the subject (Zeitschrift fur Hygiene, Band x., page 493), that cultures of the German Schweineseuche bacillus, received from the Berlin Hygienic Institute, compared with his cultures from infected swine in this country, agreed in all particulars, except that the former were de- cidedly more pathogenic for swine and for rabbits. It appears extremely probable that the form of septicaemia studied 430 BACILLI WHICH PRODUCE SEPTIOSSMIA by Davaine (1872), which he induced in the first instance by inject- ing putrid ox blood into rabbits, was due to the same pathogenic ba- cillus. The writer obtained this bacillus (1887) in Cuba from the blood of rabbits inoculated with liver tissue taken from a yellow- fever cadaver and kept for forty-eight hours in an antiseptic wrap- ping. The name which we have adopted is that proposed by Hueppe for the form of septicaemia to which it gives rise — "Septikamia hamorrhagica. " Morphology. — Short bacilli with rounded ends, from 0.6 to 0.7 p in diameter and about 1.4 ft long; sometimes united in pairs, or in chains of three or four elements. In stained preparations the ex- tremities are usually stained, while the central portion of the rod remains unstained. This " end staining" causes the rods to present the appearance of diplococci when examined with a comparatively low power, and some of the earlier observers described the microor- ganism under consideration as a micrococcus. It is quickly stained by the aniline colors usually employed, but loses its color when treated by Gram's method. Biological Characters. — A non-motile, aerobic, non-liquefy- ing bacillus. Does not form spores. Grows in various culture media at the room temperature, but more rapidly at 35° to 37° C. — the lowest temperature at which development occurs is about 13° C. Although this is an aerobic bacillus and a certain amount of oxygen is necessary for its development, it appears to grow better when the amount is somewhat restricted than it does on the surface of nutrient media. Upon gelatin plates, at the end of two or three days, small, white colonies are developed upon or near the surface ; these are finely granular and spherical, with a more or less irregular outline, and by transmitted light have a yellowish color ; later the central portion of the colonies is of a yellowish-brown color and is sur- rounded by a transparent peripheral zone. The superficial colonies are commonly smaller than those which develop a little below the surface of the gelatin. In stick cultures in nutrient gelatin the growth upon the surface consists of a thin, whitish layer in the vicinity of the point of puncture, having an irregular, jagged out- line— sometimes there is no development upon the surface ; along the line of puncture the growth consists of rather transparent, dis- crete or confluent colonies. In streak cultures upon nutrient agar, or gelatin, or blood serum the growth is limited to the immediate vicinity of the line of inoculation, and consists of finely granular, semi-transparent colonies, which form a thin, grayish-white layer with irregular, somewhat thickened margins. Upon potato no de- velopment occurs, as a rule, at the room temperature, but in the in- IN SUSCEPTIBLE ANIMALS. 43 1 cubating oven a rather thin, transparent, grayish-white or yellowish, waxy layer is developed in the course of a few days. According to Bunzl-Federn, the bacillus of fowl cholera and that of rabbit septicaemia grow upon potato, while the bacillus of Wildseuche, Schweineseuche, and Biif- felseuche do not. According to Caneva, none of the bacilli of this group grow upon potato. The same author states that the growth in milk is scanty and does not produce coagulation, while Bunzl- Federn finds that the bacillus of fowl cholera and of rabbit septi- caemia produce coagulation and the others do not. These differ- ences are not, however, consid- ered by the author last named as sufficient to establish the specific difference of the bacilli from these diff erent sources. He looks upon them rather as varieties of the same species. Bunzl-Federn has also ascertained that when cul- tivated in a peptone solution all of the bacilli of this group, with the exception of that obtained from the so-called Buffelseuche, give the reaction for phenol and for indol— the bacillus of Buffel- seuche gives the indol reaction only. Development in bouillon is rapid and causes a uniform turbidity of the fluid. Cultures of this bacillus may retain their vitality for three months or more when kept in a moist condition ; but the bacillus usually fails to grow after having been kept for a few days in a desiccated con- dition ; according to Hueppe, it may resist desiccation for fourteen days. The thermal death-point, as determined by Salmon for the bacillus of fowl cholera, is 56° C. , the time of exposure being ten minutes (55° C. with fifteen minutes' exposure — Baumgarten). It is not readily destroyed by putrefaction (Kitt). A solution of mercuric chloride of 1 :5,000 destroys it in one minute, and a three-per-cent solution of carbolic PIG. 130. —Bacillus septicaemias haemor- rhagicae; stick culture in nutrient gelatin, end of four days at 16°- 18° C. (Baumgarten ) Fia. 131.— Bacillus of Schweineseuche ; old stick culture in nutrient gela- tin. (Schutz.) FIG. 132. — Bacillus of swine plague; colonies on gelatin plate, end of seven days. X 00. (Smith.) 432 BACILLI WHICH PRODUCE SEPTICAEMIA acid in six hours (Hueppe). Pasteur (1880) has shown that when cultures of this bacillus (microbe of fowl cholera) in bouillon are kept for some time they gradually lose their pathogenic virulence, and he has ascribed this " attenuation of virulence" to the action of atmospheric oxygen. He also ascertained that the particular degree of \irulence manifested by the mother culture after a certain interval could be maintained in successive cultures made at short intervals. He was thus able to cultivate different pathogenic varieties, and to use these in making protective inoculations, by which susceptible ani- mals were preserved from the effects of virulent cultures injected subsequently. Attenuated cultures recover their virulence when inoculated into very susceptible animals. Thus a culture which would produce a non-fatal and protective attack in a chicken may, according to Pas- teur, kill a small bird, like a sparrow; and by successive inoculations from one sparrow to another the original degree of virulence may be restored, so that a minute quantity of a pure culture would be fata' to a chicken. Pathogenesis. — Pathogenic for chickens, pigeons, pheasants, sparrows, and other small birds, for rabbits and mice, also for swine (Schweineseuche), for cattle (Rinderseuche), and for deer (Wild- seuche). Subcutaneous injection of a minute quantity of a virulent culture usually kills chickens within forty-eight hours. Some time before death the fowl falls into a somnolent condition, and, with drooping wings and ruffled feathers, remains standing in one place until it dies. Infection may also occur from the ingestion of food moistened with a culture of the bacillus or soiled with the discharges from the bowels of other infected fowls. At the autopsy the mucous metnbrane of the small intestine is found to be inflamed and studded with small hsemorrhagic foci, as are also the serous membranes ; the spleen is notably enlarged. The bacilli are found in great numbers in the blood, in the various organs, and in the contents of the in- testine. In rabbits death commonly occurs in from sixteen to twenty hours, and is often preceded by convulsions. The temperature is elevated at first, but shortly before death it is reduced below the normal. The post-mortem appearances are : swelling of the spleen and lymphatic glands ; ecchymoses or diffuse haBmorrhagic infiltra- tions of the mucous membranes of the digestive and respiratory pas- sages, and in the muscles ; and at the point of inoculation a slight amount of inflammatory oedema. The bacilli are found in consider- able numbers in the blood within the vessels, or in that which has escaped into the tissues by the rupture of small veins. They are not, however, so numerous as in some other forms of septicaemia — e.g., Anthrax, mouse septicaemia — when an examination is made imme- IN SUSCEPTIBLE ANIMALS. 433 diately after death ; later the number may be greatly increased as a result of post-mortem multiplication within the vessels. The rabbit is so extremely susceptible to infection by this bacillus that inocula- tion in the cornea by a slight superficial wound usually gives rise to general infection and death. This animal may also be infected by the ingestion of food contaminated with a culture of the bacillus. It is by this means that Pasteur proposed to destroy the rabbits in Aus- tralia, which have multiplied in that country to such an extent as to constitute a veritable pest. Both in fowls and in rabbits the disease may under certain circumstances run a more protracted course — e.g., when they are inoculated with a small quantity of an attenuated cul- ture. In less susceptible animals — guinea-pigs, sheep, dogs, horses FIG. 138.— Bacillus of Sahweineseuche, in blood of rabbit. (Schutz.) — a local abscess, without general infection, may result from the sub- cutaneous injection of the bacillus ; but these animals are not entirely immune. In the infectious maladies of swine, cattle, deer, and other large animals to which reference has been made, and which are be- lieved to be due to the same bacillus, the symptoms and pathological appearances do not entirely correspond with those in the rabbit or the fowl; but the bacillus as obtained from the blood of such animals corresponds in its morphological and biological characters with Pas- teur's microbe of fowl cholera and Koch's bacillus of rabbit septi- caemia, and pure cultures from the various sources mentioned are equally fatal to rabbits and to fowls. In the larger animals pul- monary and intestinal lesions are developed, and in swine a diffused red color of the skin, similar to that observed in the disease known in Germany as Schweinerothlauf (Fr. rouget), is sometimes seen. 434 BACILLI WHICH PRODUCE SEPTICAEMIA According to Baumgarten, bacilli from Wildseuche or from Kinder- seuche inoculated into swine give rise to fatal Schweineseuche, and bacilli from any of these forms of disease, when inoculated into pigeons, produce characteristic fowl cholera ; but the bacillus as ob- tained from Schweineseuche or Wildseuche is not fatal to chickens, and the bacillus from Schweineseuche is fatal to guinea-pigs, which have but slight susceptibility to the bacillus of rabbit septicaemia. Notwithstanding these differences he agrees with Hueppe in the view that the bacilli from the various sources mentioned are specifically identical ; although evidently, if this view is adopted, we must admit that varieties exist which differ somewhat in their pathogenic power. The researches of Smith and of Moore show that " an attenuated variety of bacteria, belonging to the group of swine-plague bacteria and not distinguishable from them, inhabit the mouth and upper air passages of such domesticated animals as cattle, dogs, and cats" (Smith). 62. BACILLUS OF CHOLERA IN DUCKS. Obtained by Cornil and Toupet (1888) from the blood of ducks, in the Jardin d'Acclimation at Paris, which had died of an epidemic disease charac- terized by diarrhoea, feebleness, and muscular tremors, and which resulted fatally in two or three days. Morphology. — Does not differ in its morphology from the bacillus of fowl cholera (Bacillus septicaemias haemorrhagicse) ; short rods with rounded ends, from 1 to 1.5 ft in length and 0.5 // broad. Stains with the usual aniline colors, but not by Gram's method ; the ends stain more deeply than the central portion. Biological Characters.— An aerobic, non liquefying, non- motile bacillus. Does not form spores. Grows in the usual culture media at the room tem- peratu re. In its gro wth in various media, as well as in its morphology, Cornil and Toupet found this bacillus to correspond with the bacillus of fowl cholera. In gelatin stick cultures the growth upon the surface consists of a thin, gravish layer, and along the line of puncture as small, semi transpa- rent, slightly yellowish, spherical colonies. Upon agar, in the incubating oven, at the end of twelve hours small, lentil shaped, waxy colonies are formed, which later may have a diameter of three to four millimetres. Upon potato circular, yellowish colonies are formed, which become con- fluent and form a somewhat depressed, pale-yellow layer. Pathogenesis. — According to Cornil and Toupet, this bacillus is patho- genic for ducks, but not for chickens or pigeons, and only kills rabbits when injected in considerable quantity. Ducks die in from one to three days from subcutaneous injections, or by the ingestion of food to which the bacil- lus has been added. 63. BACILLUS OP HOG CHOLERA (Salmon and Smith). Synonyms. — Bacillus of swine plague (Billings) ; Bacillus of swine- pest (Selander). According to Smith, this bacillus was first described by Klein (1884) ; it was first obtained in pure cultures and its principal char- acters determined by Salmon and Smith (1885), and has since been IN SUSCEPTIBLE ANIMALS. 435 studied in cultures and by experimental inoculations by Selander, Billings, Frosch, Welch, Caneva, Bunzl-Federn, and others. The bacillus is found in the blood and various organs of hogs which have succumbed to the infectious disease known in this country as hog cholera ; and also in the contents of the intestine, from which it may be obtained by inoculations into rabbits, but is not easily iso- lated by the plate method owing to the large number of other bac- teria present (Smith). Morphology. — Short bacilli with rounded ends, 1.2 to 1.5 yu in length and 0.6 to 0.7 /* in breadth ; usually united in pairs. This bacillus is easily stained by the aniline colors usually em- ployed, but does not retain its color when treated by Gram's method. When the staining agent is allowed to act for a very short time the FIG. 131.— Bacillus of hog cholera; stained by Loffler's method to show flagella. x 1,000. From a photomicrograph made at the Army Medical Museum. (Gray.) ends of the rods may be stained while the central portion remains unstained. Biological Characters. — An aerobic (facultative anaerobic), non- liquefying, actively motile bacillus. In many of its characters this bacillus closely resembles the one last described (Bacillus septicaemias hsemorrhagicse), but it is distinguished from it by its active move- ments, which, according to Smith, may be still observed in cultures which have been kept for weeks or months. Does not form spores. Grows readily in various culture media at the room temperature — more rapidly in the incubating oven. Upon gelatin plates colonies are developed in from twenty-four to forty-eight hours. The deep colo- nies are spherical and homogeneous, and have a brownish color by transmitted light; they seldom exceed one-half millimetre in diameter. 436 BACILLI WHICH PRODUCE SEPTICAEMIA The superficial colonies may attain a diameter of two millimetres : they present no distinctive characters. Upon agar plates the colonies may have a diameter of four millimetres ; they have a grayish, trans- parent appearance and a shining surface. In gelatin stick cultures small, yellowish-white colonies are developed along the line of in - oculation, which may become confluent ; upon the surface a thin. pearly layer is developed about the point of inoculation, which may have a diameter of six millimetres or more. Upon potato a straw- yellow layer is developed, which later acquires a darker color. In slightly alkaline bouillon a slight cloudiness may be observed at the end of twenty -four hours, and at the end of one or two weeks, if not disturbed, a deposit is seen at the bottom of the tube and a thin, broken film may form upon the surface. The development of this bacillus in milk produces a direct solution of the casein without pre- vious coagulation ; when a solution of litmus has been added to milk it retains its blue color in presence of this bacillus, while the bacillus previously described causes it to change to red. Neither phenol nor indol is produced in solutions containing peptone (Bunzl-Federn) — another distinguishing character from the Bacillus septicaemias hsemorrhagicae. This bacillus may be cultivated in slightly acid media, which after a time acquire an alkaline reaction. In Smith's experiments this bacillus was found to resist desicca- tion from nine days to several months, according to the thickness of the layer dried upon the cover glass ; bacilli from an agar culture in some experiments failed to grow after seventeen days, and in others still gave cultures after four months. Bouillon cultures are steril- ized in four minutes by a temperature of 70° C., in fifteen minutes by 58° C., and in one hour by 54° C. (Smith). Novy has isolated from cultures of the hog-cholera bacillus a toxic basic substance which he calls susotoxin. This was obtained by Brieger's method ; it is a yellowish-brown, syrup-like liquid, which, when injected into rats in doses of 0.125 to 0.25 cubic centimetre, causes their death in less than thirty-six hours. He also obtained by precipitation with absolute alcohol, from cultures concentrated in a vacuum at 36° C., a toxalbumin which when dried was in the form of a white powder easily soluble in water. Rats died in three or four hours after re- ceiving subcutaneously a dose of 0.1 to 0.5 gramme. Pathogenesis. — Pathogenic for swine, rabbits, guinea-pigs, mice, and pigeons. In certain parts of the United States the disease known as " hog cholera " f requently prevails among swine as a fatal epidemic. It may occur as an acute and quickly fatal septicaemia, or in a more chronic form lasting from two to four weeks or even longer. In the acute form death may occur within twenty-four hours, and haem- IN SUSCEPTIBLE ANIMALS. 437 orrhagic extravasations are found upon the mucous and serous membranes and in the parenchyma of the lungs, kidneys, and lym- phatic glands. The spleen is greatly enlarged, soft, and dark in color. In the chronic form of the disease the most notable changes are found in the alimentary canal. These are most constant and characteristic in the caecum and colon, which may be studded with spherical, hard, necrotic masses or extensive diphtheritic patches. According to Smith, the hsemorrhagic and necrotic form of the dis- ease may exist at the same time in different animals of the same herd. The bacilli are found in all of the organs, and especially in the spleen, where they are associated in irregular colonies similar to those of the typhoid bacillus. Smith has demonstrated their pre- sence in urine taken from the bladder immediately after the death of the animal, and states that the kidneys are almost always in- volved, as shown by the presence of albumin and tube casts in the urine. An extremely minute quantity of a bouillon culture injected be- neath the skin of a rabbit causes its death in from seven to twelve days ; a larger quantity may produce a fatal result in five days ; in- travenous injections of very small amounts may be fatal within forty-eight hours. After a subcutaneous injection the animal re- mains in apparent good health for three or four days, after which it loses its appetite and is indisposed to move ; several days before death the temperature is suddenly elevated from 2° to 3° C., and it remains high until the fatal termination. At the autopsy the spleen is found to be enlarged and of a dark-red color ; the liver is studded with small, yellowish- white, necrotic foci; the kidneys have under- gone parenchymatous changes ; the heart is fatty ; and the intestinal mucous membrane is more or less marked with hsemorrhagic extra- vasations. The bacilli are found in all of the organs. In house mice the results of experimental inoculations are similar to those in rabbits. Guinea-pigs succumb when inoculated subcutaneously with one-tenth cubic centimetre ; pigeons require a still larger dose — about three-quarters of a cubic centimetre. Swine are killed by the intravenous injection of one to two cubic centimetres of a recent bouillon culture, but, as a rule, do not succumb to subcutaneous injections. Cultures recently obtained from diseased animals are more virulent than those which have been propagated for a consider- able time in artificial media. Smith has described a variety of the hog-cholera bacillus obtained during- an epidemic in which the disease was of longer duration — about four weeks —than is usual, and in which there was commonly found at the autopsy a diphtheritic inflammation of the mucous membrane of the stomach. This bacillus differed from the typical form by being somewhat larger and in forming considerably larger colonies in gelatin plates — two or three times 438 BACILLI WHICH PRODUCE SEPTIOEMIA as large. It also produced a greater opacity in peptonized bouillon, and in general showed a more vigorous growth in various nutrient media. It dif- fered also in its pathogenic power, as tested upon rabbits, causing- death at a later date or not at all ; and in fatal cases the swelling of the spleen and necrotic foci in the liver, produced by the first-described species, were absent. Bang (1892) has obtained a bacillus from infected swine in Denmark which corresponds with the American hog-cholera bacillus. In chronic forms of the disease pneumonia and an extensive diphtheritic process in the intestines occurred as a complication. This was found to be due to another bacillus, called by Bang " vacuole-bacillus." This produced a fatal pleuro- pneumonia when injected into the lungs in pigs. According to Bang, his *4 vacuole-bacillus " is without doubt identical with the swine-plague bacillus of Salmon and Smith, and the disease of swine studied by him was a mixed infection. The necrotic changes in the intestine, found in cases running a chronic course, are believed by Bang to be due to still another bacillus—his "necrosis-bacillus." Affanas'sieff (1892) confirms the results previously ob- tained by several independent observers as to the identity of the swine-plague bacillus of Salmon and Smith with the Loffler-Schiitz bacillus. The only difference observed was a difference in pathogenic virulence — the bacillus from America corresponding with a somewhat attenuated variety of that from Germany. Welch (1894), as a result of his extended researches, arrives at the follow- ing conclusion : "Our own conclusion as to the bacteria of Schweineseuche and of swine plague is that no difference exists between them as regards morphology, culture behavior, and pathogenic effects on rabbits, mice, and other labora- tory animals. Cultures of each occur which are also indistinguishable by inoculation of pigs. The only difference by laboratory experiment which has thus far been brought out is that there occur Schweineseuche bacilli of higher degree of virulence as tested on pigs than any swine-plague bac- teria which have hitherto been isolated from pigs in this country. Another point to be considered in this connection is that Schweineseuche occurs as an independent disease in Germany without association with hog cholera, whereas swine plague has not been shown to prevail with the same inde- pendence as an epizootic in this country." Silberschmidt (1895) arrives at a different conclusion from that reached by Smith, Welch, Bang, and others, He believes that the diseases of swine known as hog cholera, swine plague, and infectious pneumo-enteritis are all due to one and the same bacillus, which, however, varies considerably both in its morphological characters and its pathogenic power. In view of the results previously reached by equally competent bacteriologists, and especially by Smith and by Welch in this country, we are not disposed to accept the view maintained by Silberschmidt. Smith has described several varieties of the hog-cholera bacillus, and in his account of the "hog-cholera group of bacteria " shows that the Bacillus enteriditis of Gartner and the Bacillus typhi murium of Loffler belong to this group. The characters of the different varieties (or species?) belonging to the group are given by Smith in detail (United States Department of Agri- culture, Bureau of Animal Industry, Bulletin No. 6, 1894), and the follow- ing general statement is made: " If we attempt to sum up those characters which are to circumscribe the hog-cholera group of bacteria we are at once confronted by the scarcity of common character's. Pathogonesis, though of great importance from the standpoint of pathology, is probably the last character acquired and evident ly the most variable and most readily lost. If we base the unity of this group on morphological and biological characters, we are like- wise met by variations in size, absence of motility, variations in the ap- pearancr <>f th«- colonies. Then* are, however, certain underlying char- IN SUSCEPTIBLE ANIMALS. 439 acters, as expressed by the behavior of these bacteria in bouillon con- taining- dextrose, saccharose, and lactose, which I think will serve as a very important group character, differentiating such groups sharply from the colon group. I would therefore suggest that for the present all bacteria whose size approximates that of this group, which do not liquefy gelatin, and whose fermentative properties are the same as those described for this group, should be ranged under it. Future investigations into the biochemical char- acters* of these varieties or sub-species may reveal other differential charac- ters, but the time has not yet come when such laborious work will be under- taken o» a sufficiently extensive scale to be of any service in differentiating varieties and sub-species." Selander in 1890, and Metschnikoff in 1892, have reported a rapid increase in virulence of the bacillus of hog cholera by successive inoculations in rabbits* or pigeons. Moore (1894) has shown that this is a mistake, and that the bacteriologists named probably did not experiment with cultures of the hog-cholera bacillus, as they supposed, but that their experiments were made with the bacillus of swine plague — Bacillus septicaemias hemprrhagi- cae — which when passed through a series of rabbits attains a notable increase in pathogenic virulence. In a recent article, Klein, of London (1895) says: " The bacillus of English swine plague, which I described in 1884, in Virchow's Archiv, as shown by Smith and Welch, is identical with the bacillus of American hog cholera." 64. BACILLUS OF BELFANTI AND PASCAROLA. Synonym. — Impf tetanusbacillus. Obtained by Belfanti and Pascarola (1888) from the pus of wounds in an individual who succumbed to tetanus. Morphology. — Bacilli with rounded ends, sometimes so short as to resemble micrococci ; resemble the Bacillus septicaemiae haemorrhagicae (fowl cholera). Stains with the usual aniline colors and also by Gram's method. The ends are commonly more deeply stained than the central portion. Biological Characters.— An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates yel- lowish-gray, finely granular, spherical colonies with smooth outlines are developed. In gelatin stick cultures, at 18° to 25° CM at the end of twenty- four hours small, spherical colonies are developed along the line of punc- ture, which are isolated or closely crowded; upon the surface a rather thin, shining, grayish- white, iridescent, circular layer is formed ; gas is given off which has not a disagreeable odor. Upon the surface of agar elevated, shining, gray colonies develop along the impfstrich, or a gray, shining band is formed which increases in thickness but not in breadth — usually less than one-half centimetre broad. Old cultures give off an acid odor. Upon blood serum a thin, white layer is developed along the line of inoculation. Upon potato a thin, white, varnish- like layer is formed. Pathogenesis.— Very pathogenic for rabbits, guinea-pigs, white mice, and sparrows. Not pathogenic for chickens, pigeons, or geese. 05. BACILLUS OF SWINE PLAGUE, MARSEILLES. Synonyms. — Bacillus der Schweineseuche, Marseilles (Rietsch and Jobert) ; Bacillus der Frettchenseuche — ferret disease (Eberth 440 BACILLI WHICH PRODUCE SEPTICAEMIA and Schimmelbusch) ; Bacillus der Amerikanischen Rinderseuche (Caneva) ; Bacillus of spontaneous rabbit septicaBmia (Eberth). The researches of Caneva and of Bunzl-Federn agree as to the identity of the bacillus obtained by Rietsch and Jobert (1887) from swine attacked with a fatal epidemic disease in Marseilles, and the bacillus found by Eberth and Schimmelbusch (1889) in the blood of ferrets suffering from a fatal form of septicasmia studied by them. The first-named bacteriologist also identifies a bacillus supposed by Billings to be the cause of "Texas fever" in cattle (" Ameri- kanische Rinderseuche ") and the bacillus of swine plague (Billings) with the above. Bunzl-Federn obtained cultures of Billings' swine- plague bacillus at two different times. He identifies the one first re- ceived with the bacillus now under consideration, and the other with the bacillus of hog cholera (Salmon).1 Morphology. — Bacilli with rounded ends, about twice as long as broad, and one-third smaller than the bacillus of typhoid fever (Eberth and Schimmelbusch). The bacillus of hog cholera is shorter and more slender than the Marseilles bacillus, and the bacillus of Loffler and Schiitz (No. 61) is still smaller (Rietsch and Jobert). In stained preparations the extremities of the rods are usually deeply stained, while the central portion remains unstained — "polar staining." By Loffler's method of staining the presence of flagella may be demonstrated (Frosch). Stains readily with the aniline dyes usually employed, but does not retain its color when treated by Gram's method. Biological Characters. — An aerobic (facultative anaerobic), non-liquefying, actively motile bacillus. Grows readily at the room temperature, and is distinguished from the bacillus of septi- caemia hoemorrhagica by its active movements and more rapid and abundant development in the various culture media usually em- ployed. It is distinguished from the bacillus of hog cholera (No. 63) by producing phenol and indol in solutions containing peptone, by causing coagulation of milk, and by producing an acid reaction in this fluid. Grows in culture media having an acid reaction. Rietsch and Jobert give the following account of the characters of growth in various culture media, as compared with the bacillus of hog cholera and the bacillus of Schweineseuche (Loffler, Schiitz), No. 61 : 1 The author named says : " With reference to the bacillus of swine plague (Billings), I obtained, as did Caneva, a decided production of acid in the cultures first sent by Billings ; but upon testing later cultures received directly from Bil- lings and from other sources, the result was exactly the opposite— viz., a decided production of alkali in milk and identity with the hog-cholera bacillus of Salmon." IN SUSCEPTIBLE ANIMALS. 441 Gelatin streak cultures. At the end of twenty-four hours this bacillus had developed considerably, while the growth of the hog- cholera bacillus was scarcely to be discerned with the naked eye, and the bacillus of Schweineseuche did not form a visible growth until the end of forty-eight hours. After several days the bacillus of swine plague (Marseilles) formed an opaque, yellowish-white streak, which, when examined with a low-power lens, had a brown color by transmitted light and a bluish-white color by reflected light. The streak of the Lomer-Schiitz bacillus was not so thick and not so opaque, and was made up of small, nearly transparent colonies ; the hog-cholera bacillus came between the other two. Upon blood serum, agar, and glycerin-agar the Marseilles bacillus grew more rapidly than the other two, forming a layer which was opaque and of a white color, with bluish and reddish reflections. Upon potato it formed a thick, opaque, yellowish layer, while the growth of the hog-cholera bacillus was much thinner and that of the Loffler-Schutz bacillus scarcely to be seen. In bouillon the Loffler-Schutz bacillus, at the end of three days at 37° C., had not produced any perceptible cloudiness, while the Marseilles bacillus at the end of twenty-four hours had caused the fluid to be clouded, a film of bacteria had formed upon the surface and a deposit at the bottom of the tube ; the hog-cholera bacillus produced a less degree of opacity in the bouillon. Pathogenesis. — This bacillus is pathogenic for sparrows and other small birds when injected beneath the skin in small amounts, and also for pigeons in a longer time — five to fourteen days. Frosch reports a negative result from subcutaneous injections into rabbits, guinea-pigs, mice, and pigeons, but his cultures appear to have be- come attenuated, as the recent cultures of Eberth and Schimmelbusch were fatal to pigeons in four out of five experiments. Two rabbits were inoculated subcutaneously by Rietsch and Jobert with half a Pravaz syringef ul of a pure culture of the Marseilles bacillus ; one of these died on the sixth day and the other survived. In sparrows, which succumb in from twenty-four to thirty-six hours after receiving a small amount of a pure culture in the breast muscle, the bacillus is present in the blood in large numbers, and a purulent pleuritis and pericarditis is found at the autopsy. In the ferrets from which Eberth and Schimmelbusch obtained their cultures the bacillus was not present in the blood in sufficient numbers to be readily demonstrated by microscopical examination, but it was ob- tained in pure cultures from the liver, spleen, and lungs. The prin- cipal pathological appearances noted were enlargement of the spleen and pneumonia. Caneva reports that the Marseilles bacillus injected into white mice gives rise to an extensive abscess at the point of in- oculation, but does not kill adult animals. In a young mouse which 31 442 BACILLI WHICH PRODUCE SEPTICAEMIA succumbed to such an injection the bacilli were not generally dis- tributed in the tissues, but were found as emboli in the smaller capil- laries. This bacillus, then, is distinguished from the similar bacilli previously described (Nbs. Gl and 63) by its comparatively slight pathogenic power, as well as by its more vigorous growth in culture media, and the other characters heretofore mentioned. 66. BACILLUS SEPTICUS AGRIGENUS. Obtained by Nicolaier from soil which had been manured. Moi°phology. —Resembles the bacillus of fowl cholera and of rabbit sep- ticaemia, of which it is perhaps a variety, but is usually somewhat longer. It also sometimes shows the end-staining characteristic of Bacillus septicse- miae haemorrhagicae, but not so constantly and not so sharply defined. Biological Characters.— An aerobic, (non liquefying ?), non- motile ba- cillus. Does not form spores. In gelatin plate cultures spherical, finely granular colonies are developed having a yellowish-brown central portion, which is separated by a dark ring from a grayish brown marginal zone; later this difference in color dis- appears and the colonies become more decidedly granular. In stick cultures tne growth consists of a thin layer which is not at all characteristic. Pathogenesis. — Small quantities of a pure culture injected into the ear vein of a rabbit cause its death in from twenty-four to thirty six hours; pathogenic also for house mice and for field mice. At the autopsy no notable pathological changes are observed. The bacilli are found in blood from the neart and in the capillaries of the various organs, but are not so numerous as in rabbit septicaemia; they show a special inclination to adhere to the margins of the red blood corpuscles. 67. BACILLUS ERYSIPELATOS SUIS. Synonyms. — Bacillus of hog erysipelas; Bacillus des Schweine- rothlauf (Loffler, Schiitz) ; Bacille du rouget du pore (Pasteur) ; Ba- cillus of mouse septicaemia; Bacillus murisepticus (Flugge) ; Bacil- lus des Mauseseptikamie (Koch). The bacillus of mouse septicaemia, first described by Koch (1878), resembles so closely in its morphology, characters of growth, and pathogenic power the bacillus of Schweinerothlauf of Loffler and Schiitz (1885) that they can scarcely be considered as distinct spe- cies, although, from slight differences which have been observed, they are perhaps entitled to separate consideration as varieties of the same species. Fliigge, Eisenberg, Frankel, and other authors, while recognizing the fact that the bacilli from the two sources closely re- semble each other, apparently do not consider them identical, and describe them separately. Baumgarten, on the other hand, describes them under one heading and considers it highly probable that they are identical, although he also admits slight differences in the morphological characters and growth in culture media. These differences are, however, no greater than we have in artificially pro- duced varieties of other well-known microorganisms, and we think IN SUSCEPTIBLE ANIMALS. 443 it best to follow Baumgarten in describing them under a single heading. Koch first obtained this bacillus by injecting putrefying blood or flesh infusion, during the first days of putrefactive change, beneath the skin of mice. A certain proportion of the animals experimented FIG. 135.— Bacillus of mouse septicaemia in leucocytes from blood of mouse, x 700. (Koch.) upon contracted a fatal form of septicaemia, and the bacillus under consideration was found in their blood. The bacillus of Schweine- rothlauf was obtained by Loffler and by Schiitz from the blood and various organs of swine which had succumbed to the infectious malady known in Garmany as rothlauf and in France as rouget. Morphology. — Extremely minute bacilli, about 1 /* in length and 0.2 /^ in diameter. The Schweinerothlauf bacilli are described as somewhat thicker and longer by Fliigge, by Frankel, and by Eisen- berg, but Baumgarten states that they are somewhat more slender and Fro. 136.— Bacillus of rouget, from a pure culture. X 1,000. From a photomicrograph. (Roux.) on the average shorter than the bacillus of mouse septicaemia. The bacilli are solitary, or in pairs the elements of which are often united at an angle; occasionally a chain of three or four elements may be observed, and in old cultures the bacilli may grow out into short 444 BACILLI WHICH PRODUCE SEPTICAEMIA threads which are straight or more or less curved and twisted. Small refractive bodies may sometimes be distinguished in the rods, and these have been supposed by some authors to be spores, but this has not been demonstrated. This bacillus stains readily with the ordi- nary aniline staining agents and also by Gram's method. Biological Characters. — A facultative an- aerobic, non-liquefying bacillus. According to Schottelius, the rothlauf bacilli are sometimes motile, but Flugge states that other observers have not seen them in active motion. Frankel says they have the power of voluntary motion. Eisenberg says that the bacillus of mouse septi- ca3ima is motionless, and Frankel says they " seem to be incapable of voluntary motion." Baumgar- ten remarks: "Whether the bacilli exhibit vol- untary movements has not been determined." Although this bacillus is not strictly anaerobic, it grows better in the absence of oxygen than in its presence. Development occurs in various cul- ture media at the room temperature, but is more rapid in the culture oven. In gelatin stick cul- tures no development occurs upon the surface, but the growth along the line of puncture is very characteristic; this consists of a delicate, cloud- like, radiating growth, which extends, in the course of a few days, almost to the walls of the test tube. The rothlauf bacillus does not extend so rapidly through the gelatin, and the branching, cloud-like growth is not as delicate; Flugge compares it to the brush of bristles used for cleansing test tubes. Fio. 187.— Bacillus of mouse septicaemia; culture in nutrient gela- tin, end of four days at 18° C. (Baumgarten.) Fio. 138.— Bacillus of mouse septicaemia; single colony in nutrient gelatin. X 80. (Flugge.) In old cultures in nutrient gelatin a slight softening of the gelatin occurs along the line of growth, and as a result of evaporation and desiccation a funnel-shaped cavity is formed in the culture medium in the course of two or-three weeks. In gelatin plates colonies are IN SUSCEPTIBLE ANIMALS. 445 developed in the course of two or three days in the deeper layers of the gelatin, but not upon the surface ; these are nebulous, grayish- blue, radiating masses, which are so delicate as to be scarcely visi- ble without the aid of a lens or a dark background. Under a low power they appear as branching feathery masses, which have been compared by Flugge to the radiating growth of "bone corpuscles," In older cultures they coalesce and cause a nebulous opacity of the whole plate, which has a bluish-gray lustre. Upon the surface of nutrient agar or blood serum a very scanty development occurs along the line of inoculation. No growth occurs upon potato. In bouillon the bacilli cause a slight cloudiness at the outset, and later a scanty, grayish- white deposit upon the bottom of the test tube ; no film is formed upon the surface. The thermal death-point of this bacillus, as determined by the writer (1887), is 58° C., the time of exposure being ten minutes. In the experiments of Bolton it was destroyed in two hours by mercuric chloride solution in the proportion of 1 : 10,000 ; by carbolic acid and by sulphate of copper in one-per-cent solution. These results are opposed to the view that the minute refractive granules which may sometimes be seen in the interior of the rods are reproductive spores, for all known spores have a much greater resisting power to heat and the chemical agents named. Pathogenesis. — Pathogenic for swine, rabbits, white mice, house mice, pigeons, and sparrows. Field mice, guinea-pigs, and chickens are immune. Swine may be infected by the ingestion of food containing the rothlauf bacillus, as has been demonstrated by allowing them to eat the intestine of an animal which had recently succumbed to the dis- ease, and also by the subcutaneous injection of pure cultures. The disease usually terminates fatally within three or four days, and sometimes in less than twenty-four hours. It is characterized by fever, debility, loss of appetite, and by the appearance upon the sur- face of the body of red patches, which gradually extend and become confluent, producing after a time a uniform dark-red or brown color of the entire surface. The discharges from the bowels frequently contain bloody mucus. At the autopsy, in acute cases, the spleen is notably enlarged, and the liver and kidneys are likely to be more or less swollen, as are also the lymphatic glands, especially those of the mesentery; the gastric and intestinal mucous membranes are usually inflamed and spotted with hsemorrhagic extravasations ; the serous membranes also may be inflamed, and the cavities of the pleura3, pericardium, and peritoneum usually contain more or less fluid. The bacilli are found in the blood vessels throughout the 446 BACILLI WHICH PRODUCE SEPTICAEMIA body and are especially numerous in the interior of the leucocytes. Cornevin and Kitt have shown that the contents of the intestine also contain the bacilli in large numbers, and the disease appears to be propagated among swine principally by the contamination of their food with the alvine discharges of diseased animals. Pigeons are very susceptible to the pathogenic action of this ba- cillus, and usually die within three or four days after inoculation with a pure culture. Rabbits are not so susceptible, although a certain proportion die from general infection after being inoculated in the ear. The first effect of such an inoculation is to produce an erysipelatous inflammation. When the animal recovers it is subse- quently immune. White mice and house mice are extremely susceptible, but field Fro. 189.— Section of dlaphrasrm of a mouse dead from mouse septicaemia, showing bacilli in a capillary blood vessel. (Baumgarten.) mice are immune. This remarkable fact was first ascertained by Koch by experiments with his bacillus of mouse septicaemia. House mice which have been inoculated with a minute quantity of a pure culture of the rothlauf , or mouse septicaemia, bacillus, die in from forty to sixty hours. The animal is usually found dead in a sitting position, with its back strongly curved, and for many hours before death it remains quietly sitting in the same position ; the eyes are glued together by a sticky secretion from the conjunctival mucous membrane. At the autopsy the spleen is found to be very much en- larged, and there may be a slight amount of oedema at the point of inoculation. The bacilli are found in the blood vessels generally, and are very IN SUSCEPTIBLE ANIMALS. 447 numerous in the interior of the leucocytes, which are sometimes com- pletely filled with them. Pasteur's first studies relating to the etiology of "rouget" were made, in collaboration with Chamberlain, Roux, and Thuillier, in 1882. His description of the microorganism to which he attributed the disease does not correspond with that subsequently isolated by Loffler an I by Schiitz ; but the last-named bacteriologists, and Schot- telius also, found the characteristic rothlauf bacillus in cultures from his laboratory which had baan prepared for the protective inoculation of swine — " vaccins." Pasteur found, by experimental inoculations of his bacillus of rouget into pigeons, that the virulence of his cul- tures was increased by successive inoculations through a series of these birds, as shown by the oocurrenca of daath at an earlier date, and also by the fact that blood taken from the last pigeon in a series was more virulent for swine than that from the first or from an in- fected pig. On the other hand, the virulence was diminished by in- oculations into rabbits ; and, by passing the bacillus through a series of these animals, a vaccine was obtained which produced a com- paratively mild and non-fatal attack in swine. In practice the U33 of two different vaccines is recommended, a mild — " attenuated " —virus being first inoculated, and, after an interval of twelve days, a second vaccine having greater pathogenic potency. These inocula- tions have been extensively practised in France, and that immunity from the disease may be secured in this way is well established, hav- ing been confirmed in Germany by Schiitz, by Lydtin, and by Schot- telius. There is, however, some doubt as to the practical value of the method, inasmuch as a certain number of the inoculated animals die, and there appears to be danger that the disease may be spread by the alvine discharges of inoculated animals. In a region where the annual losses from tho disease are considerable, and where the soil is, perhaps, thoroughly infected with rothlauf bacilli, protective inoculations probably afford the best security against loss. But under other circumstances the quarantine of infected animals and thorough disinfection of the localities in which cases have occurred will probably prove a better mode of procedure. 68. BACILLUS COPROGENES PAKVUS. Synonym.— Mauseseptikamieahnlicher Bacillus (Eisenberg). Obtained by Bienstock from human faeces. Morphology. — A very minute bacillus, which is but little longer than it is broad, and might easily be mistaken for a raicrococcus. Biological Characters.— Grows very slowly on nutrient gelatin, forming a scarcely visible film along the line of inoculation, which at the end of several weeks is scarcely one millimetre wide. Is not motile. Pathogenesis. — In white mice an extensive O3dema is developed at the 448 BACILLI WHICH PRODUCE SEPTICAEMIA. point of inoculation at the end of ten or twelve hours, and the animal dies within thirty-six hours. The bacilli are found in great numbers in the effused serum at the point of inoculation and in comparatively small num- bers in the blood. A rabbit inoculated with a pure culture obtained from a mouse died at the end of eight days. The inoculation, which was made in the ear, gave rise to a local erysipelatous inflammation. 69. BACILLUS CAVICIDA. Synonym.— Brieger's bacillus. Probably a pathogenic variety of Bac- terium coli commune of Escherich. Obtained by Brieger (1884) from human fasces. Morphology — Small bacilli, about twice as long as broad, which closely resemble the colon bacillus of Escherich (Bacterium coli commune). Biological Characters. — An aerobic (facultative anaerobic), non-liquefy- ing bacillus. The growth in gelatin plate cultures is said to be very characteristic, the colonies being " in the form of very beautifully grouped, whitish, concentric rings, which are arranged like the ccales upon the back of a turtle" (Eisen- bergi. The writer has studied cultures of this bacillus brought from the bacteriological laboratories of Germany, side by side with cultures of the Bacterium coli commune of Escherich, and has found no appreciable differ- ences in the colonies in gelatin plates, or in the growth in various culture media. Upon potato it grows rapidly in the incubating oven, forming a dirty-yellow, moist layer. Pathogenesis. — This bacillus, as first obtained by Brieger, was character- ized by being very pathogenic for guinea-pigs, which were invariably killed, within seventy-two hours, by the subcutaneous injection of a minute quan- tity of a pure culture. The bacillus was found in great numbers in the blood of animals which succumbed to an experimental inoculation. The writer's experiments with this bacillus, made in 1889, indicate that its patho- genic power had become attenuated, inasmuch as considerable quantities of a pure culture injected into guinea-pigs did not cause the death of the ani- mals— culture used came originally from Germany. Not pathogenic for rabbits or for mice. 70. BACILLUS CAVICIPA HAVANIENSIS. This bacillus was obtained by the writer from the contents of the intestine of a yellow-fever cadaver, in Havana, 1889, through inoculated guinea-pigs. Morphology. — A bacillus with rounded ends, from two to three p long and about 0.7 // broad, frequently united in pairs. Stains readily with the ordinary aniline colors. Biological Characters. — An aerobic and fac- ultative anaerobic, non -liquefying, actively mo- tile bacillus. In gelatin stick cultures the growth upon the surface is very scanty and thin, not extending far from the point of puncture ; along the line of puncture are developed small, translucent, pearl- like, spherical colonies, which later become opaque and sometimes granular. In gelatin roll tubes, Fio. i40.-Baciiius cavicida at the end of twenty-four hours at 22° C., Havaniensis; from a potato the deep colonies are very small spheres, of a pale culture, x 1,000. Fromapho- straw color ; later they become opaque, light brown tomicrograph. (Sternberg.) spheres, or may have a dark central mass sur- rounded by a transparent zone. The superficial colonies at the end of five days are small, translucent masses of a pale straw color towards the centre, with thin and irregular margins, sometimes with IN SUSCEPTIBLE ANIMALS. 449 a central light-brown nucleus ; at the end of ten days the deep colonies are still quite small, of a brown color, and opaque. In glycerin-agar roll tubes, at the end of twenty-four hours, the deep colo- nies are in the form of a Biconvex lens, and appear spherical when viewed in face and biconvex when seen from the side ; they have a straw color by transmitted light and are bluish- white by reflected light ; the superficial colonies are translucent, with a bluish white lustre, On potato, at 22° C., at the end of forty eight hours there is a thin, dirty- yellow growth of limited extent; at the end of ten days there is a thin, gamboge yellow layer and little masses of the same color; the growth is quite thin, with irregular outlines, and is confined to the vicinity of the impfstrich. \ Grows in nutrient agar containing 0.2 per cent of hydrochloric acid. Thermal death point 55° C. Grows in agua coco without forming gas, and causes this liquid and bouillon to become slightly translucent— not milky. Pathogenesis. — Pathogenic for guinea-pigs, less so for rabbits. Guinea- pigs inoculated subcutaneously with a few drops of a pure culture die in ten or twelve hours from general infection. There is usually a considerable effusion of bloody serum in the vicinity of the point of inoculation, and the spleen is more or less enlarged. 71. BACILLUS CRASSUS SPUTIGENUS, Obtained by Kreibohm (1886) from the sputum of two individuals, and once in scrapings from the tongue. Morphology. — Short, thick bacilli, of oblong form, with rounded corners, en bent or twisted — "sausage-shaped." Immediately after division the bacilli are about one-half longer than they are broad, but before dividing FIG. 141.— Bacillus crassus sputigenus, from blood of mouse, x 700. (Flug^e.) again they may attain a length of three to four times the breadth. Irregular forms with swollen ends or uneven contour are frequently seen. This bacillus is quickly stained by the ordinary aniline colors and also by Gram's method. Biological Characters.— An aerobic, non-liquefying (non-motile ?) ba- cillus. Grows in various culture media at the room temperature— more rapidly in the incubating oven. "Appears to form spores at 65 U. (Fliigge). In gelatin plates, at the end of thirty-six hours, grayish- white colonies are developed, which soon reach the surface of the gelatin and spread out as 450 BACILLI WHICH PRODUCE SEPTICAEMIA round, viscid, grayish- white drops, which project considerably above the surface of the culture medium. Under a low magnifying power recent colo- nies appear as spherical, grayish -brown discs, the surface of which is marked with dark points or lines. The superficial colonies are more transparent, have irregular outlines, and the surface, especially near the margins, is coarsely granular. The development in stick cultures is very rapid and re- sembles that of Friedlander's bacillus — " nail-shaped " growth. Upon potato the growth is also similar to that of Friedlander's bacillus, and consists of a thick, grayish-white, moist, and shining layer. Pathogenesis.—yLice inoculated with a small quantity of a pure culture die from acute septicaemia in about forty-ei^ht hours. The bacilli are found in blood from the heart and from the various organs — most numerous in the liver. Rabbits are killed within forty eight hours by intravenous injec- tion of a small quantity, and the blood contains the bacillus in great num- bers. Larger amounts injected into the circulation of rabbits or dogs cause death in a few hours (three to ten), preceded by diarrhoea, and in some in- stances bloody discharges from the bowels. At the autopsy an acute gastro- enteritis is found. 72. BACILLUS PYOGENES FCETIDUS. Obtained by Passet (1885) from an abscess of the anus. Morphology.— Short bacilli with rounded ends, 1.45 jn long and 0.58 n broad ; usually associated in pairs or in short chains. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Grows rapidly in the usual culture media at the room temperature. In the interior of the rods, in stained preparations, one or two unstained, spherical places may sometimes be seen, which have been supposed to be spores (?). The independent motion exhibited by this bacillus is not very active. In gelatin plates white colonies are developed at the end of twenty-four hours, which upon the surface spread out as grayish-white plaques, having a dia- meter sometimes of one centimetre ; these are thickest in the centre and of a whitish color ; the colonies may become confluent. In gelatin stick cul- tures the growth upon the surface, at the end of twenty-four hours, consists of a thin, grayish- white layer with rather thick, irregular margins; along the line of puncture more or less crowded colonies. Upon potato >the bacillus forms an abundant, shining, pale-brown layer. The cultures give off a dis- agreeable putrefactive odor. According to Eisenberg, mice and guinea-pigs are killed in twenty-four hours by injections beneath the skin or into the cavity of the abdomen, and numerous bacilli are found in the blood. 73. PROTEUS HOMINIS CAPSULATUS. Obtained by Bordoni-Uffreduzzi (1887) from two cadavers presenting the pathological appearances of the so-called " Hadernkrankheit." Morphology. — Bacilli, varying considerably in dimensions; somewhat thicker than the anthrax bacillus; often swollen in the middle or at the ex- tremities: more or less curved; isolated, united in pairs or in long filaments; in stained preparations from agar cultures or from blood the bacilli are sur- rounded by a "capsule." Stains with the usual aniline colors and also by Gram's method. Biological Characters. — An aerobic (facultative anaerobic ?), non-lique- jying* non-motile bacillus. Formation of spores not observed. Grows in the usual culture media at the room temperature. At a temperature of 15° to 17° C. long filaments are formed, in which the bacilli are surrounded with a rapsulo; at 22 to 24 " C. the bacilli are for the most part isolated, but few fila- ments being formed ; at 32° to 37° C. the bacilli are so short as to resemble micrococci; development ceases at a temperature of 8° and is very slow at 16 C. IN SUSCEPTIBLE ANIMALS. 451 Fro. 148,— Proteus hominis capsulatus, from liver of mouse. X 1,000. (Bordoni.Uffre- duzzi.) This bacillus grows as well m an acid medium as in one which is slightly alkaline. In gelatin plates, at the end of eighteen to twenty-four hour/ colonies are formed which under a low power are seen to be spherical and to contain a quantity of shining granules; the following day, at a tempera- ture ot 15 to 17 C., the colonies may be as large as a phi's head and still remain spnerical or slightly oval, but the outline is no longer so uniform, and between the shining points in the interior a confused network may be seen ; as the colony becomes larger it is raised above the surface of the gela- tin, becomes opaque, and has a pearly lustre like that of Friedlander's bacil- lus. In gelatin stick cultures the growth resembles that of Friedlan- der's bacillus — " nail-shaped growth." Upon the surface of nutrient agar a rapidly extending, semi transparent layer is formed. Upon potato, at 15° to 17° C., at the end of twenty-four hours transparent drops are seen in the vicinity of the point of inocula- tion, and later a moist, shining, color- less layer, of tough consistence, is formed, which gradually extends over the surface. The growth upon blood serum resembles that upon nutrient agar, and the blood serum is not liquefied'. In liquid blood serum or in bouillon the bacilli are isolated — not in filaments ; they cause a clouding of the liquid, and an abundant deposit accumulates at the bottom of the tube, while a film of bacilli forms upon the surface. The cultures never give off a putrefactive odor. Pathogenesis.— Pathogenic for dogs and for mice, less so for rabbits and for guinea-pigs. Agar cultures grown in the incubating oven at 32° to 37 C. are more pathogenic than cultures in gelatin at the room temperature. A small quantity of a recent culture injected subcutaneously in mice causes their death in from one to four days, according to the quantity and age of the culture; the recent cultures are most virulent. When the animal lives more than twenty -four hours it has a mucous diarrhoea. At the autopsy the spleen is found to be much enlarged and dark in color ; the lymphatic glands are also swollen and haemorrhagic, the liver and kidneys hyperaemic; in the vicinity of the point of inoculation is a subcutaneous cedema of jelly- like appearance and numerous punctiform haemorrhages are seen. The ba- cillus is found in great numbers in the effused serum from the subcutaneous tissues, in the blood, the contents of the intestine, and in the parenchyma of the various organs. When examined at once the bacilli in the subcutaneous oedema and in the lymphatic glands are usually quite short, and even spherical, while in the blood they are somewhat longer and may appear as short fila- ments with swollen ends, surrounded by a capsule. When the examination is made some time after the death of the animal longer filaments are quite numerous. Rabbits and guinea-pigs are killed by the intravenous injection of comparatively small amounts of a recent culture, but quite large doses are required to produce a fatal result when the injection is made beneath the skin. From two to three cubic centimetres of a recent culture injected into the circulation of a dog give rise to symptoms of toxaemia, and the ani- mal usually dies on the second day. At the autopsy the abdominal organs are found to be hyperaemic, the mucous membrane of the intestine swollen, red in color, and covered with bloody mucus. The bacillus is found in the blood and in the various organs. When smaller doses are injected into a vein (a few drops) the animal, after a few hours, has a mucous diarrhoea and 452 BACILLI WHICH PRODUCE SEPTICAEMIA vomiting, or efforts to vomit. Death usually occurs at the end of two or three days. At the autopsy the spleen is found to be normal, the other or- gans slightly hyperaemic, and the intestinal mucous membrane in a state of catarrhal inflammation. The bacilli are found in the blood and in the vari- ous organs in considerable numbers. 74. PROTEUS CAPSULATUS SEPTICUS. Obtained by Banti (1888) from a case of " acute hsemorrhagic infection/' According to Banti, this is possibly identical with the preceding species- Proteus hominis capsulatus — but in some respects more nearly resembles Friedlander's bacillus. 75. BACILLUS ENTERITIDIS. Obtained by Gartner (1888) from the tissues of a cow which was killed in consequence of an attack charactei ized by a mucous diarrhoea, and also from the spleen of a man who died twelve hours after eating the flesh of this animal. Morphology. — Short bacilli, about twice as long as broad, frequently united in pairs; chains of four to six elements are sometimes seen. Stains with the usual aniline colors, and presents the peculiarity of staining deeply at one end while the remainder of the rod is but slightly stained. When two bacilli are united the deeply stained ends are in apposi- tion. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Spore formation not determined. Grows in the usual culture media at the room temperature. Upon gelatin plates pale-gray, superficial colonies are formed at the end of twenty-four hours; under a low power these are seen to be coarsely granular and transparent; the central portion usually pre- sents a greenish color; deep colonies are spherical, indistinctly granular, and of a brownish color ; in older colonies a marginal transparent zone is seen which appears to be made up of minute fragments of glass of a pale- brown color. In gelatin stick cultures but slight development occurs along the line of puncture ; upon the surface a thick, grayish-white layer is formed, which after a time becomes very much wrinkled. Upon the surface of agar, at 37° C., at the end of eighteen to twenty 'hours a grayish- yellow layer has formed. Upon potato a moist, shining, yellowish-gray layer is developed. The growth upon blood serum is rapid in the form of a gray layer along the line of inoculation. Pathogenesis. — White mice and house mice usually die in from one to three days when fed with a pure culture of this bacillus. Rabbits and gui- nea-pigs die in from two to five days from subcutaneous injections— less pathogenic for pigeons and canary birds. Dogs, cats, chickens, and sparrows are immune. A. goat died in twenty hours after receiving an intravenous injection of two cubic centimetres or a culture in blood serum. The princi- pal pathological appearance consists in an intense inflammation of the in- testinal mucous membrane. The bacilli are found in blood from the heart and also in the contents of the stomach. 76. BACILLUS OF GROUSE DISEASE. Obtained by Klein (1889) from the lungs and liver of grouse which had succumbed to an epidemic disease. Morphology.— Bacilli with rounded ends, from 0.8 to 1.6 //long; may also be seen as spherical or oval cells 0.6 // long and 0.4 /* thick; solitary, in pairs, or in chains of three to four elements. Stains best with Weigert's solution of methylene blue in aniline water. Biological Characters — An aerobic, non-liquefying, non-motile bacillus. Spore formation not observed. Grows in the usual culture media at the IN SUSCEPTIBLE ANIMALS. 453 room temperature — better in the incubating oven. Upon gelatin plates, at 20° C., at the end of twenty-four hours small, angular, transparent scales may be seen upon the surface with a low- power lens; at the end of three or four days these form flat, more or less irregular, shining, gray colonies, with thin and of ten dentate margins ; these colonies may become confluent and form a dry, scaly layer which by reflected light has a peculiar, fatty lustre In gelatin stick cultures the superficial growth is in the form of a trans- parent, dry, grayish layer with dentate margins, not more than three to five millimetres in diameter. Upon agar, at 36° to 37° C., a thin, whitish-gray, dry layer is formed. Pathogenesis. — Pathogenic for mice, for guinea-pigs, for linnets, and for green-finches; less so for sparrows. Chickens, pigeons, and rabbits, accord- ing to Klein, are immune. Of eight mice inoculated subcutaneously with one or two drops of a bouillon culture, six died within forty- eight hours and two recovered. Out of eight guinea-pigs inoculated in the same way four died in forty-eight hours and two recovered. At the autopsy the lungs and liver were found to be hyperaemic, the spleen not enlarged. The bacilli were present in large numbers in blood from the heart and in the lungs. 77. BACILLUS GALLINARUM. Obtained by Klein (1889) from the blood of chickens which succumbed to an epidemic disease resembling u fowl cholera." The bacillus is believed by Klein not to be identical with Pasteur's bacillus of fowl cholera, and is said not to be pathogenic for rabbits, which would seem to differentiate it from this bacillus ( Bacillus septicaemias haemorrhagicae). Morphology. — Bacilli with rounded ends, from 0.8 to 2 n long and 0.3 to 0.4 u thick ; often in pairs. Stains with the usual aniline colors. Biological Characters. — An aerobic, non-liquefying, non-motile bacillus. Does not form spores. Grows in the usual culture media at the room tem- perature—better in the incubating oven. Upon gelatin plates forms grayish- white, superficial colonies, which later present the appearance of flat, homo- geneous, whitish discs with thin edges and irregular margins, and by transmitted light have a brownish color. The deep colonies are small and spherical, and have a brownish color by transmitted light. In gelatin stick cultures a thin, gray layer with irregular margins and of limited extent forms upon the surface, and a scanty growth occurs along the line of punc- ture in the form of a grayish- white line. Upon the surface of agar, at 37° C., a thin, gray layer with irregular margins has developed at the end of twenty-four hours ; later this extends over the entire surface as a thin, gray- ish-white layer. No growth occurs upon potato at 37° C. In bouillon, at 37° C., development occurs, with clouding of the bouillon, within twenty -four hours; later a deposit consisting of bacilli is seen at the bottom of the tube, but no film forms upon the surface. Pathogenesis. —Chickens inoculated subcutaneously with a pure culture die in from twenty-four hours to eight or nine days. Pigeons and rabbits are immune. 78. BACILLUS SMARAGDINUS FCETIDUS. Obtained by Reimann (1887) from the nasal secretions in a case of ozaena. Morphology. — Small, slender, slightly curved bacilli, about half as large as the tubercle bacilli; usually arranged in parallel groups. Biological Characters.— Anaerobic and facultative anaerobic, liquefying bacillus. Spore formation not observed. Grows slowly at the room tem- perature in the usual culture media— more rapidly at 37° C. In gelatin stick cultures development occurs along tha line of puncture, and at the end of forty-eight hours a slight liquefaction, in form of a funnel, occurs near the 454 BACILLI WHICH PRODUCE SEPTICAEMIA surface ; after the eighth day liquefaction progresses more rapidly. About the sixth day a bright-green color is recognized in the upper part of the tube by reflected light. Upon agar plates, at 37° C., at the end of forty-eight hours minute colonies are formed, of irregular form, which have a white color with a shade of green ; in older colonies the central portion may be finely granular and brownish yellow in color, while the marginal zone is more transparent; the agar has by reflected light a deep emerald green color. In agar stick cidtures, at the end of twenty hours, an abundant development has occurred without color; at the end of forty-eight hours the culture me- dium is of a bright green color throughout ; later the color changes to brown. A dirty-yellow layer forms upon the surface of the agar. Upon potato, at 37° C., a dark- brown layer forms in the vicinity of the line of inoculation; later this is chocolate brown. The cultures in gelatin and agar give off a peculiar, penetrating odor similar to that of jasmin. Pathogenesis. — Pathogenic for rabbits when injected into a vein or sub- cutaneously. Death occurs in from thirty six to forty- eight hours. At the autopsy haemorrhagic extravasations are found beneath the pericardium and the pleurae; abscesses in the lungs and liver. The bacilli are found in the blood and in the various organs in large numbers. 79. BACILLUS PNEUMOSEPTICUS. Obtained by Babes (1889) from the blood and tissues of an individual who died of septic pneumonia. Morphology. — Small, straight bacilli about 0.2 // thick. Stains with the usual aniline colors, but not by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non-lique- fying, non-motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates superfi- cial colonies are formed which are flat, irregular in outline, whitish, shining, and semi transparent ; under a low power finger like offshoots are seen about the periphery. In gelatin stick cultures an abundant development occurs along the line of puncture; the colonies give off a strong sperm-like odor. Upon the surface of agar small, whitish, flat, shining colonies with ill de- fined outlines are formed, which soon become confluent arid cover the sur- face; an abundant white deposit is seen in the condensation water. Upon potato a moist, white layer is formed. Upon blood serum circular, whitish, transparent colonies are formed along the line of inoculation, which soon coalesce. Pathogenesis. — Very pathogenic for rabbits, guinea-pigs, and mice when injected subcutaneously in small amount. The animals die in from two to three days without any noticeable local inflammation and with symptoms of septicaemia. The lungs and spleen are found to be hyperaemic. The bacilli are found in the blood free, or sometimes enclosed in the leucocytes; they are only found in small numbers in the capillaries of the internal organs. Cultures gradually lose their virulence when propagated in artificial media. 80. BACILLUS CAPSULATU8. Obtained by Pfeiffer (1889) from the blood of a guinea pig which died spontaneously. Morphology.— Thick bacilli with rounded ends, usually two or three times as long as broad; often united in chains of two or three elements; may prow out into homogeneous filaments. Stained preparations show the ba- cilli to be enveloped in an oval capsule which may be considerably broader than the bacilli themselves— two to five times as broad ; where several ba- cilli are united they are surrounded by a single capsular envelope. Stains with the usual aniline colors, but not by Gram's method. In pre- parations which are deeply stained with hot f uchsin or gentian violet solu- IN SUSCEPTIBLE ANIMALS. 455 tion the capsule is so deeply stained that the bacillus is hidden; by careful treatment with a weak solution of acetic acid the capsule may be differen- tiated as a pale-red or violet envelope surrounding the deeply stained bacilli. Biotogical Characters. — An aer- obic and facultative anaerobic, non liquefying, non motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. The cul- tures in agar or upon potato are very viscid and draw out into long threads when touched with the pla- tinum needle; the blood of an ani- mal killed by inoculation with this bacillus has the same viscid charac- ter. Upon gelatin plates minute colonies are first visible at the end of twenty four to thirty-six hours; later the deep colonies are white, oval masses the size of a pin's head ; the superficial colonies attain the size of a lentil, and are flattened, hemispherical masses with a porce- lain white color. In gelatin stick Fia 143-~ Bacillus capsulatus, from peritoneal Cultures growth occurs to the bot- exudate of an inoculated guinea-pig, x 1,000. torn of the line of puncture, and on ^°* a photomicrograph. (Ffeiffer.) the surface a shining white, circular, arched mass forms around the point of puncture, resembling the growth of Friedlander's bacillus. Upon the surface of agar, at 37° C , at the end of twenty-four hours a thick, soft layer of a pure white color is formed, which is very viscid and resembles the growth of Micrococcus tetragenus upon the same medium. Upon potato an abundant and viscid, shining, yellowish- white layer is quickly developed. Pathogenesis. — Pathogenic for white mice and for house mice, which die at the end of two or three days after being inoculated at the root of the tail with a small quantity of a pure culture. Inoculation from mouse to mouse increases the virulence of the cultures. At the autopsy the superficial veins are distended with blood, the inguinal glands enlarged, the spleen consid- erably enlarged, the liver and kidneys hyperaemic, the intestine pale, the heart distended with blood, which usually is very viscid and is drawn out into threads when touched with the platinum needle. The bacilli are found in the blood and in all of the organs, in the contents of the peritoneum and pleurae, and in the exudate in the vicinity of the point of inoculation. Pathogenic also for guinea pigs and for pigeons; guinea-pigs are infallibly killed within thirty-six hours by the injection of a single drop of a bouillon culture, twenty-four hours old, into the cavity of the abdomen; the blood contains the bacillus in enormous numbers, as does the viscid fluid found in the peritoneal cavity. Rabbits do not succumb to intraperitoneal or subcu- taneous inoculations, but are killed by the intravenous injection of one cubic centimetre of a recent bouillon culture. Putrefactive changes occur very quickly in animals killed by inoculation with this bacillus. 81. BACILLUS HYDROPHILTJS FUSCUS. Obtained by Sanarelli (1891) from the lymph of frogs suffering from a fatal infectious disease. Morphology. — Bacilli with rounded ends, usually from 1 to 3 H in length; often short oval; may grow out into filaments of 12 to 20 jit in length. Biological Characters. — An aerobic, liquefying, motile bacillus. Grows in the usual culture media at the room temperature. In gelatin stick cul- 456 BACILLI WHICH PRODUCE SEPTICAEMIA u, and often appear to be surrounded by a capsule. In fresh cultures the bacilli are often in form of IN SUSCEPTIBLE ANIMALS. 459 a figure 8, and are only stained at the point of contact of the two segments. In potato cultures they are sometimes elongated and swollen at one ex- tremity. Stains with the usual aniline colors and by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non-lique- fying, actively motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature — more rapidly at 37° C. In gelatin stick cultures yellowish- white colonies are developed along the line of puncture ; at the bottom these may have a diameter of one to two millime- tres, and they have a brown color. Upon the surface an irregular, lobulated, whitish, translucent, paraffin-like layer is developed. At the end of eight days the surface growth consists of large, confluent, transparent plaques, with irregular outlines and crenated, elevated margins ; along the line of puncture large, separate, lenticular or spherical colonies are seen ; these have a brownish-white color. At the end of two months the surface growth is concentric and still more transparent, while the colonies near the surface have become almost brown. Upon the surface of agar, at 37° C., a narrow band is developed along the line of inoculation ; above, this is composed of transparent, shining, flat, round colonies having a diameter of one milli- metre or more ; below, the colonies are confluent and form a transparent, whitish layer. In glycerin-agar development is still more abundant, and may already be perceived at the end of twelve hours. Crystals are seen below the surface in agar cultures and about the superficial colonies in gela- tin. Upon potato a uniform, thin, grayish, verv transparent layer is de- veloped, which sometimes has a brownish-gray tint. At the end of a few days the potato acquires a brownish color. In bouillon cloudiness of the medium is apparent at the end of ten hours ; twenty-four hours later a whitish precipitate is seen at the bottom of the tube, which is more abun- dant when the culture medium contains glucose; later a thin pellicle is seen upon the surface and the bouillon acquires a yellowish color. Pathogenesis. — Recent cultures are pathogenic for rabbits, guinea-pigs, pigeons, and mice, which die from general infection in from two to four days. Old cultures are less virulent. 87. BACILLUS OF LUCET. Obtained by Lucet (1891) from chickens and turkeys suffering from an infectious form of septicaemia characterized by dysenteric discharges — " Dy- senterie epizootique des poules et des dindes." Resembles Bacillus gallinarum of Klein, and is perhaps identical with this microorganism. Morphology.— Short bacilli, from 1.2 to 1.8 /* long, usually in pairs. Stains with the usual aniline colors, but not by Gram's method. Biological Characters.— An aerobic and facultative anaerobic, non-lique- fying, non-motilebaicillus. Spore formation not observed. Grows slowly in the usual culture media at the room temperature — more rapidly at 37° C. In gelatin plates small, shining, moist, white, circular colonies are devel- oped, which look like little drops of wax ; later these increase in size, and especially in thickness, forming hemispherical masses. In gelatin stick cul- tures grayish, punctiform colonies are developed along the line of puncture, and upon the surface a circular, prominent, whitish plaque. Streak cultures upon the surface of gelatin are in the form of a dirty-white or grayish- white, moist streak, with regular margins, limited to the line of inoculation, but increasing in thickness until it breaks loose and slips down the oblique sur- face of the culture medium. The deposit which collects in this way acquires, as it becomes old, in the deepest portion a reddish color. Upon agar it forms a thick, yellowish- white, mucus-like layer with straight or slightly dentate margins. In bouillon it produces a decided clouding of the liquid, and an abundant grayish, pulverulent sediment accumulates at the bottom of the tube ; the bouillon after a time becomes transparent above this sediment and 460 BACILLI WHICH PRODUCE SEPTICAEMIA. is viscid, drawing out into threads. In the absence of oxygen the characters of growth are the same as in its presence. The cultures acquire an alkaline reaction ; they are sterilized by exposure for ten minutes to a temperature of 60° C. Does not grow upon potato. Pathogenesis. — Pathogenic for chickens and turkeys. Not pathogenic for pigeons, guinea-pigs, or rabbits when injected subcutaneously or into the peritoneal cavity, but kills rabbits when injected into a vein. In the in- fected fowls the bacilli are found in small numbers in the blood, more nu- merous in the kidneys and liver, still more numerous in the spleen, and in enormous numbers in the intestinal mucus, where in acute cases it is found almost in a pure culture. Fowls do not contract the disease as a result of the ingestion of grains soiled with cultures of the bacillus, but become in- fected when fed with animal food to which a pure culture has been added. 88. CAPSULE BACILLUS OF LOEB. Obtained from a case of keratomalacia infantum by inoculating culture media with a little of the softened exudate in the cornea. Morphology. — Resembles Bacillus capsulatus of Pfeiffer, but this is said to be somewhat larger and thicker. In the blood of mice, however, both bacilli vary considerably in size, and according to Loeb it was not possible to determine with certainty that one bacillus was, on the average, larger than the other. In staining reactions, also, no difference was observed — both bacilli stain with the usual aniline colors, and under certain circumstances the centre of the rods is less deeply stained than the extremities. Biological Characters. — An aerobic and facultative anaerobic, non-lique- fying, non-motile bacillus. Grows in the usual culture media at the room temperature. In its growth in culture media it closely resembles Bacillus capsulatus of Pfeiffer (No. 80). Pathogenesis. — Pathogenic for mice and for guinea-pigs, but not for rab- bits and pigeons ; Pf eiffer's bacillus is pathogenic for these animals. PLATE VII. BACILLUS OP GLANDERS. IG. 1. — Bacillus mallei from the liver of a field mouse, cover -glass pre- paration. (Loffler.) FIG. 2.— Bacillus mallei from a recent culture upon blood serum- (Lof fler.) FIG. 3. — Bacillus mallei in section of spleen of a field mouse dead from glanders. (Loffler.) FlG 4.— Culture of glanders bacillus upon cooked potato. (Loftier.) r....,.,, i. :..i. VMKi?(Vs IJACTKKIOLOCVY. Plate '% Fig.l. Fig. 2. X " • 4 ^ H ACII.LITS OF GLAND EKS (I.OKFFLER) XIII. PATHOGENIC AEROBIC BACILLI NOT DESCRIBED IN PREVIOUS SECTIONS. A CONSIDERABLE number of saprophytic bacilli are pathogenic for small animals when injected into the circulation, or subcutaneously, or into a serous cavity in considerable quantity — one to five cubic centimetres or more — but fail to produce any appreciable effect when introduced into the bodies of these animals in minute doses, and do not multiply in the blood to any considerable extent, al- though in fatal cases they may usually be recovered in cultures from the blood and tissues. These bacilli are pathogenic by reason of the toxic ptomaines produced by them, or because of local inflammatory processes which they induce, or for both of these reasons combined. Some of them may also, under certain circumstances, multiply in the blood and thus give rise to septicaemia as well as to toxaemia ; this is the case, for example, with the " colon bacillus " of Escher- ich. When injected in considerable quantity into the circulation of a guinea-pig it causes the death of the animal within twenty-four hours, and the bacillus is found in the blood in great numbers ; but minute amounts injected into a vein, or larger amounts injected subcutaneousiy, do not usually produce general infection. It is, therefore, not included among the "bacilli which produce septi- caemia in susceptible animals/' There is reason to believe, however, that under certain circumstances this bacillus may have sufficient pathogenic potency to produce a genuine septicaemia in guinea-pigs. Thus the original cultures of Brieger's bacillus, which appears to be a variety or the colon bacillus, are reported to have produced fatal septicaemia in guinea-pigs when injected subcutaneousiy in small amounts. A strict division into pathogenic bacilli which produce general blood infection — septicaemia — and those which produce a fatal result owing to the production of toxic chemical substances is not possible; for many pathogenic bacteria produce general infection when injected in comparatively large doses, and at the same time give rise to symptoms of toxaemia ; or general infection may occur in animals of one species, and fatal toxaemia without septicaemia in 462 PATHOGENIC AEROBIC BACILLI those of another species. Many of the bacilli described in the pre- sent section are common saprophytes, which have been shown by laboratory experiments to be pathogenic for certain animals when introduced into their bodies in a certain amount, which differs greatly for different bacteria and for different species of animals. The ex- periments of Cheyne and others show how largely the pathogenic power of saprophytic bacteria depends upon the quantity of a cul- ture which is injected, as well as upon the age of the culture and the seat of the inoculation — in the blood, the abdominal cavity, the subcutaneous tissues, or the muscles. And the bacteriologist named has also shown that pathogenic power depends, in some instances at least, upon the combined action of the toxic substances introduced in the first instance and of the living bacteria. Thus Cheyne found that one-tenth of a cubic centimetre of a bouillon culture of Proteus vulgaris injected into the dorsal muscles of a rabbit infallibly caused its death within forty-eight hours, but when the dose was reduced to one-fortieth cubic centimetre the animal recovered. But if to this amount (one-fortieth cubic centimetre) he added one cubic cen- timetre of a sterilized (by heat) culture of the same bacillus instead of diluting with distilled water, and injected the mixture into the dorsal muscles of a rabbit, death occurred in every experiment within forty-eight hours. The sterilized culture injected by itself produced no effect in this dose (one cubic centimetre), and Cheyne believes that the fatal result in these experiments was due to the fact that the toxic products present in the sterilized culture over- came the natural resisting powers of the tissues and enabled the bacillus to multiply over a larger area than would otherwise have been the case. As a result of this, toxic substances were produced in the body of the animal in sufficient quantity to cause general toxae- mia and death ; whereas the bacilli alone, in the dose mentioned, were not able to invade the tissues in the vicinity of the point of inoculation, and gave rise to a local abscess only. The same ex- planation is probably true for very many of the saprophytic bacteria which have been shown to possess pathogenic power ; and it is prob- able that many of those which are now classed by bacteriologists as non-pathogenic would prove to be pathogenic in the same way if thoroughly tested upon various species of animals, although it might be necessary to use unusually large doses to accomplish the same result. 89. BACILLUS COLI COMMUNIS. Synonyms. — Bacterium coli commune (Escherich) ; Colon bacillus of Escherich ; Emmerich's bacillus (Bacillus Neapolitanus). Prob* ably identical with Bacillus cavicida (Brieger's bacillus). NOT DESCRIBED IN PREVIOUS SECTIONS. 463 Obtained by Emmerich (1885) from the blood, various organs, and the alvine discharges of cholera patients at Naples ; by Weisser (1886) from normal and abnormal human faeces, from the air, and from putrefying infusions ; by Escherich (1886) from the fasces of healthy children ; since shown to be commonly present in the alvine discharges of healthy men, and probably of many of the lower ani- mals. Found by the writer'in the blood and various organs of yellow- fever cadavers, in Havana (1888 and 1889). Numerous varieties have been cultivated by different bacteriolo- gists, which vary in pathogenic power and to some extent in their growth in various culture media ; but the differences described are not sufficiently characteristic or constant to justify us in considering them as distinct species. Morphology. — Differs considerably in its morphology as obtained from different sources and in various culture media. The typical form is that of short rods with rounded ends, from two to three /* in length and 0.4 to 0.6 yu broad ; but under certain cir- cumstances the length does not exceed the breadth — about 0. 5 /* — and it might be mistaken for a micrococ- cus ; again the prevailing form in a culture is a short oval ; filaments of five /* or more in length are often observed in cultures, associated with short rods or oval FIG. 146.— Ba- cells. The bacilli are frequently united in pairs. The £"™& co" j^J; presence of spores has not been demonstrated. In un- (Escherich.) favorable culture media the bacilli, in stained prepara- tions, may present unstained places, which are supposed by Escherich to be due to degenerative changes in the protoplasm. Under certain circumstances some of the rods in a pure culture have been observed by Escherich to present spherical, unstained portions at one or both extremities, which closely resemble spores, but which he was not able to stain by the methods usually employed for staining spores, and which he is inclined to regard as " involution forms." This bacillus stains readily with the aniline colors usually em- ployed by bacteriologists, but quickly parts with its color when treated with iodine solution — Gram's method — or with diluted al- cohol. Biological Characters. — An aerobic and facultative anaerobic, non-liquefying bacillus. Sometimes exhibits independent move- ments, which are not very active. One rod of a pair, in a hanging- drop culture, may advance slowly with a to-and-fro movement, while the other follows as if attached to it by an invisible band (Escherich). The writer's personal observations lead him to believe that, as a rule, this bacillus does not exhibit independent movements. Does not form spores. Grows in various culture media at the room 464 PATHOGENIC AEROBIC BACILLI temperature — more rapidly in the incubating oven. Grows in a de- cidedly acid medium. In gelatin plates colonies are developed in from twenty-four to forty-eight hours, which va,ry considerably in their appearance ac- cording to their age, and in different cultures in the same medium. The deep colonies are usually spherical and at first are transparent, homogeneous, and of a pale-straw or amber color by transmitted light ; later they frequently have a dark-brown, opaque central por- tion surrounded by a more transparent peripheral zone ; or they may be coarsely granular and opaque ; sometimes they have a long-oval or "whetstone" form. The superficial colonies differ still more in appearance ; very young colonies by transmitted light often resemble little drops of water or fragments of broken glass ; when they have sufficient space for their development they quickly increase in size, and may attain a diameter of three to four centimetres ; the central portion is thickest, and is often marked by a spherical nucleus of a dark-brown color when the colony has started below the surface of the gelatin ; the margins are thin and transparent, the thickness gradually increasing towards the centre, as does also the color, which by transmitted light varies from light straw color or amber to a dark brown. The outlines of superficial colonies are more or less irregular, and the surface may be marked by ridges, fissures, or concentric rings, or may be granular. The writer has observed colonies re- sembling a rosette, or a daisy with expanded petals. Escherich speaks of colonies which present star-shaped figures surrounded by concentric rings. In gelatin stick cultures the growth upon the surface is rather dry, and may be quite thin, extending over the entire surface of the gelatin, or it may be thicker with irregular, leaf -like outlines and with superficial incrustations or concentric annular markings. An abundant development occurs all along the line of puncture, which in the deeper portion of the gelatin is made up of more or less closely crowded colonies ; these are white by reflected light, and of an am- ber or light-brown color by transmitted light ; later they may become granular and opaque. Frequently a diffused cloudy appearance is observed near the surface of the gelatin, and under certain circum- stances branching, moss-like tufts develop at intervals along the line of growth. One or more gas bubbles may often be seen in recent stick cultures in gelatin. Upon nutrient agar and blood serum, in the incubating oven, an abundant, soft, white layer is quickly developed. Upon potato an abundant, soft, shining layer of a brownish-yellow color is developed. The growth upon potato differs considerably, according to the age of the potato. According to Escherich, upon old potatoes there may NOT DESCRIBED IN PREVIOUS SECTIONS. 465 be no growth, or it may be scanty and of a white color. In milk, at 37° C., an acid reaction and coagulation of the casein are produced at the end of eight or ten days. In the absence of oxygen this bacillus is able to grow in solutions containing grape sugar (Escherich). In bouillon it grows rapidly, producing a milky opacity of the culture liquid. The thermal death-point of Emmerich/s bacillus, and of the colon bacillus from faeces, was found by Weisser to be 60° C., the time of exposure being ten minutes. The writer has obtained corre- sponding results. Weisser found that when the bacilli from a bouil- lon culture were dried upon thin glass covers they failed to grow FIG. 147. F;o. 148. FIG. 147.— Bacillus coli communis in nutrient gelatin containing twenty percent of gelatin, end of two week*, showing moss-like tufts along the line of growth. (Sternberg.) FIG. 148.— A portion of the growth shown in Fig 147, at a, magnified about sir diameters. From a photograph. (Sternberg.) after twenty-four hours. These results give confirmation to the view that the bacillus under consideration does not form spores. This view receives further support from the experiments of Wal- liczek (1894), who found that when dried upon pieces of sterile filter paper the bacillus failed to grow at the end of eighteen hours. Pathogenesis. — Comparatively small amounts of a pure culture of the colon bacillus injected into the circulation of a guinea-pig usually cause the death of the animal in from one to three days, and the bacillus is found in considerable numbers in its blood. But when 4GG PATHOGENIC AEROBIC BACILLI injected subcutaneously or into the peritoneal cavity of rabbits or guinea-pigs, a fatal termination depends largely on the quantity in- jected ; and although the bacillus may be obtained in cultures from the blood and the parenchyma of the various organs, it is not present in large numbers, and death appears to be due to toxaemia rather than to septicaemia. Mice are not susceptible to infection by subcutaneous injections. Small quantities injected beneath the skin of guinea-pigs usually produce a local abscess only ; larger amounts — two to five cubic centimetres — frequently produce a fatal result, with symptoms and pathological appearances corresponding with those resulting from intravenous injection. These are fever, developed soon after the injection, diarrhoea, and symptoms of collapse appearing shortly before death. At the autopsy the liver and spleen appear normal, or nearly so ; the kidneys are congested and may present scattered punctiform ecchymoses (Weisser). According to Escherich, the spleen is often somewhat enlarged. The small intestine is hyper- semic, especially in its upper portion, and the peritoneal layer pre- sents a rosy color ; the mucous membrane gives evidence of more or less intense catarrhal inflammation, and contains mucus, often slightly mixed with blood. In rabbits death occurs at a somewhat later date, and diarrhoea is a common symptom. In dogs the subcu- taneous injection of a considerable quantity of a pure culture may give rise to an extensive local abscess. In human pathology the colon bacillus plays an important role. It is concerned in the etiology of a considerable proportion of the cases of cystitis and of pyelonephritis, and peritonitis resulting from perforation. It appears to be the cause of certain affections of the anal region (Hartmann and Lieffring). It has been obtained in pure culture from abscesses in various parts of the body, from the valves of the heart in endocarditis, from the pleural cavity in empyema, etc. It has also been found in the blood, as a result of general infection following cystitis and pyelonephritis (Sittmann and Barnow). Varieties. — Booker, in his extended studies relating to the bac- teria present in the faeces of infants suffering from summer diarrhoea, has isolated seven varieties " which closely resemble Bacterium coli commune in morphology and growth in agar, neutral gelatin, and potato, but by means of other tests a distinction can be made between them." Some of the pathogenic bacteria heretofore described are also closely allied to the " colon bacillus " and by some bacteriologists are supposed to belong to the same group— i.e., to be varieties of the same species rather than independent species with fixed characters. Whatever may t>e the remote relationship, the typhoid group, the hog- NOT DESCRIBED IN PREVIOUS SECTIONS. 467 cholera group, the Bacillus typhi murium of Loffler, the bacillus of Laser, the Bacillus euteritidis of Gartner, and other similar bacilli appear to be differentiated from one another by characters which justify their description under separate names. Still it is difficult to fix upon any one of these characters to which specific value can be attached ; and, in view of the many varieties found in nature or pro- duced artificially in laboratory experiments, we are not justified in asserting that our classification of these low organisms has any sub- stantial scientific foundation. The difficulties attending an attempt to establish specific characters are well illustrated by the extensive literature relating to the differentiation of bacilli belonging to the typhoid group from those belonging to the colon group. The main points upon which the distinction must depend have been referred to in the section devoted to the typhoid bacillus. Fremlin (1893) has made a comparative study of the colon bacil- lus from various sources. He finds the common characters of gas production in media containing sugar and coagulation of milk. Cul- tivated from different animals the morphology is the same, but there are differences as regards motility. The most active movements are said to be exhibited in the bacillus from man, while the variety ob- tained from the intestines of rabbits showed scarcely any movements. The different varieties displayed considerable differences in their growth upon potato. Dreyfuss (1894) finds decided differences in the pathogenic viru- lence of the colon bacillus from healthy individuals and from those suffering from various intestinal disorders. A culture from the dis- charges of a fatal case of cholera nostras proved to be exceptionally virulent — tested by intraperitoneal injections in guinea-pigs. Gilbert (1895), as a result of his extended researches, concludes that there are five principal types among the bacilli most nearly related to the colon bacillus: 1st. Bacilli which differ from the colon bacillus by their being non-motile. This type includes two varieties : one gives thick yellowish colonies upon gelatin plates and numerous gas bubbles on potato — this is the bacille lactique of Pasteur and the Bacillus lactis aerogenes of Escherich ; the other gives thin, bluish-white colonies and includes the bacille de rendocardite of Gilbert and Lion. 3d. Bacilli which differ from the colon bacillus by the fact that cultures do not give the indol reaction. 3d. Bacilli which do not cause the fermentation of lactose. 4th. Bacilli which are not motile and do not ferment lactose. 5th. Bacilli which are not motile, do not give the indol reaction, and do not ferment lactose. Theobald Smith (1895) gives the following account of his method of detecting bacilli of the "colon group " in water : 468 PATHOGENIC AEROBIC BACILLI 4 * The method followed by the writer in the general bacteriological exam- ination of water consists, first, in the preparation of gelatin plates for the usual enumeration ; and, second, in the addition to every one of ten fermen- tation tubes, containing a one-per-cent dextrose bouillon, a certain quantity of water. This is added most easily by first diluting the water, so that one or two cubic centimetres are equivalent to the quantity which it is desired to add to each tube. Pipettes graduated by drops are convenient, but not so accurate. In case of ground water it is well to prepare in addition a flask containing fifty to one hundred cubic centimetres of the water, and an equal, or greater, quantity of bouillon, to which sugar is not added. Plates may be prepared from this flask after sixteen to twenty -four hours. When gas begins to ap- pear in the fermentation tubes, the amount accumulated at the end of each twenty-four hours should be marked with a glass pencil on the tube. From these tubes, which contain fifty to sixty per cent of gas on the third day, and are very strongly acid, plates may be prepared to confirm the indications of Bacillus coli. This, however, is not essential, for the writer has found as yet no species having these fermentative characters which is not one of the following : Bacillus coli, Bacillus lactis aerogenes, Bacillus enteriditis, Bacil- lus typhi murium, Bacillus cholera? suis. The three last-mentioned species are probably as rare in water as Bacillus typhosus itself. * ' My own experience coincides with that of Matthews when he states that ninety -two per cent of all bacteria in ground water are suppressed in the thermostat. While the addition of 0.5 cubic centimetre, or even more, of such water may fail to produce cloudiness in any of the series of fermenta- tion tubes, the same quantity, or less, of surface water never fails to infect the tubes." BACILLUS d OF BOOKER. " Found in two cases of cholera infantum and the predominating form in one serious case of catarrhal enteritis. 14 Morphology.— Resembles Bacterium coli commune. ** Groivth in Colonies. — Gelatin : Colonies grow luxuriantly in gelatin,and thrive in acid and sugar gelatin equally as well as in neutral gelatin. In the latter the colonies closely resemble, but are not identical with, the Bac- terium coli commune. In acid gelatin they ditt'er very much from Bacterium coli commune. The colonies spread extensively and are bluish-white with concentric rings. Slightly magnified, they have a large, uniform, yellow central zone surrounded by a Border composed of perpendicular threads placed thickly together. Sometimes a series of these rings appear with inter- vening yellow rings.- "Agar: The colonies are round, spread out, and blue or bluish- white. Slightly magnified, they have a pale-yellow color. ' Stab Cultures — Gelatin: In sugar gelatin the surface growth has a nearly colorless centre surrounded by a thick border with an outer edge of fine, hair-like fringe ; the growth along the line of inoculation is fine and deli- cate. In neutral gelatin the growth is not so luxuriant as on sugar gelatin ; on the surface it is thick and white, with a delicate stalk in the depth. "Agar: Thick white surface growth with a well-developed stalk in the depth. "Potato: Luxuriant yellow, glistening, moist, and slightly raised sur- face, with well-defined Orders. 11 Action on Milk. — Coagulated into a gelatinous coagulum in twenty-four hours at 88* G., and into a solid clot in two days. " Milk Litmus Reaction. — Milk colored blue with litmus is changed to light pink in twenty-four hours at 38° C. The pink color gradually fades, and by the second or third day is white or cream color with a thin layer of pint on top. The pink color extends in a few days about one-half down the clot. NOT DESCRIBED IN PREVIOUS SECTIONS. 469 Temperature.— Grows best about 38° C. " Spores have not been observed. " Gas Production. — Gas bubbles are produced in milk; not observed on potato." BACILLUS 6 OF BOOKER. * ' Found as the predominating form in two cases of dysentery one of which was fatal and the other a mild case. " Morphology. — Resembles Bacterium coli commune. " Growth in Colonies. — Gelatin : The colony growth varies considerably with slight difference in the gelatin. In ten-per-cent neural gelatin the colo- nies resemble those of Bacterium coli commune. On the second or third day, when the colonies have just broken through the surface and are spread out, it is impossible to distinguish one variety from the other, but as the colonies grow older a difference can generally be recognized. In sugar and acid gelatin the colonies have a clear centre with white border ; slightly magnified, a uniform brown centre surrounded by a brown zone composed of fine, needle- like rays perpendicular to the border. After cultivating for a few generations on acid and sugar gelatin the colonies cease to develop, and either grow in very small colonies or do not grow at all. The activity is re- gained if cultivated on neutral gelatin. " Agar : Colonies are large, round, and have a mother-of-pearl appearance. Slightly magnified, a uniform yellow color. " Stab Cultures. — Agar: Luxuriant, nearly colorless surface growth, with well-developed stalk along the line of inoculation in the depth. "Potato: Golden-yellow, glistening, slightly raised surface with well-de- fined borders. ' ' Action on Milk.— Milk becomes gelatinous in twenty-four hours at 38° C. , and in a few days a solid coagulum is formed. Milk colored blue with lit- mus is reduced to white or cream color in twenty-four to forty-eight hours at 38° C., with a thin layer of pink at the top of the culture. The pink color gradually extends lower in the coagulum. " Temperature.— Thrives best at about 38° C. " Spores have not been observed. " Gas Production.— Occurs in milk, but not seen in potato cultures. " Relation to Gelatin. — Does not liquefy gelatin. " Resemblance. — Resembles Bacterium coli commune and bacillus d; dif- fering from the former in the character of the colony growth on acid and sugar gelatin, and in ceasing to develop in these media after several genera- tions. It differs from bacillus d in this latter respect." BACILLUS / OF BOOKER. " Found in one case of cholera infantum and one case of catarrhal ente- ritis. " Morphology. — Resembles Bacterium coli commune. " Growth in Colonies.— Gelatin: It is difficult to distinguish the colony growth from the Bacterium coli commune. There is often a difference in the colonies planted at the same time and kept under similar conditions, but it is not very marked nor always the same kind of difference. The tendency to concentric rings is greater in this variety. The colonies develop some- what better on neutral and sugar gelatin than on acid gelatin. ' ' Agar : The colonies are large, round, and bluish- white. Slightly magni- fied, a light-yellow color. 4 ' Stab Cultures. —Gelatin : The culture is spread over the surface and has a mist-like appearance ; in the depth along the line of inoculation is a deli- cate stalk. , "Agar: Thick, luxuriant, white surface growth, with a well-developed stalk along the line of inoculation in the depth. 470 PATHOGENIC AEROBIC BACILLI " Potato : Bright-yellow, glistening, moist surface with well-defined bor- ders, and but slightly raised above the siirrounding potato. " Action on Milk and Litmus Reaction. — Milk is coagulated into a solid clot in twenty-four hours at 38° C. Milk colored blue with litmus is changed to pink in twenty-four hours at 38° C. , and in forty-eight hours is reduced to white or cream color with a thin pink layer on top. " Gas Production. — Gas bubbles arise in milk cultures, but they have not been observed on potato cultures. " Temperature.— Grows better at 38° C. '* Spores have not been observed. "Relation to Gelatin.— Does not liquefy gelatin. "Resemblance. — It closely resembles Bacterium coli commune and Brie- ger's bacillus in the character of its growth upon different media, but is readily- distinguished from both, as is also Brieger's bacillus from the Bacterium coli commune, by the following differential test recently made known by Dr. Mall. Yellow elastic tissue from the ligamentum nuchae of an ox is cut into fine bits and placed in test tubes containing water with ten-per-cent bouillon and one-per-cent sugar, and sterilized from one and one-half to two hours at a time for three consecutive days. Into this is inoculated two species of bacteria, one of which is the bacterium under observation, the other a bacillus found in garden earth. The latter bacillus is anaerobic, grows in hydrogen, nitrogen, and ordinary illuminating gas, in the bottom of bouillon, in the depth but not on the surface of agar stab cultures, and not at all in gelatin stab cultures. It has a spore in one end making a knob bacillus. Different species of bacteria — Streptococcus Indicus, tetragenus, cholera, swine plague, Bacterium lactis aerogenes, Bacterium coli commune, Brieger's bacillus, and a number of varieties of bacteria which I have iso- lated from the faeces — were inoculated with the head bacillus into the above- described elastic- tissue tubes. The tubes inoculated with Brieger's bacillus developed a beautiful purple tint, which started as a narrow ring at the top of the culture, gradually extending downward and deepening in color until the whole tube had a dark-purple color. This color reaction began in five to fourteen days, and was constantly present in a large number of tests. Tubes inoculated with bacillus / gave a much fainter purple color, which was longer in appearing and never became so dark as with Brieger's bacillus. "Tubes inoculated with the other species of bacteria above mentioned gave no color change and remained similar to control. Bacillus / also shows a slight difference from Bacterium coli commune in coagulating milk and re- ducing litmus more rapidly, and appears to produce moreactive fermentation in milk. Like Brieger's bacillus, the gelatin colonies more frequently show a concentric arrangement than those of the Bacterium coli commune," BACILLUS g OF BOOKER. " Found in one case of serious gastro-enteric catarrh. It was not in large quantity. ' Morphology and Biological Characters. — In morphology, character of growth on agar, gelatin, and potato, it resembles Bacterium coli commune. " Action on Milk and Litmus Reaction. — Milk is not coagulated, and milk colored blue with litmus is changed to pink in a few days, and holds this color. These characteristics distinguish it from the Bacterium coli com- mune. " Gas Production.— Not observed in milk or potato cultures. " AWf/f/oH t<> Crltttin.— Dors not liquefy gelatin.'1 NOT DESCRIBED IN PREVIOUS SECTIONS. 471 BACILLUS k OF BOOKER. "Found in one case of mild dysentery, not in large quantity. " Morphology. — Resembles Bacterium coli commune. " Growth in Colonies. — Gelatin: In plain neutral gelatin the colonies re- semble those of Bacterium coli commune. In sugar gelatin the colonies are white and spread extensively. Slightly magnified, they have a round, dark centre surrounded by a yellow, loose zone with an outer white rim ; later the whole colony has a uniform yellow color and is not compact. " Agar : Colonies are white, round, and large. Slightly magnified, they are brownish-yellow. " Stab Cultures. — Nothing characteristic in gelatin and agar. "Potato culture is yellow, dry, and slightly raised, with well-defined bor- ders. " Action on Milk and Litmus Reaction. — Milk is coagulated into a solid clot in two days at 38° C. Milk colored blue with litmus is changed to pink in twenty-four hours. " Gas Production. — Occurs in milk; not observed on potato. "Relation to Gelatin. — Does not liquefy gelatin.'- BACILLUS k OF BOOKER " Found in two cases of cholera infantum and one of catarrhal enteritis. "Morphology. — Resembles Bacterium coli commune. ' ' Growth in Colonies. — Gelatin : In neutral gelatin the colonies cannot be distinguished from those of Bacterium coli commune. In acid gelatin the colonies do not spread so extensively as those of Bacterium coli commune, and they have a decided concentric arrangement, a wide white centre sur- rounded by a narrow, transparent blue ring, and outside of this a white bor- der. Slightly magnified, the colonies have an irregular, yellowish-brown centre mottled over with dark spots and surrounded by a light-yellow ring bordered by a brownish-yellow wreath,, ' ' Agar : Colonies are large, round, and bluish-white. Slightly magnified, a light brownish-yellow color. *' Stab Cultures. — Gelatin : In sugar gelatin the surface growth is exten- sive, nearly colorless, and has a rough, misty appearance. In the depth if a delicate growth. In plain neutral gelatin the surface growth is bluish- white, thick, and not so extensively spread ; the growth in the depth is also thicker. " Potato culture is moist, dirty-cream color, has raised surface and defined border. " Action on Milk. — Milk becomes gelatinous in twenty-four hours at 38° C., and a solid clot in two days. Milk colored blue with litmus is changed to pink in twenty- four hours, and reduced to white with a pink layer on top in two days," BACILLUS U OF BOOKER,, "Found in large quantity, but not the predominating form, in one case of chronic gastro-enteric catarrh— extremely emaciated. " Morphology. —Resembles Bacterium coli communeo " Growth in Colonies.— Gelatin: In neutral gelatin the colonies are spread out and have a frosty or ground-glass appearance. The centre is blue and border white, but both have the ground-glass appearance. Slightly magni- fied, the central part is light yellow and the border brown with a rough, fur- rowed surface. In acid gelatin the white border is wider and the surface is rougher. "Agar: Colonies are round, blue, or bluish-white, and spread out. Under the microscope they have a light-yellow color. "Stab Cultures. — Gelatin: Has a rough, nearly colorless surface growth, and a thick stalk in the depth along the line of inoculation. ' ' Agar : Thick white surface growth with well-developed stalk in the depth. 472 PATHOGENIC AEROBIC BACILLI " Action on Milk and Litmus Reaction. — Milk remains liquid, and milk colored blue with litmus is changed to pink. ** Gas Production. — Not observed in milk or potato cultures. " Relation to Gelatin. — Does not liquefy gelatin. "Spores have not been noticed." Bacillus Coli Communis in Peritonitis. — The researches of A. Frankel show that Bacillus coli communis may be obtained in pure cultures from the exudate into the peritoneal cavity in a considerable proportion of the cases of peritonitis, and there is good reason for believing that in these cases it was the cause of the inflammatory process. Thirty-one cases were examined by Frankel, with the fol- lowing result: Pure cultures of Bacillus coli communis were obtained in nine cases ; of Streptococcus (pyogenes ?) in seven ; of Bacillus lactis aerogenes in two ; of " diplococcus pneumonise " in one ; of Staphylococcus pyogenes aureus in one. Of the remaining eleven cases, seven gave mixed cultures, and in three of these Bacillus coli communis was the most abundant species. The author referred to has also shown that pure cultures of Bacillus coli communis injected into the cavity of the abdomen of rabbits cause a typical peritonitis. The present writer has frequently obtained the same result in experi- ments made with this bacillus. It would appear, therefore, that the peritonitis which so constantly results from wounds of the intestine is probably due, to a considerable extent, to the introduction of this microorganism from the lumen of the intestine, where it is con- stantly found, into the peritoneal cavity, where the conditions are favorable for its rapid development. 90. BACILLUS LACTIS AEROGENES. Obtained by Escherich (1886) from the contents of the small intestine of children and animals fed upon milk ; in smaller numbers from the faeces of milk-fed children, and in one instance from uncooked cow's milk. v Morphology. — Short rods with rounded ends, from 1 to t£l \f 2 H in length and from 0.1 to 0.5 /* broad ; short oval and 99 spherical forms are also frequently observed, and, under t** f t certain circumstances, longer rods — 3 /' — may be developed : usually united in pairs, and occasionally in chains contain- ing several elements. In some of the larger cells Escherich has observed unstained spaces, but was not able to obtain any evidence that these represent spores. •heri h This bacillus stains readily with the ordinary aniline colors, but does not retain its color when treated by Gram's method. Biological Characters. — An aerobic (facultative anaerobic), non-liquefy- ing, non motile bacillus. Does not form spores. Grows in various culture media at the room temperature— more rapidly in the incubating oven. Upon gelatin plates, at the end of twenty-four hours, small white colonies are developed. Upon the surface these form hemispherical, soft, shinin" masses which, examined under the microscope, are found to be homogeneous NOT DESCRIBED IN PREVIOUS SECTIONS. 473 and opaque, with a whitish, lustre by reflected light. The deep colonies are spherical and opaque and attain a considerable size. In gelatin stick cul- tures the growth resembles that of Friedlander's bacillus — i.e., an abundant growth along the line of puncture and a rounded mass upon the surface, forming a " nail-shaped " growth. In old cultures the upper portion of the gelatin is sometimes clouded, and numerous gas bubbles may form in the gelatin. Upon the surface of nutrient agar an abundant, soft, white layer is developed. Upon old potatoes, in the incubating oven, at the end of twenty-four hours a yellowish -white layer, several millimetres thick, is developed, which is of paste-like consistence and contains about the peri- phery a considerable number of small gas bubbles ; this layer increases in dimensions, has an irregular outline, and larger and more numerous gas bubbles are developed about the periphery, some the size of a pea; later the whole surface of the potato is covered with a creamy, semi-fluid mass filled with gas bubbles. On young potatoes the development is different; a rather luxuriant, thick, white or pale yellow layer is formed, which is tolerably dry and has irregular margins ; the surface is smooth and shining, and a few minute gas bubbles only are formed after several days. Pathogenesis. — Injections of a considerable quantity of a pure culture into the circulation of rabbits and of guinea-pigs give rise to a fatal result within forty-eight hours. In his first publication relating to " the bacteria found in the dejecta of infants afflic ted with summer diarrhoea," Booker has described a bacillus which he designates by the letter B, which closely resembles Bacillus lactis aerogenes and is probably identical with it. He says : " Summary of Bacillus B. — Found nearly constantly in cholera infan- tum and catarrhal enteritis, and generally the predominating form. It appeared in larger quantities in the more serious cases. It was not found in the dysenteric or healthy faeces. It resembles the description of the Ba- cillus lactis aerogenes, but the resemblance does not appear sufficient to con- stitute an identity, and, in the absence of a culture of the latter for com- parison, it is considered a distinct variety for the following reasons : Bacillus B is uniformly larger, its ends are not so sharply rounded, and in all culture media long, thick filaments are seen, and many of the bacilli have the pro- toplasm gathered in the centre, leaving the poles clear. There is some difference in their colony growth on gelatin, and in gelatin stick cultures bacillus B does not show the nail-form growth with marked end swelling in the depth. In potato cultures the Bacillus lactis aerogenes shows a differ- ence between old and new potatoes, while bacillus B does not show any difference. "Bacillus B possesses decided pathogenic properties, which was shown both by hypodermic injections and feeding with milk cultures." 91. BACILLUS C OF BOOKER. Found by Booker (1889) in a case of cholera infantum. Morphology. — Resembles Bacillus lactis aerogenes of Escherich. Biological Characters. — Resembles Bacillus lactis aerogenes, but differs from it in not coagulating milk; the growth on potato also is more luxuri- ant and the surface is more thickly covered with gas bubbles. BACILLI OF JEFFRIES. Jeffries, in a study of the alvine discharges of children suffering from summer diarrhoea, isolated a number of bacilli resembling Bacillus coli communis and Bacillus lactis aerogenes of Escherich. He says: 44 While Brieger's bacillus and the lactic acid bacillus of Escherich were not found, a whole group of species in growth, form, and general physiology closely resembling them have been isolated. This group is represented by bacilli A, G, J, K, P, S, Z ; they seem to form a genus ; the form is very 33 474 PATHOGENIC AEROBIC BACILLI much alike. All are good anaerobic growers ; all form gas ; all turn milk distinctly acid ; and all closely resemble one another in pure cultures. Many would doubtless class these altogether as one species ; but if species are to be recognized at all, we must come to recognizing every fixed difference as constituting a species. 41 This group occurred — always very abundantly— in eighteen out of the twenty two cases of summer diarrhoea, and is also well represented among the kittens. They are, however, so much like the harmless forms found by Escherich that they may for the present be laid aside as of no specific sig- nificance. They are also almost the only forms tested which failed to pro- duce intestinal troubles in kittens. Excluding these, there is no species, or group of species, left either generally occurring or in sufficient numbers to be regarded as the specific pathogenic plant of summer diarrhoaa." 92. BACILLUS ACIDIFORMANS. Obtained by the writer (1888) from a fragment of yellow-fever liver pre- served for forty-eight hours in an antiseptic wrapping ; since obtained from Fio. 150. Fia. 151. X 1,000. From a photomicrograph* Fio. 151— Bacillus acidiformaos, from a potato culture. (Steinberg ) Fio. m.— Culture of Bacillus acidiformans in nutrient gelatin, end of four days at 22° C. From a photograph. (Steinberg.) liver preserved in the same way from two comparative autopsies— i.e., not cases of yellow fever. Morphology.— & short bacillus with rounded corners, sometimes short oval in form ; from H to 3 // in length and about 1.2 # in breadth ; may grow out into filaments of 5 to 10 >u, or more, in length; in some cultures the short oval form predominates. Stains readily with the aniline colors usually employed, and by Gram's method. Biological Characters.— An aerobic and facultative anaerobic, non- /"/'":///"'.'/. Mtn-uu*til<>, bacillus. Dors not form spoivs. Grows rapidly at the room temperature in the usual culture media. Grows in decidedly acid media; in culture media containing glycerin or glucose it produces an abun- dant evolution of carbon dioxide, and a volatile acid is formed. t does not liquefy gelatin, and in stick cultures grows abundantly both on the surface and along the line of puncture. At the end of twenty-four hours, at 22° C., a rounded white mass is formed upon the surface, resembling NOT DESCRIBED IN PREVIOUS SECTIONS. 475 the growth of Friedlander's bacillus ; at the bottom of the line of puncture the separate colonies are spherical, opaque, and pearl-like by reflected light. Gas bubbles are formed in the gelatin. At the end of a week the surface is covered with a thick, white, semi-fluid mass. In gelatin roll tubes the superficial colonies are translucent or opaque, and circular or somewhat irregular in outline; by reflected light they are slightly iridescent ; the deep colonies are spherical, opaque, and homo- geneous. The growth upon the surface of nutrient agar is abundant and rapid, of a shining milk-white color, and cream like in consistence. An abundant development forms along the line of puncture and the culture medium is split up by gas bubbles. In glycerin-agar the evolution of gas is very abun- dant and the culture medium acquires an intensely acid reaction. On potato the growth is abundant and rapid at a temperature of 20° to 30° C., forming a thick, semi-fluid mass of a milk-white color. I have not obtained any evidence that this bacillus forms spores; the cultures are sterilized by ten minutes' exposure to a temperature of 160° F. When cultivated in bouillon to which five per cent of glycerin has been added the culture medium acquires a milky opacity, and there is a copious precipitate, of a viscid consistence, consisting of bacilli ; during the period of active development the surface is covered with gas bubbles, as in a sac- charine liquid undergoing alcoholic fermentation, and the liquid has a de- cidedly acid reaction. Pathogenesis. — Pathogenic for rabbits and for guinea pigs when injected into the cavity of the abdomen — one to two cubic centimetres of a culture in bouillon. The animal usually dies in less than twenty-four hours. The bacilli are found in the blood in rather small numbers, and are frequently seen in the interior of the leucocytes. The spleen is enlarged, the liver normal, the intestine usually hyperaemic. 93. BACILLUS CUNICULICIDA HAVANIENSIS. Obtained by the writer (1889) from the contents of the intestine of yellow- fever cadavers, and also from fragments of yellow-fever liver preserved for forty-eight hours in an antiseptic wrap- ping— my bacillus a?, Havana, 1889. Morphology. — This bacillus resembles the colon bacillus in form, but is some- what larger, from 2 to 4 // in length and from 0.8 to 1 /* in diameter ; sometimes associated in pairs ; may grow out into short filaments — not common. The ends of the rods are rounded, and under cer- tain circumstances vacuoles are seen at the extremities, especially in potato cul- tures. Stains quickly with the aniline colors usually employed, and also by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non-lique- fying bacillus. Under certain circum- FIG. 152.— Bacillus cuniculicida Havani- stances may exhibit active movements, ensis, from a single colony in nutrient gela- but is usually motionless. tin- * ^m- Frona a photomicrograph. A very curious thing with reference (Sternberg.) to this bacillus is that it presented ac- tive movements in my cultures made directly from yellow-fever cadavers, but that these movements were not constant, and that since my return to Baltimore I have not, as a rule, observed active movements in cultures from the same stock, which, however, preserved their pathogenic power and other 476 PATHOGENIC AEROBIC BACILLI characters. In Havana these movements were usually not observed in all the bacilli in a field under observation, but one and another would start from a quiescent condition on an active and erratic course ; sometimes spinning actively upon its axis, and again shooting across the field as if propelled by a nagellum. My notes indicate that cultures passed through the guinea-pig are more apt to be motile. In gelatin stick cultures the growth of bacillus x resembles that of the colon bacillus, but the colonies at the bottom of the line of puncture are more opaque and not of a clear amber color like that of colonies of the colon bacillus. Upon the surface the growth is thicker than that of the colon bacillus, and forms a milk-white, soft mass. The colonies in gelatin Esmarch roll tubes vary considerably at different times. Deep colonies are usually spherical, homogeneous, light brown in color, and more opaque than the similar colonies of the colon bacillus. At the end of a few days the deep colonies become quite opaque, and may be lobate, like a mulberry, or coarsely granular ; sometimes the deep colonies have an opaque central portion surrounded by a transparent marginal zone. In old gelatin roll tubes these deep colonies form opaque white hemi- Fio. 163. FIG. 154. Fig. 158.— Bacillus cuniculicida Havaniensis; colonies in gelatin roll tube, third day at 20° C. X 6. From a photograph. (Sternberg.) Fio. 154.— Bacillus cuniculicida Havaniensis ; colonies in gelatin roll tube, end of forty-eight hours, x 10. From a photograph. (Sternberg.) spheres projecting from the surface of the dried culture medium, and little tufts of acicular crystals are sometimes observed to project from the side of such old colonies. The superficial colonies are circular or irregular in outline, with trans- puiviit margins ;uul an opaque central portion, sometimes corrugated. They are finely granular and iridescent by reflected light, and of a milk-white color; by transmitted light they have a brownish color. Young colonies closely resemble those of the colon bacillus. This bacillus grows well at a temperature of 20° C. (68° F.), but more rapidly and luxuriantly at a higher temperature— 30° to 35° C. It grows well in agar cultures, and especially inglycerin-agar,'m which it produces some gas and an acid reaction. The growth on the surface of glycerm-agar cultures is white, cream-like in consistence, and quite abun- dant. It grows well in an agar or gelatin medium made acid by the addition of 0.2 per cent (1: 500) of hydrochloric acid. NOT DESCRIBED IN PREVIOUS SECTIONS. 477 In cocoanut water it multiplies rapidly, producing a milky opacity of the previously transparent fluid, an acid reaction, and an evolution of carbon dioxide. On potato it produces a thick layer, which may cover the entire surface in three or four days, and which has a dirty-white, cream- white, or pinkish- white color and cream-like consistence. The growth upon potato varies at different times, evidently owing to differences in the potato. When stained preparations are examined with the full light of the Abbe condenser the ends of some of the rods appear to be cut away, leaving a con- cave extremity ; but by using a small diaphragm to obtain definition it will be seen that the cell wall extends beyond the stained portion of the rod and includes what appears to be a vacuole. There is no reason to believe that this appearance is due to the presence of an end spore, for the supposed vacuole is not refractive, as a spore would be, and my experiments on the thermal death-point of this bacillus indicate that it does not form spores. Cultures are sterilized by exposure for ten minutes to a temperature of 160° F. (71.2° C.). Pathogenesis. — Very pathogenic for rabbits when injected into the cavity of the abdomen. Injections of a small quantity of a pure culture into the ear vein or subcutaneously generally give a negative result. Injections of from one to five cubic centimetres of a culture in bouillon, blood serum, or agua coco, into the cavity of the abdomen, frequently prove fatal to rabbits in a few hours — two to six. The negative results obtained in injecting cultures beneath the skin or into the ear vein of rabbits show that this bacillus does not induce a fatal septicaemia in these animals, and the fatal result when injections are made into the peritoneal cavity does not appear to be due to an invasion of the blood, but rather to the local effect upon the peritoneum, together with the toxic action of the chemical products resulting from its growth. It is true that I have always been able to recover the bacillus from the liver, or from blood obtained from one of the cavities of the heart, even in animals which succumb within a few hours to an injection made into the cavity of the abdomen. But the direct examination of the blood shows that the bacilli are present in very small numbers, and leads me to believe that the bacillus does not multiply, to any considerable extent at least, in the circulating fluid. The spleen is not enlarged, as is the case in anthrax, rabbit septicaemia, and other diseases in which the pathogenic microorganism multiplies abun- dantly in the blood. On the other hand, there is evidence of local inflammation in the peri- toneal cavity. When death occurs within a few hours the peritoneum is more or less hyperaemic and there is a considerable quantity of straw-colored fluid in the cavity of tbe abdomen. When the animal lives for twenty hours or more there is a decided peritonitis with a fibrinous exudation upon the surface of the liver and intestine. Usually the liver, in animals which die within twenty-four hours, is full of blood, rather soft, and dark in color. In a single instance I found the liver to be of a light color and loaded with fat. The rapidly fatal effect in those cases in which I have injected two or more cubic centimetres of a culture into the cavity of the abdomen has led me to suppose that death results from the toxic effects of a ptomaine con- tained in the culture at the time of injection. The symptoms also give sup- port to this supposition. The animal quickly becomes feeble and indisposed to move, and some time before death lies helpless upon its side, breathing regularly, but is too feeble to get up on its feet when disturbed. Death some- times occurs in convulsions, but more frequently without— apparently from heart failure. Pathogenic also for guinea-pigs when injected into the cavity of the abdomen, but death does not occur in so short a time — eighteen to twenty hours. Subcutaneous injections of one-half to one cubic centimetre gave a 478 PATHOGENIC AEROBIC BACILLI negative result in eleven out of thirteen guinea-pigs inoculated — two died within twenty-four hours. 94. BACILLUS LEPORIS LETHALIS. Obtained by Dr. Paul Gibier (1888) from the contents of the intestine of yellow-fever patients ; also by the writer from the same source (1888, 1889) in exceptional cases and in comparatively small numbers. Named and de- scribed by present writer. Morphology. — Bacilli with rounded ends, from 1 to 3 UL in length and about 0.5 u in breadth. The length may vary in the same culture from a short oval to rods which are two or three times as long as broad, or it may grow out into flexible filaments of considerable length. In recent cultures the bacilli are frequently united in pairs. Stains readily with the aniline colors usually employed. In cultures which are several days old, or in recent cultures when the stained prepara- tion is washed in alcohol, the ends of the rods are commonly more deeply stained than the central portion — " end staining"; and in old cultures some of the bacilli are very faintly stained. Biological Characters. — Anaerobic, liquefying, actively motile bacillus. Does not form spores. In gelatin stick cultures, at the end of twenty-four hours at a tempe- rature of 20° to 22° C., there is an abundant development along the line of puncture and commencing liquefaction at the surface. Later the liquefaction is funnel-shaped, and there is an opaque white central core along the line of puncture, with liquefied gelatin around it. Liquefaction progresses most rapidly at the surface, and in the course of three or four days the upper por- tion of the gelatin for a distance of half an inch or more is completely lique- fied, and an opaque white mass, composed of bacilli, rests upon the surface of the unliquefied portion. In gelatin roll tubes the young colonies upon the surface are transparent and resemble somewhat small fragments of broken glass; later liquefaction occurs rapidly. Deep colonies in gelatin roll tubes, or at the bottom of stick cultures, are spherical, translucent, and of a pale straw color. Upon the surface of nutrient agar it grows rapidly, forming a rather thin, translucent, shining, white layer, which covers the entire surface at the end of two or three days at a temperature of 20° C. Upon potato the growth is rapid and thin, covering the entire surface, and is of a pale-yellow color. This bacillus grows at a comparatively low temperature, and its vitality is not destroyed by exposure for an hour and a half in a freezing mixture at 15° C. below zero (5° F.). Decided growth occurred in a stick culture in gelatin exposed in Balti- more during the month of January in an attic room. During the twenty- two days of exposure the highest temperature, taken at 9 A.M. each day, was 11° C., and the lowest 2° C. At a temperature of 16° to 20° C. develop- ment in a favorable culture medium is rapid. There is no evidence that this bacillus forms spores ; cultures are sterilized by exposure to a temperature of 60° C. for ten minutes. Coagulated blood serum is liquefied by this bacillus. It retains its vitality for a long time in old cultures, having grown freely when replanted at the end of a year from a hermetically sealed tube containing a pure culture ia blood serum. Pathogenefds.— This bacillus is very pathogenic for rabbits when injected into the cavity of the abdomen in quantities of one cubic centimetre or more ; it is less pathogenic for guinea-pigs, and is not pathogenic for white rats when iniected subcutaneously. Gelatin cultures seem to possess more in- tense pathogenic power than bouillon cultures, and cultures from the blood of an animal recently dead as the result of an inoculation are more potent NOT DESCRIBED IN PREVIOUS SECTIONS. 479 than those from my original stock which had not been passed through a susceptible animal. The mode of death in rabbits is quite characteristic. A couple of hours after receiving in the cavity of the abdomen two or three cubic centimetres of a liquefied gelatin culture the animal becomes quiet and indisposed to eat or move about. Soon after it becomes somnolent, the head drooping for- ward and after a time restiug between the front legs, with the nose on the floor of its cage. It can be roused from this condition, and raises its head in an indifferent and stupid way when pushed or shaken, but soon drops off again into a profound sleep. Frequently the animals die in a sitting posi- tion, with their nose resting upon the floor of the cage between the front legs. I have not seen this lethargic condition produced by inoculations with any other microorganism. Convulsions sometimes occur at the moment of death. The time of death depends upon the potency of the culture and its quan- tity as compared with the size of the animal. From three to four cubi« centimetres of a liquefied gelatin culture usually kill a rabbit in from three to seven hours. The rapidity with which death occurs when a considerable quantity of a liquefied gelatin culture is injected into the cavity of the abdomen, and the somnolence which precedes death, give rise to the supposition that t^e lethal effect is due to the presence of a toxic chemical substance rather than to a multiplication of the bacillus in the body of the animal. And this view is supported by the fact that animals frequently recover when the dose admin- istered is comparatively small and especially when it is injected subcuta- neously. In all cases in which death occurs, even when but a few hours have elapsed since the inoculation was made, I have recovered the bacillus in cultures made from blood obtained from the heart or the interior of the liver, and, as stated, these cultures appear to have a greater virulence than those not passed through the rabbit. In sections of the liver and kidney stained with Loffler's solution of methylene blue the bacilli are seen, and are often in rather long-jointed fil- aments. 95. BACILLUS PYOCYANEUS. vxj. ^fc Jy^ ^ 2. "] ] Synonyms. — Bacillus of green pus ; Microbe du pus bleu; Bacil- len des grunblauen Eiters ; Bacterium aeruginosum. Obtained by Gessard (1882) from, pus having a green or blue color, and since carefully studied by Gessard, Charrin, and others. This bacillus appears to be a widely distributed saprophyte, which is found occasionally in the purulent discharges from open wounds, and some- times in perspiration and serous wound secretions (Gessard) . The writer obtained it, in one instance, FIG. 155. — Bacillus in cultures from the liver of a yellow-fever cada- X7°°' ver (Havana, 1888). Morphology. — A slender bacillus with rounded ends, somewhat thicker than the Bacillus murisepticus and of about the same length (Fliigge) ; frequently united in pairs, or chains of four to six elements; occasionally grows out into filaments. Biological Characters. — An aerobic, liquefying, motile bacil- lus. Grows readily in various culture media at the room tempera- 480 PATHOGENIC AEROBIC BACILLI ture — more rapidly in the incubating oven. Is a facultative anae- robic (Frankel). Does not form spores. The thermal death-point, as determined by the writer, is 56° C., the time of exposure being ten minutes. In gelatin plate cultures colonies are quickly developed, which give to the medium a fluorescent green color ; at the end of two or three days liquefaction commences around each colony, and usually the gelatin is completely liquefied by the fifth day. Before liquefaction commences the deep colonies, under a low power, appear as spherical, granular masses, with a serrated margin, and have a yellowish-green color ; the superficial colonies are quite thin and finely granular ; at the centre, where they are thickest, they have a greenish color, which fades out towards the periphery. In stick cultures in nutrient gelatin development is most abun- dant near the surface, and causes at first liquefaction in the form of a shallow funnel ; later the liquefied gelatin is separated from that which is not liquefied by a horizontal plane, and a viscid, yel- lowish-white mass, composed of bacilli, accumulates upon this sur- face, which gradually has a lower level as liquefaction progresses ; the transparent, liquefied gelatin above is covered with a delicate, yellowish-green film, and the entire medium has a fluorescent green color. Upon nutrient agar a rather abundant, moist, greenish-white layer is developed, and the medium acquires a bright green-color, which subsequently changes to olive green. Upon potato a viscid or rather dry, yellowish-green or brown layer is formed, and the potato beneath and immediately around the growth has a green color when freely exposed to the air or to the vapors of ammonia. In milk the casein is first precipitated and then gradually dissolved, while at the same time ammonia is developed. The green pigment is formed only in the presence of oxygen; it is soluble in chloroform and may be obtained from a pure solution in long, blue needles ; acids change the blue color to red, and reducing substances to yellow. According to Ledderhose, it is an aromatic compound resembling anthracene, and is not toxic. According to Gessard's latest researches (1890), two different pigments are produced by this bacillus, one of a fluorescent green and the other — pyocyanin — of a blue color. Cultivated in egg albumin the fluorescent green pigment, which changes to brown with time, is alone produced. In bouillon and in media containing peptone or gelatin both pigments are formed, and the pyocyanin may be obtained separately by dissolving it in chloroform. In an alkaline solution of peptone (two per cent) to which five per cent of glycerin has been added the blue pigment alone is formed. Pathogenesis. — The experiments of Ledderhose, Bouchard, and others show that this bacillus is pathogenic for guinea-pigs and rab- bits. Subcutaneous or intraperitoneal injections of recent cultures — NOT DESCRIBED IN PREVIOUS SECTIONS. 481 one cubic centimetre or more of a culture in bouillon — usually cause the death of the animal in from twelve to thirty-six hours. An ex- tensive inflammatory oedema and purulent infiltration of the tissues result from subcutaneous inoculations, and a sero-fibrinous or puru- lent peritonitis is induced by the introduction of the bacillus into the peritoneal cavity. The bacillus is found in the serous or purulent fluid in the subcutaneous tissues or abdominal cavity, and also in the blood and various organs, from which it can be recovered in pure cultures, although not present in great numbers, as is the case in the various forms of septicaemia heretofore described. When smaller amounts are injected subcutaneously the animal usually recovers after the formation of a local abscess, and it is subsequently immune when inoculated with doses which would be fatal to an unprotected animal. Immunity may also be secured by the injection of a con- siderable quantity of a sterilized culture. Bouchard has also pro- duced immunity in rabbits by injecting into them the filtered urine of other rabbits which had been inoculated with a virulent culture of the bacillus. It has been shown by Bouchard, and by Charrin and Guignard, that in rabbits which have been inoculated with a culture of the anthrax bacillus a fatal result may be prevented by soon after inoculating the same animals with a pure culture of the Bacillus pyocyaneus. The experiments of Woodhead and Wood indicate that the antidotal effect is due to chemical products of the growth of the bacillus, and not to an antagonism of the living bacterial cells. They were able to obtain similar results by the injection of sterilized cul- tures of Bacillus pyocyaneus, made soon after infection with the anthrax bacillus. Schimmelbusch (1894) reports that in researches made by Muh- sam this bacillus was found in the axilla, the anal region, or the in- guinal fold in fifty per cent of the healthy individuals examined. Its presence in wounds greatly delays the process of repair and may give rise to a general depression of the vital powers from the ab- sorption of its toxic products. Schimmelbush states that a physician injected 0.5 cubic centimetre of sterilized (by heat) culture into his forearm. That as a result of this injection, after a few hours he had a slight chill, followed by fever, which at the end of twelve hours reached 38.8° ; an erysipelatous - like swelling of the forearm oc- curred, and the glands in the axilla were swollen and painful. Re- covery occurred without the formation of an abscess. Buchner has related a similar case. Krannhals (1894) refers to seven cases in which a general pyocy- aneus infection in man was found, and adds an eighth from his own experience. In this the Bacillus pyocyaneus was obtained, post mor- 34 482 PATHOGENIC AEROBIC BACILLI tern, from green pus in the pleural cavity, from serum in the peri- cardial sac, and from the spleen, in pure culture. Martha, Gruber, Maggiora, Gradenigo, Kossel, and Rohrer have reported cases in which the Bacillus pyocyaneus has been obtained in pure cultures from pus obtained from the tympanic cavity in middle- ear disease. Kossel (1894) relates several cases in his own experience which led him to the conclusion that, in children, the Bacillus pyocy- aneus, through general blood infection or indirectly through the absorption of its toxic products, may be the cause of death. The following varieties of this bacillus have been described by bacteriologists : BACILLUS PYOCYANEUS /? (P. Ernst). Found in pus from bandages colored green. Morphology.— Slender bacilli from 2 to 4 //long — occasionally 5 to 6 n — and from 0.5 to 0.75 //broad; sometimes united in pairs, or chains of three elements. Biological Characters. — An aerobic, liquefying, actively motile, chro- mogenic bacillus. Produces a yellowish-green pigment; when old cul- tures are shaken up with chloroform and this is allowed to stand, three layers are formed — an upper, clouded, dirty-yellow layer ; below this is a milky, pale-green layer ; and at the bottom a transparent, azure-blue layer. Spore formation has not been demonstrated. Grows in the usual culture media at the room temperature— more rapidly at 35° C. Upon gelatin plates colonies are formed resembling those of the well-known Bacillus pyocyaneus, but liquefaction is more rapid. In gelatin stick cultures funnel-shaped liquefaction occurs at the upper -part of the line of puncture by the third day, and progresses more rapidly than is the case with. Bacillus pyocyaneus under the same circumstances ; on the fifth day a bluish-green color is de- veloped; by the twelfth day liquefaction has obliterated the entire line of growth and extends to the margins of the tube; the liquefied gelatin for a depth of about one centimetre has a dark emerald-green color, and a film consisting of bacilli is seen upon the surface. Upon the surface of agar a flat, greenish- white, dry layer is formed along the line of inoculation, and the agar around, at the end of a week, acquires a bluish-green color. Upon potato, at the end of three days, an abundant dry layer of a fawn brown color has developed ; this is surrounded by a pale-green coloration of the potato, and at points where the surface is fissured, an intense dark-green color is developed; the growth on potato has a more or less wrinkled appear- ance ; when one of the fawn-colored colonies is touched with the platinum needle, the point touched, at the end of two to five minutes, acquires an in- tense dark leaf-green color, which reaches its maximum intensity in about ten minutes, and has faded out again at the end of half an hour. Ernst con- siders this "chameleon phenomenon" the most characteristic distinction between the bacillus under consideration and Bacillus pyocyaneus. In milk a green color is developed at the surface, the casein is precipitated and sub- sequently peptonized. Bacillus pyocyaneus peri car ditidis. Found by H. C. Ernst in fluid obtained by tapping the pericardial sac of a man aged forty- seven years. Fluid was drawn from the pericardial sac on four dif- ferent occasions. The man subsequently "eloped." Ernst gives the following description of this bacillus : NOT DESCRIBED IN PREVIOUS SECTIONS. 483 ORIGIN. — Pericardial fluid, containing also bacilli of tuberculosis. FORM AND ARRANGEMENT. — Small straight bacilli, with rounded ends, three or four times as long as broad, and on most media slightly larger than the Bacillus pyocyaneus of Gessard, occurring -within the cells in the origi- nal fluid, and sometimes showing two or three end to end, but never observed in long chains. MOTILITY. — Actively motile in hanging-drop culture. No cilia or flagel- la have been demonstrated. GROWTH — Gelatin : Plates. — Colonies appear at the end of thirty-six to forty-eight hours as fine white points in the interior, and upon the surface of the medium ; edges are sharply defined ; soon there appears a circular zone of liquefaction, finally passing through the stratum of the medium with the colony at the bottom. Under a low power the centre of the colony may be of a brownish color. On the second day a greenish tinge may be seen about the individual colonies on the surface which spreads through the entire medium. The plates may always be distinguished from those of the Bacillus pyocyaneus of Gessard by the bluish-green when contrasted with the yellowish-green color of this latter. Gelatin: Needle Cultures. — At the end of twenty-four hours a small, saucer-shaped depression of liquefaction at upper end of needle track, which gradually spreads and deepens until the liquefaction extends straight across the tube, and about half-way down the needle track. A bluish-green fluores- cence appears about the liquefied portion at the very upper part of the gela- tin, later changing into a yellowish green. The colony is deposited as a yellowish, heavy sediment at the bottom of the liquefied portion, the upper part of which is clear. A small, whitish growth occurs along the remainder of the needle track. Old cultures, in which a certain amount of evapora- tion has occurred, assume a very dark greenish-black color. Agar-agar. — Along the needle track appears aflat, dry colony of a dirty grayish- white color spreading out upon each side of the needle track and growing at first upon the surface of the water of condensation, later depos- iting a white sediment at the bottom. From the first there may be detected, by reflected light, a metallic lustre on the surface of the colony in places, which metallic sheen later spreads over the whole colony and furnishes a marked differentiating point. In addition to this, within twenty-four to forty-eight hours at 37° C., there appears a green fluorescence throughout the whole of the medium, which increases slowly to a marked bluish-green color, and never assumes the nut-brown of the Bacillus pyocyaneus of Gessard upon the same medium. The colony is not especially viscid. Potato. — There appears a reddish-brown colony along the needle track, elevated and moist, confined to the line of the needle. It presents no change of color upon touching with the needle, but certain specimens (as do some of the Bacillus pyocyaneus) develop later a heavy green color extending over the whole surface of the potato, which later changes almost to black. Bouillon.— Twenty-four hours at 37° C. gives a growth, especially on the surface, which is a wrinkled scum ; no cloudiness of the bouillon, and a very faint greenish fluorescence one centimetre below the surface. At this time it differs from the Bacillus pyocyaneus of Gessard, in that the latter shows cloudiness of the medium all through. Later the same cloudiness appears in bouillon cultures of this new bacillus, together with a whitish sediment de- posited at the bottom of the tube, and then the cultures are indistinguishable from each other. The same changes, but slower, occur at room tempera- ture. Peptone.~One, 3.5, and six-per-cent solution. Twenty-four hours at 37° C. gives a faint bluish tinge at upper edge of medium with very faint cloudi- ness ; later (in one or two weeks) there forms a marked scum upon the sur- face that is difficult to break up by shaking, and the whole medium assumes a grass-green color of more or less intensity, and not seen on other similar bacilli. The shape and size of the organism, under the microscope, differ 484 PATHOGENIC AEROBIC BACILLI very markedly in this medium from any other bacilli examined. The same changes are to be seen at room temperature, but more slowly. Egg- Albumin: Plain. — Twenty four hours at 37° C., yellowish-white, very prof use growth all along the needle track ; yellowish-green spreading out liom it almost to sides of tube, and in the condensation water as well. The growth has no especial distinguishing characteristics. Irregular lique- faction occurs, but the growth at no time differs in any marked way from other varieties of the Bacillus pyocyaneus. Blood Serum. — Twenty-four hours at 37° C. shows flat, moist colony with bluish-green fluorescence in its neighborhood. Liquefaction begins early and goes on slowly until complete in from one to two weeks, with an increasing intensity of color which becomes markedly blue, and eventually almost black. Milk.— Behaves as do the other bacteria. BEHAVIOR TO TEMPERATURE. — Grows at 15°-25° C. slowly; much more freely at 35J-38° C., when it produces the coloi more quickly. RAPIDITY OF GROWTH. — Moderate. SPORE-PRODUCTION. — Not observed. NEED OF AIR. — Does not grow under mica. Facultatively anaerobic, but does not produce color except with free access of oxygen. GAS-PRODUCTION. — Produces faint foul odor. BEHAVIOR TO GELATIN.— Liquefies gelatin slowly. COLOR-PRODUCTION. — Produces a bluish-green color which in old cul- tures changes almost to a black. Upon the addition of acids (both vegetable and mineral) to cultures the color changes to red, and upon the addition of alkalies a bright grass-green appears. This reaction is best seen in bouillon and gelatin cultures, but occurs in other media as well, notably blood-serum. BEHAVIOR TO ANILINE DYES.— Stains easily and well with any of the aniline dyes usually employed, and by Gram's method. MICROSCOPIC APPEARANCE IN DIFFERENT MEDIA. — Under the micro- scope, its general appearance on various media is of a rod larger than the Bacillus pyocyaneus. In peptone cultures this difference is verv marked. In this case, the Bacillus pyocyaneus tested appeared as very short, oval, bacilli, almost like micrococci, while the new bacillus showed as a long, fine rod, from four to six times as long as broad — length about one-half the diameter of a red-blood corpuscle — and arranged sometimes two or three end to end. These same cultures transferred to gelatin became indistin- guishable from each other in size. PATHOGENESIS.— Injections of small quantities (0.5 centimetre) of a bouil- lon culture twenty-four hours old into the abdominal cavity of rabbits and guinea-pigs, killed fifty per cent in from twenty-four to thirty-six hours. Autopsy showed general congestion of abdominal viscera, slight effusion into the peritoneal cavity, and cover-glass preparations and cultures showed the bacilli in the effusion in the abdominal cavity, as well as in the blood from the heart and various organs. 96. BACILLUS OP FIOCCA. Found by Fiocca in the saliva of cats and dogs. Closely resembles the influenza bacillus of Preiffer and of Canon. Morphology. — Resembles the bacillus of rabbit septicaemia, but is only half as large— from 0.2 to 0.33 n in breadth. The length is but little greater than the breadth. Usually seen in pairs, closely resembling diplococci. When cultivated on potato it appears to be a micrococcus, but in the blood of infected animals and in bouillon cultures it is seen to be a short bacillus. Stains with difficulty with the usual aniline colors, but is readily stained by Ehrlich's method or with Ziehl's solution. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Spore formation not observed. Grows best NOT DESCRIBED IN PREVIOUS SECTIONS. 485 at 37° C. and does not develop at temperatures below 15° C. In agar plates, at 37° C., small, punctiform colonies are developed at the end of twenty-four hours; these do not increase in size later; under the microscope the deep colonies are seen to be spherical, granular, and dark yellow in color ; the superficial colonies are more or less round, with irregular outlines, trans- parent, slightly granular, and often have a shining nucleus at the centre. Upon gelatin plates the colonies have a similar appearance, but are not vis- ible in less than four or five days. In streak cultures upon the surface of agar small, punctiform colonies are seen along the track of the needle at the end of twenty-four hours, resembling fine dewdrops; the following day these colonies are a little larger and less transparent; they remain distinct, especially along the margins of the line of growth. Upon potato a very thin, transparent layer is developed, which does not change the appearance of the surface of the potato, but slightly increases its resistance to the plati- num needle. In bouillon small flocculi. suspended in the clear liquid, are developed within twenty -four hours; these subsequently sink to the bottom. Milk is not coagulated by this bacillus, and no gas is produced in media containing sugar. Pathogenesis. — Pathogenic for rabbits, guinea-pigs, young rats, and mice, in which animals it produces general infection, and death- in rabbits— at the end of twenty-four hours. The bacillus is found in the blood in great numbers. 97. PROTEUS VULGARIS. Obtained by Hauser (1885) from putrefying animal substances, and since shown to be one of the most common and widely distrib- uted putrefactive bacteria. This and the other species of Proteus FIG. 156.— Proteus vulgaris; '* swarming islands " from a gelatin culture. X 285. (Hauser.) described by the same bacteriologist (Proteus mirabilis, Proteus Zen- keri) have no doubt frequently been encountered by previous observ- ers, and are among the species formerly included under the name 66 Bacterium termo," which was applied to any minute motile bacilli found in putrefying infusions. Morphology. — Bacilli with rounded ends, about 0.6 // broad, and 486 PATHOGENIC AEROBIC BACILLI varying greatly in length, being sometimes short oval, and at others from 1.25 to 3.75 /* in length ; also grow out into flexible filaments, which may be more or less wavy or spiral in form. The short rods are commonly seen in pairs ; they have terminal flagella ; involution forms are frequently seen, the most common being spherical bodies about 1.6 //in diameter. In old cultures in bouillon, or in cultures made in meat infusion in the incubating oven, the short oval forms greatly predominate, but in recent cultures in nutrient gelatin fila- ments of considerable length are encountered in association with shorter rods. Stains readily with fuchsin or gentian violet — not so well with the brown aniline colors ; does not stain by Gram's method (Cheyne). Biological Characters. — Anaerobic and. facultative anaerobic, liquefying, motile bacillus. Grows rapidly in the usual culture media at the room temperature. The growth upon gelatin plates (five per cent of gelatin) at the room temperature is very characteristic ; at the end of six or eight hours small depressions in the gelatin are observed, which contain liquefied gelatin and grayish- white masses of bacilli. Under a low power these depressions are seen to be surrounded by a marginal zone consisting of two or three layers, outside of which is a zone of a single layer, from which amoeba-like processes extend upon the sur- face of the gelatin. These processes are constantly undergoing changes in their form and position, and may become separated from the mother colony, or remain temporarily attached to it by a narrow thread consisting of bacilli ; after a time the entire surface of the gelatin is covered with wandering, amoeba-like colonies ; these rapidly cause liquefaction, which by the end of twenty-four to forty- eight hours has reached a depth of one millimetre or more over the entire surface. The deep colonies also are surrounded by processes projecting into the gelatin, which may be observed to suddenly ad- vance and again to be retracted towards the central zoogloea-like mass. Liquefaction around the colony rapidly progresses, and actively motile rods and spiral filaments may be seen about the peri- phery of this liquefied gelatin, while about it is a radiating crown of irregular processes, some of which may be screw-like or corkscrew- formed. In ten-per-cent gelatin the migration of surface colonies, above described, is not observed. In gelatin stick cultures liquefac- tion occurs along the entire line of puncture, and soon the contents of the tube are completely liquefied ; near the surface of the liquefied gelatin the growing bacilli form a grayish-white cloudiness, and at the bottom of the tube an abundant flocculent deposit is formed. Upon the surface of nutrient agar a rapidly extending, moist, thin, grayish- white layer is formed. Upon potato this bacillus produces a NOT DESCRIBED IN PREVIOUS SECTIONS. 487 dirty-white, moist layer. The cultures in media containing albumin or gelatin have a putrefactive odor and acquire a strongly alkaline reaction. A temperature of 20° to 24° C. is most favorable for the growth of this bacillus. It is a facultative anaerobic and grows in an atmosphere of hydrogen or of carbon dioxide, although not so rapidly as in the presence of oxygen. The movements are often ex- tremely active and difficult to follow under the microscope ; again they may be quite deliberate, or the bacilli may remain motionless for a time and again dart off in active motion. The long terminal flagella may sometimes be discerned by means of a good objective and careful manipulation of the light. Pathogenesis. — Pathogenic for rabbits and for guinea-pigs when injected into the circulation, into the cavity of the abdomen, or sub- cutaneously in considerable quantity. Cultures in nutrient gelatin are said by Cheyne to be more pathogenic (toxic) than those in bouil- lon. When injected into the muscles of rabbits a much smaller dose produces a fatal result than when injected subcutaneously. In Cheyne's experiments, made in London (1886), one-tenth cubic centimetre of a liquefied gelatin culture, injected into the dorsal muscles, was invariably fatal in from twenty-four to thirty-six hours; a dose of one-twentieth cubic centimetre, injected in the same way, usually caused death; while one- fortieth cubic centimetre gave rise to an extensive local abscess, and the animals died at the end of six or eight weeks. Doses of less than one-five-hundredth cubic centimetre produced no effect. Cheyne estimates that one cubic centimetre of a culture in nutrient gelatin contains 4,500,000,000 bacilli, and, conse- quently, that a smaller number than 9,000,000 produced no effect when injected into the muscular tissue of rabbits. Injections into the sub- cutaneous connective tissues of a dose twice as large as that which in- variably proved fatal when injected into the muscles usually caused an extensive abscess, but did not kill the animal; and, after re- covery from the effects of such an injection, the rabbit was found to be immune against a similar dose injected into the muscles. Foa and Bonome have succeeded in producing immunity against the effects of virulent cultures of this bacillus by inoculating rabbits with filtered cultures, and also by injecting beneath the skin of these ani- mals a solution of neurin, which they believe to be the principal toxic product present in the cultures. Proteus Vulgar is in Cholera Infantum. — The extended re- searches of Booker have led him to the conclusion that this bacillus plays an important part in the production of the morbid symptoms which characterize cholera infantum. Proteus vulgaris was found in the alvine discharges in a considerable proportion of the cases ex- amined, but was not found in the faeces of healthy infants. " The 488 PATHOGENIC AEROBIC BACILLI prominent symptoms in the cases of cholera infantum in which the proteus bacteria were found were drowsiness, stupor, emaciation and great reduction in flesh, more or less collapse, frequent vomiting and purging, with watery and generally offensive stools." The researches of Krogius, Schnitzler, Schmidt and Aschoff, and others, show that in cases of c}'stitis and of pyelonephritis this bacil- lus is frequently found in pure cultures, or associated with other bac- teria. The authors last named state that in sixty cases of cystitis reported by various authors the colon bacillus was found in pure cul- tures, and in thirteen cases the proteus of Hauser. Next to Bacillus coli communis Proteus vulgar is appears to be the microorganism most frequently concerned in the etiology of pyelonephritis. Levy (1895) isolated from sour yeast a bacillus, which he identified as "Proteus Hauseri," and made numerous experiments on dogs to test its pathogenic power. From five to ten cubic centimetres of a liquefied gelatin culture injected into the circulation, through a vein, caused the typical symptoms of "sepsin poisoning," as formerly de- scribed by Bergmann and Schmeideberg (1868). In two dogs which died at the end of forty-eight hours the intestinal tract was found in a condition of intense hemorrhagic infiltration. The spleen and glands of the mesentery were much enlarged. But a bacteriological examination gave an entirely negative result, showing that death resulted from toxemia and not from septicaemia. Further experi- ments showed that the dried precipitate obtained from liquefied gela- tin cultures, by the addition of alcohol, had the same pathogenic action on dogs, rabbits, and mice as cultures containing the living bacilli. That a similar pathogenic effect is produced in man by the products of growth of this bacillus was shown by the following facts: While conducting his experiments Levy had an opportunity to make a bacteriological examination in the case of a man who died after a brief attack of cholera morbus. From the vomited material and the stools he obtained a pure culture of proteus ; but the blood, collected at the autopsy, was sterile. In the mean time seventeen other per- sons who had eaten at the same restaurant were taken sick in the same way. Upon an examination at the restaurant it was found that the bottom of the ice chest in which the proprietor kept his meats was covered with a slimy, brown layer, which gave off a disagreeable odor. Cultures from this gave the proteus as the principal micro- organism present. Levy concludes from his own investigations and those of other bacteriologists that in so-called " flesh-poisoning " bac- teria of this group are chiefly at fault, and that the pathogenic effects are due to toxic products evolved during their development. NOT DESCRIBED IN PREVIOUS SECTIONS. 489 98. PROTEUS OF KARLINSKI. Synonym. — Bacillus murisepticus pleomorphus (Karlinski). Probably identical with Proteus vulgaris of Hauser. Obtained by Karlinski (1889) from a fibro-purulent uterine discharge, and from abscesses in the uterus and its appendages in a puerperal woman. Morphology. —Resembles Proteus vulgaris of Hauser in its morphology, and presents various forms under different circumstances relating to the culture medium, the temperature, age of culture, etc. — sometimes as spheri cal or short oval cells, at others as longer or shorter rods or spiral filaments; usually as bacilli with round ends two and a half times as long as thick, often united in pairs. Stains with the usual aniline colors, but not by Gram's method. Biological Characters. — An aerobic arid facultative anaerobic, liquefy- ing, motile bacillus. Spore formation not observed. Grows rapidly in the usual culture media at the room temperature. In gelatin plate cultures, at the end of ten hours, small colonies are developed which nave well-defined outlines, are oval or whetstone-shaped, of a light-brown color by transmitted light and white by reflected light, with a somewhat darker margin and a smooth surface, sometimes marked by shallow clef ts ; at the end' of twenty hours the colonies commence to have irregular margins, and the surface of the gelatin above them is marked by concentric rings. At the end of thirty hours the colonies have formed a bulb-shaped liquefaction of the gelatin, and delicate, ray like offshoots are seen around the margin. At the end of two days the bulbous cavities are about one and a half millimetres in diameter and contain a cloudy, grayish -white liquid ; they are surrounded by a moist- looking, gray, irregular marginal zone. In gelatin stick cultures, at the end of twenty-four hours, a funnel shaped liquefaction of the gelatin occurs near the surface, and a grayish- white, cloudy mass is developed along the line of puncture; at the end of forty- eight hours a sac-like pouch of liquefied gela- tin has formed, and in the course of four or five days the gelatin is entirely liquefied. Upon agar plates the colonies are at first oval in form and white by reflected light, or pale brown by transmitted light ; at the end of thirty hours the surface becomes wrinkled or folded and is surrounded by radiat- ing, delicately twisted offshoots. Upon the surface of agar a white layer is developed. Upon potato a whitish-gray, soft, homogeneous layer, which after standing along time has a darker 'color. Upon blood serum a thin, grayish-white layer is formed and the serum is rapidly liquefied. Gelatin cultures acquire a strongly alkaline reaction and give off a disagreeable odor resembling that of butyric acid. Pathogenesis. — White mice inoculated at the root of the tail die in from twenty-two to twenty-four hours ; the spleen is greatly enlarged; the bacilli are found in blood from the various organs — less numerous in blood from the heart. Field mice and house mice are less susceptible. Subcutaneous injections in rabbits may give rise to local inflammation and also to general infection. In white rats and guinea-pigs a local abscess may result from a subcutaneous inoculation, 99. PROTEUS MIRABILIS. Obtained by Hauser (1885) from putrefying animal substances. Morphology. — Bacilli resembling very closely the preceding species (Pro- teus vulgaris), but presenting more numerous involution forms, which may be spherical, pear-shaped, or spermatozoa-like, etc. The bacilli are about 0 6 // in diameter and vary greatly in length, being sometimes nearly spheri- cal, or forming rods of 2 to 3.75 // in length, or long filaments. Biological Characters. — An aerobic said facultative anaerobic, liquefy- ing, motile bacillus. Spore formation has not been observed. Grows in the usual culture media at the room temperature. Does not liquefy gelatin as 490 PATHOGENIC AEROBIC BACILLI rapidly as Proteus vulgaris. Upon gelatin plates, at the end of twelve hours, superficial colonies of two to three millimetres in diameter are formed ; under a low power these appear finely granular and brownish in color, and have an irregular outline; outgrowths from the margin extend in various directions and form new colonies, which may be attached for a time by a long and slender thread consisting of bacilli. The movement of these new colonies is not as pronounced as in the case of the preceding species, and Fio. 157.—" Swarming islands " of Proteus mirabilis, from a gelatin culture, x 285. (Hauser.) they are characterized by the presence of numerous distorted bacilli — invo- lution forms. The deep colonies form spiral zoogloea masses. In gelatin stick cultures the whole surface is first covered with threads and islands of bacilli, which after a time form an anastomosing network, and finally a confluent layer which at the end of forty-eight hours is rather thick, FIG. 158.— Spiral zooglosa from a culture of Proteus mirabilis. X 95. (.Hauser.) with a moist, shining surface and grayish color, and appears to be perforated with numerous small, sieve-like openings. These thinner and transparent places disappear after a time, and at the end of two or three days liquefac- tion of the gelatin commences; complete liquefaction does not occur until the fifth or sixth day, or even later. Along the line of puncture finely gran- ular colonies are first formed, from which long threads are given off, which form after a short time a tolerably broad zone of threads and spiral zoogloea masses. NOT DESCRIBED IN PREVIOUS SECTIONS. 491 Pathogenesis. — In Hauser's experiments filtered cultures (two to six cubic centimetres), injected into the circulation or into the cavity of the abdomen in rabbits, caused fatal toxaemia. 100. PROTEUS ZENKERI. Obtained by Hauser (1885) from putrefying animal substances. Morphology. — Bacilli which vary greatly in length — average about 1. 65 //, and about 0.4 ju broad. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon the surface of nutrient gelatin a laminated mass forms about the point of puncture, from the peri- phery of which offshoots are given off, at the extremities of which colonies are formed, as in the case of Proteus mirabilis. Gradually a rather thick, grayish-white, opaque layer is formed, which covers the entire surface of the gelatin and is easily detached from it. This species is distinguished from the two preceding by the fact that it does not liquefy gelatin or blood serum and does not give off a decided putrefactive odor when cultivated in these media. Pathogenesis. — Considerable quantities injected into small fl.tmna.1a give rise to local abscesses and to symptoms of toxaemia. 101. PROTEUS SEPTICUS. Obtained by Babes (1889) from the mucous membrane of the intestine and the various organs of a boy who died of septicaemia. Morphology. — Bacilli about 0 4/z broad and varying greatly in length; slightlv curved rods or flexible filaments, often associated in loose chains. Stains by the usual aniline colors and by Gram's method. Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. In gelatin plates centres of liquefaction are quickly formed and rapidly extend. The spherical, liquefied places have at first a wavy or dentate outline, and are surrounded by a branching, transparent, granular margin which rapidly extends in advance of the liquefaction. In stick cul- tures in nutrient gelatin liquefaction of the entire con tents of the tube may take place within twenty-four hours, or a broad, liquefied sac is formed along the line of puncture. Gelatin cultures give off a very disagreeable odor. Upon the surface of nutrient agar, at 37° C., a peculiar, thick net- work extends over the surface in the course of a few hours. Upon potato an elevated, brownish-white, shining layer is formed. Blood serum is lique- fied by this bacillus. Pathogenesis. — Pathogenic for mice, less so for rabbits. In mice death occurs in from one to three days after the subcutaneous injection of a small quantity of a pure culture ; the bacilli are present in the blood in small numbers. 102. PROTEUS LETHALIS. Synonym. — Proteus bei Lungengangran des Menschen (Babes). Obtained by Babes (1889) from the spleen and gangrenous portions of the lung of a man who died of septicaemia. Morphology — Short rods with round ends, from 0.8 to 1.5 >u thick ; often swollen in the middle, like a lemon or a flask ; forms short, flexible filaments which also present similar swellings. Stains with the usual aniline colors and also by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- fying, motile bacillus. Not observed to form spores. Grows in the usual culture media at the room temperature. In gelatin plates forms hemi- spherical, elevated, whitish, translucent colonies, which later send out 492 PATHOGENIC AEROBIC BACILLI coarse branches which ramify over the surface of the gelatin. A similar growth is observed upon the surface of gelatin stick cultures, and an abun- dant development takes place along the line of puncture. Upon nutrient agar a thick, opaque, slightly yellowish layer is formed. Upon potato a moist, shining, brownish layer is developed, and the potato acquires a brownish color. Upon blood serum the growth is less abundant than on agar; the blood serum is not liquefied. This bacillus grows rapidly at the room temperature; it is destroyed by a temperature of 80° C., and presum- ably does not form spores. Pathogenesis. — Recent cultures are very pathogenic for mice and for rabbits, less so for guinea pigs. The subcutaneous injection of a small quantity of a pure culture kills susceptible animals in two or three days. More or less oedema is found at the point of inoculation. Injections into the rectum of rabbits gave rise to haemorrhagic enteritis, peritonitis, and death at the end of four days. 103. BACILLUS A OF BOOKER. "Obtained by Booker (1889) from the alvine discharges of children suffer- ing from cholera infantum. Morphology. — Bacilli with round ends, varying greatly in length, usually three to four /* long and 0 7 JJ. broad (in recent agar cultures). In older cul- tures the bacilli are shorter and smaller. Biological Characters. — An aerobic and facultative anaerobic, lique- fying, motile bacillus. Grows at the room temperature in the usual culture media. In gelatin plates colonies are visible at the end of twenty-four hours; under the microscope these are nearly colorless, and liquefaction soon occurs around them. In gelatin stick cultures complete liquefaction occurs in three or four days. Upon agar a colorless layer covering the entire surface is developed in tnree or four days, and an abundant development occurs along the line of puncture. Agar colonies have a bluish look, and are surrounded by an indistinct halo which shades off gradually into the surrounding agar ; under a low power the colonies are light-brown and the borders indistinct; the surface has a delicate, wavy appearance. Upon po- tato the growth is luxuriant and of a dirty-brown color. Blood serum is liquefied by this bacillus. Milk is coagulated into a gelatinous mass having an alkaline reaction ; later the coagulum is dissolved. Pathogenesis. — Mice and guinea-pigs fed with cultures in milk die in from one to eight days. 104. BACILLUS ENDOCARDITIDIS GRISEUS. Obtained by Weichselbaum (1888) from the affected valves in a case of endocarditis recurrens ulcerosa. Morphology. — Short rods with rounded or somewhat pointed ends, about two to three times as long as broad— of about the same dimensions as the bacillus of typhoid fever." Stains with the usual aniline colors and also by Gram's method; the longer rods from old cultures are irregularly stained. Biological Characters. — An aerobic, non liquefy ing, motile bacillus. Refractive bodies may be seen in some of the rods, which resemble spores and are stained by the method of Ernst, but they do not show the resistance of known spores to physical and chemical agents. Grows well in the usual culture media at trie room temperature. Upon gelatin plates colonies are formed which resemble those of Friedlander's bacillus, but which gradually acquire a gray or grayish-white color. The prominent, convex, superficial colonies under a low power are finely granular and grayish brown in color; the deep colonies are yellowish-brown in color, have slightly notched mar- gins, and the surface is covered with minute projections. In stick cultures NOT DESCRIBED IN PREVIOUS SECTIONS. 493 a rather thin, circular layer forms about the point of puncture; this has the appearance of stearin ; later it becomes grayish- white and the margins are marked by radiating lines. Upon the surface of nutrient agar a similar growth occurs which has a pale-brown or reddish-gray color. Upon potato in the incubating oven an abundant development occurs, forming a dry looking layer of a grayish-brown color and having irregularly notched mar- gins. Upon blood serum an abundant, grayish-white growth of cream-like consistence forms along the impfstrich; later this has a reddish gray color. This bacillus grows to the bottom of the line of puncture in stick cultures, and is no doubt a facultative anaerobic. Pathogenesis. — Pathogenic for white mice and for guinea-pigs. 105. BACILLUS ENDOCARDITIDIS CAPSULATUS. Obtained by Weichselbaum (1888) from thrombi and embolic infarctions in the spleen and kidneys of a man who died from endocarditis with forma- tion of thrombi. Morphology. — Resembles Friedlander's bacillus, and is frequently sur- rounded by a capsule, which may be stained ; also forms long, curved fila- ments, in the protoplasm of which vacuoles may be observed in stained pre- parations. Stains with the usual aniline colors, but not by Gram's method; by staining with fuchsin and carefully decolorizing with diluted alcohol the presence of a capsule may be demonstrated. Biological Characters.— An aerobic, non -liquefying bacillus. Grows in the usual culture media at the room temperature. In gelatin stick cultures development occurs along the line of puncture, and on the surface as a rather thin, white, dry layer which resembles stearin. In agar plates the superficial colonies are thin, about two millimetres in diameter and gray in color ; under a low power the margins are trans- parent and colorless, and the centre resembles the deep colonies; these are very small and grayish white in color ; under a low power the surface is seen to be covered with tooth-like, projecting masses, the margin is dentate and has a pale-yellow color, while the centre is yellowish-brown. Pathogenesis. — Rabbits are killed by the injection of a considerable quan- tity of a pure culture into the cavity of the abdomen or subcutaneously. 106. BACILLUS OF LESAGE. Obtained by Lesage (1887) from the green-colored discharges of infants suffering from " green diarrhoea," and supposed to be the cause of this com- plaint (?). According to Baumgarten, this bacillus is probably identical with a well-known pigment-producing saprophyte— the Bacillus fluorescens non liquefaciens. Morphology. — Small bacilli with round ends, about 2 4 /* long and 0.75 to I/* broad ; in old cultures may grow out into long filaments. Stains with the usual aniline colors, but not by Gram's method. Biological Characters — An aerobic, non-liquefying (slight liquefaction in old cultures), motile bacillus. Forms spores. Grows slowly at the room temperature in the usual culture media, more rapidly at 25° to 35° C. Upon gelatin plates superficial colonies are formed which have irregularly dentate, leaf-like margins and a smooth surface ; they produce a greenish color in the gelatin. In gelatin stick cultures a thin, smooth, transparent, greenish layer forms upon the surface, and in the course of four or five days the gela- tin has acquired throughout a bright-green color. Upon potato a dark- green layer is formed. The cultures have the odor of old urine. Pathogenesis. — The injection of a considerable quantity of a pure culture into the ear vein of a rabbit is said to have produced green diarrhoea, and the same result was obtained by mixing cultures with the food of these ani- mals. These results have not yet been confirmed by other investigators. 494 PATHOGENIC AEROBIC BACILLI 107. BACILLUS OF DEMME. Obtained by Demme (1888) from the fluid contents of the tumors and pustules of erythema nodosum, and also from the blood of the affected indi- vidual. Morphology.— Bacilli with round ends, from 2.2 to 2.5 // long and 0.5 to 0.7/u broad; usually collected in smaller or larger groups. Stains with the usual aniline colors and by Gram's method. Biological Characters. — An aerobic (facultative anaerobic?) bacillus, which does not grow in nutrient gelatin at the room temperature. Grows in nutrient agar at 35° to 37° C. Forms spores. In agar plates, at 35° to 37° C., smooth, spherical, shining white colonies are formed in from forty-eight to sixty hours, whicli at the end of six or seven days may have the size of a small coin — five centimes; these are marked by lines radiating from the centre, which are slightly elevated above the surface of the colony and have a silvery lustre by obliquely reflected light; the margins of the colony are fringe-like, and after ten or twelve days conical offshoots are given off from this thready margin. In agar stick cultures growth occurs along the line of puncture in the form of a thorny column which has a paraffin-like lustre. Pathogenesis. — According to Demme, when injected subcutaneously into guinea-pigs, or by rubbing pure cultures into the scarified skin, an eruption occurs which resembles that of erythema nodosum and is followed by a gangrenous condition of the skin. Rabbits, dogs, and goats proved to be refractory. 108. BACILLUS CEDEMATIS AEROBICUS. Synonym. — A new bacillus of malignant osdema (Klein). Obtained from garden earth by inoculation in guinea-pigs. Morphology. — Bacilli from 0.8 to 2.4 // in length and 0.7 u thick; grow out into long filaments. Stains with the usual aniline colors, but not by Gram's method. Biological diameters. — An aerobic and facultative anaerobic, non-lique- fying, motile bacillus. Does not form spores. Grows at the room tempera- ture in the usual culture media. Upon gelatin plates, at the end of twenty- four hours, small, gray, punctiform colonies are developed ; at the end of forty-eight hours the superficial colonies are seen as flat, grayish, transparent plaques, the margins of which are thin and irregularly notched ; these attain a diameter of several millimetres in the course of a few days. The deep colo- nies do not exceed the diameter of a pin's head ; they remain spherical, and by transmitted light have a brownish color. In gelatin stick cultures a, white line of growth is developed along the track of the inoculating needle, and at the bottom of this isolated, punctiform colonies are seen ; upon the surface a flat, thin, transparent, grayish layer with a dentate margin is developed. Upon the surface of agar a smeary, grayish-white stripe is de- veloped along the impfstrich. Alkaline bouillon, at the end of twenty-four hours at 37J C., is densely clouded, and later contains numerous flocculi, but no pellicle upon the surface ; at the end of twenty-four hours the reaction becomes strongly alkaline. Upon potato a viscid, yellowish stripe is devel- oped along the line of inoculation. In deep cultures in nutrient gelatin gas bubbles are developed in from twenty-four to forty-eight hours; these are attached to the isolated colonies. Pathogenic for guinea-pigs, rabbits, and white mice. The animals die within twenty four hours — when very small quantities are injected subcu- taneously into guinea-pigs they may live for two or three days and sometimes recover. The lethal dose of a bouillon culture is from one-fourth to one- half cubic centimetre, but one drop of the cedematous fluid from the subcu- taneous connective tissue of an inoculated animal is infallibly fatal. In guinea-pigs an extensive inflammatory oedema is produced by subcutaneous inoculations ; the spleen is but slightly enlarged. In rabbits but slight oedema NOT DESCRIBED IN PREVIOUS SECTIONS. 495 and a small spleen. In mice no oedema and a slightly enlarged spleen. The bacilli are found in the blood of the heart in small numbers, and are some- what more numerous in the spleen, especially in mice. 109. BACILLUS OF LETZERICH. Obtained by Letzerich (1887) from the urine of children suffering from "nephritis interstitialis primaria." Etiological relation not satisfactorily demonstrated. Morphology. — Bacilli with round ends, straight or slightly curved, often forming filaments. Stains with the usual aniline colors. Biological Characters.— An aerobic, liquefying bacillus. Forms spores. Grows rapidly in nutrient gelatin at a comparatively low temperature — best at 14° C. Upon gelatin plates, at 14° C., complete liquefaction has occurred in from thirty-six to forty-eight hours, and a thin, white film covers the surface of the liquefied gelatin; the same in gelatin stick cultures. Pathogenesis. — Rabbits injected in the cavity of the abdomen are said to die in about fourteen days. The autopsy shows an extensive abscess, en- largement and congestion of the kidneys, enlarged spleen, etc. The bacilli are found in great numbers in all of the organs. 110. BACILLUS OP SCHIMMELBUSCH. Obtained by Schimmelbusch (1889) from the necrotic tissues at the boun- dary line of the still living tissues in cancrum oris, or noma. Etiological relation not proved. Morphology. — Small bacilli with round ends; often united in pairs; may grow out into long filaments. Stains best with an aqueous solution of gentian violet; does not stain by Gram's method. Biological Characters. — An aerobic, non-liquefying bacillus. Grows in the usual culture media at the room temperature— better in the incubating oven at 30° to 37° C. Upon gelatin plates forms below the surface spheri- cal, finely granular, grayish-white colonies, which come to the surface and form elevated masses with slightly dentate margins and an irregularly cleft surface. In gelatin stick cultures the growth along the line of inoculation is coarsely granular ; upon the surface a broad, flat layer. Upon the sur- face of agar, in twenty-four hours at 37° C., a grayish- white layer along the line of inoculation, which is smooth and about three millimetres in breadth. Upon potato, at the end of two weeks, a broad, moist, grayish- white layer from two to three millimetres wide. Upon coagulated ascitic fluid, at the end of twenty-four hours, a thin layer along the impfstrich, from which lateral offshoots are given off. Pathogenesis.— Cultures injected subcutaneously into rabbits produced local abscesses only ; not pathogenic for mice or pigeons. 111. BACILLUS FCETIDUS OZ^EN^E. Obtained by Hajek (1888) from the nasal secretions of patients with ozae- na. Etiological relation not proved. Morphology. — Short bacilli, but little longer than broad; usually in pairs, or in chains of six to ten elements. Stains with Loffler's solution of methylene blue or solutions of aniline colors in aniline water — not so well in aqueous solutions; does not stain by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates the colonies, at the end of thirty-six hours, are scarcely visible, with well-defined but 496 PATHOGENIC AEROBIC BACILLI somewhat irregular outlines; later liquefaction commences and crater-like depressions in the gelatin are formed, in which a gas bubble is seen ; com- plete liquefaction occurs in the course of a few days. In gelatin stick cul- tures liquefaction occurs all along the line of inoculation, and is complete at the end of from eight to fourteen days. Upon agar plates the colonies are granular in the centre, and the margins, under a low power, are seen to be fringed. Upon the surface of agar a moist, slimy layer is formed along the impfstrich. Upon, potato, at the end of twenty-four hours, a yellowish- brown layer is formed. Upon blood serum development is rapid in the form of a whitish layer, which extends over the whole surface. The cultures, and especially those kept in the incubating oven, give off a disagreeable putrefactive odor, which is most intense in the blood-serum cultures. Pathogenesis. — Pathogenic for mice. When injected subcutaneously into rabbits it gives rise to intense local inflammation and progressive gan- grene of the connective tissue. 112. BACILLUS OF LUMNITZER. Obtained by Lumnitzer (1888) from the bronchial secretions of persons suffering from *' putrid bronchitis." Etiological relation not demonstrated. Morphology. — Bacilli with round ends, from 1.5 to 2n long, somewhat curved. Stains with the usual aniline colors. Biological Characters. — An aerobic, motile bacillus. Does not grow in nutrient gelatin at the room temperature. Grows slowly upon agar and more rapidly upon blood serum at 36° to 38° C. Forms spores. Upon agar plates, at 37° C , small, grayish-white colonies are formed in two or three uays; upon the surface these form hemispherical masses which slowly in- crease in size. At the end of six or seven days the cultures give off a dis- agreeable odor, quite like that given off by the sputum of the cases of putrid bronchitis from which the bacillus was obtained. Upon the surface of blood serum the growth is rapid and forms grayish-white, shining colonies, of about one millimetre in diameter, which become confluent at the end of about four days, and cover the entire surface in eight or nine days. Pathogenesis. — Causes a purulent inflammation when injected into the lungs of rabbits, which involves the bronchial tubes, the blood vessels, and the pulmonary alveoli ; when injected subcutaneously produces inflamma- tion and necrosis of the tissues. 113. BACILLUS OF TOMMASOLI. Obtained by Tommasoli (1889) from the hairs of the head of a patient suf- fering from a form of sycosis supposed to be due to the presence of this parasite (?). Morphology.— Short, straight bacilli, with round ends, from 1 to 1.8 /* long- and from 0.25 to 0.3 n broad ; often united in chains containing four to six elements. Stains with the usual aniline colors. Biological Characters. — An aerobic, non-liquefying, non-motile bacil- lus. Does not form spores. Grows slowly at the room temperature in the usual culture media. Upon gelatin plates, at the end of four days, the deep colonies are seen as small, white points, the superficial colonies as smooth discs of a grayish color. At the end of a month the deep colonies may be as large as a mustard seed; the superficial are thin, shining, and slimy, and have a diameter of one to two millimetres. In gelatin stick cultures a con- vex, shining, white mass is developed at the point of inoculation, and along the line of puncture in the course of five or six days a white line of growth is seen which consists of closely crowded, small colonies. Upon agar the development is very slow, and forms at first thin, slimy, grayish-white patches which are distributed along the impfstrich ; later these become con- T DESCRIBED IN PREVIOUS SECTIONS. 497 fluent and form, shining, wavy stripes. Upon potato the development is more rapid and forms elevated, sharply denned colonies, of granular ap- pearance and of a chamois-yellowish-white color ; later these become conflu- ent; the potato acquires a dark-gray color and the culture gives off an in- tensely disagreeable odor. Pathogenesis. — Pure cultures rubbed into the skin of man produce, at the end of twenty-four hours, intense itching, redness, and a vesicular erup- tion about the hairs ; at the end of three days small pustules are formed, from which pure cultures may be recovered (Tommasoli) . Subcutaneous in- jection into a rabbit produced no other result than the formation of a small abscess. 114. BACILLUS OF SCHOU. Obtained by Schou (1885) in rabbits suffering from vagus pneumonia resulting from section of the vagi ; found also in the buccal secretions of a healthy rabbit — one out of twenty-five examined. Morphology. — Described as elliptical cocci, or diplococci, or as short, thick bacilli. Stains with the aniline colors usually employed, but not by Gram's method. Biological Characters. — An aerobic, liquefying, motile bacillus. Grows in the usual culture media at the room temperature. In gelatin plates forms spherical, opaque, granular colonies having a slightly rough surface. At the end of twenty -four hours, under the microscope, active movements are observed in these colonies, which are surrounded by a zone of diverging rays. In gelatin stick cultures liquefaction quickly occurs, and a copious white deposit, consisting of bacilli, is seen at the bottom of the tube. Pathogenesis. — Pure cultures injected into the trachea, the pleural cavity, or the lungs are said to have produced fatal pneumonia in rabbits; a similar result was obtained from inhalation experiments. 115. BACILLUS NECROPHORUS. Obtained by Lofner (1884) from rabbits which had been inoculated in the anterior chamber of the eye with small fragments of a broad condyloma. Morphology. — Bacilli of various lengths, often forming long, slender, wavy filaments. Biological Characters. — Does not grow in the ordinary culture media, but may be cultivated in neutral rabbit bouillon ; a less favorable medium is blood serum from the horse. When small fragments of the organs of an infected animal are placed in rabbit bouillon they become enveloped, in the course of three or four days, in a cotton-like mass of filaments ; later white flocculi are distributed tlirough the medium, which consist of similar fila- ments loosely interlaced. The filaments may present swellings here and there, which are supposed to represent involution forms. Pathogenesis. — Rabbits inoculated in the ear or in the anterior chamber of the eye with the flocculi from a bouillon culture, or with a small frag- ment of one of the organs of an infected animal, usually die at the end of eight days. At the autopsy a necrotic, cheesy process is found at the point of inoculation, and purulent foci, surrounded by inflamed or necrotic areas, in the lungs ; also purulent collections in the myocardium ; these were the principal pathological changes, but sometimes nodules were found in the abdominal viscera. The slender bacilli described were found in all of these localized centres of infection. Pathogenic also for white mice, which usually died in six days after being inoculated subcutaneously. 116. BACILLUS COPROGENES FCETIDUS. Synonym. — Darmbacillus of Schottelius. Obtained by Schottelius (1885) from the intestinal contents of pigs which had died of Schweiiierothlauf (rouget) . 35 49b PATHOGENIC AEROBIC BACILLI Morphology. —Resembles Bacillus subtilis, but is shorter, with rounded ends. Biological Characters. — An aerobic, non-liquefying, non-motile bacillus. Forms spores in presence of oxygen in the course of three or four days at the room temperature ; these are oval in form«and are arranged in rows; when they germinate this occurs in a direction perpendicular to their long axis and to that of the filament in which they developed ; as a result of this the newly formed rods lie parallel to each other. In gelatin stick cul- tures the growth upon the surface consists of a thin, transparent, grayish layer; along the line of puncture crowded, pale-yellow colonies are de- veloped. The cultures give off an intense putrefactive odor. 'Upon potato a dry, grayish layer is formed, which may be about 0.5 millimetre in thick- ness. Pathogenesis. — Not pathogenic for mice or for rabbits when injected in small amounts, but in considerable quantities causes fatal toxasmia in rabbits. 117. BACILLUS OXYTOCUS PERNICIOSUS. Obtained by Wyssokowitsch from milk which had been standing for a long time. Morphology. — Short bacilli with rounded ends, somewhat thicker and shorter than the lactic acid bacillus. Biological Characters. — An aerobic, non-liquefying bacillus. In gela- tin plates the deep colonies are small, spherical, finely granular, and of a yellowish or brownish-yellow color. The superficial colonies are hemi- spherical masses of a grayish- white color— by transmitted light, light-brown. They may have a diameter of one and one-half millimetres. In gelatin stick cultures the growth is at first "nail-like" ; later it ex- tends over the entire surface of the gelatin. It causes coagulation of milk, with a sour reaction, within twenty-four hours. The cultures are without odor. Pathogenesis. — Small doses are not pathogenic for mice or for rabbits, but considerable quantities injected into the circulation of rabbits cause their death in from three to twenty-two hours. Soon after the injection an abun- dant diarrhoea is developed. At the autopsy a hsemorrhagic inflammation of the intestinal mucous membrane is the principal pathological appearance observed. 118. BACILLUS SAPROGENES II. Obtained by Rosenbach (1884) from the perspiration of foul-smelling feet. Morphology. — Short bacilli with rounded ends. Biological Characters. — Aerobic and facultative anaerobic. Characters pf growth in gelatin, motility, etc. , not given. Streak cultures upon the surface of nutrient agar, at the end of twenty- four hours, cause the entire surface to be covered with minute, transparent colonies, which later become confluent and gradually somewhat opaque, forming a viscid, whitish gray layer. The odor of cultures resembles that of perspiring feet. Causes putrefaction of albuminous substances in the pre- sence of oxygen, with evolution of stinking gases. In the absence of oxygen putrefactive changes also occurred, but less rapidly. Pathogenesis. — When injected in considerable quantity into the knee joint or into the pleural cavity of rabbits, the animals succumb in from three to five days. 119. BACILLUS OF AFANASSIEW. Obtained by Afanassiew (1887) from mucus and masses of pus coughed up by patients suffering from whooping cough. Etiological relation not demonstrated. Morphology. — Bacilli from 0.6 to 2.2 ju long; solitary, in pairs, or in short chains. NOT DESCRIBED IN PREVIOUS SECTIONS. 499 Stains with the usual aniline colors. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Forms spores. Grows at the room temperature in the usual culture media. Upon gelatin plates the colonies are spherical or oval and of a light-brown color ; under a low power they are seen to be finely granular, and later have a dark-brown color. Upon the surface of gelatin stick cultures a grayish- white layer is formed ; but slight development occurs along the line of punc- ture. Upon the surface of agar a thick, gray layer forms along the line of inoculation. Upon potato yellowish, glistening, dew-like drops are first formed along the line of inoculation, and later a rather thick, brownish layer is formed which extends rapidly over the surface. Development is most rapid in the incubating oven. Pathogenesis. — According to Afanassiew, pure cultures injected into the air passages or pulmonary parenchyma, in young dogs or in rabbits, produce bronchial catarrh, broncho-pneumonia, and attacks of spasmodic coughing resembling those of whooping cough. Death sometimes occurs. At the autopsy the bacillus is found in great numbers in the bronchial and nasal mucus. 120. PNEUMOBACILLUS LIQUEFACIENS BOVIS. Obtained by Arloing from the lung of an ox which succumbed to infectious pleuro-pneumonia. Morphology- — Slender, short bacilli, which rather resemble mi- crococci when cultivated in gelatin. Stains with the usual aniline colors. Biological Characters. — An aerobic and facultative anaerobic, liquefying, non-motile bacillus. Spore formation not observed ; is killed by exposure for fifteen to twenty minutes to a temperature of 55° C. Grows in the usual culture media at the room temperature — better at 35° C. Forms white colonies in gelatin plates, and causes rapid liquefaction of the gelatin. Upon potato grows very rapidly as a white layer, which later has a brownish color. Pathogenesis. — From one-half to one cubic centimetre of a pure culture injected beneath the skin of an ox, where the connective tis- sue is loose, causes the development of an acute abscess the size of a man's hand ; after extending for two or three days this gradually becomes smaller and recovery occurs. When larger quantities are injected a fatal termination may result. Guinea-pigs and rabbits are less susceptible, and dogs are said to be immune. The researches of Arloing seeem to have established the etiologi- cal relation of this bacillus to infectious pleuro-pneumonia of cattle. Arloing has shown (1894) that inoculations in cattle of pure cultures of the bacillus are followed by immunity quite as pronounced as that resulting from inoculations with serum from the lungs of diseased animals — method of Willems ; also that infected animals are more sensible to the action of the toxic substances in filtered cultures than healthy animals — corresponding with results obtained by use of tu- berculin and mallem. Arloing has obtained two varieties of his bacillus from the lungs of cattle, one an attenuated variety which does not liquefy gelatin. 500 PATHOGENIC AEROBIC BACILLI He has also found that the liquefying variety when cultivated in bouillon through a series of generations loses its liquefying power to a considerable extent. In the liquefying cultures the bacilli are often elongated and articulated ; in the non-liquefying the short, thick forms, with rounded ends, are most numerous. When injected in equal quantity (two cubic centimetres) under the skin of an ox the results are similar but differ in degree — the liquefying bacillus producing a more extensive local lesion. Injected into the lung the results are similar : the liquefying bacillus causes pneumonic nodules as large as an apple and an extensive pleurisy with thick fibrinous exudation infiltrated with a yellow serum ; the non-liquefying bacillus causes the development of nodules the size of an almond or of a walnut, with a limited, but characteristic, pleuritic inflammation. Robcis (1894), after discussing the results of inoculations made in the Department of the Seine with pulmonary serum, arrives at the conclusion that Arloing's method of protective inoculations with cul- tures of the Pneumobacillus liquefaciens bovis gives better results than the legal method with serum from an infected animal. Arloing prepares from the cultures of his bacillus a "lymph," corresponding with tuberculin and mallein, which he calls " pneumo- bacilline." The toxic action of this lymph corresponds with that of cultures sterilized by heat. The experiments of Guinard and Artaud (1895) show that the toxic products of the bacillus of Arloing are extremely active, and that in dogs the injection of twenty to fifty cubic centimetres of a sterilized (by heat) culture gives rise almost immediately to torpor and sometimes to vomiting and defecation; after several hours vomiting an$ bloody diarrhoea occur, the animal becomes more and more feeble, and finally, if the dose has been suffi- cient, is completely paralyzed and dies. 121. BACILLUS PSEUDOTUBERCULOSIS. Obtained by Pfeiffer (1889) from the organs of a horse suspected of hav- ing glanders and killed. Morphology. — Rather thick bacilli with round ends ; vary considerably in length — usually three to five times as long as broad. Stains with fuchsin and Lotfler's solution of methylene blue ; does not stain by Gram's method. Biological Characters.— An aerobic, non-liquefying, non-motile bacil- lus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of twenty-four hours, the superficial colonies are small, yellowish-brown plates, which in- crease rapidly in diameter; under a low power a central papilla is observed, around which the colony extends as a pale-yellow, peculiarly marbled, crys- talline disc ; the deep colonies are at first transparent, sharply denned spheres ; on the third day, under a low power, they are seen to have a dark, finely granular central portion surrounded by a transparent zone; when not crowded upon the plate they may appear as yellowish-brown, finely granu- lar, pear-shaped or lemon-shaped colonies. In gelatin stick cultures growth occurs along the line of puncture in the form of grayish-white, spherical NOT DESCRIBED IN PREVIOUS SECTIONS. 501 colonies, more or less crowded above, and often isolated below, where by transmitted light they are seen to have a brownish color ; upon the surface a grayish-white, concentric layer is formed about the point of inoculation in the course of five or six days, which later forms a disc with thickened mar- gins. Upon the surface of agar the growth along the line of inoculation is- abundant and viscid. Does not grow well upon potato. Upon blood serum forms transparent, drop-like colonies which have an opalescent appearance. Pathogenesis. — Pathogenic for rabbits, guinea-pigs, hares, white mice, and house mice. Death occurs in from six to twenty days. At the autopsy the lymphatic glands are found to be enlarged and to have undergone case- ation ; the liver and spleen are enlarged, the lungs cedematous and occasion- ally contain tuberculous-looking nodules. An abscess forms at the point of inoculation. Bacilli are found in the blood, the lymphatic glands, and the various organs. 122. BACILLUS GINGIV^E PYOGENES. Synonym. — Bacterium gingivae ^yogenes (Miller). Obtained by Miller from an alveolar abscess and from deposit around the teeth " in a filthy mouth." Morphology. — Short and thick bacilli with rounded ends, one to four times as long as broad ; occur singly or in pairs. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing bacillus. Grows rapidly in the usual culture media. Upon gelatin plates it forms spherical colonies at the end of twenty-four hours, which have a yellowish color and well-defined margin ; at the end of forty-eight hours liquefaction has progressed so far that the colonies have become con- fluent. In gelatin stick cultures liquefaction occurs rapidly in the form of a funnel, at the bottom of which a white deposit is formed. Upon the surface of agar a thick, moist growth occurs along the line of inoculation, which under the microscope has a slightly greenish-yellow tint and a fibrillated structure. Pathogenesis. — Pathogenic for rabbits, guinea-pigs, and for white mice, when injected into the cavity of the abdomen in comparatively small amounts (0.25 cubic centimetre). At the autopsy peritonitis, sometimes purulent, is observed. Death occurs in from ten to twenty-four hours. The bacilli are found in the blood in small numbers. Subcutaneous injections in the animals mentioned produce a local abscess only. 123. BACILLUS DENTALIS VIRIDANS. Found by Miller in the superficial layers of carious dentine. Morphology. — Slightly curved bacilli with pointed ends; solitary or in pairs. Biological CJiaracters. — An aerobic and facultative anaerobic, non- liquefying bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. In gelatin plates the colonies are spherical, and under a low power are colorless or have a slightly yellow tint; when not crowded they may present two or three concentric rings. In gela- tin stick cultures growth occurs both upon the surface and along the line of puncture. Gelatin cultures acquire an opalescent-green color. Upon the surface of agar a thin growth with irregular margins occurs along the impf- strich ; this is bluish by transmitted light and greenish-gray by reflected light — colorless under the microscope. Pathogenesis. — Injections into the cavity of the abdomen of white mice or of guinea-pigs usually cause fatal peritonitis in from one to six days ; the bacilli are only found in the blood in small numbers, by the culture method. Subcutaneous injections in the animals mentioned produce severe local in- flammation arid suppuration. 124. BACILLUS PULP.E PYOGENES. Obtained by Miller from gangrenous tooth pulp. 502 PATHOGENIC AEROBIC BACILLI Morphology. — Slightly curved bacilli with pointed ends; solitary or in pairs, or in chains of four to eight elements. Biological Characters.— An aerobic and facultative anaerobic, liquefy- ing bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. In gelatin plates large, spherical, opaque, yellowish-brown colonies are formed. In gelatin stick cultures liquefaction occurs in the upper part of the tube and gradually extends downward, the liquefied gelatin being separated from the non-liquefied by a horizontal plane. Pathogenesis. — Small quantities of a pure culture injected into the abdo- minal cavity of white mice proved fatal to these animals in from eighteen to thirty hours. 125. BACILLUS SEPTICUS KERATOMALACI^. Obtained by Babes (1889) from the broken-down corneal tissues and from the various organs of a child which died of septicaemia following keratoma- lacia. Stains with the usual aniline colors ; deeply colored granules may often be seen at the extremities of the rods, or in the middle, in preparations stained with Lofner's solution. Morphology. — Short, thick bacilli, thinning out at the ends; often united in pairs ; may be surrounded by a capsule. Biological Characters.— An aerobic and facultative anaerobic, non- liquefying bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates forms white, slightly elevated, flat colonies with finely dentate margins. In gelatin stick cultures the growth is abundant both on the surface and aiong the line of puncture ; gas bubbles are formed in the gelatin. Upon the surface of agar the growth along the line of inoculation is leaf-like, finely dentate, some- what opalescent, and the culture has a^slightly ammoniacal odor. Upon blood serum a semi-transparent, glistening film is formed, which has dentate margins. Pathogenesis. — Pathogenic for rabbits and mice, less so for birds; not pathogenic for guinea-pigs. The animals die in from three to seven days. Inoculated into the cornea it causes a purulent keratitis. 126. BACILLUS SEPTICUS ACUMINATUS. Obtained by Babes (1889) from the blood, the umbilical stump, and the various organs of a child which died five days after birth, apparently from septic infection. Morphology. — Bacilli with lancet-shaped ends, somewhat resembling the bacillus of mouse septicaemia, but thicker. Often shows unstained places in the middle of the rods in stained preparations. Stains readily with the usual aniline colors. Biological Characters. — An aerobic bacillus; does not grow in gelatin at the room temperature. Spore formation not observed. Grows upon blood serum and upon nutrient agar at 37° C., in form of small, flat, circular, transparent, shining colonies, which become confluent and later form a yel- lowish layer. Blood serum is the most favorable medium. Pathogenesis. — Pathogenic for rabbits and guinea-pigs, not for mice. The animals die in from two to six days, and the bacilli are found in their blood u ml in the various organs. 127. BACILLUS SEPTICUS ULCERIS GAXGR^ENOSI. Obtained by Babes (1889) from the blood and various organs of a boy \vli<> died from septicaemia following gangrene of the skin, etc. Morphology. — Bacilli with round ends, oval or rod-shaped, about 0.5 to 0.6 u thick. I NOT DESCRIBED IN PREVIOUS SECTIONS. 503 Biological Characters. — An aerobic, liquefying, motile bacillus. Does not form spores. Grows in the usual culture media at the room temperature. In gelatin stick cultures a sac-formed liquefaction occurs and a yellow de- posit is seen at the bottom of the liquefied gelatin ; gas bubbles are given off from the culture. Upon the surface of agar development occurs along the line of inoculation in the form of flat, grayish-yellow, transparent, varnish- like plaques. Upon potato, after several days, a brownish, shining, moist, transparent film is formed. Upon the surface of blood serum smooth, yellowish, transparent colonies are formed, under which the blood serum is softened, allowing these to sink below the surface. Pathogenesis. — Pathogenic for mice and for guinea-pigs, which die in from one to two days. An abscess forms at the point of inoculation, which is covered with a dry, retracted crust. 128. BACILLUS OF TRICOMI. Obtained by Tricomi (1886) from a case of senile gangrene. Morphology. — Bacilli with round ends, about three /* long and one u thick, solitary o- in pairs ; sometimes one end of a rod shows a club-shaped thickening. Stains with the usual aniline colors and by Gram's method. * Biological Characters. -An aerobic, liquefying, non-motile bacillus. Forms spores. Grows in the usual culture media at the room temperature — better at 37° C. f Upon gelatin plates, at the end of twenty-four hours, the colonies are spherical, finely granular, and of a dirty-yellow color ; after from thirty.-six to forty-eight hours liquefaction of the surrounding gelatin occurs. In gela- tin stick cultures closely crowded, small, white colonies are formed along the line of puncture ; at the end of forty-eight hours liquefaction com- mences in funnel form, with formation of an air bubble above — like the cholera spirillum; later the entire gelatin is liquefied and becomes trans- parent, while a dirty-white collection of bacilli is seen at the bottom of the tube. Upon the surface of agar a white layer with irregular margins is formed, which later extends over the entire surface as a homogeneous, rather thin membranous film. \Jyon potato, at 37° C., dirty- white, milky colonies are formed, which later become confluent. Upon blood serum the growth is similar to that upon agar. Pathogenesis. — The subcutaneous injection of one-half to one cubic centi- metre of a gelatin culture is said by Tricomi to produce in rabbits and in guinea-pigs a gangrenous process resembling senile gangrene in man. The subcutaneous connective tissue is infiltrated with a foul-smelling serum, the muscles are soft and gray, and a portion of the skin has a mummified ap- pearance. The gangrene extends over the abdomen, and death occurs in guinea-pigs in two to three days, in rabbits after four days, in house mice at the end of twenty-four hours ; white mice are said to be immune. 129. BACILLUS ALBUS CADAVERIS. Obtained by Strassmann and Strieker (1888) from the blood of two cada- vers four days after death. Morphology.— Bacilli about two and one-half /* long and 0.75 fi broad; also grow out into filaments of six fit or longer. Stains with the usual aniline colors and by Gram's method. Biological Characters.— Anaerobic, liquefying, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room tem- perature. In gelatin plates small, spherical, yellowish colonies are formed during the first twenty-four hours ; later a radiating outgrowth occurs from the periphery, and liquefaction of the gelatin takes place. In gelatin stick cultures liquefaction begins within forty-eight hours, and forms a long fun- nel, at the opening of which is a cavity containing air ; the liquefied gela- 504 PATHOGENIC AEROBIC BACILLI tin is transparent, and a deposit of thick, granular masses accumulates at the bottom of the tube. Upon the surface of agar a thick, white layer is formed, which later is wrinkled and after a time gives off a putrefactive odor. Gela- tin cultures give off an odor of sulphuretted hydrogen. Upon potato a soft, white or pale-yellow layer is formed, which in places is made up of small granules. The potato around the growth has a bluish-brown color. Pathogenesis. — Subcutaneous injection of a small quantity (0.1 cubic centimetre) of a liquefied gelatin culture is fatal to mice in about six hours ; the animals become comatose before death, and at the autopsy putrefactive changes are already observed ; the bacillus can be recovered from the blood in cultures. Sterilized cultures also prove fatal to mice. Pathogenic also for guinea-pigs, which die in, about twenty hours after receiving a subcuta- neous inoculation. 130. BACILLUS VARICOSUS CONJUNCTIVE. Obtained by Gombert (1889) from the healthy conjunctival sac of man. Morphology. — Large bacilli with round ends, from two to eight n long and about one JJL broad; the shorter bacilli are often constricted in the middle. Stains with the usual aniline colors. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, non-motile bacillus. Grows very slowly in nutrient gelatin at 22° C. ; rapidly in agar and upon potato at 37° C. In gelatin stick cultures, at the end of twenty-four hours, a circular layer haying a grayish- white centre is developed upon the surface, and a scarcely visible grayish- white thread along the line of puncture. Liquefaction extends gradually from the surface without clouding or changing the gelatin, so that at the end of two weeks the gelatin is entirely liquefied without giving any other evidence of the pre- sence of the microorganism. Upon agar plates, at 37° C., the deep colonies have a diameter of about four millimetres by the end of the fourth day; under a low power they are seen to be covered with minute, irregular, thorn- like projections, which subsequently increase in size ; the centre of the colony is granular and opaque. The superficial colonies, under a low power, are seen to have an opaque central nucleus surrounded by a yellowish, finely granu- lar, transparent peripheral zone; later the central portion is irregular and semi-opaque, surrounded by a broad marginal zone which consists of twisted and bent tapering offshoots having a dark contour. Upon the surface of agar a thin, white, dry, very adherent film is formed ; a thick, white film forms upon the surface of the condensation water. Upon potato develop- ment is rapid at 37° C., forming at first a dry, white layer, which at the end of ten days covers the entire surface ; it then has an irregular surface and fringed margins, is smooth, dry, and after a time has a reddish-brown color. Pathogenesis. — When inoculated into the cornea of rabbits a grayish- white cloudiness is developed in twenty-four hours, around which the cornea is highlv vascular ; the animal recovers without the formation of an abscess. Injected into the conjunctiva it causes an intense hyperaemia. 131. BACILLUS MENINGITIDIS PURULENTJE. Obtained by Neumann and Schaffer (1887) from pus from beneath the pia mater in an individual who died of purulent meningitis. Morphology. — Bacilli about two u long and 0.6 to 0.7/* broad; often grow out into long filaments, especially in gelatin cultures. Stains with the usual aniline colors, but not by Gram's method. Biological Characters.— An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Does not form spores. Grows in the usual culture media at the room temperature — better in the incubating oven. Upon gelatin plates the deep colonies, under a low power, are homogeneous, round or oval, pale brown, and with a smooth contour; the superficial colonies are NOT DESCRIBED IX PREVIOUS SECTIONS. 505 thin, moist, and transparent in appearance; later they have a grayish color, a coarsely granular surface, and are made up of flap-like layers. In gelatin stick cultures the superficial growth consists of broad, grayish layers, and a grayish-yellow growth is seen along the line of puncture, made up of crowded colonies. Upon agar plates, at the end of twenty-four hours at 37° C., thin colonies are developed, which have a granular surface, a smooth, more or less irregular outline, and a pale-brown color in the centre. Upon potato a scanty, moist, white layer develops along the line of inoculation. Upon blood serum, at 37° C. , at the end of twenty-four hours a moist, shining layer about four millimetres broad is developed along the impf strich ; this is gra- nular at the margins, and later more or less fissured. Pathogenesis. —Subcutaneous injection produces in dogs, rabbits, guinea- pigs, and white mice a purulent inflammation in the vicinity of the point of inoculation. 132. BACILLUS SEPTICUS VESIOE. Obtained by Clado (1887) from the urine of a person suffering from cys- titis. Morphology. — Bacilli with round ends, 1 6 to 2 ji long and 0.5 jn thick; never united in pairs or chains. Stains with the usual aniline colors and also by Gram's method. Biological Characters.— An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Forms spores. Grows in the usual culture media at the room temperature. Upon gelatin plates small, spherical or oval colonies are developed throughout the gelatin, which rarely exceed the size of a pin's head ; these are transparent, and yellowish-white in color ; under a low power the centre is seen to be dark gray and is surrounded by a well-defined marginal zone of a pale-yellow color. In gelatin stick cultures the growth along the line of puncture is first seen as a delicate, whitish thread ; at the end of six or seven days it is made up of lenticular colonies, one-third as large as a pin's head, arranged in two lines like piles of coin. Upon the surface the growth is scanty and consists of a thin layer around the point of inoculation, which has a jagged contour. Upon the surface of agar development is slow and forms a grayish- white stripe along the impf strich. Upon potato a flat, dry, chestnut-brown layer is formed. Pathogenesis. — Pathogenic for rabbits, guinea-pigs, and mice. Death appears to result from the toxic products formed, as well as from the multi- plication of the bacilli in the inoculated animals. 133. BACILLUS OF GESSNER.' Synonym. — Bacterium tholoideum (Gessner). Obtained by Gessner from the contents of the intestine of healthy persons. Resembles in its morphology and in its growth in culture media Bacillus lactis aerogenes of Escherich. Pathogenic for mice and for guinea-pigs. 134. BACILLUS CHROMO-AROMATICUS. Obtained by Galtier (1888) from a pig which died from a general infec- tious malady characterized by broncho-pneumonia, pleuritis, enteritis, and swelling of the lymphatic glands. Morphology. — -Bacilli of medium size with rounded ends. Stains with the usual aniline colors. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Not observed to form spores. Grows in the usual cul- ture media at the room temperature — better in the incubating oven. The cultures all produce a green or brown pigment and have an aromatic odor. In gelatin stick cultures a yellowish-white layer is formed upon the surface of the liquefied gelatin, which has a bright-green color ; a yellowish-white 506 PATHOGENIC AEROBIC BACILLI deposit accumulates at the bottom of the tube. Upon the surface of agar whitish colonies are formed, which coalesce to form a thin layer. Upon potato a tolerably thick, somewhat iridescent, brown layer is formed, which extends over the entire surface. In bouillon, at the end of twenty-four to forty-eight hours at 37° C., a greenish-yellow color is developed, first near the surface and later extending throughout the fluid, which acquires the color of a dilute solution of sulphate of copper ; a whitish film forms upon the surface. In anaerobic cultures the color is a pale brown instead of green. Pathogenesis. — Rabbits die at the end of two to three weeks after receiv- ing an intravenous injection. At the autopsy they are found to have pneu- monia with pleuritis and pericarditis. 135. BACILLUS CANALIS CAPSULATUS. Obtained by Mori (1888) from sewer water. ? Morphology. — Bacilli with round ends, elliptical or rod-shape in form, and from 0.9 to 1.6 jn thick ; often surrounded with a broad capsule, which is always seen in preparations from the blood or tissues of an infected ani- mal ; sometimes in pairs with the acute ends of the rods in apposition, and surrounded by a single capsule. Stains with the usual aniline colors, but not by Gram's method. Biological Characters.— An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates hemispherical, porcelain- white, sharply defined colonies, resembling those of Friedlander's bacillus, are developed at the end of twenty -four hours. In gelatin stick cultures development occurs along the line of puncture and upon the surface, forming a u nail-shaped " growth similar to that of Fried- lander's bacillus (Bacillus pneumonise) in the same medium. Upon agar a viscid and abundant growth is formed in the incubating oven at 37° C. "Upon potato an abundant development in the form of a yellowish, moist, vis- cid layer, with irregular outlines. In bouillon, at the end of three or four days, a white film forms on the surface, especially in contact with the test tube. Pathogenesis. — Mice die in two to three days after receiving a subcutane- ous injection. Guinea-pigs and rabbits are immune. 136. BACILLUS CANALIS PARVUS. Obtained by Mori (1888) from sewer water. Morphology. — Bacilli with round ends, from 2 to 5 n long and 0.8 to 1 ft broad. Stains with the usual aniline colors, but not by Gram's method ; the ends of the rods are more deeply stained than the central portion. Biological Characters. — An aerobic, non-liquefying, non-motile ba<-il lus. Not observed to form spores. Grows very slowly at the room tempera- ture— more rapidly at 37° C. Upon gelatin plates, at the end of two to three weeks, extremely minute, homogeneous, pale-yellow colonies are developed. In gelatin stick cultures a thin, yellowish layer forms upon the surface at the end of three weeks. Upon the surface of agar, at 37° C. , a dry, yellow- ish layer with jagged outlines is developed in two or three days. No growth occurs upon potato. Upon blood serum a thin, pale-green, dry layer is formed. Pathogenesis.— Mice die in from sixteen to thirty hours after receiving a subcutaneous inoculation, guinea-pigs in about two days. 137. BACILLUS INDIGOGENUS. Obtained by Alvarez (1887) from an infusion of the leaves of the indigo plant. Morphology. — Bacilli with round ends, about 3 // long and 1.5 /* thick, NOT DESCRIBED IN PREVIOUS SECTIONS. 507 often united in chains of six to eight elements. The cells are surrounded by a transparent capsule resembling that of Friedlander's bacillus. Biological Characters. — An aerobic, motile bacillus. Upon agar, at 37° C. , a yellowish- white layer is quickly developed and there is production of gas. According to Alvarez, this bacillus develops an indigo-blue color in a sterilized infusion of the leaves of the indigo plant. Pathogenesis. — Guinea pigs die in from three to twelve hours from the intravenous injection of a pure culture. 138. BACILLUS OF KARTULIS. Obtained by Koch (1883) and by Kartulis from the conjunctiyal secre- tions of persons suffering from a form of infectious catarrhal conjunctivitis which prevails in Egypt. Morphology. — Resembles the bacillus of mouse septicaemia (Bacillus mu- risepticus) in its form and dimensions. Stains with the usual aniline colors. Biological Characters. — An aerobic bacillus. Does not grow in nutri- ent gelatin at the room temperature. Upon the surface of nutrient agar, at 28° to 30° C., at the end of thirty to forty hours small, grayish- white points are developed along the impfstrich ; later these become confluent and form an elevated, shining, dark-colored layer with irregular and often jagged margins. Pathogenesis. — Out of six experimental inoculations, with pure cultures, made by Kartulis in the eyes of healthy individuals, four gave a negative result, one produced a catarrhal inflammation lasting for a week, in an e^e which was blind from a previous attack of sclerochoroiditis, and one a con- junctivitis lasting for ten days in a perfectly healthy eye. 139. BACILLUS OF UTPADEL. Obtained by Utpadel (1887) from the wards of a military hospital at Augs- burg— in the ' ' Zwischendeckenf iillung " ; also by Gessner from the contents of the small intestine in man. Morphology. — Bacilli with round ends, 1.25 to 1.5 n long and 0.75 to 1 /* thick ; often united in pairs or in chains of three elements. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Grows in the usual culture media at the room temperature. Spore forma- tion not observed. Upon gelatin plates the superficial colonies are elevated and sometimes conical, and of a milk-white color. The deep colonies are round or oval ; the centre is dark green and is surrounded by a brownish- green peripheral zone. Upon the surface of agar a yellowish-white layer is developed very slowly. The growth upon gelatin is rapid. Pathogenesis. — When injected subcutaneously into cats, guinea-pigs, or mice it produces an extensive inflammatpry oedema, resulting in the death of the animals. 140. BACILLUS ALVEI. Synonym. — Bacillus of foul brood (of bees). Obtained by Cheshire and Cheyiie (1885) from the larvae in hives infected with " foul brood." The larvae in the interior of cells in the comb die and become almost fluid as a result of parasitic invasion by this bacillus. Morphology. — Bacilli with rounded ends, from 2.5 to 5 ju in length (aver- age about 3.6 ju) and 0.8 u in diameter. Grow out into filaments and form large oval spores which have a greater diameter than the rods in which they are developed — 1.07 n. Stains readily with the aniline colors usually employed, also by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, liquefy- 508 PATHOGENIC AEROBIC BACILLI ing, motile bacillus. Forms endogenous spores. Grows readily in the usual culture media at the room temperature. In gelatin plates small, round or oval colonies are formed, which later become pear-shaped ; a branching outgrowth occurs about the margins of the colonies, and especially from the small end of the pear-shaped mass. In streak cultures upon the surface of gelatin growth occurs first along the impf- strich, and from this an outgrowth occurs consisting of bacilli in a single row or in several parallel rows, and forming irregular or circular figures, from which other similar outgrowths occur; the branching outgrowths may anastomose. The gelatin is liquefied in the vicinity of these lines of growth, forming a network of channels. A similar growth is seen upon the surface of gelatin stick cultured, and along the line of puncture white, irregular masses are formed, from which rather coarse branches are given off which often have a club-shaped extremity. In older cultures the finer branches disappear, so that the secondary centres of growth are disconnected from the original colonies ; complete liquefaction of the gelatin occurs in about two weeks; the liquefied gelatin has a yellowish color and peculiar odor. Upon the surface of nutrient agar, at 37° C. , a white layer is formed. Upon potato the development is slow and results in the formation of a dry, yellowish layer. In milk coagulation first occurs, and the coagulum is subsequently dissolved; a slightly acid reaction is produced. This bacillus grows best in the incubating ovea at 37°, and does not develop at temperatures below 16° C. The spores require for their destruction a temperature of 100° C. main- tained for four minutes (determined by the writer, 1887). Pathogenesis. — The introduction of pure cultures of this bacillus into hives occupied by healthy swarms causes them to become infected with foul brood; grown bees also become infected when given food containing the ba- cillus (Cheshire) . Mice injected subcutaneously with a considerable quan- tity die within twenty-four hours, guinea-pigs in six days (Eisenberg). Small amounts injected beneath the skin of mice or rabbits produce no appa- rent result. 141. BACILLUS OP ACNE CONTAGIOSA OF HORSES. Obtained by Dieckerhoff and Grawitz (1885) from pus and dried scales from the pustules of " acne conta^iosa " of horses. Morphology.— Short rods, straight or slightly bent, 0.2 ju in diameter. Stains best with an aqueous solution of fuchsin, and also by Gram's method ; does not stain well with Loftier 's alkaline solution of methvlene blue. Biological Characters. — Anaerobic, non-liquefying bacillus. In gelatin stick cultures a very scanty growth occurs along the line of puncture ; upon the surface a white mass forms about the point of puncture. Upon blood serum and nutrient agar an abundant growth at the end of twenty-four hours at 37° C., consisting of white colonies along the impfstrich, which later have a yellowish-gray color. The growth is more abundant and rapid upon blood serum than upon other media. Pathogenesis. — Pure cultures of the bacillus described are said by Diecker- hoff and Grawitz to produce typical acne pustules when rubbed into the skin of horses, calves, sheep, and dogs. When rubbed into the intact skin of guinea-pigs a phlegmonous erysipelatous inflammation was produced, and the animal died at the end of forty-eight hours with symptoms of toxemia. Subcutaneous injections in guinea-pigs caused toxaemia and death at the end of twenty-four hours. A.t the autopsy a haemorrhage infiltration of the in- testinal mucous membrane was observed ; the bacilli were not found in the internal organs. In rabbits pure cultures rubbed into the intact skin caused a development of pustules and a severe inflammation of the subcutaneous connective tissue, t'n >i 1 1 w 1 1 ich the animal usually recovered. Subcutaneous injections in rabbits sometimes caused a fatal toxiemia. House mice, field mice, and white mice were not affected by the application of cultures, by NOT DESCRIBED IN PREVIOUS SECTIONS. 509 rubbing, to the uninjured skin, but succumbed to subcutaneous injections in twenty-four hours or between the fifth and tenth days. Those which died at a late date presented the pathological appearances which characterize pysemia. 142. BACILLUS NO. I OF ROTH. Obtained by Roth (1890) from old rags. Resembles Bacillus coli com- munis and Brieger's bacillus in its morphology and growth in various culture media, but, according to Roth, is distinguished from these bacilli by the fact that colonies upon gelatin plates are thicker and more opaque. Pathogenesis. — Pathogenic for rabbits and for guinea-pigs when injected into the cavity of the abdomen; death usually occurs within twenty-four hours. The spleen is greatly enlarged, and the bacilli are found in cultures from the blood and various organs. 143. BACILLUS NO. II OF ROTH. Obtained by Roth (1890) from old rags. Morphology. — Bacilli with round ends, 0.6 to 1 jo, broad and two to four times as long. Stains with the usual aniline colors. When stained by Gram's method it is decolorized by alcohol. Biological Characters.— An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Grows in the usual culture media at the room temperature. Upon gelatin plates colonies resembling those of the <;olon bacillus are developed at the end of twenty-four hours ; on the third day small, drop-like, shining, bluish-white colonies, around the periphery of which a commencing extension upon the surface of the gelatin is seen. Older colonies are seldom more than one-half centimetre in diameter, and are some- what thicker than this ; they are nearly transparent. Upon the surface of gelatin stick cultures a rather moist, yellowish-white layer with dentate margins is developed. Upon potato a colorless layer is developed, which later has a grayish color. Pathogenic for rabbits and guinea-pigs when injected into the abdominal cavity. 144. BACILLUS OF OKADA. Obtained by Okada (1891) from dust between the boards of a floor. Morphology. — Short rods with round ends, about as long as Bacillus murisepticus, but somewhat thicker — about twice as long as thick ; solitary or in pairs; in old cultures may grow out into filaments. Stains with the usual aniline colors, but not by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Does not form spores. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of two to three days, small, white, spherical colonies are developed. Under the microscope these are seen to be granular, pale-brown in color, and with slightly jagged margins ; the superficial colonies after several days are con- siderably elevated above the level of the gelatin. In gelatin stick cultures development occurs as a white thread along the line of puncture, and upon the surface as a flat, milk-white layer which does not extend to the walls of the test tube. Upon agar, at 37° C., the growth is rapid and the surface is nearly covered at the end of eighteen hours with a milk-white layer ; the con- densation water is filled with a viscid mass of bacilli. Upon blood serum the growth is shining and almost transparent. In bouillon development is rapid, clouding the fluid throughout, and a cream-like layer forms upon the surface. Pathogenesis. — Rabbits and guinea-pigs die in about twenty hours after receiving a subcutaneous injection of a half-syringeful of a bouillon cul- ture, or from a small quantity (two ose) from a gelatin or agar culture. In 510 PATHOGENIC AEROBIC BACILLI mice a minute quantity of a pure culture invariably proved fatal in about twenty hours. Four hours after the inoculation an abundant secretion from the lachrymal glands occurs, and soon after the eyes become completely closed. According to Okada, this bacillus is differentiated from the bacillus of Briefer, and from Emmerich's bacillus which it greatly resembles, by the fact that it does not grow upon potato. 145. BACILLUS OF PURPURA H^MORRHAGICA OF TIZZONI AND GIOVANNINI. Obtained by Tizzoni and Giovannini (1889) from the blood of two children who died of purpura haemorrhagica following impetigo. Morphology.— Bacilli with round ends, from 0.75 to 1.3 fit long and 0.2 to 0.4 fj. broad; often seen in pairs or in groups like streptococci. Stains with the usual aniline colors, but not by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates the colonies at first resemble those of Streptococcus pyogenes. Upon the surface small, opaque points are seen at the end of forty-eight hours, which at the end of four to five days develop into spherical, yellowish-gray colonies with irregular margins, surrounded by a growth resembling tufts of curly hair. Upon agar the growth is similar, but more rapid and of a pale color, often with a central nucleus surrounded by a net-like marginal zone. Upon blood serum the growth is similar to that upon agar. Upon potato, at 37° C., a limited development occurs about the point of inoculation, which has a dark-yellow color. The cultures give off a very penetrating odor. Pathogenesis. — Pathogenic for dogs, rabbits, and guinea-pigs when in- jected subcutaiieously. Not pathogenic for white mice or pigeons. The symptoms resulting from a subcutaneous injection are said to be fever, al- buminuria and, in some cases, anuria, haemorrhagic spots upon the skin, convulsions : death occurs in from one to three days. At the autopsy there are found oedema about the point of inoculation, haemorrhages in the skin and muscles, and sometimes in the internal organs and in serous cavities; the blood does not coagulate. The bacilli are found in the subcutaneous con- nective tissue, but not in the blood or in the various organs. Sections show coagulation necrosis of the liver cells and of the renal epithelium. 146. BACILLUS OF PURPURA H^EMORRHAGICA OF BABES. Obtained by Babes (1890) from the spleen and lungs of an individual who died from purpura haemorrhagica with symptoms of septicaemia. Resembles the bacillus previously described by Tizzoni and Giovannini, and still more that of Kolb ; but, according to Babes, differs in some respects from both of these, although they all belong evidently to the same group. Morphology. — Bacilli with rounded ends, oval or pear-shaped, about 0.3 p thick, surrounded by a narrow capsule. Stains with the aniline colors, but not deeply, and still less intensely by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Does not form spores. Grows in the usual culture media at the room temperature. In gelatin stick cultures, at the end of three days, a thin, transparent, irregular layer has developed upon the surface, and a whitish, punctate stripe along the line of inoculation. In agar stick cultures an abundant development occurs along the line of punc- ture, and at the end of three days the growth upon the surface consists of small, moist, transparent drops; later of larger, flat, shining, yellowish- white plaques which have ill-defined margins. Upon blood serum the de- velopment is somewhat more abundant in the form of small, white, moist colonies one to two millimetres broad. Upon potato, at the end of three days, moist, whitish drops with ill-defined margins. NOT DESCRIBED IN PREVIOUS SECTIONS. 511 Pathogenesis. — Inoculations in the conjunctivas of rabbits produce ecchy- moses of the conjunctiva. At the autopsy numerous hasmorrhagic extrava- sations are found in all the organs, especially in the lungs and liver; the spleen is enlarged ; the bacilli can be recovered in pure cultures from the various organs. Old cultures proved to have lost their virulence. Patho- genic for mice, which die from general infection in the course of a few days ; the spleen is enlarged, and haemorrhages in the serous membranes are usually seen. 147. BACILLUS OF PURPURA H^MORRHAGICA OF KOLB. Obtained by Kolb (1891) from the various organs of three individuals who died in from two to four days from attacks characterized by suddenly developed feveV, purpura, and albuminous urine. Morphology. — Oval bacilli, usually in pairs, 0.8 to l.Syulong andO.8/* broad, surrounded by a narrow capsule, which is only seen distinctly in preparations from the organs. Stains with the aniline, colors, but not deeply, and still more feebly by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying non-motile bacillus. Does not form spores. Grows iri the usual culture media at the room temperature. In gelatin stick cultures, at the end of four days, a very small, thin, hyaline growth is seen about the point of inoculation. The development is more abundant along the line of puncture. Upon the surface of agar a thin layer is formed with smooth margins. Upon potato, at the end of three to four days, a whitish, moist, shining stripe is seen along the impfstrich which is about three millimetres broad. Pathogenesis. — Injections of 0.5 to 1 cubic centimetre of a bouillon culture into the abdominal cavity of rabbits cause symptoms of general in- fection in the course of a few days, and not infrequently haemorrhagic ex- travasations are seen in the ear muscles. More than one cubic centimetre may cause death in from one to three days. At the autopsy haemorrhagic extravasations are found in the subcutaneous tissues and in the serous and mucous membranes. The blood has little disposition to coagulate; the bacillus may be recovered in pure cultures from the various organs. In guinea-pigs local ecchymoses are sometimes produced, otherwise not natho- geiiic for this animal. Pathogenic for mice, which die from general infec- tion, after being inoculated with a small quantity of a pure culture, in from two to three days; spleen enlarged; lymphatic glands often haemorrhagic. Not fatal to dogs, but animals which were inoculated with one cubic centi- metre of a bouillon culture and subsequently killed proved to have haemor- rhagic extravasations in the various organs. 148. BACILLUS HEMINECROBIOPHILUS. Obtained by Arloing (1889) from a caseous lymphatic gland in a guinea-pig. Morphology. — Bacilli which vary greatly in length and are sometimes so short as to resemble micrococci ("polymorphous"); usually from one to four n long; in anaerobic cultures from eight to twenty #. Stains with the usual aniline colors. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, slightly motile bacillus. Spore formation not observed. Grows rapidly in the usual culture media — best in the incubating oven at 35° C. The growth upon the surface of gelatin has a yellowish color. Upon potato a yellowish-white layer is developed. Pathogenesis. — According to Arloing, this bacillus is not pathogenic when injected into healthy tissues in dogs, sheep, guinea-pigs, and rabbits, but when the tissues have previously been injured it produces a local oedema and necrotic changes, accompanied by gas formation. This is not peculiar to the microorganism described by Arloing, which appears to be one of the Proteus group. 512 PATHOGENIC AEROBIC BACILLI 149. STREPTOCOCCUS CONGLOMERATE (Kurth). Obtained by Kurth (1890) from cases of scarlet fever. Morphology. — As obtained from bouillon cultures it consists of masses made up of chains of cocci; free chains are only occasionally seen. Biological Characters. — This streptococcus is said to differ from Strepto- coccus pyogenes and various other previously described streptococci by the fact that in bouillon cultures, at a temperature of 37° C. , it forms at the bot- tom of the tube smooth, round, and very firm white scales, or a single flat layer which is not disintegrated when the tube is slightly agitated ; other streptococci are said to form a loose deposit which is either entirely broken up or forms viscid threads when the tube is gently rotated. Pathogenesis. — Very pathogenic for mice. * 150. BACILLUS CAPSULATUS Mucosus (Fasching). Obtained from the nasal secretion in two cases of influenza. Morphology.— Bacilli from 3 to 4 /* long and 0.75 to 1 /* thick, enveloped in a capsule containing one to four individuals. Biological Characters. — An aerobic and facultative anaerobic, non- motile, non-liquefying bacillus. Does not form spores. Grows in the usual culture media at 18° to 35° C. Upon gelatin plates circular, milk-white colo- nies are developed ; these have a faint aromatic odor and are cupped upon the upper surface ; they resemble drops of mucus about the size of a pin's head. In stick cultures in gelatin a nail-like growth, like that of Friedlander's bacil- lus, is seen, and there is a formation of gas. Stains with the usual aniline colors, but not by Gram's method. Pathogenesis. — White mice and field mice die from general infection in from thirty-six to forty-eight hours after inoculation ; they also suffer from conjunctivitis. Not pathogenic for rabbits or for pigeons. 151. BACILLUS PYOGENES SOLI. Obtained by Bolton from garden earth by inoculation into a rat. Found in association with the tetanus bacillus in pus from the inoculation wound. Morphology. — Closely resembles the bacillus of diphtheria. " It presents the same irregularities of shape, and the transverse, unstained clear spares in stained preparations, as the diphtheria bacillus. The individual bacilli vary greatly in length and thickness, and many of them are bent and nar- rower through the middle than at the poles." Stains readily with the usual aniline colors, but takes the stain irregularly, sometimes showing deeply stained spots which may be perfectly round. Does not stain by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- Uquefying, non-motile bacillus. Spore formation not observed with cer- tainty — highly refractive ovoid bodies are sometimes met with, but these do not seem to be specially resistant to heat. In gelatin roll tubes very small, spherical colonies are developed, which under a low power are seen to !><• finely granular and to have a lemon-yellow color. Grows best in a slight 1 y acid medium — very slowly at the room temperature. In gelatin stick cul- tures isolated colonies are formed along the line of puncture. Scanty growt h on potato or blood serum. Bolton says : " I have rarely succeeded in getting a growth in agar." Pathogenesis. — Subcutaneous inoculations in rats, gray mice, rabbits, and usually in white mice produce an abscess at the point of inoculation. Injections into the ear veins of rabbits sometimes give rise to multiple ab- scesses, especially in the joints and kidneys. " The abscesses following sub- cutaneous inoculation form very quickly, within twenty-four hours, and run NOT DESCRIBED IN PREVIOUS SECTIONS. 513 a longer or shorter course, from forty -eight hours to eight or ten days, in direct proportion to the amount of the culture introduced. The animals do not seem to suffer any inconvenience, as a rule, and after the abscess is opened suppuration ceases. The organism is found aggregated in small and large, irregular clumps in the pus, many of them lying in the pus corpuscles. It seems to form metastatic abscesses only under exceptional circumstances, such as when injected directly into the blood. Otherwise the abscess remains strictly confined to the seat of inoculation in rabbits, white rats, and gray mice." 152. BACILLUS VENENOSUS. Obtained by Vaughan from water. Morphology. — Bacilli with rounded ends, two to four times as long as broad. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile bacillus. Spore formation not mentioned. Grows rapidly in the usual culture media at the room temperature — also at 38° C. On gelatin plates small, white, spherical colonies — sometimes slightly yel- low; the superficial colonies are elevated above the surface of the gelatin. In gelatin tubes an abundant growth occurs along the line of puncture and slowly extends upon the surface. In cultures from the spleen of an inocu- lated animal the growth upon the surface is less marked. On agar a thin, white layer is formed. On potato a light-brown, moist growth. In recent cultures from the spleen of an inoculated animal the growth upon potato may be invisible. Grows abundantly both in Parietti's solution and in Uf- felmann's gelatin. Pathogenesis. —Pathogenic for rats, mice, guinea-pigs, and rabbits. 153. BACILLUS VENENOSUS BREVIS. Obtained by Vaughan from water. Morphology. — Short, thick bacilli, about twice as long as broad; in old cultures grows out into threads. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile bacillus. Spore formation not mentioned. Grows rapidly in the usual culture media at the room temperature— also at 38° C. On gelatin plates forms small, round colonies with concentric rings; the deeper colonies are generally yellowish or brown ; the surface colonies are elevated and spread but little. In gelatin tubes grows along the line of puncture and spreads slowly upon the surface, finally reaching the sides of the tube. Upon agar a thin, white layer is formed. On potato a thick and moist, light-brown growth. When kept for fourteen days or longer at 40° C. there is an invisible growth upon potato. Grows abundantly in Parietti's solution and slowly in Uffelmann's gelatin. Pathogenesis. — Pathogenic for rats, mice, guinea-pigs, and rabbits. 154. BACILLUS VENENOSUS INVISIBILIS. Obtained by Vaughan from water. Morphology. — A slender bacillus with rounded ends, from two to four times as long as broad. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not mentioned. Grows slowly in the usual culture media at the room temperature — also at 38° C. On gela- tin plates small, granular, yellowish colonies are developed ; the superficial colonies are coarsely granular and very irregular in size and outline. In gelatin tubes grows slowly both on the surface and along the line of punc- ture ; scarcely visible at end of three days. On agar a very thin, white 3« 514 PATHOGENIC AEROBIC BACILLI growth. On potato the growth is sometimes invisible ; on some potatoes a light-brown layer may be developed. Grows well both in Parietti's solution and in Uffelmann's gelatin. Pathogenesis. — Pathogenic, but in less degree than Bacillus venenosus. 155. BACILLUS VENENOSUS LIQUEFACIENS. Obtained by Vaughan from water. Morphology. — Bacilli with rounded ends, one and one-half to twice as long as broad. Biological Characters. — An aerobic and facultative anaerobic, lique- fying, motile bacillus. Spore formation not mentioned. Grows rapidly in the usual culture media at the room temperature— also at 38° C. On gehttni plates the deep colonies are finely granular, spherical, and yellowish iu color; superficial colonies elevated and spread over the surface. In gelatin tubes grows abundantly along the line of puncture and spreads slowly over the surface; liquefaction commences in from four to six weeks. On agar a thin, white growth. On potato a moist, light-brown or yellowish growth. When kept for fourteen days or longer on spleen tissue it forms an invisible growth on potato. Grows abundantly both in Parietti's solution and in Uffelmann's gelatin. Pathogenesis. — Pathogenic for mice, rats, guinea-pigs, and rabbits. 156. BACILLUS AEROGENES CAPSULATUS. Found by Welch in the blood vessels of a patient with thoracic aneurism opening externally ; autopsy made in cool weather eight hours after death — the vessels found full of gas bubbles. Morphology. — Straight or slightly curved bacilli with slightly rounded or sometimes square-cut ends ; a little thicker than Bacillus aiithracis, and varying in length — average length 3 to 6 fj. ; long threads and chains are oc- casionally seen. The bacilli, both from cultures and in the animal body, are enclosed in a transparent capsule. Biological Characters. — An anaerobic, non-motile, non-liquefying ba- cillus. Does not form spores. Grows in the usual culture media, in the ab- sence of oxygen, at the room temperature, and produces an abundant de- velopment of gas in all. In nutrient gelatin there is no marked liquefaction, but the gelatin is slightly peptonized. In agar, colonies are developed which are usually one to two millimetres in diameter, but may attain a diameter of one centimetre ; they are grayish- white in color and in the form of flattened spheres, ovals, or irregular masses, beset with little projections or hair-like processes. Bouillon is rendered diffusely cloudy, with an abundant white sediment. Milk is coagulated in one or two days. The cultures in agar and bouillon have a faint odor, comparable to that of stale glue. Upon potato & pale grayish-white layer is developed; growth occurs at 18° to 20° C., but is much more rapid at 30° to 37° C. Bouillon cultures are sterilized by ex- posure to a temperature of 58° C. for ten minutes. Pathogenesis. — "Quantities up to 2.5 cubic centimetres of fresh bouillon cultures were injected into the circulation of rabbits without an y apparent effect, except in one instance in which a pregnant rabbit was killed, by the injection or one cubic centimetre, in twenty -one hours. If the animal is killed shortly after the iniection the bacilli develop rapidly after death, with an abundant formation of gas in the blood vessels and organs, especially t lie liver. At temperatures of 18° to 20J C. the vessels, organs, and serous cavi- ties may be full of gas in eighteen to twenty-four hours, and at tempera- tures or 30° to 32° C. in four to six hours, when one cubic centimetre of a bouillon culture has been injected into the circulation shortly before death." It is suggested by Welch and Nuttall that in some of the cases in which death has been attributed to the entrance of air into the veins, the gas NOT DESCRIBED IN PREVIOUS SECTIONS. 515 found at the autopsy may not have been atmospheric air, but may have been produced by this or some similar microorganism entering the circulation and developing after death. 157. BACILLUS OF CANON AND PIELICKE. "Found by Canon and Pielicke (1892) in the blood of fourteen patients with measles, and supposed to be the etiological agent in this disease. Morphology. — Bacilli varying greatly in size; sometimes the length is equal to the diameter of a red blood corpuscle, others are quite short and resemble diplococci ; often united in pairs. Stained by Canon, in blood drawn from the finger, by the use of the fol- lowing solution : Concentrated aqueous solution of methylene blue, forty cubic centimetres ; one-quarter-per-cent solution of eosin in seventy-per-cent alcohol, twenty cubic centimetres; distilled water, forty cubic centimetres. The preparations were first placed in absolute alcohol for five to ten minutes, then placed in the staining solution in the incubating oven at 37° C. from six to twenty hours. Some of the bacilli do not stain uniformly, but present the appearance of stained spots alternating with unstained portions. Biological Characters not determined. Does not grow in glycerin-agar or in blood serum. In bouillon inoculated with blood from the finger of a measles patient, bacilli were obtained in three cultures which resembled the bacillus found in the blood, and which failed to grow when transplanted to- glycerin-agar, blood serum, or bouillon. At first the bouillon remained clear, with a sediment at the bottom partly made up of the inoculated blood ; after several days a faint cloudiness was noticed and small flocculi formed. In these bouillon cultures the bacilli had various forms and dimensions, some of them exceeding in length those found in stained preparations from the blood. They appeared to have a slight independent motion. The bacilli in these bouillon cultures did not stain by Gram's method. The bacilli re- ferred to were found in the blood preparations in varying numbers — some- times very few, and at others the first field examined was crowded. They were found during the whole course of the disease, and in one case three days after the fever had disappeared. They were also found in the secre- tions from the nose and conjunctiva of measles patients. 158. BACILLUS SANGUINIS TYPHI. Obtained (1892) by Brannan and Cheesman from the blood of typhus- fever patients. "The blood, obtained under strict antiseptic precautions from the six living patients, was streaked on six-per-cent glycerin-agar plates, and smeared on sterilized cover glasses by Dr. Brannan and brought at once to the laboratory. The cover-glass smears from all the cases, being' dried at once in the air, were fixed in alcohol and stained in Czenzynski's solution for eighteen hours at room temperature. Although all of these covers were examined throughout with a one-sixteenth homogeneous immer- sion lens in the most careful manner, in only about one-half of them a few blue-stained bacilli were found, never more than eight or ten on a cover." Morphology. — Bacilli with round ends, from 1 to 2. 5 ju long and 0.5 to 0.8 u broad ; solitary or in pairs, and occasionally in chains containing six to eight elements; often club-shaped, or ovoid in recent cultures. Stains with the usual aniline colors and by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- motile bacillus. Does not form spores. Does not grow at a lower tempera- ture than 27° C. Grows best upon blood serum at 37.5° C. Upon glycerin- agar plates colonies are developed which at the end of eighteen hours appear as minute, bluish-gray, translucent spots, the diameter of which does not exceed 0.25 millimetre ; later the colonies appear dry and scaly, they are flat, more opaque, and whiter, and do not exceed two millimetres in 510 PATHOGENIC AEROBIC BACILLI diameter. Under a low power the recent colonies are seen to be granular, to have a sinuous and sharply defined margin and a pale-brown color which is more intense at the centre and in scattered points upon the surface. When magnified one hundred diameters the surface appears to be coarsely granular, and coarse, irregular spiculae are seen about the margin. In glycerin-agar tubes, at 37. 5° C., growth occurs upon the surface and along the line of puncture as small, white, isolated colonies. Upon blood serum a slightly elevated, white, shining layer is developed. In milk a white deposit is formed at the bottom of the tube and the milk undergoes no apparent change. On potato no visible growth was obtained. Pathogenesis.— "Inoculations of cultures of the bacillus obtained from two of the cases were made in eight rabbits, two guinea-pigs, and two white mice. All the animals showed marked emaciation, and, with the exception ot two rabbits, all the animals experimented upon died in from ten to twenty- nine days. The inoculated bacillus was obtained from the heart's blood of two of the rabbits that died." Fia. 159. FlO. 160. FIG. 159.— Bacillus gracilis cadaveris, from a gelatin culture. X 1,000. From a photomicro- graph. (Sternberg.) FIG. 160.— Bacillus gracilis; colonies in gelatin roll tube, end of forty-eight hours. X 12. From a, photograph. (Sternberg ) 159. BACILLUS GRACILIS CADAYERIS (Sternberg). Obtained (1889) from a fragment of liver, of man, kept for forty-eight hours in an antiseptic wrapping. Morphology. — Bacilli about 1 // broad and 2 ft long, associated in long chains. Biological Characters. — An aerobic and facultative anaerobic, non- motile, non-liquefying bacillus. Spore formation not observed. In gelatin roll-tubes the deep colonies are opaque and spherical ; superficial colonies circular or slightly irregular in outline, white in color, and opaque or slightly translucent. In gelatin stick cultures, at 22° C., at the end of fiv<> days a rather thick, white mass at tho point of puncture, covering one-third of tin- sin-face, and closely crowded, opaque colonies at bottom of line of puncturr. with slander, branching outgrow! li above. In nutrient agar, at the end of five days at 22° C., a milk-white growth upon the surface and opaque growth to bottom of line of puncture. On potato, at end of five days at 22° C., rather thick, cream-white growth witn irregular margins along the impfstrich. Cultures in bouillon have a milky opacity and a very disagree- able odor. Grows in agna coco without formation of r lumpy. Upon agar, at 37° C. , a soft, shining layer is formed at the end of twenty- four hours ; this consists of fine drops, which are colorless by reflected li<£ht and grayish-white bv transmitted light. Usually these little drops do not run together. In milk, development occurs without coagulation or produc- tion of acid. Does not grow on potato. Pathogenesis. — Pathogenic for rabbits, guinea-pigs, white mice, and pig- eons. Subcutaneous injections of 0.4 to 1 cubic centimetre of a recent bouil- lon culture give rise to septicaemia and death — in rabbits from twelve hours to three days, in guinea-pigs in from two to four days. 166. BACILLUS OF BUBONIC PLAGUE (Kitasato). Discovered by Kitasato (1894) in the blood of living patients, and in the buboes, blood, and organs of those who had recently died from the infectious malady known as bubonic plague. Kitasato was sent to Hong- Kong by the Japanese Government for the purpose of inves- tigating this disease. According to Lowson the bacilli are found in the faeces, in the contents of the buboes, and in the blood. Morphology. — In his preliminary note, Kitasato described tin- plague bacilli as "rods with rounded ends," which are readily stained by the ordinary aniline dyes, the poles being stained darker than the middle part, especially in blood preparations, and present- ing a capsule sometimes well marked, sometimes indistinct. Yersin, who was sent by the French Government to study the bubonic plague at Hong-Kong, arrived in that city on the 15th of June, 1894. He describes the bacillus found in the contents of the buboes as being short and thick, with rounded ends, staining easily with the aniline colors, but not by Gram's method. " The extremities NOT DESCRIBED IN PREVIOUS SECTIONS. 521 stain more intensely than the centre, so that they often present a clear space in the middle. Sometimes the bacilli appear to be sur- rounded by a capsule. ... In bouillon the bacillus has a very char- acteristic appearance, resembling the cultures of the streptococcus of erysipelas — a clear liquid with grumous deposits on the walls and at the bottom of the tube. These cultures examined under the micro- scope show veritable chains of short bacilli, presenting in places a considerable spherical enlargement." Biological Characters. — We quote from Kitasato's preliminary report as follows : The bacilli show very little movement, and those grown in the incubator, in beef -tea, make the medium somewhat cloudy. The growth of the bacilli is strongest on blood serum at the normal temperature of the human body (34° C.) ; under these conditions they develop luxuriantly and form a col- ony moist in consistence and of a yellowish-gray color ; they do not liquefy the serum. On agar-agar jelly (the best is good glycerin agar) they also grow freely. The different colonies are of a whitish-gray color and by re- flected light have a bluish appearance ; under the microscope they appear moist and in rounded patches with uneven edges ; at first they appear every- where as if piled up with "glass-wool," later as if haying dense, large cen- tres. If a cover-glass preparation is made from a cultivation on agar-agar, and, after haying been stained, is observed under the microscope, long threads of bacilli are seen, which might, by careless inspection, be mistaken for a coccus chain, but are recognized with certainty as " threads of bacilli " under closer observation. The growth on agar-gelatin is similar to that on agar-agar ; in a puncture cultivation at the ordinary temperature after a few days they are found growing as a fine dust in little points alongside the puncture, but with very little growth on the surface. Whether these ba- cilli are able to liquefy ordinary gelatin or not I am at present unable to de- cide, as the temperature of Hong-Kong ranges so high that the employment of simple nutritive gelatin is out of the question. I shall give further infor- mation on this question later. On potatoes at a temperature of from 28° to 30° C., there was no growth after ten days' observation, but at a tempera- ture of 37° C. the bacilli developed sparingly after a few days ; the growth was whitish-gray in color and exsiccated. As mentioned before, the bacilli grow best at a temperature of from 38° to 39° C. ; at how low a temperature growth is possible I am unable at present to state. So far I have been un- able to observe the formation of spores. Experiments on Animals. — Mice, rats, guinea-pigs, and rabbits are sus- ceptible to inoculation. If these animals are inoculated with pure culti- vations, or with the blood of a plague patient in which the bacilli have been observed, or with the contents of a bubo, or with pieces of internal organs, or even with the contents of the intestine, they begin to become ill in from one to two days, according to the size of the animal. Their eyes become wa- tery, they begin to show disinclination for any effort, later they avoid their food, and hide quietly in a corner of the cage. The temperature rises to 41.5° C., and with convulsive symptoms they die in from two to five days. I must observe that in Hong-Kong I could only obtain small guinea-pigs . (weight from one hundred to one hundred and fifty grammes) and small rabbits (from two hundred to two hundred and fifty grammes). If I could have experimented upon larger animals it is possible that life would have been prolonged somewhat beyond the periods mentioned above. The parts around the point of inoculation are infiltrated with a reddish gelatinous exudation, the spleen is enlarged, sometimes there is a swelling of the lym- phatic glands, and in all the organs the bacilli are found. The results found 522 PATHOGENIC AEROBIC BACILLI after death in animals are very similar to those found in anthrax and in oedema malignum. Pigeons do not appear to be susceptible to the influence of the bacilli. I made experiments by feeding some mice and guinea-pigs with pure cultivations of the bacillus and with small pieces of the internal organs : the result was, such animals perished in a few days under the same symptoms as those which had been inoculated. In all the internal organs of animals so destroyed I found the bacilli. With the dust of dwelling- houses from which the plague-stricken had been removed, I made sev- eral experiments upon animals. Some of the animals died from tetanus. In one case only a guinea-pig died with plague symptoms, and in this ani- mal the same bacilli were found in the internal organs as in those of plague patients who had succumbed. These experiments with the dust from infected houses I shall certainly continue. Many rats and mice at present die spontaneously in Hong-Kong. I examined some of them. In the inter- nal organs of a mouse I discovered the same bacilli. Experiments with Desiccation, — The contents of a bubo in which the bacilli were present in great numbers were wiped over cover glasses (per- fectly cleansed by heat and alcohol), and some of these cover-glasses were dried in the air of a room at a temperature ranging from 28° to 30° C. Oth- ers I exposed directly to the sun's rays, and from among them, after an expo- sure of from one, two, and three hours up to six days, I removed some parts. putting such portions in beef -tea and placing them in the incubator. Those which had been standing in the room from one to thirty-six hours showed a pretty good growth in the incubator, but those which had been in the room for more than four days were unable to show any growth even after one week's incubation. Those exposed directly to the sun were all destroyed after from three to four hours. Further cultivations on serum were treated exactly like the contents of the bubo with very similar results. Experiments with Heat. — Beef -tea cultivations which had been heated for thirty minutes in a water bath up to 80° C. were destroyed; at 100° C., in the vapor apparatus they were destroyed in a few minutes. Yersin reports that when fragments of the spleen or liver of animals which have died of the plague are fed to rats and mice they usually become infected and die, and the bacillus is found in their organs, lymphatic glands, and blood. He also demonstrated the pres- ence of the bacilli in dead rats found in the houses or streets of Hong-Kong. 167. BACILLUS PISCICIDUS AGILIS (Sieber). Discovered by Sieber (1895) in infected fish, which died of an epidemic disease in the laboratory of Professor Nencki, at St. Petersburg. Morphology. — Short bacilli, often united in pairs. Biological Characters. — An aerobic and facultative anaerobic, motile, liquefying bacillus. In old cultures in bouillon spores are developed. Grows at temperatures of from 12° to 37.5° C. Thermal death point, 00 to 65° C. On gelatin and agar plates forms granular, grayish, or yellowish colonies, which appear to be made up of three concentric rings — the outer one having a jagged outline. Gas is developed during the growth of the bacillus — carbon dioxide and methyl merecaptaii in small amount. Upon potato it f<»nns yellowish-brown, pearl -like colonies. Causes coagulation of milk. Retains its vitality and virulence for months in well or river water. Pathogenesis. — Pathogenic for fish, frogs, guinea-pigs, rabbits, mice, and dogs (not for birds). Old cultures are more pathogenic than recent ones, and gelatin cultures are the most active. Frogs are killed in half an hour by 0.1 cubic centimetre of a bouillon culture six days old. Filtered cultures are as toxic as those containing the living bacillus ; they give with iro NOT DESCRIBED IN PREVIOUS SECTIONS. 523 11 chloride a characteristic color reaction — an intense red color. Sieber has obtained from his cultures an extremely toxic alkaloid in the form of a hydrochlorate. Two litres of filtered culture gave 0.1 gramme of this salt. An aqueous solution of this killed a frog in fifteen minutes in the dose of 0.0035 gramme. 168. BACILLUS OF MERESHKOWSKY. Obtained by Mereshkowsky (1894) from infected animals (Spermophilus musicus) which died from an epidemic malady developed in his laboratory. Morphology. — Closely resembles Loffler's Bacillus typhi murium. Biological Characters.— An aerobic, motile, non-liquefying bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature — best at 37.5° C. In bouillon, at the end of twenty-four hours, the medium is clouded and a white pellicle is seen upon the surface, which breaks up into small flocculi and falls to the bottom when the tube is slightly shaken. On gelatin plates minute, slightly granular, pale-brown colonies may be seen, under a low power at the end of twenty-four hours ; on the sscond day these are visible as white spheres, which under the micro- scope have a pale-brown color and a more or less transparent, peripheral zone. In media containing glucose 110 gas is developed. The growth upon agar and potato presents nothing characteristic. Pathogenesis. — Pathogenic for Zieselmausen (Spermophilus musicus), for Spermophilus guttatus, for squirrels (Sciurus vulgaris) for house mice, for field mice (Arvicola arvalis). Not pathogenic for man or for the domes- tic animals tested, horse, swine, sheep, fowls. Mereshkowsky proposes to use cultures of this bacillus for the extermination of field mice, which die in from one to ten days after being fed upon biscuit wet with a bouillon cul- ture. 169. BACILLUS OF EMMERICH AND WEIBEL. Obtained by Emmerich and Weibel (1894) from infected trout in ponds belonging to an establishment for raising these fish. The disease appeared as a superficial ' ' f urunculosis with secondary development of abscesses con- taining bloody pus." Death occurred in from twelve to twenty days. The pustules and secondary abscesses and blood from the heart and various or- gans contained bacilli, which proved to be the cause of the infectious malady. Morphology. — Bacilli about as long as the typhoid bacillus, but not so thick, very frequently united in pairs ; occasionally grows out into filaments. Biological Characters. — An aerobic and facultative anaerobic, lique- fying, non-motile bacillus. Does not form spores. Thermal death point, 60° C. Stains with the usual aniline colors but not by Gram's method. Grows best at 10° to 15° C. The growth in gelatin is quite characteristic. At the end of two or three days, in gelatin plates, at the room temperature, small white colonies are developed ; in four or five days small gas bubbles or ex- cavations are seen, at the bottom of which lie the scale-like or rosetta-formed colonies. The margin of the colonies is irregular and later jagged. At first the colonies are grayish- white or yellowish, later brownish. The superficial colonies have a peculiar lustre. In gelatin stick cultures, colo- nies develop along the line of puncture, which at first resemble the growth of Streptococcus pyogenes, and no development is seen on the surface. At the end of five to seven days in place of the line of colonies is seen a channel filled with air, or gas developed by the separate colonies, the bubbles from which coalesce. The funnel formed in this way is somewhat larger above, and at the bottom contains a whitish sediment consisting of bacteria con- tained in a few drops of liquefied gelatin. Along the sides of the funnel bubble-like cavities may frequently be seen, at the bottom of which the bac- teria have accumulated. In bouillon a slight cloudiness is seen near the surf ace, on the walls of the test tube; when .slightly shaken this falls to the 524 PATHOGENIC AEROBIC BACILLI bottom, leaving- the bouillon entirely clear. In agar-agar tubes, a veil- like stripe develops along the line of puncture, and a grayish-yellow, moist layer, with irregular outlines upon the surface. After some weeks this acquires a brown color. No growth occurs upon potato. No development occurs in the incubating oven at 37° C. Pathogenesis. — Trout became infected and died through direct infection, subcutaneous or intramuscular inoculations, or through the addition of cul- tures to the* water in which they were kept, or by placing infected fish in the same tank with healthy ones. 170. GAS-FORMING AEROBIC BACILLUS OF LASER. Obtained by Laser (1892) from a piece of liver and lung from a calf which died of an infectious disease. Morphology. — " Short bacilli." Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile, gas-producing bacillus. Stains by Gram's method. Spore formation not observed. In gelatin stick cultures development oc- curs on the surface in the form of a button-like mass, and along the line of puncture colonies are formed which may be separate below. The colonies in gelatin and agar plates are not characteristic. Upon agar, at 37° C. a slimy, moist, shining layer is developed which covers the entire surface. In stick cultures in gelatin or agar containing glucose an abundant develop- ment occurs, attended with an evolution of gas. In bouillon, in the incubat- ing oven, a uniform cloudiness of the culture medium is seen at the end of twenty-four hours, and the bacilli gradually sink to the bottom of the tube. Upon potato in the incubating oven, a shining, white layer is developed over the entire surface ; on potato kept at the room temperature a thick, grayish-yellow layer in the middle, which gradually becomes more decidedly yellow, while the potato around this growth has at first a violet shimmer, and later an intense violet color. Pathogenesis. — The limited number of experiments made on mice, rab- bits, and guinea-pigs resulted in the death of some of the animals, while others recovered. (This appears to be a bacillus of the colon group, which differs but little from Bacillus coli communis. G. M. S.) 171. BACILLUS OF BECK. Synonym. — Der Bacillus der Brustseuche beim Kaninchen. Obtained by Beck (1892) from rabbits which died of an infectious malady in the Institut fiir Infectionskrankheiten, in Berlin. Morphology. — Very small and slender bacilli, about twice as long and twice as thick as the influenza bacillus ; somewhat pointed at the extremities ; show a tendency to grow out into filaments. Biological Characters. — An aerobic (strict) non-liquefying, non-motile bacillus. Spore formation not observed. Grows at the room- temperature and more vigorously at 38° C. Does not stain by Gram's method. Thermal death point, 50° C. (five minutes). Resists desiccation, at the room tempera- ture, for seventeen days, at 37° C. for three days. On gelatin plates, at the end of forty-eight hours, small, finely granular, glass-like colonies are developed ; older colonies have a pale-brown appear ance. In gelatin stick cultures a granular growth of a white color is seen along the line of puncture. Upon agar, at 37° C., an abundant development occurs in twenty -four hours. The line of puncture seen from above is gray- ish-white, by transmitted light bluish and porcelain-like with a brownish tint. On agar plates the colonies have a yellowish-gray appearance ; the margin of the finely granular colonies is sharply defined. In agar cultures several days old the colonies are sticky and may be picked up as a compact mass, or drawn out into threads. In bouillon, at 37° C., there is a slight NOT DESCRIBED IN PREVIOUS SECTIONS. 525 cloudiness at the end of twenty-four hours ; later the bouillon is clear and a white sediment is seen at the bottom of the tube. In bouillon cultures especially, the bacillus grows out into long filaments. Pathogenesis. — From 0.25 to 1 cubic centimetre of a bouillon culture injected into the pleural cavity of a rabbit caused a development of all of the symptoms of influenza (Brustseuche)— viz. , elevation of temperature at the end of five or six hours, cough, nasal discharge, dyspnoea, and death — usually in from three to five days. The autopsy showed a distinct pleuro- fneumonia and a general blood infection by the bacillus in question, njections into the circulation also give rise to the symptoms of influenza, including pneumonia, and to death at the end of from ten to fourteen days. Subcutaneous injections resulted in the development of an abscess and of ex- tensive necrosis of the tissues, but did not cause a general blood infection. Guinea-pigs were somewhat less susceptible than rabbits, but injections into the pleural cavity produced similar symptoms and death at a later date. White mice and house mice, as a result of intra peritoneal injections, died within two or three days from general blood infection. 172. BACILLUS BOVIS MORBIFICANS Obtained by Basenau (1893) from the flesh of a cow, which is supposed to have died from puerperal fever and was condemned by the inspector at the slaughter-house in Amsterdam. Morphology. — Short bacilli, with rounded ends, two to two and one-half times as long as broad, usually united in pairs. 0.3 to 0.4 ^ broad. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile bacillus. Does not stain by Gram's method. Does not form spores — is killed in one minute by exposure to a temperature of 70° C. Does not coagulate milk. In media containing glucose causes a moderate development of gas. Grows at a temperature of 9° C., best in incubating oven at 37° C. In bouillon, at 37° C., a uniform clouding of the medium occurs in twenty-four hours ; later, a thin, smooth pellicle forms 011 the surface, this is readily broken up by gentle agitation and falls to the bottom, where a grayish- white mass accumulates. In gelatin stick cultures a slender, yellowish-white growth is seen along the line of puncture, and a white, thick layer, with more or less irregular outlines, is slowly developed on the surface. In streak cultures the growth is like that of the ' ' colon bacillus. " Upon agar, at 37° C., at the end of twenty- four hours, an abundant grayish-white layer is developed. Upon potato it grows more slowly and forms a soft, yellow layer, which never acquires a brown color. Pathogenesis. — Causes a fatal infection in mice, white rats, guinea-pigs, rabbits, and calves. Mice and guinea-pigs succumb to subcutaneous injec- tions, rabbits to intra-peritoiieal infection, and calves to intraperitoneal injec- tions, or from the iiigestion of milk containing the bacilli. Young guinea- pigs may be infected through the mother's milk. (This bacillus belongs to the "colon group" and is probably a pathogenic variety of Bacillus coli communis. — G. M. S.) 173. BACILLUS PISCICIDUS (Fischel and Enoch). Obtained by Fischel and Enoch (1892) from an infected carp. Morphology. — Bacilli solitary or in chains of four to five elements, long- and ' \JL m V/J.1.CW1.AJ.U \SJL i\_f»-t.i \J\S AA T V> V/ L\s JJL J.V^XX lAJj J. • ^ to 3/* long and 0.25// thick. Stains by the usual aniline colors and by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- motile, liquefying bacillus. Forms spores. In gelatin plates forms round colonies of a pale yellowish-brown color, having a slightly toothed border and a granular surface. At the end of twenty-four hours a narrow zone of 526 PATHOGENIC AEROBIC BACILLI liquefaction can be discerned around the colonies, and at the end of about ten days the gelatin is entirely liquefied. In gelatin stick cultures a scanty growth is seen along the line of inoculation at the end of twelve hours ; the growth upon the surface is rapid, and liquefaction commences at the end of twenty-four hours. Upon agar, at 37° C. at the end of eighteen hours, a thin granular layer is seen, which consists of small, pale-gray colonies. In agar stick cultures a scanty growth occurs along the line of puncture, which does not increase after thirty-six hours. Upon the surface the growthis abundant, forming, at the end of five days a tolerably thick grayish-white layer. No growth occurs upon potato at the room temperature, but at 37° C. a tolera- ly thick, sticky layer of a grayish-white color is developed in three or four days. In bouillon, at 37° C., the medium is clouded at the end of twelve hours, and a thin pellicle is seen upon the surface at the end of thirty-six hours ; this falls to the bottom when the tube is slightly agitated. At the end of four days development has ceased, and the bouillon is again transpar- ent, while a flocculent deposit is seen at the bottom of the tube. The bouillon gives off a penetrating odor, like that of burnt milk. The same odor is given off from cultures in milk, which is peptonized by the action of the bacillus. At the end of twenty days, at 37° C., the entire contents of the tube have be- come transparent. Pathogenesis. —Produces a fatal infectious disease in fish ("gold carp'1) when inoculated beneath the skin ; also pathogenic for mice and for guinea- pigs. 174. BACILLUS PYOGENES FILIFORMIS (Flexner). Obtained by Flexner (1895) from the interior of the uterus and from an exudate in the pericardial and pleural cavities, of a rabbit which died on the fifth day after parturition! Morphology.— Pleomorphous cocci-like forms, short or long bacilli, and long threads are seen in cover slips prepared from the exudate. "Very few of the bacilli stain regularly ; for the most part brightly stained spots appear between stained areas. An outer membrane always stains, enclosing the stained dots in a colorless ground. The threads, as a rule, present delicate, sinuous, and wavy outlines ; the short forms are straight with rounded ends." Biological Characters. — All attempts to cultivate this bacillus in the usual media, either in the presence of oxygen or in an atmosphere of hydro- gen, proved unsuccessful. But successive cultures were made by inoculations in the pleural cavity of rabbits — a bit of pleural exudate suspended in bouil- lon was used for this purpose. The bacillus was also propagated upon the lungs, heart, uterus, and kidney of healthy rabbits. The organs were re- moved with great care to prevent contamination and placed in sterilized test tubes. Transplantations from these cultures were only successful for one or two generations. Better results were obtained by cultivating the bacillus upon the one-third to one-half grown foetuses of rabbits. Pathogenesis. — " Considerable variations were observed according as the inoculations were made into the pleural cavity, the peritoneal cavity, the sub- cutaneous tissue, beneath the dura mater, or directly into the circulation. The inoculations gave positive results in all cases except a few, in which they were made subcutaneously. The death of the animal occurred soonest when inoculation was made beneath the dura mater. A small portion of the skull was trephined, care being taken to exclude rxt raucous microorganisms, and a drop of the pleural fluid or a speck of the librinous oxiidato \vas introduced beneath (lie membranes, care, being1 taken not to injure the brain. These animals, which quickly recovered from the effects of the operation, died on an average about twelve hours after the inoculation. . . . " The pleural inoculations were followed by death, as before stated, in ev- ery instance, the death of the animal occurring upon the third or fourth day. The appearances presented at the autopsy were for the most part an exact NOT DESCRIBED IN PREVIOUS SECTIONS. 527 reproduction of those observed in the animal which had succumbed to the natural disease. Upon the side of inoculation a thick, grayish-yellow, shaggy membrane covered the pleural surfaces, being at times four or five millime- tres in thickness. The pleural cavity contained several cubic centimetres of a clear haemoglobin-colored fluid, the lung for the most part being com- pressed. At times smaller or larger areas of lobular pneumonia would be present ; and, as a rule, the inflammation was not limited to the serous mem- brane of the side of inoculation, but extended into the opposite pleural cavity and into the pericardial sac. However, in these situations the process was, as a rule, less intense, the solid exudate being less considerable, and in the case of the opposite pleural cavity sometimes entirely wanting. The super- ficial vessels, however, were injected and the serous surface of the affected membrane covered with a slimy, clear fluid. In addition to this the oppo- site pleural cavity always contained a similar pink serum to that described upon the side of inoculation. "The study of the exudate upon the side of inoculation as well as the fluid contained in the opposite pleural cavity and in the pericardium showed the same organisms as had been introduced." 175. BACILLUS OF UNNA AND HODARA. Obtained by Hodara (1894) from the contents of acne pustules — ** in enor- mous masses in the comedones of true acne." Morphology.— Small bacilli, from 0.3 to 0.7 t* long and 0.3 ft thick. When stained, by Unna's method, with methylene-blue-glycerin ether, or with methylene-blue-tannin solution, they are seen to be surrounded by a homo- geneous, jelly-like mass which is stained pale violet by the first method and green by the second. The bacilli are sometimes united in chains of two or three elements, and single rods may present in the middle an unstained zone with deeply stained extremities. Biological Characters.— Not determined. 176. PROTEUS FLUORESCENS (Jaeger). Obtained by Jaeger (1892) from the liver, spleen, and kidneys of fatal cases of infectious icterus ("Weil's disease"). Morphology.— A. pleomorphous bacillus of theproteus group ; in the same culture cocci-like elements, short rods either straight or curved, and long filaments are seen. Biological Characters. — An aerobic and facultat ive anaerobic, motile, liquefying, chromogenic bacillus. Is not to be distinguished from Proteus vulgaris except by the fact that it produces an intense fluorescent-green pig- ment. Jaeger says that cultures which originally failed to liquefy gelatin and produced the fluorescent-green pigment, at the end of two and a half years had lost the property of producing pigment and had acquired the property of liquefying gelatin, and could not be distinguished from Proteus vulgaris. But when these cultures were kept at a lower temperature they gradually regained their former characters. Pathogenesis.— Pathogenic for mice and for pigeons; but the virulence of cultures proved to be very variable. 177. MICROCOCCUS INSECTORUM (Burrill). Obtained by Burrill (1883) from the alimentary canal of infected "chinch bugs" (Blissus leucopterus). , Morphology.— Oval or spherical (micrococci ?) bacteria, usually in pairs, but sometimes in chains of four to eight elements. ' ' Undivided segments vary from 0.8 to 1.6 /« in length, with a uniform width of 0.65 p. " (Forbes). 528 PATHOGENIC AEROBIC BACILLI Biological Characters.— Forbes (1891) says: "I have lately succeeded, in conjunction with Professor Burrill, in making pure cultures in consid- erable numbers in both animal and vegetable media." . . . "We have ob- tained successful cultures in all the neutral and alkaline fluids and in none of the*acid ones." Non-motile and does not form spores. 178. BACILLUS MONACH^E (v. Tubeuf). Obtained by v. Tubeuf (1892) from infected caterpillars of Liparis monacha. Morphological and Biological Characters. — Short, motile, aerobic, non- liquefying bacilli, which grow in the usual culture media at the room tem- perature. 179. MICROCOCCUS OF BRUCE. Obtained by Bruce (1892) from the spleen, post-mortem — of cases of so- called "Malta fever." Morphology. — Micrococci, about .33 p in diameter, solitary or in pairs — never in chains. Biological Characters. — An aerobic, non-liquefying, micrococcus. Does not stain by Gram's method. Grows best in nutrient agar. In stab cultures no growth is seen for several days. "At length the growth appears as pearly- white spots scattered around the point of puncture and minute, round, white colonies are also seen along the course of the needle track " ; these increase in size and after some weeks a rosette-shaped growth is seen upon the surface, and the growth along the line of puncture has a yellowish-brown color. At the end of nine or ten days, at 37° C., some of the colonies on the surface of nutrient agar are as large as No. 4 shot; by transmitted light they have a yellowish color at the centre, and the periphery is bluish-white ; by reflected light they have a milky-white color. At 25° C. colonies first become visible at the end of about seven days, at 37° C. in three to four days. Does not grow upon potato. Very scanty growth upon nutrient gelatin at 22° C. at the end of a month. Pathogenesis. — Pathogenic for monkeys, which suffer from fever as a result of subcutaneous inoculations and usually (three out of four experi- mented upon) die in from thirteen to twenty-one days. The spleen is found to be enlarged and contains the micrococcus. Not pathogenic for mice, guinea-pigs, or rabbits. 180. BACILLI OF GUILLEBEAU (a, 6, and C). Obtained by Guillebeau from the milk of cows suffering from mastitis, and found by Freudenreich to produce an abnormal fermentation of cheese, characterized by the presence of large cavities ("boursouflement") and by a very bad taste. BACILLUS a. Morphology. — Varies considerably in size, and may resemble a micrococ- cus in form ; usually 1 // broad and 1 to 2 ft- long. Stains with the usual aniline colors, but rather feebly ; does not stain by Gram's method. Biological Characters.— A.naerobics,ndfact anaerobic, slightly motile, non-liquefyingl>au in length. Biological Characters.— AIL an- aerobic, non-motile bacillus; not cultivated in nutrient gelatin; not observed to form spores. Bacillus cadaveris is a strict anae- robic and is difficult to cultivate. I have succeeded best with nutrient agar containing five per cent of glycerin, removing the oxygen thoroughly by passing a stream of hydrogen through the liquefied me- dium. The colonies in a glycerin- agar roll tube (containing hydrogen and hermetically sealed) are opaque, irregular in outline, granular, and of a white color by reflected light. The culture medium acquires an acid reaction as a result of the de- velopment of the bacillus. Liver tissue containing- this bacillus, after having been kept in an anti- septic wrapping for forty-eight hours, has a fresh appearance, a very acid re- action, and is without any putrefactive odor. FIG. 168.— Bacillus cadaveris, from an anae- robic culture in glycerin-agar. x 1,000. From a photomicrograph. (Sternberg.) 542 PATHOGENIC ANAEROBIC BACILLI. tissue containing this bacillus is very pathogenic for guinea-pigs when injected subcutaneously, and causes an extensive in- flammatory oedema extending from the point of inoculation. Pure cul- tures of the bacillus are less pathogenic, and the few experiments which I made in Havana gave a somewhat contradictory result, recovery having occurred in one guinea-pig which received a subcutaneous injection of ten minims of liquid from an anaerobic culture in glycerin-agar, while another died at the end of twenty hours from a subcutaneous injection of three minims, with extensive inflammatory oedema in the vicinity of the point of inoculation. 188. BACILLUS OF SYMPTOMATIC ANTHRAX. Synonyms. — Rauschbrandbacillus, Ger. ; Bacille du charbon symptomatique, Fr. First described by Bellinger and Feser (1878); carefully studied and its principal characters determined by Arloing, Cornevin, and Thomas (1880-83). FIG. 169. Fio. 170. FIG. 169.— Bacillus of symptomatic anthrax, from an agar culture, x 1,000. From a photomi- crograph. (FrSnkel and Pfeiffer.) FIG. 170.— Bacillus of symptomatic anthrax, from muscles of inoculated guinea-pig. From a photomicrograph. (Roux.) Found in the affected tissues of animals— principally cattle— suf- fering from ' ' black leg, " ' ' quarter evil, " or symptomatic anthrax (Fr. , "charbon symptomatique"; Ger., "Rauschbrand"). The disease prevails during the summer months in various parts of Europe, and is characterized by the appearance of irregular, emphysematous swellings of the subcutaneous tissue and muscles, especially over the quarters, hence the name "quarter evil." The muscles in the PATHOGENIC ANAEROBIC BACILLI. 543 affected areas have a dark color and contain a bloody serum in which the bacillus is found. Morphology. — Bacilli with rounded ends, from three to five /* long and 0.5 to 0.6 /* broad ; sometimes united in pairs, but do not grow out into filaments. The spores are oval, somewhat flattened on one side, thicker than the bacilli, and lie near the middle of the rods, but a little nearer to one extremity. The bacilli containing spores are somewhat spindle-formed (Kitasato). "Involution forms" are quite common in old cultures or in unfavorable media ; in such cultures variously distorted and often greatly enlarged bacilli may be seen, some being greatly swollen in the middle — spindle- shaped. When properly stained, by Loffler's method, a number of flagella are seen around the periphery of the cells. Stains with the aniline colors usually em- ployed, but not by Gram's method. Spore-bear- ing bacilli may be double-stained by first stain- ing the spores by ZiehTs method, and then the bacilli with a solution of methylene blue. Biological Characters. — An anaerobic, liq- uefying, mot i '1 e bacillus. Forms spores. Grows at the room temperature in the usual culture media, in the absence of oxygen, in an atmosphere of hy- drogen, but not in carbon dioxide. This bacillus grows more rapidly and abundantly in nutrient agar or gelatin to which 1.5 to 2 per cent of grape sugar or five per cent of glycerin has been added. Colonies in gelatin, in an atmosphere of hydrogen, are at first spherical, with irregular out- lines and a wart-like surface ; later the gelatin is liquefied around them, and radiating filaments grow out into the gelatin, so that by transmitted light they present the appearance of an opaque central mass with an irregular surface surrounded by rays. In stick cultures in nutrient gelatin, at 20° to 25° C., at the end of two or three days development occurs at the bottom of the line of puncture to within about two fingers' breadth of the surface ; the gelatin is slowly liquefied and considerable gas is formed. In old cultures the growth and liquefaction of the gelatin extend nearly to the sur- face. In agar stick cultures, in the incubating oven, develop- ment begins within a day or two and extends to within one finger's breadth of the surface ; considerable gas is evolved, and FIG. in. — Bacillus of symptomatic an- thrax; lon^ stick cul- ture in nutrient gela- tin, ten days at 18°- 90° C. CKitasato.) 544 PATHOGENIC ANAEROBIC BACILLI. the cultures have a peculiar, acid, penetrating odor. Development is most rapid at 36° to 38° C., but may occur at a temperature of 16° to 18° C. — not lower than 14°. Spores are quickly formed in cul- tures kept in the incubating oven — not so quickly at the room tem- perature. These withstand a temperature of 80° C. maintained for an hour, but are killed in five minutes by a temperature of 100° C. (in steam). In the bodies of infected animals spores are not formed until after the death of the animal, at the end of twenty-four to forty- eight hours (Kitasato). The spores are destroyed by a five-per-cent solution of carbolic acid in ten hours, and the bacilli, in the absence of spores, in five minutes ; a 1 : 1,000 solution of mercuric chloride destroys the spores in two hours (Kitasato). According to Kitasato, certain shining bodies of irregular form, which stain readily with the aniline colors, are to be seen in the rods as they are found in the bloody serum from an animal recently dead ; but these are not spores, as some bacterio- logists have supposed. Pathogenesis. — Cattle, which are immune against malignant oedema, are most subject to infection by the bacillus of symptomatic anthrax, and the disease produced by this anaerobic bacillus prevails almost entirely among them ; horses are not attacked spontaneously — i.e., by accidental infection — and when inoculated with a culture of this bacillus present only a limited local reaction. Swine, dogs, rab- bits, fowls, and pigeons have but slight susceptibility, but the re- searches of Arloing, Cornevin, and Thomas, and of Roger show that by the addition of a twenty-per-cent solution of lactic acid to a cul- ture its virulence is greatly increased, and animals which have but little susceptibility, like the rabbit or the mouse, succumb to such in- jections ; similar results were obtained by Roger by the simultaneous injection of sterilized or non-sterilized cultures of Bacillus prodigiosus or of Proteus vulgaris. The guinea-pig is the most susceptible ani- mal. When inoculated subcutaneously with a small quantity of a pure culture, or with spores attached to a silk thread, it dies in from twenty-four to thirty-six hours. At the autopsy a bloody serum is found in the subcutaneous tissues in the vicinity of the point of in- oculation, and the muscles present a dark-red or black appearance similar to that in cattle affected with " black leg." The internal or- gans present no notable pathological changes. Immediately after death the bacilli are found only in the effused serum and the affected tissues near the point of inoculation, but later they multiply in the cadaver and are found throughout the body. According to Kitasato, the cultures in solid media preserve their virulence for an indefinite period, but cultures in a bouillon made from the flesh of guinea-pigs soon lose their virulence. Cultures are readily attenuated by heat PATHOGENIC ANAEROBIC BACILLI. 545 according to the method of Toussaint and Chauveau ; a temperature of 42° to 43° C. is suitable. The pathogenic virulence of spores may also be attenuated by subjecting them to dry heat — a temperature of 80° to 100° C. maintained for several hours. For the production of immunity in cattle Arloing, Cornevin, and Thomas recommend the use of a dried powder of the muscles of animals which have suc- cumbed to the disease, and which has been subjected to a suitable temperature to insure attenuation of the pathogenic virulence of the spores contained in it. Kitt, who has made extended experiments with this bacillus, recommends that the muscles be first dried at 32° to 35° C. and then powdered. Two vaccines are then prepared — a stronger vaccine by exposure of a portion of the powder to a tem- perature of 85° to 90° C. for six hours, and a weaker vaccine by ex- posure for six hours to a temperature of 100° to 104° C. (dry heat), Inoculations made with this attenuated virus — the weakest first and subsequently the least attenuated — give rise to a local reaction of moderate intensity, and the animal is subsequently immune from the effects of the most virulent material. Immunity may also be secured by intravenous inoculations ; or, in guinea-pigs, by inoculations with bouillon cultures which have been kept for a few days and as a re- sult have lost their original virulence, or with cultures kept in an in- cubating oven at a temperature of 42° to 43° C. ; or by inoculation with a very minute quantity of a pure culture ; or by an inoculation made into the extremity of the tail ; or by inoculations with filtered cultures (Roux and Chamberlain), or with cultures sterilized by heat (Kitasato). It has been claimed (Roux) that animals which have been made immune against symptomatic anthrax are also immune against malignant oedema. But in a carefully conducted series of experiments Kitasato was unable to confirm this ; he found that guinea-pigs which had an immunity against the most virulent cul- tures of the Rauschbrand bacillus succumbed invariably to malig- nant oedema when inoculated subcutaneously with the bacillus of malignant oedema. Klein (1894) has obtained from the spleen of sheep a bacillus which corresponds with the bacillus of malignant oedema in every respect, except that it proved to be without pathogenic power — "a non-virulent variety of the Rauschbrand bacillus" (Klein). 189. BACILLUS CEDEMATIS MALIGNI NO. II. (Novy). Obtained by Novy (1894) from the subcutaneous oedema in guinea-pigs which were inoculated with a solution of milk-nuclein, which had been pre- pared from fresh casein. Morphology. — Bacilli with rounded ends, usually solitary, from 2.5 to 5 fj- long and from 0.8 to 0.9 // broad. Occasionally short and straight fila- ments, 8 to 14 // long, are seen — very rarely these reach a length of 22 to 35 n. 546 PATHOGENIC ANAEROBIC BACILLI. Long and slender spiral filaments are found in pure cultures which are be- lieved to be gigantic flagella. These are seen in preparations stained with gentian violet as unstained spiral filaments, usually from 17 to 25 n long ; some are of uniform thickness and others spindle-formed, having a thickness of 1.7 to 2.6 p in the middle, and tapering to a scarcely visible line at the ex- tremities. These flagella are readily stained by Loffler's method. They are attached to the periphery of the rods, as in the typhoid bacillus. In artifi- cial cultures they are usually from 40 to 50 /" long. With reference to the peculiar spindle-formed bodies found in the cultures Novy says : " As to the character of these gigantic flagella little can be said. Loftier, who, so far as I know, was the first to observe these singular forms, regarded them as bun- dles or collections of flagella." Although at first inclined to doubt this, Novy says, in a postscript to his paper, that an examination of photo-micrographs, which had been made to accompany it, convinces him that Loffler's explanation is probably correct. Biological Characters. — An anaerobic, motile bacillus. The motions are not active, but consist in a very moderate to-and-fro swinging motion. Does not form spores. Does not grow at the room temperature. Grows at tem- peratures of 24° to 38° C. The best media for its development are slightly alkaline bouillon, gelatin, or agar, containing two per cent of glucose. May be cultivated in a vacuum or in an atmosphere of hydrogen, carbon dioxide, or illuminating gas. Also in long stick cultures in agar. In glucose-agar plates colonies develop in fifteen hours at 38 C. These appear as small, white masses the size of a pin's head, which, under the microscope, appear to be made up of thickly felted threads. The smaller colonies appear as a network of branching lires, very similar to the colonies of the tetanus ba- cillus ; larger colonies have a dark centre, with an irregular, fringed margin, and are surrounded by delicate filaments. In glucose-agar stick cultures growth occurs along the line of puncture to within one cubic centimetre of the surface, but is not as abundant as the growth of the bacillus of malig- nant oedema or of symptomatic anthrax. At 38° C. development occurs within twelve to sixteen hours, and has reached its maximum at the end of twenty- four hours. An abundant development of gas occurs, which splits up the agar and forces the upper portion towards the top of the tube. The develop- ment of gas is most abundant in alkaline media, being almost absent in media having a neutral or acid reaction. The most favorable medium is a fresh al- kaline bouillon containing two per cent of gelatin, of glucose, and of pep- tone. Pathogenesis. — Pathogenic for rabbits, guinea-pigs, white mice, white rats, pigeons, and cats. Death usually results in from twelve to thirty-six hours after the subcutaneous injection of one-tenth to one-fourth cubic centimetre of a pure culture. At the autopsy an extensive subcutaneous cedema is found extending from the point of inoculation. The fluid in the brawny connective tissue is usually colorless, sometimes of a pale-red color. A small amount of gas is commonly present. The pleura! cavities contain an enormous amount of serous exudate, which at first is fluid, but when the autopsy is delayed be- comes gelatinous. In rabbits and guinea-pigs the amount of this serum ob- tained from the pleural cavities may be from fifty to sixty cubic centime! r» «s. The bacilli are usually not very numerous in this serum from the subcuta- neous tissues and pleural cavity. Kerry (1894) has described a " new pathogenic anaerobic bacillus " which resembles that of Novy in several particulars. It does not grow at the ropin temperature, does not form spores, and is pathogenic for mice, rats, rabbits, and guinea-pigs ; it forms "very long and thick flagella, which may be sjn'r- alia gechlangelt." This bacillus was obtained from a guinea-pig inoculated with dried blood (suspended in water containing lactic acid and glucose) which had been obtained from a cow that was supposed to have died of Rauschbrand. PATHOGENIC ANAEROBIC BACILLI. 547 190. BACILLUS PHLEGMONES EMPHYSEMATOS^E (E. Frankel). Obtained by Frankel (1893) from four cases of "gas phlegmon." Morphology. — Short, thick bacilli, with round ends, about as thick as the anthrax bacillus, usually united in pairs ; long filaments are seen in gela- tin cultures, and in the tissues of infected guinea-pigs. Biological Characters. — An anaerobic, non-motile, liquefying bacillus. Spores are occasionally seen in agar cultures ; these are spherical and lo- cated in the slightly swollen extremities of the rods. In glucose-agar (one per cent glucose) plates, in an atmosphere of hydrogen at 37° C., gas bub- bles are seen upon and below the surface; these may have a diameter of one centimetre, and small bubbles are often attached to the larger ones. In other places the agar is split open ; in others still, colonies are developed without the formation of gas ; these are round, with a dark-brown centre and paler margin. In stick cultures in agar containing one per cent of glucose the growth, at 37° C., is abundant at the end of twenty-four hours all along the line of puncture ; the agar is split up by gas, and bubbles often accumulate on the surf ace. In gelatin cultures, in an atmosphere of hydro- gen at the end of two or three days, small, round, brownish-yellow, slightly granular colonies are developed, which later appear to lie in an air bubble. In gelatin stick cultures growth is first seen one to two centimetres below the surface ; after several days spherical colonies are developed along the line of puncture, and at times liquefaction is seen, while at others gas bubbles are developed. Upon blood serum, in hydrogen, an abundant development occurs with formation of gas ; these cultures give off a fetid odor. In milk coagulation occurs, but no gas is developed. The bacillus dies out in agar cultures within two or three days, unless they are preserved in hydrogen. In gelatin cultures it survives for several months. Pathogenesis. — In guinea-pigs subcutaneous inoculation gives rise to the development of a gas phlegmon, and usually to the death of the animal. Not pathogenic for mice or for rabbits when injected into the circulation. NOTES RELATING TO THE PREVIOUSLY DESCRIBED ANAEROBIC BACILLI. Tetanus Bacillus.— Brieger and Cohn, in investigations (1893) relating to the toxic products of the tetanus bacillus, have arrived at the following re- sults : The cultures were made in veal bouillon containing one per cent of peptone and one- fifth per cent of chloride of sodium. Large quantities of the cultures in this medium were filtered through porcelain filters. The active substance was precipitated from the filtrate by means of a saturated solution of ammonium sulphate. By adding this salt in excess the precipi- tate is made to rise to the surface and is skimmed off with a platinum spatula. The liquid is removed by placing this upon porous porcelain plates and the crude toxin is dried in a vacuum. It still contains 6.5 per cent of ammo- nium sulphate. The tetanus bouillon after filtration is said to be fatal to mice in the dose of 0.00005 cubic centimetre. A litre of this bouillon gave about one gramme of the dried precipitate, which produced characteristic tet- anic symptoms and death when injected into mice in the dose of 0.0000001 gramme. Kitasato, in his experiments, had previously obtained a tetanus bouillon which was five times as toxic as that used by Brieger and Cohn in their experiments, and which killed mice in the dose of 0.00001 cubic centi- metre. The dried precipitate obtained by Brieger and Cohn contained vari- ous impurities, including a certain amount of ammonium sulphate, but was found to kill susceptible animals in the proportion of 0.0000066 gramme per kilogramme of body weight. It was purified without loss of toxic power by placing it in a dialyzer in running water for from twenty-four to forty-eight hours, after which it was 548 PATHOGENIC ANAEROBIC BACILLI. dried in vacuo at 20° to 22° C. The purified toxin as thus obtained had a slightly yellowish color, and was in the form of transparent scales, which were odorless, tasted like gum acacia, and were easily soluble in water. The chemical reactions of this purified toxin, according to Brieger and Cohn, show that it is not a true albuminous body. When injected beneath the skin of a mouse weighing fifteen grammes, in the dose of 0.00000005 gramme, it causes its death, and one-fifth of this amount gave rise to tetanic symptoms from which the animal recovered after a time. The lethal dose for a man weigh- ing seventy kilogrammes is estimated by the authors named to be 0.00023 gramme (6.23 milligramme). Comparing this with the most deadly vege- table alkaloids known it is nearly six hundred times as potent as atropin and one hundred and fifty times as potent as strychnin. Fermi and Pernossi (1894), as a result of an elaborate research, have deter- mined many of the chemical characters of the tetanus toxin. "When in solu- tion it is destroyed by a comparatively low temperature (55° C. for one hour) and by exposure to direct sunlight, but the dry powder resists a temperature of 120° C. It has not the properties of an alkaloid, as it is not dissolved by any of the usual solvents of these bodies— the only solvent thus far discovered is said to be water. It resembles the albumins and peptones in its failure to pass through a dialyzing membrane. The authors last referred to conclude their summary of results as follows : "The appended table shows that the tetanus poison, like that of diphtheria, in its behavior as regards the action of light, heat, chemical agents, and dialysis, as also its solvents, the agents which precipitate it, and its action upon living animals, closely resembles the poisons of serpents (Naja tripudians, Crotalus, etc.). As to the chemical nature of this group of substances, we can at present only say that they rather have the characters of colloidal sub- stances than otherwise, and more nearly resemble the albuminoid bodies than the bases. We do not, however, reject the very probable hypothesis that these toxins are acids or bases, or other very unstable, peculiar substances which are closely united with colloidal substances, as is the case, for example, with the alkali and acid albumins and so many other albuminous bodies." While the exact nature of the toxic substance contained in tetanus cul- tures has not been determined, we probably cannot, at present, do better than to continue to speak of it as a '* toxalbumin." Symbiosis. — Car bone and Perrero (1895), in a case of so-called rheumatic tetanus, in which there was no evidence of a wound through which infection might have occurred, obtained the tetanus bacillus, by inoculations in mice, from an exudate into the larger bronchial tubes. The micrococcus of pneu- monia was also present, and the authors named report that as a result of asso- ciation with this coccus the tetanus bacillus is able to grow in the presence of oxygen. Other bacteriologists had previously reported that the tetanus bacil- lus is able to grow in mixed cultures in the presence of oxygen, and this has been confirmed by the recent researches of Kedrowski (1895). Righi ( 1 6 claims that the tetanus bacillus may acquire the faculty of growing in the presence of oxygen, when it is gradually habituated to the presence of this gas. Penzo has observed a similar modification in the biological characters of the bacillus of malignant oedema ; and Kitt (1895) has succeeded in obtain- ing aerobic cultures or the bacillus of symptomatic anthrax. He says : ' ' The bouillon cultures do not always develop anaerobic ; one must inoculate sev- eral half-litre flasks and place them in the incubating oven ; some remain clear and without evidence of development, however long they are kept ; others begin to ferment at the end of two days. Sometimes this formation of gas only lasts for a day ; again, with more vigorous development, it may last for several days, and the contents of the flasks have the appearance of bubbl i ii^r champagne or 1 1 V /.s.s- bier. When a culture is once obtained in this way there is no difficulty in making a series of aerobic cultures. STERNBERG'S BACTERIOLOGY. PI., I r- \!|| Spirillum Obonnoiori in blooil of two inotilu-x • niociilu t CM! iifli-r ri'iiM'v.il ol sleen. XV. PATHOGENIC SPIRILLA. 191. SPIRILLUM OBERMEIERI. Synonyms. — Spirochsete Obermeieri ; Spirillum of relapsing fe- ver ; Die Recurrensspirochate. Discovered by Obermeier (1873) in the blood of persons suffering from relapsing fever. This spirillum is present, in very great numbers, in the blood of relapsing-fever patients during the febrile paroxysms. It has not been found under any other circumstances, and its etiological rela- tion to the disease with which it is associated is generally admitted. Morphology. — Very slender, flexible, spiral or wavy filaments, with pointed ends ; from sixteen to forty j* in length and consider- ably thinner than the cholera spirillum — about 0.1 /*. Koch has demonstrated the presence of flagella (Eisenberg). Stains readily with the aniline colors, especially with fuchsin, Bismarck brown, and in Lofner's solution of methylene blue. Biological Characters. — An aerobic, motile spirillum which has not been cultivated in artificial media. This spirillum appears to be a strict parasite, whose habitat is the blood of man. The disap- pearance of the parasite from the blood soon after the termination of a febrile paroxysm, and its reappearance during subsequent par- oxysms, have led to the inference that it must form spores, but this has not been demonstrated. In fresh preparations from the blood the spirillum exhibits active progressive movements, accompanied by very rapid rotation in the long axis of the spiral filaments, or by undulatory movements. The movements are so vigorous that the comparatively large red blood corpuscles are seen, under the micro- scope, to be thrown about by the slender spiral filaments, which it is difficult to see in unstained preparations. When preserved in a one- half -per-cent salt solution they continue to exhibit active movements for a considerable time. Efforts to cultivate this spirillum in artificial media have thus far been unsuccessful, although Koch has observed an increase in the length of the spirilla and the formation of a tangled mass of filaments. 550 PATHOGENIC SPIRILLA. In experiments made by Heydenreich the spirillum was found to preserve its vitality (motility) for fourteen days at a temperature of Fio. 172.— Spirillum Obermeieri in blood of man. x 1,000. From a photomicrograph. (FrSnkel and Pf eiffer. ) 16° to 22° 0., for twenty hours at 37°, and at 42.5° for two or three hours only. Pathogenesis. — Causes in man the disease known as relapsing fever. Munch and Moczutkowsky have produced typical relapsing Fio. 173.— Spirillum Obermeieri in blood of an inoculated ape. x 700. (Koch. fever in healthy persons by inoculating them with blood containing the spirillum of Obermeier. The spirilla are found in the blood dur- ing the febrile paroxysm, and for a day or two, at the outside, after PATHOGENIC SPIRILLA. 551 its termination ; sometimes they are present in great numbers, and at others can only be found by searching several microscopic fields; they are not present in the various secretions — urine, sweat, saliva, etc. In fatal cases the principal pathological changes are found in the spleen, which is greatly enlarged, and in the liver and marrow of the bones, which contain inflammatory and necrotic foci. Koch and Carter have succeeded in transmitting the disease to monkeys by subcutaneous inoculations with small amounts of defibrinated blood containing the spirillum. After an incubation period of seve- ral days typical febrile paroxysms were developed, during which the actively motile spirilla were found in the blood in large numbers. Blood from one animal, taken during the attack, induced a similar febrile paroxysm when inoculated into another of the same species — relapses, such as characterize the disease in man, were not observed. One attack did not preserve the animals experimented upon from a similar attack when they were again inoculated after an interval of a few days. Soudakewitch (1891) has made successful inoculation experiments in monkeys, and has shown that in monkeys from which the spleen has previously been removed the spirilla continue to multiply very abundantly in the blood and the disease has a fatal termination, whereas in monkeys from which the spleen has not been removed the spirilla disappear from the blood within a few days after the access of the febrile paroxysm and the animal recovers. 192. SPIRILLUM ANSERUM. Synonym. — Spirochaeta anserina (Sakharoff). Obtained by Sakharoff (1890) from the blood of geese affected by a fatal form of septicaemia due to this spirillum. This disease prevails among geese in Caucasia, especially in swampy regions, appearing annually and destroy- ing a large number of the domestic geese. Morphology. — Resembles the spirillum of relapsing fever. The long and flexible spiral filaments, when the disease is at its height, are often seen in interlaced masses, around the margins of which radiate single filaments which by their movements cause the whole mass to change its place, as if it were a single organism. These masses are sometimes so large that a single one occupies the entire field of the microscope. Stains with the usual aniline colors. Biological Characters. — An aerobic, motile spirillum. Not cultivated in artificial media. The movements are very active, resembling those of Spirillum Obermeieri, but cease in an hour or two in preparations made from the blood of geese containing it. Pathogenesis. —A small quantity of blood from an infected goose inocu- lated into a healthy animal of the same species induces the disease after a period of incubation of four to five days. The infected goose ceases to eat, becomes apathetic, remaining in one place, and usually dies at the end of a week ; the temperature is increased, and in some cases there is diarrhoea. The spirilla are found in the blood at the outset of the malady, but after death they are not seen either in the blood or in the various organs. The heart and the liver are found to have undergone a fatty degeneration, and yellowish, cheesy granules the size of a millet seed are seen upon the surface of these organs. The spleen is soft and easily broken up by the fingers. 552 PATHOGENIC SPIRILLA. Inoculations into chickens and pigeons were without result ; in one chicken the spirilla were found in the blood on the fourth day after inocula- tion, but the fowl recovered. 193. SPIRILLUM CHOLERA ASIATICS. Synonyms. — Spirillum (" bacillus ") of cholera ; Comma bacillus of Koch ; Kommabacillus der Cholera Asiaticae ; Bacille-virgule cholerigene. Discovered by Koch (1884) in the excreta of cholera patients and in the contents of the intestine of recent cadavers. The researches of Koch, made in Egypt and in India (1884), and subsequent researches by bacteriologists in various parts of the world, show that this spirillum — so-called " comma bacillus" — is con- stantly present in the contents of the intestine of cholera patients during the height of the disease, and that it is not found in the con- tents of the intestine of healthy persons or of those suffering from * J; FIG. 174. FIG. 176. FIG. 174.— Spirillum choleras Asiaticse. X 1,000. From a photomicrograph. (Koch.) FIG. 175.— Spirillum cholera Asiatic®, involution forms. X 700. (Van Ermengem.) other diseases than cholera. The etiological relation of this spiril- lum to Asiatic cholera is now generally admitted by bacteriologists. Morphology. — Slightly curved rods with rounded ends, from 0.8 to 2 /tin length and about 0.3 to 0.4 /tin breadth. The rods are usually but slightly curved, like a comma, but are occasionally in the form of a half -circle, or two united rods curved in opposite directions may form an S-shaped figure. Under certain circum- stances the curved rods grow out into long, spiral filaments, which may consist of numerous spiral turns, and in hanging-drop cultures the S-shaped figures may also be seen to form the commencement of a spiral ; in stained preparations the spiral character of the long filaments is often obliterated, or nearly so. When development is very rapid the short, curved rods or S-shaped spirals only are seen ; but in hanging-drop cultures, or in media in which the develop. PATHOGENIC SPIRILLA. 55S ment is retarded by an unfavorable temperature, the presence of a little alcohol, etc., the long, spiral filaments are quite numerous, and bacteriologists generally agree that the so-called " comma bacillus n is really only a fragment of a true spirillum. By Loffler's method of staining the rods may be seen to have a single terminal flagel- lum. In old cultures the bacilli frequently lose their characteristic form and become variously swollen and distorted — involution forms. Hueppe has described the appearance of spherical bodies in the course of the spiral filaments, which he believes to be reproductive elements — so-called arthrospores. Stains with the aniline colors usually employed, but not as quick- ly as many other bacteria ; an aqueous solution of fuchsin is the FIG. 176. FIG. 177. FIG. 176.— Spirillum cholerse Asiatic®; colonies upon gelatin plate, end of thirty hours. X 100. Photograph by Frankel and Pfeiffer. FIG. 177.— Spirillum cholera Asiatic®, from a gelatin culture, x 1,000. From a photomicro- graph. (Frankel and Pfeiffer.) most reliable staining agent; is decolorized by iodine solution — Gram's method. Sections may be stained with Loffler's solution. Biological Characters. — An aerobic (facultative anaerobic), liquefying, motile spirillum. Grows in the usual culture media at the room temperature — more rapidly in the incubating oven. Does not grow at a temperature above 42° or below 14° C. Does not form endogenous spores (forms arthrospores, according to Hueppe ?). In gelatin plate cultures, at 22° C., at the end of twenty-four hours small, white colonies may be perceived in the depths of the gelatin ; these grow towards the surface and cause liquefaction of the gelatin in the form of a funnel which gradually increases in 39 554 PATHOGENIC SPIRILLA. depth, and at the bottom of which is seen the colony in the form of a small, white mass ; as a result of this the plates on the second or third day appear to be perforated with numerous small holes ; later the gelatin is entirely liquefied. Under a low power the young colonies, before liquefaction has commenced, present a rather characteristic appearance ; they are of a white or pale-yellow color, and have a more or less irregular outline, the margins being rough and uneven; the texture is coarsely granular, and the surface looks as if it were covered with little fragments of broken glass, while the colony has a shining appearance ; when liquefaction commences an ill-defined halo is first seen to surround the granular colony, which by transmitted light has a peculiar roseate hue. In stick cultures in nutrient gelatin development occurs all along the line of inoculation, FIG. 178.- Colonies of the cholera spirillum; a, end of twenty hours; 6, end of thirty hours ; c, end of forty- eight hours; d, after liquefaction of the gelatin. (Flugge.) Fio. 179.-Spirillum choleras Asiaticae; a, one day old; 6, three days old; c, fourdays old; d, five days old ; e, seven days old ; /, 10 days old. From photographs by Koch. but liquefaction of the gelatin first occurs only near the surface ; on the second day, at 22° C., a short funnel is formed which has a comparatively narrow mouth, and the upper portion of which con- tains air, while just below this is a whitish, viscid mass ; later the funnel increases in depth and diameter, and at the end of from four to six days may reach the edge of the test tube ; in from eight to fourteen days the upper two-thirds of the gelatin is completely lique- fied. Owing to the slight liquefaction which occurs along the line of growth during the first three or four days, the central mass which PATHOGENIC SPIRILLA. 555 had formed along the line of inoculation settles down as a curled or irregularly bent, yellowish-white thread in the lower part of a slender tube filled with liquefied gelatin, the upper part of which widens out and is continuous with the funnel above. Upon the sur- face of nutrient agar a moist, shining, white layer is formed along the line of inoculation — impfstrich. Blood serum is slowly liquefied by this spirillum. Upon the surface of cooked potato, in the incu- bating oven, a rather thin and semi-transparent brown or grayish- brown layer is developed. In bouillon the development is rapid and abundant, especially in the incubating oven ; the fluid is only slightly FIG. ItiO.— Cultures in nutrient gelatin, at the room temperature (16° to 18° C.), at the com- mencement of the fourth day; a, Spirillum choleree Asiatic®; 6, Spirillum tyrogenum; c, Spirillum of Finkler and Prior. (Baumgarten.) clouded, but the spirilla accumulate at the surface, forming a wrin- kled membranous layer. Sterilized milk is also a favorable culture medium. In general this spirillum grows in any liquid containing a small quantity of organic pabulum and having a slightly alkaline reaction. An acid reaction of the culture medium prevents its de- velopment, as a rule, but it has the power of gradually accommo- dating itself to the presence of vegetable acids, and grows upon potatoes— in the incubator only — which have a slightly acid reaction. Abundant development occurs in bouillon which has been diluted with eight or ten parts of water, and the experiments of Wolffhugel 556 PATHOGENIC SPIRILLA. and Riedel show that it also multiplies to some extent in sterilized river or well water, and that it preserves its vitality in such water for several months. But in milk or water which contains other bac- teria it dies out in a few days. Gruber and Schottelius have shown, however, that in bouillon which is greatly diluted the cholera spiril- lum may take the precedence of the common saprophytic bacteria, and that they form upon the surface of such a medium the charac- teristic wrinkled film. Koch found in his early investigations that - rapid multiplication may occur upon the surface of moist linen, and ' also demonstrated the presence of this spirillum in the foul water of a " tank " in India which was used by the natives for drinking purposes. In the experiments of Bolton (1886) the cholera spirillum was found to multiply abundantly in distilled water to which bouillon was added in the proportion of fifteen to twenty-five parts in one thousand. The thermal death-point of the cholera spirillum in recent cul- tures in flesh-peptone-gelatin, as determined by the writer (1887), is 52° C., the time of exposure being four minutes ; a few colonies only developed after exposure to a temperature of 50° for ten minutes. In Kitasato's experiments (1889) ten or even fifteen minutes7 expo- sure to a temperature of 55° C. was not always successful in destroy- ing the vitality of the spirillum, although in certain cultures exposure to 50° for fifteen minutes was successful. He was not, however, able to find any difference between old and recent cultures as regards resistance to heat or to desiccation. In a moist condition this spiril- lum retains its vitality for months — as much as nine months in agar and about two months in liquefied gelatin. It is quickly destroyed by desiccation, as first determined by Koch, who found that it did not grow after two or three hours when dried in a thin film on a glass cover. In Kitasato's experiments (1889) the duration of vital- ity was found to vary from a few hours to thirteen days, the differ- ence depending largely upon the thickness of the film. When dried upon silk threads they may retain their vitality for a considerably longer time (Kitasato). Very numerous experiments have been made to determine the amount of various disinfecting agents re- quired to destroy the vitality of this microorganism. We give be- low the results recently reported by Boer (1890), whose experiments were made in Koch's laboratory. Experiments upon a culture in bouillon kept for twenty-four hours in the incubating oven, time of exposure two hours : hydrochloric acid, 1 : 1,350 ; sulphuric acid, 1 : 1,300 ; caustic soda, 1 : 150 ; ammonia, 1 : 350 ; mercuric cyanide, 1 : 60,000 ; gold and sodium chloride, 1 : 1,000 ; silver nitrate, 1: 4,000; arsenite of soda, 1 : 400 ; malachite green, 1 : 5,000 ; methyl violet, 1 : 1,000 ; carbolic acid, 1 : 400 ; creolin, 1 : 3,000 ; lysol, 1 : 500. In PATHOGENIC SPIRILLA. 557 Bolton's experiments (1887) mercuric chloride was effective in two hours in the proportion of 1 : 10,000 ; sulphate of copper, 1 : 500. The low thermal death-point and comparatively slight resisting power for desiccation and chemical agents indicate that this spiril- lum does not form spores, and most bacteriologists agree that this is the case. Hueppe, however, has described a mode of spore for- mation which is different from that which occurs among the bacilli, viz. , the formation of so-called arthrospores ; these are said to be developed in the course of the spiral threads, not as endogenous re- fractive spores, but as spherical bodies which have a somewhat greater diameter than the filament and are somewhat more refrac- tive. This mode of spore formation has not been observed by Kita- sato and other bacteriologists who have given attention to the Ques- tion, and cannot be considered as established. In competition with the ordinary putrefactive bacteria the cholera spirillum soon disap- pears, and, as determined by Neffelrnan and by Kitasato, they only survive for a few days when mixed with normal faeces. A test for the presence of the cholera spirillum has been found by Bujwid and by Dunham in the reddish- violet color produced in bouillon cultures containing peptone, or in cultures in nutrient gela- tin, when a small quantity of sulphuric acid is added to the culture. According to Frankel, this test serves to distinguish it from the ordi- nary bacteria of the intestine and from the Finkler-Prior spirillum, but not from MetschnikofFs spirillum (" vibrio"). The reaction is shown by bouillon cultures which have been in the incubating oven for ten or twelve hours, and by gelatin cultures in which liquefac- tion has occurred. The sulphuric acid used should be quite pure ; the color quickly appears and is reddish- violet or purplish-red. Ac- cording to Salkowski, the red color is due to the well-known indol reaction, which in cultures of the cholera spirillum is exceptionally intense and rapid in its development. A test which is said to dis- tinguish cultures of the cholera spirillum from the spirillum of De- neke and that of Finkler-Prior, has been proposed by Cahen. This consists in adding a solution of litmus to the bouillon and in making the culture at 37° C. The cholera cultures show on the following day a decoloration which does not occur at this temperature with the other spirilla named. For determining as promptly as possible whether certain suspected excreta contain cholera spirilla, a little of the material maybe used to inoculate greatly diluted bouillon, gelatin plates being made at the same time. At the end of ten or twelve hours the cholera spiril- lum, if present, will already have formed a characteristic wrinkled film upon the surface ; a little of this should be used to start a new culture in diluted bouillon, and a series of gelatin plates made from 558 PATHOGENIC SPIRILLA. it, after which the color test may be applied. The result of this, in connection with the morphology of the microorganisms forming the film and the character of growth in the gelatin plates, will estab- lish the diagnosis if the cholera spirillum is present in considerable numbers. If but few are present in the original material it may be necessary to make two or more series of plates and bouillon cultures before a pure culture can be obtained and a positive diagnosis made. Brieger has succeeded in isolating several toxic ptomaines from cultures of the cholera bacillus, some of which had previously been obtained from other sources — cadaverin, putrescin, creatinin, me- thyl-guanidin. In addition to these he obtained two toxic sub- stances not previously known. One of these is a diamin, resembling trimethylenediamin ; it gave rise to cramps and muscular tremor in inoculated animals. The other poison reduced the frequency of the heart's action and the temperature of the body in the animals sub- jected to experiment. In more recent researches made by Brieger and Frankel (1890) a toxalbumin was obtained from cholera cultures which, when injected subcutaneously into guinea-pigs, caused their death in two or three days, but had no effect upon rabbits. Pfeiffer in 1892 published his extended researches relating to the cholera poison. He finds that recent aerobic cultures of the cholera spirillum contain a specific toxic substance which is fatal to guinea- pigs in extremely small doses. This substance stands in close rela- tion with the bacterial cells and is perhaps an integral part of the same. The spirilla may be killed by chloroform, thymol, or by desi- cation without apparent injury to the toxic potency of this sub- stance. It is destroyed, however, by absolute alcohol, by concen- trated solutions of neutral salts, and by the boiling temperature, and secondary toxic products are formed which have a similar physio- logical action but are from ten to twenty times less potent. Similar toxic substances were obtained by Pfeiffer from cultures of Finkler- Prior's spirillum and from Spirillum Metschnikovi. The spirillum is not found in the blood or in the various organs of individuals who have succumbed to an attack of cholera, but it is constantly found in the alvine discharges during life and in the con- tents of the intestine examined immediately after death ; frequently in almost a pure culture in the colorless " rice-water" discharges. It is evident, therefore, that if we accept it as the etiological agent in this disease, the morbid phenomena must be ascribed to the absorption of toxic substances formed during its multiplication in the intestine. In cases which terminated fatally after a very brief sickness Koch found but slight changes in the mucous membrane of the intestine, which was slightly swollen and reddened ; but in more protracted cases the follicles and Peyer's patches were reddened around their margins, and PATHOGENIC SPIRILLA. §59 an invasion of the mucous membrane by the " comma bacilli " was observed in properly stained sections ; they penetrated especially the follicles of Lieberkiihn, and in some cases were seen between the epithelium and basement membrane. As a rule, the spirillum is not present in vomited matters, but Koch found it in small numbers in two cases and Nicati and Rietsch in three. In about one hundred cases in which Koch examined the excreta, or the contents of the in- testine of recent cadavers, during his stay in Egypt, in India, and in Toulon, his " comma bacillus" was constantly found, and other ob- servers have fully confirmed him in this particular — Mcati and Rietsch in thirty-one cases examined at Marseilles ; Pf eiffer, twelve cases in Paris ; Schottelius in cases examined in Turin ; Ceci in .-•-'" ^^^wr - ;£*-- .. v^7" FIG. 181. — Section through mucous membrane of intestine from cholera cadaver; a tubular gland (a) is cut obliquely; in the interior of this (6), and between the epithelial and basement membrane, are numerous spirilla. X 600. (Tlugge.) Genoa, etc. On the other hand, very numerous control experiments made by Koch and others show that it is not present in the alvine discharges of healthy persons or in the contents of the intestine of those who die from other diseases. In the writer's extended bacte- riological studies of the excreta, and contents of the intestine of ca- davers, in yellow fever, he has not once encountered any microor- ganism resembling the cholera spirillum. As none of the lower animals are liable to contract cholera during the prevalence of an epidemic, or as a result of the ingestion of food contaminated with choleraic excreta, we have no reason to expect that pure cultures of the spirillum introduced by subcutaneous inocu- lation or by the mouth will give rise in them to a typical attack of 560 PATHOGENIC SPIRILLA. cholera. Moreover, it has been shown by experiment that this spi- rillum is very sensitive to the action of acids, and is quickly de- stroyed by the acid secretions of the stomach, of man or the lower animals, when the functions of this organ are normally performed. By a special method of procedure, however, Nicati and Bietsch, and Koch, have succeeded in producing in guinea-pigs choleraic symp- toms and death. The first-named investigators injected cultures of the spirillum into the duodenum, after first ligating the biliary duct; the animals experimented upon died, and the intestinal contents con- tained the spirillum in large numbers. The fact that this procedure involves a serious operation which alone might be fatal, detracts from the value of the results obtained. Koch's experiments on guinea-pigs are more satisfactory, and, having been fully controlled by comparative experiments, show that the " comma bacillus " is pathogenic for these animals when introduced in a living condition into the intestine. This was accomplished by first neutralizing the contents of the stomach with a solution of carbonate of soda — five cubic centimetres of a five-per-cent solution, injected into the stomach through a pharyngeal catheter. For the purpose of restraining in- testinal peristalsis the animal also receives, in the cavity of the abdo- men, a tolerably large dose of laudanum — one gramme tincture of opium to two hundred grammes of body weight. The animals are completely narcotized by this dose for about half an hour, but re- cover from it without showing any ill effects. Soon after the ad- ministration of the opium a bouillon culture of the cholera spirillum is injected into the stomach through a pharyngeal catheter. As a result of this procedure the animal shows an indisposition to eat and other signs of sickness, its posterior extremities become weak and apparently paralyzed, and, as a rule, death occurs within forty-eight hours. At the autopsy the small intestine is found to be congested and is filled with a watery fluid containing the spirillum in great numbers. Comparatively large quantities of a pure culture injected into the abdominal cavity of rabbits or of mice often produce a fatal result within two or three hours ; and Nicati and Bietsch have ob- tained experimental evidence of the pathogenic power of filtered cul- tures not less than eight days old. The most satisfactory evidence that this spirillum is able to produce cholera in man is afforded by an accidental infection which occurred in Berlin (1884), in the case of a young man who was one of the attendants at the Imperial Board of Health when cholera cultures were being made for the instruction of students. Through some neglect the spirillum appears to have been introduced into his intestine, for he suffered a typical attack of cholera, attended by thirst, frequent watery discharges, cramps in the extremities, and partial suppression of urine. Fortunately he PATHOGENIC SPIRILLA. 561 recovered ; but the genuine nature of the attack was shown by the symptoms and by the abundant presence of the " comma bacillus" in the colorless, watery discharges from his bowels. Nicati and Rietsch observed a certain degree of attenuation in the pathogenic power of the spirillum after it had been cultivated for a considerable time at 20° to 25° C. ; and the observation has since been made that cultures which have been kept up from Koch's original stock have no longer the primitive pathogenic potency. Cunningham, as a result of researches made in Calcutta (1891), arrives at the conclusion that Koch's "comma bacillus" cannot be accepted as the specific etiological agent in this disease. This conclusion is based upon the results of his own bacteriological studies, which may be summed up as follows : First, in many un- doubted cases of cholera he has failed to find comma bacilli. Sec- ond, in one case he found three different species. Third, in one case the reaction with acids could not be obtained. From sixteen cases in which Cunningham made cultures he obtained ten different vari- eties of comma bacilli, the characters of which he gives in his pub- lished report. It may be that in India, which appears to be the permanent habitat of the cholera spirillum, many varieties of this microorganism exist ; but extended researches made in the laborato- ries of Europe show that Cunningham is mistaken in supposing that spirilla resembling Koch's " comma bacillus " are commonly present in the intestine of healthy persons. The view advocated is that during the attack these spirilla are found in increased numbers be- cause conditions are more favorable for their development, but that they have no etiological import. The writer would remark that, in very extended researches made in the United States and in Cuba, he has never found any microorganism resembling Koch's cholera spi- rillum in the faeces of patients with yellow fever or of healthy indi- viduals, or in the intestinal contents of yellow-fever cadavers. 194:. SPIRILLUM OF FINKLER AND PRIOR. Synonym. — Vibrio proteus. Obtained by Finkler and Prior (1884) from the faeces of patients with cholera noslras, after allowing the dejecta to stand for some days. Subse- quent researches have not sustained the view that this spirillum is the speci- fic cause of cholera morbus. Morphology.— Resembles the spirillum of Asiatic cholera, but the curved segments (" bacilli" ) are somewhat longer and thicker and not so uniform in diameter, the central portion being usually thicker than the somewhat pointed ends ; forms spiral filaments, which are not as numerous, and are usually shorter than those formed by the cholera spirillum. In unfavorable media involution forms are common — large oval, spherical, or spindle shaped cells, etc. Has a single flagellum at one end of the curved segments, which is from one to one and one-half times as long as these. Stains with the usual aniline colors — best with an aqueous solution of fuchsin. 562 PATHOGENIC SPIRILLA. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile spirillum. Spore formation not demonstrated. Grows in the usual culture media at the room temperature. Upon gelatin plates small, white, punctiform colonies are developed at the end of twenty four hours, n to be finely granular and yellowish or which under the microscope are seen yellowish-brown in color ; liquefaction of the gelatin around these colonies progresses rapidly, and at the end of forty-eight hours is usually complete in plates where they are numerous. Isolated colonies on the second day form saucer-shaped depressions in the gelatin the size of lentils, having a sharply defined border. In gelatin stick cultures liquefaction progresses much more rapidly than in similar cultures of the cholera spirillum, and a stocking- shaped pouch of liquefied gelatin is already seen on the second day, which rapidly increases in dimensions, so that by the end of a week the gelatin is usually completely liquefied ; upon the surface of the liquefied medium a whitisn film is seen. Upon agar a moist, slimy layer, covering the entire surface, is quickly developed. The growth in blood serum is rapid and d FIG. 182. FIG. 183. FIG. 184. FIG. 188.— Spirillum of Finkler and Prior, from a gelatin culture. X 1,000. From a photomicro- graph. CFriinkel and Pfeiffer.) FIG. 188.— Spirillum of Finkler and Prior; colonies upon gelatin plate; a, end of sixteen hours; 5, end of twenty-four hours; c, end of thirty-six hours. X 80. (FlQ«ge ) FIG. 184.— Spirillum of Finkler and Prior; culture in nutrient gelatin; c, two days old; d, four days old. (Flugge.) causes liquefaction of the medium. Upon potato this spirillum grows at the room temperature and produces a slimy, gravish-yellow, glistening layer, which soon extends over the entire surface. The cholera spirillum does not grow upon potato at the room temperature. The cultures of the Fink In- Prior spirillum give off a tolerably strong putrefactive odor, and, according to Buchner, in media containing sugar an acid reaction is produced as a re- sult of their development. They have a greater resistance to desiccation tl uin the cholera spirillum. Pathogenesis. — Pathogenic for guinea-pigs when injected into the stomach by Koch's method, after previous injection of a solution of car- bonate of soda, but a smaller proportion of the animals die from sucli injec- tions (Koch). At the autopsy the intestine is pale, and its watery contents, PATHOGENIC SPIRILLA. 563 which contain the spirilla in great numbers, have a penetrating-, putrefactive odor. 195. SPIRILLUM TYROGENUM. Synonyms — Spirillum of Deneke; Kasespirillen. Obtained by Deneke (1885) from old cheese. Morphology. — Curved rods and long, spiral filaments resembling the spirilla of Asiatic cholera. The diameter of the curved segments is some- what less than that of the cholera spirillum, and the turns in the spiral fila- ments are lower and closer together. The diame- ter of the "commas" is uniform throughout, so that this spirillum more closely resembles the ~ cholera spirillum than does that of Finkler and Prior. Stains with the usual aniline colors— best with an aqueous solution of f uchsin. Biological Characters. — An aerobic and fac- ultative anaerobic, liquefying, motile spirillum. Spore formation not demonstrated. Grows in the usual culture media at the room temperature FIG. 185. — Spirillum tyroge- — more rapidly than the cholera spirillum and num. x 700. (Flugge.) less so than that of Finkler and Prior. Upon gelatin plates small, punctiform colonies are developed, which on the second day are about the size of a pin's head and have a yellowish color ; under the microscope they are seen to be coarsely granular, of a yellowish -green color in the centre and paler towards the margins. The outlines of the colo- nies are sharply defined at first, but later, when liquefaction has commenced, the sharp contour is no longer seen. At first liquefaction of the gelatin causes funnel-shaped cavities resembling those formed by the cholera spirillum, but lique- faction is more rapid. In gelatin stick cultures . liquefaction occurs all along the line of punc- ture, and the spirilla sink to the bottom of the FIG i86.-SPiriliumtyrogenum; liquefied gelatin in the form of a coiled mass, colonies in gelatin plate; a, end while a thin, yellowish layer forms upon the of sixteen hours; 6, end of twen- surface ; complete liquefaction usually occurs in ty-four hours; c, end of thirty- about two weeks, Upon the surface of agar a thin, yellowish layer forms along the impf- strich. Upon potato, at a temperature of 37° C., a thin, yellow layer is usually developed (not always— Eisenberg) ; this contains, as a rule, beautifully formed, long, spiral filaments. Pathogenesis. — Pathogenic for guinea-pigs when introduced into the stomach by Koch's method; three out of fifteen animals treated in this way succumbed. 196. SPIRILLUM METSCHNIKOVI. Synonym. — Vibrio Metschnikovi (Gameleia). Obtained by Gameleia (1888) from the intestinal contents of chickens dying of an infectious disease which prevails in certain parts of Russia dur- ing the summer months, and which in some respects resembles fowl cholera. The experiments of Gameleia show that the spirillum under consideration is the cause of the disease referred to, which he calls gastro-enteritis cholerica. Morphology. — Curved rods with rounded ends, and spiral filaments; the curved segments are usually somewhat shorter, thicker, and more decidedly curved than the " comma bacillus " of Koch. The size differs very consid- erably in the blood of inoculated pigeons, the diameter being sometimes twice as great as that of the cholera spirillum, and at others about the same. A single, long, undulating flagellum maybe seen at one extremity of the spiral filaments or curved rods in properly stained preparations. 564 PATHOGENIC SPIRILLA. Stains with the usual aniline colors, but not by Gram's method. Biological Characters. — An aerobic (facultative anaerobic ?), liquefy- ing, motile spirillum. According to Gamaleia, endogenous spores are formed by this spirillum; but Pfeiffer does not confirm this observation, and it must , be considered extremely doubtful in view of the slight resistance to heat— killed in five minutes by a temperature of 50° C. Grows in the usual culture media at the room temperature. Upon gelatin plates small, white, puncti- form colonies are developed at the end of twelve to six- teen hours ; these rapidly increase in size and cause lique- faction of the gelatin, which is, however, much more rapid with some than with others. At the end of three days large, saucer-like areas of liquefaction may be seen, resem- bling that produced by the Finkler-Prior spirillum and the contents of which are turbid, while other colonies have produced small, funnel-shaped cavities filled with trans- parent, liquefied gelatin and resembling colonies of the cholera spirillum of the same age. Under the microscope the larger liquefied areas are seen to contain yellow ish- brown granular masses which are in active movement, and the margins are surrounded by a border of radiating fila- ments. In gelatin stick cultures the growth resembles that of the cholera spirillum, but the development is more rap- id. Upon agar, at 37° C., a yellowish layer resembling that formed by the cholera spirillum is quickly developed. Upon potato no growth occurs at the room temperature, but at 37° C. a yellowish-brown or chocolate-colored layer is formed, which closely resembles that produced by the cholera spirillum under the same circumstances. In bouil- lon, at 37° C., development is extremely rapid, and the liquid becomes clouded and opaque, having a grayish- white color, while a thin, wrinkled film forms upon the surface. When muriatic or sulphuric acid is added to a culture in peptonized bouillon a red color is produced similar to that froduced in cultures of the cholera spirillum, and even more pronounced, n milk, at 35° C. , rapid development occurs, and the milk is coagulated at the end of a week ; the precipitated casein accumulates at the bottom of the tube in irregular masses and is not redissplved. The milk acquires a strongly acid reaction and the spirilla quickly perish. Patliogenesis. — Pathogenic for chickens, pigeons, and guinea-pigs; rab- bits and mice are refractory except for very large doses. Chickens suffering from the infectious disease caused by this spirillum remain quiet and somno- lent, with ruffled feathers ; thev have diarrhoea ; the temperature is not ele- vated above the normal, as is the case in chicken cholera. At the autopsy the most constant appearance is hyperaemia of the entire alimentary canal A grayish-yellow liquid, more or less mixed with blood, is found in con- siderable quantity in the small intestine ; the spleen is not enlarged and the organs generally are normal in appearance. In adult chickens the spirillum is not found in the blood, but in young- ones its presence may be verified by the culture method and by inoculation into pigeons, which die in from twelve to twenty hours after being inoculated with two to four cubic cen- timetres. The pathological appearances in pigeons correspond with those found in chickens, but usually the spirillum is found in great numbers in blood taken from the heart. A few drops of a pure culture inoculated sub cutaneously in pigeons or injected into the muscles cause their death in eight to twelve hours. Gameleia claims that the virulence of cultures is greatly increased by successive inoculations in pigeons, but Pfeiffer has shown that very minute doses are fatal to pigeons and that no decided in- crease of virulence occurs as a result of successive inoculations. According to Gameldia, chickens may be infected by giving them food contaminated FIG 187.-Spiri)- lum Metschnikovi; culture in nutrient gelatin, end of forty- eight hours From a photograph. (Fran- kel and Pfeiffer.) PATHOGENIC SPIRILLA. 565 with the cultures of the spirillum, but pigeons resist infection in this way. Guinea-pigs usually die in from twenty to twenty-four hours after receiving a subcutaneous inoculation ; at the autopsy an extensive subcutaneous oedema is found in the vicinity of the point of inoculation, and a superficial necrosis may be observed ; the blood and the organs generally contain the " vibrio " in great numbers, showing that the animals die from general in- fection— acute septicaemia. When infection occurs in these animals by way of the stomach the intestine will be found highly inflamed and its liquid con- tents will contain numerous spirilla. Gameleia has shown that pigeons and guinea-pigs may be made immune by inoculating them with sterilized cultures of the spirillum — sterilized by heat at 100° C. Old cultures contain more of the toxic substance than those of recent date. Thus two to three cubic centimetres of a culture twenty days old will kill a guinea-pig when injected subcutaneously, while five cubic centimetres of a culture five days old usually fail to do so. According to Pfeiffer, old cultures have a decidedly alkaline reaction, and their toxic power is neutralized by the addition of sulphuric acid. Gameleia has claimed that by passing the cholera spirillum of Koch through a series of pigeons, by successive inoculation, its pathogenic power is greatly increased, and that when sterilized cultures of this virulent vari- ety of the ' ' comma bacillus " are injected into pigeons they become immune against the pathogenic action of the " vibrio Metschnikoff , " and the reverse. Pfeiffer (1889), in an extended and carefully conducted research, was not able to obtain any evidence in support of this claim. NOTES RELATING TO THE PATHOGENIC SPIRILLA. During the past three or four years quite a number of spirilla have been obtained from various sources which resemble more or less closely the spirillum of Asiatic cholera. It appears probable that some of these are in fact varieties of Koch's " comma bacillus " which have undergone various modifications as a result of the con- ditions under which they have maintained their existence as sapro- phytes. Others are evidently essentially different, and have no very near relationship to the cholera spirillum. The principal points of difference between these recently described spirilla and Spirillum cholerse Asiatics are given in the following resume, for which we are indebted to Dieudonne (1894). "Since the outbreak of cholera in 1892, various vibrios have been de- scribed which resemble more or less closely the cholera vibrio. When these are tested as to their morphological characters, growth in peptone solutions, in gelatin and agar plates, cholera-red reaction, and pathogenic power, they may be divided, at the outset, into two groups : viz., such vibrios as show only a remote resemblance to the cholera vibrio, and therefore are easily dif- ferentiated from it, and such as present only minor differences or none at all that have been demonstrated. * ' To the first group belongs the spirillum isolated by Russell from sea water — Spirillum marinum — which rapidly liquefies gelatin and does not grow at the body temperature. Renon isolated from water, obtained at Bil- lancourt, a vibrio which likewise quickly liquefies gelatin, but is not patho- genic for guinea-pigs, either by subcutaneous or intraperitoneal inoculation. Gimther, in examining the Spree water, found a vibrio which, upon gelatin plates, formed circular colonies with smooth margins, very finely granular and of a brown color. This vibrio did not give the indol reaction, and all infection experiments gave a negative result. Gunther named this sapro- 566 PATHOGENIC SPIRILLA. phyte Vibrio aquatilis. About the same time (1892) Kiessling obtained from water, from Blankenese, a vibrio which presented similar characters and probably is identical with that of Gunther. Weibel obtained from well-water a vibrio which liquefies gelatin more rapidly than the cholera vibrio ; its pathogenic action was not tested. Bujwid (1893) isolated from Weichsel water a vibrio which at low temperatures (12° C.) grew almost the same as the cholera vibrio, but at higher temperatures was easily distinguished from it. Bujwid's assistant, Orlowski, found in a well at Lubin a very similar vibrio. Loffler (1893) obtained from the Peene water a vibrio which at 37° C. grows rapidlv and liquefies gelatin very rapidly, like the Finkler-Prior spirillum. Fokker (1893), from water of the harbor at Groningen, obtained a vibrio which rapidly liquefied gelatin and occasionally gave the indol re- action. Injections into the peritoneal cavity of mice and guinea-pigs gave a negative result. Fokker supposes that this is an attenuated cholera bacil- lus, because it forms the same ensyme as cholera bacteria, and when culti- vated for three months its characters, especially its peptonizing power, had changed. Fischer (1893) found in the stools of a woman suffering from diar- rhoea a vibrio which in gelatin cultures resembled that of Fiiikler and Prior. In bouillon and peptone solution it caused clouding and formation of a pellicle, but only gave a slight indol reaction. A portion of the mice in- oculated subcutaneously had after a time abscesses, from the contents of which Fischer was able to cultivate his vibrio, which he named Vibrio helco- genes. Vogler (1893), in an extended series of examinations of faeces, found a vibrio which showed many points of resemblance to the cholera vibrio in its growth in gelatin. But it constantly gave a negative indol reaction, and was not pathogenic for guinea-pigs when injected into the peritoneal cavity. Bleisch obtained from the dejecta of a man who died with choleraic symptoms a bacterium which upon gelatin plates grew at first like the cholera bacillus, but was distinguished from it by many points of difference in other respects : short rods, sometimes bent, but never showing spiral forms. It gave the cholera-red reaction. Wolf (1883) obtained from cervical secretion, from a woman suffering from chronic endometritis, a comma-formed bacillus, which in its growth on gelatin plates resembled the cholera vibrio. The liquefac- tion was, however, much more rapid, a culture a day old being as far ad- vanced as a cholera culture of three to four days. The addition of sulphuric acid to a bouillon culture caused a faint rose-red color, which upon standing changed to brown. The addition of sulphuric acid and potassium iodide paste did not cause a blue color, so there was no formation of nitrites. Bonhoff (1893), in water from Stolpe, in Pommerania, discovered two vibrios, one of which in the first twenty-four hours grew like the cholera vibrio, but did not give the cholera-red reaction. Out of four guinea-pigs inoculated one only died with cholera-like symptoms. The other vibrio gave the cholera-red reac- tion, but did not liquefy gelatin and was very inconstant as regards its patho- genic power. Zorkendorfer (1893) isolated a vibrio from the stools of a woman who died with choleraic symptoms, which ac first grew upon gelatin plates like the cholera vibrio, but after the second day liquefied the gelatin very rapidly, so that it could no longer be taken for the same. The indol reaction was constantly absent, and it was not pathogenic for guinea-pigs, rabbits, or pigeons. Blackstein (1893) obtained from trie water of the Seine a comma bacillus which resembled the cholera vibrio in many particulars, but was distinguished by the finer granulation and more opaque appearance of its colonies. Sanarelli (1893), by the use of special media, isolated from the water of the Seine and of the Marne no less man thirty-two vibrios, four of which resembled the cholera vibrio in giving the indol reaction. Three others gave the indol reaction after eight days ; the remainder did not give it at all, or only very faintly. The vibrios which upon a first inoculation gave no results or only very slight evidence of pathogenic power, when carried through a series of animals caused a fatal infection. When a sterilized cul- ture of the colon bacillus was injected at the same time death always oc- PATHOGENIC SPIRILLA. 567 curred. Sanarelli believes that these vibrios must have had a common ori- gin—from the dejecta of cholera patients. Fischer (1894) has described a number of vibrios from sea-water which are distinguished from the cholera vibrio especially by a preference for media containing sea- water. Finally, the vibrios found in water, referred to by Koch (' Ueber den augenblicklichen Stand der Cholera-diagnose,' Zeitschr. fur Hygiene, Bd. xiv., page 319), belong here. "Quite different from these is a second group of vibrios which in their in- vestigation offered great and often almost insuperable difficulties for the differential diagnosis. Here, first of all, is the Vibrio Berolinensis, found by Neisser in August, 1893, and described by Rubner, Neisser, and Giinther. This was isolated from water which had previously contained cholera vibrios, for which reason Dunbar considers it not impossible that this is a genuine cholera vibrio, somewhat changed perhaps by long-continued development in water. Neither in its morphology nor in its behavior in gelatin stick cul- tures, in milk and other media, could it be distinguished from the genuine comma bacillus ; the indol reaction and pathogenic action upon guinea-pigs were the same ; on the contrary, a differentiation was easily made in gelatin plate cultures. At the end of twenty -four hours it formed small, spherical, finely granular colonies, which at the end of forty-eight hours were not yet visible to the naked eye. Heider (1893) isolated from the water of the Donau canal a vibrio which he called Vibrio Danubicus. This resembles the chol- era vibrio fully in its morphology. As a distinguishing character it was found that this vibrio, in thinly planted plates, forms flat, superficial colo- nies having irregularly rounded margins and other slight differences ; also the pathogenic action upon mice inoculated subcutaneously, and the ease with which guinea-pigs are infected by way of the respiratory passages. It is worthy of note that the day after the sample was taken a man was taken sick with cholera who had worked on the Donau the day before — on the principal stream at a place far below the junction of the canal. Dunbar (1893) found vibrios in the Elbe, in the Rhine, in the Pegnitz, and in the Amstel at Amster- dam. These presented no decided characters by which he was able to differ- entiate them from the cholera vibrio. The most careful comparative investi- gations did not lead to the discovery of any points of difference which had not already been observed in genuine cholera cultures. Everything, there- fore, indicated that these were genuine cholera bacilli, especially as these vibrios disappeared from the rivers when cholera ceased to prevail. It was first possible through an observation of Kutscher's to differentiate a portion of these water bacteria, and certain vibrios isolated from the discharges of persons suspected of having cholera from cultures of the cholera spirillum. In the presence of oxygen, at a suitable temperature, they give off a greenish- white phosphorescence. ' 'As phosphorescence has never been observed in undoubted cholera cul- tures, we can assert with tolerable certainty that such phosphorescent vibrios are not genuine cholera bacteria. But as this phosphorescent property was inconstant in thirty-eight out of sixty-eight cultures, Dunbar believes that some reserve must be exercised in accepting this as evidence that these are not genuine cholera vibrios. Maassen (1894) gives as a further distinguishing character of these phosphorescent vibrios the fact that they form a strong, usually wrinkled pellicle in bouillon, of proper alkalinity, containing gly- cerin or carbohydrates (cane sugar, lactose) ; also that in such media the formation of indol and a subsequent return to an alkaline reaction may be observed. " As already stated, Sanarelli isolated from Seine water a considerable number of vibrios, and among them four — viz. : one from St. Cloud, Point- du-Jour, Gennevilliers No. 5, and Versailles (Seine), which after twenty-four hours gave a distinct indol reaction and were more or less pathogenic for guinea-pigs (the one from St. Cloud was also pathogenic for pigeons}. Ivan- off (1893) describes a vibrio which he isolated from the faeces of a patient with 568 PATHOGENIC SPIRILLA. typhoid fever. But as the discharges had been mixed with Berlin hydrant water, Ivanoff admits the possibility that his vibrio came from this water. It closely resembles the cholera vibrio, but is distinguished by its colonies in gelatin plates, which, at the end of twenty -four to thirty-six hours, in place of the usual coarse granulation of cholera colonies shows a distinct formation of filaments. Morphologically the vibrio is distinguished by a decided ten- dency to preserve the spiral form, and especially by its size. Celli and San- tori (1893) describe a Vibrio romanus, which they isolated from twelve undoubted cases of cholera. This does not give the indol reaction, is not pathogenic for animals, and does not grow in bouillon or agar at 37° C. This is considered by the authors named an atypical variety of the cholera vibrio, especially as the distinguishing characters did not prove to be perma- nent. After eight months' cultivation the cultures gave the indol reaction, but the pathogenic power was still almost absent. Recently Chantemesse (1894) has described a vibrio which he found in the spring of 1894 during the chol- era epidemic at Lisbon. This differed in many particulars from the genuine cholera vibrio, resembling more closely the vibrio of Finkler-Prior. As in the Lisbon epidemic, with a large number taken sick, only one death occurred, and in view of the results of the bacteriological examination, Chantemesse supposes this to have been an epidemic of cholera nostras. Finally, Pfuhl (1894) found a vibrio in the north harbor of Berlin which from its growth in gelatin and pathogenesis for pigeons he believes to be identical with Vibrio Metschnikovi." To the list of vibrios above referred to as resembling more or less closely the cholera spirillum we must add those described by Cun- ningham (1894) and obtained by him from the discharges of cholera patients. He has described " thirteen distinct forms obtained from cases of cholera and one of non-choleraic origin." Pfeiffer and Issaeff (1894), in a recent publication, report that they have found a sensitive test for the differentiation of these vibrios in the specific character of cholera immunity. They found that guinea- pigs which were immunized against cholera infection have a lasting immunity, and that the serum of such immunized animals has a specific action in protecting against infection by genuine cholera vib- rios only, while for other species it has no action different from that of the blood serum of normal animals. In all cases where the cholera serum acted specifically the vibrios were promptly destroyed, while in cases where this specific action was absent the injected vibrios multiplied rapidly and caused the death of the animal. By means of this method the vibrios isolated from water — the phosphorescent vibrios of Dunbar, Vibrio Danubicus, Cholera Massanah — are shown to be distinct species, while the vibrio of Ivanoff behaves like the genuine cholera vibrio. In a subsequent paper Pfeiffer reports the interesting fact that a trace of highly active cholera serum, added to a culture of the cholera spirillum, when injected into the peritoneal cavity of a guinea-pig, within a surprisingly brief time causes the destruction of the cholera vibrios ; whereas no such effect is produced upon other species. A similar destruction occurs when cholera vib- rios are injected into the abdominal cavity of immunized guinea- PATHOGENIC SPIRILLA. 569 pigs. The researches of Dunbar (1894) indicate that Pfeiffer's test is not so reliable as he supposed ; and also that phosphorescence can- not be relied upon for distinguishing similar water bacteria from genuine cholera vibrios. Rumpel has reported the fact that two un- doubted cultures of the cholera spirillum, from different sources, after being passed through pigeons and cultivated for some time in arti- ficial media, showed phosphorescence. One of these cultures was ob- tained originally from the discharges of Dr. Oergel, who was a vic- tim to cholera from laboratory infection (case reported by Reincke, in the Deutsche medicinisclie Woclienschrift, No. 41, 1894). Another case of supposed laboratory infection, in which recovery occurred, is reported by Lazarus, in the Berliner medicinische Wochenschrift, 1893, page 1,241. That cholera vibrios may be present in the alimentary canal of healthy individuals without giving rise to any symptoms of ill-health appears to be demonstrated. In support of this conclusion we quote as follows from a recent paper by Abel and Claussen : " In Wehlau (East Prussia), in the autumn of 1894, seven cases of cholera occurred about the same time. The members of the family were at once isolated and their faeces examined almost daily. Of especial interest were seventeen individuals who belonged to families in which three fatal cases occurred. Of these seventeen persons, who were not sick at all or only had for a brief time a diarrhoea, thirteen had cholera vibrios in their discharges for a considerable time. As the table shows, many of these comma bacilli were not found in dis- charges every day, but were obtained again after being absent" (in the cultures) " for a day or two." Abel and Claussen (1895), as a result of very extended experi- ments, arrive at the conclusion that cholera vibrios in faaces as a rule do not survive longer than twenty days, and often cannot be ob- tained after two or three days; exceptionally they were obtained in cultures at the end of thirty days — Karlinsky and Dunbar have re- ported finding them at the end of fifty-two days and four months. Karlinsky (1895) has also reported that upon woollen and linen goods, cotton batting and wool, which were soaked in the discharges of cholera patients and preserved from drying by being wrapped in waxed paper, the cholera vibrio retained its vitality for from twelve to two hundred and seventeen days. The researches of Kasansky (1895) show that the cholera spiril- lum is not destroyed by alow temperature (—30 C.) and that it even resists repeated freezing and thawing — three or four times. Behring and Ransom (1895) as a result of an extended experi- mental research, arrive at the conclusion that cholera cultures from which the bacteria have been removed have specific toxic properties, 40 570 PATHOGENIC SPIRILLA. and cause symptoms similar to those which result from the intro- duction into guinea-pigs of the living bacteria ; that from these fil- tered cultures a solid substance can be obtained having the same toxic properties, and that from susceptible animals which have been treated with this toxic substance a serum can be obtained which is active not only against the cholera poison, but against the cholera vibrio. These results support those previously reached by other bacteriologists and lead to the hope that a specific treatment of the disease may be successfully employed. The results obtained by Haffkine in India are favorable to the view that his method of prophy- laxis, by the subcutaneous injection of virulent cholera cultures, has a real value. PLATE IX. FIG. 1.— Bacillus diphtherise (Klebs-Loffler) from culture on blood serum. Stained with Loffler's solution of methylene blue. X 1,000. Photomicro- graph by oil lamp. (Borden.) FIG. 2.— Micrococcus gonorrhoeas in urethral pus. Stained with Loffler's solution of methylene blue. X 1,000. Photomicrograph by oil lamp (Borden.) FIG. 3. — Bacillus tuberculosis in sputum. X 1,000. Photomicrograph by oil lamp. (Borden.) FIG. 4. --Bacillus typhi abdominalis, from agar culture. X 1,000. Photo- micrograph by oil lamp. (Borden.) FIG. 5. — Streptococcus pyogenes (longus). X 1,000. Photomicrograph made at the Army Medical Museum by sunlight. (Gray.) FIG. 6. — Bacillus mallei. X 1,000. Photomicrograph made at the Army Medical Museum by sunlight. (Gray.) PLATE IX. STERNBERG'S BACTERIOLOGY v - Fig. 2. FiK. 3. Fig. 4. rv-LM / PATHOGENIC BACTERIA. XVI. BACTERIA IN INFECTIOUS DISEASES. IN the present chapter we shall give a brief account of the re- searches which have been made relating to the presence of bacteria in various infectious diseases of man and the lower animals, and in localized infections which have been supposed, on more or less satis- factory evidence, to be due to their presence. For convenience of reference we shall arrange these diseases in alphabetical order. ABSCESSES. The bacteria principally concerned in the causation of acute abscesses are : Staphylococcus pyogenes aureus (No. 1), Staphylococcus pyogenes albus (No. 2), and Streptococcus pyogenes (No. 5). For details, see Sec. IV. The following species have also been found in acute abscesses, sometimes in pure culture : Staphylococcus pyogenes citreus (No. 3), Micrococcus tetra- genus (No. 18), Micrococcus pneumonia) crouposje (No. 8), Bacillus coli communis (No. 89), Bacillus typhi abdominalis (No. 46), Bacillus pyogenes fcetidus (No. 72) obtained from an abscess near the anus by Passet, Bacil- lus pyogenes fcetidus liquefaciens, from a brain abscess following otitis media, by Lanz. Passet also found in two abscesses out of thirty-three examined a micrococcus named by him Micrococcus cereus albus, and in a single case his Micrococcus cereus flavus. Recent researches show that next to the micrococci mentioned above as commonly found in the pus of acute abscesses (Nos. 1, 2, 5). The micrococ- cus of croupous pneumonia, the colon bacillus, and the bacillus of typhoid fever are most frequently present — the last mentioned in abscesses follow- ing typhoid fever. In the so-called "cold" abscesses, due to tubercular in- fection of glands, the tubercle bacillus is usually the only microorganism present. See also Bubo, Mastitis, Otitis Media. ACNE. Hodara (1894) finds in the pustules of acne a small bacillus (No. 175) which he believes to be the cause of the disease. It is said to be found at the base and central portion of the comedones, while cocci and flask-shaped bacilli are found in the superficial portion. In pseudo-acne pustules this bacillus was not found. ACNE CONTAGIOSA OF HORSES. Dieckerhoff and Grawitz believe the cause of ' * acne contagiosa " in horses to be a bacillus described by them (No. 141). 572 BACTERIA IX INFECTIOUS DISEASES. ALOPECIA. Robinson (1888) claims to have found, in sections from the diseased skin in a case of alopecia areata, micrococci having- a diameter of about 0.8 /*, usu- ally united in pairs and associated in zooglcea masses. They were located for the most part in the lymph spaces of the central portion o'f the chorium. They stained with the usual aniline colors and also by Gram's method. No culture or inoculation experiments were made. Kasauli (1889) obtained from the margins of the affected patches in alope- cia areata a bacillus about two to three times as long as broad, and which formed spores. It was attached to hail's withdrawn from the diseased patches, and was easily cultivated in various media. Yaillard and Vincent (1890), in a form of alopecia resembling favus, ob- tained by cultivation, from hairs pulled out from the diseased patches, a mi- crococcus ; this was also found in the hair follicles in stained sections. The diameter of this micrococcus was about 1 n ; it was easily stained with the aniline colors and by Gram's method ; grew in nutrient gelatin, causing liquefaction ; did not grow well upon potato ; was pathogenic for mice. When applied to the surface of the body of guinea-pigs or rabbits, by rub- bing, alopecia resulted similar to that in the cases from which the micrococ- cus was first obtained. Hollborn (1895) thinks it probable that alopecia areata is due to a micro- scopic fungus described by him, which bears some resemblance to Trichophy- ton tonsurans. Elliott (1895) believes that the most frequent cause of alopecia praematura is some form or grade of eczema seborrhoicum. See Eczema. ANGINA. Although the pus cocci are frequently found in the secretions from the mouth, nares, and fauces of healthy persons, there can be but little doubt that they are concerned in the etiology of angina, and of catarrhal or pseudo- diphtheritic inflammations of mucous membranes elsewhere. Dornberger (1894) reports that in fortv-five per cent of the healthy indi- viduals examined streptococci were found. In 78.9 per cent of the cases of angina Streptococcus longus was found, but never in pure cultures ; in aii- gina phlegmonosa Streptococcus brevis was present ; in seven cases of acute catarrhal angina streptococci were found five times, and in chronic catarrhal angina in one-half the cases. Plaut (1894) in five cases of severe angina found Miller's bacillus in large numbers in the exudate in the fauces, and believes that it was the cause of the inflammation of the mucous membrane. Goldschneider (1893) found in the angina of scarlet fever streptococci only in seven cases, and staphylococci alone in fourteen. No difference was observed in the exudate in the cases belonging to the two groups, but tlu> streptococcus angina was more severe and ran a more protracted ccnu>«; (average duration 12.6 days). In eight cases streptococci and staphylococci were associated— these had an average duration of thirteen days. Booker (1892) found streptococci in the angina of scarlet fever and measles, associated in some cases with staphylococci. ANTHRAX. Due to the presence of Bacillus anthracis (No. 45) in the blood and tissues of infected animals — or in malignant pustule and in " wool-sorters' disease " in man. BACTERIA IN INFECTIOUS DISEASES. 573 APPENDICITIS. Hodenpyl (1893) in ten cases of appendicitis in which a bacterio- logical examination was made found Bacillus coli communis in pure culture, and in one case the same bacillus associated with Strepto- coccus pyogenes. Including his own cases with twenty-four re- corded by other investigators the colon bacillus was the only micro- organism present in thirty-two out of the thirty-five cases. ARTHRITIS. In arthritis following pneumonia the Micrococcus pneumonia? crou posse has been found in pure culture by several bacteriologists — Boulloche, Schwartz, Picque and Veillon, Brunner. In gonorrhoeal arthritis the gonococcus has been found by Bordoni-Uffreduzzi, Pal- tauf, Lindemann, Neisser, and others. Manley (1894) saj's: "In the most virulent cases which have come under my own care the as- pirated fluid was found to contain no gonococci ; while in other cases which ran a mild course, it was said that the gonococcus and some- times the diplococcus were seen in large numbers." In suppurative arthritis following scarlet fever streptococci have been found in pus from the affected joints by Babes, Kankin, Len- harz, and by Bellingham Smith (1895). BERI-BERI. Lacerda (1887) claims to have demonstrated the presence of cocci, some- times united in chains, in the blood and tissues of persons affected with beri- beri, and also to have produced in rabbits, by inoculation with his cultures, certain symptoms resembling those which characterize this disease. Pekelharing and Winkler (1887) have also obtained by cultivation, from the blood of patients with beri-beri, various forms of bacteria, but princi- pally cocci ; these are described as being usually associated in pairs or in ir- regular groups, as forming a milk-white mass upon agar, and as liquefying gelatin. According to the authors named, injection into rabbits of cultures of this coccus gave rise to multiple nerve degeneration, such as is seen in cases of beri-beri in man. Eykmann (1888) failed to obtain cultures from the blood of patients with beri-beri, but demonstrated the presence of slender bacilli similar to those which Pekelharing and Winkler encountered in some of their cases. These failed to grow in the usual culture media. In his latest communication upon the subject Pekelharing says that in twelve cases out of fifteen he obtained cultures of micrococci, and bacilli in three out of fifteen. From his inoculation experiments he concludes that the micrococci found are the cause of the morbid phenomena which charac- terize the disease. When in Eio de Janeiro (1887) the writer collected blood from the finger from four typical cases of beri-beri, selected by Dr. Lacerda, and introduced it into the usual culture media. The result of this experiment was negative, agreeing in this regard with the results obtained by Eykmann. Musso and Morelli (1893) report that they obtained from the blood, sub- 574 BACTERIA IX INFECTIOUS DISEASES. cutaneous oedema, ascitic fluid, etc., of two persons who died of beri-beri, a micrococcus, which, when injected into rabbits, caused their death in from forty days to four months, with symptoms similar to those of beri-beri. Their micrococcus is from 0.8 to 2.4 // in diameter ; in pairs or in chains ; stains by Gram's method and liquefies gelatin- BISKRA BUTTON. See Micrococcus of Heydenreich (No. 26). BRONCHITIS. Lumnitzer (1888) obtained from the sputum of a patient with putrid bron- chitis a bacillus which proved to be pathogenic for mice and for rabbits, and the cultures of which gave off a characteristic odor, similar to that of the putrid bronchial secretion in his patient (No. 112). Picohini (1889), in three cases of " croupous bronchitis," made culture ex- periments and isolated three different micrococci ; one developed upon nutri- ent gelatin as a grayish- white mass and did not liquefy ; one as a reddish- gray mass, also non-liquefying ; the third form was always associated with these two. Bernabei (1895) has found the bacillus of Lumnitzer in a number of cases of putrid bronchitis, and believes it to be the cause of the disease. Alfieri (1894) has also reported a case in which a bacillus was found which appears to be the same. Hitzig (1895) obtained two bacilli resembling the colon bacil- lus f rom a case of putrid bronchitis investigated by him. BRONCHO-PNEUMONIA. Netter (1892) has made a bacteriological study of 95 fatal cases of broncho-pneumonia, 53 adults and 42 children. Of the adult cases 39 gave a pure culture of a single species, which in 15 was the mi- crococcus of croupous pneumonia, in 12 Streptococcus pyogenes, in !> Fried lander's bacillus, in 3 staphylococci. In 14 cases of mixed in- fection the micrococcus of pneumonia and staphylococci were found in 5 ; the pneumonia coccus and streptococci in 3 ; the pneumonia coccus with Friedlander's bacillus in 2 ; pneumonia cocci, strepto- cocci, and staphylococci in 1. In 42 cases in children 25 were simple and 17 mixed infection; in 10 pneumonia cocci only, in 8 streptococci, in 5 staphylococci, in 2 Friedlander's bacillus. In the mixed infec- tions pneumonia cocci and streptococci in 5, streptococci and staphylo- cocci in 5, streptococci and Friedlander's bacillus in 3 ; pneumonia cocci, streptococci, and staphylococci in 2, pneumonia cocci and staphylococci in 1, pneumonia cocci and Friedlander's bacillus in 1. In broncho-pneumonia following epidemic influenza (8 cases) the pneumonia coccus was found in 1, streptococci in 1, Friedlander's bacillus in 2, pneumonia coccus and streptococcus in 1, streptococcus and staphylococcus in 1. BUBO. Hoffa (1886) in 2^ cases of inguinal bubo examined found Staphy- BACTERIA IN INFECTIOUS DISEASES. 575 lococcus pyogenes aureus in 10, Staphylococcus pyogenes albus in 9, and Staphylococcus pyogenes citreus in 3. Later researches indicate that, as a rule, the pus cocci are not present in the pus from an unopened inguinal bubo following chan- croid. In this regard Ducrey, Krefting, and Spietschka are in ac- cord. The last-named author also arrives at the conclusion that the streptobacillus found in chancroidal ulcers is not present in the pus of unopened buboes, and that this is not virulent. Inoculation ex- periments with such pus gave a negative result, and the most careful microscopical investigation failed to reveal the presence of micro- organisms of any kind. Cheinisse (1894) also had a negative result from inoculations with the pus from buboes except in one case in which the bacillus of Ducrey was, by exception, demonstrated to be present. He finds that while the pus from unopened buboes is usually sterile it sometimes contains the ordinary pyogenic micro- cocci. BUBONIC PLAGUE. The bacillus found by Kitasato (1894) and by Yersin (1894) in the contents of the buboes and in the blood of infected animals is no doubt the cause of this infectious malady (see No. 166). CARCINOMA. Various bacteria have occasionally been found in carcinomatous growths, and especially in those which have undergone ulceration ; but that any one of these bears an etiological relation to such malignant tumors has not been demonstrated. CEREBRO-SPINAL MENINGITIS. Various microorganisms have been found by bacteriologists in the exudate of cerebro-spinal meningitis, and there seems to be but little doubt that the meningeal inflammation is due to their presence, as the bacteria usually found are pathogenic for certain of the lower animals, and when introduced into a serous cavity they give rise to a fibrinous or purulent inflammatory process. The researches of Weichselbaum, Netter, and others show that Micrococcus pneu- monia crouposa3 (" diplococcus pneumonia ") is the microorganism most frequently found, and next to this the Diplococcus intercel- lularis meningitidis of Weichselbaum. Streptococcus pyogenes has also been found in a certain proportion of the cases — four out of twenty-five cases of purulent meningitis studied by Netter. Bonome, in a series of cases studied by him, obtained a micro- coccus closely resembling Micrococcus pneumonia crouposa3, but 576 BACTERIA IN INFECTIOUS DISEASES. which he believes not to be identical with it (see Micrococcus of Bonome, No. 40). Jaeger (1894) from a study of the literature arrives at the conclu- sion that in from sixty to seventy per cent of the recorded cases of idiopathic cerebro-spinal meningitis the pneumonia coccus (" Diplo- coccus lanceolatus") has been found. His own researches lead him to believe that the " diplococcus intercellularis " of Weichselbaum is the cause of genuine epidemic meningitis, and that the pneumonia coccus may be present also as a secondary infection. Sporadic cases may be due to streptococcus infection, to tubercular infection, or to pneumococcus infection. In meningitis secondary to middle-ear dis- ease the pneumonia coccus is the usual infectious agent. Sherer (1894) reports three cases of leptomeningitis purulenta in nursing in- fants in which the Bacillus coli communis was found in pure cultures. The infection is supposed to have occurred by bathing the infants in water contaminated by their own discharges. In a later communi- cation (1895) the same author gives an account of an epidemic of cerebro-spinal meningitis among soldiers in which the infectious agent was Diplococcus intercellularis meningitidis (No. 9). This diplococcus was found in the nasal secretions of the infected individ- uals during life, as well as in the exudate from the inflamed men- inges, obtained post mortem. Centanni (1893) has described "a new microorganism of meningitis" under the name Bacillus aerogenes meningitidis (No. 181). CHALAZION. Deyl (1893) was unable to cause the development of chalazion by introduc- ing1 a culture of staphylococci into the mouths of the Meibomian glands in man and rabbits. In fifteen cases of incipient sty in which he made bacterio- logical examinations, he found a bacillus which he believes to be concerned in the etiology of this localized infection. Landwehr (1894) found in one case almost a pure culture of Micrococcus tetragenus. He arrives at the conclusion that in a certain proportion of tbe cases the tubercle bacillus is the etiological agent, but admits that this has not been demonstrated, and that inoculation experiments in susceptible animals with the contents of the sty have always given negative results. CHANCROID. Ducrey, in an extended research (1890), was not able to cultivate any specific microorganism from the pus of soft chancres, or of buboes resulting from these ulcers. Various common microorgan- isms were obtained in cultures from the chancroidal ulcers, but a negative result was obtained in his cultures from the pus of buboes. In pustules developed upon the arm from the inoculation of chan- croidal virus he found constantly a bacillus which did not grow in artificial cultures. This was about 1.48 /* long and 0.5 /* thick, with BACTERIA IN INFECTIOUS DISEASES. 577 round ends. It was readily stained with a solution of fuchsin, but not by Gram's method. Unna, in 1892, reported that in five cases of chancroid he had found a strepto-bacillus, which on account of its numbers and situa- tion in the tissues involved would probably prove to be the specific cause of this localized infection. Quinquaud (1892) confirmed Unna as to the presence of a strepto-bacillus, but neither of the bacteriolo- gists named succeeded in obtaining this bacillus in cultures. Since this date numerous papers have been published with reference to the presence of this and other bacilli in chancroidal ulcers. In his latest communication (1895) Unna maintains that the bacillus of Ducrey is in fact identical with his strepto-bacillus. He says that the pres- ence of this bacillus has now been demonstrated in more than one hundred cases; and, on the other hand, it has never been found in pus from other sources. It is readily stained by Gram's method, and this serves to distinguish it from the only similar strepto-bacillus — which is often found in serpinginous chancroid and especially about the margins. Unna asserts that this strepto-bacillus is constantly present, that it is the only microorganism in the chancroidal tissue, and that it has not been found elsewhere. He therefore feels justified in concluding that it is the specific etiological agent, although it has not as yet been obtained in pure cultures. Spietschka (1894) also reports the presence of strepto-bacilli in chancroidal ulcers, and says that the bacilli seen by him correspond with those found by Unna and later by Peterson, as to size, arrange- ment, and location, but that they have rounded ends and a constric- tion in the middle and do not stain by Gram's method — i.e., they correspond with the bacillus described by Ducrey. Peterson (1894) has also found the bacillus of Ducrey in his cases, in St. Petersburg, and thinks there can be no doubt that it is the specific etiological agent. Krefting has never failed to find this bacillus in chancroidal virus, and in inoculations made with such virus the quicker and more intense the result the more numerous were the bacilli found to be. For the staining of cover-glass preparations Krefting recommends the methylene blue solution of Sahli : Aqua destillata, 40 cubic centi- metres; saturated aqueous solution of methylene blue, 24 cubic centi- metres; solution of borax (five per cent) 16 cubic centimetres. Ac- cording to Krefting the bacilli are from 1.5 to 2 v- long, and from 0.5 to 1 /j. broad. Ducrey describes his bacilli as short, thick rods, with round ends, and at times a slight constriction in the middle. Unna describes his bacilli as short rods, l£ to 2 v- long and £ /* broad, ar- ranged in chains of four to ten elements. These chains are con- stantly ' found in the lymph spaces, between the cells — never in the leucocytes or blood-vessels. 578 BACTERIA IN INFECTIOUS DISEASES. CHOLERA ASIATICA. The etiological relation of Koch's "comma bacillus" to cholera is now generally accepted by bacteriologists and pathologists. But recent researches show that Spirillum cholera Asiatics (No. 180) does not always present identical biological characters when obtained from different cases of epidemic cholera; and that very similar spirilla are sometimes found as saprophytes in river water, and in the alvine discharges of healthy persons. We call attention to the fact that these cholera-like spirilla have for the most part been found in Europe, where epidemic cholera has been widely diffused during the past few years. It is probable that a considerable number of them, at least, are saprophytic varieties of the genuine cholera spiril- lum. CHOLERA INFANTUM. The researches of Booker and of Jeffries do not support the idea that cholera infantum is due to the presence of a specific micro- organism in the intestine, but rather that the symptoms are due to the absorption of toxic products formed in the alimentary canal, or in the child's food before it is ingested, as a result of the multiplica- tion and ferment action of various microorganisms, and especially of certain putrefactive bacteria. The common putrefactive bacillus, Proteus vulgaris, and other species nearly related to this, were found by Booker in a considerable proportion of his cases, and he is dis- posed to believe that these putrefactive bacteria play an important part in the development of the morbid phenomena which characterize the disease. Jeffries, after reviewing the various theories which have been advanced in explanation of the etiology of cholera in- fantum, says : " Bacteria I believe to be at the bottom of the disease — that is, rule bacteria out of all foods and the alimentary canal, and summer diarrhoea would cease to be." Upon another page of bis memoir he says : " Passing a step further, the symptoms, pathology, and etiology of summer diarrhoea stand in close relationship with the group of symptoms first clearly brought to light by Panum as putrid infection. The animals poisoned by the injection of putrid fluids, sterile or not, sicken and die with two variable groups of symptoms: one referable to the nervous system, the other to the in- testines, diarrhoea being a prominent symptom, and the autopsy re- vealing inflammatory changes in the intestine." CHOLERA NOSTRAS. What has been said above with reference to cholera infantum applies as well to cholera uostras. This has not been shown to be BACTERIA IN INFECTIOUS DISEASES. 579 due to the presence in the alimentary canal of a particular micro- organism ; but it can scarcely be doubted that the morbid phenomena are induced by the development of toxic substances as a result of the ferment action of various species of bacteria. Finkler and Prior (1884) obtained from the fa?ces of patients with cholera nostras a spirillum which they supposed to be the specific cause of this disease, but subsequent researches have not confirmed their conclusion. Thus, in seven cases studied by bacteriological methods in Koch's laboratory during the years 1885, 1886, and 1887, the spirillum of Finkler and Prior was not found in a single instance (Frank). In an epidemic of cholera nostras, in which five cases out of seven proved fatal, Carp (1893) was not able to find spirilla in the dis- charges from the bowels, or in the drinking-water to which the out- break was ascribed. The drinking-water was, however, found to be very bad and to contain "fa3ces bacilli." Kirchner (1892) in sixteen cases of cholera nostras examined failed to find the spirillum of Finkler and Prior — in two cases he found a spirillum which failed to grow in gelatin plates and in three a streptococcus. Ruete and Enoch (1894) in a fatal case of cholera nostras obtained from the small intestine a spirillum which they identified as that of Finkler and Prior. The authors named state that researches made in the laboratories of Koch, Hueppe, and Baumgarten show that " Mil- ler's bacillus," which is occasionally found in the mouths of healthy persons, is identical with the spirillum of Finkler and Prior. Grube (1887) has reported a fatal case of cholera nostras in which this spirillum was present in the intestine, and Lustig (1887) reports two fatal cases of cholera in which it was associated with Koch's "comma bacillus." In view of the facts stated, and of the patho- genic properties of this spirillum, we have the same reasons for sup- posing that it is the cause of those cases of cholera nostras in which it is found, as for assuming the etiological relation of the Spirillum cholera Asiaticse. But it is evident that cholera nostras is not a specific disease due to the pathogenic action of a single microorgan- ism. On the other hand, the experimental evidence indicates that in this disease, and in cholera infantum, summer diarrhoea, and other gastro-intestinal disorders, the toxic products developed by various bacteria may give rise to the symptoms characterizing these diseases. CONJUNCTIVITIS. The various forms of conjunctivitis have been ascribed to the specific action of bacteria. That this is true as regards gonorrhceal ophthalmia is now generally admitted, and there is some reason to 580 BACTERIA IN INFECTIOUS DISEASES. believe that the bacillus discovered by Koch and studied by Kartulis (see Bacillus of Kartulis) is the cause of one form of " Egyptian ca- tarrhal conjunctivitis." The non-infectious forms of conjunctivitis can scarcely be supposed to be due to the action of specific micro- organisms; but it is probable that an inflammation resulting from any cause, such as a chemical or mechanical irritant, may be ag- gravated and become chronic as a result of the presence of various microorganisms, and especially of the pyogenic micrococci. Kain (1892) from a case of croupous conjunctivitis obtained a bacillus which when introduced into the conjunctival sacs of rabbits is said to have caused a purulent, membranous inflammation. Wilbrand, Sanger, and Staelin (1893) have investigated an epi- demic of conjunctivitis in patients at their eye clinic in Hamburg with the following results: "With a high degree of probability we may conclude that a diplococcus plays the principal role in the eti- ology of this epidemic. As already indicated, these diplococci in smear preparations resemble the gonococcus of Neisser, and were rec- ognized as such by all unprejudiced and competent observers among our colleagues; but this decision soon proved to be erroneous, inas- much as inoculations in the urethra of two men with the secretion from two severe cases, at the outset of the epidemic, gave a com- pletely negative result. Further, the diplococci lying in the cells are distinguished from gonococci by the fact that they stain by Gram's method, and that they show an evident growth in nutrient gelatin." In their culture experiments the authors named obtained four differ- ent diplococci — viz., Micrococcus flavus desidens, a common, non- pathogenic species, found in the air and in water ; Micrococcus sub- flavus, a micrococcus closely resembling the " trachomacoccus " of Michel ; and a diplococcus which they believe to be new and which proved to be pathogenic for animals. They are not, however, cer- tain whether any one of these corresponds with the diplococcus found in the pus cells, and which, unlike the gonococcus, does not stain by Gram's method. Certain cases were characterized by the presence of bacilli and the absence of diplococci. The bacillus found in these cases, within the pus cells, corresponded with the bacillus first dis- covered by Koch in cases of Egyptian ophthalmia (Bacillus of Kartu- lis, No. 138). 4 'CORN-STALK DISEASE" IN CATTLE. Billings in 1888 ascribed this disease to a bacillus, and Burrill (1889) sub- sequently described "a bacterial disease of corn." According to Billings the bacillus of Burrill is identical with that to which he ascribes the " corn- stalk disease" of cattle. Pyle (1893) says: "Comparing the two germs in cultivation, I doubt their identity, though I recognize their great similarity in developing. On slides they present no marked difference, to me, in appear- BACTERIA IN INFECTIOUS DISEASES. 581 aiice." Pyle further says: "Dr. Billings lias made many successful experi- ments in connection with this disease. He has succeeded in causing- death in susceptible animals by feeding corn-stalk leaves and tops of corn supposed to be diseased with the ' bacterial disease of corn ' which Dr. Burrill has so completely described. From animals thus destroyed Dr. Billings obtained pure cultures of the * corn-stalk disease.' With these cultures he destroyed susceptible animals by inoculation." CORYZA. Hajek found in the secretions of acute nasal catarrh a large diplococcus, called by him " Diplococcus coryzae," and probably identical with the diplo- coccus previously obtained by Klebs from the same source. This was most abundant during the early stage of the disease, when the secretion from the nasal mucous membrane was thin and abundant ; later various other micro- organisms were encountered in greater numbers, and among them Fried- lander's bacillus. There is no satisfactory evidence that the diplococcus of Hajek or any other known bacteria are directly concerned in the etiology of this affection. To what extent chronic nasal catarrh is due to the action of microorganisms is also uncertain, but it appears probable that they play an important part in maintaining such inflammations ; and in ozaena the offen- sive odor of the nasal secretions is no doubt due to the presence of certain bacteria, whatever may be the relation of these to the morbid process which gives rise to the chronic discharge. (See Bacillus foetidus ozsena? of Hajek.) CYSTITIS. The extensive researches which have been made during the past few years show that the presence of bacteria in the healthy bladder does not induce cystitis, but that when the mucous membrane is in- jured by mechanical violence, or by the presence of a foreign body, cystitis is likely to result from the introduction of bacteria, and that the Bacillus coli communis is most frequently concerned in the de- velopment of chronic inflammation of the bladder. In the extended researches of Rovsing — thirty cases of cystitis — the following results were obtained : In one case diagnosed as cysti- tis no bacteria were found ; in three cases culture experiments gave a negative result, but the tubercle bacillus was found in the urine by microscopical examination — in these cases the urine was strongly acid; in twenty-six cases the urine was ammoniacal, and in all of these bacteria were found — usually but a single species. All of these grew in the usual culture media except the tubercle bacillus, which in two cases was associated with some other species, and all pro- duced alkaline fermentation in sterile urine when added to it in pure cultures. The following species were found: Tubercle bacil- lus, Staphylococcus pyogenes aureus, Staphylococcus pyogenes albus, Staphylococcus pyogenes citreus, Streptococcus pyogenes urese (n. sp.), Diplococcus pyogenes urese (n. sp.), .Coccobacillus pyogenes ureaa (n. sp.), Micrococcus pyogenes ureae flavus (n. sp.), Diplococ- cus urea3 trifoliatus (n. sp.), Streptococcus urese rugosus (n. sp.), Diplococcus urea3 (n. sp.), Coccobacteria urese (n. sp.). 582 BACTERIA IN INFECTIOUS DISEASES. Pure cultures of all of these species introduced into the bladder of rabbits failed to induce cystitis, even when injected in consider- able quantities. But when retention of urine was effected artificially for six to twelve hours, allowing time for ammoniacal fermentation to occur, cystitis was developed. When the pyogenic species were introduced under these circumstances, a suppurative inflammation of the mucous membrane occurred ; the non-pyogenic species caused a catarrhal cystitis. Rovsing records the important fact, as bearing upon the etiology of cystitis, that in twenty of the cases examined the bladder had been invaded by the finger or by instruments prior to the development of cystitis. Lundstrom (1800) isolated from alkaline urine obtained from pa- tients with cystitis two species of staphylococci — Staphylococcus urese candidus and Staphylococcus urese liquef aciens ; from albu- minous, acid urine he obtained Streptococcus pyogenes. Krogius ob- tained from the urine of individuals suffering from cystitis a bacillus which he calls Urobacillus liquefaciens septicus. Schnitzler (ISiMi) found the same bacillus, or one very similar to it, in thirteen out of twenty cases of purulent cystitis examined by him. In eight of these cases it was obtained from the urine in pure cultures, and in five it was associated with other bacteria. In twelve of these twenty cases the cystitis resulted directly from catheterization ; in the others it occurred in individuals suffering from stricture or from calculus. When cultures of this bacillus were injected into a vein in rab- bits, the animals died in from three to eight days, and in every in- stance an intense nephritis was observed at the autopsy — twice with the formation of small abscesses. The bacillus was found in the blood and the organs generally. Injections into the bladder of rab- bits almost always gave rise to a severe purulent cystitis — large rab- bits were selected and great care taken not to injure the mucous membrane of the bladder. Schnitzler was not able to induce cystitis in rabbits by injecting in the same way considerable quantities of a culture of Staphylococcus pyogenes aureus. Guyon (1888) did not succeed in inducing cystitis by the injection of pure cultures of various microorganisms into the bladder, unless he at the same time produced an artificial retention of urine. His experimental results are therefore in accord with those of Rovsing, who found that without mechanical injury, or artificial retention until ammoniacal fermentation had occurred, no results followed his injections into the bladder. According to Schmidt and Aschoff (1893) subsequent researches indicate that some of the species described by Rovsing as being new are in fact varieties of Bacillus coli communis. The identification of the Bacillus pyogenes of Albarran and Halle BACTERIA IN INFECTIOUS DISEASES. 583 (Bacterie septique of Clado) with Bacillus coli communis was first made by Krogius (1891), and about the same time by Achard and Renault. In twelve cases of cystitis, six of which were complicated with ascending nephritis, Krogius demonstrated the presence of Bacillus coli communis, and showed that in its growth in culture media it corresponded with the Bacillus pyogenes of previous authors. In a second communication Krogius states that in twenty-two cases of cystitis studied by him he obtained Bacillus coli communis sixteen times, and of these fourteen times in pure culture. He also calls at- tention to the fact that in those cases where no other microorganism was associated with the colon bacillus the urine was always acid — a statement which is sustained by the subsequent researches of Schmidt and Aschoff. He also gives details with reference to the pleomorphism of this bacillus and differences in the appearance of gelatin cultures from different sources, the growth being sometimes opaque and sometimes transparent. When cultures of this bacillus were injected into the bladder of rabbits and retained by ligating the urethra, an intense cystitis was developed in from twenty to thirty hours. Injections into the ureter gave a result similar to that subse- quently reported by Schmidt and Aschoff. The animal died in about two days, and pyelitis, together with more or less necrosis of the renal epithelium, was found at the autopsy. Reblaub (1892) ob- tained Bacillus coli communis in pure culture in six out of sixteen cases of cystitis examined. In their latest publication Achard and Renault arrive at the con- cluson that there are some differences between their " urobacillus " and Bacillus coli communis, which they state as follows: 1. Upon most media, especially upon malt agar, the growth is more luxuriant. 2. Cultures of the urobacillus upon potato appear grayish white, very luxuriant, and have many gas bubbles. 3. The urobacillus develops much gas even in gelatin and agar cultures containing little sugar. Morelle (1892) and Denys (1892) in their bacteriological re- searches obtained from numerous cases of cystitis a bacillus which they identified with Bacillus lactis aerogenes of Escherich. But the last-mentioned author has since stated that this bacillus presents varieties which cannot be distinguished from the typical cultures of Bacillus coli communis. The recent researches referred to having shown that Bacillus coli communis is very commonly present in the urine in cases of cystitis, and often in pure cultures, its etiological relation to the disease in question seems probable ; and this view is further sustained by exper- 584 BACTERIA IN INFECTIOUS DISEASES. iments upon animals and by the demonstrated fact that retention of urine per se does not give rise to inflammation of the bladder. But this is not the only microorganism which is capable of causing a cystitis when introduced into a bladder which has suffered some kind of mechanical injury or has been subjected to the action of chemical irritants contained in the urine. The researches of Krogius, Schnitz- ler, and of Schmidt and Aschoff show that next to the colon ba- cillus the microorganisms most commonly found in cases of cystitis and of pyelonephritis is a proteus (Proteus vulgaris?). At the date of the publication of the monograph of Schmidt and Aschoff the Bacillus coli communis had been found in pure culture in sixty cases of cystitis, and the proteus in thirteen cases. An important point to be kept in view is the fact that when Ba- cillus coli communis is found in the urine in pure culture, this fluid is more or less acid, as the bacillus in question does not give rise to alkaline fermentation, at least not under the conditions found in the bladder and in the absence of retention. But when proteus is present the urine is almost always ammoniacal. DENGUE. McLaughlin (1886) has claimed to find micrococci in the blood of patients suffering from dengue. No satisfactory evidence of their etiological relation has been presented, and his observations have not yet been confirmed by other investigators. DENTAL CARIES. The extended researches of Miller lead him to the conclusion that dental caries is due to various microorganisms described by him. In a paper pub- lished in 1894 his conclusions are formulated as follows : "1. In infectious processes in the pulp, almost without exception, we have a mixed infection, and cocci and bacilli are found with about equal fre- quency ; somewhat less frequently, long slender filaments and spiral forms are encountered. At times very peculiar forms are seen ; spore-bearing rods and filaments are occasionally encountered. "2. The microscopical examination of cover-glass preparations justifies the view that micrococci are especially concerned in the production of pus. "3. The bacteria make their way to the pulp principally through the carious dentine, and a thin layer of hard dentine does not protect it with cer- tainty from infection. Infection of the pulp through the blood-vessels may IM- possible in certain cases, but has not been demonstrated. "4. The pulp is predisposed to infection by the action of products formed in the carious dentine (acids-ptomaines). "5. In disease of the pulp bacteria are chiefly concerned which cannot be cultivated. "(>. Various bacilli which can be cultivated have been found in di- pulp, but for the most part they are non-pathogenic. "7. The typical pyogenic cocci, Streptococcus pyogenes aureus and allms, and Streptococcus pyogenes, are seldom found in pus from the pulp, the contrary, various cocci are found, ('specially a group of nearly related snecies, which cause pus-formation in mice. This question has not yet been cleared up. BACTERIA IN INFECTIOUS DISEASES. 585 "8. A micrococcus which I could identify with the micrococcus of spu- tum septicaemia, i.e., the pneumococcus, in spite of numerous experiments on animals, I have as yet failed to find. At best we can only speak of a va- riety of the pneumococcus. "9. The action of the pulp cocci is greatly increased by putrefactive proc- esses. A putrid pulp, whether bacteria may be obtained from it in pure cultures or not, is always a dangerous infectious material. ' ' 10. Putrid decomposition of the tooth pulp is caused by various bacteria, and the putrid products are not always the same. In addition to gaseous products (NHs, SH2) there are various other substances, the nature of which has not been determined." DIARRHOEA. In the green diarrhoea of infants Lesage obtained a bacillus (No. 106) which he supposed to be the cause of the malady. His bacillus is probably identical with Bacillus fluorescens non-liquefaciens. Vogler (1893) obtained from a diarrhceal stool a vibrio different from that of cholera, and which was not pathogenic for guinea-pigs. We have referred to the researches of Booker and of Jeffries under the heading "Cholera Iiifantum." Bajinsky (1894) agrees with Fliigge in believing that the toxins produced by bacteria are the usual cause of summer diarrhoea in children, a view in which we fully concur. But there is no reason to suppose that any particular micro- organism of this class has a specific role in the etiology of affections of this class. Probably bacilli of the colon group and of the prpteus group are more frequently than any others responsible for gastro-intestinal troubles in chil- dren. They are very widely distributed and multiply with great rapidity under favorable temperature conditions in milk or other articles of liquid food. - DIARRHCEA (INFECTIOUS) IN CALVES. Jensen (1892) has investigated a fatal infectious disease of calves, charac- terized by diarrhoea, etc., and concludes that it is due to a bacillus which cor- responds with Bacillus coli communis in all respects, except in its increased virulence. In the contents of the intestine of calves which have recently succumbed to the malady, the bacillus is found almost in pure culture ; also in the inflamed mucous membrane, in the hyperaemic mesenteric glands, and in the blood and various organs. Calves fed with a culture of this bacillus invariably died within two or three clays, and the bacilli were found in almost pure culture in the contents of the intestine, and in great numbers in the blood and organs. The subcutaneous injection of four cubic centimetres of a bouillon culture caused fatal septi- caemia. DIPHTHERIA. The Klebs-Loffler bacillus (No. 47) is now generally recognized as the specific infectious agent in diphtheria. DIPHTHERIA IN CALVES. Due to Loffler's Bacillus diphtheriae vitulorum (No. 50). DIPHTHERIA IN PIGEONS. Due to Lb'ffler's Bacillus diphtheriae columbrarum (No. 49). 41 586 BACTERIA IN INFECTIOUS DISEASES. DOGS, INFECTIOUS DISEASES OF. According to Schantyr (1893) the so-called "distemper" in dogs includes three different infectious diseases — "abdominal typhus, typhoid, and genuine distemper." In several cases of so-called distemper (" staupe ") he obtained a bacillus, previously described by Semmer, which reproduced the disease when inoculated into healthy animals. This bacillus closely resembles Bacillus typhi abdominalis, and is perhaps identical with it, but its virulence for ani- mals is greater. In typhoid of dogs he found in the blood and various or- gans small bacilli, which stained by Gram's method and also closely resem- bled the bacillus of typhoid fever in man. Young dogs died within a short time when inoculated with cultures of this bacillus. In thirteen cases of genuine distemper, Schantyr found in the blood and organs a great number of bacilli, from 1 to 2 p> long, mostly associated in groups, which he was not able to cultivate in the usual media. Once only he obtained an agar culture and from this a culture on blood serum. Two out of three dogs inoculated with this culture died of distemper. Galli-Valerio (1895) has reviewed the literature relating to the etiology of distemper in dogs, and reports the results of his own investigations. He found constantly in young dogs which succumbed to the disease an oval ba- cillus, 1.25 to 2. 5 n long, and 0.31 /* thick. This was present in the lungs, the brain, and the spinal marrow, but never in the blood. In gelatin cul- tures gas bubbles appeared along the line of puncture within twenty-four hours, and a small, white, wax-like point was developed on the surface ; later along and narrow funnel was seen, but this did not contain liquefied gelatin. The bacillus was readily stained with the aniline colors and by Grain's method. A young dog, five months old, succumbed to a subcutaneous inocu- lation, at the end of eighteen days, with all the symptoms of distemper. And the bacillus was recovered from the pustules, the lungs, brain, and spinal marrow. DYSENTERY. While the evidence seems to support the view that certain cases of dysen- tery are due to the presence of the amoeba coli, this parasite is not found in others. Thus in twenty cases studied by Maggiori (1893) it was only found in one ; and in an epidemic of dysentery in Japan, studied by Ogata, amoebae were not found in the discharges. In the epidemic observed oy Maggiori, three deaths occurred out of two thousand and one cases ; the duration of the disease was from six to twelve days. The bacteriological examination dem- onstrated the presence of Bacillus coli communis in great numbers, and in most of the cases of Proteus vulgaris, but not in great abundance ; in six cases Bacillus fluorescens liquefaciens was found ; in two Staphylocoecus Syogenes aureus ; in one Staphylococcus pyogenes albus ; in fiveout of eleven acillus pyocyaneus in small numbers. The colon bacillus and Bacillus py<>- cyaneus proved to be very virulent. Ogata found in the recent discharges, in great numbers, fine, short bacilli, which liquefied gelatin and stained l»y Gram's method. This bacillus proved to be pathogenic for animals, and is believed by Ogata to have been the cause of the epidemic observed by him. Arnaud (1894) investigated sixty cases of tropical dysentery, and arrives at the conclusion that it is due to a pathogenic variety of the colon bacillus. I dogs inoculated in the rectum with his cultures of this bacillus had dysentery as a result of these inoculations, with characteristic ulceration of the colon. Laveran ((1893), also, only found amoebae in one case out of ten of " Euro- pean dysentery " studied by him A bacillus which was apparently identical with Bacillus coli communis was present in great numbers. Bertram! and Baucher (1893) have studied an epidemic among the troops stationed at Cher- bourg, and arrive at the conclusion that no one of the microorganisms found by them can be considered as specific for the disease. They found among BACTERIA IN INFECTIOUS DISEASES. 587 other bacteria present in the dysenteric discharges : Bacillus coli communis, Bacillus pyocyaneus, Bacillus fluorescens liquefaciens, Staphylococcus pyo- genes aureus, and Bacillus cedematis maligni. ECLAMPSIA. G-erdes (1892) obtained from the kidneys, lungs, liver, and blood from the aorta of two fatal cases of puerperal convulsions a bacillus which he supposed to be the cause of the disease. Hofmeister (1892) has shown that the bacillus of Gerdes was, in fact, the well-known saprophyte, Proteus vulgaris. Haeg- ler (1892) in examinations of the blood of cases of puerperal eclampsia always had a negative result, but in the urine various bacteria were found — in one case Proteus vulgaris, in one Micrococcus urese, in one Staphylococcus pyo- genes albus, in one a diplococcus which was probably identical with the micro- coccus of croupous pneumonia. Doderlein (1893) also failed to find bacteria of any kind in the blood of patients with puerperal eclampsia — or in the urine. Bar and Renon (1894), in three cases in which a bacteriological examination of the liver was made immediately after death, found in one Staphylococcus pyogenes albus and aureus ; in the other cases cultures from the liver re- mained sterile. Combemale and Bue (1892) report that in four cases, in which there was albuminuria, oedema, and disturbance of vision, a bac- teriological examination of the blood demonstrated the presence of Staphy- lococcus pyogenes albus, alone or associated with Streptococcus pyogenes albus. ECZEMA. Unna and Tommasoli (1889) have described three species of micrococci and six bacilli obtained from cases of eczema seborrhoicum which they re- garded as new, viz. : Bacillus liquefaciens fluorescens minutissimus, Bacillus aureus, Bacillus spiriferus, Bacillus albicans pateriformis, Bacillus ovatus minutissimus, Ascobacillus citreus, Diplococcus citreus liquefaciens, Diplo- coccus liquefaciens tardus, Diplococcus albicans tardus. Merrill (1895) has made a bacteriological study of fifty cases of eczema seborrhoicum. In two cases the result was negative, "it being impossible to obtain any growth from the scales by any method." In the remaining forty- eight cases bacteria were obtained in cultures, made at the room temperature. "Pure cultures obtained in the experiments showed three distinct varieties of bacteria which may be designated Nos. 1, 2, and 3. In thirty-one cases all three were present ; in seven only Nos. 1 and 2 ; in two, Nos. 1 and 3 ; in five, No. 1 ; and in one, No. 3 alone. Staphylococcus pyogenes aureus was also obtained in one case and Bacillus fluorescens liquefaciens minutis- simus in three. The bacteria found are described as follows : " Variety 1. — Small diplococci, single or in irregular groups. The parts forming each diplococcus are round or only slightly oval. The germs are aerobic, noii -liquefying, and iion-chromogenic. At 70° F. they grow rap- idly. On gelatin plates the deep-seated colonies remain about the size of an ordinary pin's head for weeks. The superficial colonies are round, white, with slightly raised surfaces, and smooth or somewhat irregular borders. In its growth the colony adheres very nearly to its circular form. After the first week the centre begins to turn darker, and with increasing age of the colony the whole surface, hitherto smooth, begins to be wrinkled and the edges become irregular, as though the evaporation of the water caused con- traction. At the end of three weeks growth seems to stop, and the colony changes from its original white color to a dusky brown. On agar-agar the appearances closely resemble those of the gelatin colonies, except that it is slower in its growth and its surface has a whiter lustre. On potato the growth begins to be visible on the second day. On the fifth day it is cream white, smooth, raised about a tenth of an inch, and its edges are irregular 588 BACTERIA IN INFECTIOUS DISEASES. and scalloped. At this time it covers about two-thirds of the surface of a potato stick half an inch in diameter. After the first week the growth is slow, and at the age of three weeks its size only equals that of the iirst week, but the colony itself is shrivelled, dried, and dark in color. In milk the cul- ture had on the second day a slight greenish tinge, which, by the fifth day. had disappeared. The upper quarter inch of the milk seems slightly thicker, but no other change is visible to the naked eye. " Variety 2. — In. appearance it is almost identical with Variety 1, except that it seems more oval in form. This diplococcus is aerobic, non-liquefy- ing, and chromogenic. As in Variety 1, the ordinary changes of tempera- ture, as occur from the rotation of the seasons of the year, retard or acceler- ate the growth of the culture. On Petri dishes of gelatin, the minute, round, yellow colonies appear on the third or the fourth day. Those on the surface grow slowly, are slightly raised, and have smooth borders. After the first week's growth the centre shows a deeper orange color. On agar' 'agar, the growth is slightly lustrous, thicker, and of a light orange color. On potato, a deep golden layer develops, which is well raised and has irreg- ular borders. In milk, this diplococcus grows as Variety 1 does, except that after ten days the upper layer of the milk is thickened and has turned the same golden color mentioned. In stab cultures of Varieties 1, and 2 the growth adheres pretty closely to the puncture line, gradually spreading down it and over the surface. " Variety 3. — A bacillus with rounded ends, single, in pairs, or in short or long chains. It is aerobic and anaerobic, motile, liquefying, and non- chromogenic. In gelatin tubes a grayish- white growth commences on the second day. In smear cultures a pit of liquefied gelatin is formed, and, re- maining of the same irregular shape as the smear, it gradually deepens and contains at the bottom a whitish sediment. In stab cultures the resulting pit is the shape of the puncture and contains the same white sediment. On aga r- agar the growth is whitish, its surface raised, without lustre, and its border indented. " Inoculation experiments were attempted in twelve cases. The site of inoculation chosen was the hairy scalp, or over the sternum. After thor- oughly sterilizing the skin, two or three hairs were pulled out and the skin slightly abraded, as in vaccination ; portions of an actively growing culture were then rubbed in with a sterilized platinum needle. With No. 3 two attempts were made and both failed. With No. 1 five attempts were made. Of these, one was a failure. In the four others the edges of the inoculation spots began to grow slightly reddened from the fourth to the sixth day, and small scales formed on the surface. By the seventh to the tenth day t lie spots had increased in size and were covered with dry white scales. Scales taken from these spots and placed in suitable culture media in each case gave rise to pure cultures of diplococcus No. 1. Variety No. 2 was used ome. On the sixth day yellowish scales appeared over the surface. They grew slightly more marked on the tenth aay, and the lesion then closely resem- bled certain typical forms of seborrhceic eczema. Diplococcus No. II. was found in the cultivations from these scales. The last four inoculations were made with both No. 1 and No. 2. Of these, one was a failure. Another showed a small spot covered with a few branny scales, too small to allow of any conclusions being drawn. In the other two achange began on the fourth day. The bases began to redden, and typical, crumbly, greasy scales began to cover the surfaces and pile up in the centres. On the eighth day the spots were an eighth of an inch across, and represented patches of seborrhoeic eczema. Both Nos. 1 and 2 could be cultivated from these scales. It is possible that Variety No. 2 may have given the yellow color to those seal. •-. as it was absent in the successful cases 'using No. 1. "The result of the twelve inoculation experiments, therefore, are : Five failures. Seven cases in which definite lesions were produced." BACTERIA IN INFECTIOUS DISEASES. 589 ECZEMA EPIZOOTICA. monym. — Foot and mouth disease. Tnis is an infectious disease of horned cattle, characterized by a vesicular eruption in the mouth and about the feet. It affects also sheep and pigs and may be communicated to man. Schottelius (1892) has described a microorganism which he thinks may bear an etiological relation to the disease. His inoculation experiments do- not, however, sustain this view, inasmuch as the characteristic vesicles were- never developed in inoculated calves, and experiments upon other animals- gave a negative result. In young cattle small doses (one cubic centimetre) of a bouillon culture gave rise to a slight fever and loss of appetite, while larger doses produced an intense fever, salivation, and great debility. But recovery occurred at the end of five or six days without any aphthous erup- tion. Schottelius obtained from the clear contents of the vesicles in the. mouth various bacteria which he believes to have been accidentally present. After making a considerable number of culture experiments his attention was attracted by a spherical microorganism, united in chains, which grew very slowly in the ordinary culture media. This he describes as follows : The individual cells vary greatly in diameter, and are considerably larger than known micrococci ; they are associated in longer or shorter chains, and are endowed with active movements. According to Schottelius, they be- long to the " streptocyt en " rather than to the streptococci. They do not stain readily with methylene blue, but may be stained with gentian violet and by Gram's method. Development does not occur at temperatures below 37° to 39° C. The most suitable culture medium was found to be bouillon or glycerin agar to which formate of soda had been added (amount?). Growth occurred in an atmosphere of CO2 as well as in atmospheric air. Upon, plates of nutrient agar — containing glycerin and formate of soda — at 37° C., very delicate, almost transparent colonies developed ; they were of a pearl- gray color, with an irregular, rosette-like margin ; in the course of several weeks they attained a diameter of one to one and one-half millimetres. Upon potato a scanty, grayish-white, dry layer is developed. Under the most favorable conditions the development was very slow — not more rapid than that of the tubercle bacillus. Kurth (1894) obtained from the contents of vesicles on the udders of in- fected animals seven different microorganisms, six of which were not con- stantly present, while the seventh was found in great numbers in all cases, with one exception, and was also present in the saliva. This was a streptococcus, named by Kurth Streptococcus involutus. Pure cultures of this streptococcus were rubbed into the mouths of young sheep and calves without result. Sanfelice (1894) in an extended research verified the pres- ence of the Streptococcus involutus of Kurth in the aphtiious vesicles and superficial erosions of the tongue in infected animals. But his inoculations of pure cultures into susceptible animals gave a negative result, and he con- cludes that this streptococcus is not concerned in the etiology of the disease. Piano and Fiorentini (1895) arrive at the conclusion that the disease is not due to any microorganism belonging to the schizomycetes ; but that it is probably due to an amoeboid microorganism which is found in the contents of the vesicles and in the blood of infected animals. This conclusion is in accord with the views of Schottelius and of Behla, and will probably prove to be well founded. EMPYEMA. "A. Frankel (1888), as a result of his bacteriological studies in twelve cases of empyema, divides the cases into four groups. In one group of three cases Streptococcus pyogenes was the only microor- 590 BACTERIA IN INFECTIOUS DISEASES. ganism obtained in his cultures or seen in stained preparations of pus from the pleural cavity. In a second group of three cases, oc- curring in the course of a pneumonia, the only microorganism pres- ent was " diplococcus pneumonia? " (Micrococcus pneumonia? crou- posa?). The third group included four cases of tubercular emp3rema; in one of these tubercle bacilli only were found in pus from the pleural cavity, in one case streptococci were found, and in two no microorganisms were found. In the fourth group of two cases the empyema resulted from the opening of an abscess into the pleural cavity, and streptococci were found in the pus. Netter, in a series of forty-six cases examined by him, found Mi- crococcus pneumonia? crouposa? in fourteen. Koplik (1890) found the same microorganism in seven cases examined by him, and Strep- tococcus pyogenes in two cases. Weintrand (1893) has reported a case of empyema following ty- phoid fever, in which the typhoid bacillus in pure culture was found in pus drawn from the pleural cavity by means of a syringe. Prudden (1893) found microorganisms in every case examined by him (twenty-four) ; in seven cases out of eight Streptococcus pyo- genes was present in pus obtained from the pleural cavity. In the cases of metapneumonic empyema the germ most commonly present (in nine cases out of eleven) was the Micrococcus lanceolatus (pneu- mococcus). In four cases of foetid empyema various bacilli were found. Staphylococcus pyogenes aureus was present in one case only. Levy (1895), from a review of the literature of the subject in connection with his own observations, arrives at the conclusion that Streptococcus pyogenes is the usual cause of purulent inflammation of the pleura — found in sixty per cent of the cases. ENDOCARDITIS. The experimental evidence relating to endocarditis is similar to that in cystitis. The injection of the microorganisms found attached to the diseased structures into the circulation of lower animals does not produce endocarditis unless the valves have been previously in- jured by mechanical violence or by chemical irritants. If some doubt remains among pathologists as to the etiological relation of the microorganisms found, the serious secondary results of the mycotic invasion are well established. In a series of twenty-nine cases stud- ied by Weichselbaum (1885-1888) the following results were ob- tained : In eight the result of culture experiments and microscopical examination was negative; in seven "diplococcus pneumonia?" (Mi- crococcus pneumonia? crouposa?) was found; in six Streptococcus BACTERIA IN INFECTIOUS DISEASES. 591 pyogenes ; in two Staphylococcus pyogenes aureus ; in two Bacillus endocarditidis griseus ( Weichselbaum) ; in one Micrococcus endo- carditidis rugatus (Weichselbaum); in one Bacillus endocarditidis capsulatus (Weichselbaum); in two cases a bacillus which he did not succeed in cultivating. For further details see the descriptions of microorganisms referred to. Howard (1893) reports a case of acute ulcerative endocarditis in which the diphtheria bacillus was present in pure culture — also ob- tained in cultures from the liver, spleen, and kidneys. In a case of malignant endocarditis in a patient with gonorrhoea and gonorrhoeal rheumatism, Leyden (1893) found the gonococcus in the vegetations upon the valves. Banti (1894) in 22 cases examined obtained Streptococcus pyogenes in 7, Staphylococcus pyogenes aureus in 1, these two microorganisms associated in 3, the micrococcus of pneu- monia in 8 ; in 2 no bacteria were found. Dessy (1894) also exam- ined 22 cases and had a negative result in 2. In the cases in which bacteria were present he found " Diplococcus lanceolatus cap- sulatus " (Micrococcus pneumonias crouposaa) in 8, Streptococcus py- ogenes in 7, Staphylococcus pyogenes aureus in 1, Staphylococcus pyogenes aureus and Streptococcus pyogenes associated in 3, Staphy- lococcus pyogenes albus and Diplococcus lanceolatus in 1. ENDOMETRITIS. La Place (1892) has reported the results of his bacteriological investiga- tions of the secretions of the endometrium and cervix uteri in health and dis- ease. He found numerous bacteria in the normal secretions, but vastly more in secretions from the inflamed endometrium, "the superficial exfoliating cells also containing them." "In chronic endometritis the secretions con- tain about as many infectious organisms, the mucous membrane and fibrous tissue become greatly hypertrophied under the continued development of these organisms, and whether this chronic condition be simple or gonor- rhceal, we find the germs both in the epithelium and fibrous tissue." The microorganisms obtained from the secretions of women with endocervicitis were the ordinary pus cocci, Bacillus pyocyaneus, and certain other bacteria designated by the letters, x, y, and z. Wolf (1893), in the secretions from the uterus in eight women suffering from endometritis, found micrococci to be most numerous ; but in two cases bacilli were found, and in one a vibrio somewhat resembling Koch's "com- ma bacillus." This he describes under the name of Bacillus choleroides. ERYSIPELAS. Due to infection by Streptococcus pyogenes (No. 5) . ERYTHEMA. Cordua (1885) obtained from a series of cases of an erysipelatoid skin affection of the fingers and hands, which he identified as corresponding with erythema exudativum multiforme of Hebra, a micrococcus resembling Staphylococcus pyogenes albus in its biological characters, but which he de- 592 BACTERIA IN INFECTIOUS DISEASES. scribes as being three to four times as large. Inoculations in animals were without result, but two inoculations upon his own hand produced a dark-red tumefaction in the vicinity of the point of inoculation resembling that in the individuals from whom he obtained his cultures. In two cases of " polymorphous erythema" Haushalter (1887) isolated a streptococcus which did not produce an erysipelatous inflammation when in- oculated into the ear of rabbits, and which he supposed to be a different species (?) from the now better known Streptococcus pyogenes. In five cases of erythema nodosum in children Demme obtained a bacillus which his inocu- lation experiments proved to be pathogenic, and which was perhaps con- cerned in the etiology of the skin affection from which his cultures were ob- tained (see Bacillus of Demme, No. 107). Finger (1892) has reported a case in which there was also an extensive diphtheritic process in the throat, and metastatic abscesses in the kidneys and myocardium from which Streptococcus pyogenes was obtained in pure cultures. In the erythema papules, also, were found great masses of strep- tococci, exclusively in the blood-vessels and filling the capillaries of the pap- illary bodies as if by an injection mass. In erysipelas the streptococcus is not found in the blood-vessels, but invades the lymph channels. FARCY IN CATTLE. See Bacillus of Nocard (No. 60). FISH, INFECTIOUS DISEASES OF. See Bacillus piscicidus (No. 173), Bacillus piscicidus agilis (No. 167), Bacil- lus of Emmerich and Weibel (No. 169). FOOT AND MOUTH DISEASE. See Eczema epizob'tica. FOWL CHOLERA. Due to infection by Bacillus septicaemias hemorrhagicae (No. 61). FURUNCULOSIS. Due to infection by the ordinary pus cocci (Nos. 1, 2, 5), and especially by Staphylococcus pyogenes aureus. FROGS, INFECTIOUS DISEASES OF. See Bacillus hydrophilus fuscus, of Sanarelli (No. 81). GANGRENE. When the vital resistance of the tissues is impaired by malnutrition and pressure, or by an impaired blood supply from any cause, an invasion by saprophytic bacteria is liable to occur and a more or less extensive gangrene results. It is probable that the infectious disease known as ''hospital gan- grene" is due to common saprophytes which have attained increased patho- genic virulence as a result of special conditions relating to their environment in suppurating wounds. This has not, however, been demonstrated, and itn possible that the development of an epidemic of hospital gangrene is due to the introduction of some pathogenic microorganism different from those usually found in the secretions of wounds and which has the power of invad- ing healthy tissues when introduced into an open wound. BACTERIA IN INFECTIOUS DISEASES. 593 GAS PHLEGMON. In four cases of so-called gas phlegmon Frankel (E.) found an anaerobic bacillus named by him Bacillus phlegmones emphysematosse. Cultures of this bacillus gave rise to a similar process when injected subcutaneously in guinea-pigs. In a case reported by Bunge (1894) Bacillus coli com- munis is believed to have been the infectious agent to which the develop- ment of the gas phlegmon was due. GLANDERS. Due to infection by Bacillus mallei (No. 56). GONORRHOEA. Due to infection by Micrococcus gonorrhoeas (No. 6) — "Gono- coccus" of Neisser. GRANULOMA FUNGOIDES (MYCOSIS FUNGOIDES). Rindfleisch (1885) and Auspitz (1885) report the presence of streptococci in the capillary vessels of the papillary body and of the subcutaneous tissue in the affected localities in cases of this disease. That the streptococcus differs from Streptococcus pyogenes, as Auspitz supposes, has not been definitely established. GROUSE DISEASE. See Bacillus of grouse disease, of Klein (No. 76). HOG CHOLERA. Due to infection by a motile bacillus of the "colon group" — Bacillus of hog cholera, of Salmon and Smith (No. 63). HOG ERYSIPELAS. Due to infection by Bacillus erysipelatos suis (No. 67). HYDROPHOBIA. Notwithstanding the extended researches made, especially in Pasteur's laboratory, the etiology of hydrophobia still remains unsettled. It has been demonstrated by experiment that the virus of the disease is located in the brain, spinal marrow, and nerves of animals which have succumbed to the disease, as well as in the salivary secretions of rabid animals, and that the disease may be transmitted by intravenous inoculation, or by introducing a small quantity of virus beneath the dura mater, with greater certainty than by subcutaneous inoculations. But the exact nature of this virus has not been determined. The fact that a considerable interval elapses after inoculation before the first symptoms are developed indicates that there is a multiplica- tion of the virus in the body of the infected animal ; and this is further shown by the fact that after death the entire brain and spinal marrow of the animal have a virulence equal to that of the material with which it was in- oculated in the first instance. The writer's experiments (1887) show that this virulence is neutralized by a temperature of 60° C. maintained for ten min- utes—a temperature which is fatal to all known pathogenic bacteria in the absence of spores. But recent experiments show that certain toxic products 594 BACTERIA IN INFECTIOUS DISEASES. of bacterial growth are destroyed by the same temperature, We are, there- fore, not justified in assuming that the morbid phenomena are directly due to the presence of a living microorganism ; and, indeed, it seems probable, from what we already know, that the symptoms developed and the death of the animal are due to the action of a potent chemical poison of the class known as toxalbumins. But, if this is true, we have still to account for the production of the toxic albuminoid substance, and, in the present state of knowledge, have no other way to explain its increase in the body of the in- fected animal than the supposition that a specific, living germ is present in the virulent material, the introduction of which into the body of a suscep- tible animal gives rise to morbid phenomena characterizing1 an attack of rabies. Pasteur and his associates have thus far failed to demonstrate the pre- sence of microorganisms in the virulent tissues of animals which have suc- cumbed to an attack of rabies. Babes has obtained micrococci in cultures from the brain and spinal cord of rabid animals, and states in his article on hydrophobia in u Les Bacteries" (second edition, page 791) that pure cultures of the second and third generations induced rabies in susceptible animals ; but his own later researches do not appear to have established the etiological re- lation of this micrococcus. Gibier (1884) has reported the presence of spherical refractive granules, resembling micrococci, in the brain of rabid animals, which he demonstrated by rubbing up a little of the cerebral substance with distilled water. As these supposed micrococci did not stain with the usual aniline colors and were not cultivated, it appears very doubtful whether the refractive granules seen were really microorganisms. Fol (1885) claims to have demonstrated the presence of minute cocci, 0.2 u in diameter, in sections of spinal cord from rabid animals, by Weigert's method of staining. The cords were hardened in a solution of bichromate of potash and sulphate of copper, colored with a solution of hsematoxylon, and decolorized in a solution of ferrocyanide of potash and borax. The writer (1887) has made similar preparations, carefully following the method as described by Fol, but was not able to demonstrate the presence of microorganisms in the numerous sections made. Nor have the observations of Fol been confirmed by the researches of other bacteriologists who have given their attention to the subject since the publication of his paper. With reference to the results of Pasteur's protective inoculations, we may say that it is now pretty generally admitted that the published statistics demonstrate the prophylactic value of the method as practised at the Pasteur Institute in Paris. ICTERUS. Karlinsky (1890), in a series of five cases of " infectious icterus " attended with fever, found in the blood, during the height of the attack, curved bacilli from two to six u long and one-third to one n broad, which were readily stained by the usual aniline colors, but not by Gram's method. These he did not succeed in cultivating in any of the culture media usually employed. Ducamp (1890) has also given an account of a " slight epidemic of infec- tious icterus," which he supposes to have been due to microorganisms. In "Weil's disease," which is characterized by fever and icterus, and is believed to be an infectious malady, Jaeger (1892) has obtained a bacillus which he considers the specific infectious agents in the disease — his Proteus fluorescens. Vincent (1893) in a case of icterus with fever, which ended fatally in forty-eight hours, obtained cultures of Bacillus coli commuiiis from the blood and various organs. BACTERIA IN INFECTIOUS DISEASES. 595 INFLUENZA. Epidemic influenza (" la grippe") is due to infection by the Bacil- lus of influenza (No. 52). INFLUENZA OF HORSES. Dieudonne (1892), in an epidemic of influenza among horses, found in the nasal secretions of infected animals a micrococcus resembling that of crqup- ous pneumonia in man. He did not succeed in cultivating this micrococcus in nutrient gelatin. Schutz (1888) had previously cultivated a streptococcus from the lymphatic glands of horses suffering from epidemic influenza (Druse des Pferdes) which he believes to be the specific infectious agent in this dis- ease (see Streptococcus coryzaa contagiosse equorum, No. 33). INSECTS, INFECTIOUS DISEASES OF. The infectious disease of bees known as "foul brood" is due to Bacillus alvei (No. 14€). Pebrine, an infectious disease of silkworms, is due to in- fection by " Nosema bombycis" (No. 25). Another infectious disease of silk- worms (la flacherie) is believed by Bechamp to be due to infection by Strepto- coccus bombycis (No. 24). v. Tubeuf (1892) has obtained from infected caterpillars of Liparis monacha a motile bacillus — Bacillus monachae (No. 178). An infectious disease of the "chinch bug" (Blissus leucopterus) is be- lieved to be due to Micrococcus insectorum (No. 177). KERATITIS. Bach (1895) as the result of his investigations arrives at the con- clusion that Ulcus corn 86 serpens is due to invasion of the cornea by microorganisms, and that such invasion is almost always secondary to a traumatism, with loss of substance. The most common infec- tious agents are the pyogenic staphylococci and Streptococcus pyo- genes, but certain other bacteria are occasionally concerned in the localized infectious process. The researches of Gasparrini, Bassi (1893), Cuenod (1895), and others indicate that the "diplococcus pneumonia" is not infrequently concerned in the etiology of puru- lent keratitis, and this is confirmed by the researches of Uhthoff (1895). The last-named author investigated 50 cases of purulent keratitis in man with the following result : 35 were cases of typical ulcus corna3 ; 2 of hypopyonkeratitis, not of a serpiginous character ; 3 of keratoma lacia; and 4 of panophthalmia following corneal infec- tion. In 24 cases of typical ulcus cornse serpens the diplococcus of pneumonia was found alone, also in 2 cases of panophthalmia ; in 7 cases the pneumonia coccus was found in association with other microorganisms — 4 of these were cases of ulcus cornaB serpens; in 13 cases, 4 of which were typical ulcus cornse, the pneumonia coccus was not found, but staphylococci or other bacteria were present ; in 3 cases of keratomalacia streptococci were found. Loeb (1891) ob- 596 BACTERIA IN INFECTIOUS DISEASES. tained from a case of keratomalacia a capsule bacillus resembling that of Pfeiffer, wbich was pathogenic for mice and for guinea-pigs. The bacillus of Friedlander has also been found by Etienue and by Yerson and Gabrielides (1894) in "ulcus comas septicum." LEPROSY. No satisfactory experimental demonstration that the Bacillus leprae is the cause of the disease with which it is associated has yet been made ; but there is very little doubt among bacteriologists and pathologists that such is the case. For the facts relating to its pres- ence in leprous tissues, its morphology, etc., the reader is referred to the descriptive account of Bacillus leprse (No. 53, page 394). LEUCOCYTH^KMIA. Pawlowsky (1892) in four cases of leucocythsemia found i» the blood a few short bacilli, with round ends, which showed polar staining (with methylene blue solution). He did not succeed in cultivating them in the usual media, but in a mixture of bouillon and blood serum a granular de- posit was seen at the end of four days, and transplantation from this to gly- cerin-agar (plates) gave colonies, at 37° C., in three or four days. These were small, round, and of a grayish-yellow color. Inoculations in rabbits gave a negative result. LUPUS. Due to infection by Bacillus tuberculosis (No. 53). MADURA FOOT. Le Dantec (1894) arrives at the conclusion that the variety of madura foot in which the characteristic masses are black is probably due to a bacillus found by him in these " grains." This bacillus differs from the streptothnx previously described by Vincent, and supposed by him to be the cause of the malady. It is difficult of cultivation, and inoculation experiments in rab- bits and guinea-pigs gave a negative result. LYMPHANGITIS. Lymphangitis of the extremities, according to Verneuil and Clado, is due to infection by Streptococcus pyogenes. Fiscner and Levy (1893) as a result of their investigations arrive at a different conclusion. In 8 cases tlu-y found Staphylococcus pyogenes albus in 5, Staphylococcus pyogenes am VMS in 1, Bacillus coli communis in 1, Staphylococcus pyogenes aureus and Staphylococcus pyogenes albus associated in 1. In abscesses following lym- phangitis (8) Staphylococcus pyogenes albus was found in 4, Streptococcus pyogenes in 2, Staphylococcus pyogenes albus and Staphylococcus pyogrm s aureus associated in 1 ; Staphylococcus pyogenes albus and Streptococcus pyogenes in 1. MALARIA. Klebs and Tommasi-Crudeli, as a result of researches made by then i in the vicinity of Rome (18/9), announced the discovery of a bacillus which they supposed to be the cause of malarial fevers— their Bacillus malariae. BACTERIA IN INFECTIOUS DISEASES. 597 The writer repeated their experiments the following year (1880) in the vicin- ity of New Orleans, and reported as follows : "Among1 the organisms found upon the surface of swamp mud near New Orleans, and in the gutters within the city limits, are some which closely resemble, and perhaps are identical with, the Bacillus malarias of Klebs and Tommasi-Crudeli; but there is no satisfactory evidence that these or any of the other bacterial organisms found in such situations, when in- jected beneath the skin of a rabbit, give rise to a malarial fever corre- sponding with the ordinary paludal fevers to which man is subject. " The evidence upon which Klebs and Tommasi-Crudeli have based their claim of the discovery of a Bacillus malarise cannot be accepted as sufficient; (a) because in their experiments and in my own the temperature curve in the rabbits experimented upon has in no case exhibited a marked and dis- tinctive paroxysmal character ; (6) because healthy rabbits sometimes exhi- bit diurnal variations of temperature (resulting apparently from changes in the external temperature) as marked as those shown in their charts ; (c) be- cause changes in the spleen such as they describe are not evidence of death from malarial fever, inasmuch as similar changes occur in the spleens of rabbits dead from septicaemia produced by the subcutaneous injection of human saliva; (d) because the presence of dark-colored pigment in the spleen of a rabbit cannot be taken as evidence of death from malarial fever, inasmuch as this is frequently found in the spleens of septicaemic rabbits." Later researches have also failed to confirm the supposed discovery of Klebs and Tommasi-Crudeli, and it is now generally admitted that there is no satisfactory evidence in favor of the view that microorganisms of this class are concerned in the etiology of the malarial fevers. On the other hand, we have now very extended observations which indicate that the blood parasite discovered by Laveran (1881) in the blood of patients suffering from various forms of malarial fever bears an etiological relation to fevers of this class. This haematozodn belongs to quite a different class of microorgan- isms. It was first described by Laveran as the Oscillaria malariee, but is more frequently spoken of at present as the Plasmodium malarias. MALTA FEVER. In twelve out of thirteen cases of "Malta fever "Bruce (1892) found a micrococcus which he believes to be the cause of this fever. See Micrococ- cus of Bruce (No. 179). MALIGNANT CEDEMA. See Bacillus oedematis maligni (No. 186). MASTITIS. In ten cases of puerperal mastitis Bumm (1886) found Staphy- lococcus pyogenes aureus in seven and Streptococcus pyogenes in three. In a case reported by Sarpert (1894) diplococci were found corresponding in their morphological characters with the gonococcus —the patient was suffering from gonorrhoea. MASTITIS IN COWS. Bovine mastitis is usually due to infection by streptococci, which are not always the same, although possibly varieties of the same species. See Strep- tococcus of Nocard and Mollereau (No. 31), Micrococcus of Kitt (No. 21), Streptococcus agalactia; coiitagiosae (No. 45). Streptococcus mastitis spor- adise (No. 45). See also Bacilli of G-uillebeau (No. 180). 598 BACTERIA IN INFECTIOUS DISEASES. Lucet (1891) in twenty-two cows suffering from mastitis obtained in twelve cases a motile bacillus, from 1 to 2 p long, which did not liquefy gelatin and caused a development of gas in culture media (Bacillus coli communis ?). MASTITIS IN SHEEP. See Micrococcus of gangrenous mastitis in sheep (No. 30). MEASLES. The etiology of measles and of the specific eruptive febrile diseases gener- ally still remains unsettled. The occasional presence of micrococci in the blood of patients with measles, which has been noted, is without doubt due to a secondary or mixed infection by one of the common pyogenic micro- cocci. In pneumonia occurring during the course of an attack of measles the Micrococcus pneumonia? crouposae is usually found in the pulmonary exudate. No great importance can be attached to the observations made, with ref- erence to the presence of microorganisms in this disease, prior to the intro- duction of Koch's plate method and the use of solid culture media for the differentiation of bacteria similar in their morphology. In 1892 Canon and Pielicke, of Berlin, announced the discovery of a minute bacillus in the blood of patients (fourteen) with measles, but their discovery, so far as the writer knows, has not been confirmed by more recent investigations. See Bacillus of Canon and Pielicke (No. 157). MENINGITIS. See Cerebro-spinal Meningitis. MICE, INFECTIOUS DISEASES OF. See Bacillus typhi murium (No. 84) and Bacillus of Laser (No. 83) ; also Bacillus erysipelatos suis (No. 67), which appears to be identical with Koch's Bacillus of septicaemia in mice ; also Bacillus murisepticus pleomorphus (No. 98). NEPHRITIS. The various microorganisms which have occasionally been found in the urine of cases of chronic nephritis are probably not directly related to the renal disease. Numerous observations are on record which snow that patho- genic microorganisms present in the blood or tissues may find their way into the urine during the course of the acute infectious diseases. In these cases it is probable that the passage of bacteria into the urine depends upon struc- tural changes in the kidneys, due to the presence of pathogenic bacteria or to the action of their toxic products. Pernice and Scagliosi (1894) have stud- ied the development of nephritis in guinea-pigs, dogs, and white mice, into which they injected bouillon cultures of various pathogenic bacteria — An- thrax bacillus, Bacillus pyocyaneus, Staphylococcus pyogenes aureus, Ba- cillus prodigiosus. They also injected filtered cultures of these bacilli. As a result of their experiments they conclude that the appearance of microor- ganisms in the urine in acute infectious diseases depends upon pathological anatomical changes in the kidneys, which may result either from the jm s ence of the bacteria or from the action of their toxic products. Pathogenic bacteria are not infrequently found in the urine in the acute infectious dis- eases of man — e.g., typhoid fever, pneumonia, septicaemia ; and in certain cases of mixed infection bacteria may be found in the urine which have no BACTERIA IN INFECTIOUS DISEASES. 599 direct etiological relation to the specific infectious disease from which the pa- tient is suffering — e.gr., staphylococci in cases of measles, or streptococci in cases of diphtheria. Ascending nephritis is an infectious process, usually due to Bacillus coli communis (See pyelonephritis). Letzerich (1887) has described a form of nephritis which he ascribes to a bacillus found by him in the urine and in sections of the kidneys of rabbits inoculated with a culture of this bacillus. Lustgarten arid Manneberg (1887) in three cases of acute Bright's disease found streptococci in the urine, which they suppose to have had an etiologi- cal relation to the renal disease. The following year Manneberg reported eleven additional cases, in eight of which he found the same streptococcus,- which he believes to be different from Streptococcus pyogenes, but this can- not be considered as established. Nor has he shown that the streptococcus obtained by him from the urine was present in the kidneys of his patients, or that pure cultures of this streptococcus produce acute nephritis when in- oculated into lower animals. OPHTHALMIA. Although various pathogenic bacteria are frequently found in healthy eyes, there can be no doubt that acute and chronic inflamma- tions here, as elsewhere, are commonly due to the presence of micro- organisms. In gonorrhoeal ophthalmia the " gonococcus " of Neisser is the infectious agent. According to Fuchs (1894) a considerable proportion of the cases of so-called Egyptian ophthalmia are due to infection by the gonococcus, while in another group of cases the in- fectious agent is the bacillus of Koch and Kartulis (No. 138). Cer- tain cases are also due to a mixed infection resulting from the pres- ence of both of these pathogenic microorganisms. Demetriades (1894) says that the gonococcus found in cases of Egyptian ophthalmia is much smaller than that encountered in cases of gonorrhoea ; but that it is the same was demonstrated by Kartulis, who introduced pus from the eye of a patient, containing this coccus, into the urethra of a native, who developed a typical gonorrhoea at the end of twenty- four hours as a result of the inoculation. Perles (1895) has made numerous inoculation experiments in the eyes of rabbits and reports the following results : Pure cultures of Bacillus subtilis, of the cholera spirillum, and of various non-patho- genic saprophytes, introduced into the anterior chamber or the vit- reous, caused no perceptible changes. Typhoid bacilli introduced into the anterior chamber caused hypopyon and in the vitreous an abscess. Streptococci gave rise to an exudate in the anterior cham- ber and to pus formation in the vitreous. Diphtheria bacilli caused a purulent exudate in the anterior chamber with a moderate kerato- iritis, and abscess formation when introduced into the vitreous. Friedlander's bacillus, in the vitreous, caused a severe panophthal- mitis, which led to rupture of the eyeball at the end of sixteen hours ; 600 BACTERIA IN INFECTIOUS DISEASES. in the anterior* chamber the result was similar, but not so rapidly induced. No infection occurs through the uninjured conjunctiva. When the pneumonia coccus was introduced into the eye of a rabbit general infection and death from septica3mia quickly followed. Ac- cording to Gasparrini the micrococcus of pneumonia is found in the conjunctival sac in a large proportion of healthy eyes — in 8 out of lo of the 100 students examined by him. When injected into the vit- reous or anterior chamber, in rabbits, fresh cultures gave rise to a .panophthalmia, and cultures four or five days old to a plastic iritis or to atrophy of the eye from a chronic infectious process. In cases of kerato-hypopyon in man (21) and of panophthalmia (4) this mi- crococcus was found, and in six of the first-mentioned cases it was so virulent that it killed rabbits, when injected subcutaneously, in from twenty-two to thirty-six hours. In seven other cases it was found in pure culture, but proved not to be virulent — i.e., did not kill rabbits. In eight cases it was associated with staphylococci. Sattler, in a case of panophthalmia resulting from injury by a splinter of wood, ob- tained cultures of Bacillus pyocyaneus; Randolph reports a case caused by Bacillus coli communis; Wagenmann states that in most cases in which purulent infiltration of the vitreous follows a perfora- ting wound of the eye the ordinary pus cocci are found. In the me- tastatic eye affections occurring in the course of puerperal septi- ca3mia, Herrnheiser (1892) obtained in two cases (of retinitis septica) very virulent cultures of Streptococcus pyogenes and in one a culture of Staphlyococcus pyogenes aureus. In a case of metastatic pan- ophthalmia, occurring in a man aged sixty-seven, after an attack of pneumonia, the micrococcus of pneumonia was found. Accord- ing to Axenfeld (1894) the last-mentioned microorganism is a fre- quent cause of purulent metastatic ophthalmia. OSTEOMYELITIS AND PERIOSTITIS. The evidence with reference to the presence of Staphylococcus pyogenes aureus in acute osteomyelitis and its probable etiological relation to the cases in which it is found, is given in the article de- scriptive of this microorganism; but the researches of Kraske (lSs»>) and of Lannelongue and Achard (1890) show that the "golden sta- phylococcus " is not always found in osteomyelitis. The last-named investigators, in a series of thirteen cases, found Staphylococcus py< >- genes aureus in four only, and in one of these Staphylococcus pyo- genes albus was also present; in three cases Staphylococcus pyogem s albus was obtained in pure cultures; in two cases it was associated with Streptococcus pyogenes; and in two cases a streptococcus ^ as found which resembled Streptococcus pyogenes and yet differed from BACTERIA IN INFECTIOUS DISEASES. 601 it in some particulars. The same bacteriologists found the pneu- monia coccus in two cases in children. Fisher and Levy (1893) also report two cases, in children, in which this micrococcus was found — one a fatal case of meningitis. In two other cases streptococcus pyogenes was obtained in pure cultures. The typhoid bacillus has also been found by several investigators — Ebermayer, Orion0, Colzi, Ullmann. It is therefore evident that osteomyelitis cannot be con- sidered a specific affection; on the other hand, as in abscesses de- veloped in the cellular tissue, in glands, or in the various organs, it is to be regarded as a localized infectious process which may be induced by various pathogenic microorganisms which through some channel have effected a lodgment in the blood or tissues of the body. The exciting cause of a peri osteal inflammation is, no doubt, not in- frequently an injury of some kind. Chronic periostitis and osteo- myelitis are developed in a similar way as a result of a localized tubercular infection. OTITIS MEDIA. In otitis media various microorganisms have been found in pus obtained by paracentesis of the tympanum, as well as in the chronic discharge after perforation ; and there can be but little doubt that these microorganisms are responsible, directly or indirectly, for the inflammatory process and pus formation. The following species are most frequently found in the purulent discharge in recent cases of otitis media : Micrococcus pneumonia crouposa3 (" Diplococcus pneu- moniaa "), Streptococcus pyogenes, Staphylococcus pyogenes albus, Staphylococcus pyogenes aureus, Friedlander's bacillus. The fol- lowing have also been found occasionally: Staphylococcus tenuis, Bacillus tenuis, Micrococcus tetragenus, Bacillus pyocyaneus. According to Zaufal, Micrococcus pneumonia crouposa3 is most frequently found in cases which result from exposure to cold, while the ordinary pus cocci are more frequently found in otitis which is secondary to specific febrile diseases. Martha (1892) reports two cases in which Bacillus pyocyaneus was present in pure culture — in fifty-one other cases examined this bacillus was not found. This bacillus has also been found occasion- ally by other investigators— Pes and Gradenigo (1894), Hartmann (1894), Kossel (1894). Scheibe (1892) in sixteen cases of mastoid abscess following mid- dle-ear disease found the micrococcus of pneumonia in six, Strepto- coccus pyogenes in five, Staphylococci in one, and an undetermined micrococcus in one. Stern (1895) in thirty cases of chronic purulent otitis media made bacteriological examinations with the following result : Staphylococ- 42 602 BACTERIA IN INFECTIOUS DISEASES. cus pyogenes albus was found in six, Staphylococcus pyogenes aureus in two, Streptococcus pyogenes in three, Bacillus coli communis in one, and various bacilli, vibrios, and cocci in the remaining cases; some of these were fluorescent, some produced a foetid odor, etc. OZ^ENA. The researches of Thost, Klamann, Hajek, and others show that Friedlander's bacillus is present in the nasal secretions in a consider- able proportion of the cases of ozsena, but its etiological relation to the morbid condition which gives rise to the offensive discharge has not been established. Thost found this bacillus in twelve out of seventeen cases studied by him, and frequently almost in a pure culture; but he also found it in rhinitis from syphilitic ulceration, from polypus, and in simple coryza. Hajek found Friedlander's bacillus in seven out of ten cases stud- ied by him, but it was associated with various other species of bac- teria, and especially with the pyogenic micrococci and with Bacillus fluorescens liquefaciens. He also obtained almost constantly his Bacillus fcetidus ozsense (No. Ill), which appears to have been the cause of the foetid odor of the nasal discharge. Marsano (1890) in ten cases of ozsena found a capsule bacillus in the nasal secretions which closely resembles Friedlander's bacillus, but which he believes not to be identical with it. Abel (1893) in sixteen cases of " oza3na simplex " found a similar capsule bacillus, but he arrives at the conclusion that it is not iden- tical with Friedlander's bacillus, and believes it to be the specific cause of rhinitis atrophicans fcetida. According to Abel his bacillus resembles Pfeiffer's capsule bacillus^ (No. 80) more closely than it does that of Friedlander, and it is almost identical with the capsule bacillus of Fasching (No. 150). This bacillus is described by Abel under the name of Bacillus mucosus Ozsena?. It is said to be differ- entiated from the bacillus of rhinoscleroma and Bacillus sputigenus crassus of Kreibohm, by the fact that it does not stain by Gram's method. Strazza (1893) in twenty-five cases examined found a capsule bacillus constantly associated with streptococci and staphylococci. This bacillus was not found in cases of rhinitis chronica simplex or of rhinitis syphilitica. According to Strazza, also, this bacillus is differentiated from the bacillus of rhinoscleroma bjr the fact that it does not stain by Gram's method ; it is said to resemble Pfeiffer's bacillus in cultures, but to be somewhat smaller. Loewenberg (1894) has called attention to the fact that he re- BACTERIA IN INFECTIOUS DISEASES. 603 ported in 1881 that he had always found in oza3na a microbe sur- rounded by a capsule and not staining by Gram's method. He now says that this microbe corresponds with Friedlander's bacillus in form and staining reactions, but differs from it in other particulars as follows : It shows a very scanty development in milk and causes no perceptible change in this medium. Friedlander's bacillus, on the other hand, coagulates milk and its growth is often attended with an evolution of gas. The two bacilli also give rise to different odors. The bacillus of Friedlander gives off from gelatin and bouillon cul- tures an odor of trimethylamine and old gelatin plates give off the odor of old cheese. Cultures of the bacillus of ozaBna, on the con- trary, give off an agreeable odor. The offensive odor characteristic of oza3na is never given off from cultures of this bacillus. Loewen- berg concludes from his investigations that the microbe of ozsena is specifically distinct from the bacillus of Friendlander and that it bears an etiological relation to this disease. PANARITIUM. According- to Saint-Sevrin (1894) panaritium (Panaris des pecheurs) is very common among the fisherman of the island of Newfoundland and of the North Sea. His researches lead him to conclude that it is due to infection by a micrococcus which produces a red pigment (microbe rouge de la sardine) in association with an anaerobic bacillus. The coccus is from 0.5 to 0.6 /* in diameter and is usually seen in pairs ; it liquefies gelatin and produces a carmine-red pigment. It is probable that the common pus cocci are usually concerned in the etiology of "felons." In a case reported by Huber Staphylococcus pyogenes albus was obtained in pure culture from the pus of a panaritium and also in blood obtained from a finger of the opposite hand. Bernheim obtained the colon bacillus from the pus of a panaritium developed in the course of an attack of typhoid fever. PAROTITIS. No demonstration of a specific microorganism in mumps has been made, but in non-specific, suppurative parotitis one or other of the pyogenic micro- cocci appears to be the cause of the inflammation and pus formation. In parotitis occurring as a complication of pneumonia Micrococcus pneumonias crouposse has been found as the only microorganism in pus from the inflamed gland (Testi, Duplay). Letzerich in 1895 made a preliminary communication in which he claimed to have discovered microorganisms in the blood of patients with mumps. These he describes as "large, round spores;" no bacilli were found. In a fatal case of typhoid fever in which a suppurative inflammation of the parotid gland was found, Janowski (1895) obtained a pure culture of Ba- cillus typhi abdominalis from the pus of the parotid abscess. PEMPHIGUS. Demme (1886) has cultivated a diplococcus from a case of acute pemphi- gus which possibly is related to this disease (see Micrococcus of Demme, No. 27, page 331). The same coccus was found by Dahnhardt in a similar case. 604 BACTERIA IN INFECTIOUS DISEASES. Strelitz (1892) obtained in cultures from pemphigus vesicles a micrococcus which corresponded in every respect with Staphylococcus pyogenes aureus. Inoculation of this micrococcus in his own arm caused the development of typical pemphigus bullae. PERICARDITIS. Pericarditis is a localized infectious process due to various patho- genic microorganisms. In two cases reported by Barbacci (1892) the micrococcus of pneumonia was found to be the infectious agent. Paviot (1894) reports a fatal case of purulent pericarditis in which a diplococcus was found resembling Friedlander's bacillus. Ernst (1893) obtained from the pericardial sac, in a case in which the tu- bercle bacillus was also present, a variety of Bacillus pyocyaneus. In " uraemic pericarditis " Banti failed, in four cases, to find any microorganism in fluid obtained from the pericardial sac. In pericarditis occurring in general septica3mic infection the microorganism to which the general infection is due will probably be found in the pericardial sac, and when it occurs as a complication of one of the specific infectious diseases in which bacteria are usually not found in the blood — eruptive fevers — it is probably due to a mixed infection with one of the common pus cocci. In chronic tubercular pericarditis the tubercle bacillus is the infectious agent. PERITONITIS. That peritonitis usually results from the presence of microorgan- isms in the cavity of the abdomen seems to be well established by ex- perimental evidence and by bacteriological researches in cases of this disease. Mechanical irritants, like finely powdered glass (writer's experiments) , introduced into the cavity of the abdomen of rabbits, do not cause peritonitis unless microorganisms are introduced at the same time ; the minute fragments of glass become encysted and the animal remains in good health. But Pernice has shown that peri- tonitis may be induced in rabbits and in guinea-pigs by injecting into the cavity of the abdomen various chemical substances, such as concentrated mineral acids, acetic acid, phenol, nitrate of silver, etc. It is also demonstrated by numerous experiments that pure cultures of various bacteria injected into the cavity of the abdomen of the animals mentioned may produce a fibrinous or a purulent peritonitis. Among these is the Bacillus coli communis, which is constantly present in the intestine of healthy persons; and in peritonitis follow- ing perforation of the bowels this bacillus is responsible, in part at least, for the intense peritoneal inflammation which so quickly occurs. In puerperal peritonitis the pus cocci, and especially Streptococcus pyogenes, appear to be the usual cause of the inflammatory process. BACTERIA IN INFECTIOUS DISEASES. 605 Weichselbaum has observed two cases of primary peritonitis and pleuritis apparently induced by Micrococcus pneumonias crou posse, as this microorganism was found in the exudate into the peritoneal cavity. The same author, in a case of peritonitis resulting from rupture of the spleen in the course of typhoid fever, obtained a pure culture of the typhoid bacillus from the peritoneal cavity. The re- sults of A. FrankePs researches (1891) are as follows : In thirty-one cases examined pure cultures were obtained in twenty, viz. : Bacil- lus coli communis, nine times; streptococci, seven times; Bacillus lactis aerogenes, twice; Micrococcus pneumonia crouposas, once; Staphylococcus pyogenes aureus, once. In three cases Bacillus coli communis was present in association with other bacilli, and in four cases the bacteriological examination gave a negative result. Frankel has also shown that pure cultures of Bacillus coli com- munis injected into the cavity of the abdomen of rabbits cause a typical peritonitis. The present writer has frequently obtained the same result in experiments made with this bacillus. It would ap- pear, therefore, that the peritonitis which so constantly results from wounds of the intestine is probably due, to a considerable extent, to the introduction of this microorganism from the lumen of the intes- tine, where it is constantly found, into the peritoneal cavity, where the conditions are favorable for its rapid development. Malvoz in 1893 found Bacillus coli communis, for the most part in pure cultures, in five out of seven cases examined by him ; in the other two cases he found Streptococcus pyogenes in one and a bacil- lus which appeared to be identical with Bacillus typhi abdominalis in one. Barbacci (1892) in two cases in which meningitis was also present (in one endocarditis also) found the micrococcus of pneu- monia in pure cultures. Le Gendre (1895) has reported a case in which the same microorganism was alone present, and states that in a search of the literature he finds eleven recorded cases due to this micrococcus; of these eight terminated fatally. Tavel and Lanz (1893) in a series of seventy-two cases examined found bacteria re- sembling the colon bacillus in thirty-one. Flexner (1893) reports a case of peritonitis caused by Proteus vulgaris. Tubercular peri- tonitis is, of course, due to infection by the tubercle bacillus. PLANTS, INFECTIOUS DISEASES OF. The infectious diseases of plants are, for the most part, due to parasitic fungi, but several infectious plant diseases have been shown to depend upon the presence of bacteria in the diseased tissues, and in others this has been claimed by investigators upon more or less satisfactory evidence. The lim- its of the present volume only admit of an enumeration of the most impor- tant of these bacteria : Micrococcus amylovorus (Burrill) is believed to be the cause of "pear blight;" Bacillus sorghi (Kellerman and Swingle) of "sorghum blight;'* 606 BACTERIA IN INFECTIOUS DISEASES. Bacillus hyacinth! septicus (Heinz) of an infectious disease of hyacinths ; Ba- cillus amylobacter of potato rot (Nassfaule) ; Bacillus tracheiphilus (E. F. Smith) of blight in melons and other cucurbitacese. PLEURITIS, The usual infectious agent in acute fibrinous pleurisy accompany- ing pneumonia is Micrococcus pneumonia? crouposa? (No. 8). Net- ter (1892) reports that in 66 cases of genuine fibrinous pleurisy in which he has made bacteriological researches since 1886, he has always found this micrococcus. In cases in which culture experi- ments give a negative result, this is, according to Netter, due to the fact that the micrococci are apt to perish at the crisis of the disease. These cases do not usually result in empyema and run a more favor- able course than those in which the pus cocci are present. Prudden (1893) in 21 cases of sero-fibrinous pleurisy failed to find any bac- teria in the exudate in 1*2, and found the micrococcus of pneumonia in 2 only. Lemoine (1895) also reports that in 28 cases, out of 32 examined by him, the exudate was entirely sterile; in 4 cases he found Staphylococcus pyogenes albus. The remaining cases were of tubercular origin. Levy (1895), in reviewing the literature of the subject, arrives at the conclusion that the micrococcus of pneumonia is the usual cause of pleurisy in children and of metapneumonic pleurisy, but that in metastatic, pysemic, pleuritic inflammation streptococci or staphylo- cocci are the usual infectious agents. Pleurisy due to streptococcus or staphlyococcus infection is not in all cases attended with pus for- mation ; the exudate, in a certain proportion of the cases, may remain serous (Levy, Ludwig, Goldschneider) . The micrococcus of pneumonia was found by Jakowski (1892) in 21 out of 34 cases in which pure cultures were obtained ; of the re- maining cases streptococci were found in 10, Staphylococcus pyogenes aureus in 1, and the tubercle bacillus in 2. In 14 cases of mixed infection Staphylococcus pyogenes aureus and albus were found in 6, Friedlander's bacillus and streptococci in 1; the micrococcus of pneumonia with streptococci in 1, with Staphylococcus pyogenes albus in 2, and with Staphylococcus pyogenes aureus in 1. In 7 cases no bacteria were found. According to Jakowski those cases in which no bacteria are obtained in cultures are usually due to tubercular infection. Goldschneider (1892) reports 4 cases of serous pleurisy, in 3 of which he found Streptococcus pyogenes and in 1 Staphylococcus pyogenes aureus. Bordoni-Uffreduzzi (1895) has re- ported a case of double pleurisy in a girl, aged twelve, who was assaulted by an individual with gonorrhoea, in which the gonococ- cus was the only microorganism present in the pleural exudate. BACTERIA IN INFECTIOUS DISEASES. 60? In pleurisy occurring as a complication of typhoid fever the ty- phoid bacillus has been found in the exudate (sometimes serous and sometimes purulent) by several bacteriologists. Bacillus coli com- munis has also been found (Albarran and Halle). According to the statistics of Flemming about 41 per cent of the fatal cases of pleurisy (424 cases examined) are due to tubercular infection. PLEURO-PNEUMONIA OF CATTLE. The evidence appears to be satisfactory that this infectious disease is due to the Pneumobacillus liquefaciens bovis of Arloing (No. 120). PLEURO-PNEUMONIA (SEPTIC) OF CALVES. An infectious disease of calves, described by Galtier as a septic pleuro- pneumonia, or pneumo-enteritis, is apparently due to the Pneumobacillus septicus (No. 182) of the author named. PNEUMONIA. The usual infectious agent in croupous pneumonia is Micrococcus pneumonise crouposa3 (No. 8). Friedlander's bacillus and other mi- croorganisms have been found in a comparatively small proportion of the cases ; but it is probable that some of these at least were due to a mixed infection and that the specific infectious agent was over- looked. (See also Broncho-pneumonia.) PNEUMONIA IN HORSES. See Diplococcus of pneumonia in horses, of Schutz, No. 32. PNEUMO-ENTERITIS OF SWINE. See Swine plague. PSEUDO-LEUKEMIA. Various microorganisms have been found in connection with pseudo-leu- kaemia, but no one of these has been shown to bear a specific etiological re- lation to the disease. In certain cases diagnosed as pseudo-leukaemia tuber- cular infection of the lymphatic glands and spleen has been found at the autopsy (Weishaupt). In a case reported by Verdelli (1891) the ordinary pyogenic micrococci were obtained from the blood and lymphatic glands. Gabbi and Barbacci (1892) report a case in which a virulent variety of the colon bacillus was obtained from blood drawn from a finger, and from the spleen and lymphatic glands after death. In another case no microorgan- isms could be found. Traversa (1893) obtained a streptococcus (probably Streptococcus pyogenes) in pure cultures from the blood, in a case which came under his observation. Grossi (1893), in a case in which he had an opportunity to make a post-mortem examination, failed to find any microor- ganisms in the blood, the lymphatic glands, or in serum from the cedematous tissues. 608 BACTERIA IN INFECTIOUS DISEASES. PSEUDO-TUBERCULOSIS. Preisz (1894) has compared the bacillus of pseudo-tuberculosis described by Npcard with that of Pfeiffer, of Parietti, and of Zagari, and finds them identical (see Bacillus pseudo-tuberculosis, No. 121). A different bacillus of pseudo-tuberculosis was obtained by Preisz from an infected sheep (No. 183). Kutscher (1894) has described a bacillus which produces a pseudo-tubercu- losis in mice, under the name Bacillus pseudo-tubercularis murium (No. 184). Eberth (1886) has described a bacillus of pseudo-tuberculosis in rabbits which appears to be identical with the microorganism of ** zooglcea-tuberculosis " of Malasses, Vignal, and Chantemesse. PUERPERAL FEVER. Puerperal fever is usually due to infection by Streptococcus pyo- genes (No. 5), and in fatal cases this microorganism is almost always found. In a few fatal cases Staphylococcus aureus has been found in pure cultures (Brieger, Fehling, Doderlein, and others), and in non-fatal cases of a comparatively mild character staphylococci are not infrequently the cause of the infection and accompanying fever. Doderlein has reported an epidemic, of limited extent, in which there was a mixed infection by streptococci and staphylococci. Sanger, Kronig (1893), and others have reported cases in which fever, devel- oped during the puerperium, was apparently due to the presence of gonococci in the uterus. Von Frangue (1893) and Eisenhart (1884) have reported cases in which Bacillus coli communis was the infectious agent. PURPURA H^EMORRHAGICA. See account of bacilli found in purpura ha3morrhagica by Babes (No. 146), Kolb (No. 147), and Tizzoni and Giovannini (No. 145). PYAEMIA. Pathologists at the present day include all cases of general blood infection under the name septicaemia, and localized infections have special names — e.g., mastitis, pleuritis, peritonitis, metastatic abscess, etc. When the toxic products of pathogenic bacteria are absorbed from the surface of a suppurat- ing wound, from an abscess cavity, or from any localized focus of infection, we have a septic toxaemia, which manifests itself by more or less fever, and by various symptoms connected with the nervous system, etc. The use of these terms in the sense indicated seems to do away with the necessity for using the old term pyaemia. PYELONEPHRITIS. In ascending nephritis or pyelonephritis, which is very commonly secondary to a cystitis of long standing, there is good reason to be- lieve that the inflammatory changes and pus formation depend upon the presence of certain bacteria which are found in the urine of such BACTERIA IN INFECTIOUS DISEASES. 609 patients during life and in the diseased kidney removed by surgical operation or post-mortem. And recent researches show that the Ba- cillus coli communis, which is constantly present in the intestine of healthy individuals, is found more frequently than any other micro- organism in the so-called "surgical kidney." The most important and comprehensive work upon the bacteriology of pyelonephritis is that of Schmidt and Aschoff, published in Jena in 1893. The authors named give a complete resume of the literature of the subject, and a full report of fourteen cases of pyelonephritis, in which they have made bacteriological investigations. They also report a series of experiments upon rabbits, in which injections of a pure culture of the Bacillus coli com- munis were made into the left ureter, after tying it below the point of in- jection. The ligature was removed after the injection had been made, and the wound in the abdominal wall, which had been made with antiseptic pre- cautions, was closed. Some of the animals so treated died in from twelve hours to four or five days, while others survived and were killed on the sev- enth and ninth day. The left kidney, especially in the cases which survived the operation for several days, was found to be two or three times as large as the right and to present all the evidences of parenchymatous inflammation. The pelvis of the kidney contained more or less ammoniacal urine, pus, and bacilli ; the parenchyma gave evidence of diffuse inflammation and contained numerous bacilli. As a rule, a pure culture of the Bacillus coli communis was obtained from the inflamed kidney. A similar experiment was made with a species of proteus (vulgaris ?), and with a similar result. The animal died at the end of two days. The left kid- ney was twice as large as the right, the surface of a deep-red color dotted with numerous white spots ; the parenchyma had a striped appearance on section and a greenish color in the vicinity of the pelvis, which contained ammoniacal and bloody urine. A putrefactive odor was given off from the or- gan. Proteus in pure culture was recovered from the interior of the kidnev. Some of the earlier observers have described non-liquefying bacteria ob- tained from the bladder in cases of chronic cystitis or of pyelonephritis fol- lowing cystitis, which, according to Schmidt and Aschoff, correspond in morphological and biological characters with Bacillus coli communis, and were no doubt identical with it. They believe that the bacillus described by Clado (1887) under the name of " Bacterie septique," and subsequently found byAlbarran and Halle in forty-seven out of fifty cases ^ of cystitis (fifteen times in pure culture), and called by them "Bacille pyogene," is in fact the Bacillus coli communis. Schmidt and Aschoff say that the changes found in the kidneys of rabbits after the injection of Bacillus coli communis into the ligated ureter correspond with those seen in the "surgical kidney" of man. They were surprised at the rapidity with which the bacilli penetrated the urinary tubules. The first changes in the parenchyma of the organ occurred at the end of thirty-six hours, and at the end of five to seven days these changes had reached their extreme development. They evidently depended upon the invasion of the urinary tubules by bacilli. This conclusion corresponds with that reached in previous researches by Albarran, Achard and Renault, and by Krogius. No doubt cystitis and ascending pyelonephritis are usually caused by microorganisms introduced through the urethra into a bladder which is ren- dered susceptible to infection by mechanical violence or chemical irritation. The most frequent cause of such local infection is the Bacillus coli communis, which is constantly present in the intestine and upon the external surface in the vicinity of the anus, from whence it may easily be transported to the in- terior of the bladder by catheters, etc. , used by the patients themselves or by their medical attendants. 610 BACTERIA IN INFECTIOUS DISEASES. PYOSALPINX. The researches of Zweifel (1892) show that a certain proportion of the cases of pyosalpinx are due to the presence of the gonococcus; in other cases the infectious agent is Streptococcus pyogenes, and in a few cases the micrococcus of pneumonia has been found in pus from the tubes removed by operation. In seventy-one cases of pyosalpinx or of salpingo-oophoritis, which were examined by Zweifel after oper- ation, the gonococcus was found eight times and streptococci three times, while in one the micrococcus of pneumonia was present. Menge (1892) found gonococci, and no other microorganisms, in three cases. Zweifel believes that in many cases in which the gonococcus is not found it was the infectious agent to which the in- flammation and pus formation was due, but that its presence can only be demonstrated in recent cases, as it soon dies out. RELAPSING FEVER. Due to infection by Spirillum Obermeieri (No. 191). RHEUMATIC FEVER. The symptoms and complications of acute rheumatism indicate that it is an infectious disease, and the researches of bacteriologists give some support to this view. Singer (1895) in seventeen cases investigated found Staphylococcus pyogenes albus in the urine in ten, and in two cases in the blood; in three cases Streptococcus pyo- genes was found in the urine alone and in two cases in association with Staphylococcus pyogenes albus; in one case Staphylococcus pyogenes aureus was obtained from the urine, and in one, compli- cated with cystitis, Bacillus coli communis. According to Singer the microorganisms found were in large numbers during the acute stage of the disease and disappeared when convalescence was established. On the contrary, Chvostek (1895) failed to find microorganisms in the urine in nine out of twelve cases examined by him; in three cases he obtained micrococci — Staphylococcus pyogenes albus in one, Diplococcus ureas in one, and an undetermined coccus in one. The same author reports that in numerous examinations of the contents of the inflamed joints, both in acute and chronic cases, he failed to find bacteria of any kind. Sahli (1892) refers to the uniformly nega- tive results obtained by different bacteriologists who have made cul- tures from fluid obtained from the inflamed joints, and reports a fatal case in which he also failed to obtain cultures from the fluid in the affected joints, but in which Staphylococcus pyogenes citreus was obtained in cultures from the blood, the sy no vial membrane of the BACTERIA IN INFECTIOUS DISEASES. 611 affected joints, the fibrinous exudate upon the pericardium, the endo- cardial vegetations, and the swollen bronchial glands. It is evident that in this case there was a general infection, and it seems probable that the joint inflammation was due to the same microorganism as that of the other tissues involved in the infectious process. It is pos- sible, however, that the so-called complications of acute rheumatism result from a mixed infection. Saint-Germain (1893), in inoculation experiments made with attenuated cultures of staphylococci into the circulation in young animals, was able to produce joint inflamma- tions with effusion of serum into the joint cavity, but this serum proved to be sterile. In cases of subacute or chronic articular rheu- matism Bouchard and Charrin report (1891) that they have fre- quently obtained cultures of Staphylococcus pyogenes albus from fluid drawn from the affected joints. Sacaze (1894) calls attention to the fact that acute rheumatism is sometimes preceded by a lesion through which general infection may have occurred, and he records a case fol- lowing a suppurating wound, from the pus of which Staphylococcus pyogenes albus was obtained in pure culture, also several cases occur- ring in individuals with hypertrophy of the tonsils, in which he supposes that this was the channel of infection. If, as appears probable, acute rheumatism is due to infection by the ordinary pus cocci, we may suppose that this occurs in conse- quence of a loss of the natural immunity which in a normal condi- tion of health protects man from invasion by these micrococci, which are constantly present (especially Staphylococcus pyogenes albus) upon the surface of the body and of mucous membranes. In the writer's work on " Immunity, Protective Inoculations, and Serum- Therapy " (1895) the following conclusion is reached with reference to the explanation of natural immunity : " The experimental evidence submitted considered in connection with the extensive literature relating to "phagocytosis," leads us to the conclusion that natural immunity is due to a germicidal sub- stance present in the blood serum which has its origin (chiefly at least) in the leucocytes and is soluble only in an alkaline medium." Now, in acute rheumatism there is an excess of acid in the system, and it seems quite probable that, as a result of this, the natural im- munity against infection by these micrococci is neutralized. RHINITIS FIBRINOSA. In a considerable number of cases of fibrinous rhinitis, reported by vari- ous authors, the Klebs-Lb'ffler diphtheria bacillus has been demonstrated to be present — Baginsky, Park, Abbott, Stamm, Concetti, Gerber, and Podack (1895), and others. V ery curiously the diphtheritic process, as a rule, does not extend to the fauces, and the disease runs a favorable course, although virulent diphtheria bacilli may be obtained from the fibrinous exudate. 612 BACTERIA IN INFECTIOUS DISEASES. RHINOSCLEROMA. This appears to be a localized infectious process, due to the presence of the Bacillus of rhinoscleroma (No. 58). SCARLET FEVER. The specific infectious agent in scarlet fever has not been demonstrated. In the diphtheritic exudate frequently seen in the angina of scarlet fever a streptococcus is commonly found which appears to be identical with Strep- tococcus pyogenes; and in the secondary affections which occur in the course of this disease or during convalescence, when suppuration occurs, one or the other of the common pyogenic micrococci is usually found and is doubtless the cause of the local inflammatory process. (See Otitis media.) Crajkowski (1895) has reported that he found in fifteen cases, in which he examined the blood of scarlet fever patients, a diplococcus present in com- paratively small numbers — seldom more than one or two in a microscopic field. This diplococcus does not stain by Gram's method and in general stains feebly and quickly loses its color. Cultures were obtained in bouillon and upon solid media (in the incubating oven ?), but not in gelatin. The develop- ment is said to be slow, and the colonies resemble small drops of dew — not more than one-third to one-half of a millimetre in diameter. Pathogenic for mice, but not for rabbits. Crajkowski does not claim that the etiologk-al relation of this diplococcus to scarlet fever has been demonstrated. His dried blood preparations were stained by the method of Chencinsky. SCORBUTIS. In an epidemic of scurvy in a cavalry regiment Babes (1894) found in every case, in the necrotic margin of the mucous membrane of the gums, a slender, pointed, and bent bacillus, resembling the tubercle bacillus ; this did not stain by Gram's method. This bacillus grew in nutrient agar at 37 C., and the cultures injected into rabbits caused a hemorrhagic septicaemia and death. SEPTICAEMIA. Septicaemia in man is usually due to infection, through a wound or mucous membrane denuded of its epithelium, by a virulent variety of one of the common pus cocci — Streptococcus pyogenes (No. 5), or Staphylococcus pyogenes aureus (No. 1). Canon (1894) has reported the results of his bacteriological researches in seventy cases of "septi- caemia, pyaemia, and osteomyelitis." He divides his cases into three groups. In the first group ('20) microorganisms were present in the blood without metastases, in the second (20) microorganisms were present and metastatic foci of infection were found; in the third group there were metastatic foci but no microorganisms were found in the blood. In the first group of cases, in blood obtained post mortem from a vein in the arm, streptococci were found in a ma- jority of the cases, staphylococci in a smaller number, the pneumonia coccus in one, and Bacillus coli communis in one. In this group the blood was examined in seven cases during life and in three of them with a positive result. In eleven cases of various origin, in which BACTERIA IN INFECTIOUS DISEASES. 613 metastatic foci were found, streptococci or staphylococci were usu- ally found in the blood post mortem, and in four out of five cases the blood was examined during life with a positive result. In five cases of osteomyelitis blood examinations showed that Staphylococ- cus pyogenes aureus was usually present. (See also Osteoymelitis.) Petruschky (1894) in his extended researches obtained positive results in seventeen cases in which the blood was examined during life (eight non-fatal cases). Streptococci were found in fourteen of these cases, Staphylococcus pyogenes aureus in two, and Staphylococ- cus pyogenes albus in one. In puerperal septicaemia Streptococcus pyogenes is the usual infectious agent. (See Puerperal Fever.) In gangrenous septicaemia (septicemie gangreneuse or gazeuse of French authors) the bacillus of malignant oedema (No. 186) is the usual infectious agent, but this is a localized infectious process rather than a general blood infection. Certain cases of so-called purpura haemorrhagica are probably due to general blood infection by pathogenic bacilli (see Bacillus of Tizzoni and Giovannini, No. 145, Bacillus of Babes, No. 146, and Ba- cillus of Kolb, No. 147), and von Dungern has described a case of haemorrhagic septica3mia in a new-born child due to infection by a capsule bacillus (No. 164). Septiccemia in cattle (Rinderseuche) is due to infection by Bacil- lus septica3mia3 baemorrhagicae (No. 61), as is also septicaemia in deer (Wildseuche), in swine (Schweineseuche), and in rabbits (Kaninchen- septikamie, Koch) . The same bacillus is the cause of the infectious disease of fowls known as " fowl cholera. ' ' Other bacteria producing septicaemia in fowls are bacillus of Lucet (No. 87) and Bacillus gallinarum of Klein (No. 77). Septiccemia in ducks is caused by the bacillus of Cornil and Toupet (No. 62) ; in geese by Spirillum anserum (No. 192) ; in frogs by Bacillus hydrophilus fuscus (No. 81); in fish by Bacillus piscicidus agilis (No. 167), and Bacillus of Emmerich and Weibel (No. 169) ; in grouse by Bacillus of grouse disease (No. 76), in parrots by Streptococcus perniciosus psittacorum (No. 43) ; in mice by numerous bacteria, including Bacillus erysipel- atos suis (No. 67), Bacillus typhi murium (No. 84), Bacillus of Laser (No. 83), and Bacillus of Mereshkowski (No. 168); in rabbits by very many pathogenic bacteria from various sources including Bacil- lus septicaemia haemorrhagicae (No. 61), Micrococcus pneumoniae crouposae (No. 8), Bacillus anthracis (No. 45), Bacillus cuniculicida Havaniensis (No. 93), Bacillus leporis lethalis (No. 94) ; in swine by Bacillus septicaemiae haemorrhagicae (No. 61), Bacillus of swine plague, Marseilles (No. 65), and Bacillus of hog cholera (No. 63). 614 BACTERIA IN INFECTIOUS DISEASES. SILKWORMS, INFECTIOUS DISEASES OF. See Streptococcus bombycis (No. 24) and Nozema bombycis (No. 25). STOMATITIS. Schimmelbusch (1889), Lingard (1888) and Foote (1893) have described bacilli obtained by them from the necrotic tissues in cases of iioma. The bacillus of Lingard, obtained from five cases, appears to be identical with that of Schimmelbusch (No. 110). In the case reported by Foote the bacilli found differed from the bacillus of Schimmelbusch as shown by the fact that they stained by Gram's method. SYMPTOMATIC ANTHRAX. Symptomatic anthrax ("blackleg," "quarter evil," Ger., Rausch- brand) is due to infection by an anaerobic bacillus (see Bacillus of Symptomatic anthrax, No. 188). SYPHILIS. The etiology of syphilis has not been determined by the researches of bacteriologists. For an account of the microorganisms which have been en- countered in syphilitic lesions the reader is referred to the article on the Bacillus of Lustgarten (No. 55). TETANUS. Due to infection by Bacillus tetani (No. 185). OF CATTLE. Billings (1888) has announced the discovery of a bacillus in the blood of cattle suffering from Texas fever, which he supposed to be the cause of this disease, but the investigations of other bacteriologists have failed to confirm the alleged discovery. It appears probable that a mistake in diagnosis was made, and that the disease studied by Billings was an infectious form of sep- ticaemia in cattle similar to the Kinderseuche of German authors. The mi- croorganism which he has described as coming from the blood of the infected animals resembles in its morphology Bacillus septicaemia' li;i -UK >r- rhagicse (No. 61), and, if not identical with this widely distributed species, appears to be very nearly related to it. The researches of Smith and other bacteriologists connected with the United States Department of Agriculture appear to have elucidated the etiol- ogy of this disease, and to show that it is due to a blood parasite belonging to the protozoa (Pyrosoma bigeminum of Smith). TRACHOMA. Fuchs (1894) as a result of his investigations arrives at the con- clusion that trachoma is frequently due to infection by the gono- coccus. He believes that in acute cases the transfer of the infectious secretions causes an acute gonorrhceal ophthalmia; and that when BACTERIA IN INFECTIOUS DISEASES. 615 this becomes chronic a similar transfer of infectious material gives rise to the chronic inflammatory process known as trachoma. Hoor (1895) also ascribes trachoma to infection by gonococci, and believes that in their etiology papillary trachoma (blennorrhcea chronica) and granular trachoma (trachoma verum) are identical. TYPHOID FEVER. The etiological relation of Bacillus typhi abdominalis (No. 46) to typhoid fever is now generally admitted by pathologists and bacteri- ologists. TYPHUS FEVER. The etiology of typhus fever has not been determined in a definite man- ner. Hlava (1888) has described a " streptobacillus " which he supposes to be concerned in the etiology of this extremely contagious disease ; but it lias been shown by other investigators that this bacillus is not constantly present, and there is no satisfactory evidence that it is the specific infectious agent. Thoinet and Calmette (1892) examined the blood in seven cases and were unable to find the streptobacillus of Hlava ; their cultures from the spleen of living patients and from the spleen and blood from the heart of recent cadav- ers gave a negative result. They found, however, in blood examined under the microscope abnormal elements, in the form of motile granules and fila- ments, which were sometimes adherent to the red blood corpuscles. Lewa- schoff in 1892 claimed to have found in the blood of typhus patients motile micrococci having long spiral flagella. In a subsequent epidemic Lewa- schoff (1894) claims to have found the same microorganism in one hundred and eighteen cases examined, and also to have obtained in cultures from blood taken from the spleen or from the finger the same microorganism, sometimes solitary and provided with flagella, and sometimes in chains. Weinshal (1892) in ten cases examined by the method recommended by Lewaschoff was unable to find the microorganism described by him or any other. Hlava (1893) in his more recent researches has not obtained his streptobacillus in cultures made soon after death (ten cases) ; but he obtained various micro- organisms in his cultures from the spleen, lungs, etc., which he concludes are not directly concerned in the etiology of the disease — streptococci, staphy- lococci, pseudo-diphtheria bacilli, and a proteus ("Vibrio proteus ruber"). He also observed in the blood and spleen bodies resembling the spores of yeast fungi. Dubieff and Bruhl (1893) in nine cases examined (six post-mortem) found in small numbers in the blood and spleen a diplococcus, called by them Dip- lococcus exaiithematicus. This is said to have been difficult to cultivate and to have been present in enormous numbers in mucus from the nose and throat and from pneumonic foci in the lungs. Calmette (1893), in a detailed account of the microorganism previously described by himself and Thoinet, reports that this is especially abundant in splenic pulp obtained by an aspirating syringe during life. The bodies found are actively motile, and from 2 to 3 /^ in diameter, having sometimes a fili- form appendage from 4 to 5 \i in length. Long spiral filaments are also occa- sionally seen ; these are from 20 to 30 \L long and 1 to 2 fj. thick ; they are ac- tively motile. Cultures from blood were obtained in media containing sugar or acidified with lactic or tartaric acid. This microorganism is believed by Calmette to be a microscopic fungus, belonging to the ascomycetes, or to the genus Ustilago. Curtis and Combemale (1893) in cultures from blood drawn from the finger, in twelve cases, had invariably a negative result ; in three 616 BACTERIA IN INFECTIOUS DISEASES. cases out of six in which cultures were made from material obtained from the spleen, post mortem, a very minute diplococcus developed, at 37 C. This formed a grayish layer upon the surface of nutrient agar at the end of two or three days. TUBERCULOSIS. The various forms of tubercular infection in man are due to a single specific infectious agent — Bacillus tuberculosis (No. 53). Tu- berculosis in cattle is due to infection by the same bacillus. The bacillus which produces tuberculosis in fowls closely resembles that of human tuberculosis, but owing to slight differences is described under a separate heading — Bacillus tuberculosis gallinarum (No. 54). VARICELLA. Various microorganisms have been found in the contents of the vesicles and pustules of varicella, but there is no evidence that any one of these bears an etiological relation to this specific eruptive fever. VARIOLA AND VACCINIA. The etiology of small-pox still remains undetermined. The common pus cocci and various other microorganisms are found in the characteristic pus- tular eruption, and various microorganisms have been isolated from vaccine vesicles ; but no one of these has been shown to possess the specific pathogenic power of unfiltered lymph from the same source. The experiments of Cars- tens and Coert show that the specific virulence of vaccine lymph is destroyed by ten minutes' exposure to a temperature of 54° C. And the writer's experi- ments show that various disinfecting agents tested — chlorine, sulphur dioxide, nitrous acid — destroy the infective virulence of lymph dried upon ivory points in about the same proportion as is required for the destruction of some of the best-known pathogenic bacteria. But this does not prove that viru- lence depends upon the presence of a living microrganism, however probable this appears, for certain toxalbumins are likewise destroyed by a correspond- ingly low temperature and by the action of various chemical disinfectants. Nikolsky (1892) obtained from the base of the pustules of small-pox a mo- tile, liquefying, spore-producing bacillus which when introduced into ilie peritoneal cavity of rabbits is said to have given rise to a pustular eruption ; the bacillus was recovered in cultures from these pustules. Grigorijew (1889) in three cases found a small bacillus, twice as long as thick, which slowly liquefied gelatin, and did not coagulate milk. Besser (1893) givrs an account of the microorganisms found in the pustules of variola and adds to the list a bacillus found by himself in a single case. This does not grow in gelatin at the room temperature and is more slender than the bacillus of Grigoriew. The results of the researches of Martin (1891) have been reported by Ernst (1893). He obtained various bacteria from the lymph of vat-cine vesicles, and among these was a bacillus which he believed to be the specific infectious agent. This he was able to cultivate upon the surface of sterilized blond serum of the ox, at 37° C., and his cultures of the ninth generation are said to have produced typical cow-pox when inoculated upon calves. He says : 4 'The material 'takes' with the same certainty as the lymph from the vesicle. BACTERIA IN INFECTIOUS DISEASES. 617 pure, but contained quite a variety of bacteria. In every instance the blood serum was liquefied." As to the morphology he says : "The bacterium va- ries in form according1 to the various conditions of its nutritive environment and the consequent rate of its development. The most constant and preva- lent form is a short, fine bacillus with rounded or nearly square ends. Those parts of the culture where the nutriment is apparently exhausted show the same "bacilli in short chains, longer bacilli, and bacilli much enlarged at one end or the middle, as if in preparation, for spore formation." As Martin's cul- tures were not pure we have no evidence that his successful inoculations were due to the particular bacillus which attracted his attention. Possibly a microorganism of another class was also present and was carried over from one culture to another. Ruete and Enoch (1893) have also reported success- ful vaccinations in the calf with a micrococcus which they cultivated from vaccine lymph. Buttersack (1893) as a result of his researches arrived at the conclusion that there are numerous minute elements in vaccine lymph which do not stain and are sometimes arranged in chains. The subsequent researches of Landmann (1894) and others indicate that the supposed microorganisms of Buttersack are non-living, albuminoid granules, artificially produced by his method of investigation. This view is confirmed by the investigations of Draer (1894). Guarnieri (1892), Monti (1894), Piana and Galli-Valerio (1894), and Clarke (1895), have observed amoeboid microorganisms in the pustules of variola and in vaccine lymph which may prove to be the specific infectious agent in this disease. These are described by Guarnieri under the name Cytorycetes variolas and Cytorycetes vacciniae. According to Clarke these amoeboid para- sites belong to the Sprozoa. E. Pfeiffer (1895) has studied this parasite by inoculations into the cornea of rabbits, guinea-pigs, and calves. WHOOPING-COUGH. No satisfactory demonstration has yet been made of the specific infectious agent in whooping-cough. Hitter (1892) has obtained from the nasal and bronchial secretions in cases (eighteen) of whooping-cough a small diplococcus which he believes to be the cause of the disease. This is aerobic, stains by Gram's method, and grows upon nutrient agar in the incubating oven — not in bouillon, in gelatin, or 011 potato. Cohn and Neumann (1893) in 18 cases in which they made a careful re- search by approved methods were only able to demonstrate the presence of Ritter's diplococcus in one; in two other cases somewhat similar diplococci were found. The bacillus described by Afanassiew in 1887 (No. 119) was also seen occasionally, but was evidently not the specific infectious agent. The micro- organism most constantly found was a streptococcus, apparently identical with Streptococcus pyogenes. This was present in 20 cases out of 25 exam- ined, and in 12 of these it was obtained from bronchial mucus in such num- bers as almost to constitute a pure culture. YELLOW FEVER. The results of investigations made by the writer in Cuba during the sum- mers of 1888 and 1889 are given in the following summary statement from the Transactions of the Tenth International Medical Congress (Berlin, 1890) : Bacterial Researches in Yellow Fever. The report relates to investigations made in Havana, Cuba, during the summers of 1888 and 1889, in Decatur, Alabama, during the autumn of 1888, 43 618 BACTERIA IN INFECTIOUS DISEASES. and in the pathological and biological laboratories of the Johns Hopkins University during the winters of 1888 and 1889. Forty-two autopsies were made in typical cases of yellow fever and seven- teen autopsies in other diseases for comparative researches. Aerobic and anaerobic cultures were made from the blood, the liver, the kidney, the urine, the stomach, and the intestine. The experimental data recorded in this report show that: The specific infectious agent in yellow fever has not been demonstrated. The most approved bacteriological methods fail to demonstrate the con- stant presence of any particular microorganism in the blood and tissues of yellow-fever cadavers. The microorganisms which are sometimes obtained in cultures from the blood and tissues are present in comparatively small numbers ; and the one most frequently found (Bacterium coli commune) is present in the intestine of healthy individuals, and consequently its occasional presence cannot have any etiological import. A few scattered bacilli are present in the liver, and probably in other or- gans, at the moment of death. This is shown by preserving portions of liver, obtained at a recent autopsy, in an antiseptic wrapping. At the end of twenty-four to forty-eight hours the interior of a piece of liver so preserved contains a large number of bacilli of various species, the most abundant being those heretofore mentioned as occasionally found in fresh liver tissue, viz. , Bacterium coli commune and Bacillus cadaveris. Blood, urine, and crushed liver tissue obtained from a recent autopsy are not pathogenic in moderate amounts for rabbits or guinea-pigs. Liver tissue preserved in an antiseptic wrapping at a temperature of 28° to 30° C., for forty eight hours, is very pathogenic for guinea-pigs when in- jected subcutaneous! y. This pathogenic power appears to be due to the microorganisms present and to the toxic products developed as a result of their growth. It is not peculiar to yellow fever, inasmuch as material preserved in the same way at comparative autopsies, in which death resulted from accident or other diseases, has given a similar result. Having failed to demonstrate the presence of a specific "germ" in the blood and tissues, it seems probable that it is to be found in the alimentary canal, as is the case in cholera. But the extended researches made, and re- corded in the present report, show that the contents of the intestines of yel- low-fever cases contain a great variety of bacilli, and not a nearly pure cul- ture of a single species, as is the case in recent and typical cases of cholera. Comparatively few liquefying bacilli are found in the faeces discharged during life, or in the intestinal contents collected soon after death from yel- low-fever cadavers. On the other hand, non-liquefying bacilli are very abundant. The one most constantly and abundantly present is the Bacterium coli commune of Escherich. This is associated with various other bacilli, some of which are strict anaerobics and some facultative anaerobics. Among the facultative anaerobics is one — my Bacillus X — which has been isolated by the culture method in a considerable number of cases and may have been present in all. This bacillus has not been encountered in the comparative experiments made. It is very pathogenic for rabbits when in- jected into the cavity of the abdomen. It is possible that this bacillus is concerned in the etiology of yellow fever, but no satisfactory evidence that this is the case has been obtained by experi- ments on the lower animals, and it has not been found in such numbers as to warrant the inference that it is the veritable infectious agent. All other microorganisms obtained in pure cultures from yellow-fever cadavers appear to be excluded, either by having been identified with known species, or by having been found in comparative researches made outside of BACTERIA IN INFECTIOUS DISEASES. 619 the area of yellow fever prevalence, or by the fact that they have only been found in small numbers and in a limited number of cases. l Finally we remark that many facts relating to the origin and extension of yellow-fever epidemics give support to the inference that the specific in- fectious agent is present in the dejecta of those suffering from the disease, and that accumulations of faecal matter, and of other organic material of ani- mal origin, furnish a suitable nidus for the development of the "germ" when climatic conditions are favorable for its growth. It may be that such a nidus is essential, and that the culture media usually employed by bacteriologists do not afford a suitable soil for this par- ticular microbe. It is also possible that its development depends upon the presence of other microorganisms found in faecal matter, which give rise to chemical products required for the development of this one. Some of the microorganisms present in the dejecta of yellow-fever pa- tients, as shown by stained smear preparations, have not developed in the cultures made, either aerobic or anaerobic. One extremely slender filiform bacillus, which can only be seen with high powers and which is quite abun- dant in some of my preparations, has never been obtained in the cultures made, and no doubt there are others in the same category. That the yellow-fever germ is a strict anaerobic, or that it will only grow in a special nidus, may be inferred from certain facts relating to the exten- sion of epidemics. There is rio evidence that yellow fever is propagated by contamination of the supply of drinking water, as frequently, and probably usually, occurs in the case of typhoid fever and cholera. Moreover, epidemics extend in a more deliberate manner and are restricted within a more definite area than is the case with cholera and typhoid fever. It is usually at least ten days or two weeks after the arrival of an infected vessel or of a person sick with the disease before cases of local origin occur ; and these cases occur in the imme- diate vicinity of the imported case or infected vessel. When the disease has effected a lodgment the area of infection extends slowly and usually has well-defined boundaries. In towns and cities having a common water sup- ply one portion remains perfectly healthy, while another, and usually the most filthy portion, may be decimated by the scourge. The experimental evidence recorded, and the facts just stated, seem to justify the recommendation that the dejecta of yellow-fever patients should be regarded as infectious material, and that such material should never be thrown into privy vaults or upon the soil until it has been completely disin- fected. This rule thoroughly enforced, together with an efficient quarantine ser- vice and proper attention to the sanitary police of our exposed seaport cities, would, I believe, effectually prevent this pestilential disease from again ob- taining a foothold within the limits of the United States. 1 The possibility, of course, remains that the specific infectious agent in yellow fever may belong to an entirely different class of microorganisms from the bacteria, or that it may be ultra-microscopic or not capable of demonstration in the tissues by the staining methods usually employed by bacteriologists. PART FOURTH. SAPEOPHYTES. I. BACTERIA IN THE AIR. II. BACTERIA IN WATER. III. BACTERIA IN THE SOIL. IV. BACTERIA ON THE SURFACE OF THE BODY AND OF EX- POSED Mucous MEMBRANES. V. BACTERIA OF THE STOMACH AND INTESTINE. VI. BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL FROM VARIOUS SOURCES. VII. BACTERIA IN ARTICLES OF FOOD. BACTERIA IN THE AIR. THE saprophytic bacteria are found wherever the organic material which serves as their pabulum is exposed to the air under conditions favorable to their growth. The essential conditions are presence of moisture and a suitable temperature. The organic material may be in solution in water or in the form of moist masses of animal or vegetable origin, and the temperature may vary within considerable limits — 0° to 70° C. But the species which takes the precedence will depend largely upon special conditions. Thus certain species multi- ply abundantly in water which contains comparatively little organic pabulum, and others require a culture medium rich in albuminous material or in carbohydrates ; some grow at a comparatively low or high temperature, while others thrive only at a temperature of 20° to 40° C. or have a still more limited range ; some require an abun- dant supply of oxygen, and others will not grow in the presence of this gas. Our statement that saprophytic bacteria are found wherever the organic material which serves as their pabulum is exposed to the air — under suitable conditions — relates to the fact that it is through the air that these bacteria are distributed and brought in contact with exposed material. It is a matter of common laboratory experi- ence that sterilized organic liquids quickly undergo putrefactive de- composition when freely exposed to the air, and may be preserved in- definitely when protected from the germs suspended in the air by means of a cotton air filter. But the organic pabulum required for the nourishment of these bacteria is not found in the air in any con- siderable amount, and if they ever multiply in the atmosphere it must be under very exceptional conditions. Their presence is due to the fact that they are wafted from surfaces where they exist in a desiccated condition, and, owing to their levity, are carried by the wind to distant localities. But, under the law of gravitation, when not exposed to the action of currents of air they constantly fall again upon exposed surfaces, which, if moist, retain them, or from which, if dry, they are again wafted by the next current of air. Under these circumstances it is easy to understand why, as deter- 624 BACTERIA IN THE AIR. mined by investigation^ more bacteria are found near the surface of the earth than at some distance above the surface, more over the land than over the ocean, more in cities with their dust-covered streets than in the country with its grass-covered fields. Careful experiments have shown that bacteria do not find their way into the atmosphere from the surface of liquids, unless portions of the liquid containing them are projected into the air by some mechanical means, such as the bursting of bubbles of gas. Cultures of pathogenic bacteria freely exposed to the air in laboratories do not endanger the health of those who work over them; but if such a cul- ture is spilled upon the floor and allowed to remain without disin- fection, when it is desiccated the bacteria contained in it will form part of the dust of the room and might be dangerous to its occupants. Bacteria do not escape into the air from the surface of the fluid contents of sewers and cesspools, but changes of level may cause a deposit upon surfaces, which is rich in bacteria, and when dried this ma- terial is easily carried into the atmosphere by currents of air. Tyndall's experiments (1869) show that in a closed receptacle in which the air is perfectly still all suspended particles are af- ter a time deposited on the floor of the closed air chamber. And common experience de- monstrates the fact that the dust of the at- mosphere is carried by the wind from ex- posed surf aces and again deposited when the air is at rest. This dust as deposited, for example, in our dwellings contains innu- merable bacteria in a desiccated condition, and the smallest quantity of it introduced into a sterile organic liquid will cause it to undergo putrefactive decomposition, and by bacteriological methods it will be found to contain various species of bacteria. Such dust also contains the spores of various mould fungi which are present in the atmo- sphere, usually in greater numbers than the bacteria. The mould fungi are air pL-snts which vegetate upon the surface of moist organic material and form innumerable spores, which are easily wafted into the air, both on account of their low specific gravity and minute size, and because they FIG. 188. — Penicillum glau- cum; m, mycelium, from which is given off a branching pedicle bearing spores. X 160. BACTERIA IN THE AIR. 625 are borne upon projecting pedicles by which they are removed from the moist material upon which and in which the mycelium develops (Fig. 188), and, being dry, are easily carried away by currents of air. Bacteriologists have given much attention to the study of the mi- croorganisms suspended in the atmosphere, with especial reference to hygienic questions. The methods and results of these investigations will be considered in the present section. Pasteur (1860) demonstrated the presence of living bacteria in the atmosphere by aspirating a considerable quantity of air through a filter of gun-cotton or of asbestos contained in a glass tube. By dis- solving the gun-cotton in alcohol and ether he was able to demon- strate the presence of various microorganisms by a microscopical ex- amination of the sediment, and by placing the asbestos filters in sterilized culture media he proved that living germs had been filtered out of the air passed through them. FIG. 189. A method employed by several of the earlier investigators con- sisted in the collection of atmospheric moisture precipitated as dew upon a surface cooled by a freezing mixture. This was found to con- tain living bacteria of various forms. The examination of rain water, which in falling washes the suspended particles from the atmosphere, gave similar results. The first systematic attempts to study the microorganisms of the air were made by Maddox (1870) and by Cunningham (1873), who used an aeroscope which was a modification of one previously de- scribed by Pouchet. In the earlier researches of Miquel a similar aeroscope was used. This is shown in Fig. 189. The opening to the cylindrical tube A is kept facing the wind by means of a wind vane, and when the wind is blowing a current passes through a small aper- ture in a funnel-shaped partition which is properly placed in the cylindrical tube. A glass slide, upon the lower surface of which a 626 BACTERIA IN THE AIR. mixture of glycerin and glucose has been placed, is adjusted near the opening of the funnel, at a distance of about three millimetres, so that the air escaping through the small orifice is projected against it. By this arrangement a considerable number of the microorganisms present in the air, as well as suspended particles of all kinds, are ar- rested upon the surface of the slide and can be examined under the microscope or studied by bacteriological methods. But an aeroscope of this kind gives no precise information as to the number of living germs contained in a definite quantity of air. The microscopical ex- amination also fails to differentiate the bacteria from particles of various kinds which resemble them in shape, and the microorgan- isms seen are for the most part spores of various fungi mingled with pollen grains, vegetable fibres, plant hairs, starch granules, and amorphous granular material. Another method, which has been employed by Cohn, Pasteur, Miquel, and others, consists in the aspiration of a definite quantity of air through a culture liquid, which is then placed in an incubating oven for the development of microorganisms washed out of the air which has been passed through it. This method shows that bacteria of different species are present, but gives no information as to their relative number, and requires further researches by the plate method to determine the characters of the several species in pure cultures. A far simpler method consists in the exposure of a solid culture medium, which has been carefully sterilized and allowed to cool on a glass plate or in a Petri's dish, for a short time in the air to be ex- amined. Bacteria and mould fungi deposited from the air adhere to the surface of the moist culture medium, and form colonies when the plate, enclosed in a covered glass dish, is placed in the incubating oven. The number of these colonies which develop after exposure in the air for a given time enables us to estimate in a rough way the num- ber of microorganisms present in the air of the locality where tin- exposure was made ; and the variety of species is determined by ex- amining the separate colonies, each of which is, as a rule, developed from a single germ. By exposing a number of plates at different times this method enables us to determine what species are m< >st abundant in a given locality and the comparative number in dif- ferent localities, as determined by counting the colonies after ex- posure for a definite time — e.g., ten minutes. Of course we will only obtain evidence of the presence of such aerobic bacteria as will grow in our culture medium. The anaerobic bacteria may be studied by placing plates exposed in a similar way in an atmosphere of hydro- gen. Bacteria which grow slowly and only under special conditions, like the tubercle bacillus, would be likely to escape observation, as the mould fungi and common saprophytes would take complete pos- BACTERIA IN THE AIR. 627 session of the surface of the culture medium before the others had formed visible colonies. Students will do well to employ this simple and satisfactory method for the purpose of making themselves familiar with the more common atmospheric organisms, and they will find the shallow glass dishes with a cover, known as Petri's dishes, very convenient for the purpose. These dishes should be sterilized in the hot-air oven and sufficient sterile nutrient gelatin or agar poured into them to cover the bottom. After the culture medium has be- come solid by cooling, the exposure may be made by simply remov- ing the cover and replacing it at the end of the time fixed upon. FIG. 190. To determine in a more exact way the number of microorganisms contained in a given quantity of air will require other methods. But we may say, en passant, that such a determination is usually not of great scientific importance. The number is subject to constant fluc- tuations in the same locality, depending upon the force and direction of the wind. If we have on one side of our laboratory a dusty street and on the other a green field, more bacteria will naturally be found when the wind blows from the direction of the street than when it comes from the opposite direction ; or, if the air is filled with dust from recently sweeping the room, we may expect to find very 628 BACTERIA IN THE AIR. many more than when the room has been undisturbed for some time. The painstaking researches which have already been made have es- tablished in a general way the most important facts relating to the distribution of atmospheric bacteria, but have failed to show any de- finite relation between the number of atmospheric bacteria and the prevalence of epidemic diseases. In the apparatus of Hesse, Fig. 190, a glass tube, having a diameter of four to five centimetres and a length of half a metre to a metre, is employed. In use this is sup- ported upon a tripod, as shown in the figure, and air is drawn through it by a water aspirator consisting of two flasks, also shown. The upper flask being filled with water, this flows into the lower flask by siphon action, and upon reversing the position of the flasks number one is again filled. By repeating this operation as many times as desired a quantity of air corresponding with the amount of water passed from the upper to the lower flask is slowly aspirated through the horizontal glass tube. The microorganisms present are deposited upon nutrient gelatin previously allowed to cool upon the lower portion of the large glass tube. The air enters through a small opening in a piece of sheet rubber which is tied over the extremity of the horizontal tube, and before the aspiration is commenced this opening is covered by another piece of sheet rubber tied over the first. Experience shows that when the air is slowly aspirated most of the germs contained in it are deposited near the end of the tube through which it enters. The colonies which develop upon the nu- trient gelatin show the number and character of living microorgan- isms contained in the measured quantity of air aspirated through the apparatus. The method with a soluble filter of pulverized sugar, to be described hereafter, is preferable when exact results are desired; and for the purpose of determining the relative abundance and the variety of microorganisms present in the atmosphere of a given lo- cality the exposure of nutrient gelatin in Petri's dishes is far simpler, and, as a rule, will furnish all the information that is of real value. In his extended researches made at the laboratory of Montsouri, in Paris, Miquel has used various forms of apparatus and has i >1 >- tained interesting results ; but his method of ensemencements frafr tionnes requires a great expenditure of time and patience, and the more recent method with soluble filters is to be preferred. In his latest modification of the method referred to Miquel used a flask like that shown in Fig. 191. From twenty to forty cubic cen- timetres of distilled water are introduced into this flask. The cap A contains a cotton air filter and is fitted to the neck of the flask by a ground joint. This is removed during the experiment. The tube C is connected with an aspirator. It contains two cotton or asbestos BACTERIA IN THE AIR. 629 Fio. 191. filters, c and b. The cap being removed and the aspirator attached, the air is drawn through the water, by which suspended germs are arrested ; or if not they are caught by the inner cotton plug b. The sealed point of the tube B is now broken off, and the contents of the flask equally divided in thirty to forty tubes containing bouillon, which are placed in the incubating oven. Twenty-five cubic centimetres of bouillon are also introduced into the flask, and the cotton plug b is pushed into it so that any bacteria arrested by it may develop. If one-fourth or one-fifth of the bouillon tubes show a development of bacteria it is in- ferred that each culture originated from a single germ, and the number present in the amount of air drawn through the flask is estimated from the number of tubes in which development occurs. The method adopted by Straus and Wiirtz is more convenient and more reliable in its results. This consists in passing the air by means of an aspirator through liquefied nutrient gelatin or agar. The ap- paratus shown in Fig. 192 is used for this purpose. Two cotton plugs are placed in the tube B, to which the aspirator is attached, and after the determined quantity of air has been passed through the liquefied medium the inner plug is pushed down with a sterilized platinum needle so as to wash out in the culture medium any germs arrested by it. Finally the gelatin or agar is solidified upon the walls of the tube A by rotating it upon a block of ice or under a stream of cold water. It is now put aside for the development of colonies, which are counted to determine the number of germs pre- sent in the quantity of air passed through the liquefied culture medium. The main difficulty with this apparatus is found in the fact that the nutrient gelatin foams when air is bubbled through it ; for this reason an agar medium is to be preferred. In using this it will be neces- sary to place the liquefied agar in a bath main- tained at 40° C. Foaming of the gelatin is pre- vented by adding a drop of olive oil before ster- ilization in the steam sterilizer. But this inter- feres with the transparency of the medium. In the earlier experiments upon atmospheric organisms Pasteur used a filter of asbestos, which was subsequently washed out in a FlO. 192. 630 BACTERIA IN THE AIR. culture liquid. A filter of this kind washed out in liquefied gelatin or nutrient agar would give more satisfactory results, as the culture medium could be poured upon plates or spread upon the walls of a test tube and the colonies counted in the usual way. Petri prefers to use a filter of sand, which he finds by experiment arrests the mi- croorganisms suspended in the atmosphere, and which is subsequently distributed through the culture medium. The sand used is such as has been passed through a wire sieve having openings of 0. 5 millimetre in diameter. This is sterilized by heat, and is supported in a cylin- drical glass tube by small wire-net baskets. The complete arrangement is shown in Fig. 193. Two sand filters, cl and C2, are used, the lower one of which serves as a control to prove that all microorganisms present in the air have been arrested by the upper one. The upper filter is protected, until the aspirator attached to the tube h is put in operation, by a sterile cotton plug, not shown in the figure which represents the filter in use. Petri uses a hand air pump as an aspirator, and passes one hundred litres of air through the sand in from ten to twenty minutes. The sand from the two filters is then distributed in shallow glass dishes and liquefied gelatin is poured over it ; this is allowed to sol- idify and is put aside for the development of colonies. The principal objection to this method is the presence of the opaque particles of sand in the culture medium. This objection has been overcome by the use of soluble filters, a method first employed by Pasteur and since perfected by Sedgwick and by Miquel. The most useful material for the purpose appears to be cane sugar, which can be sterilized in the hot-air oven at 150° C. without undergoing any change in its physical characters. Loaf sugar is pulver- ized in a mortar and passed through two sieves in order to remove the coarser grains and the very fine powder, leaving for use a powder having grains of about one-half millimetre in diameter. This powdered sugar is placed in a glass tube provided with a cap having a ground joint and a cot- ton plug to serve as an air filter (A, Fig. 104), or in a tube such as is shown at B, having the end drawn out and hermetically sealed. Two cotton plugs are placed at the lower portion of the tube, at a and at b. Fio. 193. BACTERIA IN THE AIR. 631 Glass tubing having a diameter of about five millimetres is used in making these tubes, and from one to two grammes of powdered sugar is a suitable quantity to use as a filter. The whole apparatus is steril- ized for an hour at 150° C. in a hot-air oven after the pulverized sugar has been introduced. Before using 'it will be necessary to pack the sugar against the supporting plug a by gently striking the lower end of the tube, held in a vertical position, upon some horizon- tal surface ; and during aspiration the tube must remain in a vertical J\ position, or nearly so, in order that the sugar may properly fill its entire calibre. The aspirator is attached to the lower end of the tube by a piece of rubber tubing. When the tube B is used the sealed extremity is broken off at the moment that the aspirator is set in action, and it is again sealed in a flame after the desired amount of air has been passed through the filter. The next step consists in dis- solving the sugar in distilled water or in liquefied gelatin. To insure the removal of all the sugar the cot- ton plug a may be pushed out with a sterilized glass rod, after removing b with forceps. From fifty to five hun- dred cubic centimetres of distilled water, contained in an Erlenmeyer flask and carefully sterilized, may be used, the amount required depending upon circumstances relating to the conditions of the experiment. By adding five or ten cubic centimetres 01 & KJ 1> of this water, containing the sugar FlG 194 FlG> 195 and microorganisms arrested by it, to nutrient gelatin or agar liquefied by heat, and then making Es- march roll tubes, the number of germs in the entire quantity is easily estimated by counting the colonies which develop in the roll tubes. Sedgwick and Tucker, in a communication made to the Boston Society of Arts, January 12th, 1888, were the first to propose the use of a soluble filter of granulated sugar for collecting atmospheric germs. Their complete apparatus consists ,of an exhausted receiver, from which a given quantity of air is withdrawn by means of an air pump. A vacuum gauge is attached to the receiver, which is coupled 632 BACTERIA IN THE AIR. with the glass tube containing the granulated -sugar filter by a piece of rubber tubing. Instead of transferring the soluble filter to gela- tin in test tubes, they use a large glass cylinder having a slender stem, in which the sugar is placed (Fig. 195). After the aspiration liquefied gelatin is introduced into the large glass cylinder, which is held in a horizontal position ; the sterilized cotton plug is then re- placed in the mouth of the cylinder, the sugar is pushed into the liquefied gelatin and dissolved, and by rotating the cylinder upon a block of ice the gelatin is spread upon its walls as in an Esmarch roll tube. For convenience in counting the colonies lines are drawn upon the surface of the cylinder, dividing it into squares of uniform di- mensions. GENERAL RESULTS OF RESEARCHES MADE. As already stated, the presence of bacteria in the atmosphere de- pends upon their being wafted by currents of air from surfaces where they are present in a desiccated condition. That they are not carried away from moist surfaces is shown by the fact that expired air from the human lungs does 'not contain microorganisms, although the in- spired air may have contained considerable numbers, and there are always a vast number present in the salivary secretions. The moist mucous membrane of the respiratory passages constitutes a germ trap which is much more efficient than the glass slide smeared with glycerin used in some of the aeroscopes heretofore described, for it is a far more extended surface. As a matter of fact, most of the sus- pended particles in inspired air are deposited before the current of air passes through the larynx. Air which passes over large bodies of water is also purified of its germs and other suspended particles. The researches of Fischer show that at a considerable distance from the land no germs an- found in the atmosphere over the ocean, and that it is only upon ap- proaching land that their presence is manifested by the development of colonies upon properly exposed gelatin plates. Uffelmann found, in his researches, that in the open fields th) has shown that the ripening of certain kinds of cheese (fromages mous) is probably due to Oidium lactis. Meats, even when salted and smoked, may contain living patho- genic bacteria which were present prior to the death of the animal, and, when not properly preserved, are of course liable to be invaded by putrefactive bacteria. BACTERIA IN ARTICLES OF FOOD. 681 The researches of Foster (1889) show that the typhoid bacillus, the pus cocci, the tubercle bacillus, and the bacillus of swine plague resist the action of a saturated solution of salt for weeks and even for months; and the same observer found that the ordinary processes of salting and smoking did not destroy the tubercle bacillus in the flesh of a cow which had succumbed to tuberculosis. Beu has made cul- tures from a large number of specimens of fresh, salted, and smoked meats and fish, with the general result that the fresh and salted meats were found to contain a limited number of bacteria of various species, and that smoking for several days did not insure the destruction of these microorganisms. In specimens of sausage six days' smoking did not destroy a liquefying bacillus which was present, but at the end of six weeks' exposure to smoke this bacillus no longer grew, while a non-liquefying bacillus present in the same specimen had not been destroyed. Fourteen days' smoking sufficed to destroy all the microorganisms in a specimen of bacon, but this was not sufficient for the interior portions of a ham. Among the bacteria obtained by Beu from smoked meats he mentions the following : Staphylococcus cereus albus, Proteus vulgaris, Staphylococcus pyogenes aureus, Ba- cillus liquefaciens viridis, etc. The number of colonies which de- veloped from a fragment, the size of a mustard seed to that of a flax- seed, taken from the interior of the meats examined, was usually small; and the presence of a few scattered bacteria of these common species has no significance from a sanitary point of view, except as showing that pathogenic bacteria may survive in infected meats after they have been exposed to the usual processes of salting and smoking. Petri, in experiments upon the bacillus of swine plague (Schweine- rothlauf), arrived at the following results : The flesh of swine which died of this disease preserved its infec- tious properties after having been preserved in brine for several months, and the same flesh salted or pickled for a month and then smoked for fourteen days contained the rothlauf bacillus in a living and unattenuated condition. At the end of three months virulent rothlauf bacilli were still obtained from a smoked ham, but they were no longer found at the end of six months. Schrank (1888) has made cultures from both the albumin and the yolk of fresh eggs, and finds that they are free from bacteria. He thinks that, as a rule, putrefactive bacteria obtain access to the inte- rior through injured places in the shell, although exceptionally the egg may be infected with them in the oviduct of the fowl. The usual bacteria concerned in the putrefactive changes in eggs are, according to the author mentioned, a variety of Proteus vulgaris and Bacillus fluorescens putidus. Zorkendorfer (1893) has cultivated from rotten eggs sixteen dif- 47 G82 BACTERIA IN ARTICLES OF FOOD. ferent bacilli, all of which are described in detail and none of which were found to correspond with previously described species as given in Eisenberg's Bacteriological Diagnosis. Peters (1889) has studied the flora of the "sauerteig" used in Germany as yeast for leavening bread. In addition to the numerous cells of three species of Saccharomyces, he finds that bacilli are present in great numbers, as shown by direct microscopical examination and culture experiments. He describes five species, designated Bacillus A, B, C, D, and E, which are commonly present, and to which the acid fermentation of the dough is ascribed. In Graham bread which had undergone changes making it unfit to eat, Kratschmer and Niemilowicz have found the Bacillus mes- entericus vulgatus, which appears to have been the cause of the fermentation, which was produced in bread having a slightly alka- line reaction by inoculating it with a pure culture of this bacillus. The infected bread has a brownish color, a peculiar odor, and be- comes sticky and viscid. Uffelmann (1890) has also studied the bacteria in spoiled rye bread, and obtained, in addition to common mould fungi, Bacillus mesentericus vulgatus and Bacillus liodermus. Waldo (1894) has shown that baking does not sterilize bread. This was to have been expected in the case of the spores of bacilli, but it is somewhat surprising to find that two species of Sarcina and two micrococci survived the baking process. In all Waldo obtained thirteen species of bacteria from the interior of sixty-two loaves examined. Bacillus subtilis and allied spore-forming bacilli were most frequently found, and the statement is made that a loaf " from a low-class, dirty bakery will almost invariably contain more living bacteria (or their spores) than one from a good, clean bakery." Lehmann (1894) under the name Bacillus levans has described a microorganism which closely resembles Bacillus coli communis. This was obtained from sour dough, and was believed to be the cause of the acid fermentation which so often interferes with success in ob- taining sweet and wholesome bread. When a culture of this bacil- lus was added to flour and water, without the addition of yeast, an active fermentation occurred and the dough became acid. INDEX. ABRIN, experiments of Ehrlich, 270 Abscesses, etiology of, 571 formation of, 222, 275, 276 Micrococeus pneumonise croupos* in, 313 Acetic acid, germicidal action of, 178 production of, 134 Acetone, antiseptic value of, 193 Acids, germicidal action of, 176-179 Acne, etiology of, 571 contagiosa of horses, bacillus of, 508, 571 Acute rheumatism, etiology of, 610 Ae"roscopes, 625 Agar-agar, 43 filtration of, 44 Agar-gelatin, 43 Agitation, germicidal action of, 15& Agua coco, as a culture medium, 39 Air, bacteria in, 623-635 filter of cotton, 4 Alcohol, germie&al value of, 193 Alexins, 273 Alkalie% germicidal value of, 179-181 Alkaline fermentation of urine, 137 Alopecia, etiology of, 572 Afum, antiseptic value of, 184 Aluminium acetate, antiseptic value of, 184 Ammonia, germicidal value of, 180 liberation of, 140 oxidation of, 141 Ammonium carbonate, antiseptic value of, 184 chloride, antiseptic value of, 184 fluosilicate, antiseptic value of, 184 sulphate, antiseptic value of, 184 Anaerobic bacteria, cultivation of, 78-85 bacilli, 531-548 cultures, 79 cultures, Buchner's method, 83 cultures, Esmarch's method, 82 cultures, Franker s method, 80 cultures, Liborius' method, 83 cultures, Stern berg's method, 81 Anaesthetics, use of, 97 Angina, etiology of, 572 Aniline dyes, germicidal value of, 193 oil, antiseptic value of, 194 Anthrax, etiology of, 339 bacillus, discovery of, 6 bacillus, toxic products of, 147 Antiseptic action, conditions governing, 162 value, how determined, 161 Antiseptics, comparative value of, 182, 183 definition of, 160 Antitoxin of diphtheria, results of treat- ment, 386 of pneumonia, 268 of snake venom, 269 of tetanus, 268 of tetanus, from milk, 271 Antitoxins, 266-272 do not dialyze, 273 Appendicitis, etiology of, 573 Arsenious acid, germicidal value of, 179 Arthritis, etiology of, 573 Arthrospores, 120 Ascococcus, 17, 21 Johnei, 327 Aseptol, germicidal value of, 193, 194 Asporogenous varieties, 126 Attenuation of virulence, 127-129 by antiseptics, 128 by heat, 128 BACILLI, morphology of, 22 Bacillus, generic characters, 18 A of Booker, 492 acidiformans, 474 of acne contagiosa of horses, 508 aeTogenes capsulatus, 514 aeTogenes meningitides, 529 albus cadaveris, 503 alvei, 507 anthracis, 340 anthracis, biological characters, 341 anthracis, morphology, 340 anthracis, pathogenesis, 345 anthracis, spore formation, 341, 343 anthracis, toxic products, 345, 348 anthracis, varieties of, 343 of Babes and Oprescu, 458 of Beck, 524 of Belfanti and Pascarola, 439 bovis morbificans, 525 684 INDEX. Bacillus of bubonic plague, 520 of Bunzl Federn, 520 cadaveris, 541 canalis capsulatus, 506 canal is parvus, 506 of Canon and Pielicke, 515 capsulatus, 454 capsulatus mucosus, 512 cavicida, 448 cavicida Havaniensis, 448 of Cazal and Vaillard, 458 of chancroid, 576 "Bacillus" of cholera, 552 of cholera in ducks, 434 cholerae galliuarum, 429 Bacillus of Chiari, 517 chromo-aromaticus, 505 . coli communis, 462-474 coli communis in bread, 682 coli communis in cystitis, 583 coli communis in peritonitis, 472, 605 coli communis in pyelonephritis, 609 coli communis from stomach, 672 coli communis, varieties of, 466-472 coprogenes foetid us, 497 coprogenes parvus, 447 crassus sputigenus, 449 cuniculicida, 429 cuniculicida Havaniensis, 47 of Demme, 494 dentalis viridans, 501 diphtheriae, 375-380 diphtheria?, biological characters, 375 diphtheria, branching forms, 384 diphtheriae in fauces of healthy per- sons, 385 diphtheria), morphology of, 375 diphtheria!, pathogenesis, 377 diphtheria?, persistence after recov- ery, 384, 385 diphtheria;, toxic products of, 379 diphtheria, varieties of, 380 diphtheriae columbrarum, 382 diphtheriae vitulorum, 383 of Ducrey, 576 of von Dungern, 519 of Emmerich and Weibel, 523 endocarditidis capsulatus, 493 endocarditidis griseus, 493 cntcritidis. 452 erysipelatos suis, 442 erysipelatos suis, pathogenesis, 445 of Eve and Lingard, 424 of Fiocca, 484 f of fowl cholera. 1*.".' of Frettenseuche, 439 of Friedlandcr, 308 galliiiarum, 45o of Gerdcs, 5H7 of Qessncr, 505 Bacillus of Gibier, 478 gingivae pyogenes, 501 of Gplasz, 425 gracilis cadaveris, 516 of grouse disease, 452 of Guillebcau, «, l>, and c, 528, 529 of Harris, 517 heminecrobiophilus, 511 of hog cholera, 434 of hog cholera, varieties of, 438 liominis capsulatus, 450 hydrophilus fuscus, 455 indigogenus, 507 of influenza, 388 of influenza, biological characters, 389 of influenza, pathogenesis, 389 of intestinal diphtheria in rabbits, 384 of Jeffries, 474 of Kartulis, 507 of Koubasoff, 426 lactis aeTogenes, 472 of Laser, 457 of Laser, gas-forming aerobic, 524 leporis lethalis, 478 leprae, 414-416 of Lesage, 493 of Letzerich, 495 of Loeb, 460 of Lucet, 459 of Lumnitzer, 496 of Lustgarten, 422 malaria', 596 mallei, 417-422 mallei, pathogenesis, 419 meningitidis purulenta?, 504 of Mereshkowsky, 523 monachae, 528 mucosus ozaenae, 518 murisepticus, 442 Neapolitanus, 462 necrophorus, 497 of Nicolaier, 517 of Nocard, 426 oadematis afrobicus, 494 oedematis maligni, 537 cedematis maligni No. 11 (Novy), 545 of Okada. 509 phlegmones emphysematosa?, 547 piscicidus, 525 piscicidus agilis, 522 pneumonia', 308 pneumosepticus, 454 prodigiosus, pigment production, 132 pseudo-tuberculosis, 500, 530 pseudo-tuberculosis muriuin, 530 pulpa1 pyogenes, 501 of purpura ha'morrhagica of Babes, 510 INDEX. 685 Bacillus of purpura haemorrhagica of Kolb, 511 of purpura haemorrhagica of Tizzoni and Giovannini, 510 pyocyaneus, 479-484 pyocyaneus ft (P. Ernst), 482 pyocyaneus, in otitis media, 482 pyocyaneus, pathogenesis, 480 pyocyaneus, pigments produced by, 131 pyocyaneus pericarditidis, 482 pyogenes filiformis, 526 pyogenes fcetidus, 450 pyogenes soli, 512 of rabbit septicaemia, 429 of rhinoscleroma, 425 of Rinderseuche, 440 No. I. of Roth, 509 No. II. of Roth, 509 salivarius septicus, 310 sanguinis typhi, 515 of Schimmelbusch, 495 of Schou, 497 septicaemiae haemorrhagicae, 429-434 septicaemiae haemorrrhagicae, attenu- ation of, 432 septicaemiae hsemorrhagicae, patho- genesis of, 432 septicus acuminatus, 502 septicus agrigenus, 442 septicus kerato-malaciae, 502 septicus sputigenus, 310 septicus ulceris gangraenosi, 502 septicus vesicae, 505 smaragdinus foetidus, 453 of swine plague, Marseilles, 439 of symptomatic anthrax, 542 tenuis sputigenus, 457 tetani, 531 tetani, aerobic cultures of, 548 tetani, cultivation of, 533 tetani, pathogenesis, 534 of Tornmasoli, 497 of Tricomi, 503 tuberculosis, 393 tuberculosis, action of sunlight on, 403 tuberculosis, attenuation of viru- lence, 404 tuberculosis, biological characters, 397 tuberculosis, branching forms of, 412 tuberculosis, in bronchial glands of healthy persons, 413 tuberculosis, cultivation of, 399 ^tuberculosis, duration of vitality, 403 tuberculosis, morphology of, 394 tuberculosis, pathogenesis, 408 tuberculosis, spore formation (?), 398 tuberculosis, staining of the, 394 Bacillus tuberculosis, thermal death - point, 154 tuberculosis, toxic products of, 405 tuberculosis galliriarum, 410 typhi abdominalis, 358 typhi abdominalis, biological char- acters, 359 typhi abdominalis, detection in water, 365-368 typhi abdominalis, flagella of, 359 typhi abdominalis, morphology of, 358 typhi abdominalis, pathogenesis, 364 typhi abdominalis, pus production by, 369 typhi abdominalis, thermal death- point, 363 typhi abdominalis, toxic products of, 363 typhi murium, 457 typhosus, 358 of Unna and Hodara, 527 of Utpadel, 507 varicosus conjunctivae, 504 venenosus, 513 veneuosus brevis, 513 venenosus invisibilis, 513 venenosus liquet'aciens, 514 Bacteria in the air, methods of collect- ing, 625-631 in the air, results of researches, 632 in the air, in school -rooms, 635 in the air, species found, 634 chemical composition of, 121 of mouth, 661. 666 of mouth, peptonizing action of, 663 in the soil, 652-657 in the soil, kinds of, 654, 656 in the soil, method of studying, 652 in the soil, number of, 653 structure of, 115 on surface of the body, 658 thermal death -point of, 150 in tissues, methods of staining, 34 in water, 636-651 in water, collection of water, 637 in water, enumeration of, 639, 641 in water, species found, 648 in wounds, 276 Bacteridie du Charbon. 340 Bacterie septique, 583 Bacteriology, literature of, 8 Bacterium, 18 ae"ruginosum, 479 coli commune, 462 termo, 676 Barium chloride, antiseptic value of, 184 Bees, infectious disease of, 507 Beggiatoa, 19 Benzene, germicidal value of, 194 Benzoic acid, germicidal value of, 179 Benzo-naphthol, germicidal value of, 202 Beri-beri, etiology of, 573 686 INDEX. Binary division, 117 Biological characters, modifications of, 126-131 Biskra button, etiology of, 330 Blood serum, as a culture medium, 75 serum, coagulation of, 56 serum, collection of, 37 serum, germicidal action of, 204, 236 serum, sterilization of, 55 Booker's bacilli, 468-472 Boracic acid, germicidal value of, 178 Bouillon, 41 Bread, bacteria in, 682 Brieger's bacillus, 448 Bromine, germicidal action of, 174 Bronchitis, bacteria in, 496, 574 Broncho-pneumonia, bacteria in, 574 Brownian movement, 117 Bubo, bacteria in, 574 Bubonic plague, bacillus of, 520 plague bacillus, discovery of, 8 Bttffelseuche, bacillus of, 429 Butter, bacteria of, 680 " cheesy, " bacteria of, 680 rancid, bacteria of, 680 Butyric acid, germicidal value of, 179 acid, production of, 134 CADAVERIN, 143 Cadavers, bacteria of, 674 Calcium chloride, antiseptic value of, 184 hydroxide, germicidal value of, 180 hypochlorite, germicidal value of, 184 light for photographing bacteria, 106 Camphor, antiseptic value of, 194 Capsule bacilli, 517, 518, 519 bacilli in ozana, 603 composition of, 116 Carbol-fuchsin solution, 29 Carbolic acid, germicidal value of, 195 Carbon, how obtained, 123 dioxide, not a germicide, 170 Carbonic oxide, not a germicide, 171 Carcinoma, bacteria in, 575 Catarrh, nasal, 294 Catarrhal inflammations, 226 Caterpillars, infectious disease of, 338 Caucasian milk ferment, 136 Cell membrane, 115, 116 Cerebro-spinal meningitis, etiology of, 575 Cervix uteri, bacteria in, 665 Chalazion, bacteria in, 576 Chancroid, etiology of, 57(1 Charbon, etiology of, 339 symptomatique, bacillus of, 542 Cheese, bacteria in, 680 ripening of, 680 Chemiotaxis, 247 Chloral hydrate, antiseptic value of, 185 Chlorine, germicidal action of, 173 Chloroform, germicidal action of, 173 Cholera, Asiatic, etiology of, 578 in ducks, bacillus of, 434 in fowls, bacillus of, 429 infantum, bacteria in, 468-471, 578 infautum, etiology of, 487 immunity, 568 nostras, bacteria in, 561, 578 des poules, bacillus of, 429 ptomaines, 146 spirillum, biological characters, 553 spirillum, cholera-red reaction, 557 spirillum, duration of vitality in earth, 655 spirillum, duration of vitality in water, 647 spirillum, morphological characters, 552 spirillum, pathogenesis, 560 spirillum, thermal death-point, 556 spirillum, toxic products of, 558 spirillum in water, detection of, 650 in swine, bacillus of, 434 Cholin, 144 Chromic acid, germicidal action of, 177 Chromogenes, 14 Chromogenic bacteria, 132 bacteria in milk, 678 Chronic infectious diseases, etiology of, 392 Citric acid, germicidal action of, 178 Cladpthrix, 19, 24 Clarification of culture media, 42, 43 Classification, 10-19 biological, 13 morphological, 13 of Baumgarten, 12 of Colin, 11 of Davaine, 3, 10 of Dujardin, 3 of Ehrenberg, 3, 10 of Hoffmann, 10 of Nageli, 11 of Zopf, 12 Clostridium, 18 Coal-tar products, antiseptic action of, 193-203 Coffee infusion, germicidal value of, 196 Cohn's solution, 40, 123 Cold, germicidal action of, 149 Colon bacillus, 462 bacillus in water, detection of, 650 Colonies of bacteria, 70 counting, 640 Comma bacillus of Koch, 552 Conjunctiva, bacteria of, 659 Conjunctivitis, bacteria in, 294, 507, 579 Contact preparations, 27 "Corn-stalk disease, " etiology of, 580 Cory/a, bacteria in, 581 Cover-glass preparations, 25 Crenothrix, 19 Creolin, germicidal value of, 197 INDEX. 687 Creosote, germicidal value of, 197 Cresol, germicidal value of, 197 Culture media, 37-49 media, filtration of, 42 media, liquid, 38-41 media, natural, 37 media, reaction of, 124 media, solid, 41-49 Cultures in liquid media, 60-66 on potato, 76 in solid media, 67-77 Cultivation of anaerobic bacteria, 78-85 Cupric chloride, antiseptic value of, 185 sulphate, germicidal value of, 185 Cystitis, bacteria in, 505, 581 DARMBACILLUS of Schottelius, 497 Decolorization, 28 Defensive proteids, 236 Dengue, bacteria in, 584 Dental caries, bacteria in, 501, 584 Desiccation, germicidal action of, 155 Diaphtherin, germicidal value of, 198 Diarrlioea, bacteria in, 585 in calves, 585 green, of infants, 493 summer, etiology of, 473 Dimensions of bacteria, 20 Dimethylamine, 144 Diphtheria bacillus, antitoxin of, 380 antitoxin, results of treatment, 386 bacillus, pseudo-, 380 discovery of, 7 etiology of, 371 immunity, 379 mixed infection, 385 toxin, action of, 225 toxalbumin, 146 Diphtheritic inflammations, 225 Diplococcus, 17 intercellularis meningitidis, 322 of pneumonia in horses, 334 pneumoniae, 310 Disinfecting solutions, standard, 208 Disinfection of clothing, bedding, etc., 209 of dead bodies, 209 in diphtheria, 218 of excreta, 208, 213-217 of the hands, 212 of merchandise and the mails, 210 practical directions for, 208-218 of privy vaults, 217 of rags, 210 of railway cars, 210 of ships, 210 of sick-room, hospital wards, etc., 209 by steam, 210 Disinfektol, germicidal value of, 198 Distemper in dogs, etiology of, 586 Distilled water, bacteria in, 645 Dogs, infectious diseases of, 586 Double staining, 28 Drop cultures, 62 Dust, bacteria in, 634, 635 of streets, bacteria in, 656 Dysentery, bacteria in, 586 ECLAMPSIA, bacteria in, 587 Eczema, bacteria in, 587 epizoOtica, bacteria in, 589 Eggs as a culture medium, 40 rotten, bacteria of, 682 Egyptian ophthalmia, bacteria in, 507, 580, 599 Ehrlich's stain, 29 Ehrlich-Weigert method, 30 Electric light, germicidal action of, 158 light for photographing bacteria, 105 Electricity, germicidal action of, 158 Eisner's method, 367 Emmerich's bacillus, 462 Empyema, etiology of, 589 Endocarditis, ulcerative, 283 etiology of, 590 Endogenous spores, 118 Endometritis, bacteria in, 591 Endosporium, 119 Environment, changes due to, 222 Enzymes, 131 Epidermis, bacteria of, 659 Van Ermengen's method, 33 Erlenmeyer flasks, 61 Erythema, bacteria in, 591 nodosum, bacillus of Demme, 494 v. Esmarch's roll tubes, 74 Essential oils, antiseptic value of, 198 Ether, germicidal value of, 198 Eucalyptol, antiseptic value of, 199 Euphorin, antiseptic value of, 200 Exospprium, 119 Experiments upon animals, 94-100 Eye, inoculations in, 97 FACULTATIVE anaerobics, 16 parasites, 15, 125 Faeces, bacteria of, 671 Fermentation tube, 66 Ferric chloride, antiseptic value of, 185 Ferrous sulphate, antiseptic value of, 185 Filtration of culture media, 42 Finkler and Prior, spirillum of, 561 Fiocca's method, 32 Fish, infectious diseases of, 522, 523, 525 " Fixing " on cover glass, 26 Flagella, 116 methods of staining, 32 Flesh-peptone-gelatin, 41, 67 peptone solution, 41 Foot and mouth disease, bacteria in, 589 Formaldehyde, germicidal value of, 200 Formalin, germicidal value of, 200 Formic acid, germicidal value of, 179 Foul brood, bacillus of, 507 f>88 INDEX. Fowl cholera, bacillus of, 429 Freezing, germicidal action of, 149 Friedlander's bacillus, 309 method, 30 Fungi, spores of, in the air, 624 Furunculosis, etiology of, 592 GABBETT'S method, 30 Gallic acid, germicidal value of, 179 Gangrene, bacteria in, 503, 592 hospital, 224 Gas phlegmon, etiology of, 593 Gases, germicidal action of, 168-173 Gaslight for photographing bacteria, 107 Gastric juice, germicidal action of, 668 Gelatin, liquefaction of, 132 nutrient, preparation of, 41 Germicidal action, conditions governing, 165-167 value, tests of, 163-165 Glanders, bacillus of, 417 bacillus, discovery of, 7 diagnosis of, 421 Glycerin-agar, 43 Gold chloride, germicidal value of, 186 Gonococcus, 295 discovery of, 7 Gram's method, 29, 35 Granuloma fungoides, 593 Green pus, bacillus of, 479 Growth, conditions of, 122-125 Guaiacol, germicidal value of, 200 H.EMATOCOCCUS bovis, 334 Hasmoglobinuria of cattle, etiology of, 334 Hail, bacteria in, 642 Haloid elements, germicidal action of, 173-175 Heat, dry, action of, 150 germicidal action of, 149 moist, action of, 150 Heterogenesis, 5 Hog erysipelas, bacillus of, 442 Holtz' method, 365 Hot air sterilizer, 52 Hydrant water, bacteria in, 644 Ilydrocele fluid, as a culture medium, 40 Hydrochloric acid, germicidal action of, 177 Hydrofluoric acid, germicidal action of, 175 Hydrogen apparatus, 83 not a germicide, 169 peroxide, germicidal action of, 169 Hydrophobia, etiology of, 593 inoculations, Pasteur's method, 97, 246 Hydrosulphuric acid, germicidal action of, 171 acid, production of, 138 Hydroxylamine, germicidal value of, 200 ICE, bacteria in, 643 Ichthyol, germicidal value of, 200 Icterus, infectious, bacteria in, 594 Immunity, acquired, 242 acquired, explanation of, 274 alkalinity of blood a factor, 238 Buclmer's experiments, 236, 237 of carnivora, 234 due to antitoxins, 266 exhaustion theory, 249 Hankin's experiments, 237 through mother's milk, 271 neutralized by depressing agencies, 241 neutralized by chemical substances, 240 Pasteur's method of producing, 245- race, 234 relative value of, 241 retention theory, 251 by sterilized cultures, 245 from vaccination, 244 in various diseases of man, 243 vital resistance theory, 252 Impf tetanus bacillus, 439 Incubating ovens, 86, 87 Incubator of D' Arson val, 92 Indol, germicidal value of, 201 Infection, channels of, 229-232 general, 226 influence of quantity, 227 localized, 223 mixed, 228 rapidity of, 230 secondary, 227 through intestine, 230 through lungs, 230 through unbroken skin, 229 through wounds, 229 Infectious diseases, bacteria in, 571-619 Influenza bacillus, discovery of, 8 etiology of, 387-391 bacillus, pseudo-, 391 of horses, etiology of, 334, 595 Infusoria, 3 Inoculation experiments, conditions to be observed, 98 Inoculations, technique of, 95, 96 Insects, infectious diseases of, 595 Intestine, bacteria of, 670, 673 Involution forms, 23 Iodine, germicidal action of, 173 trichloride, germicidal action of, 174 lodoform, germicidal action of, 174 ether, germicidal action of, 175 lodol, germicidal action of, 175 Izal, germicidal value of, 201 JEQUIRITY solution, 47 KASESPIRILLEN, 563 Herat itis, etiology of, 595 Koch's plate method, 72 syringe, 95 INDEX. 689 Kruse's method, 77 Kiihne's method, 35 LACTIC acid, germicidal action of, 178 acid, production of, 134 Lake water, bacteria in, 643 Lanolin, germicidal value of, 201 Lead chloride, antiseptic value of, 186 nitrate, antiseptic value of, 186 Leprosy bacillus, discovery of, 7 etiology of, 414 Leptothrix, 19 Leucocytha?mia, bacteria in, 596 Leuconostoc, 17 Light, germicidal action of, 155 Liquefaction of gelatin, 132 Liquefying bacteria, characters of growth, 70 Literature of bacteriology, 8 Lithium chloride, antiseptic value of, 186 Lochial discharge, bacteria of, 664 Loffler's method, 32 solution, 29 Loretin, germicidal value of, 201 Lymphangitis, bacteria in, 596 Lysol, germicidal value of, 201 MADURA foot, bacteria in, 596 Malachite green, germicidal value of, 194 Malarial diseases, etiology of, 596 Malic acid, germicidal value of, 179 Malignant oedema, 537 oedema, immunity from, 540 Mallein, 148, 422 " Malta fever, " micrococci in, 528, 597 Manganese protochloride, antiseptic value of, 186 Marsh gas, production of, 138 Mastitis, bacteria in, 597 bovine, etiology of, 329, 333, 338, 528, 597 in sheep, etiology of, 332 Measles, bacteria in, 515, 598 Measurements of bacteria, 20 Meat infusions, 40 Meats, bacteria in, 680 smoked, bacteria in, 681 Meatus urinarius, bacteria of, 664, 666 Meconium, bacteria in, 670 Meningitis, bacteria in, 337, 504, 529 Micrococcus pneumonia? crouposae in, 313 Mercuric chloride, germicidal value of, 186 cyanide, germicidal value of, 188 iodide, antiseptic value of, 188 Mercury, oxides of, antiseptic value of, 189 Merismopedia, 17, 22 Metallic salts, germicidal value of, 182-192 Metchnikoff theory, 256. 258-265 Methane, germicidal action of, 171 Methylamin, 144 Methyl -guanidin, 145 Mice, infectious diseases of, 598 Micrococci, general characters of, 17 morphology of, 21 Micrococcus, generic characters, 17 of Almquist, 337 askoformans, 327 botryogenus, 327 of bovine mastitis, 329 of bovine pneumonia, 329 of Bruce, 528 of Demme, 331 endocarditidis rugatus, 332 No. II. of Fischel, 336 of Forbes, 338 of gangrenous mastitis in sheep, 332 gingivae pyogenes, 335 gonorrhoea3, 295 gonorrhceae, cultivation of, 297 gonorrhoea?, pathogenesis, 298 of Heydenreich, 330 insectorum, 527 of Kirchner, 336 lanceolatus, 310 of Manfredi, 328 ovatus, 330 Pasteuri, 310 pneumonia? crouposae, 310 pneumonia? crouposa? in abscesses, 313 pneumonia? crouposa?, action of germicides on, 316 pneumonia? crouposa?, biological characters of, 314 pneumonia? crouposa? in empyema, 590 pneumonia? crouposae in endocardi- tis, 313, 590 pneumonias crouposa? in healthy eyes, 600 pneumonia? crouposa?, immunity from, 316, 320 pneumonia? crouposa? in menin- gitis, 312 pneumonia? crouposae, morphology of, 314 pneumonia? crouposa? in otitis media, 313, 601 pneumonia? crouposae, pathogenesis, 318 pneumonia? crouposae in pleuritis, 606 pneumonias crouposse in pneumonic sputum, 311 pneumonia? crouposa? in purulent keratitis, 595 pneumonia? crouposa? in saliva, 310 pneumonias crouposae, varieties of, 317 690 INDEX. Micrococcus of progressive abscess for- mation in rabbits, 323 of progressive tissue necrosis in mice, 323 of pyaemia in rabbits, 324 pyogenes tennis, 286 salivarius septicus, 324 of septicaemia in rabbits, 324 subflavus, 324 tetragenus, 326 of trachoma ( ?) , 325 Micromillimetre, 20 Microzyma bombycis, 330 Milk, antitoxin in, 270 bacteria in, 232, 677 bitter, bacteria of, 680 chromogenic bacteria in, 678 coagulation of, 677 as a culture medium, 39 fermentation of, 136, 677 of healthy women, bacteria in, 679 tubercle bacillus in, 679 viscous fermentation of, 678 Milzbrandbacillus, 340 Modes of action, 221-228 MOller's method, 32 Morphia hydrochlorate, antiseptic value of, 189 Morphology, 20-24 Mould fungi, in the air, 624 Mouse septicaemia, bacillus of, 442 Mouth, bacteria in, 661, 666 Movements due to flagella, 117 character of, 117 Mucous membranes, bacteria of, 659 Mucus, germicidal action of, 206 Muscarin, 144 Mustard, oil of, antiseptic value, 202 Mykoprotein, 121 Mytilotoxin, 145 NAPHTHOL, germicidal value of, 201 Nasal catarrh, 294 mucus, bacteria in, 660 Natural immunity, explanation of, 239 Neisser's method, 31 Nephritis, bacteria in, 495, 598 Neuridin, 143 Neurin, 144 Nickel sulphate, antiseptic value of, 189 Nitrates, reduction of, 140 Nitric acid, germicidal action of, 177 Nitrification, 140 Nitrogen, how obtained, 123 dioxide, germicidal action of, 171 Nitrous acid, germicidal action of, 177 oxide, not a germicide, 171 Non- liquefy ing bacteria, characters of growth, 68 Nose, bacteria in, 660, 666 Nosema bombycis, 330 Nosophen, germicidal action of, 175 Nucleins, germicidal action of, 206, 239 Nucleus in bacteria, 115 staining of, 116 Nutrient agar, 43 agar, filtration of, 44 agar, preparation of, 46 OIDIUM lactis, in cheese, 680 Oleic acid, germicidal value of, 179 Ophthalmia, etiology of, 599 Orthpchromatic plates, 103 Osmic acid, germicidal action of, 177 Osteomyelitis, 282, 600 Otitis media, Bacillus pyocyaneus in, 482 media, etiology of, 293, 601 media, Micrococcus pneumonia crouposae in, 313 Oxalic acid, germicidal action of, 178 Oxygen, germicidal action of, 168 how obtained, 122 Ozaena, bacteria in, 496, 518, 602 Ozone, germicidal action of, 168 PANARITIUM, etiology of, 603 Panhistophytpn ovatum, 330 Panophthalmia, bacteria in, 600 Papin's digester, 54 Parasites, 15, 221 facultative, 124 strict, 124 Parasitism, 124 Parietti's method, 366 Parotitis, bacteria in, 603 Parrot disease, etiology of, 338 Pasteur-Chamberland filter, 57 Pasteur's flasks, 61 method of producing immunity, 245 solution, 40, 123 Pathogenes, 14 Pathogenic bacteria, 15 bacteria in water, 646 bacteria in water, detection of, 650 bacteria in water, duration of vital- ity. 646 power, explanation of, 222 saprophytes, 462 Pebrine, etiology of, 330 Pemphigus, bacteria in, 331, 603 neonatorum, etiology of, 337 Peppermint, oil of, antiseptic value, 202 Peptonizing action of bacteria in saliva, 663 ferment, 132 Peptotoxin, 145 Pericarditis, etiology of, 604 Periostitis, etiology of, 600 Peritonitis, etiology of, 472, 604 Petri's dishes, 73 Phagocytosis, 255 Phenol, germicidal value of, 195 Phosphorescence, 141 Phosphoric acid, germicidal action of, 177 Photographing bacteria, 101-111 INDEX. 691 Photographing bacteria, amplification, bacteria, apparatus required, 103 Photomicrographs, value of, 101 Photomicrography, Borden's method, 109 by calcium light, 106 developing solution, 111 by electric light, 105 by gaslight, 107 by oil-light, 108 Stern berg's method, 107 by sunlight, 105 Phragmidiothrix, 19 Phylaxins, 221 Physical agents, influence of, 149-159 Pigment production, 130 Pittield's method, 33 Plants, infectious diseases of, 605 Plate method of Koch, 639 Platinum bichloride, antiseptic value of, 189 Pleuritis, etiology of, 606 Pleuro-pneumonia bacillus, discovery of, 8 of cattle, etiology of, 499 of cattle, protective inoculations in, 500 in calves, bacteria in, 529 Pneumobacillus liquefaciens bovis, 499 septicus, 529 Pneumococcus (Friedlander) , 308 Pneumonia antitoxin, 268 bovine, etiology of, 329 croupous, etiology of, 300-307, 607 in horses, etiology of, 334 micrococcus, discovery of, 7 Post-mortem examinations, 99 Potassium acetate, antiseptic value of, 189 arsenite, antiseptic value of, 189 bichromate, antiseptic value of, 189 bromide, antiseptic value of, 189 carbonate, antiseptic value of, 189 chlorate, antiseptic value of, 189 chromate, antiseptic value of, 189 cyanide, antiseptic value of, 189 hydroxide, germicidal value of, 179 iodide, antiseptic value of, 189 permanganate, germicidal value of, 190 Potato paste, preparation of, 48 Pregl's method, 36 Pressure, germicidal action of, 159 regulator of Moitessier, 88 Products of vital activity, 130-142 Proteus capsulatus septicus, 452 fluorescens, 527 Hauseri, 488 of Karlinsky, 489 lethalis, 491 mirabilis, 489 septicus, 491 Proteus vulgaris, 485 vulgaris in cholera infantum, 487 vulgaris in cystitis, 584 vulgaris in pyelonephritis, 609 Zenkeri, 491 Protoplasm of bacteria, 116 granules in, 116 Pseudo-diphtheritic bacillus, 380 Pseudo-diplococcus pneumonias, 335 Pseudo-leukaemia, bacteria in, 607 Pseudo-tuberculosis, bacteria in, 608 Ptomaines, non -toxic, 143 production of, 222 toxic, 144 Puerperal fever, etiology of, 608 Pure cultures, 37 cultures obtained by inoculation, 100 Purpura hrcmorrhagica, bacteria in, 510, 511, 613 Pus cocci, in inflammations of mucous membranes, 293 cocci in otitis media, 293 formation, 223, 275 Putrefaction, bacteria of, 675 products of, 139 Putrescin, 144 PyaBmia, definition of, 608 Pyelonephritis, etiology of, 608 Pyocyanin, 131, 480 Pyogenic bacteria, 275-299 Pyoktanin, germicidal value of, 194 Pyosalpinx, bacteria in, 610 QUININE hydrochlorate, antiseptic action of, 190 sulphate, antiseptic value of, 190 RABBIT septicaemia, bacillus of, 429 Rain-water, bacteria in, 642 Ranvier's moist chamber, 63 Rauschbrand, bacillus of, 542 Relapsing fever, etiology of, 549 fever inoculation experiments, 551 fever spirillum, discovery of, 7 Reproduction, by binary division, 117 by spores, 118 rapidity of, 118 Rheumatic fever, etiology of, 610 Rhinitis fibrinosa, bacteria in, 611 Rhinoscleroma, bacillus of, 425 Ricin, experiments of Ehrlich, 270 Rinderseuche, bacillus of, 429 Roll tubes, v. Esmarch's, 74 Rothlauf, bacillus of, 442 Rotz bacillus, 417 Rouget, bacillus of, 442 protective inoculations, 447 SALICYLIC acid, germicidal action of, 178 Saliva, bacteria in, 662 germicidal action of, 663 Salomonson's method, 77 602 INDEX. Salt, not a germicide, 681 Saprin, 144 Saprol, antiseptic value of, 202 Saprophytes, 15 Saprophytic bacteria, where found, 623 Sarcina, 17, 22 Sarcinae, from stomach, 672 Scarlet fever, bacteria in, 612 Scheinfaden, 118 Schultz's method, 46 Schweineseuche, bacillus of, 429 Scorbutus, bacteria in, 612 Screens, colored in photomicrography. 103, 106 Sea- water, bacteria in, 647, 651 Sepsin poisoning, 488 Septicaemia, 226 bacteria in, 612 in cattle, bacteria in, 613 definition of, 428 in fowls, bacteria in, 613 hnmorrhagica, bacilli in, 458 in swine, bacteria in, 613 Sewers, bacteria in, 644 Silicate jelly, 47 Silkworm, diseases of, 330 Silver chloride, germicidal value of. 190 nitrate, germicidal value of, 190 Skatol, antiseptic value of, 202 Small-pox, antitoxin of, 274 Smear preparations, 26 Smoke, antiseptic value of, 202 Snake venom, antitoxin of, 269 Snow, bacteria in, 642 Soap, germicidal value of, 181 Sodium borate, antiseptic value of, 191 carbonate, antiseptic value of, 191 chloride, antiseptic value of, 191 hydroxide, germicidal value of, 180 hyposulphite, antiseptic value of, 191 sulphite, antiseptic value of, 191 Solid culture media, 41 Soluble ferments, 140 Sozoiodol acid, germicidal action of, 175 Spirilla, morphology of, 24 Spirillum, generic characters, 18 an serum, 551 cholera? Asiatic*, 552-561 choleras Asiatics, varieties of, 565 differential diagnosis, 568 of Deneke, 563 of Finkler and Prior, 561 Metchnikovi, 563 Obermeieri, 549 tyrogenum, 563 Spirochaete, 18 anserina, 551 Obermeieri, 549 Spirulina, 18 Spontaneous generation, 4 Spores, discovery of, 5 Spores, formation of. 118 germination of, 119 location of, 119 methods of staining, 31 thermal death -point of, 153 Sputum, examination of, for tubercle bacilli, 395 Stab cultures, 87 Staining bacteria to photograph, 103 bacteria in tissues, 34 of cover-glass preparations, 27 tlagella, 32 methods, 25-36 spores, 31 Staphylococci, 21 Staphylococcus aureus, toxic products of, 147 epidermidis albus, 284 pyogenes albus, 284 pyogenes albus in "stitch abscess, !r 285 pyogenes aureus, 277-284 pyogenes aureus, action of germici- dal agents, 278 pyogenes aureus, pathogenesis, 280 pyogenes aureus, pus production by, 281 pyogenes citreus, 285 pyosepticus, 337 salivarius pyogenes, 323 Steam, sterilization by, 51 sterilizers, 53 Sterilization, 5 of culture media, 50-59 by discontinuous heating, 51 by dry heat, 52 by moist heat, 51 by filtration, 56 Sterilizers, hot air, 52 steam, 53 Sternberg's bulbs, 64 Stick cultures, 67 cultures, long, for anaCrobics, 7& Stomach, bacteria of, 668 dilated, bacteria of, 672 Stomatitis, bacteria in, 614 Streak cultures, 75 Streptococci, 22 Streptococcus, generic characters, 17 agalactiau contagiosae, 338 articulorum, 290 bombycis, 330 of Bonome, 337 brevis, 287 conglomeratus, 512 coryza1 contagions equorum, 334 erysipelatos, 286 lanceolatus Pasteuri, 310 longus, 287 of JVIunneberg, 331 of mastitis in cows, 333 perniciosus psittacorum, 338 pyogenes, 286 INDEX. 693 Streptococcus pyogenes, action of ger- micides upon, 289 pyogenes, in diphtheria, 290 pyogenes, pathogenic action of, 289 pyogenes, in puerperal fever, 291 pyogenes, in ulcerative endocardi- tis, 290 pyogenes malignus, 292 septicus, 330 septicus liquefaciens, 335 Structure of bacteria, 115 Sulphur dioxide, germicidal action of, 172 Sulphuric acid, germicidal action of, 176 Sulphurous acid, germicidal action of, 176 Sunlight, germicidal action of, 155 for photographing bacteria, 105 Susceptibility, 233 of young animals, 233 Swine plague, bacillus of, 429 Sycosis, bacteria in, 496 Symbiosis, 125, 548 Symptomatic anthrax, bacillus of, 542 immunity from, 545 Syphilis, bacteria in, 422-425 TAXNIC acid, germicidal value of, 179 Tartaric acid, germicidal value of, 179 Temperature, limits of growth, 123 Tetanin. 146, 533 Tetanotoxin, 146, 534 Tetanus antitoxin, 268, 537 bacillus, discovery of, 8 bacillus of. 531-537 bacillus, toxic products of, 547 immunitv, 536 Tetrads, 22 Texas fever, of cattle, etiology of, 614 Thermal death-point of bacteria, 151 Thermo-regulator of Bohr, 88 of Roux, 92-93 of Reichert, 88 Thermo-regulators, 78-92 electro-magnetic, 90, 93 Thymic acid, germicidal value of, 179 Thymol, antiseptic value of, 203 Thymus bouillon, 246 Tin chloride, antiseptic value of, 191 Tobacco smoke, antiseptic value of, 203 Torula chains, 22 Toxaemia, definition of, 428 Toxalbumins, 146 vegetable, 270 Trachoma, etiology of, 294, 325, 614 Trikresol, germicidal value of, 198 Trimethylamine, 144 Tubercle bacilli, dead, pathogenic action of, 413 bacilli in dust, 634, 635 bacillus, attenuation of virulence, 413 bacillus, demonstration of, in spu- tum. 414 bacillus, discovery of, 392 Tubercle bacillus, methods of staining, 30 Tuberculin, 148 Koch's method of preparing, 406 test for cattle, 407 Tuberculosis, acquired immunity from, 407, 413 in cattle, prevalence of, 410 Turpentine, oil of, antiseptic value, 202 Typhoid bacillus, discovery of, 7 bacillus, duration of vitality in water, 647 bacillus, experiments on animals, 353 bacillus, toxic products of, 145, 147, 356 bacillus in water, detection of, 650 bacillus, where found, 352 fever, etiology of, 349-357 infection, predisposing causes, 369 Typhotoxin, 145 Tyrotoxicon, 145, 678 Typhus fever, bacteria in, 555, 616 ULCUS corn*, etiology of, 595 Underclothing, bacteria attached to, 656 Urea, fermentation of, 136 Urine, bacteria in, 232 as a culture medium, 39 germicidal action of, 207 Urobacillus liquefaciens septieus, 582 VACCINIA, bacteria in, 616 Vagina, bacteria in, 664, 665 Valerianic acid, germicidal value of, 179 Varicella, bacteria in, 616 Variola, bacteria in, 616 Vegetable infusions as culture media, 41 Vibrio, 18 of Asiatic cholera, 552 Metchnikovi, 563 proteus, 561 Vibrioniens, 3 Vibrion septique, 537 Virulence, attenuation of, 127, 129 recovery of, 129 Viscous fermentation, 137 fermentation in milk, 678 WASH- WATER, bacteria in, 659 Water, pathogenic bacteria in, 646 Wells, bacteria in, 644 Whooping-cough, bacteria in, 617 Wildseuche, bacillus of, 429 Wool-sorter's disease, 339 Wurtz's method, 367 YELLOW-FEVER, bacteria in, 617 ZIEIIL-NEELSON method, 30, 35 Ziehl's solution, 29 Zinc chloride, antiseptic value of, 191 sulphate, antiseptic value of, 192 Zooglcea, 21 Zymogenes, 14 THE LIBRARY UNIVERSITY OF CALIFORNIA San Francisco Medical Center THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW Books not returned on time are subject to fines according to the Library Lending Code. Books not in demand may be renewed if application is made before expiration of I6an period. RETURNED APR 14 1969 CEIPI 30m-10,'61 (C3941s4)4128 QR46 Sternberg, G.M. S83 Text-book of bacteriology 1896 6011)5